characterization of structure-function...
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CHARACTERIZATION OF STRUCTURE-FUNCTION CORRELATIONS OF NOVEL JAK2 SMALL MOLECULE INHIBITORS AND THEIR MECHANISMS OF ACTION
By
ANURIMA MAJUMDER
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2011
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© 2011 Anurima Majumder
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To my Mom, Dad and Arjun for their constant love and support
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ACKNOWLEDGMENTS
I would like to acknowledge all those people who have made this dissertation
possible and because of whom my graduate experience has been one that I will cherish
forever. First, I would like to thank my mentor, Dr. Peter P. Sayeski. I consider myself
fortunate to have had Peter as my advisor. His patience, support, encouragement and
constant guidance throughout my graduate studies has helped me overcome problems
and achieve my goals. He not only taught me research techniques, but also guided me
on how to improve my scientific writing and presentation skills. Peter has been more
than just a mentor for graduate studies by being readily available to help me out with
research problems, career options as well as any other questions that I have had.
Second, I would like to thank the members of my supervisory committee, Drs. Kathleen
Shiverick, Thomas Rowe, Brian Law and William Slayton, for their invaluable guidance,
insight and advice through the course of my graduate studies.
I would also like to thank a few people who have taught me or helped me with
various techniques over these past four years. Special thanks to Andrew Magis and
Lakshmanan Govindasamy for helping me with the molecular docking studies. I would
also like to thank Marjorie Chow and Kanchana Karrupiah. The two-dimensional gel
electrophoresis studies would not have been possible without them. In addition, I also
thank Doug Smith and Steve McClellan for their help with fluorescent microscopy and
flow cytometry, respectively.
I would next like to thank all past and present members of the Sayeski Lab:
Jacqueline Sayyah, Robert Blair, Nicholas Figueroa, Shigeharu Tsuda, Dr. Sung Park,
Annet Kirabo, Kavitha Gnanasambandan and Rebekah Baskin. They not only provided
an environment conducive to learning and research but, also made lab life a lot more
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enjoyable. I am going to miss all the fun stuff we did together both inside and outside
lab. Special thanks to my friends for their support and companionship during my stay in
Gainesville. Thank you for being there for me when times were stressful.
Lastly, I would like thank my family for their unending advice, encouragement, love
and support without which I would never have been able to complete my graduate
studies. I am deeply indebted to my parents for their constant wisdom and
encouragement. I have come so far in life only because of them and the values that
they have instilled in me. I would also like to thank my little sister, Anusmita, for
reducing my stress by providing comic relief. I am also grateful to my in-laws for their
support and encouragement. Finally, thanks to my husband, Arjun, for bearing with me
and my mood swings and keeping me sane during times of stress. His love,
understanding and support have been invaluable.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 9
LIST OF FIGURES ........................................................................................................ 10
LIST OF ABBREVIATIONS ........................................................................................... 12
ABSTRACT ................................................................................................................... 13
CHAPTER
1 INTRODUCTION .................................................................................................... 16
Tyrosine Kinases .................................................................................................... 16 Jak2 Tyrosine Kinase.............................................................................................. 17
History .............................................................................................................. 17 Structure ........................................................................................................... 18
Jak/STAT Signaling Paradigm .......................................................................... 20 Jak2 and Cancer ..................................................................................................... 21
Jak2 in Solid tumors ......................................................................................... 21 Jak2 in Hematological Malignancies ................................................................ 22
Jak2 chromosomal translocations and hematological malignancies .......... 23 Jak2 point mutations and hematological malignancies .............................. 23
Myeloproliferative Neoplasms ................................................................................. 24 Jak2 Mutations in Myeloproliferative Neoplasms .............................................. 25
Jak2 Inhibitors for the Treatment of Myeloproliferative Neoplasms .................. 27 Rationale for Studies............................................................................................... 34
2 A46, A BENZOTHIOPHENE DERIVED COMPOUND, SUPPRESSES JAK2-MEDIATED PATHOLOGIC CELL GROWTH .......................................................... 42
Materials and Methods............................................................................................ 43 Drug.................................................................................................................. 43
Cell Culture ....................................................................................................... 44 Cell Proliferation Assay .................................................................................... 44
Enzyme-linked Immunosorbent Assay ............................................................. 44 Cell Cycle Assay .............................................................................................. 44
Apoptosis Assay ............................................................................................... 45 Western Blotting ............................................................................................... 45
Colony Formation Assay .................................................................................. 45 Clonogenic Assay ............................................................................................. 46
Statistical Analysis ............................................................................................ 46 Results .................................................................................................................... 46
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A46 Inhibits Jak2-V617F-dependent Cell Proliferation ..................................... 46 A46 Inhibits the Jak/STAT Signaling Pathway .................................................. 48
A46 Induces G1/S Cell Cycle Arrest in HEL Cells ............................................ 49 A46 Induces Apoptosis in HEL Cells ................................................................ 50
A46 Suppresses Cytokine-independent Pathologic Cell Growth of Jak2-V617F Positive Bone Marrow Cells, ex vivo .................................................. 52
Discussion .............................................................................................................. 53
3 STRUCTURE-FUNCTION CORRELATION OF G6, A NOVEL SMALL MOLECULE INHIBITOR OF JAK2: INDISPENSABILITY OF THE STILBENOID CORE ..................................................................................................................... 64
Experimental Procedures ........................................................................................ 65 Drugs ................................................................................................................ 65
Cell Culture ....................................................................................................... 66 Cell Proliferation Assay .................................................................................... 66
Enzyme-linked Immunosorbent Assay ............................................................. 66 Cell Lysis and Immunoprecipitation .................................................................. 66
Western Blotting ............................................................................................... 67 Apoptosis Assay ............................................................................................... 67
Real Time PCR Analysis .................................................................................. 68 Patient Sample ................................................................................................. 68
Colony Forming Unit-Erythroid Colony Formation Assay ................................. 68 Computational Docking .................................................................................... 69
Statistical Analysis ............................................................................................ 70 Results .................................................................................................................... 70
A Stilbenoid Core Is Essential for Time- and Dose-dependent Inhibition of Jak2-V617F-dependent Cell Growth ............................................................. 70
Indispensability of the Stilbenoid core in Decreasing Phosphorylation of STAT3 and STAT5 ........................................................................................ 72
Induction of Apoptosis in HEL Cells by G6 and its Derivatives ......................... 73 Stilbenoid Core Bearing Derivatives of G6 Suppress Pathologic Cell Growth
of Patient-derived Bone Marrow Cells, ex vivo .............................................. 74 Computational Docking of G6 and its Derivatives into the ATP Binding
Pocket of the Jak2 Kinase Domain ............................................................... 75 Discussion .............................................................................................................. 77
4 CELL DEATH INDUCED BY THE JAK2 INHIBITOR, G6, CORRELATES WITH CLEAVAGE OF VIMENTIN FILAMENTS ............................................................... 90
Experimental Procedures ........................................................................................ 91 Drugs ................................................................................................................ 91
Reagents .......................................................................................................... 92 Cell Culture ....................................................................................................... 92
2-D Differential In Gel Electrophoresis (2-D DIGE) .......................................... 92 Cell Lysis .......................................................................................................... 94
Western Blotting ............................................................................................... 94
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Immunofluorescence ........................................................................................ 95 Cell Proliferation Assay .................................................................................... 95
In vivo Animal Model ........................................................................................ 95 Bone Marrow Immunohistochemistry ............................................................... 96
Statistical Analysis ............................................................................................ 96 Results .................................................................................................................... 96
G6 Treatment Induces Time- and Dose-Dependent Degradation of Vimentin . 96 G6 Treatment Induces Marked Reorganization of Vimentin Intermediate
Filaments within Cells.................................................................................... 98 G6-induced Cleavage of Vimentin is Jak2-mediated ........................................ 99
G6-induced Cleavage of Vimentin is Independent of de novo Protein Synthesis and Caspase Activity, but Calpain-dependent .............................. 99
The Mobilization of Calcium is Essential and Sufficient for the Cleavage of the Intermediate Filament Protein Vimentin ................................................ 101
Cleavage of Vimentin is Sufficient to Reduce HEL Cell Viability .................... 102 G6 Treatment Decreases the Levels of Vimentin Protein, in vivo ................... 103
Discussion ............................................................................................................ 104
5 CONCLUSIONS AND PERSPECTIVES ............................................................... 116
Importance of Designing/Identifying a Jak2 Specific Inhibitor ............................... 117 Characterization of the Novel Jak2 Inhibitor, A46 ................................................. 119
Stilbenes and Benzothiophenes as Potential Scaffolds for the Design of Novel Jak2 Small Molecule Inhibitors .......................................................................... 120
Comparison of Two Novel Jak2 Inhibitors, G6 and A46........................................ 122 Potential of Jak2 Inhibitor Therapy for Myeloproliferative Neoplasms .................. 124
Characterization of the Link Between G6-induced Cell Death and Cleavage of Vimentin............................................................................................................. 129
LIST OF REFERENCES ............................................................................................. 134
BIOGRAPHICAL SKETCH .......................................................................................... 150
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LIST OF TABLES
Table page 1-1 Preclinical characterization of Jak2 inhibitors ..................................................... 39
1-2 Clinical characterization of Jak2 inhibitors .......................................................... 40
2-1 Chemical characteristics of A46 ......................................................................... 57
3-1 Percent growth inhibition of G6 and its derivatives ............................................. 80
5-1 GI50 of other Jak2 inhibitors in HEL cells .......................................................... 133
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LIST OF FIGURES
Figure page 1-1 Tyrosine kinase catalyzed phospho-transferase reaction ................................... 36
1-2 Schematic representation of the Jak2 protein showing the regions where the somatic mutations identified in myeloproliferative neoplasm (MPN) patients are commonly found ........................................................................................... 37
1-3 Schematic representation of the Jak/STAT signaling paradigm ......................... 38
2-1 A46 inhibits Jak2-V617F-dependent cell proliferation ......................................... 58
2-2 A46 inhibits the Jak/STAT signaling pathway ..................................................... 59
2-3 A46 arrests HEL cells in G1 phase of the cell cycle ........................................... 60
2-4 A46 induces apoptosis in HEL cells .................................................................... 61
2-5 A46-induced apoptosis is mediated by the down regulation/cleavage of Bcl-2 family proteins Bim, Bax and Bid ........................................................................ 62
2-6 A46 suppresses cytokine-independent pathologic cell growth of Jak2-V617F positive bone marrow cells, ex vivo .................................................................... 63
3-1 Time- and dose-dependent effect of G6 and its derivatives on the HEL cell proliferation and Jak2 phosphorylation ............................................................... 81
3-2 Dose-dependent effect of G6 and its derivatives on the proliferation of Ba/F3-EpoR-Jak2-V617F cells ...................................................................................... 82
3-3 Inhibition of phosphorylation of STAT3 and STAT5 by G6 and its derivatives .... 83
3-4 Induction of apoptosis in HEL cells by G6 and its derivatives ............................. 84
3-5 Treatment with G6 or its stilbenoid derivatives leads to HEL cell death via the intrinsic apoptotic pathway.................................................................................. 85
3-6 Suppression of Jak2-V617F mediated pathologic cell growth in patient-derived bone marrow cells by G6 and its derivatives, ex vivo............................. 86
3-7 Molecular surface representation of the ATP binding site of Jak2 ...................... 87
3-8 Molecular docking of G6 and its derivatives into the ATP binding pocket of Jak2 .................................................................................................................... 88
3-9 Docking of G6 and its derivatives into the ATP binding pocket of Jak2 .............. 89
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4-1 Identification of vimentin as a differentially expressed protein between vehicle treated and G6 treated HEL cells ..................................................................... 108
4-2 G6 treatment induces time- and dose-dependent degradation of vimentin ...... 109
4-3 G6 treatment induces marked reorganization of vimentin intermediate filaments within cells ......................................................................................... 110
4-4 G6-induced cleavage of vimentin is Jak2-mediated ......................................... 111
4-5 G6-induced cleavage of vimentin is independent of de novo protein synthesis and caspase activity, but calpain-dependent .................................................... 112
4-6 Mobilization of calcium is essential and sufficient for the cleavage of vimentin 113
4-7 Cleavage of vimentin is sufficient to reduce HEL cell viability........................... 114
4-8 G6 treatment decreases the levels of vimentin protein, in vivo ......................... 115
5-1 Surface representation of the structure of Jak2, indicating possible sites for inhibitor targeting .............................................................................................. 132
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LIST OF ABBREVIATIONS
Jak2 Janus Kinase 2
JH1 Jak Homology 1
JH2 Jak Homology 2
SH2 Scr Homology 2
FERM 4.1 Ezrin Radixin Moesin
GPCR G-protein Coupled Receptor
STAT Signal Transducers and Activators of Transcription
MPN Myeloproliferative Neoplasms
PV Polycythemia Vera
ET Essential Thrombocythemia
PMF Primary Myelofibrosis
HEL Human Erythroleukemia
DMSO Dimethyl Sulfoxide
ELISA Enzyme-linked Immunosorbent Assay
G1/S Gap1/Synthesis
DTT Dithiothreitol
SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
FITC Fluorescein Isothiocyanate
IDPN Beta, beta’-iminodipropionitrile
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
CHARACTERIZATION OF STRUCTURE-FUNCTION CORRELATIONS OF NOVEL JAK2 SMALL MOLECULE INHIBITORS AND THEIR MECHANISMS OF ACTION
By
Anurima Majumder
August 2011
Chair: Peter P. Sayeski Major: Medical Sciences-Physiology and Pharmacology
Jak2 is a cytoplasmic tyrosine kinase that is involved in signaling via a diverse
range of ligands, including cytokines and growth factors. Hyperkinetic Jak2 tyrosine
kinase has been linked to various neoplastic disorders, including solid tumors and
hematological malignancies. In 2005, a Jak2 gain-of function mutation, Jak2-V617F,
was identified in a majority of patients with myeloproliferative neoplasms (MPNs). This
discovery spurred a great deal of interest in identifying small molecule inhibitors that
target Jak2, as effective inhibitors may have significant therapeutic potential for Jak2-
mediated pathologies and may also serve as useful research tools to study Jak2-
mediated signaling in general. In this dissertation, we first characterize a novel
benzothiophene based small molecule Jak2 inhibitor, A46, which we have identified by
structure-based drug design. We show that A46 specifically inhibited the proliferation of
Jak2-V617F expressing cells in both a time- and dose-dependent manner. Cells
exposed to 10 µM of A46 for 48 hours or more were unable to recover after drug
removal. A46 also caused a corresponding decrease in the phosphorylation of Jak2
and STAT3 proteins within these cells. Cell growth inhibition correlated with an induction
of cell cycle arrest and promotion of apoptosis. Moreover, we report that this compound
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inhibited the pathologic growth of primary Jak2-V617F expressing bone marrow cells,
ex vivo. Collectively, our data demonstrate that the benzothiophene based compound,
A46, suppresses Jak2-mediated pathogenesis, thereby making it a potential candidate
drug against Jak2-mediated disorders.
Our lab has previously identified a novel Jak2 inhibitor called G6 using structure-
based drug design. G6 showed promising results in preclinical studies and inhibited
Jak2 tyrosine kinase-mediated pathologic cell growth in vitro, ex vivo and in vivo. Here,
we identified a structure-function correlation of this compound. Specifically, we showed
that the stilbenoid core in G6 is essential for its ability to i) inhibit Jak2-V617F-
dependent cell proliferation, ii) suppress phosphorylation of key signaling molecules
involved in the Jak/STAT pathway, such as Jak2, STAT3 and STAT5, iii) induce
apoptosis in HEL cells via the intrinsic apoptotic pathway, iv) inhibit pathologic growth of
patient-derived Jak2-V617F-positive bone marrow cells, ex vivo and v) form strong
docking interactions with the ATP-binding pocket of Jak2 kinase domain. As such, we
demonstrated that G6 has a stilbenoid core that is indispensable for maintaining its Jak2
inhibitory potential.
Having shown that G6 specifically inhibits Jak2 kinase activity and suppresses
Jak2-mediated cellular proliferation, we next wanted to elucidate the molecular and
biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation. For
this, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12
hours with either vehicle control or 25 µM of the drug and compared protein expression
profiles using two-dimensional gel electrophoresis. One differentially expressed protein
identified by electrospray mass spectroscopy was the intermediate filament protein,
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vimentin. It was present in DMSO treated cells, but absent in G6 treated cells. HEL cells
treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as
a marked reorganization of vimentin intermediate filaments within intact cells. In a
mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the
levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be
Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium
mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show
that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL
cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-
mediated pathogenic cell growth is concomitant with the disruption of intracellular
vimentin filaments. As such, this work describes a novel pathway for the targeting of
Jak2-mediated pathological cell growth.
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CHAPTER 1
INTRODUCTION
Tyrosine Kinases
Tyrosine kinases are enzymes that catalyze the transfer of the γ-phosphate of
ATP to tyrosine residues of protein substrates (Fig. 1-1). These enzymes are critical
components of important signaling pathways that regulate vital physiological processes
such as cell proliferation, survival, differentiation and apoptosis. Abnormal tyrosine
kinase activity has however been linked to cancer, immunodeficiency, artherosclerosis,
cardiovascular disease etc.
Tyrosine kinases can be divided into two broad subfamilies; receptor tyrosine
kinases (RTKs) and non-receptor tyrosine kinases (NRTKs). The RTKs usually possess
a ligand binding extracellular domain, a transmembrane domain and a catalytic
intracellular kinase domain. These membrane-spanning kinases, therefore, have
intrinsic kinase activity and can undergo autophosphorylation upon activation by ligand
binding. The RTK family includes the insulin receptor and several growth factor
receptors, such as epidermal growth factor receptor and platelet-derived growth factor
receptor [1]. The NRTKs are usually cytoplasmic and possess a kinase domain, but lack
receptor-like features such as extracellular and transmembrane domains [1]. However,
these tyrosine kinases have additional domains that enable them to interact with other
signaling molecules, such as 1) an N-terminal myristylation/palmitoylation domain for
anchoring the kinase to the plasma membrane, 2) a pleckstrin homology (PH) domain
for binding to lipids, 3) an SH2 domain to bind phospho-tyrosines on other proteins, and
Portion of this chapter has been reproduced from Jak2 Inhibitors for the treatment of Myeloproliferative Neoplasms. Drugs of the Future 2010; 35(8): 651-660 with permission from 2010 Prous Science, S. A. U. or its licensors.
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4) an SH3 domain to bind to proline-rich sequences on other proteins [2]. Unlike growth
factor receptors, cytokine receptors do not have an intrinsic kinase domain. However,
these receptors associate with non-receptor tyrosine kinases and can thereby induce
tyrosine phosphorylation of the receptors and a variety of cellular proteins [3]. The
NRTK family includes the Src, Abl, Csk and FAK kinase subfamilies among many
others. The Janus kinases form a distinct family of cytoplasmic tyrosine kinases that
have a critical role to play in diverse signaling pathways that are essential for the normal
physiology and function.
Jak2 Tyrosine Kinase
History
Jak2, a cytosolic protein of approximately 130 kDa in mass, is a member of the
Janus family of cytoplasmic tyrosine kinases. Other known members of this family are
Tyk2, Jak1 and Jak3. These kinases are ubiquitously expressed in a variety of different
cell types with the exception of Jak3, which is predominantly expressed in cells of
hematopoietic origin [4]. Tyk2 (Tyrosine kinase 2), the first member of the Janus
tyrosine kinase family to be cloned, was identified by screening a T-cell cDNA library
using the sequence of the c-fms tyrosine kinase domain via a low stringency
hybridization technique [5]. The kinase domains of Jak1 and Jak2 were cloned by
polymerase chain reaction (PCR) using degenerate oligonucleotides complementary to
highly conserved kinase domains of the Src family members [6]. These kinase domain
sequences were then used to clone the full length Jak1 and Jak2 proteins [7, 8]. Jak3
was also cloned using similar PCR-based techniques [9, 10]. Initially, this family was
named as ‘Just Another Kinase’. However, subsequent sequence homology studies
revealed that a unique feature of all the members of this family of tyrosine kinases is
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that they have two potential kinase domains at their C-terminus. Thus, this family was
renamed as Janus Kinase after Janus, the Roman God with two opposite faces.
Structure
The unique structure of the Jak kinases clearly distinguishes them from other
members of the protein tyrosine kinase family. Similar to other members of the Jak
family, Jak2 has seven highly conserved Jak homology (JH) domains, JH1 through JH7,
numbered from the C- to the N-terminus, respectively [11] (Fig. 1-2). A hallmark feature
of all Janus kinases is the presence of two domains (JH1 and JH2) in the carboxyl half
of the protein that both have extensive homology to the kinase domain of other protein
tyrosine kinases. However, only the JH1 domain has functional kinase activity whereas
the JH2, or pseudokinase domain, has an important role in limiting the activity of the
kinase domain. The JH1 domain is a highly conserved kinase domain which contains
the activation loop, the critical sites of primary phosphorylation (Tyr1007 and Tyr 1008),
and the ATP-binding region. Phosphorylation of Tyr1007 residue in the activation loop is
essential for maximal Jak2 kinase activity [12]. The JH2 domain, which is adjacent to
the JH1 domain, negatively regulates both the basal as well as the ligand induced levels
of phosphotransferase activity [13, 14] by physically interacting with the JH1 domain
[15]. Even though this domain is structurally homologous to the JH1 domain, it lacks
certain critical amino acid residues that are essential for functional kinase activity and
hence is not catalytically active [16].
The amino half of Jak2 consists of domains JH3 through JH7. The JH3-JH4 region
weakly resembles the canonical Src Homology 2 (SH2) domain [17] and is hence called
the SH2-like domain, but its function is yet to be elucidated [18]. The JH4-JH7 domains
are N-terminal to the putative SH2 domain and are collectively called the FERM (4.1,
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Ezrin, Radixin, Moesin) domain. This domain is primarily involved in the binding to and
crosstalk with other cellular proteins [19]. For example, regions within the JH6 and JH7
domains mediate interactions with cell surface receptors by associating with conserved
Box 1 structural motifs on the cytokine receptors [20, 21].
The three-dimensional crystal structure of the entire Jak2 molecule has not yet
been resolved. However, a portion of the Jak2 kinase domain has been crystallized
recently [22]. This crystal structure gives important insights into the precise atomic
architecture of the Jak2 tyrosine kinase domain. Structurally, the kinase domain is
highly conserved among all tyrosine kinases thereby making it challenging to
specifically target Jak2 over the other kinases. However, this paper reports that the
Jak2 kinase domain has some unique features that can help distinguish it from the
kinase domains of other tyrosine kinases and thus help in achieving specificity. Firstly,
Jak family members possess an extra loop structure between amino acids 1056 and
1078, called the Jak2 insertion loop, which is relatively mobile and solvent accessible
[22]. The serine at position 1056 is conserved between all members of the Jak family
and is solvent exposed, suggesting that this residue might have an important role to
play in phosphorylation-dependent regulatory role in Jak2 function [22]. Analysis of the
Jak2 kinase domain crystal structure shows that the swing of the N-terminal lobe
towards the C-terminal lobe markedly narrows the active site of Jak2, thereby making it
more constricted and closed when compared to the active site of other kinases [22].
Comparisons of the electrostatic surface potentials around the ATP-binding sites
suggest that Jak2 is more positively charged than Jak1, whereas Jak3 is less charged
overall [23]. These structural differences between Jak2 and other closely related
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kinases, though subtle, might prove useful in achieving selectively for Jak2 over other
kinases. Recent crystal structures of the Jak2 kinase domain have undoubtedly
improved our understanding of the structure, function and regulation of Jak2 kinase.
However, future crystal structure analysis of the entire Jak2 molecule will be critical in
further developing our knowledge about these kinases and in attaining selectivity for
them.
Jak/STAT Signaling Paradigm
Jak2 is an important downstream signaling molecule activated by a variety of
cytokines, growth factors and G-protein coupled receptor (GPCR) ligands. An overview
of the canonical Jak/STAT signaling pathway, as activated by cytokines and growth
factors, is shown in Fig. 1-3. Briefly, signaling is initiated by binding of a ligand to its
cognate cell surface receptor resulting in receptor dimerization. Receptor dimerization
brings the receptor-associated Jak proteins in close proximity to each other allowing
them to transphosphorylate one another on specific tyrosine residues; Tyr 1007/1008 in
the case of Jak2 [12]. An activated Jak can in turn phosphorylate specific tyrosine
residues on the cytoplasmic tails of the receptors, thereby creating docking sites for
SH2-domain containing proteins such as the Signal Transducers and Activators of
Transcription (STAT) proteins. Receptor-bound STAT monomers are then
phosphorylated by Jak2 on specific tyrosine residues. Phosphorylated STATs form
homo- or hetero-dimers which translocate to the nucleus where they bind to gene
promoter elements to modulate gene transcription. Thus, Jak/STAT signaling results in
a signal cascade from extracellular binding and activation of a cell surface receptor to
changes in gene transcription in the nucleus.
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The important role that Jak kinases play in mediating cytokine signaling was first
characterized using mutant cell lines that did not respond to interferons. Analysis of this
particular mutant cell line showed that it lacked the tyrosine kinase Tyk2 and expressing
this kinase exogenously restored the ability of this cell to respond to interferons [24].
Specific Jak kinases, either alone or in combination with other Jak kinases, is
preferentially activated depending on the type of receptor that is being stimulated. For
example, binding of erythropoietin (Epo) to its cognate receptor activates Jak2 [25],
whereas binding of interleukin-3 (IL-3), interleukin-5 (IL-5) or granulocyte macrophage-
colony stimulating factor (GM-CSF) to their associated receptors activate both Jak2 and
Jak1 [26-28]. Thus, it was shown that Jak2 can associate with diverse cytokine and
growth factor receptors. Subsequently, it was shown that Jak2 is a versatile signaling
molecule that can also be activated by G-protein coupled receptors, such as the AT1
receptor [29], and by non-conventional ligands, such as reactive oxygen species [30].
The Jak/STAT signaling pathway can modulate cell growth, survival, differentiation and
death and is therefore critical for maintaining normal physiology. Deregulation of this
signal transduction pathway has been implicated in various disease states. On one
hand, Jak2 knockout mice die embryonically at day 12.5 due to lack of definitive
erythropoiesis [31, 32], suggesting that this kinase has a non-redundant role to play in
the erythropoiesis process. On the other side, hyperactive Jak2 signaling promotes cell
proliferation and prevents apoptosis, thereby leading to various neoplastic disorders,
including solid cancers and hematological malignancies.
Jak2 and Cancer
Jak2 in Solid tumors
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A deregulated Jak/STAT signaling pathway has been linked to several solid
tumors, including breast, prostate, ovarian, head and neck cancers [33-38]. Jak2 can
cause constitutive tyrosine phosphorylation of ErbB2, an oncogene over expressed in
human breast cancer, leading to tumor progression and metastasis [39]. It is also known
that overexpression of BRCA1 leads to a constitutively active Jak2-STAT3 signaling in
human prostate cancer cells [40]. Epidermal growth factor stimulation constitutively
activates STAT3, thereby leading to head and neck carcinogenesis by activation of anti-
apoptotic mechanism [37]. Additionally, it has been demonstrated that hyperactivation of
the Jak/STAT3 signal transduction pathway contributes directly to ovarian cancer
growth and invasiveness [38].
Jak2 in Hematological Malignancies
Apart from its role in promoting growth and metastasis of solid tumors, a
constitutively active Jak kinase signaling has also been implicated in various
hematological malignancies. Hematopoiesis, the process of generating blood cells from
a self-renewing population of multipotent hematopoietic stem cells, is regulated by a
group of soluble factors called cytokines. The binding of cytokines to their cognate
receptors initiates signal transduction pathways that govern survival, proliferation,
differentiation, and apoptosis of the hematopoietic cells. A deregulation of these
signaling pathways can lead to the development of different hematological disorders,
such as leukemias and myeloproliferative neoplasms. Cytokine receptors lack an
intergral cytoplasmic kinase domain and hence signal via association with cytoplasmic
tyrosine kinases, such as the Jak kinase family members [3, 41]. Jak2 is widely involved
in cytokine signaling. Cytokines, such as erythropoietin, thrombopoietin, growth
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hormone, prolactin and GM-CSF, signal exclusively through Jak2 [42, 43]. Hence, it is
unsurprising that hyper activation of Jak2 kinase activity can cause overproduction of
blood cells, thereby leading to the development of neoplastic hematological
malignancies.
Jak2 chromosomal translocations and hematological malignancies
Several studies have linked specific Jak2 chromosomal translocations to human
neoplastic growth. For example, an abnormal fusion protein that has the helix-loop-helix
dimerization domain of TEL, an ETS family transcription factor, fused to the catalytic
domain of Jak2 was detected in a patient with early B-precursor acute lymphobastic
leukemia and another diagnosed with atypical chronic myeloid leukemia [44, 45]. The
phenotypes of these two patients were diverse because of the fact that the TEL-Jak2
fusion proteins had resulted from two distinct translocation events; a t(9:12)(p24;p13) in
the former case and an a t(9:15:12)(p24;q15;p13) in the latter. However, in both cases,
the translocation event resulted in constitutive activation of the Jak2 kinase domain and
its downstream signaling pathways [46]. Recent studies have also reported the
presence of a BCR-Jak2 fusion protein arising from a t(9:22)(p24;q11.2) translocation in
patients with typical and atypical chronic myelogenous leukemia [47-49]. In other
instances, a t(8:9) translocation event results in a PCM1-Jak2 fusion protein that
contains the coiled coil domains of PCM1 and the tyrosine kinase domain of Jak2 [50-
52]. This fusion protein has been implicated in several hematological disorders including
acute erythroid leukemia, atypical chronic myeloid leukemia, T-cell lymphoma, and
myeloproliferative neoplasms.
Jak2 point mutations and hematological malignancies
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In addition to chromosomal translocations, point mutations in the Jak2 allele also
cause constitutive Jak2 activation and subsequent activation of its downstream
signaling pathways. Substitution and deletion mutations in Jak2 have been detected in
many patients with different hematological disorders. For example, Jak2 amino acids
682-686 were found to be deleted (Jak2-∆IREED) in a patient with Down syndrome and
B-cell precursor acute lymphoblastic leukemia [53]. In another instance, a novel Jak2-
T875N mutation was identified in an actue megakaryoblastic leukemic cell line using a
combination of mass spectrometry and growth inhibition assays [54]. Jak2-K607N [55]
and Jak2-L611S [56] are other Jak2 point mutations that have been identified in patients
with acute myeloid leukemia and acute lymphoblastic leukemia, respectively. Overall,
these studies demonstrate that the Jak2 locus is susceptible to diverse genetic
altrerations that hyper activate the catalytic activity of Jak2 kinase, thereby leading to
the development of hematological malignancies.
Myeloproliferative Neoplasms
Myeloproliferative Neoplasms (MPNs) are a group of heterogeneous diseases
arising from a transformed hematopoietic stem cell and characterized by the presence
of excessive numbers of one or more terminally differentiated blood cells of the myeloid
lineage such as erythrocytes, thrombocytes or white blood cells. These differentiated
cells spread and accumulate in the bone marrow as well as in the peripheral blood and
other sites of extramedullary hematopoiesis due to excess proliferation and/or
decreased apoptosis of hematopoietic progenitors. MPNs have a high propensity to
progress to malignant leukemias. Excess production of red blood cells is a hallmark of
polycythemia vera (PV) and too many platelets is a classical characteristic of essential
25
thrombocythemia (ET) whereas primary myelofibrosis (PMF) is characterized by bone
marrow fibrosis and impaired hematopoietic stem cell function.
Jak2 Mutations in Myeloproliferative Neoplasms
The year 2005 was a landmark year for the field of MPNs as five different groups,
working independently, identified a somatic Jak2 Val 617 to Phe substitution mutation
(Jak2-V617F) in a large number of patients with myeloproliferative neoplasms (MPNs)
[57-61]. It is interesting to note that each group used a different approach to arrive at the
result. The group led by William Vainchenker [57] began by culturing erythroid cells from
PV patients in the absence of exogenous cytokines. The cells were then treated with
different small molecule inhibitors in an effort to identify the factors and signaling
pathways required for PV erythroid cell proliferation. It was observed that Jak2 inhibition
can suppress PV erythroid proliferation and that siRNA mediated silencing of Jak2 can
also inhibit erythroid cell proliferation. This led them to sequence JAK2 in PV patient
samples and hence the presence of the Jak2-V617F mutation was discovered [57].
Robert Kralovics and his group [59] [62] followed up on the important discovery that loss
of heterozygosity at the 9p24 chromosomal locus as a result of uniparental disomy
(UPD) is present in a large number of PV patients [62]. Genes in this region of UPD
were sequenced in MPN patient samples and the Jak2-V617F allele was detected [59].
Two other groups used candidate gene approaches to identify the Jak2-V617F
mutation. Anthony Green and coworkers [58] were sequencing genes involved in
signaling pathways known to be implicated in oncogenic transformations when they
discovered this mutant allele in MPN. On the other hand, Gary Gilliland’s group [60]
detected the presence of the Jak2-V617F mutation while sequencing conserved
domains of all tyrosine kinases on a large set of MPN patients who had submitted
26
samples via an internet-based collection protocol. Joe Zhao’s group [61] performed a
large scale DNA sequencing analysis of candidate tyrosine kinases and phosphatases
and thereby identified the presence of the gain-of-function Jak2 mutation in a majority of
PV patients.
These five groundbreaking studies, as well as other subsequent studies, reported
the presence of the Jak2-V617F mutation in over 90% of patients with PV and about
50% of patients with ET and PMF. A guanine to thymine mutation acquired in the
hematopoietic stem cells results in a substitution of valine to phenylalanine at codon
617 in the exon 14 of the JH2 pseudokinase domain of Jak2. It is believed that this
mutation at position 617 in the autoinhibitory pseudokinase domain of Jak2 allows the
kinase to evade inhibition and leads to a constitutively active Jak-STAT signaling
pathway [62, 63] and induces cytokine independent growth of cells [57, 60]. The co-
expression of Jak2-V617F and an ectopic erythropoietin receptor (EpoR) in the IL-3-
dependent hematopoietic cell line Ba/F3 transforms these cells to cytokine independent
growth [57, 60, [63]. Subsequently, it has also been shown that expression of this
mutation in both murine bone marrow transplant models [64, 65] as well as transgenic
models [66-71] is sufficient for the development of MPN-like phenotypes in recipient
mice. These reports strongly suggest that the Jak2-V617F mutation plays a critical role
in the pathogenesis of myeloproliferative neoplasms.
Although the Jak2-V617F mutation on exon 14 is the predominant disease-
associated allele in myeloproliferative neoplasms, several other Jak2 exon 14
mutations, such as C616Y [72] and D620E [73], have been identified in V617F-
negative, MPN patients. Moreover, about sixteen different Jak2 exon 12 mutations have
27
been detected to date in Jak2-V617F-negative PV patients [74]. These exon 12
mutations are usually insertions, deletions or substitutions in the Jak2 sequence
spanning amino acids 538-543 (Fig. 1-2). This region is highly conserved and links the
SH2-like and the pseudokinase domains of Jak2. A recent study suggests that this
linker region acts as a switch in relaying signals from receptor binding to Jak2 kinase
activation by flexing the pseudokinase domain hinge [75]. Finally, a subset of patients
with Jak2-V617F-negative ET and PMF were found to carry MPLW515L/K mutations in
the thrombopoietin receptor, which also leads to the deregulation of the Jak/STAT
signaling pathway via activation of wild type Jak2 protein [76, 77]. Collectively, these
studies clearly demonstrate that hyperkinetic Jak2 has a crucial role to play in the
pathogenesis of MPNs as well as in a number of hematological malignancies.
Therefore, the clinical development of Jak2 inhibitors is of potential value for patients
with these hematological disorders and is currently an area of active research.
Jak2 Inhibitors for the Treatment of Myeloproliferative Neoplasms
Currently available treatment options for MPN patients are limited. For PV/ET
patients, the available therapies aim to reduce myeloproliferation and
thrombotic/hemorrhagic complications associated with it. As such, a PV/ET patient is
mostly treated with phlebotomy/plateletpheresis or myelosuppressive agents such as
hydroxyurea [78-80]. On the other hand, in PMF patients, treatment is directed towards
management of symptoms such as anemia and splenomegaly. Therefore, agents that
can improve cytopenia (erythropoietin, androgens) or induce myelosuppression (such
as hydroxyurea, thalidomide) are routinely used for treating these individuals [80, 81]. In
certain cases, more invasive techniques, such as splenectomy or radiation, are used to
treat severe splenomegaly in MPN patients. Unfortunately, these treatment strategies
28
are more palliative and not curative in nature. The only therapy that has been shown to
be curative in patients with myeloproliferative neoplasms is stem cell transplantation.
Although this approach is potentially curative, risks associated with it, such as graft-
versus-host disease and nonrelapse mortality, severely limit its application for therapy
[81]. Given the critical role that constitutive Jak2 tyrosine kinase activity plays in the
pathophysiology of myeloproliferative neoplasms, it became an attractive molecule for
therapeutic targeting and prompted researchers to start the search for potent and
selective Jak2 small molecule inhibitors. Interestingly, the first widely available Jak2
inhibitor dates back to 1995 when Meydan et al. identified AG490 via a high throughput
screen of potential tyrosine kinase inhibitors [82]. However, it was subsequently
reported that although AG490 was a potent Jak2 inhibitor, it had poor specificity [83].
The discovery of the Jak2-V617F mutation in 2005 and its identification in a vast
majority of patients with MPNs provided a new thrust to the search for identification of
small molecule inhibitors that specifically target Jak2. Given the fact that the Jak2-
V617F mutation leads to sustained kinase signaling, similar to the effect of the BCR-
ABL fusion protein in chronic myelogenous leukemia (CML), it was expected that a Jak2
inhibitor would be as effective in MPN therapy as imatinib is in CML [84]. Moreover, in
2006, the resolution of a crystal structure encoding a portion of the Jak2 kinase domain
provided a valuable tool for designing inhibitors that could be highly specific for Jak2
[22].
Several pharmaceutical companies have developed Jak2 inhibitors that are
currently under study for the treatment of MPNs. The clinical trials conducted thus far
have focused on patients with PMF because it is the most serious condition among the
29
different MPNs. PMF patients are characterized by fibrous scar tissue in their bone
marrow, impaired hematopoiesis and high levels of pro-inflammatory cytokines and
growth factors. These symptoms significantly reduce the quality of life of these patients
and as mentioned above, effective therapies are limited for these patients. The results
from four clinical trials in these patients are publicly available; the inhibitors tested were
INCB018424, CEP-701, TG101348, and XL019 (Fig. 4).
INCB018424 is a potent Jak2 inhibitor which has an in-vitro IC50 of 2.8 nM [85]. In
primary cultures, this inhibitor has an ability to suppress erythroid progenitor colony
formation from Jak2-V617F positive PV patients with an IC50 of 67 nM vs. healthy
donors (IC50 > 400 nM). In a mouse model of Jak2-V617F mediated MPN, treatment
with 180 mg/kg/day of INCB018424 significantly decreased splenomegaly, reduced
levels of some circulating cytokines, preferentially eliminated neoplastic cells, and
increased animal survival without myelosuppressive or immunosuppressive effects. In
clinical phase I/II trials, this drug was generally well tolerated; the maximum tolerated
dose (MTD) was 25 mg PO BID [86]. INCB018424 treatment resulted in reduced
splenomegaly in over 93% of the patients irrespective of Jak2 mutational status [87].
Treatment with this drug inhibited production of pro-inflammatory cytokines in all PMF
patients [88]. There was also significant improvement in quality of life and weight of the
patients with treatment [86]. However, the Jak2-V617F allele burden showed only a
modest decrease of 13% in the marrow and 9% in the peripheral blood [89], suggesting
that this drug might be inhibiting downstream Jak/STAT signaling in cells with
hyperkinetic Jak2 rather than altering the mutant allele burden. There were also no
reports of improvement or normalization of either the diseased bone marrow or the
30
spleen tissues in any of the treated patients. No significant changes in fibrosis score,
bone marrow cellularity or circulating CD34+ cells were observed [90]. Finally,
thrombocytopenia and myelo-suppression were the most common side effects of drug
treatment [86]. Phase III clinical studies for this drug have recently been initiated
(www.clinicaltrials.gov).
CEP-701 (also called lestaurtinib), an indolocarbazole alkaloid, is an orally
available tyrosine kinase inhibitor that has anti-Jak2 tyrosine kinase activity. This drug is
a known inhibitor of Fms-like tyrosine kinase 3 (FLT3) and is currently being evaluated
in clinical trials for treating acute myelogenous leukemia patients with FLT3 mutations
[91, 92]. Lestaurtinib inhibits Jak2 in vitro, ex vivo, and in vivo [93]. It potently inhibits
Jak2 kinase activity in vitro with an IC50 of 1 nM and suppresses ex vivo erythroid colony
formation from primary CD34+ cells isolated from MPN patients at a concentration of
100 nM. CEP-701 treatment is also able to reduce phosphorylation of STAT3, STAT5
and other downstream signaling molecules of the Jak/STAT signaling pathway in vitro.
With respect to in vivo studies, this drug was able to inhibit the proliferation of Jak2-
V617F bearing HEL 92.1.7 cells xenografted into nude mice at a dose of 30 mg/kg BID.
The results of a phase II clinical study of CEP-701, with twenty two Jak2-V617F positive
PMF patients, were recently published [94]. These patients were treated with an oral
dose of 80 mg twice a day. 27% of the patients (6 out of 22) showed clinical
improvement with this treatment (i.e. the lowest level of response as per the
International Working Group for Myelofibrosis Research and Treatment criteria). The
responding patients included three who only showed a reduction in spleen size, two
who achieved transfusion independency, but only one who had a reduction in spleen
31
size in conjunction with improvement in cytopenia [94]. Median time for response was
about three months and the duration of the response was fourteen months or higher.
There was an observed decrease in the levels of phosphorylated STAT3 from baseline
levels in the responding patients [94]. Most critically, no improvements were seen either
in the marrow fibrosis or in the Jak2-V617F allele burden in any of the treated patients.
Finally, 36% of the treated patients (8 out of 22) experienced toxic effects; principally,
myelo-suppression (anemia and thrombocytopenia) and gastrointestinal problems such
as diarrhea and nausea [94].
TG101348 is a potent and selective inhibitor of Jak2. It is an ATP-competitive
inhibitor that shows a high selectivity for Jak2 over other Jak family members [65, 95].
Among all the Jak2 inhibitors that are currently in clinical trials, TG101348 seems to be
the most selective for Jak2. Previous studies with this compound have shown that it
inhibits Jak2 kinase activity with an IC50 of 3 nM in vitro [65, 95]. Furthermore, it was
found to suppress erythroid colony formation from primary progenitor cells with an IC50
of about 300-600 nM [96]. The in vivo therapeutic efficacy of TG101348 in a murine
model of Jak2-V617F mediated PV was also evaluated in a study which found that mice
treated with 120 mg/kg BID showed a decrease in Jak2V617F mutant allele burden,
hematocrit values, leukocyte counts and spleen sizes [65]. TG101348 was also
evaluated in a phase I/II dose escalation study with twenty eight myelofibrosis patients
[97, 98]. The maximum tolerable dose was found to be 680 mg/day, taken once daily.
64% of the patients showed a decrease in spleen size of more than 50%. Fourteen
patients, who initially had leukocytosis, experienced improvement in their WBC counts
post treatment. Mutant allele burden was found to decrease in 32% of the Jak2-V617F
32
positive patients enrolled in the study. The most frequent toxicities observed were
nausea/vomiting (64%) and diarrhea (50%) [97]. No changes in levels of plasma
cytokines were detected in patients treated with this drug [98]. Thrombocytopenia,
neutropenia and anemia were the other side effects that correlated with drug treatment
[97].
XL019 inhibits Jak2 kinase activity with an IC50 of 2 nM. In EPO stimulated primary
erythroid ex vivo cultures, STAT5 phosphorylation was inhibited with an IC50 of 64 nM
[99]. In in vivo studies using the HEL cell xenograft model, treatment with this inhibitor
reduced tumor growth in a dose-dependent manner; 60% inhibition at a dosage of 200
mg/kg BID and 70% inhibition at a dosage of 300 mg/kg BID relative to vehicle control
treated animals [99]. The effect of XL019 administration on human subjects was tested
in a phase I/II clinical study in patients with PMF post-PV or post-ET [100]. To date, 21
patients have been studied over multiple doses ranging from 25 mg to 300 mg, using
different schedules of administration (either once daily or three times weekly). Adverse
neurotoxicity was observed in patients receiving doses >100 mg (65). However, the
drug was well tolerated at the lower doses of 25 to 50 mg daily or 25 mg three times
weekly. A greater than 50% reduction in spleen size was achieved in 42% of the
patients. Reduction in leukocytosis and circulating blast cells and improvement of
anemia, pruritis, and fatigue were also observed [100]. The drug associated toxicities
were mostly related to adverse neurological effects such as peripheral neuropathy,
formication, balance disorder and confusion [99]. Adverse hematological side effects
were however not seen in treated patients. Although XL019 showed some beneficial
effects, it has been withdrawn from clinical trials due to the neurotoxicity concerns.
33
A summary of the preclinical data pertaining to INCB018424, CEP-701,
TG101348, and XL019 is shown in Table 1-1 and a summary of the clinical trial
regimens for the same four drugs is displayed in Table 1-2. Data from these initial
reports about the clinical trials of Jak2 inhibitors show that the good preclinical
efficiencies demonstrated by these compounds in murine models of myeloproliferative
neoplasms did not translate to clinical trials in human patient populations. However,
there are also a number of other Jak2 inhibitors that are currently being evaluated in
early clinical trials, such as SB1518 and CYT387 (www.clinicaltrials.gov).
SB1518 is a potent, selective, orally active, ATP competitive Jak2 inhibitor that
inhibits Jak2-V617F activity with an IC50 of 19 nM (66). This compound was found to
inhibit the proliferation of Ba/F3 cells that had been transfected with erythropoietin
receptor (EpoR) and mutant Jak2-V617F with an IC50 of 81 nM [101]. In a mouse model
of MPD, established by injection of Ba/F3-JAK2V617F cells into nude mice, SB1518 was
found to produce therapeutic effects such as normalization of elevated white blood
cell count and reduction of GFP-labeled BaF3 cells in the peripheral blood, resolution of
hepatosplenomegaly, reduction of phospho-STAT5 in diseased organs, prolonged
survival and alleviation of terminal-stage anemia and thrombocytopenia [101].
CYT387, an aminopyrimidine derivative, inhibits Jak1/Jak2 with an IC50 of 11 and
18 nM respectively [102]. It inhibits the growth of Ba/F3-JAK2V617F and human
erythroleukemia (HEL) cells (IC50 ~1500 nM). The drug selectively suppressed the in
vitro growth of erythroid colonies harboring JAK2-V617F from polycythemia vera (PV)
patients, an effect that was attenuated by exogenous erythropoietin [102]. In a murine
MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and
34
restored normal levels of inflammatory cytokines. Furthermore, treatment with this drug
was able to reduce the Jak2-V617F allelic burden, but not eliminate the mutant cells
[103]. With a number of other putative Jak2 inhibitors currently in various stages of
preclinical studies, it is hopeful that the list of Jak inhibitors in clinical trials will grow
significantly in the years to come and a potent and specific small molecule inhibitor that
is able to cure Jak2-mediated pathologies will soon be identified.
Rationale for Studies
Jak2, a cytoplasmic tyrosine kinase, has a critical role to play in hematopoiesis
and activating mutations in this kinase are associated with a number of hematological
disorders. Recently, several somatic point mutations, such as the Jak2-V617F, that
constitutively activate the Jak/STAT signaling pathway have been identified in the
majority of patients with myeloproliferative neoplasms (MPNs). This discovery made
Jak2 an attractive molecule for therapeutic targeting in MPNs and has been the driving
force behind the development of small molecule inhibitors that specifically target Jak2.
Since then, many different Jak2 inhibitors have been identified using a variety of
screening techniques and have been characterized preclinically as well as clinically.
However, as we have discussed in the previous section, initial reports from the clinical
trials of Jak2 inhibitors have not lived up to the expectations. These inhibitors were able
to alleviate symptoms in patients, but failed to actually reverse the progression of the
disease. On the basis of these reports from early clinical trials of Jak2 inhibitors, we can
conclude that there is still an unmet clinical need for Jak2 inhibitors that can
successfully cure patients with myeloproliferative neoplasms.
The goal of the study presented in the following chapters is to identify and
characterize the structure-function correlation of novel Jak2 inhibitors. Here, we first
35
present a study in which we identify and characterize a novel benzothiophene-based
Jak2 inhibitor, called A46. Using structure-based virtual screening, our group recently
identified a novel small molecule inhibitor of Jak2 named G6 [104]. We showed that G6
has a specific inhibitory effect on Jak2 kinase activity as measured by in vitro enzyme
assays and an immunoassay ELISA [104]. We also demonstrated that this compound
exhibits exceptional therapeutic efficieny, in vivo [105]. Here, we perform structure-
function analysis of this novel Jak2 inhibitor, G6. Additionally, we also attempt to
elucidate the molecular and biochemical mechanisms by which G6 exerts its anti-Jak2
activity. The overall aim of the study presented here is to better understand the
mechanism of action of novel Jak2 inhbitors – both from a structural as well as a
functional point of view.
36
Figure 1-1. Tyrosine kinase catalyzed phospho-transferase reaction. Tyrosine kinases catalyze the transfer of the γ-phosphate of ATP to tyrosine residues of protein substrate.
Protein Substrate Protein Substrate
ATP ADP
Tyrosine Kinase
37
Figure 1-2. Schematic representation of the Jak2 protein showing the regions where
the somatic mutations identified in myeloproliferative neoplasm (MPN) patients are commonly found. Jak2 has seven highly conserved Jak homology or JH domains, JH1-JH7. The Jak2-V617F point mutation, the predominant disease-associated allele in MPNs, is on exon 14 of the JH2 or pseudokinase domain of Jak2. Other exon 14 mutations have also been identified in MPN patients who lack the V617F mutation. Additionally, about sixteen different exon 12 mutations have been detected in Jak2-V617F-negative PV patients. These mutations cluster in a region spanning from amino acids 538-543.
38
Figure 1-3. Schematic representation of the Jak/STAT signaling paradigm. (a) Binding of the ligand to its cognate cell-surface receptor causes receptor dimerization. (b) Receptor dimerization brings the receptor associated Jak molecules in close proximity, such that they can trasphosphorylate one another on specific tyrosine residues (c) Active Jaks can now phosphorylate tyrosine residues on the cytoplasmic tail of the receptor. (d) STAT proteins bind to these phosphorylated tyrosine residues on the receptor and are also phosphorylated by receptor associated active Jaks. (e) Phosphorylated STAT proteins form dimers which translocate to the nucleus. (f) STAT dimers bind to the promoter region of specific target genes and modulate gene transcription, producing biological effects such as cell proliferation, differentiation, survival and growth.
39
Table 1-1. Preclinical characterization of Jak2 inhibitors. The Jak2 IC50 was derived from cell-free assays using recombinant Jak2 protein. The HEL cell IC50 was determined using in vitro cultures of the Jak2-V617F expressing HEL cells. The ex vivo IC50 values were measured in primary cultures isolated from MPN patients. The in vivo drug dosage (mg/kg) is shown along with the animal model that was employed for that specific study.
Drug Jak2 IC50
(nM) HEL cell IC50
(nM) Ex Vivo IC50
(nM)
In Vivo Dosage
(mg/kg) (Mouse model used)
INCB018424
2.8
186
67
180 daily (Ba/F3-Jak2V617F
xenograft)
CEP-701 1 30-100 100 30 twice daily (HEL xenograft)
TG101348 3 300 300-600 120 twice daily
(Jak2V617F induced PV-bone marrow
transplant)
XL019 2 60-200 64 200-300 twice daily (HEL xenograft)
40
Table 1-2. Clinical characterization of Jak2 inhibitors. Shown is a list of the four drugs tested in clinical trials and an overview of the treatment regimen. PMF – Primary myelofibrosis, post-PV/ET MF – post polycythemia vera (PV)/essential thrombocythemia (ET) myelofibrosis.
Drug Company Phase of
Development
Clinical Trial Dose (mg)
Route of Administration
Patient Population
Studied
Number of Patients
Clinical/Molecular Responses
Observed Toxicities
INCB018424 Incyte Phase I / II Phase III
25 twice daily
Oral PMF and post-PV/ET
MF
>100 Decrease in splenomegaly irrespective of
Jak2 mutational status, improved quality of life and weight, reduce
levels of inflammatory
cytokines, modest decrease in Jak2
V617F allele burden, no change
in bone marrow fibrosis
Thrombocytopenia, myelosuppression
CEP-701
Cephalon
Phase II
80 twice
daily
Oral
Jak2V617F- positive PMF
22
Reduced spleen
size, Improvement in cytopenia, two patients achieved
transfusion independency,
decrease in phospho-STAT3,
no improvement in bone marrow fibrosis, no significant
improvement in allele burden
Myelosuppression
(Anemia and thrombocytopenia),
gastrointestinal problems (diarrhea
and nausea)
41
Table 1-2. Continued
Drug Company Phase of
Development
Clinical Trial Dose (mg)
Route of Administration
Patient Population
Studied
Number of Patients
Clinical/Molecular Responses
Observed Toxicities
TG101348 TargeGen Phase I / II 680 once daily
Oral PMF and post-PV/ET
MF
28 More than 50% decrease in spleen size, reduction in Jak2-V617F allele burden, marked
reduction in leukocytosis,
improvement in constitutional symptoms like
pruritus, no change in levels of plasma
cytokines
Nausea / vomiting, diarrhea,
thrombocytopenia, neutropenia,
anemia
XL019
Exelixis
Phase I / II
(Discontinued)
25-50
once daily
Oral
Primary or post-PV/ET
MF
21
Decrease in
splenomegaly, reduction in
leukocytosis and circulating blast
cells, improvement of anemia, pruritus
and fatigue
Neurotoxicities
such as peripheral neuropathy, formication,
balance disorder and confusion
42
CHAPTER 2 A46, A BENZOTHIOPHENE DERIVED COMPOUND, SUPPRESSES JAK2-
MEDIATED PATHOLOGIC CELL GROWTH
Jak2, a member of the Janus family of cytoplasmic tyrosine kinases, is
ubiquitously expressed and is a key mediator of signal transduction and gene
transcription. A variety of cytokines, growth factors and GPCR ligands can activate
Jak2, trigger the canonical Jak/STAT signaling cascade and cause physiological effects
such as regulation of cell survival, development, growth, and proliferation. The critical
role of Jak2 in embryonic development has been demonstrated by gene deletion studies
in which Jak2 knock-out mice were found to be embryonic lethal due to absence of
definitive erythropoiesis that led to profound anemia [31, 32]. Aberrant Jak2 activity has,
however, been associated with several different pathological conditions. Constitutive
activation of the Jak-STAT pathway promotes aberrant cell proliferation and can lead to
the development of hematological malignancies and myeloproliferative neoplasms
(MPNs) [44, 45, 48]. MPNs include three pathogenetically related disorders;
polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis
(PMF). These disorders arise from a transformed hematopoietic stem cell and are
characterized by increased production of blood cells of the myeloid origin.
A somatic Jak2 mutation (Jak2-V617F) was identified in patients with
myeloproliferative neoplasms including 90% of polycythemia vera (PV) patients and
about 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis
(PMF) [57-61]. A valine to phenylalanine substitution at codon 617 (V617F) in the
pseudokinase domain of Jak2 in hematopoietic stem cells results in a constitutively
active Jak-STAT signaling pathway that induces cytokine hypersensitivity and growth
factor independent growth of these cells. The presence of this mutation has also been
43
detected in other hematological malignancies such as acute myeloid leukemia, chronic
myelomonocytic leukemia and chronic neutrophilic leukemia [106]. The co-expression of
Jak2-V617F and an ectopic erythropoietin receptor (EpoR) in the IL-3-dependent
hematopoietic cell line Ba/F3, transforms these cells to cytokine independent growth
[63]. Subsequently, it has also been shown that expression of this mutation in both
murine bone marrow transplant models [64, 107] as well as transgenic models [66, 67,
70, 71] is sufficient for the development of MPN-like phenotypes in recipient mice.
These reports strongly suggest that the Jak2-V617F mutation plays a causative role in
the pathogenesis of myeloproliferative neoplasms.
Unfortunately, as of present, there are no effective treatment options available for
these patients. Specifically, current treatment options are directed towards management
of symptoms and are palliative rather than being curative. The presence of the Jak2-
V617F mutation in a majority of MPN patients suggests that identification of Jak2
specific inhibitors is an important step towards developing an effective targeted therapy
for MPNs. Our laboratory has been actively involved in the identification of Jak2
inhibitors and has previously reported three small molecules with anti-Jak2 activity [104,
105, 108, 109]. Here, we used structure-based drug design to identify a benzothiophene
based structure, 1-benzothiophen-2-yl-(4-dimethylaminophenyl)methanol (termed as
A46), and show that this compound suppresses Jak2-mediated pathological cell growth
in vitro and ex vivo.
Materials and Methods
Drug
The small molecule A46 was obtained from the National Cancer
Institute/Developmental Therapeutics Program (NCI/DTP). The drug was solubilized in
44
dimethyl sulfoxide (DMSO) at a concentration of 10 mM. The drug was stored at -20⁰C
in small aliquots to avoid repeated freeze thaw cycles.
Cell Culture
HEL and Raji cells were purchased from the American Type Culture Collection
(ATCC) and CMK cells from the German Collection of Microorganisms and Cell
Cultures DSMZ. SET-2 cells were a kind gift from Dr. Gary Reuther at the Moffitt Cancer
Center and Research Institute, Tampa, FL. These cells were cultured in RPMI 1640
(Mediatech) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin
and L-glutamine at 37⁰C and 5% CO2.
Cell Proliferation Assay
HEL, Raji, CMK and SET-2 cells were plated in 96-well plates at a concentration of
approximately 5 × 104 cells per well. The cells were then treated with either 0.25%
DMSO or A46 for the indicated periods of time or concentrations. Cell viability was
assessed by trypan blue exclusion staining or an MTS assay as indicated.
Enzyme-linked Immunosorbent Assay
HEL cells, treated with either 0.25% DMSO or increasing doses of A46 for 48 hrs,
were lysed and analyzed by Enzyme-linked Immunosorbent Assay (ELISA) for detection
of phospho-Jak2 and phospho-STAT5 protein levels. For this, Jak2 [pY1007/pY1008]
and STAT5b [pY699] ELISA kits (Invitrogen) were used according to the manufacturer’s
protocol.
Cell Cycle Assay
The CycleTESTTM PLUS DNA Reagent Kit (BD Biosciences) was used to analyze
the DNA obtained from HEL cells suspensions. The cells were first incubated with
45
0.25% DMSO or increasing doses of A46 for 48 hrs and then analyzed using a
FACSCalibur flow cytometer (BD Biosciences) as per the manufacturer’s protocol.
Apoptosis Assay
Induction of apoptosis in HEL cells was determined with the FITC AnnexinV
Apoptosis Detection Kit (BD Pharmigen) as per the manufacturer’s protocol. The cells
were incubated with either 0.25% DMSO or increasing doses of A46 for 48 hrs, stained
with AnnexinV and Propidium Iodide (PI) and then analyzed using a FACSCalibur flow
cytometer (BD Biosciences) to determine the percentage of cells undergoing apoptosis.
Western Blotting
HEL cells were treated with the indicated concentrations of A46 for 24 hrs. The
cells (~ 1 x 107) were then lysed in 0.8 ml of ice-cold RIPA buffer and protein
concentration was determined using a Bradford assay (Bio-Rad). Protein samples (~50
μg) were separated by gel electrophoresis. The separated proteins were then
transferred to nitrocellulose membranes and blotted with the indicated antibodies. All
the antibodies used were purchased from Cell Signaling Technology except for Cyclin
D1 which was from Santa Cruz Biotechnology. Western blots were visualized using the
enhanced chemi-luminescence system (Perkin-Elmer).
Colony Formation Assay
Following an Institutional Review Board approved protocol, a residual bone
marrow aspirate was obtained from a de-identified Jak2-V617F positive female
diagnosed with Essential Thrombocythemia. The marrow-derived mononuclear cells
were first washed in Iscove’s Modified Dulbecco’s Medium (IMDM) and then cultured in
human methylcellulose complete medium lacking erythropoietin and thrombopoietin
(R&D Systems). Cells were plated in 35-mm petri dishes at a concentration of
46
approximately 4 × 105 cells/ml in the absence or presence of increasing doses of A46.
Thrombopoietin (50 ng/ml) was added to the indicated samples. The dishes were
incubated at 37⁰C and 5% CO2 in a humidified atmosphere for 14 days following which
the number of colony forming units-megakaryocytes (CFU-Megs) were counted.
Clonogenic Assay
Bone marrow cells from Jak2-V617F transgenic mice [67] were harvested,
cultured ex vivo in Iscove’s Modified Dulbecco’s Medium (IMDM), and treated with 25
µM of A46 for the indicated periods of time. Post treatment, the drug was washed away
and the cells (approximately 3 x 104 cells/ 35 mm dish) were plated in MethoCult
medium lacking cytokines. Five days later, the number of granulocyte/macrophage and
erythroid colony forming units were counted and plotted as a function of treatment
group.
Statistical Analysis
Results are expressed as mean +/- SEM. For statistical evaluation of time-
dependent responses to A46, a two-way analysis of variance (ANOVA) was used. For
the analysis of inhibition of phosphorylation, induction of cell cycle arrest, induction of
apoptosis and suppression of pathologic cell growth and clonogenic potential ex vivo, a
Student’s t-test was employed. Data were assumed to be statistically significant when p
< 0.05.
Results
A46 Inhibits Jak2-V617F-dependent Cell Proliferation
Using an in silico structure based drug design approach, we screened a library of
small molecules from the NCI/DTP repository as described previously [109] and
identified a small molecule, termed A46, which bound the ATP-binding pocket of Jak2
47
with a favorable energy score. Table 2-1 lists the chemical name, NSC number,
chemical structure, and molecular weight of this compound. We next wanted to test the
ability of this compound to suppress Jak2-V617-mediated cell proliferation, in vitro. For
this, we used the human erythroleukemia (HEL 92.1.7) cell line, which is homozygous
for the Jak2-V617F mutation [110] and has a transformed proliferative phenotype that is
driven by constitutively active Jak2-V617F signaling [111]. HEL cells were treated either
with DMSO or with 10 µM of A46 for increasing periods of time. Viable cell numbers for
each treatment were determined using trypan blue exclusion and hemocytometer.
When compared to vehicle treated cells, we found that A46 significantly reduced viable
cell numbers in a time-dependent manner (Fig. 2-1A).
We next wanted to determine if the suppressive effects of A46 treatment on HEL
cell growth were reversible. For this, HEL cells were first exposed to 10 µM of A46 for 0,
8, 12, 24, 48 and 72 hrs. At the end of each time point, the drug was washed away and
the cells were resuspended in fresh growth medium. They were then allowed to grow for
an additional 72 hrs in the absence of the inhibitor. Viable cell numbers were
determined at the end of the 72 hour recovery period. We found that ~19 hrs of initial
exposure to A46 prevented 50% of the cells from recovering from the treatment (Fig. 2-
1B). For cells that had been treated with A46 for 48 hrs or more, fewer than 20% were
able to recover after removal of the drug (Fig. 2-1B), suggesting that after a finite time of
initial treatment, the effect of the drug on HEL cell growth is largely irreversible.
To determine the specificity of A46 for Jak2-V617F-dependent cell growth, the
drug was applied to four different cell lines and cell viability was assessed. The cell
lines were (i) HEL (erythroleukemia) cells that are homozygous for the activating Jak2-
48
V617F mutation, (ii) Raji (Burkitt lymphoma) cells that have a translocation between the
c-Myc gene on chromosome 8 and the heavy chain locus on chromosome 14, (iii) SET-
2 (essential thrombocythemia) cells that are heterozygous for the Jak2-V617F mutation,
and (iv) CMK (acute megakaryocytic leukemia) cells that harbor a Jak3-A572V
mutation. Each of these cell lines was treated with increasing doses of A46. After 72 hrs
of treatment, we found that A46 markedly inhibited the growth of the Jak2-V617F
expressing HEL (Fig. 2-1C & D) and SET-2 (Fig. 2-1C) cells in a dose dependent
manner with a GI50 of ~ 400 nM for both cell types. The viability of Raji cells (Fig. 2-1C &
D) was, however, not affected by A46 treatment (GI50 > 25,000 nM). On the other hand,
the Jak3-A572V expressing CMK cells had intermediate susceptibility to the drug (Fig.
2-1D) with a GI50 of ~2,500 nM, suggesting that A46 is selective for Jak2-V617F
expressing cell lines over Jak3 and c-Myc mutated cell lines. Collectively, the data in
Fig. 2-1 demonstrate that the novel small molecule inhibitor A46 selectively inhibits
Jak2-V617F dependent in vitro cell growth, and this inhibitory effect of the drug on HEL
cell growth is largely irreversible after a determinate period of initial drug exposure.
A46 Inhibits the Jak/STAT Signaling Pathway
Phosphorylation of Jak2 on tyrosine residues 1007/1008 is concomitant with
increased Jak2 kinase activity [12]. Activated Jak2 can in turn phosphorylate and
activate downstream signaling targets such as STAT5. Hence, we next wanted to see if
the A46 mediated reduction in HEL cell numbers directly correlated with the
suppression of the Jak/STAT signaling pathway. This study is important in order to
eliminate the possibility that A46 exerts its cell growth inhibitory effect via mechanisms
that are independent of the Jak/STAT signaling pathway. For this, we treated HEL cells
with increasing concentrations of A46 for 48 hours and then measured the levels of
49
phospho-Jak2 and phospho-STAT5 proteins in these cells using an ELISA-based
assay. It is important to note that the phospho-protein levels were measured 48 h after
drug exposure rather than the 72 h used in Fig. 2-1 because far fewer viable cells exist
at the longer time point. We observed that A46 significantly reduced levels of both
phospho-Jak2 (Fig. 2-2A) and phospho-STAT5 (Fig. 2-2B) proteins in a dose-
dependent manner. These reductions in levels of phosphorylated proteins of the
Jak/STAT signaling pathway correlated well with the A46-mediated reductions in HEL
cell numbers. Thus, the data in Fig. 2-2 indicate that A46 inhibits HEL cell growth by
directly down regulating the phosphorylation of key signaling molecules of the Jak/STAT
pathway; namely, Jak2 and STAT5.
A46 Induces G1/S Cell Cycle Arrest in HEL Cells
To determine the mechanism of A46-mediated suppression of HEL cell growth, we
first analyzed the cell cycle distribution of cells as a function of drug treatment. Here, we
treated HEL cells with increasing doses of A46 for 48 hours and then performed cell
cycle analysis via flow cytometry. Again, analysis was performed 48 h after drug
exposure rather than the 72 h used in Fig. 2-1 because far fewer viable cells exist at the
longer time point. We found that exposure to A46 increased the percentage of cells in
G1 phase and correspondingly decreased the percentage of cells in S phase, and this
effect was dose-dependent (Fig. 2-3A).
Cell cycle progression is driven and regulated by the cyclic expression of
cyclin/CDK complexes [112]. Cyclin D1 levels play a critical role in promoting the
progression of the cell cycle from G1 to S phase [113] and is also known to be a
downstream target of STAT5 transcriptional regulation [114]. Therefore, in order to
confirm the induction of G1 phase arrest in these cells, we performed anti-cyclin D1
50
western blot analysis on HEL cells that had been treated with increasing doses of A46
for 24 hours. We performed our western blot analysis 24 hrs after drug exposure rather
the 48 hrs used in Figs. 2-3A because we wanted to ensure there were sufficient
numbers of viable cells and hence, sufficient levels of cellular protein to analyze. We
found that A46 treatment decreased the levels of cyclin D1 protein in a dose-dependent
manner (Fig. 2-3B, upper panel), thereby confirming the A46-induced arrest of HEL
cells in the G1 phase of the cell cycle. Analysis of the same samples with an anti β-actin
antibody was used as a loading control (Fig. 2-3B, bottom panel). Overall, the data in
Fig. 2-3 demonstrate that A46 treatment induces a G1/S cell cycle arrest in HEL cells
via the down regulation of cyclin D1.
A46 Induces Apoptosis in HEL Cells
The Jak/STAT signaling pathway is known to have direct effects on cell survival
and apoptosis (28). Having shown that A46 inhibited HEL cell growth (Fig 2-1) and
induced cell cycle arrest (Fig. 2-3), we next wanted to determine if this drug causes
apoptotic cell death in HEL cells. For this, we used Annexin V/Propidium Iodide (PI)
double staining and flow cytometry. The treatment conditions were the same as in Fig.
2-3A. The data in Fig. 2-4A shows representative apoptosis assay staining profiles from
one experiment while Fig 2-4B is a quantitative graph of two independent experiments
showing the percentage of cells in early apoptosis (Annexin V positive/PI negative)
plotted as a function of treatment condition. These data show that A46 induces
apoptosis in HEL cells in a dose-dependent manner.
Cleavage of procaspases converts these proteins into their functionally active
forms. Active caspases can then act on and cleave their substrates, such as the DNA
repair enzyme PARP, thereby affecting the integrity of the cell and triggering apoptosis
51
[115]. Cleavage of procaspases and PARP are therefore considered as hallmarks of
apoptotic induction. In order to confirm the induction of apoptosis by A46, we analyzed
the effect of A46 treatment on the cleavage of procaspase 3 and its downstream
substrate, PARP. HEL cells were treated with increasing doses of A46 for 24 hours and
procaspase-3 and PARP protein levels were measured via western blot analysis (Fig. 2-
5A). The samples were also blotted with an anti β-actin antibody to demonstrate equal
protein loading across all lanes (Fig. 2-5A). We found that A46 induced a dose-
dependent cleavage of PARP and a decrease in procaspase 3 expression, thereby
corroborating the data from the Annexin V/PI apoptosis assay (Fig. 2-4A & 2-4B).
Having confirmed the ability of A46 to induce apoptosis in HEL cells, we next
wanted to determine the mechanism by which this drug causes apoptotic cell death.
Apoptosis is regulated by members of the Bcl-2 family, many of which are direct
downstream gene targets of the Jak/STAT signaling pathway (28). Therefore, we next
monitored the expression of Bcl-2 family members in HEL cells exposed to different
doses of A46 for 24 hours via western blot analysis. We observed an A46-induced
dose-dependent decrease in expression BimEL, a pro-apoptotic member of the Bcl-2
family (Fig. 2-5B). Although Bim is a pro-apoptotic protein, its disappearance/down
regulation has been shown to be associated with the generation of cleaved pro-
apoptotic forms that positively regulate and amplify apoptotic signaling [116, 117].
Another pro-apoptotic Bcl-2 family protein, Bax, was also down regulated dose-
dependently in response to A46 treatment, although to a much lesser extent (Fig. 2-5B).
Cleavage of Bax in response to apoptotic cues is not uncommon and is known to
enhance its cell death function [118, 119]. Additionally, we found that A46 decreased
52
the levels of the inactive precursor form of Bid (Fig. 2-5B), which is yet another pro-
apoptotic protein that is activated upon cleavage [120]. Levels of anti-apoptotic Bcl-2
protein did not change with A46 treatment (Fig. 2-5B). Lastly, the samples were blotted
with an anti β-actin antibody to demonstrate equal protein loading across all lanes (Fig.
2-5B). In summary, the data in Figs. 2-4 and 2-5 indicate that A46 induces apoptotic cell
death in HEL cells via the down regulation/cleavage of Bcl-2 family proteins Bim, Bax
and Bid. Overall, from the data in Figs. 2-3, 2-4, and 2-5, we can conclude that A46
inhibits HEL cell proliferation by arresting the cells at G1/S transition and inducing
apoptosis.
A46 Suppresses Cytokine-independent Pathologic Cell Growth of Jak2-V617F Positive Bone Marrow Cells, ex vivo
We next wanted to determine the ability of A46 to inhibit the pathologic cell growth
of patient-derived Jak2-V617F positive bone marrow cells, ex vivo. For this,
mononuclear cells isolated from the bone-marrow of a female essential
thrombocythemia (ET) patient, who was Jak2-V617F positive, were cultured in medium
lacking thrombopoietin (TPO) and in the absence or presence of increasing doses of
A46. Hematopoietic progenitor cells isolated from a normal individual will be unable to
grow in the absence of cytokine. However, the V617F positive progenitor cells from the
patient will be able to grow under such conditions because the Jak2-V617F mutation
confers thrombopoietin-independent growth. Additionally, bone marrow cells from MPN
patients are known to be hypersensitive to cytokine stimulation [121]. Hence, the ET
patient derived bone marrow cells were also cultured in the presence or absence of the
exogenously added thrombopoietin. The results obtained show that treatment of the
mutant bone marrow cells with A46 significantly suppressed pathologic cell growth in a
53
dose-dependent manner (Fig. 2-6A). We also observed that exposure of these patient
derived marrow cells to exogenous thrombopoietin significantly increased the number of
colonies formed (Fig. 2-6A), suggesting that these mutant cells from the marrow of an
ET patient are indeed hypersensitive to cytokine.
We also carried out a clonogenic assay to determine if A46 could inhibit the
cytokine-independent colony forming ability of bone marrow cells obtained from Jak2-
V617F transgenic mice [67]. Jak2-V617F positive bone marrow cells were harvested
from these transgenic animals, cultured ex vivo, and then exposed to 25 µM of A46 for
the indicated periods of time. At the end of this initial treatment period, the drug was
washed away and the cells were plated in semi-solid MethoCult medium lacking
cytokines. Five days later, the number of granulocyte-macrophage (GM) and erythroid
(E) colony-forming units in each treatment group were counted and plotted as a function
of time of initial exposure to the drug. We found that A46 significantly reduced the
number of CFU-GMs and CFU-Es in a time-dependent manner (Fig. 2-6B), suggesting
that this drug can suppress the cytokine-independent colony forming ability of Jak2-
V617F expressing murine bone marrow cells. As such, data in Fig. 2-6 demonstrate that
A46 inhibits Jak2-V617F-dependent pathologic cell growth of patient derived bone
marrow cells and also suppresses the clonogenic growth potential of Jak2-V617F-
positive bone marrow cells from MPN mice, ex vivo.
Discussion
Aberrant Jak2 kinase activity has been linked to human disorders including
several hematological malignancies and the MPNs [44, 45, 48]. A Jak2 gain-of-function
mutation (Jak2-V617F) has been detected in over 90% of patients with PV and about
50% of patients with ET and PMF [57-61]. Jak2-V617F-negative MPN patients often
54
carry other activating mutations either in Jak2 [72-74] or in upstream signaling
molecules, such as the thrombopoietin receptor, MPL [76, 77]. The critical role of Jak2
in the pathogenesis of these disorders coupled with the fact that there are no curative
treatment options currently available for MPN patients, has generated considerable
amount of interest in developing Jak2 inhibitors as potential therapeutics for MPNs.
In this study, we characterized a novel benzothiophene inhibitor of Jak2 that was
identified by in silico structure based drug screening. We show that this compound
inhibits Jak2-V617F mediated cell proliferation both time and dose dependently (Fig.
1A, C & D) and this inhibitory effect of the drug on HEL cell growth is largely irreversible
after ~48 hrs of initial exposure (Fig. 1B). An important parameter used in evaluating the
effectiveness and safety of a drug is its specificity since more selective drugs can be
safely administered at higher doses without causing severe side-effects. In the past,
Jak2 inhibitors have been reported to have non-specific off target effects. AG490, one of
the first Jak2 inhibitors identified, was a potent Jak2 inhibitor, but had poor specificity
[82, 83, 122]. Our data here show that the Jak2-V617F positive cell lines, HEL and
SET2, are significantly more sensitive to inhibition by A46 (GI50 ~300 nM, Fig. 1C) when
compared to Jak3-dependent CMK cells (GI50 ~2,500 nM, Fig. 1D) or c-myc-dependent
Raji cells (GI50 > 25,000 nM, Fig. 1C & D), thereby suggesting that A46 selectively
inhibits the proliferation of Jak2-V617F-dependent cell lines while having little to no
effect on other cells lines whose proliferation is driven by Jak2-independent
mechanisms.
We have also shown that the A46 induced cell growth inhibition correlates with
direct suppression of the Jak/STAT signaling (Fig. 2A & B) which eliminates the
55
possibility that A46 might be exerting its cell growth inhibitory effect via mechanisms
that are independent of the Jak/STAT signaling pathway. Our results indicate that the
mechanism by which A46 suppresses Jak2-V617F-mediated HEL cell proliferation is via
the induction of both G1/S cell cycle arrest (Fig. 3) and apoptosis (Fig. 4). Deregulation
of the Jak/STAT signaling promotes cell proliferation and blocks apoptosis resulting in
disease pathogenesis. Hence, a better understanding of the mechanism of Jak2-
inhibition induced cell death may lead to the development of more effective therapeutic
strategies for treating MPN patients, including combination therapies of Jak2 inhibitor
and apoptotic modulator mimetics. Here, we show that A46 induces apoptotic cell death
in HEL cells via the down regulation/cleavage of Bcl-2 family proteins Bim, Bax and Bid.
There are conflicting reports on the effect of apoptosis induction on the expression of
the proapoptotic protein Bim; some studies suggest that Bim is up regulated during
apoptosis [123, 124] whereas others suggest that Bim is cleaved during apoptosis
generating active and proapoptotic cleaved fragments (30, 31).
Jak2 small molecule inhibitors currently represent a diverse number of chemical
structures including pyrazines, pyrimidines, azaindoles, aminoindazoles, deazapurines,
stilbenes, benzoxazoles, and quinoxalines [125]. Our work here is significant in that it is
the first report indicating that benzothiophene based compounds, such as A46, possess
Jak2 inhibitory potential. Benzothiophenes are heterocyclic structures known to have
pro- and anti-estrogenic [126], anti-lipoxygenase [127] and anti-fungal properties [128].
Not surprisingly, several approved pharmaceuticals including raloxifene, zileuton, and
sertaconazole have benzothiophene based chemical structures. Molecules with a
benzothiophene core have also been reported to inhibit tubulin, cysteine and serine
56
proteases (such as cathepsins K and L and thrombin), herpes simplex virus type I
replication and opioid receptor analgesics [129]. These heterocyclic structures are
relatively stable and their reactive site allows for subsequent derivatization, suggesting
that A46 is quite amenable to future lead optimization. As such, benzothiophenes may
be a new class of scaffolds for Jak2 small molecule inhibitors. In summary, our data
collectively show that the novel benzothiophene small molecule inhibitor of Jak2, A46,
inhibits Jak2-V617F-mediated pathological cell growth in vitro and ex vivo. As such, this
compound may perhaps serve as a lead therapeutic agent for the treatment of Jak2-
V617F mediated pathogenesis.
57
Table 2-1. Chemical characteristics of A46. A46 is shown here along with its chemical name, NSC #, chemical structure, and molecular weight.
Compound Name
Chemical Name NSC # Structure Molecular
Weight
A46 1-benzothiophen-2-yl-(4-
dimethylaminophenyl)methanol 40282
283.3874
58
Figure 2-1. A46 inhibits Jak2-V617F-dependent cell proliferation. A) HEL cells were treated with either DMSO or 10 μM A46 for 0, 24, 48 or 72 hours. Viable cell numbers for each treatment were determined by trypan blue exclusion staining using a hemocytometer. Each sample was measured in triplicate. Shown is one of two sets of representative results. *p < 0.05 with respect to DMSO. B) HEL cells were treated with 10 μM A46 for 0, 8, 12, 24, 48 and 72 hours. At the end of each treatment, the cells were washed, placed in fresh medium and cultured in the absence of the inhibitor for an additional 72 hours. The number of viable cells in each sample was then determined. Each sample was measured in triplicate. Shown are the means ± S.E. from two independent experiments. HEL (C & D), SET-2 (C), Raji (C & D) and CMK (D) were treated with increasing doses of A46 for 72 hours. The percent viable cells from each condition were determined either by trypan blue exclusion staining (Fig. 1C) or via an automated cell viability assay using the MTS reagent and a plate reader (Fig. 1D). Each sample was measured in triplicate.
59
Figure 2-2. A46 inhibits the Jak/STAT signaling pathway. HEL cells were treated with 0, 0.09, 0.3, 0.9, 3, 9 and 30 μM of A46 for 48 hours. The cell lysates were then analyzed by ELISA for the measurement of phospho-Jak2 [pY1007/pY1008] (A) and phospho-STAT5 [pY699] (B) protein levels. Each condition was run in triplicate. Shown are the means ± S.D. of two independent experiments.
60
Figure 2-3. A46 arrests HEL cells in G1 phase of the cell cycle. HEL cells were treated with 0, 0.1, 0.3, 1, 3, 10 and 30 μM of A46. A) After 48 hrs of treatment, cell cycle distribution of treated cells was determined as a function of drug treatment using flow cytometry. Shown are the means ± S.E. of two independent experiments. B) Following 24 hrs of treatment with the indicated concentrations of A46, cells were lysed and analyzed by immunoblotting with an anti-cyclin D1 (top) or an anti-β-actin antibody (bottom). Shown is one of two representative results.
61
Figure 2-4. A46 induces apoptosis in HEL cells. HEL cells were treated with 0, 0.1, 0.3,
1, 3, 10 and 30 μM of A46 for 48 hours, stained with annexin V-FITC and propidium iodide and then analyzed by flow cytometry to determine the level of apoptosis in the treated cells. A) Shown are representative flow cytometry profiles from one of two independent experiments. B) Quantification of the percentage of cells in early apoptosis as a function of drug treatment. Shown are the means ± S.E. of two independent experiments.
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Figure 2-5. A46-induced apoptosis is mediated by the down regulation/cleavage of Bcl-2 family proteins Bim, Bax and Bid. HEL cells were treated with 0, 0.1, 0.3, 1, 3, 10 and 30 μM of A46 for 24 hours. The whole cell lysates were then analyzed by western blotting with a series of antibodies as indicated: A) anti-poly(ADP-ribose) polymerase (PARP), anti-caspase 3 and anti-β-actin, B) anti-Bim, -Bax, -Bid, -Bcl-2 and -β-actin. Shown are the results from one of two independent experiments.
63
Figure 2-6. A46 suppresses cytokine-independent pathologic cell growth of Jak2-
V617F positive bone marrow cells, ex vivo. A) Patient-derived bone marrow mononuclear cells were cultured in semisolid medium in the presence of increasing doses of A46, with or without thrombopoietin (TPO). At the end of 14 days, the numbers of megakaryocyte colony-forming units (CFU-Megs) were counted and plotted as a function of treatment condition. Each condition was measured in duplicate. *p < 0.05 with respect to 0 μM A46 (-TPO), #p < 0.05 with respect to 0 μM A46 (+TPO), and §p < 0.05 (+ TPO) vs. (-TPO). B) Jak2-V617F-positive bone marrow cells from transgenic mice were treated with 25 μM A46 for 0, 12 and 24 hours, ex vivo. At the end of each treatment, the drug was washed away and the cells were cultured in drug-free semi-solid medium lacking cytokines for another 5 days. The numbers of granulocyte-macrophage (GM) and erythroid (E) colony-forming units in each sample were counted and plotted as a function of treatment condition. Each sample was measured in duplicate. Shown is one of two representative experiments. *p < 0.05 vs. 0 hr for CFU-GM, #p < 0.05 vs. 0 hr for CFU-E.
64
CHAPTER 3 STRUCTURE-FUNCTION CORRELATION OF G6, A NOVEL SMALL MOLECULE
INHIBITOR OF JAK2: INDISPENSABILITY OF THE STILBENOID CORE
Jak2 plays a critical role in animal development, as mice that are devoid of a
functional Jak2 allele die during embryonic development due to a lack of hematopoiesis
[31, 32]. Deregulation of the Jak/STAT signaling pathway promotes cell growth and
prevents apoptosis in a variety of solid tumors and hematological malignancies such as
acute lymphoid leukemia and chronic myeloid leukemia [45, 48, 52, 130]. Additionally, a
somatic Jak2 mutation (Jak2-V617F) is found in a high number of myeloproliferative
neoplasm (MPN) patients including 90% of polycythemia vera (PV) patients and
approximately 50% of patients with essential thrombocythemia (ET) and primary
myelofibrosis (PM) [57-61]. MPNs are a group of heterogeneous diseases arising from a
transformed hematopoietic stem cell and characterized by excessive numbers of one or
more terminally differentiated blood cells of the myeloid lineage such as erythrocytes,
thrombocytes or white blood cells. A guanine to thymine mutation in hematopoietic stem
cells results in a substitution of valine to phenylalanine at codon 617 in exon 14 of the
JH2 pseudokinase domain of Jak2. It is believed that this mutation allows the kinase to
evade negative feedback inhibition thereby leading to a constitutively active Jak/STAT
signaling pathway characterized by growth factor independent cell growth [57, 60].
Given the critical role that Jak2 plays in the pathophysiology of MPNs,
identification of specific Jak2 inhibitors has become an important step towards the
development of an effective targeted therapy for these disorders. Using structure-based
This research was originally published in the Journal of Biological Chemistry. Structure-function correlation of G6, a novel small molecule inhibitor of Jak2: indispensability of the stilbenoid core. J Biol Chem. 2010; 285(41): 31399-407. © The American Society for Biochemistry and Molecular Biology.
65
virtual screening, our group recently identified a novel small molecule inhibitor of Jak2
named G6 [104]. We showed that G6 has a specific inhibitory effect on Jak2 kinase
activity as measured by in vitro enzyme assays and an immunoassay ELISA [104].
Examination of the chemical structure of G6 revealed the presence of a central
stilbenoid core. Stilbenoids are a group of naturally occurring compounds having a wide
range of biological activities. For example, resveratrol, piceatannol, 3,4,5,4′-
tetramethoxystilbene and 3,5,4′-trimethoxy-trans-stilbene, are all stilbenoids and are
known to have cytotoxic, anti-proliferative, pro-apoptotic, anti-angiogenic or tumour
suppressive effects [131-135].
We hypothesized that the central stilbenoid core in the G6 structure has a critical
role to play in mediating its Jak2 inhibitory potential. To test this, five derivative
compounds of G6, namely D21, D23, D25, D28 and D30, having structural similarity to
the original lead compound, were procured from the National Cancer Institute. Two of
these compounds, D28 and D30, have a stilbenoid core present in their chemical
structure similar to G6 whereas the three other compounds, D21, D23 and D25, lack the
stilbenoid core. We report here that the core stilbenoid structure present in G6 is
essential for maintaining its ability to inhibit Jak2 kinase activity.
Experimental Procedures
Drugs
G6 and its five structurally related derivative compounds (D21, D23, D25, D28 and
D30) were obtained from the National Cancer Institute/Developmental Therapeutics
Program (NCI/DTP) which maintains a repository of approximately 140,000 compounds.
Each compound was solublized in dimethyl sulfoxide at a concentration of 10 mM and
stored at -20⁰C.
66
Cell Culture
Human Erythroleukemia (HEL) cells were purchased from the American Type
Culture Collection. Ba/F3-EpoR-Jak2-V617F cells were created as described before
[136]. Both cell lines were cultured in RPMI 1640 (Mediatech) supplemented with 10%
fetal bovine serum (FBS), penicillin, streptomycin and L-glutamine at 37⁰C and 5% CO2.
Cell Proliferation Assay
HEL cells or Ba/F3-EpoR-Jak2-V617F cells were plated in 96-well plates and
treated with either 0.25% DMSO or varying concentrations of G6 and its derivatives for
the indicated periods of time. Cell viability was assessed either by trypan blue exclusion
staining or by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-
sulfophenyl)- 2H-tetrazolium, inner salt] (MTS) (Promega) as per the manufacturer’s
protocol.
Enzyme-linked Immunosorbent Assay
HEL cells were treated with either 0.25% DMSO or 25 μM of the inhibitor
compounds for 48 hrs and the cell lysates were analyzed by ELISA for detection of
phoshpho-Jak2, phospho-STAT3 and phospho-STAT5. Jak2 [pY1007/pY1008], STAT3
[pY705] and STAT5b [pY699] ELISA kits were purchased from Invitrogen and used
according to the manufacturer’s protocol.
Cell Lysis and Immunoprecipitation
HEL cells were treated with the different drugs for the indicated periods of time.
Cells (~ 107) were then lysed in 0.8 ml of ice-cold RIPA buffer and protein concentration
was determined using a Bradford assay (Bio-Rad). Cell lysates (~2500 μg) were then
immunoprecipitated by incubating with 2 μg of the appropriate antibody and 20 μl of
Protein A/G beads (Santa Cruz Biotechnology) for 4 hrs at 4⁰C with constant shaking.
67
The protein complexes were washed thrice with IP wash buffer (25 mM Tris pH 7.5, 150
mM NaCl, and 0.1% Triton X-100) and then resuspended in SDS sample buffer.
Immunoprecipitated proteins were separated by SDS-PAGE and then transferred onto
nitrocellulose membranes. The anti-STAT3 and anti-STAT5 antibodies used for
immunoprecipitation were from Santa Cruz Biotechnology. For whole cell protein
lysates, ~50 μg of soluble protein was separated via SDS-PAGE and then transferred to
nitrocellulose membranes for analysis by western blotting.
Western Blotting
Nitrocellulose membranes were first blocked with 5% milk/TBST solution and then
probed with the different primary antibodies. The immuno-reactive bands were then
visualized using the enhanced chemi-luminescence system (Western Lightning Ultra,
Perkin-Elmer). The following antibodies were used at the indicated dilutions: phospho-
STAT3 (Santa Cruz Biotechnology and Cell Signaling at 1:500 each), STAT3 (Santa
Cruz Biotechnology, 1:1000), phospho-STAT5 (Cell Signaling, 1:500), STAT5 (Santa
Cruz Biotechnology, 1:1000), and STAT1 (Santa Cruz Biotechnology, 1:1000). The
following were all obtained from Cell Signaling and used at a 1:500 dilution; poly (ADP-
ribose) polymerase (PARP), Bcl-2, Bax, Bim, and Bid.
Apoptosis Assay
Induction of apoptosis in HEL cells was determined with the FITC AnnexinV
Apoptosis detection kit (BD Pharmigen) per the manufacturer’s protocol. The cells were
incubated with 0.25% DMSO or 25 μM of inhibitors for 48 hrs and then analyzed using a
FACSCalibur flow cytometer (BD Biosciences).
68
Real Time PCR Analysis
The mRNA levels of Bcl-xL and GAPDH were measured by quantitative real-time
PCR analysis. Total RNA was extracted from HEL cells, treated with 25 μM of the
different drugs for 8 and 24 hours, using the RNeasy Mini Kit (Qiagen) as per
manufacturer’s protocol. 2 μg of each RNA sample was reverse transcribed into cDNA
in a final reaction volume of 20 μl using the High Capacity cDNA Reverse Transcription
Kit (Applied Biosystems). TaqMan Gene Expression Assays (Applied Biosystems)
Hs02758991_g1 (GAPDH) and Hs00236329_m1 (Bcl-xL) were used to detect the levels
of expression of these genes. GAPDH gene expression was used as an internal loading
control. The real-time polymerase chain reaction (PCR) was then performed with
TaqMan universal PCR Master Mix (Applied Biosystems) in a final reaction volume of
20 μl in a StepOne Real-Time PCR System as per manufacturer’s protocol.
Patient Sample
Bone marrow aspirates consisting of mononuclear cells were obtained from a de-
identified Jak2-V617F positive female diagnosed with polycythemia vera (WHO criteria)
at the UF & Shands Teaching Hospital as per an Institutional Review Board approved
protocol.
Colony Forming Unit-Erythroid Colony Formation Assay
Marrow-derived mononuclear cells were washed in IMDM and cultured in human
methylcellulose complete medium without Epo (R&D Systems) at a concentration of
approximately 4 × 105 cells/ml in the presence or absence of G6 or its structurally
related derivatives. 0.9 mL of the cultures was placed in 35-mm petri dishes and
incubated at 37⁰C and 5% CO2 in a humidified atmosphere for 14 days following which
the number of erythroid colony forming units (CFU-E) were counted.
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Computational Docking
The molecular docking program DOCK6 [137, 138] was used to study the
interactions of Jak2 with ATP, the ATP analog ACP (adenosine-5′-[β, γ-methylene]
triphosphate), G6, and the structurally related derivative compounds. The template used
was the crystal structure of Jak2 kinase domain in complex with the Jak2 inhibitor 5B3
(PDB ID: 3E64) [139]. The coordinates for ATP, ACP and each of the small molecules
were generated and energy minimized using PRODRG [140]. Subsequently, CHIMERA
[141] was used to add atomic charges to these small molecules. After removing the 5B3
molecule, the coordinates of the Jak2 kinase domain were saved in PDB format. To
prepare the protein for docking, hydrogen atoms and partial electrostatic charges were
first added to the molecule. A molecular surface of the Jak2 kinase domain was then
generated using the DMS tool in CHIMERA. The program SPHGEN was used to
generate spheres around the active site for identification of the target pocket on the
protein for small molecule docking. The grid file, necessary for rapid grid-based energy
score evaluation, was then generated around the identified docking site using the GRID
module in DOCK6. Each compound was docked in 1000 different orientations using
flexible dock and a net energy GRID score was generated for each, based on the van
der Waals and electrostatic interactions between the compound and the residues in the
binding pocket. The favorable binding orientations were selected on the basis of the
energy GRID scores generated for each orientation, wherein a favorable binding
orientation would have a more negative score. Finally, the docking results were
analyzed visually using COOT [142] and CHIMERA. Surface electrostatic potentials
were calculated using APBS [143] and PYMOL [144] was used to generate the figures.
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Statistical Analysis
For statistical evaluation of time-dependent responses to the different inhibitor
compounds, a two-way analysis of variance was used. For analysis of inhibition of
phosphorylation, induction of apoptosis, modulation of gene expression and
suppression of pathologic cell growth ex vivo, a Student’s t-test was employed. Data
were assumed to be statistically significant when p < 0.05.
Results
A Stilbenoid Core Is Essential for Time- and Dose-dependent Inhibition of Jak2-V617F-dependent Cell Growth
The human erythroleukemia (HEL 92.1.7) cell line is homozygous for the Jak2-
V617F mutation and this gain-of-function mutation is responsible for its transformed
phenotype [110, 145]. Proliferation of HEL cells is mediated by the constitutively active
Jak2-V617F signaling which promotes a G1/S phase transition, thereby leading to
increased cellular proliferation [111]. G6 and its five structurally related derivatives were
therefore first analyzed for their ability to inhibit the Jak2-V617F dependent proliferation
of HEL cells. Viable cell numbers were determined by trypan blue exclusion and
hemocytometer after 72 hrs. Each sample was measured in triplicate. Inhibition by G6
was arbitrarily set at 100% and the percent inhibition for all the other compounds
relative to G6 was defined as 1.00 - (Δ drug / Δ vehicle control). Table 3-1 summarizes
the percent growth inhibition for each of the six compounds. We found that the stilbene-
containing derivatives (D28 and D30) had high growth inhibition potentials whereas
those compounds lacking the stilbenoid core (D21, D23 and D25) had low growth
inhibition potentials.
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To determine the ability of each of these compounds to inhibit Jak2-V617F
mediated HEL cell proliferation, cells were treated either for varying periods of time or
with increasing concentrations of G6 or its derivatives. Viable cell numbers for each
treatment were determined. When compared to vehicle treated cells, we found that G6
and its stilbenoid derivatives (D28 and D30) significantly reduced viable cell numbers in
a time-dependent manner whereas the non-stilbenoid derivatives (D21, D23 and D25)
did not (Fig. 3-1A). We also found that G6 and the two stilbenoid derivatives (D28 and
D30) markedly inhibited the growth of HEL cells in a dose-dependent manner (Fig. 3-
1B) whereas the three non-stilbenoid derivatives (D21, D23, and D25) showed no
growth inhibition of HEL cells (Fig. 3-1C).
Phosphorylation of Jak2 at tyrosine residues 1007/1008 is concomitant with higher
kinase activity and increased cellular proliferation [61]. Therefore, we next wanted to
determine whether the presence of the stilbenoid core is critical for reduction of
phospho-Jak2 levels within treated cells. Phospho-Jak2 levels were measured 48 hrs
after drug exposure rather the 72 hrs used in Figs. 3-1A – C as far fewer viable cells
exist at the longer time point. Exposure of HEL cells to stilbenoid core bearing
compounds (G6, D28, and D30) significantly decreased the levels of phospho-Jak2
(Fig. 3-1D) when compared to those derivatives that lack the stilbenoid core (D21, D23,
and D25).
We next wanted to determine if the ability of G6 to inhibit Jak2-V617F mediated
cell proliferation was valid for other cell lines with constitutively active Jak2. For this, we
studied the ability of these compounds to inhibit Ba/F3 cells stably expressing Jak2-
V617F. The introduction of the Jak2-V617F cDNA into these cells via retroviral
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transduction confers cytokine independent growth that is entirely Jak2-V617F
dependent [136]. Cells were treated with various doses of the different drugs and viable
cell numbers were assessed after 72 hrs using an MTS assay. G6 and its stilbenoid
derivatives (D28 and D30) showed significant inhibition of cell growth in a dose
dependent manner (Fig. 3-2A) when compared to the non-stilbenoid derivatives (D21,
D23 and D25) (Fig. 3-2B). Collectively, these data demonstrate that the stilbenoid core
is essential for maintaining the ability of G6 to inhibit Jak2-V617F dependent cell growth
and the reduced cell growth correlates with significantly reduced levels of phospho-
Jak2.
Indispensability of the Stilbenoid core in Decreasing Phosphorylation of STAT3 and STAT5
In the canonical Jak/STAT signalling pathway, an active Jak2 phosphorylates
STAT proteins such as STAT3 and STAT5, which ultimately translocate to the nucleus
and modulate gene transcription [60]. Hence, we wanted to determine if the stilbenoid
core is critical for the inhibition of STAT phosphorylation. HEL cells were treated for 48
hrs with the different compounds and phospho-STAT levels were determined. G6 and
its stilbenoid derivatives (D28 and D30) significantly decreased phospho-STAT3 levels
as measured by ELISA (Fig. 3-3A) and western blot analysis (Fig. 3-3B). Similarly, only
the stilbenoid containing compounds (G6, D28 and D30) significantly decreased
phospho-STAT5 levels as measured by ELISA (Fig. 3-3C) and western blot analysis
(Fig. 3-3D). As such, these data confirm the ability of G6 and its stilbenioid derivatives
to downregulate the phosphorylation of key signaling molecules involved in Jak2-
dependent pathological cell growth; namely, STAT3, and STAT5.
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Induction of Apoptosis in HEL Cells by G6 and its Derivatives
Deregulation of the Jak/STAT signaling pathway is known to promote cell
proliferation and prevent apoptosis in several different cancers [146]. Given that G6 and
its stilbenoid core containing derivatives inhibited Jak2-V617F dependent cell
proliferation and Jak/STAT activation, we next wanted to determine whether these
drugs induce apoptotic death. HEL cells treated with G6 and the stilbeniod derivatives
(D28 and D30) exhibited a significant increase in the percentage of cells in early
apoptosis when compared to the DMSO or the non-stilbenoid (D21, D23, and D25)
treated cells (Fig. 3-4A). Fig 3-4B is a quantitative graph of four independent
experiments showing the amount of apoptosis plotted as a function of treatment
condition. We observed that the percentage of cells in early apoptosis increased from
7.45% in the DMSO treated control to 27.8% in G6 treated, 31.3% in D28 treated and
34.2% in D30 treated HEL cells, whereas it remained almost unchanged for the non-
stilbenoid treated cells (Fig. 3-4B).
Jak2/STAT signaling is known to positively regulate cell growth by directly
increasing expression of the anti-apoptotic marker, Bcl-xL, via STAT-binding elements
present in its promoter region [114, 147]. To determine if the presence of the stilbenoid
core correlates with reduced levels of Bcl-xL, we measured Bcl-xL mRNA levels in cells
treated with the different compounds. The stilbenoids (G6, D28 and D30), significantly
decreased Bcl-xL expression in HEL cells when compared to DMSO or the non-
stilbenoids (D21, D23 and D25) both at 8 hrs (Fig. 3-5A) and 24 hrs (Fig. 3-5B) of
treatment. As such, these data indicate that the presence of the stilbenoid core does in
fact correlate with markedly reduced levels of the proliferative marker, Bcl-xL.
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The intrinsic apoptotic pathway is regulated by members of the Bcl-2 family [148].
Therefore, we next monitored the expression of Bcl-2 family members in HEL cells
treated with 25 μM of the different compounds for 24 hours. As expected, the stilbenoid
derivates (G6, D28 and D30) induced greater cleavage of PARP, a marker of apoptosis,
when compared to the non-stilbenoids (D21, D23 and D25) (Fig. 3-5C). We also
observed a stilbenoid dependent increase in the protein levels of Bim, a pro-apoptotic
member of the Bcl-2 family (Fig. 3-5D).
The pro-apoptotic protein, Bid, is present in the cytosol as an inactive precursor.
Upon proteolytic cleavage, its active form is generated, which translocates to the
mitochondria and induces mitochondrial damage and destabilization [120]. We found
that the stilbenoid compounds decreased the levels of the inactive precursor form of
Bid, a hallmark of Bid cleavage and subsequent apoptosis. We found that the protein
levels of Bax and Bcl-2 did not change with any treatment (Fig. 3-5D). Lastly, the
samples were blotted with an anti-STAT1 antibody to demonstrate equal protein loading
across all lanes (Fig. 3-5D). Overall, the data in Figs. 3-4 and 3-5 indicate that the
stilbenoid core of G6 is critical for its ability to induce apoptotic cell death in HEL cells
via the intrinsic apoptotic pathway by downregulation of anti-apoptotic Bcl-xL, up-
regulation of pro-apoptotic Bim, and cleavage of Bid.
Stilbenoid Core Bearing Derivatives of G6 Suppress Pathologic Cell Growth of Patient-derived Bone Marrow Cells, ex vivo
We next wanted to determine whether the stilbene core is also essential for its
ability to inhibit pathologic cell growth of patient-derived bone marrow cells, ex vivo. For
this, we used a colony formation assay which measures the number of erythroid colony
forming units (CFU-E) that are produced when marrow derived stem cells are cultured,
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ex vivo. Stem cells isolated from a normal individual will be unable to grow in the
absence of exogenously added cytokine. However, the Jak2-V617F mutation confers
cytokine independent, cell growth. Here, mononuclear cells isolated from the bone-
marrow of a Jak2-V617F positive female polycythemia vera patient, were cultured in
medium lacking erythropoietin. DMSO or 5 μM of inhibitor was added as indicated. Our
previous work has shown that 5 μM of G6 inhibits ~50% of the cytokine independent
growth of Jak2-V617F expressing, marrow derived stem cells [104]. The results here
show that treatment of the primary cultures with either G6 or the stilbenoids (D28 and
D30) significantly blocked cytokine-independent pathologic cell growth when compared
to the DMSO and non-stilbenoid (D21, D23, and D25) treated samples (Fig. 3-6). As
such, data in Fig. 3-6 demonstrate that the stilbenoid core of G6 is essential for
reducing Jak2-dependent pathologic cell growth of human bone marrow mononuclear
cells cultured ex vivo.
Computational Docking of G6 and its Derivatives into the ATP Binding Pocket of the Jak2 Kinase Domain
Using the known structure of the Jak2 kinase domain [139], ATP, the ATP analog
ACP, G6, and each of its five structurally related derivatives, were docked into the ATP
binding pocket. The goal was to analyze the potential interactions of these compounds
with amino acids in this binding region. The ATP binding pocket of Jak2 and the
important residues clustered in this pocket have been described previously [16, 22].
Generation of the electrostatic surface potential for the Jak2 kinase domain showed the
predicted presence of acidic and basic patch residues clustered at the ATP binding
pocket (Fig. 3-7). We found that both ATP and ACP docked extremely well into this
pocket (Fig. 3-8A) with GRID scores of -76.81 kcal/mole and -62.37 kcal/mole,
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respectively. Furthermore, the docked molecules showed excellent correlation with the
known crystal structures of ACP and ATP complexed with kinase domains of other
proteins [149-152].
We found that the stilbenoids had higher binding affinities when compared to the
non-stilbenoids as indicated by their more negative energy scores. G6, D28, D30, D21,
D23 and D25 had energy scores of -75.46, -63.24, -72.26, -27.69, -38.90 and -48.91
kcal/mole, respectively. The docking orientations of G6, D28 and D30 into the pocket
(Fig. 3-8B) were very similar to that of ATP/ACP (Fig. 3-7) and distinctly different from
that of the non-stilbenoids (Fig. 3-8C). The docking conformation of each of the
stilbenoid inhibitors into the ATP binding pocket also correlated well (r.m.s.d < 2Å) with
the previously reported structure of another Jak2 inhibitor, 5B3, in complex with the
Jak2 kinase domain (Fig. 3-8D).
Examination of the most favorable docking orientations for G6 and each of its
derivatives showed the presence of hydrogen bonds (< 3.5Å) and van der Waals
interactions between the stilbenoid derivatives and the ATP binding pocket of Jak2 (Fig.
3-9). Specifically, the stilbenoid core containing derivatives exhibited van der Waals
interactions with many of the hydrophobic residues in the binding pocket such as V863,
L855, L983, L932, Y931, A880 and V911. The stilbenoid containing derivatives also
formed hydrogen bond interactions with several important surrounding residues
including D994, R980, E930 and L932. The non-stilbenoid derivatives however, did not
show any such hydrogen bond interactions with these critical residues of the Jak2 ATP
binding pocket. Overall, these data demonstrate the importance of the stilbenoid core
77
structure for stronger docking interactions of the drug with the ATP binding pocket of
Jak2.
Discussion
Somatic Jak2 mutations, such as the Jak2-V617F, which result in deregulated
Jak/STAT signaling, have been identified in numerous patients with myeloproliferative
neoplasms and are known to play a primary role in the pathogenesis of these diseases
[57-61]. Therefore, identification of novel compounds having inhibitory effects against
hyperkinetic Jak2 has become an attractive therapeutic strategy in MPN. Using
structure-based virtual screening, our group recently identified a novel Jak2 small
molecule inhibitor called G6 [104].
In this report, we describe a structure-function correlation of this inhibitor
compound. We demonstrate that it has a central stilbenoid core in its structure which is
indispensible for maintaining its ability to inhibit Jak2 kinase activity. G6 and its
stilbenoid core containing derivatives (D28 and D30) effectively inhibit Jak2-V617F
mediated HEL cell proliferation in a time- and dose-dependent manner.
Correspondingly, they are also capable of suppressing the phosphorylation of Jak2,
STAT3 and STAT5 proteins. Furthermore, these stilbenoids significantly induce
apoptosis in treated cells via the intrinsic pathway and possess the ability to block ex
vivo pathologic cell growth of bone marrow cells isolated from a Jak2-V617F positive
MPN patient.
Stilbenes are a group of compounds with a wide range of diverse biological
activities. Stilbenoids, such as resveratrol, piceatannol and diethylstilbestrol, are
reported to have anti-proliferative, anti-oxidative, anti-neovascularization and tumor-
suppressive effects [131-135]. Resveratrol has beneficial cardiovascular effects [153]
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whereas diethylstilbestrol is known to have estrogen activity [154]. Piceatannol, a
naturally occurring phenolic stilbenoid, is the only stilbenoid that is a known protein
tyrosine kinase inhibitor. It inhibits LMP2A, a viral tyrosine kinase implicated in diseases
associated with the Epstein-Barr virus as well as the non-receptor tyrosine kinases Syk
and Lck [155, 156]. Here, we report that G6, which is also a stilbene, has anti-Jak2
tyrosine kinase activity. More importantly, the trans stilbenoid core identified in all the
active compounds is now being subjected to bioisosteric replacements [157] that would
result in new Jak2 chemotypes with improved druglike properties.
Computational docking of G6 and its structurally related derivatives into the ATP
binding pocket of Jak2 revealed important interactions between the inhibitors and
specific residues within Jak2. For example, E930 and L932 are part of the important
hinge region of the Jak2 kinase domain and are known to be involved in adenine-
binding. We found that these residues were occupied by the stilbenoid containing
compounds, but not the non-stilbenoids. Another amino acid that interacted with the
docked stilbenoid inhibitors was D994, located in the highly conserved DFG motif. This
motif is part of the critical activation loop of the kinase and our group was the first to
identify the importance of this residue as it relates to Jak2 kinase inhibition [104]. We
show here that the stilbenoid core bearing inhibitors also had a hydrogen bond
interaction with R980, which is a part of the catalytic loop and is known to be one of the
residues involved in the coordination of magnesium ions [16, 22]. Thus, the ability of the
stilbenoid derivatives to simultaneously interact, in silico, with the hinge region, the DFG
motif, and the catalytic loop suggests that they may interfere with both ATP-binding
function and activation loop phosphorylation, thereby making them potent Jak2
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inhibitors. In summary, our data collectively show that the central stilbenoid core
structure is indispensable for maintaining anti-Jak2 kinase activity of the small molecule
inhibitor G6.
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Table 3-1. Percent growth inhibition of G6 and its derivatives. G6 and its derivatives are shown along with their NSC #, chemical structure, molecular weight and % growth inhibition. Inhibition by G6 was arbitrarily set at 100% and the inhibition potential of the other compounds relative to G6 was evaluated using the relation 1.00 - (Δ drug / Δ vehicle control).
Compound Name
NSC #
Structure
MWa
% Growth Inhibition
G6
33994
438.652
100.00
D21 10618
227.3054
-5.00
D23 47911
241.3322 -1.80
D25 13109
379.5844 -0.61
D28 27647
339.4766 124.16
D30 600567
384.5606 108.32
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Figure 3-1. Time- and dose-dependent effect of G6 and its derivatives on the HEL cell
proliferation and Jak2 phosphorylation. (A) HEL cells were treated with either 0.25% DMSO or with 25 μM of G6 or its derivatives for 0, 24, 48 or 72 h. The number of viable cells in each sample was measured by trypan blue exclusion. Each sample was measured in triplicate. D23 vs. G6 (p = 1.22 × 10-10). (B) & (C) HEL cells were treated for 72 hours either with DMSO or with 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μM of the indicated compounds. The viable cell numbers for each treatment were determined in triplicate. Shown is one of two representative results. (D) HEL cells were treated with 25 μM of the different drugs for 48 h. Cells were then analyzed by ELISA for the detection of phospho-Jak2 (pY1007/pY1008). Each experiment was run in triplicate. Shown is one of two representative results. *p<0.05 with respect to DMSO, #p<0.05 with respect to non-stilbenoids.
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Figure 3-2. Dose-dependent effect of G6 and its derivatives on the proliferation of
Ba/F3-EpoR-Jak2-V617F cells. (A) & (B) Ba/F3-EpoR-Jak2-V617F cells were treated for 72 hours either with DMSO or with 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μM of the indicated compounds. The viable cell numbers for each treatment were determined in triplicate using an MTS assay. Shown is one of two representative results.
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Figure 3-3. Inhibition of phosphorylation of STAT3 and STAT5 by G6 and its
derivatives. HEL cells were treated with 25 μM of the different drugs for 48 h. Cells were then analyzed for the detection of phospho-STAT3 and phospho-STAT5 by both ELISA (A) & (C) and western blot analysis (B) & (D). Stilbenoid containing compounds (G6, D28 and D30) effectively inhibited phosphorylation of STAT3 (A) & (B), and phosphorylation of STAT5b (C) & (D). Shown is one of two representative results for each. *p<0.05 with respect to DMSO, #p<0.05 with respect to non-stilbenoids.
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Figure 3-4. Induction of apoptosis in HEL cells by G6 and its derivatives. HEL cells were treated with 25 μM of the different drugs for 48 hrs and then stained with Annexin V-FITC and propidium iodide followed by flow cytometric analysis. (A) Shown are representative flow cytometry profiles from one of four independent results. (B) Quantification of the number of cells in early apoptosis (i.e. annexin V positive and propidium iodide negative). Data shown are the mean +/- SD from four independent experiments. *p<0.05 with respect to DMSO, #p<0.05 with respect to non-stilbenoids.
85
Figure 3-5. Treatment with G6 or its stilbenoid derivatives leads to HEL cell death via the intrinsic apoptotic pathway. HEL cells were treated with 25 μM of the different drugs for either 8 hours (A) or 24 hours (B). Bcl-xL mRNA levels were normalized to those of GAPDH and plotted as the fold change over DMSO control. Each sample was run in duplicate. Shown is a one of two representative results. *p <0.05 with respect to DMSO. (C) Cells were treated with either DMSO or 25 μM of the different drugs for 24 hrs. The whole cell lysates were then analyzed by western blotting with an anti-PARP antibody. Shown is one of three representative results. (D) Cells were treated with either DMSO or 25 μM of the different drugs for 24 hrs. Whole cell lysates were then serially analyzed by western blot analysis for the indicated proteins. Shown is one of three representative results.
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Figure 3-6. Suppression of Jak2-V617F mediated pathologic cell growth in patient-derived bone marrow cells by G6 and its derivatives, ex vivo. Marrow-derived mononuclear cells were cultured in semisolid media in the presence or absence of 5 μM G6 and its structurally related derivatives. At the end of 14 days of culture, the numbers of CFU-E were counted. Each condition was measured in duplicate. *p<0.05 with respect to DMSO, #p<0.05 with respect to non-stilbenoids.
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Figure 3-7. Molecular surface representation of the ATP binding site of Jak2. Molecular surface representation of Jak2 in complex with ATP and its analog ACP, shown here as stick models. ATP is colored yellow and ACP is green.
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Figure 3-8. Molecular docking of G6 and its derivatives into the ATP binding pocket of Jak2. Molecular docking of G6 and its derivatives into the ATP binding pocket of Jak2. ATP, the ATP analog ACP, G6, and each of its structurally related derivatives were docked into the ATP binding pocket of the known crystal structure of Jak2 kinase domain (PDB ID: 3E64). (A) Coiled representation of the structure of Jak2 with ATP and ACP docked into the ATP binding pocket. The nucleotide binding loop is blue, the hinge region is cyan, the catalytic loop is magenta, and the activation loop is red. Residues within this pocket that are critical for interactions with ATP and other docked drugs have been represented as spheres. Phosphorylation of Y1007 within the activation loop is necessary for the activation of kinase activity of Jak2. ATP (yellow) and ACP (green) had strong interactions with the pocket as indicated by their highly negative GRID scores. (B) G6 (pink), D28 (grey) and D30 (yellow) docked at the ATP-binding pocket of Jak2. (C) D21 (orange), D23 (magenta) and D25 (green) docked at the ATP-binding pocket of Jak2. (D) Comparison of the docking of ATP (yellow), ATP analog ACP (green) and G6 (pink) with the crystal structure of a Jak2 inhibitor (5B3) (blue) in complex with Jak2 kinase domain showed good correlation (r.m.s.d. < 2Å) between the structures.
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Figure 3-9. Docking of G6 and its derivatives into the ATP binding pocket of Jak2. G6 (pink) and its stilbenoid derivative D28 (grey) and D30 (yellow) showed hydrogen bond interactions (< 3.5Å) with several critical residues (E930, L932, R980 and D994) that make up the ATP binding pocket of Jak2. However, the nonstilbenoid derivatives D21 (magenta), D23 (orange) and D25 (yellow) show no such hydrogen bond interactions.
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CHAPTER 4 CELL DEATH INDUCED BY THE JAK2 INHIBITOR, G6, CORRELATES WITH
CLEAVAGE OF VIMENTIN FILAMENTS
Jak2 is a member of the Janus family of cytoplasmic tyrosine kinases. Other
members of this family include Jak1, Jak3 and Tyk2. Jak2 is activated by a variety of
cytokines, growth factors and GPCR ligands, resulting in signaling cascades that
regulate cell growth, proliferation and death. Upon binding of the ligand to its specific
receptor, the receptor-associated Jak proteins are activated via a phosphorylation
event. An activated Jak can in turn phosphorylate and activate the Signal Transducers
and Activators of Transcription (STAT) family of transcription factors. Phosphorylated
STATs dimerize and translocate to the nucleus where they modulate gene transcription.
Thus, the Jak/STAT pathway results in a signal cascade from binding and activation at
the plasma membrane to changes in gene transcription in the nucleus.
Hyperkinetic Jak2 promotes aberrant cell growth and prevents apoptosis. Hence,
constitutively active Jak/STAT signaling pathway has been implicated in a variety of
neoplastic disorders. Jak2 can become constitutively active by several different gene
alterations including specific chromosomal translocations and point mutations. Jak2
chromosomal translocations such as TEL-Jak2, REL-Jak2, BCR-Jak2 and PCM1-Jak2
lead to the development of a variety of leukemias, lymphomas and myelomas [44, 45,
130, 158-161]. Additionally, an activating Jak2 point mutation (Jak2-V617F) has been
linked to the myeloproliferative neoplasms (MPN) such as polycythemia vera (PV),
essential thrombocythemia (ET) and primary myelofibrosis (PMF) [57-61]. This valine to
phenylalanine substitution mutation present in codon 617 of the autoinhibitory
pseudokinase domain of Jak2 allows the kinase to evade negative regulation thereby
making it constitutively active. MPN patients bear this mutation in their marrow derived
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stem cells and are characterized by the overproduction of terminally differentiated blood
cells of the myeloid lineage such as red cells or platelets. Current therapies for MPN
patients include phlebotomy and hydroxyurea. While these treatments alleviate some
disease symptomologies, they are not curative in any way. Therefore, there is an
unmet clinical need for Jak2 inhibitors.
Using structure-based virtual screening, our group recently identified a novel
stilbenoid small molecule inhibitor of Jak2 named G6 [104]. We subsequently showed
that G6 specifically inhibits Jak2 mediated human pathologic cell growth in vitro, ex vivo,
and in vivo [105, 162]. We also demonstrated that G6 inhibits Jak2 mediated cell
proliferation via the suppression of key signaling molecules of the Jak/STAT pathway;
the consequence of this inhibition is G1/S cell cycle arrest and apoptosis [105, 162].
Here, we sought to elucidate the molecular and biochemical mechanisms by which
G6 inhibits Jak2-mediated cellular proliferation. For this, we compared protein
expression profiles between vehicle treated and G6 treated cells using two-dimensional
gel electrophoresis. The intermediate filament protein, vimentin, was one protein that
was differentially expressed between the two conditions. We therefore hypothesized
that the mechanism by which G6 inhibits Jak2-dependent cell proliferation involves
modification of this protein. In this study, our data supports this hypothesis as we show
that G6-induced inhibition of Jak2-mediated pathogenic cell growth correlates with the
specific cleavage and cellular reorganization of vimentin.
Experimental Procedures
Drugs
G6, obtained from the National Cancer Institute/Developmental Therapeutics
Program (NCI/DTP). The drug was solublized in dimethyl sulfoxide (DMSO) at a
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concentration of 10 mM. G6 was stored at -20⁰C in small aliquots to avoid repeated
freeze thaw cycles.
Reagents
AG490, Jak Inhibitor I, PD98059 and PP2 were purchased from Calbiochem.
Cycloheximide was purchased from Fisher Scientific. Caspase Inhibitor I (Z-VAD
(OMe)-FMK), Calpain Inhibitor V (Mu-Val-HPh-CH2F, Mu = morpholinoureidyl; HPh =
homophenylalanyl), Verapamil, BAPTA-AM, A23187 and 3’,3’-iminodipropionitrile
(IDPN) were purchased from Calbiochem.
Cell Culture
Human Erythroleukemia (HEL) cells were purchased from the American Type
Culture Collection and maintained in RPMI 1640 (Mediatech) supplemented with 10%
fetal bovine serum (FBS), penicillin, streptomycin and L-glutamine at 37⁰C and 5% CO2.
2-D Differential In Gel Electrophoresis (2-D DIGE)
HEL cells were treated either with vehicle control DMSO or 25 µM G6 for 12 hours.
The cell pellets were resuspended in ice cold buffer containing 0.3% SDS, 20 mM Tris
pH 8.0, 100 mM DTT, 10 µl protease inhibitor cocktail III (Calbiochem), 5 mM MgCl2 and
250 units of benzonase. The cell suspension was homogenized by sonication. Protein
was precipitated with 9 volumes of ice cold 10% trichloroacetic acid (TCA) in acetone
overnight at 4°C. Precipitated proteins were then dissolved in solubilization buffer (7 M
urea, 2 M thiourea, 4% CHAPS, 0.2% SDS and 20 mM Tris, pH 8.0). After
centrifugation at 43,000 rpm for 30 min, solubilized protein in the supernatant was
quantified using the EZQ Protein Assay Kit (Invitrogen). Proteins (100 µg per sample)
were minimally labeled with CyDye (GE Healthcare) as per the manufacturer’s protocol.
An internal standard, which is loaded on every gel, was created by mixing equal
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amounts of protein from all samples. Proteins from the DMSO treated samples were
labeled with Cy3 (green) and the G6 treated samples were labeled with Cy5 (red). The
internal standard was labeled with Cy2 (blue).
100 µg of Cy2 labeled internal standard, 100 µg of Cy3 labeled sample, 100 µg of
Cy5 labeled sample were mixed with 200 ug unlabeled internal standard. The mixture
was used to rehydrate a 24 cm pH 3 to 11 nl IPG strip (GE Healthcare) overnight in a
rehydration buffer (solubilization buffer with 100 nM DTT containing Orange G as
tracking dye) in the dark at room temperature. Three independent replicates of each
sample were run on three strips. IEF was carried out in IPGphor3 unit (GE Healthcare)
as per manufacturer’s recommendation. Temperature was maintained at 19°C
throughout focusing.
After completion of IEF, strips were first reduced in 15 ml of 50 mM Tris-HCl pH
6.8, 6 M Urea, 30% (v/v) glycerol, 2% (w/v) SDS, 100 mM DTT for 20 min in the dark at
room temperature, then alkylated in 15 ml of 50 mM Tris-HCl pH 6.8, 6 M Urea, 30%
(v/v) glycerol, 2% SDS, 2.5% idoacetamide for 20 min. After equilibration, strips were
transferred and mounted on an 8% - 16% precast Tris Glycine polyacrylamide gel
(Jule). Electrophoresis was carried out initially at 12ºC at 10 mA/gel for one hour and
then at constant current overnight at 12 mA/gel and a limit of 150 V until dye front
reached the bottom of the plate.
Gels were then scanned with Typhoon 9400 Variable Mode Imager (GE
Healthcare). The excitation/emission wavelengths for Cy2, Cy3 and Cy5 were 488/520,
532/580 and 633/670 nm, respectively. For each gel, images for the internal standard
as well as the control and experimental conditions were acquired. The digital images
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were then analyzed with DeCyder 2D version 7.0 (GE Healthcare). Information from
replicate gels was analyzed with BVA Module (Biological Variation Analysis). Spots
were selected by setting the fold difference threshold to 1.6-fold. Statistical significance
was estimated using Student’s t-test. Protein identification using electrospray mass
spectroscopy was done at the Scripps Research Institute.
Cell Lysis
Cells (~ 107) were washed with two volumes of ice-cold PBS and then lysed in 0.8
ml of ice-cold RIPA buffer (20 mM Tris pH 7.5, 10% glycerol, 1% Triton X-100, 1%
deoxycholic acid, 0.1% SDS, 2.5 mM EDTA, 50 mM NaF, 10 mM Na4P2O7, 4 mM
benzamidine, and 10 µg/mL aprotinin). Protein concentrations in the whole cell lysates
were determined using a Bradford assay (Bio-Rad). Cell lysates were then resuspended
in SDS sample buffer. Whole cell lysates (~ 30 μg) were separated by SDS-PAGE and
then transferred onto nitrocellulose membranes for analysis by western blotting.
Western Blotting
Nitrocellulose membranes were first blocked with 5% milk/TBST solution at room
temperature and then probed first with the different indicated primary antibodies
overnight at 4°C followed by the respective secondary antibodies (1:4000, GE
Healthcare). The immuno-reactive bands were then visualized using the enhanced
chemi-luminescence system (Western Lightning Ultra, Perkin-Elmer). The following
antibodies were used at the indicated dilutions: vimentin (Abcam and BD Biosciences,
each at 1:500), STAT1 (Santa Cruz Biotechnology, 1:1000), and β-actin (Cell Signaling,
1:500).
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Immunofluorescence
HEL cells were cultured in RPMI in 100 mm dishes and treated with 25 μM G6 for
the indicated periods of time. Following treatment, the cells were centrifuged, washed
and resuspended in 1X PBS. Cells were then plated onto poly-L-lysine coated 8-
chamber slides (Santa Cruz Biotechnology) and fixed at -20°C in a mixture of 50%
methanol and 50% acetone for 10 minutes. The fixed cells were then permeabilized with
0.2% Triton X-100 and blocked with 5% goat serum for 30 minutes at room
temperature. The samples were incubated overnight at 4°C with a primary antibody of
mouse anti-vimentin (BD Biosciences, 1:100) and washed 4X with PBS the following
morning. The samples were then incubated with a FITC-conjugated anti-mouse
secondary antibody (Santa Cruz Biotechnology) for one hour at room temperature. The
cells were again washed with PBS, mounted with UltraCruz DAPI containing mounting
media (Santa Cruz Biotechnology) and sealed with a cover slip. These cells were
imaged using a 100X objective on an inverted fluorescence microscope (Olympus).
Cell Proliferation Assay
HEL cells were plated in 96-well plates and treated with either 0.25% DMSO, 30
μM G6 or 2% IDPN for the indicated periods of time. Cell viability was then assessed for
each sample by trypan blue exclusion staining and hemocytometer.
In vivo Animal Model
The xenograft model of Jak2-V617F expressing HEL cells in NOD/SCID mice has
been described previously [105]. Briefly, 3 months old NOD/SCID mice were
randomized into 5 groups (n=6 per group). One group consisted of naive animals that
did not receive any treatment. All other groups received a single tail vein injection of 2 x
106 Jak2-V617F-positive HEL cells. Three weeks after HEL cell injection, the mice
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developed symptoms of a fully penetrant bone marrow malignancy. The mice then
began receiving intraperitoneal injections of either vehicle control (DMSO) or G6 at
doses of 0.1, 1, and 10 mg/kg/day for the next 21 days. At the end of the three week
treatment period, all groups of mice were euthanized and bone marrow tissues were
fixed in 10% neutral-buffered formalin and embedded in paraffin.
Bone Marrow Immunohistochemistry
Paraffin embedded bone marrow sections from each treatment group were
analyzed by anti-vimentin immunohistochemistry. Antigen retrieval was carried out first
by microwaving at 95°C for 20 min in 1mM EDTA-NaOH solution, pH 8.0. The section
were then cooled, blocked with Protein Block (DAKO), and incubated with anti-vimentin
antibody (Abcam, 1:100) for 2 hours at room temperature. Antigen-antibody complexes
were detected using biotinylated secondary antibodies and streptavidin-peroxidase
substrate (DAKO). Stained sections were then analyzed via a standard light microscope
(Nikon) at 40X and 100X magnifications.
Statistical Analysis
For statistical evaluation of time-dependent response of HEL cell viability to G6
and IDPN, a two-way analysis of variance was used. For analysis of differential
expression of proteins in 2-D DIGE and G6-induced degradation of vimentin using
densitometry, a Student’s t-test was employed. Data were assumed to be statistically
significant when p < 0.05.
Results
G6 Treatment Induces Time- and Dose-Dependent Degradation of Vimentin
The human erythroleukemia (HEL 92.1.7) cell line is homozygous for the Jak2-
V617F mutation [110, 145]. The presence of this mutation induces constitutively active
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Jak/STAT signaling and promotes a G1/S phase transition thereby driving increased
cellular proliferation [111]. We previously demonstrated that the Jak2 inhibitor, G6,
inhibits Jak2-V617F-mediated HEL cell proliferation and induces apoptosis [105, 162].
However, the specific mechanisms by which G6 does this are not known.
To gain some insight into the mechanism by which G6 reduces cell viability, HEL
cells were treated for 12 hours with either vehicle control (0.25% DMSO) or 25 μM G6.
The protein expression profiles of these two treatment conditions were compared using
two-dimensional gel-electrophoresis (Fig. 4-1A, 4-1B, 4-1C). One spot in particular was
present in the DMSO treated cells, but significantly reduced in the G6 treated cells (Fig.
4-1D & 4-1E; marked by arrows). That spot was excised and identified using electro
spray mass spectrometry as vimentin. In a separate two-dimensional gel
electrophoresis study, we compared the protein expression profiles between HEL cells
that had been treated with either DMSO or 25 μM G6 for 24 hours. Even at this longer
time point, the spot representing vimentin was still significantly reduced in the G6
treated cells when compared to the DMSO treated cells (data not shown).
To confirm that vimentin protein levels were decreasing with G6 treatment, protein
samples from both conditions were subjected to western blot analysis with an anti-
vimentin antibody. Consistent with the mass spectroscopy data, treatment of HEL cells
with G6 resulted in the disappearance of full-length vimentin (Fig. 4-1F). Of note, we
also observed the appearance of low molecular weight fragments of vimentin in the G6
treated cells (Fig. 4-1F).
To determine whether this effect was time- and dose-dependent, HEL cells were
treated either with 25 μM of G6 for increasing lengths of time or with varying doses of
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G6 for 24 hours. Whole cell lysates were then separated on SDS-PAGE and examined
by immunoblotting with an anti-vimentin antibody. Fig. 4-2A is a representative blot
showing that full-length vimentin was cleaved into low molecular weight fragments with
G6 treatment as a function of time. The same samples were then reprobed with an anti-
β-actin antibody to confirm equal protein loading and also to demonstrate the specificity
of G6 for vimentin over other cytoskeletal proteins such as β-actin. Quantification of all
blots using densitometry confirmed the total loss of full-length vimentin protein in
response to G6 treatment over time (Fig. 4-2B). Similarly, Fig. 4-2C is a representative
blot showing a dose-dependent cleavage of full-length vimentin in response to G6 and
Fig. 4-2D is a quantification of all blots. Collectively, the data in Figs. 4-1 and 4-2
demonstrate the ability of G6 to induce specific cleavage of the intermediate filament
protein vimentin. Furthermore, this effect is both time- and dose-dependent.
G6 Treatment Induces Marked Reorganization of Vimentin Intermediate Filaments within Cells
We next wanted to study the effect of G6 treatment on structure and cellular
distribution of intracellular vimentin filaments. For this, HEL cells were treated with 25
μM G6 for 0, 24 and 36 hours and then vimentin protein expression was analyzed via
indirect immunofluorescence. For the 0 hr time point, we found that vimentin was
distributed uniformly over the cytoplasm (Fig. 4-3A, 4-3D, & 4-3G). However, for the 24
and 36 hr time points, vimentin had a punctate staining pattern in the perinuclear region
of the cell (Fig. 4-3B, 4-3E, 4-3H & 4-3C, 4-3F, 4-3I, respectively).
As such, the data in Fig. 4-3 indicate that G6 treatment induces cellular
redistribution and aggregation of vimentin intermediate filament within HEL cells.
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G6-induced Cleavage of Vimentin is Jak2-mediated
Having already demonstrated the ability of G6 to induce specific cleavage of
vimentin (Fig. 4-2), we next wanted to determine if this G6-induced cleavage was Jak2-
dependent. For this, HEL cells were treated for 24 hours with increasing concentrations
of three different Jak2 inhibitors; G6, AG490 and Jak Inhibitor I. As a control, HEL cells
were also treated with non-Jak2 inhibitors; namely, the MAPK inhibitor, PD98059 and
Src family kinase inhibitor, PP2. Whole cell lysates were separated by SDS-PAGE and
immunoblotted with an anti-vimentin antibody. We observed that the Jak2-specific
inhibitors induced cleavage of vimentin dose-dependently (Fig. 4-4A) whereas the non-
Jak2 inhibitors had no effect on full-length vimentin (Fig. 4-4B). The loading of total
protein was confirmed by immunoblotting the same cellular lysates with an anti-STAT1
antibody (Fig. 4-4A & 4B). Thus, from Fig. 4-4 we conclude that G6-induced vimentin
degradation is Jak2-mediated.
G6-induced Cleavage of Vimentin is Independent of de novo Protein Synthesis and Caspase Activity, but Calpain-dependent
Given that G6 induces specific cleavage of vimentin (Fig. 4-2), we next wanted to
determine whether this G6-induced vimentin cleavage is dependent on de novo protein
synthesis. To assess this, HEL cells were first pretreated for 4 hours with increasing
doses of cycloheximide (CHX), an inhibitor of protein biosynthesis, and then treated with
increasing concentrations of G6 for 24 hours. Cycloheximide inhibits protein synthesis
by interfering with the translation elongation process of protein biosynthesis [163].
Western blot analysis of the cell lysates from the different treatment groups showed that
exposure to increasing doses of G6 induced a dose dependent cleavage of vimentin in
HEL cells which was not be blocked by pretreatment with cycloheximide (Fig. 4-5A),
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indicating that this G6-induced cleavage process does not require de novo protein
synthesis.
Vimentin is cleaved in response to G6 treatment into low molecular weight
fragments of vimentin (Fig. 4-2) suggesting that this process is mediated by a
protease/proteolytic enzyme. Caspases are a class of intracellular cysteine proteases
with roles in cytokine maturation, inflammation and apoptosis [164]. We previously
showed that G6 induces caspase 3/7 activation in a time-dependent manner in HEL
cells [105]. It has also been reported that vimentin is a caspase substrate and can be
cleaved by some caspases in vitro [165]. Therefore, we wanted to determine if G6-
induced vimentin cleavage is caspase-mediated. For this, we first pretreated HEL cells
with the pan-caspase inhibitor, Caspase Inhibitor I (zVAD-fmk), for 4 hours before
treating them with 30 μM and 60 μM G6 for 24 hours. The effect of caspase inhibition on
G6-dependent vimentin cleavage was then studied by western blotting the cell lysates
with an anti-vimentin antibody. We found that inhibition of caspases by zVAD-fmk was
unable to prevent G6-induced cleavage of vimentin (Fig. 4-5B), but was able to
significantly reduce the G6-induced cleavage of PARP (Fig. 4-5B), a substrate known to
be cleaved by caspases [115], thereby indicating that G6-induced cleavage of vimentin
is caspase-independent.
Calpain, a calcium-dependent neutral cysteine protease [166], is yet another
protease that is known to cleave vimentin [167, 168]. Hence, to examine whether G6-
induced vimentin cleavage is calpain-mediated, we pretreated HEL cells with a calpain
inhibitor, Calpain Inhibitor V (Mu-Val-HPh-CH2F), for 4 hours before exposing them to
increasing doses of G6 for 24 hours. Immunoblotting analysis of the HEL cell lysates
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showed that calpain inhibition prevented G6-induced cleavage of vimentin in a dose-
dependent manner (Fig. 4-5C), demonstrating that the protease involved in the
cleavage of vimentin in response to G6 treatment is calpain. Overall, the data in Fig. 4-5
indicate that the G6-induced cleavage of intermediate filament protein vimentin is
independent of de novo protein synthesis and caspase activity, but dependent on
calpain protease activity.
The Mobilization of Calcium is Essential and Sufficient for the Cleavage of the Intermediate Filament Protein Vimentin
Given that calpain is a calcium-dependent cysteine protease, we next investigated
the role of calcium in the G6-induced vimentin cleavage process. Specifically, we first
examined the effect of inhibiting the flux of extracellular calcium into cells by pretreating
the cells with verapamil. Verapamil blocks Ca2+ channels, principally the L-type channel,
thereby interfering with the extracellular influx of calcium ions. HEL cells were
pretreated with 30 μM verapamil for 4 hours before exposure to 30 μM G6 for 24 hours.
Cell lysates were then immunoblotted with an anti-vimentin antibody. We found that
inhibition of extracellular calcium ion influx into the cell via blockage of L-type calcium
channels did not have any effect on G6-induced cleavage of vimentin (Fig. 4-6A).
Therefore, we next studied the effect of chelating intracellular calcium on G6-mediated
vimentin cleavage. For this, we pretreated HEL cells with 10 μM BAPTA-AM for 2 hours
before treatment with 30 μM G6 for 24 hours. BAPTA-AM is membrane-permeable ester
form of the calcium chelator BAPTA. Once inside the cell, it is hydrolyzed by cytosolic
esterases into its active form and can chelate intracellular calcium. Results from the
western blot analysis showed that chelation of intracellular calcium protected vimentin
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from G6-induced cleavage (Fig. 4-6B), indicating that intracellular calcium has a critical
role to play in mediating the G6-induced cleavage of vimentin.
In the next experiment, we examined the effect of the calcium ionophore, A23187,
on vimentin protein levels within HEL cells. A23187 is a mobile ion-carrier that forms
stable complexes with divalent cations, such as calcium, and can hence be used for
increasing intracellular levels of calcium ions. Accordingly, HEL cells were treated with
10 μM A23187 for increasing periods of time and the cellular lysates were then western
blotted using an anti-vimentin antibody. We found that increasing intracellular calcium
levels via exposure to an ionophore is sufficient to induce cleavage of vimentin in HEL
cells (Fig. 4-6C), further confirming the essential role that calcium ions play in the
vimentin cleavage process. As such, data in Fig. 4-6 demonstrate that mobilization of
intracellular calcium ions is both essential and sufficient for the cleavage of the
intermediate filament protein, vimentin.
Cleavage of Vimentin is Sufficient to Reduce HEL Cell Viability
To determine how critical vimentin is to the viability of cells, we studied the effect
of vimentin cleavage on the survival of HEL cells. The drug 3’,3’-iminodipropiontrile
(IDPN) selectively disrupts vimentin intermediate filaments [169]. Therefore, we treated
HEL cells either with vehicle control DMSO, 30 μM G6 or 2% IDPN for 0, 6, 12, 24 or 48
hours. At each time point, the number of viable cells in each condition was determined
and cell lysates from those same conditions were immunoblotted with an anti-vimentin
antibody in order to correlate decreased cell numbers with increased vimentin cleavage.
We found that treatment with both G6 and IDPN time-dependently decreased viable cell
numbers (Fig. 4-7A) and this decrease in cell viability correlated with a corresponding
time-dependent cleavage of full-length vimentin in the G6 and IDPN treated cells (Fig.
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4-7B). Overall, the data in Fig. 4-7 demonstrate that the cleavage of vimentin
intermediate filaments is sufficient to reduce the viability of Jak2-V617F expressing HEL
cells.
G6 Treatment Decreases the Levels of Vimentin Protein, in vivo
Our data thus far indicate that treatment of HEL cells with G6 results in the
degradation and subsequent loss of vimentin protein, in vitro. To determine if this is
conserved in vivo, HEL cells were injected into the tail vein of NOD/SCID mice and
allowed to engraft into the bone marrow over the ensuing 21 days at which time the
mice began receiving daily intraperitoneal injections of either vehicle control (DMSO) or
G6 at doses of 0.1, 1, and 10 mg/kg/day for the next 21 days. At the end of the three
week treatment period, all groups of mice were euthanized and bone marrow was
analyzed for vimentin protein levels via anti-vimentin immunohistochemistry.
Representative stained sections from each treatment group are shown at 40X (Fig. 4-
8A) and 100X (Fig. 4-8B) magnification. We found that when a negative control IgG
antibody was used in place of the anti-vimentin primary antibody in the immuno-
histochemical procedure, only the hematoxylin counter stain was observed. Probing the
naïve bone marrow with the anti-vimentin antibody revealed strong staining in erythroid
cells, but not myeloid cells. HEL cell injection followed by DMSO treatment resulted in a
dramatic increase in the expression of vimentin protein when compared to naïve
animals. Treatment with 0.1 mg/kg/day of G6 did not produce any observable change in
the expression level of the protein when compared to DMSO treated mice. However,
treatment with 1 and 10 mg/kg/day of G6 clearly reduced the levels of vimentin protein
to those seen in the completely naïve animals. Hence, the data in Fig. 4-8 indicate G6
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treatment reduces HEL cell-induced vimentin expression in a dose dependent manner,
in vivo.
Discussion
Our group recently identified a novel stilbenoid Jak2 small molecule inhibitor
named G6 [104]. We subsequently showed that G6 specifically inhibits Jak2 mediated
human pathologic cell growth in vitro, ex vivo, and in vivo [105, 162]. We have also
demonstrated that G6 inhibits Jak2 mediated cell proliferation via the suppression of key
signaling molecules of the Jak/STAT pathway and with the induction of G1/S cell cycle
arrest and apoptosis [105, 162]. In this report, our objective was to determine the
mechanisms by which G6 exerts its inhibitory actions. To this end, we found that G6
treatment induced a time- and dose-dependent cleavage of the intermediate filament
protein, vimentin (Fig. 4-2). G6 treatment of HEL cells resulted in the movement of
vimentin from a uniform distribution in the cytoplasm to aggregates in the perinuclear
region of the cell (Fig. 4-3). The G6-mediated cleavage of vimentin is Jak2-dependent
(Fig. 4-4) and calpain-mediated (Fig. 4-5). The mobilization of intracellular calcium is
critical for G6-mediated vimentin cleavage (Fig. 4-6) and the cleavage of vimentin, per
se, is sufficient to reduce HEL cell viability (Fig. 4-7). Lastly, the ability of G6 to cleave
vimentin is conserved in vivo (Fig. 4-8). Taken together, these results describe a novel
mechanism by which G6 exerts its inhibitory actions.
Vimentin, a member of the Type III intermediate filament protein family, is a key
component of the cytoskeleton of the cell. It plays an important role in maintaining cell
shape and integrity as well as stabilizing cytoskeletal interactions such as adhesion,
migration and signaling. Apoptosis is associated with disruption of the cytoskeletal
network and caspase and/or calpain-induced cleavage of cytoskeletal proteins, such as
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vimentin, is known to occur in response to various inducers of apoptosis [170-174].
However, studies have reported that knockdown of vimentin does not induce significant
apoptosis per se, but cleavage or degradation of vimentin potentiates the therapeutic
effects of a drug [175, 176]. These studies also report that higher levels of vimentin
expression render cells more susceptible to drug-induced apoptosis. In agreement with
these previous reports, we found that Jak2-V617F expressing HEL cells have readily
detectable levels of vimentin protein and are very susceptible to drug inhibition and
subsequent loss of cell viability. However, in contrast to the earlier reports, we found
that the loss of vimentin, either by G6 or by IDPN treatment, was sufficient to
significantly decrease cell viability (Fig. 4-7). Possible explanations for these observed
differences include the fact that the earlier reports used siRNA to knockdown vimentin
mRNA levels whereas we used pharmacological inhibitors that resulted in decreased
vimentin protein levels. In addition, G6 also reduces Jak2 kinase activity [105, 162]
which presumably vimentin siRNA treatment does not. These differences
notwithstanding, our work here is significant in that we demonstrate that G6 treatment
results in vimentin cleavage and the subsequent loss of cell viability.
With respect to the signaling pathway that facilitates G6-mediated vimentin
cleavage, our data indicates that calcium plays a critical role in this process. For
example, the G6-induced cleavage of vimentin is mediated by the calcium-dependent
protease, calpain (Fig. 4-5C). Furthermore, we show that mobilization of intracellular
calcium is both essential and sufficient for cleavage of vimentin. Our data therefore
suggest that there is a close correlation between Jak2 kinase activity and the levels of
intracellular calcium ions. This is supported by previous work which indicates that
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erythropoietin (Epo)-induced activation of its cognate receptor, EpoR, inhibits calcium-
induced neurotransmitter release via the activation of Jak2 [177]. This report also shows
that this Epo-induced inhibition of calcium activity can be blocked by treatment with a
tyrosine kinase inhibitor, such as genistein, further confirming the importance of Jak2 in
mediating calcium-induced responses. Overall, these studies demonstrate that Jak2 can
modulate intracellular calcium levels. However, the exact protein target(s) that Jak2 may
phosphorylate in the calcium signaling pathway are not known.
We have previously reported that G6 treatment potently induces apoptosis in HEL
cells via the modulation of various Bcl-2 family proteins, such as Bcl-xL, Bim and Bid
[105, 162]. Previous studies have reported that elevation of intracellular calcium levels
can trigger apoptosis via the destabilization of the mitochondrial membrane and
subsequent activation of calcium-dependent calpain proteases [178-180]. Activation of
calpains can further lead to the cleavage and activation of apoptotic regulators of the
Bcl2 family, such as Bid [181]. Therefore, these studies suggest that calcium and
calcium-dependent proteases may have an important role to play in the G6-mediated
cell death/apoptosis process.
The Jak2-V617F mutation is found in a large percentage on MPN patients [57-61].
A previous study, which compared the mRNA expression profiles between healthy
individuals and MPN patients, reported vimentin to be one gene that was differentially
expressed in these two groups [182]. Specifically, MPN patients were found to have
elevated levels of vimentin mRNA when compared to non-diseased individuals. This
increase in vimentin gene expression correlated positively with the presence of the
Jak2-V617F mutation. In other words, significant over expression of vimentin was only
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observed in patients that were homozygous for the Jak2-V617F mutation. Thus, our
data here, which describes vimentin as one protein that is down regulated in response
to Jak2-V617F inhibition by G6, is noteworthy. Furthermore, our data which show that
the cleavage of vimentin is Jak2-dependent (Fig. 4-4), imply that pharmacological
inhibition of Jak2 is sufficient to induce cleavage of vimentin and subsequent loss of cell
viability (Fig. 4-7). When taken together with the MPN microarray data [182], our work
here suggests that there may be a link between hyper activation of Jak2 kinase and
over expression of vimentin. Furthermore, vimentin expression might be a potential
biomarker for the progression of Jak2-V617F mediated pathogenesis and for disease
regression via Jak2 inhibitory therapy. In summary, our data show that G6-induced
inhibition of Jak2-mediated cell growth correlates with the cleavage and subsequent
loss of intracellular vimentin filaments in vitro and in vivo. As such, this work describes a
novel mechanistic pathway for the targeting of Jak2-mediated pathological cell growth.
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Figure 4-1. Identification of vimentin as a differentially expressed protein between
vehicle treated and G6 treated HEL cells. HEL cells were treated with either 0.25% DMSO or with 25 μM of G6 for 12h. Proteins from the internal standard (A), DMSO treated (B) and G6 treated (C) were labeled with Cy2 (blue), Cy3 (green) and Cy5 (red), respectively. (D) An overlay of the three colored images is shown. One protein spot, indicated by the arrow, was differentially expressed between the vehicle treated and G6 treated samples. (E) Analysis of the images obtained from the 2-D DIGE using DeCyder 2D predicted this differentially expressed protein to be significantly downregulated in the G6 treated samples (p=0.01). The indicated protein was excised and identified using electro spray mass spectrometry as vimentin. (F) HEL cells were treated either with DMSO or 25 μM of G6 for 24 hr and analyzed by western blotting using an anti-vimentin antibody. The same samples were also blotted with an anti-STAT1 antibody to confirm equal loading across all lanes. Shown is one of three representative images.
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Figure 4-2. G6 treatment induces time- and dose-dependent degradation of vimentin.
HEL cells were treated either with 25 μM of G6 for varying lengths of time (A) or with increasing doses of G6 for 24 hours (C). Cell lysates were then separated on SDS-PAGE and immunoblottted with an anti-vimentin antibody. The same samples were then reprobed with an anti-β-actin antibody to confirm equal protein loading and also to demonstrate the specificity of G6 for vimentin over other cytoskeletal proteins such as β-actin. Shown is one of three independent results for each. Expression of full-length vimentin was quantified using densitometry and plotted as a function of either time (B) or dose (D) of G6 treatment. Data shown are the mean ± SE from three independent experiments. *p<0.05 with respect to 0 hr (B) or 0 μM (D).
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Figure 4-3. G6 treatment induces marked reorganization of vimentin intermediate filaments within cells. HEL cells, treated with 25 μM of G6 for 0, 24 or 36 h, were analyzed via indirect immunofluorescence for changes in the cellular distribution of vimentin in response to drug treatment. Vimentin was indirectly labeled with a FITC-conjugated secondary antibody (A, B, and C). The nuclei were counter stained with DAPI (D, E, and F). The images were then merged (G, H and I). Shown is one of two representative results.
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Figure 4-4. G6-induced cleavage of vimentin is Jak2-mediated. HEL cells were treated for 24 hrs with increasing concentrations of Jak2 specific inhibitors (G6, AG490 and Jak Inhibitor I) (A) or non-Jak2 inhibitors (MAPK inhibitor, PD98059 and c-Src inhibitor, PP2) (B). Whole cell lysates were separated by SDS-PAGE and immunoblotted with an anti-vimentin antibody. Loading of protein was confirmed using an anti-STAT1 antibody. Shown is one of three representative blots.
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Figure 4-5. G6-induced cleavage of vimentin is independent of de novo protein synthesis and caspase activity, but calpain-dependent. HEL cells were pretreated for 4 hours with either cycloheximide (CHX) (A), caspase inhibitor I (zVAD) (B) or calpain inhibitor V (C) and then treated with increasing doses of G6 for 24 hours. Whole cell lysates from the different treatment groups were then analyzed by western blotting with an anti-vimentin antibody. The same lysates were also probed with an anti-β-actin antibody to confirm equal protein loading across all lanes. Shown is one of three representative results for each.
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Figure 4-6. Mobilization of calcium is essential and sufficient for the cleavage of vimentin. HEL cells were first pretreated with either 30 μM verapamil for 4 hours (A) or 10 μM BAPTA-AM for 2 hours (B) and then treated with 30 μM G6 for 24 hours. Post treatment, the cells were lysed, proteins were separated by gel electrophoresis and then immunoblotted with an anti-vimentin antibody (upper panel) or an anti-β-actin antibody (lower panel) to confirm equal protein across all lanes. (C) HEL cells were treated with 10 μM A23187 for the indicated periods of time. Cellular lysates were then probed with an anti-vimentin antibody (top panel). An anti-β-actin antibody was used as a loading control (bottom panel). Shown is a representative blot from three independent experiments for each.
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Figure 4-7. Cleavage of vimentin is sufficient to reduce HEL cell viability. HEL cells
were exposed to either vehicle control (DMSO), 30 μM G6 or 2% IDPN for 0, 6, 12, 24 or 48 hours. (A) The numbers of viable cells at each time point were determined and plotted as a function of treatment condition. (B) Cell lysates from each treatment group were collected simultaneously and analysed by immunoblotting with either an anti-vimentin antibody or an anti-β-actin antibody. Shown is one of three representative results. *p<0.05 with respect to DMSO.
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Figure 4-8. G6 treatment decreases the levels of vimentin protein, in vivo. Anti-vimentin immuno-histochemistry was carried out on bone marrow sections from the indicated groups of animals. Shown are representative stained bone marrow sections from each treatment group at 40X (A) and 100X (B) magnifications.
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CHAPTER 5
CONCLUSIONS AND PERSPECTIVES
Jak2 was first discovered in 1992 by polymerase chain reaction (PCR)-based
techniques. Since then, this protein has been linked to many critical physiological
processes as well as several pathophysiological states. Specifically, hyperkinetic Jak2
tyrosine kinase has been implicated in diverse hematological disorders, including
myeloproliferative neoplasms and hence is an emerging therapeutic target for these
disorders. In this dissertation, we characterize the structure-function correlations of two
novel Jak2 small molecule inhibitors and their mechanisms of action. We have shown
here that A46, a novel benzothiophene based compound identified in our lab,
suppresses Jak2-mediated pathogenesis, thereby making it a potential candidate drug
against Jak2-mediated disorders. Our lab has previously identified yet another small
molecule inhibitor of Jak2, called G6, which shows remarkable therapeutic efficiency
against Jak2-mediated cell proliferation in vitro and in vivo [105]. Here, we conducted
structure-function studies to demonstrate that G6 has a stilbenoid core which is
indispensable for maintaining its Jak2 inhibitory potential, suggesting that naturally
occurring compounds, such as stilbenoids, might be a good scaffold for future
development of potent Jak2 small molecule inhibitors. In addition, we also investigate
the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cell
proliferation. Specifically, we demonstrate that the intermediate filament protein vimentin
is cleaved in response to G6 treatment and show data which suggest that G6-induced
Portion of this chapter has been reproduced from Jak2 Inhibitors for the treatment of Myeloproliferative Neoplasms. Drugs of the Future 2010; 35(8): 651-660 with permission from 2010 Prous Science, S. A. U. or its licensors.
117
inhibition of Jak2-mediated pathogenic cell growth correlates with the disruption of
intracellular vimentin filaments.
This work is significant for a number of reasons. Firstly, we identify and
characterize a novel small molecule inhibitor of Jak2, A46, which may perhaps serve as
a lead therapeutic agent for the treatment of Jak2-V617F mediated pathogenesis.
Secondly, we identify stilbenes and benzothiophenes as two new potential scaffolds for
designing potent Jak2 small molecule inhibitors. Lastly, this work describes a novel
mechanistic pathway for the targeting of Jak2-mediated pathological cell growth via
inducing cleavage of vimentin. As such, our data suggest that vimentin expression
might be a potential biomarker for the progression of Jak2-V617F mediated
pathogenesis and for disease regression via Jak2 inhibitory therapy. The implications
and perspectives of the studies presented in this dissertation are discussed in further
detail below.
Importance of Designing/Identifying a Jak2 Specific Inhibitor
More than half a century ago, Dameshek first described myeloproliferative
neoplasm (MPN) in an editorial [183]. He recognized that the different MPNs were all
characterized by proliferation of cells of the myeloid lineage, which he suggested might
result from a common ‘undiscovered stimulus’. The molecular pathogenesis of these
disorders remained elusive for about five decades after that till 2005, when five different
groups, each working independently, identified the presence of a Jak2-V617F mutation
in a majority of patients with MPNs [57-61]. These five groundbreaking studies, as well
as other subsequent studies, reported the presence of the Jak2-V617F mutation in over
90% of patients with PV and about 50% of patients with ET and PMF. In vivo studies
have subsequently demonstrated that expression of this mutation in murine models is
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sufficient for the development of fully penetrant MPN in these animals [64, 66-67, 70-71,
112]. These data strongly suggest that the Jak2-V617F mutation plays a critical role in
the pathogenesis of myeloproliferative neoplasms. There are about 22 cases of PV, 24
cases of ET and 1.46 cases of PMF per 100,000 people [184, 185]. Constitutively active
Jak/STAT signaling pathway has also been linked to various forms of neoplastic
disorders, including solid cancers and hematological malignancies, such as leukemias,
myelomas and lymphomas. Unfortunately, there are currently no effective treatments
available for such Jak2-mediated pathologies. Commonly used treatment strategies for
MPNs involve the use of phlebotomy or myelosuppressive agents such as hydroxyurea
and thalidomide. However, these treatments are only palliative in nature and provide
only temporary relief without actually curing the disease. Therefore, there is currently a
need to develop molecularly targeted therapies that will be able to cure Jak2-mediated
disorders. Identification of the hyperactivating Jak2 mutations associated with these
disorders has prompted the search for potent and selective Jak2 small molecule
inhibitors and has brought us a step closer to the development of an effective and
curative therapeutic strategy for such diseases.
Elucidating the role of Jak2 in normal physiology and development is challenging
since Jak2 knockout mice die embyonically around E12.5 [31-32]. Hence, one approach
to studying the role of Jak2 in later stages of development and in adults is to use a
potent and specific small molecule inhibitor of Jak2. Therefore, identification of a good
Jak2 inhibitor will not only be useful as a therapeutic drug for Jak2-mediated disorders,
but also be an important research tool for investigating the role of Jak2 in regulating
critical physiological processes. Yet another approach to understanding the importance
119
of Jak2 in later stages of development and in adults is to induce tamoxifen-mediated
deletion of Jak2 at various stages of development and studying the effects of Jak2
deletion on tissue histology and survivability at each stage.
Characterization of the Novel Jak2 Inhibitor, A46
In the chapter 2 of this study, we show that the novel benzothiophene small
molecule inhibitor of Jak2, A46, inhibits Jak2-V617F-mediated pathological cell growth
in vitro and ex vivo. Specifically, we show that this compound inhibits Jak2-V617F
mediated cell proliferation both time and dose dependently with a GI50 of ~300 nM. The
GI50 of A46 is comparable to other Jak2 inhibitors that have moved into clinical trials,
such as TG101348 (Table 1-1) and lower than many other Jak2 inhibitors that have
been characterized preclinically (Table 6-1), suggesting that this compound can
potentially be developed into drug for use in treatment of MPN patients.
We have also demonstrated that A46 selectively inhibits the proliferation of Jak2-
V617F-dependent cell lines while having little to no effect on other cells lines whose
proliferation is driven by Jak2-independent mechanisms. Selectivity/specificity is a
desirable quality in a drug because it allows the drug to be administered in higher doses
without producing non-desirable side-effects. We have also shown that the A46 induced
cell growth inhibition correlates with direct suppression of the Jak/STAT signaling.
Additionally, our data indicate that the mechanism by which A46 suppresses Jak2-
V617F-mediated HEL cell proliferation is via the induction of both G1/S cell cycle arrest
and apoptosis. Moreover, we report that this compound inhibited the pathologic growth
of primary Jak2-V617F expressing bone marrow cells, ex vivo.
Although we have demonstrated that A46 inhibits Jak2-mediated pathological
growth in vitro and ex vivo, we next need to demonstrate the in vivo efficacy of this drug.
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For this, we can use a transgenic mouse model of Jak2-V617F driven myeloproliferative
neoplasia [67]. These trangenic mice develop fully penetrant MPN with symptoms such
as erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and
spleen, splenomegaly, reduced levels of plasma erythropoietin and thrombopoietin, and
increased levels of cytokine-independent hematopoietic progenitor cells in the
peripheral blood, spleen and bone marrow. We hypothesize that treating MPN mice with
A46 will result in reversal of the pathologic symptoms concommitant with suppression of
the hyperactive Jak/STAT signaling in these animals. In addition to the in vivo
characterization of A46, we also need to study the pharmacologic properties of this drug
including its half life and bioavailability, before it can be moved into clinical trials for
treatment of myeloproliferative neoplasms.
Stilbenes and Benzothiophenes as Potential Scaffolds for the Design of Novel Jak2 Small Molecule Inhibitors
Our group recently identified a novel stilbenoid small molecule inhibitor of Jak2
named G6 [104]. We subsequently showed that G6 specifically inhibits Jak2 mediated
human pathologic cell growth in vitro, ex vivo, and in vivo [105]. In chapter 3, we discuss
the structure activity relationship properties of this novel Jak2 inhibitor. G6 has a
stibenoid core in its chemical structure. We demonstrated that the core stilbenoid
structure present in G6 is indispensable for maintaining its ability to inhibit Jak2 kinase
activity. Specifically, we show that the stilbenoid core in G6 is essential for conserving
its ability to i) inhibit Jak2-V617F mediated cell growth, ii) suppress the phosphorylation
of key signaling molecules of the Jak/STAT signaling pathway, iii) induce apoptosis in
HEL cells, iv) suppress pathologic cell growth of Jak2-V617F expressing human bone
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marrow cells, ex vivo and v) form stronger docking interactions with the ATP-binding
pocket of Jak2 kinase domain.
Since the discovery of the Jak2-V617F mutation as the predominant mutation
present in a large proportion of MPN patients, there has been a drive to develop Jak2-
specific inhibitors as therapeutics for MPNs and other Jak2-mediated pathologies.
There have been several Jak2 inhibitors that have been identified and characterized
preclinically and clinically. However, a potent, specific and orally bioavailable Jak2
inhibitor that has the ability to cure Jak2-mediated disorders is yet to be identified.
AG490 was the first reported Jak2 inhibitor [82]. Later, it was shown that even though
this compound was a potent inhibitor of Jak2, it was highly non-specific and inhibited
several other kinases as well [83]. Consequently, it was used as a starting point for
designing more potent and/or selective Jak2 inhibitors. Specifically, AG490 was used as
a lead compound and several derivatives of it were identified. For example, the AG490
derivative WP1066 had better potency and specificity for Jak2 than AG490 [186, 187].
Similarly, LS104, which is structurally related to AG490, is a non-competitive inhibitor of
Jak2 and showed promising preclinical results and is now being evaluated in clinical
trials [188]. Recently, a variety of medicinal chemistry techniques such as library
screening, fragment-based drug discovery, scaffold morphing, molecular docking and
lead compound derivatization have been used to study structure-function correlations of
numerous Jak2 inhibitors. The objective of these studies is to identify core scaffolds and
functional groups that play crucial roles in potent Jak2 inhibition. Jak2 small molecule
inhibitors currently represent a diverse number of chemical structures including
pyrazines, pyrimidines, azaindoles, aminoindazoles, deazapurines, benzoxazoles, and
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quinoxalines [125]. Data presented in the chapters 2 and 3 of this dissertation
demonstrate that stilbenes and benzothiophenes may be a new class of scaffolds for
Jak2 small molecule inhibitors. Specifically, stilbenoids or stilbene derivatives are
naturally occurring compounds that are known to have a wide range of biological
activities including anti-proliferative, anti-oxidative, anti-neovascularization, tumor-
suppressive and anti-tyrosine kinase effects [131-135, 153, 154]. Moreover, the trans-
stilbenoid core identified in G6 and its active derivatives is amenable to bioisosteric
replacements that could potentially result in new Jak2 chemotypes with improved
druglike properties. Similarly, benzothiophenes, relatively stable heterocyclic structures,
have reactive sites that allow for subsequent derivatization, suggesting that A46 is also
quite amenable to future lead optimization.
Having demonstrated the importance of the stilbene core in maintaining G6-
mediated inhibition of Jak2, we next want to modify the positions and sizes of the
hydroxyl and amine functional groups on the core stilbene structure of G6 and study
how they impact the properties of the lead compound G6. The objective of such studies
is to see if derivatization of G6 can result in improvement of its potency, specificity or its
drug-like properties, such as bioavailability and solubility. Similar structure-activity
relationship studies also need to be done for the benzothiophene-based Jak2 inhibitor,
A46.
Comparison of Two Novel Jak2 Inhibitors, G6 and A46
In this work we have characterized a novel Jak2 inhibitor, A46. Specifically, we
show that this compound inhibits Jak2-V617F mediated HEL cell proliferation both time
and dose dependently with a GI50 of ~300 nM. On the other hand, G6, the other novel
Jak2 inhibitor, inhibits Jak2-V617F-mediated cell proliferation with a GI50 of ~4 μM [104,
123
105]. A lower GI50 suggests that A46 is a more potent inhibitor of Jak2-mediated
pathological cell growth than G6, in vitro. However, we observed an interesting
characteristic of A46. In spite of its greater potency, this compound never kills all the
cells even at the highest dose (30 μM), in vitro. The inability of A46 to completely
eliminate viable cells was also observed in the ex vivo clonogenic assay that we
performed. In comparison, exposure of HEL cells to 10 μM G6 results in complete
elimination of viable cells after 72 hours, in vitro [105]. This study also demonstrates
that G6 has exceptional therapeutic efficacy, in vivo, in a mouse model of human
erythroleukemia. At a therapeutic dose of 1mg/kg/day, G6 is able to specifically remove
mutant cells from the bone marrow and cause significant improvement in both the bone
marrow and the spleen of the treated animals [105].
Like most other Jak2 inhibitors currently known, G6 targets and binds to the ATP-
binding pocket of Jak2. However, initial studies have shown that G6 is more efficacious
in an in vivo setting than all the other Jak2 inhibitors that have been developed so far.
This raises an interesting question as to what makes G6 different from the other ATP-
site targeting Jak2 inhibitors. A possible reason for the higher efficacy of G6 might be its
unique stilbenoid chemical structure. The molecular docking studies presented in the
third chapter of this dissertation show that the stilbenoid core in G6 is critical for
maintaining its strong binding affinity for the ATP-binding pocket of Jak2. We show that
G6 has potential hydrogen bond interactions with several critical residues lining the
ATP-binding pocket, including D994, R980, E930 and L932. Potential hydrogen bond
interactions with D994 and R980 are unique to G6 and have not been identified for any
of the other Jak2 inhibitors currently available. Yet another possible reason for the
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better efficacy of G6 maybe the pharmacokinetic properties of G6. The pharmacokinetic
properties of G6 have not been characterized yet. However, it is possible that G6 might
have better bioavailability and/or ADME (Absorption, Distribution, Metabolism and
Excretions) characteristics when compared to the other inhibitors, which makes it more
efficacious than the others.
Potential of Jak2 Inhibitor Therapy for Myeloproliferative Neoplasms
The discovery of Jak2-activating mutations in the majority of MPN patients has
spurred the development of small molecule inhibitors that specifically target Jak2. To
date, a limited number of Jak2 inhibitors have been evaluated in clinical trials.
Interestingly, while these inhibitors exhibited moderate to good preclinical efficacy,
including murine models of human MPNs, those results have not translated well at the
level of clinical trials. In these trials, the primary clinical benefits observed have been
significant reduction in splenomegaly, patient weight gain and in some cases,
reductions in the Jak2 mutant allele burden and/or reduced levels of some inflammatory
cytokines. However, none of the inhibitors have yet been able to reverse the disease by
providing any improvement in the bone marrow fibrosis of the treated patients. The
reasons as to why these inhibitors have been disappointing in clinical trials are now an
area of investigative research. For example, one theory is that the genetics underlying
MPNs are more complex than first thought. For instance, it is unclear how a single point
mutation such as Jak2-V617F can lead to the development of three phenotypically
distinct disorders; namely, PV, ET, and PMF. Some hypothesize that there are
additional genetic and/or epigenetic events that contribute to the pathogenesis of these
disorders and in turn determine which of the three disorders a specific patient will
manifest [189].
125
Recently, three independent groups showed that the presence of a genetic
haplotype leads to a predisposition for acquiring the Jak2-V617F mutation and
consequently developing MPN [190-192]. Results from recent epigenetic studies
suggest that hypermethylation of the Suppressors of Cytokine Signaling (SOCS) family
proteins, negative regulators of Jak/STAT signaling, may also have a role to play in the
pathogenesis of MPNs [193]. The presence of such genetic and epigenetic factors may
determine how a specific MPN manifests itself in a given individual and in turn, how that
individual responds to a specific treatment. This also raises the possibility that we might
have to consider the use of combination therapies for effective treatment of these
disorders instead of merely Jak2 monotherapy. Lastly, although this runs contrary to
current dogmatic drives as they relate to the development of Jak2 specific inhibitors,
perhaps inhibitors of the future need to be less specific for Jak2 and instead have some
off target properties as well. In other words, dirty might be better.
Another important factor to be considered when judging the limited success of the
Jak2 inhibitor clinical trials is the patient population being studied. These early clinical
trials have all been performed with myelofibrosis patients because it is the most severe
of the three classical MPNs. However, it must be kept in mind that a drug that does not
show significant improvement in these patients might still be efficacious if given to a
patient with a less severe MPN, such as PV, ET or even early stage PMF. Hence it will
be important to design more appropriate clinical trials that can address this important
issue before final conclusions are drawn regarding the efficacy of these drugs in
humans.
126
Most of the efforts to identify Jak2 inhibitors have been focused on screening for
small molecules that target the ATP-binding pocket within the Jak2 kinase domain (Fig.
5-1A). However, since this pocket is highly conserved amongst all kinases, developing a
Jak2 inhibitor that does not inhibit other kinases is a challenge. The ATP-pocket
targeted inhibitors have another limitation as well. Specifically, they are unable to
distinguish between the wild-type and mutant Jak2 kinases. Hence, it is unsurprising
that these inhibitors, due to their potency against wild-type Jak2, cause
myelosuppression and anemia in treated patients. The occurrence of such adverse
side effects imposes an upper limit on the dosage of the drug that can be administered
to these patients.
Another dilemma is that the Jak2 activating mutations identified in MPN patients,
including V617F, are mostly present in the JH2 or pseudokinase domain of Jak2 and
not the kinase domain. Unfortunately, the crystal structure of Jak2 encoding both the
JH1 (kinase) and JH2 (pseudokinase) domains has not yet been resolved. As such, it is
currently not possible to design inhibitors that specifically target mutant forms of Jak2
over the wild-type protein. To complicate matters even further, one wonders whether an
inhibitor that is specifically designed against Jak2-V617F will be effective against the
dozens of other Jak2 mutations that are known to exist. That said, in the future, solving
the crystal structure of full length Jak2, or at least the JH1-JH2 domains, will be very
critical for the designing future Jak2 kinase inhibitors.
Attempts are also being made to design more specific Jak2 inhibitors by exploiting
the subtle differences between the kinase domain structures of Jak2 and other protein
kinases. Crystal structure analyses have shown that the ATP-binding site of the Jak
127
kinases is much more constricted when compared to the other protein tyrosine kinases
[23]. It has also been shown that the electrostatic surface potential around the active-
site within Jak2 is more positively charged than other Jak family members, such as Jak3
(74). Moreover, several residues in the hinge region (such as M929, Y931, P934,
R938), the glycine loop (such as K857), the catalytic loop (such as I982), and the
activation loop (such as E1015) may potentially confer inhibitor selectivity to Jak2 over
other tyrosine kinases including other Jak family members (48). That said, only
completion of such studies will determine whether these structure-activity-relationships
do in fact exist.
Developing allosteric inhibitors to Jak2 might be an alternate approach for specific
Jak2 targeting. There are several other possible target sites on Jak2, other than the
ATP-binding site, against which allosteric inhibitors can be designed. The JH2 domain
of Jak2 has an autoinhibitory effect on the kinase domain [13, 14]. The mechanism for
the constitutive of Jak2-V617F involves the disruption of the inhibitory interactions
between the JH1 and JH2 domains at the JH1-JH2 interface and linker regions (Fig. 5-
1B; marked by a box) [194, 195]. Hence, this region is a possible target for allosteric
inhibition of Jak2. A recent study reported that the linker between the SH2-like domain
and the JH2 domain (Fig. 5-1C; boxed region) flexes the hinge between the JH1 and
JH2, thereby altering the accessibility of the kinase domain and leading to its activation
[75]. Incidentally, the exon 12 mutations identified in MPN patients are present in this
linker. Therefore, this linker region might also be a plausible site for allosteric targeting
of Jak2. The FERM domain negatively regulates the wild-type Jak2, but positively
regulates Jak2-V617F kinase activity [196]. Mutation/deletion of this domain also
128
increases Jak2 kinase activity, further suggesting the role of the FERM domain in
regulating activity of Jak2 [197-199]. The FERM domain is also known to regulate Jak2
receptor association [20, 21]. Hence, the FERM domain might be another interesting
region to target for allosteric inhibition of Jak2. Overall, allosteric inhibitors that target
these alternative regulatory sites on Jak2 may provide more effective inhibitors that
specifically target mutant Jak2.
In summary, the causative nature of Jak2 somatic mutations in the pathogenesis
of MPNs was established in 2005. The first clinical trial testing a putative Jak2 small
molecule inhibitor for the treatment of MPNs initiated less than three years later. While
the initial data from these limited number of clinical trials have not lived up to the
expectations, it is evident that these inhibitors, even if not curative, can still have great
therapeutic benefit for MPN patients due to their ability to reduce some clinical
symptoms as well as improve the overall quality of life. However, one of the challenges
to implementing these Jak2 inhibitor therapies for MPN patients will be their prohibitive
costs. The symptoms of MPN patients are effectively managed by existing therapeutic
options, such as venesection/cytoreductive agent. Therefore, a shift towards the use of
more expensive Jak2 inhibitor therapy will be warranted only if these inhibitors show
some curative effects as opposed to just palliative ones. The issue of acquired
resistance can also become a potential problem with the use of potential Jak2 inhibitors
similar to that seen with other types of inhibitor based therapeutics, such as BCR-ABL
and FLT3 (FMS-like tyrosine kinase 3) inhibitors. In this time of rapid expectations, we
must be mindful that this area of research is still in its infancy. Current avenues of
investigation continue to expand our knowledge of Jak2 kinase and its potential
129
inhibitors. With an increased knowledge of 1) the molecular pathogenesis of MPNs, 2)
the structural properties of wild type and mutant Jak2 proteins 3) the continued
identification of structure-activity-relationships between Jak2 and putative inhibitors and
4) the development of better animal models that more closely resemble the complex
nature of MPNs observed in humans, we are confident that one day, Jak2 inhibitors will
be a viable therapy for the treatment of myeloproliferative neoplasms.
Characterization of the Link Between G6-induced Cell Death and Cleavage of Vimentin
Our data in chapter 3 showed that the novel stilbenoid Jak2 inhibitor G6
specifically inhibits Jak2-mediated cell proliferation in a time and dose-dependent
manner. Therefore, we next wanted to understand the molecular and biochemical
mechanisms by which G6 inhibits Jak2-mediated cellular proliferation. For this, we
compared protein expression profiles between vehicle treated and G6 treated cells
using two-dimensional gel electrophoresis. We identified the intermediate filament
protein, vimentin, as one protein that was differentially expressed between the two
conditions. The data in chapter 4 show that G6-induced inhibition of Jak2-mediated
pathogenic cell growth correlates with the specific cleavage and cellular reorganization
of vimentin. Specifically, we demonstrate that G6 treatment induced a time- and dose-
dependent cleavage of the intermediate filament protein, vimentin. We also show that
G6 treatment of HEL cells resulted in the change in vimentin filament organization from
a uniform distribution in the cytoplasm to aggregates in the perinuclear region of the
cell. Our data provides evidence that this G6-mediated cleavage of vimentin is Jak2-
dependent and calpain-mediated. Additionally, we demonstrate that the mobilization of
intracellular calcium is critical for G6-mediated vimentin cleavage and the cleavage of
130
vimentin, per se, is sufficient to reduce HEL cell viability. Lastly, our data also suggest
that the ability of G6 to induce cleavage of vimentin is conserved in vivo. Taken
together, these results describe a novel mechanism by which G6 exerts its inhibitory
effects.
Having established the critical role that calcium and the calcium-dependent
protease, calpain, plays in this G6-induced vimentin cleavage process, we next need to
determine the mechanism by which inhibition of Jak2 via G6 can induce calcium
mobilization and activation of calpain. For this, we need to study the effects of G6
treatment on the expression of important calcium-regulating genes, such as calmodulin
and calmodulin kinase. As of now, there is a missing link between the inhibition of Jak2
by the inhibitor G6 and the subsequent mobilization of calcium and activation of calpain,
which finally causes degradation of vimentin. Our data suggest that there is a close
correlation between Jak2 kinase activity and the levels of intracellular calcium ions.
However, the exact protein target(s) that Jak2 may phosphorylate in the calcium
signaling pathway are yet to be identified.
Having demonstrated the correlation between G6-induced cell death and cleavage
of vimentin, it is important to next determine if there is a direct causative relation
between them. For this, we need to first transfect HEL cells with a mutated vimentin
construct that is resistant to calpain cleavage and then treat them with G6. We expect
that the cells expressing this calpain-resistant vimentin protein will be protected from
cell death induced by the Jak2 inhibitor, G6. This experiment will help us in determining
if the Jak2 inhibitor, G6, induces cell death directly via the cleavage of vimentin
filaments. However, there are some technical difficulties in conducting this experiment.
131
Calpain is a calcium-dependent protease that can recognize and cleave a wide variety
of substrates and a unique substrate identification site has not yet been characterized
for this enzyme. Hence, creating a calapin-resistant vimentin construct may be
technically challenging. Currently, due to this limitation, it was not possible to establish a
direct causative link between G6-induced cell death and cleavage of vimentin. Overall,
the data presented in this dissertation characterizes the structure-activity relationships
of novel Jak2 inhibitors and provide useful insight into the mechanisms by which these
inhibitors exert their anti-Jak2 effect.
132
Figure 5-1. Surface representation of the structure of Jak2, indicating possible sites for
inhibitor targeting. (A) The ATP-binding pocket of Jak2 is marked by a box around it. This pocket has several loops/regions that are critical for the interaction of the Jak2 kinase domain with ATP. Rotating the structure shows the regions behind the ATP binding pocket (B & C). (B) The interface between the JH1 and JH2 domains, including the linker between these two domains, is indicated by the box. This interface is critical for regulating the autoinhibitory effect of the JH2 domain on the JH1/kinase domain. (C) The linker region between the FERM domain and the SH2-like domain is indicated by the box. This linker has a critical role to play in regulating the flexing of the hinge between the JH1 and JH2 domains, thereby effecting the interactions between these domains.
COLOR CODE: Pale green – FERM domain
Light pink – SH2-like domain
Light orange – JH2 (pseudokinase) domain
White – JH1 (kinase) domain
Cyan – Linker between FERM and SH2-like domains
Yellow – Linker between SH2-like and JH2 domains
Purple – Linker between JH2 and JH1 domains
Blue – Nucleotide loop
Green – Hinge region
Magenta – Catalytic loop
Red – Activation loop
133
Table 5-1. GI50 of other Jak2 inhibitors in HEL cells. Other known Jak2 inhibitors in various stages of clinical and preclinical development are listed here along with their respective GI50 s in HEL cells.
Drug HEL cell IC50
(nM)
Reference
CEP-701 30-100 [93]
TG101209 152 [200]
INCB018424 186 [85]
TG101348 300 [65]
P1 510 [201]
CYT387 1804 [102]
WP1066 2000 [202]
G6 4000 [105]
Z3 15000 [109]
134
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BIOGRAPHICAL SKETCH
Anurima Majumder was born in 1984, in the small town of Durgapur, India. Even
as a child, Anurima was inspired by her role model, her father, to pursue a career in
academics and become a professor like him. She attended Carmel High School, where
she was first inspired to pursue a career in bioscience by her biology teacher, Mrs.
Geeta Menon. Anurima went on to earn her Bachelor of Engineering degree in
biotechnology from the Bengal College of Engineering and Technology, in August 2006.
In August 2007, she moved to Gainesville, Florida and joined the Interdisciplinary
Program in Biomedical Sciences at the University of Florida in pursuit of a Ph.D. She
began her graduate dissertation research under the guidance of Dr. Peter Sayeski in
May, 2008. Her research was primarily focused on studying structure-activity
relationships of novel Jak2 small molecule inhibitors. Anurima graduated in August 2011
and is now pursuing her postdoctoral studies at the University of Iowa.