Chronic Myelogenous Leukemia (CML) - History

• 1845- The first documented clinical description.

• 1960- The discovery of Philadelphia chromosome in CML cells

• 1973- The discovery that Philadelphia chromosome results from a reciprocal translocation of chromosomes 9 and 22.

• 1986- The cloning of BCR-ABL cDNA from CML cells.

• 1980-1983 The cloning of v-Abl oncogene in A-MuLV, cloning the c-Abl proto-oncogene, locating c-Abl to chromosome 9, and demonstrating that Abl encodes a protein tyrosine kinase.

• 1970- The isolation of Abelson murine leukemia virus (A-MuLV).

• 1996- The demonstration that imatinib (Gleevec) inhibits BCR-ABL tyrosine kinase and CML growth in vitro.

• 2001- FDA approval of Gleevec for CML therapy.

• 2002-present : Gleevec resistance, CML stem cells

• 1987-present: Understanding how BCR-ABL causes CML.

Normal Chromosomes9p

9q

22p

22q

c-Abl

Bcr

ReciprocalTranslocation

Ph1Chromosome

Bcr-Abl

SH3 SH2 Y-kinase

Gag

Myristoyl groupMyristoyl group

GAGGAG SH2 Y-kinaseV-AblV-Abl

C-AblC-Abl

Circa ~1990Circa ~1990

• V-Abl, but not c-Abl, Transforms Cells.• V-Abl, but not c-Abl, is Tyr-Phosphorylated.• ∆SH3-c-Abl becomes Tyr-Phosphorylated, and can Transform Cells

• V-Abl, but not c-Abl, Transforms Cells.• V-Abl, but not c-Abl, is Tyr-Phosphorylated.• ∆SH3-c-Abl becomes Tyr-Phosphorylated, and can Transform Cells

YY

NLSsNLSs

11 22 33

Proline-rich motifsProline-rich motifs

NESNES245245 412412

CAPCAP

Abl(1b)Abl(1b)

Abl(1a)Abl(1a)

BCR-ABLBCR-ABL

v-Ablv-Abl Gag

BCR

SH3 SH2 Kinase Domain DNA Binding Actin Binding

Myristoyl groupMyristoyl group

A coiled-coil oligomerization domain of Bcr is essential for the transforming function of Bcr-Abl oncoproteins.

McWhirter JR, Galasso DL, Wang JY.

Department of Biology, University of California, San Diego, La Jolla 92093-0116.

In Philadelphia chromosome-positive human leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and chimeric Bcr-Abl proteins are produced. The fused Bcr sequences activate the tyrosine kinase, actin-binding, and transforming functions of Abl. Activation of the Abl transforming function has been shown to require two distinct domains of Bcr: domain 1 (Bcr amino acids 1 to 63) and domain 2 (Bcr amino acids 176 to 242). The amino acid sequence of domain 1 indicates that it may be a coiled-coil oligomerization domain. We show here that domain 1 of Bcr forms a homotetramer. Tetramerization of Bcr-Abl through Bcr domain 1 correlates with activation of the tyrosine kinase and F-actin-binding functions of Abl. Disruption of the coiled coil by insertional mutagenesis inactivates the oligomerization function as well as the ability of Bcr-Abl to transform Rat-1 fibroblasts or to abrogate interleukin-3 dependence in lymphoid cells. These results strongly suggest that Bcr-Abl oligomers are the active entities in transformation.

Mol Cell Biol. 1993 Dec;13(12):7587-95. (see also McWhirter and Wang, 1991)

ABL

KinaseFunctionKinase

Function

LocationCues

LocationCues

NN

CC

SH3PXXPSH3

PXXP

SH2YpXXP

SH2YpXXP

ATPATP

PeptideYXXP

PeptideYXXP

Abl tyrosine kinaseAbl tyrosine kinase

Three Nuclear Localization

Signals (NLS). One Nuclear Export Signal (NES).

DNA-binding (bubble DNA). Actin-binding (G and F).

Three Nuclear Localization

Signals (NLS). One Nuclear Export Signal (NES).

DNA-binding (bubble DNA). Actin-binding (G and F).

BCR

ABL N-terminal StructureABL N-terminal StructureABL N-terminal StructureABL N-terminal Structure

Nagar, B., et al. (2003) Nagar, B., et al. (2003) CellCell 112: 859 112: 859

YY

PD166326PD166326N-lobeN-lobe

C-lobeC-lobe

MyristateMyristateSH2SH2

SH3SH3

YY

NLSsNLSs

11 22 33

Proline-rich motifsProline-rich motifs

NESNES245245 412412

CAPCAPAbl(1b)Abl(1b)

Abl(1a)Abl(1a)

BCR-ABLBCR-ABL

v-Ablv-Abl Gag

BCR

SH3 SH2 Kinase Domain DNA Binding Actin Binding

Myristoyl groupMyristoyl group

CAPCAP

[Nagar et al, Cell, 2003][Nagar et al, Cell, 2003]

Cryrstal Structure of the N-terminal Region of Abl

• • Welch, P.J. & Wang, J.Y.J. (1993) Welch, P.J. & Wang, J.Y.J. (1993) RB binds the ATP-binding lobe of Abl kinase domain and RB binds the ATP-binding lobe of Abl kinase domain and

inhibits its kinase activityinhibits its kinase activity. . CellCell 75, 779-90. 75, 779-90.

• • Wen, S.T. & Van Etten, R.A. (1996) Wen, S.T. & Van Etten, R.A. (1996) PAG/PrdxI binds Abl SH3 domain and inhibits its kinase PAG/PrdxI binds Abl SH3 domain and inhibits its kinase

activity.activity. Genes DevGenes Dev 11, 2456-67. 11, 2456-67.

• • Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001) Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001) F-actin binds the C-terminus of Abl and inhibits its kinase F-actin binds the C-terminus of Abl and inhibits its kinase

activityactivity. . J. Biol. Cell. 276: 27104-27110J. Biol. Cell. 276: 27104-27110. .

• • Welch, P.J. & Wang, J.Y.J. (1993) Welch, P.J. & Wang, J.Y.J. (1993) RB binds the ATP-binding lobe of Abl kinase domain and RB binds the ATP-binding lobe of Abl kinase domain and

inhibits its kinase activityinhibits its kinase activity. . CellCell 75, 779-90. 75, 779-90.

• • Wen, S.T. & Van Etten, R.A. (1996) Wen, S.T. & Van Etten, R.A. (1996) PAG/PrdxI binds Abl SH3 domain and inhibits its kinase PAG/PrdxI binds Abl SH3 domain and inhibits its kinase

activity.activity. Genes DevGenes Dev 11, 2456-67. 11, 2456-67.

• • Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001) Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001) F-actin binds the C-terminus of Abl and inhibits its kinase F-actin binds the C-terminus of Abl and inhibits its kinase

activityactivity. . J. Biol. Cell. 276: 27104-27110J. Biol. Cell. 276: 27104-27110. .

ABL InhibitorsABL InhibitorsABL InhibitorsABL Inhibitors

PP

PPAblAbl

High Activity(Transforming)High Activity

(Transforming)

ActivatedActivated

Equilibrium ofLow to medium activities

Equilibrium ofLow to medium activities

Catalytically inactiveCatalytically inactive

Co-inhibitorsCo-inhibitors

UninhibitedUninhibited

AblAbl

AutoinhibitedAutoinhibited

AblAbl

CoinhibitedCoinhibited

AblAbl

AblAbl

Coinhibitor 1Coinhibitor 1

Coinhibitor 2Coinhibitor 2

A Model for the Regulation of Abl Kinase: Auto-inhibition and Co-inhibition

A Model for the Regulation of Abl Kinase: Auto-inhibition and Co-inhibition

Wang, J.Y.J.NCB, 2004Wang, J.Y.J.NCB, 2004

Implications:Implications:Partitioning of Latent Abl Kinase into

Distinct Signaling Complexesby its Co-Inhibitors

Partitioning of Latent Abl Kinase into Distinct Signaling Complexes

by its Co-Inhibitors

RB-Abl complex: Abl Transduces Signals that Disrupt

RB-Abl Interaction: e.g., RB phosphorylation, RB degradation.

RB-Abl complex: Abl Transduces Signals that Disrupt

RB-Abl Interaction: e.g., RB phosphorylation, RB degradation.

F-actin-Abl complex: Abl Transduces Signals that Disrupt

F-actin-Abl Interaction: e.g., Cell adhesion.

F-actin-Abl complex: Abl Transduces Signals that Disrupt

F-actin-Abl Interaction: e.g., Cell adhesion.

Prdx-Abl complex: Abl Transduces Signals that Disrupt

Prdx-Abl interaction: oxidative stress?

Prdx-Abl complex: Abl Transduces Signals that Disrupt

Prdx-Abl interaction: oxidative stress?

Master Protein Kinase

Signal (ligand, second messenger)

Kinase

Protein Substrates

Slave Protein Kinase

Signals(various)

Kinase

Substrates

BCR-ABL phosphorylates proteins that are substrates or non-substrates of Abl tyrosine kinase.

BCR-ABL phosphorylates proteins that are substrates or non-substrates of Abl tyrosine kinase.

• BCR-ABL contains BCR-sequences.• BCR-ABL contains BCR-sequences.

• BCR-ABL does not enter the nucleus.• BCR-ABL does not enter the nucleus.

Biological Activity Reported for BCR-ABL• Many!

• Abrogates the cytokine requirement for cell survival..•Abrogates the adhesion requirement for cell proliferation.

• Does not abrogate the serum requirement for growth.• Stimulates random motility.

• Phosphorylates different proteins in different cell lines.

Biological Activity Reported for BCR-ABL• Many!

• Abrogates the cytokine requirement for cell survival..•Abrogates the adhesion requirement for cell proliferation.

• Does not abrogate the serum requirement for growth.• Stimulates random motility.

• Phosphorylates different proteins in different cell lines.

ABL

KinaseFunctionKinase

Function

LocationCues

LocationCues

NN

CC

SH3PXXPSH3

PXXP

SH2YpXXP

SH2YpXXP

ATPATP

PeptideYXXP

PeptideYXXP

Abl tyrosine kinaseAbl tyrosine kinase

Three Nuclear Localization

Signals (NLS). One Nuclear Export Signal (NES).

DNA-binding (bubble DNA). Actin-binding (G and F).

Three Nuclear Localization

Signals (NLS). One Nuclear Export Signal (NES).

DNA-binding (bubble DNA). Actin-binding (G and F).

BCR

Copyright ©2002 American Association for Cancer Research

Cohen, M. H. et al. Clin Cancer Res 2002;8:935-942

C29H31N7O.CH4SO3, relative molecular mass is 589.7.

Imatinib mesylate is the active component of Gleevec

N Shah, C Sawyers et al. CANCER CELL 2, 2002

Imatinib-resistant Imatinib-resistant BCR-ABLBCR-ABL kinase domain mutationskinase domain mutations

❶ F317L❷ T315I❸ F359V

❹ M244V❺ G250E❻ Q252H/R❼ Y253F/H❽ E255K

M351T E355G V379I L387M H396R

10

11

12

13

9

N Shah, C Sawyers, N Shah, C Sawyers, et alet al. . Cancer CellCancer Cell 2, 2002 2, 2002

Imatinib-resistant mutations at ABL kinase domainImatinib-resistant mutations at ABL kinase domain

Mathematical models suggest Mathematical models suggest CML stem cells to be refractory to Imatinib CML stem cells to be refractory to Imatinib

F Michor, F Michor, et al. et al. Nature Nature 435, 2005435, 2005I Roeder, I Roeder, et al. et al. Nature Med Nature Med 12, 200612, 2006

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Stem cells, cancer, and cancer stem cells Tannishtha Reya, Sean J. Morrison, Michael F. Clarke andIrving L. Weissman. Nature 414, 105-111 (1 November 2001)

The current model of hematopoiesis

CML Stem Cells

* CML is a disease of the hematopoietic stem cells (HSC)

• During Chronic Phase (CP), Ph1-chromosome can be found in mature blood cells of various myeloid and lymphoid lineage.

• Gleevec is effective during CP, but ineffective if disease hasprogressed to accelerated phase (AP) or Blast Crisis (BC).

• CML stem cell identified in myeloid blast crisis patient samples. Jamieson et al, 2004

Increased Nuclear -catenin in Blast Crisis CML Granulocyte-Macrophage Progenitors

n = 6 n = 3 n = 4Jamieson et al, New Engl J Med 2004;351:657-67

CML-like phenotypes CML-like phenotypes in mHPC/p210-transplanted micein mHPC/p210-transplanted mice

mHPC/GFP mHPC/p210 87 (21) mg 782 (84) mg

n=6 n=12

Peripheral Blood smearof mHPC/p210 mice (7w)

(Wright-Giemsa staining)

Spleen (8w)

HistopathologyHistopathology(H&E staining)(H&E staining) at 8 Weeks at 8 Weeks Post-Post-transplantationtransplantation

BoneMarrow

Liver

mHPC/GFP (8w) mHPC/p210 (8w)

Spleen

Schedule of Imatinib-treatmentSchedule of Imatinib-treatmentIrradiated Irradiated 2200-recipients-recipients

Day 0 16 27 28 ~Day 0 16 27 28 ~

TransplantationTransplantation 1 x 101 x 106 6 BM cellsBM cells from leukemic mice (1from leukemic mice (100))

11 days11 days

Imatinib-treatmentImatinib-treatment AM: 50 mg/kg, p.o.AM: 50 mg/kg, p.o. PM: 100 mg/kg, p.o.PM: 100 mg/kg, p.o.

v.s. Vehicle controlv.s. Vehicle control

Effects of ImatinibEffects of Imatinib

0

10000

20000

30000

40000

WB

C (/

l)

GFP p210 Vehicle Imatinib

Vehicle Imatinib

Sp

lee

n s

ize

s (m

g)

GFP p210 Vehicle Imatinib

Vehicle Imatinib

0

200

400

600

P0.01 P0.01

GFP p210

0

20

40

60

80

100

GM

P (%

)

mHPC/GFP mHPC/p210

P0.01 P=0.11

BM

GF

P (

%)

0

2

4

6

Transplantation of Imatinib-treated Transplantation of Imatinib-treated mouse bone marrow induced leukemiamouse bone marrow induced leukemia

30-#1 (Day 38, 30)

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

587 (146) mg n=5

Spleen

BoneMarrow

GFP GFP

71.5 % 55.5 %

85.3 % 80.8 %

30-#1 (Day 38, 30) 30-#2 (Day 41, 30)

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

In the Cytoplasm: In the Cytoplasm:

BCR-ABL inhibits apoptosis.BCR-ABL inhibits apoptosis.

OncogenicOncogenic

In the Nucleus:In the Nucleus: (not where it is usually found) (not where it is usually found)

BCR-ABL induces apoptosis.BCR-ABL induces apoptosis.

Anti-oncogenicAnti-oncogenic

Vigneri & Wang, Nat. Med. 2001Vigneri & Wang, Nat. Med. 2001

AblAbl

AblAblAblF-actinF-actin

Extra Cellular Matrix, PDGFExtra Cellular Matrix, PDGF

AblAbl

Inactive

RBRB

Inactive

NLSNLSNESNES

NucleusNucleus

CytoplasmCytoplasm

OutsideOutside

actin dynamicsactin dynamics

APOPTOSISAPOPTOSIS

DNA Damage

DNA Damage

AblAblTNFTNF

TNF-induced apoptosis of mouse thymocytes requires Abl

and is blocked by Rb-MI

TNF-induced apoptosis of mouse thymocytes requires Abl

and is blocked by Rb-MI

Chau et al, MCB, 2004Chau et al, MCB, 2004

Abl contributes to TNF-induced apoptosisAbl contributes to TNF-induced apoptosis

Chau et al, MCB, 2004.Chau et al, MCB, 2004.

LMB

Covalent modification of CRM1 by Leptomycin BCovalent modification of CRM1 by Leptomycin B

T Kau, P Silver et al. Nature Reviews Cancer 4, 2004

CRM1=exportin-1

Trapping BCR-ABL in the Nucleus:Trapping BCR-ABL in the Nucleus:

cytoplasmcytoplasm NucleusNucleus

** ** ** **NLSNLS

NESNES

Leptomycin BLeptomycin B

*STI571*STI571

Nuclear BCR-ABL kinase kills:Nuclear BCR-ABL kinase kills:

cytoplasmcytoplasm NucleusNucleus

NESNES

Leptomycin BLeptomycin B

DeathDeath

ImatinibImatinib

LMBLMB

Imatinib + LMBImatinib + LMB

Count viable cells every 2 days

Count viable cells every 2 days

48 hours48 hours

16 days16 days

oror

oror

Extensive WashingExtensive Washing

Complete Media, NO DRUGS

Complete Media, NO DRUGS

TonB (+IL3)TonB (+IL3)

22 44 66 88 1010 1212 14 days14 days

non-treatednon-treated

LMBLMBImatinibImatinib

Imatinib+LMBImatinib+LMB

1x101x1088

1x101x1077

1x101x1066

1x101x1055

1x101x1044

Un-Induced forBCR-ABL Expression

Un-Induced forBCR-ABL Expression

TonB/BCR-ABL (-IL3)TonB/BCR-ABL (-IL3)

22 44 66 88 1010 1212 1414 16 days16 days

non-treatednon-treated

LMBLMBImatinibImatinib

Imatinib+LMBImatinib+LMB

1x101x1088

1x101x1066

1x101x1044

1x101x1022

00

Induced forBCR-ABLExpression

Induced forBCR-ABLExpression

TonB/BCR-ABL (+IL3)TonB/BCR-ABL (+IL3)

22 44 66 88 1010 1212 14 days14 days

non-treatednon-treated

LMBLMB

ImatinibImatinib

Imatinib+LMBImatinib+LMB

1x101x1088

1x101x1066

1x101x1044

1x101x1022

00

Induced forBCR-ABLExpression

Induced forBCR-ABLExpression

K562 (-IL3)K562 (-IL3)

33 55 77 99 1111 1313 15 days15 days

non-treatednon-treated

LMBLMB

ImatinibImatinib

Imatinib+LMBImatinib+LMB

1x101x1088

1x101x1066

1x101x1044

1x101x1022

00

Questions-

1. How can the mathematical predictiondepicted in Fig. 4A (sc panel) of the Michor et al

paper be tested in the clinic?

2. What may account for the innate resistanceof CML stem cells to imatinib?

3. What may account for the activation ofcell death by nuclear BCR-ABL?

Questions-

1. How can the mathematical predictiondepicted in Fig. 4A (sc panel) of the Michor et al

paper be tested in the clinic?

2. What may account for the innate resistanceof CML stem cells to imatinib?

3. What may account for the activation ofcell death by nuclear BCR-ABL?