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EVALUATION OF HAIR FOLLICLES AS A SURROGATE TISSUE FOR
PHARMACODYNAMIC RESPONSE IN XENOGRAFT TUMORS
A thesis presented by
Mary-Kamala Menon
To
The Department of Biology
In partial fulfillment of the requirements for the degree of
Master of Science
In the field of
Biology
Northeastern University
Boston, Massachusetts
March, 2012
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EVALUATION OF HAIR FOLLICLES AS A SURROGATE TISSUE FOR
PHARMACODYNAMIC RESPONSE IN XENOGRAFT TUMORS
By
Mary-Kamala Menon
ABSTRACT OF THESIS
Submitted in partial fulfillment of the requirements for the degree of Master of Science in
Biology in the Graduate School of Arts and Science of Northeastern University,
March 2012
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Abstract
Identification of an easily accessible and pharmacodynamically robust surrogate tissue
for tumor biopsies is essential for rapid drug development in oncology. Blood-borne markers are
the gold standard for monitoring pharmacodynamics for many diseases, although they are not
always relevant for many cancers. Plucked hair has gained attention as a surrogate tissue as it is
an epithelial tissue similar to the origin of >80% of tumors, has good drug exposure and is highly
proliferative as with many tumors. We have linked the pharmacodynamic response of hair
follicles and tumors in vivo using a mouse xenograft model to facilitate the development of
clinical pharmacodynamic biomarkers. A Notch driven xenograft model known to respond to a
gamma-secretase inhibitor was created to compare plucked hair, skin biopsies and whiskers as
surrogate tissues for tumors. Hair growth synchronization was induced in mice by depilation
followed by collection of tumor, hair, whiskers, skin and blood samples during each phase of the
hair cycle. A qPCR assay was developed for hair phase identification using published gene
expression patterns in skin biopsies as a guide. A published Notch Signature Set, in addition to a
tissue responsive Signature Sets developed in this thesis, was used to determine whether hair
growth phase affects pharmacodynamic readout. This information allowed creation of a pre-
clinical model with the potential to drive quick decisions in the clinic about target modulation
and treatment.
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Acknowledgments
I would like to thank Dr. Joel Klappenbach for undertaking the role of committee
member in addition to his enormous regular work commitments. His patience, guidance and
generosity of time was greatly appreciated and allowed this project to move forward. I want to
thank Dr. John Reilly for supporting my endeavor to obtain a Master of Science degree while
employed in his laboratory. Completion of my degree would not have been possible without his
encouragement and funding on behalf of Merck Research Laboratories. I would also like to
extend my thanks to Dr. Erin Cram for agreeing to be part of my committee and my thesis
advisor, Dr. Wendy Smith, who encouraged and made possible my desire to obtain a research
based degree part-time and outside the laboratories of Northeastern University. Thanks to Brian
Roberts and Minilik Angagaw, who dedicated precious time to train and assist me on tools and
techniques essential to this thesis. I thank my numerous colleagues who have provided me with
reagents and more importantly their time to aid me in some aspect of this project. Finally, I
would like to recognize my appreciation to my family, friends and better half, Derrick, for their
willingness to share this journey with me over the last few years.
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Table of Contents
Abstract 3
Acknowledgements 4
Table of Contents 5
List of Abbreviations 9
List of Figures 11
List of Tables 13
1. Introduction
1.1 Stages of Drug Development 14
1.2 Utility of pharmacodynamic biomarkers in surrogate tissues 15
1.3 Normal NOTCH function and signaling cascade 16
1.4 Dysfunctional signaling and cancer 17
1.5 Notch dysregulation in a T-cell lymphoblastic neoplasm xenograft model 18
1.6 GSI-Responsive Notch Gene Signature Set 19
1.7 Hair as a developing tissue and tumor response surrogate 21
1.8 Use of a mouse model for hair development research 23
1.9 Objectives 24
2. Materials and Methods
2.1 TALL-1 cell line culture 25
2.2 Mouse strain and xenograft tumor model 25
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2.3 Cell preparation for injections 26
2.4 Hair cycle synchronization induced by wax depilation 26
2.5 Mouse randomization and treatment assignment 27
2.6 Drug formulation and dosing 27
2.7 Pharmacokinetic analysis 28
2.8 Tissue collection (blood, tumor, hair, whiskers, skin) 28
2.8.1 Timeline
2.8.2 Blood collection
2.8.3 Tumor dissection
2.8.4 Hair plucking
2.8.5 Whisker collection
2.8.6 Skin biopsy collection
2.9 RNA Extraction 30
2.10 Reverse Transcription PCR and pre-amp 30
2.11 Real Time PCR 31
2.12 Analysis 31
3. Results
3.1 Original Notch Signature Score indicates hair can be used as a surrogate tissue
and supports clinical data 32
3.2 The Notch Signature Set was refined to be comprised of highly responsive genes
across all surrogate tissues 33
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3.3 Further refinement of Notch Signature Score for hair and tumor leads to a
heightened response in hair 35
3.4 Notch Signature Sets are affected by time in certain tissue types 36
3.5 Gene expression analysis used to associate time post-depilation with phases of the
hair cycle 36
3.6 A majority of hair cycle genes show the same trend in plucked hair as skin
biopsies 37
3.7 Gamma-secretase inhibitor MK-003 perturbs normal hair cycle gene expression
patterns 38
3.8 Hair growth phase does not greatly alter significance of Refined Notch Signature
1 and 2 scores in hair follicle 39
4. Discussion
4.1 Original Notch Signature Score indicates hair can be used as a surrogate tissue
and supports clinical data 39
4.2 Further refinement of score for hair and tumor 41
4.3 A majority of hair cycle genes show the same trend in hair follicle as skin 44
4.4 Gamma-secretase inhibitor perturbs normal hair cycle gene expression patterns 45
4.5 Refined Notch Signature Scores allows hair follicles to be used as a surrogate
tissue across all presumed phases of the hair growth cycle 47
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Conclusions 48
Future Directions 49
Figures 51
Tables 69
References 78
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List of Abbreviations
T-ALL-1 human T cell Acute Lymphoblastic Leukemias
DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
IACUC Institutional Animal Care and Use Committee
RNA Ribonucleic Acid
PCR Polymerase Chain Reaction
NOD SCID Non-Obese Diabetic Severe Combined Immunodeficient
RPMI media Roswell Park Memorial Institute media
FBS Fetal Bovine Serum
DPBS Dulbecco's Phosphate Buffered Saline
RT Reverse Transcription
RT Real Time
dNTP Deoxyribonucleotide
IFC Integrated Fluidic Circuits
ADAM A Disintergrin And Metalloprotease
TACE Tumor Necrosis Factor Alpha Converting Enzyme
CSL CBF1/RBP-Jκ / Suppressor of hairless / LAG-1
GSI Gamma-Secretase Inhibitor
ICN Intracellular domain of Notch
MK-003 Merck compound, gamma-secretase inhibitor
NHV Normal Healthy Volunteer
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PK Pharmacokinetic
PD Pharmacodynamic
B-CLL B-cell chronic lymphoid leukemia
qPCR Quantitative Polymerase Chain Reaction
DSL Delta, Serrate, LAG-2
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List of Figures
Figure 1 Key Players of Notch Signaling pathway 50
Figure 2 Mechanisms of Notch Mechanism 51
Figure 3 Plucked hair transcription PD biomarker (Notch Signature score) successfully
used in normal healthy volunteers 52
Figure 4 Hair Follicle Structure 53
Figure 5 Hair cycle growth phases 54
Figure 6 Timeline of cell injections and phase induction 55
Figure 7 Average PK concentrations of MK-003 dosed groups 56
Figure 8 Treatment effect at multiple times post depilation across potential surrogate
tissues 57
Figure 9 Correlations of treatment effect by Original Notch Signature Score in multiple
tissue combinations 58
Figure 10 Comparison of Original Notch Signature Score and Refined Notch Signature
Score 1 to measure treatment effects 59
Figure 11 Poorly and highly responsive genes to MK-003 across all potential surrogate
tissue types 60
Figure 12 Refined Notch Signature Score 1 resulted in improved correlation of scores
between surrogate and target tissue 61
Figure 13 Refined Notch Signature Score 2 differs from Refined Notch Signature Score 1 by
inclusion of HEY1 and exclusion of HES1 62
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Figure 14 Refined Notch Signature Score 2 shows enhanced treatment effect read-out in
hair compared to tumor in all times post depilation 63
Figure 15 Examples of genes demonstrating poor agreement of gene expression
directionality between skin biopsy and hair follicle 64
Figure 16 Examples of genes demonstrating high agreement of gene expression
directionality between skin biopsy and hair follicle 65
Figure 17 Significance of disrupted hair cycle gene expression patterns by MK-003 was
initially determined by three-way ANOVA 66
Figure 18 Presumed phase does not greatly alter significance of Refined Notch Signature 1
and 2 scores in hair follicle 67
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List of Tables
Table 1 Hair Cycle Genes 68
Table 2 Notch Signature Genes 70
Table 3 Housekeeping Genes 71
Table 4 Composition of each Notch Signature Score used to evaluate samples 72
Table 5 Treatment effects of MK-003 in hair and tumor as measured by multiple Notch
Signature Sets 73
Table 6 Time post-depilation is a strong response factor to treatment effect in hair 74
Table 7 Gene expression patterns in skin biopsies can be used to define hair cycle phases 75
Table 8 Gamma-secretase inhibitor, MK-003, perturbs normal hair cycle gene expression
patterns during anagen 76
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1. Introduction
1.1. Stages of drug Development
The journey of a molecule to a marketed drug is a long and treacherous one lasting
upwards of 10 years with a success rate wavering around 1%. The development of a single drug
is estimated to cost from $161 million to $1.8 billion, due to absorbing the costs of failed
compounds (Morgan, Grootendorst et al. 2011). The development process has many stage gates
both in pre-clinical and clinical development, any of which can halt further exploration into the
usefulness of the molecule. The earlier the development of a “failed” drug is halted, the greater
the cost savings. The cost of running preclinical studies pales in comparison to that of human
clinical trials. This makes it cost-effective to understand as early in development as possible if a
compound will not be effective or safe. Preclinical research is designed to evaluate a drug's
toxicity and pharmacological effects in vitro and in vivo using a minimum of two species of
animal models (DiMasia and Grabowskib 2007). Great consideration is given to animal models
that most accurately recapitulate the human condition for which the drug is being targeted.
Translating the preclinical findings into the clinic can be a major obstacle. One animal model
with emerging promise for preclinical studies is the mouse hair follicle model (Al-Nuaimi, Baier
et al. 2010). A thorough analysis of how the mouse hair cycle relates to that of humans is
imperative to designing an appropriate preclinical model in which murine hair is tested for
feasibility as a surrogate to a human tumor.
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1.2. Utility of pharmacodynamic biomarkers in surrogate tissues
Pharmacodynamic (PD) biomarkers in oncology drug development present unique
challenges due to safety issues associated with the invasive collection of tumor biopsies. In
addition, the heterogeneous nature of tumor biopsies reduces the reliability of biomarker
readouts. While blood-borne markers have been the gold standard for monitoring
pharmacodynamics for many diseases, blood is not a relevant peripheral tissue for many cancers.
The most common and least invasive samples collected during clinical studies include blood,
skin, hair, urine, and nails (Randall and Foster, 2007). Identification of a surrogate peripheral
tissue that is easily accessible, has good drug exposure and active growth pathways similar to
those in the tumor has proven challenging. Plucked hair has gained recent attention as a
surrogate tissue for pharmacodynamic assessment as it meets the latter criteria and is an
epithelial tissue similar to the origin of >80% of tumors (Jemal, Siegel et al., 2007). Patient
safety and the difficulty with obtaining quality and representative tumor samples make
quantification of a corresponding tumor PD response in humans impractical and introduces
unnecessary risk to the patient. To avoid placing patients at risk, we have established a mouse
xenograft model to evaluate the pharmacodynamic response of hair follicles and tumor in vivo to
facilitate the development of clinical pharmacodynamic biomarkers. This type of model can be
used as the foundation of solid tumor drug discovery for oncology, permitting evaluation of
tumors of a variety of origins against anti-oncogenic agents in an easily-accessible surrogate
tissue.
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1.3 Normal NOTCH function and signaling cascade
The highly conserved Notch signaling pathway is found in many multi-cellular organisms
and plays an important role in embryonic development. The Notch signaling cascade regulates
neuronal function and development, cell lineage specification, stem cell maintenance, induction
of terminal differentiation and many other processes (Koch and Radtke, 2007).
Notch signaling is mediated by cell to cell contact in which Notch genes encode
transmembrane bound receptors that are activated by two distinct families of transmembrane
bound ligands. Four receptors (Notch 1-4) and five ligands (Delta-like 1, 3 and 4, and Jagged 1
and 2) are found in mammals. The receptors differ in the number of EGF-like repeats and the
length of the intracellular domain (Figure 1) (Nickoloff, Osborne et al., 2003).
Synthesis of Notch receptors results in a single precursor protein that is cleaved by a
furin-like protease during transport to the cell surface (Figure 2) (O'Neil and Look, 2007). At
the cell surface, receptors are expressed as heterodimers consisting of two extracellular subunits,
one of which contains an extracellular heterodimerization domain attached to a transmembrane
domain followed by the cytoplasmic region of the receptors. The intracellular portion of the
receptors mediates cell signaling (Koch and Radtke, 2007). Activation of Notch signaling is the
result of ligand-receptor interactions between adjacent cells that results in induction of two
proteolytic cleavages. The first cleavage occurs by an ADAM-family metalloprotease, TACE
(Tumor Necrosis Factor Alpha Converting Enzyme), which cleaves the Notch receptor protein
extracellularly and allows for release of its ligand binding domain which continues to interact
with the ligand. The ligand-expressing cell endocytoses the ligand-Notch extracellular domain
complex. The portion of the receptor that remains on the Notch-expressing cell is further
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cleaved by a γ (gamma)-secretase enzyme inside the inner leaflet of the cell membrane of that
cell. The intracellular domain of the Notch protein (ICN) is released and moves to the nucleus
where it regulates gene expression by activating transcription factor, CSL (CBF1/RBP-Jk,
Suppressor of Hairless, LAG-1) (Allenspach, Maillard et al., 2002). Use of a γ-secretase
inhibitor (GSI) blocks Notch signaling by preventing the second proteolytic cleavage of the
Notch protein (Figure 2) (O'Neil and Look, 2007).
1.4 Dysfunctional signaling and cancer
Dysregulation of Notch signaling has been implicated in a number of solid cancers such
as cervical, lung, skin, adrenal gland, breast, epithelium and prostate. It also appears in
hematological malignancies like Hodgkin’s lymphomas, anaplastic large-cell non-Hodgkin’s
lymphomas, and subsets of acute myeloid leukemias and B-cell chronic lymphoid leukemia (B-
CLL) (Allenspach, Maillard et al., 2002; Nickoloff, Osborne et al., 2003). The link between
Notch and human cancer was strengthened when a 9:7 chromosomal translocation was found to
yield a truncated human Notch-1 receptor lacking the majority of the extracellular subunit
(Nickoloff, Osborne et al., 2003). This recurrent translocation was associated with T-cell
lymphoblastic leukemias (Ellisen, Bird et al., 1991). The absence of these extracellular subunits
leads to constitutive activation of the receptor and altered proliferation rates (Allenspach,
Maillard et al., 2002);(Nickoloff, Osborne et al., 2003). Use of a gamma-secretase inhibitor
(GSI) blocks the proteolytic cleavage of Notch and has anti-neoplastic effects in cells that
overexpress or constitutively express Notch, both in vitro and in in vivo xenograft models. For
example, inhibition of gamma-secretase blocks the proliferation of human pre-T-ALL (human T
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cell acute lymphoblastic leukemias) cells with constitutively active Notch-1 (Nickoloff, Osborne
et al., 2003);(Tammam, Ware et al., 2009). A panel of twenty T-ALL cell lines was evaluated for
GSI sensitivity at Merck & Co., Inc. and TALL-1 was one of five cell lines to demonstrate this
sensitivity in the form of prolonged cell cycle arrest followed by apoptosis. Rescue experiments
overexpressing the NCID confirmed inhibition of Notch signaling pathway (Rao, O'Neil et al.,
2009);(O'Neil, Grim et al., 2007).
1.5 Notch dysregulation in a T-cell lymphoblastic neoplasm xenograft model
Notch signaling plays an essential role in the T cell development from common lymphoid
progenitors. Notch1 insufficiency results in preferential intra-thymic B-cell development over T-
cell development (Radtke, Ferrero et al., 2000). By contrast, an aberrant increase in Notch1
causes preferential ectopic T-cell differentiation in the bone marrow over B-cell differentiation
(Sriuranpong, Borges et al., 2001). Such signaling eventually causes development of the lethal
CD4+/CD8+ T-cell lymphoblastic neoplasm (T-ALLs) (Weng, Nam et al., 2003). Mouse T-ALL
models have been used to understand the dysregulation of human T-cells (Tammam, Ware et al.,
2009). Treatment of human or mouse T-ALL cell lines in vitro with GSIs caused growth arrest
and apoptosis. For example, when GSI is introduced to near-end-stage T-ALL/lnk4a/Arf+/- mice,
Notch1 is inhibited and apoptosis is increased resulting in an extended survival rate of the
leukemic mice (Cullion, Draheim et al., 2009).
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1.6 GSI-Responsive Notch Gene Signature Set
Considerable effort has been applied towards developing a Notch Signature Set of genes
that serves as a transcriptional (mRNA-based) biomarker of response to Notch pathway
inhibition by gamma-secretase inhibitors (Blackman, S., Klappenbach, J., et al. 2009). A
genetically engineered in vivo breast Her2 (BH) allograft model was one model used to develop
this signature (Watters, Cheng et al., 2009). The Her2 (BH) allograft exhibited heterogeneity in
many facets such as gene expression levels, histopathology, growth rate and Notch pathway gene
expression to GSI treatment response. Taken together, this model was representative of what has
been observed in humans and thus served as an appropriate model for the human condition.
Additionally, a panel of Notch1 activating mutant T-ALL cell lines were used to identify a set of
Notch pathway related genes that were most responsive to GSI treatment (Rao, O'Neil et al.,
2009). Despite the fact that in more than 50% of T-ALL patients, somatic activating mutations
in Notch1 exist, Notch1 mutation status is not an accurate predictor of GSI sensitivity. The
compilation of the transcriptional work allowed for introduction of a Notch Signature Set into the
clinical setting with healthy volunteers. These subjects confirmed a transcriptional response to
GSI administration compared to placebo in plucked human anagen hair follicles
(ClinicalTrials.gov 2008). The signature set was refined with use of whole genome
transcriptional profiling in human plucked hair follicles performed in this study. The follicles
were from patients treated with a smaller dose of GSI than the clinically tolerated dose in order
to create the 9-gene Original Notch Signature Score, ONSS. The Notch Signature score was
dose-responsive 96 hours post administration in normal healthy volunteers (NHV) indicating its
strength as biomarker of Notch pathway inhibition (Figure 3). While in vitro cell line data
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highlights GSI-responsive genes and in vivo human data demonstrates clinical proof of concept
in human hair, no data has been generated from a pre-clinical xenograft model linking tumor
response in hair. Validation of the tumor response in hair pre-clinically must be established in
order to bridge xenograft murine models to the human condition (Blackman, 2010).
In order to confirm this finding in a pre-clinical model, we examined the effect of GSI on
the Original Notch Signature Score in a T-ALL1 xenograft mouse model. T-ALL 1 is a highly
proliferative dysregulated Notch cell line that constitutively overexpresses the Notch intracellular
domain (NCID) leading to aberrant Notch signaling strongly associated with cancer (O'Neil,
Grim et al., 2007). Samples of tumor and surrogate tissues including hair, skin, and whiskers
were collected from both vehicle and experimental GSI MK-003 treated mice. Modulation of
Notch pathway activity in the tumors was verified using the Original Notch Signature. The
Original Notch Signature has been quantified by both microarray and qPCR in GSI dosed solid
colorectal tumor LS-1034 xenograft models. There is a tight correlation of Notch Signature
changes between each technology providing confidence in detection of the Original Notch
Signature in hair by qPCR (Klappenbach, unpublished data). Dosing conditions were replicated
in these experiments to ensure reproducible expression. While there is a substantial amount of
data demonstrating the detection of Notch Gene Signature modulation in murine hair by qPCR,
there has been no evidence linking hair and tumor in mouse until now. This experiment also has
provided data that enhances our understanding of how hair cycle could affect the response of this
signature.
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1.7 Hair as a developing tissue and tumor response surrogate
Hair is a surprisingly complex tissue consisting of the fibrous shaft, which is visible
above the skin's surface, and the hair follicle that resides in the dermis. The hair shaft is
comprised of the medulla, cortex and cuticle (Schlake, Beibel et al., 2004) (Figure 4). The hair
follicle is a mini organ made up of both epithelial and mesenchymal regions and houses stem
cells that contribute to new hair growth and regeneration of the skin. The epithelial portion of the
hair follicle can be further divided into a permanent region distal to the arrector pili muscle and
the inferior region containing the hair bulb which is the most highly proliferative tissue in the
human body (Kruase, 2003).
One of the defining characteristics of mammals is the growth of hair. Hair covers nearly
every body part of the human body aside from mucous membranes and glabrous skin, though the
type and growth rate vary leading to differences in appearance and texture. The main types of
hair are androgenic (body hair), vellus and terminal. Puberty in humans induces the transition
from fetal fine, short, light vellus hairs to thick, long and dark terminal hairs in certain areas of
the body. The type of hair changes during development and by gender. The human hair cycle,
like that of all mammals, consists of anagen (growth), catagen (transitional), and telogen
(resting) phases which occur asynchronously across the body (Figure 5). The duration of the
anagen phase is highly dependent on genetics but generally can last up to 8 years in humans
while only lasting 2 weeks in mice. During this time, the papilla cells are dividing to produce
new hair fibers while the follicle is embedded in the dermal layer of the skin and functions to
nourish the strand. Approximately 85% of human scalp hairs are in anagen phase at any given
time. Once the anagen phase has terminated, hair moves into the catagen phase for about two
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weeks in humans and merely 3-7 days in mice in which the hair follicle shrinks due to halting of
proliferation and initiation of apoptosis. The papilla detaches, breaking the connection to the
blood supply. The resulting action is an upward push of the hair shaft as the terminal fibers
lengthen. The final phase of growth is telogen in which the hair and follicle remain quiescent for
1-4 months in humans and 5-7 days in mice. Roughly 10-15% of human scalp hairs are in
telogen phase. The growth cycle begins again by pushing up and out the new growing strand
with anagen once telogen completes (Porter, 2003). The murine model undergoes the same basic
follicular transformations in humans (Kligman, 1959), and presumably similar changes in gene
expression throughout the hair cycle.
Over 80% of adult tumors are epithelial in origin making the hair follicle a potentially
ideal surrogate tissue for assessing tumor response. The hair follicle contains stem cells located
in the hair follicle bulge that can regenerate the entire skin epithelium. This highlights the strong
link between these three tissue types: skin, hair and tumor, making hair a compelling surrogate
tissue (Morris, Liu et al., 2004). Hair is also the most proliferative tissue in the human body
making it a relevant surrogate tissue for compounds that modulate cellular proliferation such as
oncology targets. Its high vascularity allows for adequate drug exposure and appropriate use in
pharmacokinetic and pharmacodynamic studies. Lastly, hair follicles, unlike tumor biopsies, are
easily accessible and non-invasive, allowing for repeated sampling over time and are less
heterogeneous than the tumor tissue.
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1.8 Use of a mouse model for hair development research
Aside from not undergoing a vellus-to-terminal hair type switch during development as
humans do, the mouse is a well accepted model system for studying human hair cycle and
disorders. There is no evidence that mouse and human hair follicle differ structurally aside from
the presence of a specialized vibrissae follicle in mice that does not undergo retraction and the
bulb remodeling occurs only during catagen (Porter, 2003). Each phase of the mouse hair cycle
has been well-characterized based on morphological structures and by gene expression. The
phases have been defined either by the age of a mouse or by the number of days post wax
depilation (Muller-Rover, Handjiski et al., 2001). Depilation is the act of removing the hair
follicle from the skin (typically by wax) in order to re-initiate and synchronize hair growth. Each
method has advantages and drawbacks. Utilization of mouse age to stage hair cycles does not
require anesthetization of the animal and allows mice as young as 4 weeks of age to be used for
experimentation. The primary drawback includes a limited window during which mice can be
used for experimentation. The restriction in age is due to 1) hair being naturally synchronized
for first two cycles and 2) development occurring in waves across the body. It is also difficult to
develop suitably sized tumors in young mice. While depilation requires anesthesia and may
cause depilation-induced inflammatory responses, synchronous anagen growth and the ability to
grow larger tumors offers distinct advantages for gene expression studies linking concomitant
responses in tumor and hair (Muller-Rover, Handjiski et al., 2001). Given the experimental
tractability, the depilation model was used to study tumor/hair gene expression response in the
studies described below.
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1.9 Objectives
A foundation of pharmaceutical research lies in pre-clinical research and the fundamental
understanding of biology. Mimicking the human disease in a rodent model and ensuring that the
biological readouts of compound efficacy can be translated into the clinic are essential for
effective drug development. In this study, we examined the hair as a surrogate for tumor by
utilizing a Notch driven xenograft model (TALL-1) known to respond to the gamma-secretase
inhibitor, MK-003. We compared plucked hair to skin biopsies and whiskers as an appropriate
surrogate tissue for tumor. While the collection of hair does not require anesthetic, it can be far
more time consuming that taking a skin biopsy and may not be an option for chemotherapeutic
patients who have hair loss. Conversely, a skin biopsy does necessitate anesthetic application
though sutures are only required for a 4 mm diameter punch or larger. Whiskers were
investigated in the pre-clinical model due to a higher RNA yield compared to hair though the
clinical equivalent is nostril hair, which may not be feasible. Hair, whiskers, skin and tumor
samples were collected for gene expression analysis during multiple phases of the hair cycle in
order to understand if the phase during sample collection plays a factor in effectiveness of the
tissue as a surrogate. It is hypothesized that hair in the rapidly proliferating anagen growth phase
would mimic the tumor's gene expression patterns more closely than during the quiescent telogen
phase or regressing catagen phase. The majority of published hair cycle gene expression patterns
are derived from skin biopsies so we initially established whether the same patterns can be seen
in hair alone. Samples were assessed using a published Notch Signature Set to understand
whether hair phase affects the PD gene expression readout in skin and/or hair. This information
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allowed establishment of a pre-clinical model to validate biomarkers that can be used in the
clinic and potentially drive quick decisions in the clinic about target modulation and treatment.
2. Materials and Methods
2.1 TALL-1 cell line culture
Human T-cell leukemia cells, TALL-1, were originally obtained from DSMZ (Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH Inhoffenstr. 7b D - 38124
Braunschweig - Germany) and stocked in Merck Research Laboratory's Centralized Cell Culture
(Boston, MA). One vial at 9x106 cells/mL was thawed in 15 mL media (85% RPMI 1640 + 15%
h.i. FBS). Cells were grown in suspension at 37°C (5% CO2), scaled up to 1.21x109/ 800mL by
splitting 1:2 approximately every 3 days for 7 weeks. The average viability was 90.2%.
2.2 Mouse strain and xenograft tumor model
All animal protocols were approved by Merck Research Laboratory's IACUC (Institutional
Animal Care and Use Committee). Female NOD SCID mice age 29-35 days old were ordered
from Charles River (Wilmington, MA) in two batches and allowed to acclimate to Merck's
Animal Facility for a minimum of 3 days prior to cell injections. Mice were 36-49 days old at
time of cell injection. Thirteen mice anesthetized with isoflourane were injected with 8x106 cells
subcutaneously on right flank at each time point. Time points (-32, -29, -26, -20, -14, -7 days)
were defined relative to hair synchronization induction at Day 0 (Figure 6). Xenograft tumor
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growth was monitored for 32 days and early euthanasia of mice bearing tumors >10% body
weight or 2000mm3 was implemented.
2.3 Cell preparation for injections
TALL-1 cells were counted with Vicell and a suspension of 8x106 viable cells/mouse was
collected. Cells were pelleted by centrifugation (Sorvall Legend RT) at 1200 RPM for 5 minutes
at 4ºC. Media was aspirated, the was cell pellet washed with 1X DPBS (Invitrogen, catalog #
14190) and then collected into one 50 mL conical tube. Cells were spun at 1200 RPM for 5
minutes at 4ºC and then aspirated in 1X DPBS. The cell pellet was finally resuspended in 50%
1X DPBS 4ºC (-Mg, -Cl) and 50% Matrigel (growth factor reduced) for a final concentration of
8x107 cells/ml. The entire resuspension was placed in a round-bottomed microcentrifuge tube
and kept on ice for duration of injections. Immediately prior to each injection, the
microcentrifuge tube was inverted and 8x106 cells/100 ul were aspirated with syringe only in
order to prevent cell shearing. A cold 25G 5/8 needle was placed onto syringe prior to right
flank subcutaneous injection of each anesthetized mouse.
2.4 Hair cycle synchronization induced by wax depilation
A section of skin on the dorsal region of each mouse was shaved to 2.4 mm (standard setting 1
on Wahl 79600-2101 Lithium Ion Cordless Clipper) one day prior to depilation. Shaving was
performed to improve the effectiveness of wax adhesion to hair and reduce irritation to skin. To
perform depilation, a tin of wax from a Simple Spa Wax Warmer Kit (Sally Hansen Inc,
Uniondale, NY, USA) was warmed for approximately 25 minutes using the supplied warmer and
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stirred frequently until wax appeared opaque and easy to stir. To prevent burning of mouse skin,
wax temperature was evaluated on the technician’s wrist prior to application on each
anesthetized mouse. A thick layer of wax was applied following direction of hair growth. Wax
was removed with one swift motion against direction of hair growth once wax was tacky enough
to grip and repeated once if necessary. Mice were visually inspected one day post depilation to
confirm welfare (Yano, Brown et al., 2001).
2.5 Mouse randomization and treatment assignment
The day prior to each dosing, tumors were measured with calipers and measurements were
recorded. Mice were randomized into treatment groups (n=6) based on average tumor volume.
2.6 Drug formulation and dosing
Mice (n=5) were dosed during one of the six time points (0, 3, 6, 12, 19, 25 days post depilation)
with the GSI MK-003. A suspension of 10 mg of MK-003 in 1 mL 5% methylcellulose (vehicle)
was vortexed for 1 minute prior to probe sonication for approximately 10 seconds or until white
froth formed. This light-sensitive suspension was moved into a room temperature sonicating
water bath for ~ 15 minutes or until large pieces were broken up followed by overnight agitation
on a shaker. Administration of the compound suspension proceeded once the absence of large
particulates was confirmed. Mice were weighed immediately prior to dosing orally at 10 mL/kg.
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2.7 Pharmacokinetic analysis
Blood was collected via cardiac puncture, and approximately 100 µL of blood was transferred
into a 96-deep well plate containing 300 µL 1M citrate buffer as an anticoagulant. The mixture
was stored at -20°C until sample collection was complete. The mixture was thawed and
centrifuged, and plasma was transferred to a 96-well plate prior to pharmacokinetic analysis. The
GSI (MK-003) concentrations in mouse plasma (as described above) were analyzed by high-
pressure liquid chromatography (HPLC) using an Allegros Pump with dual arm HTC PAL auto-
sampler (Thermo Scientific, Franklin, MA,USA) equipped with a reversed-phase column
(Waters Atlantis T3, 3 µm, 2.1 ¥ 20 mm, Waters Corp., Milford, MA, USA) and linear
water/ACN gradient (5–90% organic in 3 min) containing 0.1% formic acid at a flow rate of 850
mL·min-1. The effluent from the HPLC column was introduced into an Applied Biosystems API
4000 triple quadrupole mass spectrometer (Carlsburg, CA) equipped with an electrospray
interface. Mass spectrometric analysis was performed in the positive ionization mode with the
ion spray voltage set at 5 kV. The precursor [M+H]+/product ion MS/MS transitions selected to
monitor were m/z 552.6/221.4 for GSI and 329.2/162.1 for the internal standard. The protonated
molecules were fragmented by collision-induced dissociation with nitrogen as a collision gas.
The collision energy voltage was set at 30 V and 37 V for GSI and internal standard respectively.
The data were acquired and processed by Analyst 1.4.2 software (AB/MDS Sciex).
2.8 Tissue collection (blood, tumor, hair, whiskers, skin)
2.8.1 Timeline - Tissue collection occurred 8 hours post dosing.
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2.8.2 Blood collection – Mice were anesthetized using CO2 until obvious slowing of breath.
100 µl of blood was drawn via cardiac puncture and mixed with 300 µl 1M sodium
citrate buffer. Samples were placed on ice for duration of tissue collection and stored at -
20°C. Cervical dislocation followed. Pharmacokinetic analysis was applied to determine
concentration of MK-003 in whole blood.
2.8.3 Tumor dissection – Tumors were grossly removed from euthanized mouse and weighed.
Two 3 mm^2 tumor biopsy sections were placed in 1 mL 4°C RNAlater (Ambion,
Austin, TX) overnight and then stored at -20°C until processing for RNA extraction. Two
replicates of remaining tumor were snap frozen in liquid nitrogen and stored at -80°C
until processing for protein extraction.
2.8.4 Hair plucking – 12 tufts of hair (1 tuft is defined as one 3-mm tweezerful of hair) were
collected by holding skin taut and grasping hair close to skin's surface with tweezers
(Model Mc0130bk,Rubis Switzerland). Hair was pulled out against the direction of
growth. One tuft at a time was submerged into a microfuge tube containing 500 µl
Promega SV Lysis Buffer. Once 12 tufts were collected, each microfuge tube was
vortexed for 1 minute at maximum speed and stored at -80°C until processing for RNA
extraction.
2.8.5 Whisker collection – A minimum of 10 whiskers were plucked using tweezers. Each
was visually inspected for root attachment prior to placing into 500 µl Promega Lysis
Buffer. Once an entire sample was collected, it was vortexed for 1 minute at maximum
speed and stored at -80°C until processing for RNA extraction.
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2.8.6 Skin biopsy collection – Skin samples were dissected from depilated area. Two
replicates of 3 x 4mm biopsy punches were dissected from depilated area and placed in 1
mL 4°C RNAlater overnight. Samples were stored at -20°C until processing for RNA
extraction. Two replicates of 1 x 4mm biopsy punch were flash frozen in liquid nitrogen
and stored at -80°C until processing for protein extraction. One 1cm x 2cm strip of skin
was placed in 10% neutral buffered formalin overnight at 4°C and then stored in 4°C
70% ethanol.
2.9 RNA Extraction
Total RNA from hair and whiskers was extracted using SV Total RNA Isolation System
(Promega, Madison, WI, USA). Total RNA from skin and tumor was extracted using RNeasy 96
Kit (Qiagen, Valencia, CA, USA). A NanoDrop8000 Spectrophotometer (Thermo Scientific,
Wilmington, DE) was used to measure nucleic acid concentration at 260 nm and sample purity
ratios (260nm/280nm). Samples were normalized to 50 ng/ µl in RNAse free water.
2.10 Reverse Transcription PCR and pre-amp
cDNA was prepared from total RNA using a High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems, Foster City, CA). Each 10 µl reverse transcription (RT) reaction contained
1 µl 10x RT buffer, 1 ul 10x Random Primers, 0.4 µl dNTP mix, 0.5 µl RT enzyme, 0.2 µl
RNase Inhibitor, 2 ul of RNA (50ng/ µl) and 5 µl RNase-free water. The RT conditions used are
as follows: 25°C for 10 minutes, 37°C for 2 hours, 95°C for 5 minutes and 4°C hold.
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In order to maximize limited starting material, 20 ng (10 ng/ µl) cDNA was pre-amplified with 4
µl 2X TaqMan Pre-Amp Master Mix Kit (Applied Biosystems, Carlsbad, CA), 2 µl 0.2X pooled
assay mix for 14 cycles of amplification. The pre-amplification protocol was as follows: 95°C
for 10 min., 14 cycles of 95°C x 15 seconds and 60°C x 4 minutes, finishing with a hold step at
4°C. Pre-amplified samples were diluted 1:5 with TEZero (10mM Tris, 0.1mM EDTA in
UltraPure water). 4.5 µl of each diluted sample was mixed with 5 µl 2X Taqman enzyme mix
(Applied Biosystems, Foster City, CA, USA) and 0.5 µl of DA sample loading reagent
(Fluidigm, South San Francisco, CA,USA).6 µl of this mixture was loaded into the primed
integrated fluidic circuit (IFC). Each 20X Taqman Assay (Table 1-3) was diluted 1:1 with DA
Assay Loading Reagent (Fluidigm) and 6ul was loaded into the primed IFC.
2.11 Real Time PCR
A Biomark System was used for real-time quantitative PCR (Fluidigm, South San Francisco,
CA, USA). The Biomark system allows for the 96 samples to be quantified using 96 assays with
limited starting material in a single reaction.
2.12 Analysis
Notch Signature Scores were calculated by taking the average normalized Ct of each set of genes
(Table 4) using the following formula to determine abundance of each gene: G=log10(2-normalized
Ct). The samples were normalized to respective mouse or human species specific house keeping
genes: Prpf8 and TBP. Matlab and R software was utilized to apply statistical analysis to the
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samples collected. A three-way ANOVA was run to examine the factors of time, treatment and
tissue as well as the interactions between pairs of factors.
3. Results
3.1 Original Notch Signature Score indicates hair can be used as a surrogate tissue and
supports clinical data
We introduced GSI MK-003 into a Notch-driven tumor xenograft mouse model in order
to recapitulate previous Notch targeting evidence and link hair response to tumor (Tammam,
Ware et al., 2009). We assessed the factors affecting the strength of a surrogate tissue in addition
to quantitative measurement of its strength. PK data showed no significant difference (one-way
ANOVA, P
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Original Notch Signature Scores between vehicle and compound treated groups. The difference
in scores measured how strongly Notch signaling modulation could be detected in a particular
tissue during unique times post depilation. An ideal surrogate tissue would have a similar
difference in scores to the target tissue. The average treatment effect for each tissue type across
all time points was as follows: tumor (0.19+0.05), hair (0.19+0.06), skin (0.11+0.06), whisker
(0.16+0.11). Although the number of time points was limited, linear regression analysis
indicated that hair follicle and tumor show the most highly-correlated treatment effect over time,
confirming the clinical findings that Notch Signature Score can be used to monitor a tumor
response to GSI in plucked hair (Figure 9). Other tissues showed little, if any, correlation to
tumor response at each time point.
3.2 The Notch Signature Set was refined to be comprised of highly responsive genes
across all surrogate tissues
Whole genome microarray expression profiling is the leading method by which gene
signatures are developed. While this comprehensive method is used as a discovery method, it is
not unusual to refine signatures when performed on different platforms. In order to understand if
the Original Notch Signature Score could be improved, we examined a number of additional
transcripts using qPCR to measure gene expression. The additional genes evaluated included
both hair cycle genes and Notch signaling pathway genes in order to not impose any bias towards
Notch pathway genes. As listed in Tables 1-2, 55 genes exclusive of housekeeping genes were
examined with 19 being related to the Notch pathway. A three-way ANOVA (with all two-way
interactions) was run on all samples to detect the genes most responsive to GSI treatment across
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all tissue types independent of time. A first pass in developing a de novo score was done by
setting a p value threshold of p
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3.3 Further refinement of Notch Signature Score for hair and tumor leads to a
heightened response in hair
Hair follicle and tumor share a number of physiological similarities, with the potential of
similar regulation by GSI, which is a key component of effective surrogate tissue read-out. A
three-way ANOVA was used to evaluate the factors of time, treatment, tissue along with the
interaction of these pairs. We looked at the interaction of treatment and tissue to identify genes
responsive to drug in a subset of tissues as opposed to in all tissues for the Refined Notch
Signature Score 1. A p value threshold set at
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3.4 Notch Signature Sets are affected by time in certain tissue types
We designed this experiment to collect tissue samples at a minimum of one point in each
phase of the full hair cycle. Prior to evaluating which phase occurred at each time point, we
determined whether time (proxy for hair cycle phase) significantly altered Notch score gene
expression. As the Notch signature scores were further refined, there was a general trend of
increasingly significant time points in both hair and tumor (Table 5). The number of significant
time points assessed using the Original Notch Signature Set, Refined Notch Signature Set 1 and
Refined Notch Signature Set 2 were as follows in hair follicle (1, 4, 5) and tumor (4, 3, 4). The
standard deviation of treatment effect in hair across time is a minimum of two fold higher than
that of tumor, independent of Notch signature sets used demonstrating that time post-depilation
is a strong response factor (Table 6).
3.5 Gene expression analysis used to associate time post-depilation with phases of the
hair cycle
A majority of the work describing gene expression patterns of the hair cycle has come
from skin biopsies derived from an assortment of platforms such as microarray profiling, qPCR,
immunohistochemistry and in situ hybridization. We assigned each time point to the different
phases of the hair cycle based on published gene expression in the vehicle skin biopsies and then
verified whether similar expression patterns could be seen in plucked vehicle hair follicle
(Ishimatsu-Tsuji, Moro et al., 2005; Schlake, Beibel et al., 2004; Umeda-Ikawa, Shimokawa et
al., 2009).
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Thirty hair cycle genes were examined and associated with a particular phase based on
peak expression in published data (Table 7). We noted the time points during which peak levels
of expression of each gene occurred in our experimental skin biopsies and plucked hair (Table
7). When the genes were grouped by published phase, the majority of our skin data fell within
the same timeframe bringing confidence in our method of determining phase. Based on this
analysis, we determined early anagen appears at day 0, middle anagen occurs during days 3-6,
late anagen/early catagen occurs during day 12-19, catagen at day 19 and telogen at day 25
(Table 7).
3.6 A majority of hair cycle genes show the same trend in plucked hair as skin biopsies
In order to determine how similar hair cycle gene expression patterns were between
plucked hair and skin biopsies, we examined the four intervals of collection times that overlap
between these two tissues for each hair cycle gene. The interval between days 0-3 had to be
eliminated since hair could not be collected at day 0. Directionality of gene expression in hair
was qualitatively described as increasing or decreasing and compared to that of skin for each
interval. Each interval represents 25% of the overall agreement score. The magnitude of change
for each tissue was not considered a factor due to the difficulty in normalizing gene abundance
levels across platforms in the published references. The minimum agreement score achievable
was 0% indicating hair and skin show opposite directional trends in gene expression during all
time intervals monitored while a 100% agreement score indicated identical expression
directional changes. Of the 33 hair cycle genes assayed, only Jun resulted in a 0% agreement
score (Figure 15). The 25% agreement seen in Eif5 was a result of the trend from day 19 to day
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25 (Figure 15). Mt4 and Cryba4 are two of seven genes that had a 100% agreement (Figure 16).
A minimum 75% agreement score was the threshold set by which we considered a gene to show
the same trend between these two tissue types. Nineteen of the thirty genes assayed proved to
have this acceptable agreement score (Table 7).
3.7 Gamma-secretase inhibitor MK-003 perturbs normal hair cycle gene expression
patterns
Hair phase markers were initially examined in order to correlate each time point to a
particular phase in the hair growth cycle. While pre-clinical studies always include a vehicle
group, it would be advantageous to use the same sample for treatment read-out as well as for
phase designation. In order to do this, we must rule out the possibility that the compound alters
hair cycle gene expression patterns. An ANOVA was run on all genes expressed in mouse
derived tissues in order to examine the effect of treatment. Figure 17 shows examples of hair
cycle genes in hair tissue that did (Figure 17a,e) and did not (Figure 17b,c,d) show statistically
significant modulation by treatment (P
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in 16/17 hair cycle genes with CCL7 being the deviator from this trend (Figure 17e). Down-
regulation was seen in 7/17 hair cycle genes and always occurs at day 25 (Figure 17a-e). The
result of the entire panel of hair genes assessed is shown in Table 8. The data indicates that GSI
perturbs specific hair cycle gene expression patterns though it is undetermined whether the hair
cycle phases or transitions are disregulated.
3.8 Hair growth phase does not greatly alter significance of Refined Notch Signature 1
and 2 Scores in hair follicle
Statistical analysis of treatment effect showed the Original Notch Signature Score was
statistically significant with p
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surrogate tissue though we also sampled other potential surrogate tissues. Hair showed the
closest average treatment effect to tumor as compared to other potential surrogate tissues,
implying the Notch signaling pathway is most similarly modulated in these two tissue types. The
Notch treatment effect represents how strongly the biomarker responds to drug in a particular
tissue type. The larger the treatment effect, the greater the difference of Notch Signature Scores
between compound and vehicle exposed samples. The treatment effect seen in tumor is the
standard by which we compared other surrogate tissues. The similar treatment effects between
hair and tumor by means of the Original Notch Signature score in a murine xenograft model
confirms the initial goal of demonstrating hair has the ability to show a similar response to GSI
as tumor. Skin and whiskers showed potential for being surrogate tissues, though the
consistency of treatment effect over time was not as strong as in hair.
We were eager to determine whether the vehicle and GSI treated mice would demonstrate
similar Original Notch Signature Score treatment effects in tumor independent of time post-
depilation. Because a consistent treatment effect in tumor over time was not found, two de novo
signature scores were developed. The Original Notch Signature Score was developed using cell
lines in culture so it was not surprising that the score required refinement. An ideal biomarker
set would reduce this variability of treatment effect over time seen in the Original Notch
Signature score. By refining the Original Notch Signature Score to only include genes that were
highly responsive across all tissue types collected, we were able to reduce the variability of
treatment effect in all tissues especially that of hair and tumor. Variability reduction is
evidenced by the treatment effect standard deviations in hair and tumor reduced from 0.117 to
0.063 and 0.045 to 0.038, respectively, with use of Refined Notch Signature Score 1. The
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standard deviations within a tissue type represent how effective that Notch signature score was in
capturing Notch signaling modulation across all phases of the growth cycle.
The combination of ANOVA data and visualization of each gene allowed creation of the
Refined Notch Signature Score 1. Two genes that generally responded well to GSI across all
tissues by showing a distinct separation of samples by treatment type are Hey1 and NRARP
(Figure 11). This separation by treatment can be seen by examining the log2fold change. In
contrast to this, ATOH1 and HMGCS1 did not appear to be highly regulated by GSI since there
is an overlap of dosed and vehicle animals by measure of log2fold change (Figure 11).
Correlation graphs were also used to compare the score generated in one tissue type to another
within the same mouse. It was expected that the scores themselves would not be identical since
they are a read-out of the abundance of certain Notch signaling genes in each tissue type. Yet,
the more closely correlated scores are between two tissue types, the more similarly they are
responding to the drug in terms of Notch pathway signaling.
4.2 Further refinement of score for hair and tumor
While the Refined Notch Signature Set 1 improved GSI read-out across the multiple
surrogate tissues samples, we decided to create a hair and tumor specific signature score
(Refined Notch Signature Set 2) so as to instill confidence in the signature set for the target
tissue. Hes1 is a poor indicator of GSI treatment effect in T-ALL1 xenografts and hair resulting
in the elimination of this gene from the Refined Notch Signature Score 1. While both Hes1 and
Hey1 are canonical Notch target genes and members of the basic helix-loop-helix transcription
factor family, they appear to show different expression patterns across tissues in response to GSI,
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at least with the qPCR assays we used to assess changes in transcript abundance. Hey1, unlike
Hes1, shows strong GSI treatment effects in both hair follicle and tumor. Evidence exists that
signaling to Hes1 is not indicative of Notch pathway activation (Sang, Roberts et al. 2010). In
the context of metastatic exocrine cells, GSI chemical inhibition of Notch signaling leads to
decreased Hey1 expression and unchanged Hes1 expression (Rooman, De Medts et al., 2006).
The Refined Notch Signature Score 2 is the only score that shows a consistently larger
treatment effect in hair follicle compared to tumor (Figure 14). Notch signaling has been
implicated in hair regulatory functions, which may provide insight to this finding (Hu, Lefort et
al.). While one could interpret Refined Notch Signature Score 2 to be a more sensitive score, it
could also be seen as exaggerating the response and hence providing false indication of the
efficacy of the drug. An alternative score might balance sensitivity and magnitude of response to
the drug in both the primary and surrogate tissue.
While it is important for the treatment factor to overwhelmingly drive the response in
surrogate tissue, other variables influence the response. The time post-depilation during which
samples were collected altered the Notch score in hair follicles independent of the gene sets used.
This variation led us to investigate whether the point in the hair cycle during which hair was
plucked determined the ability of hair follicles to serve as a surrogate tissue. Examination of the
treatment effect variability in each tissue type showed that tumor was not affected by time post
depilation as strongly as hair. We had no reason to believe the act of depilating the mouse's
dorsal region would influence Notch signaling in the tumor though we did think it would affect
the hair for two reasons. The first reason is that Notch signaling plays a role in hair
development, which is being re-initiated by depilation. The second is that drastic changes in hair
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proliferation rates occur over the course of the hair cycle, which we believe we have
encompassed. The times chosen were assumed to span the hair cycle and this was verified using
gene expression analysis (Aubin-Houzelstein, G. 2011).
The skin data reproduced the hair-phase dependent gene expression patterns in the
literature for hair cycle development. Unlike the published references, we did not collect telogen
control samples on the day of depilation, which limited the number of samples collected during
this phase. The SCID-NOD immunocompromised strain of mice we used allowed xenograft
generation, which was different from the majority of published studies that use of C57/B6. The
C57/B6 strain of mouse demonstrates characteristic changes in skin color and thickness that
permit easy identification of hair phase. In order to prevent tumor size from exceeding 10% of
the mouse body weight, we choose not to collect samples past the first reported day of telogen.
Telogen phase been reported by some sources as 25 days post depilation and while others show
telogen occurs at day 28 post depilation (Muller-Rover, Handjiski et al., 2001; Umeda-Ikawa,
Shimokawa et al., 2009). Due to conflicting reports of telogen phase, we may have only spanned
one day of telogen during its early induction when we sampled on day 25 at study termination. It
is also possible that the hair cycle phases in the SCID-NOD mice vary from the C57/B6 mice or
Mini rats referenced (Ishimatsu-Tsuji, Moro et al., 2005; Umeda-Ikawa, Shimokawa et al.,
2009). The species and/or strain could explain why the telogen associated genes were not
peaking in expression at day 25 but may have if a slightly later time point was included. Our
analysis was adjusted to note abundance levels at each time point rather than following the
referenced method of normalizing each gene to telogen expression levels (Ishimatsu-Tsuji, Moro
et al., 2005; Umeda-Ikawa, Shimokawa et al., 2009). Additionally, the literature phase of hair
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growth association was derived from DNA, mRNA and protein expression, while our data was
solely mRNA based, leading to a potential discrepancy. All of our experimental samples were
induced into hair synchronization at this point supporting our finding that early anagen occurred
during Day 0.
4.3 A majority of hair cycle genes show the same trend in hair follicle as skin
The majority of work published on hair development makes use of skin biopsies
embedded with hair, not plucked hair follicles. Skin has been used for its ease of sampling,
greater protein and nucleic acid yield, enhanced control of hair orientation for histology
purposes, and potential avoidance of a wound healing response (Argyris, 1968). One concern
with skin biopsies is that gene expression analysis of changes in embedded hair follicles will be
“diluted” by mRNA from the dermis layer. Hair follicles are proposed as an alternative to skin
biopsies, which require anesthesia and can require post-collection sutures. It is also challenging
to guarantee that the biopsy will contain enough or any hair follicles. Hair follicles are the most
proliferative tissue in the body and therefore could potentially yield a more robust response than
skin (Krause and Foitzik, 2006).
We were initially surprised to see that only 19/30 genes showed the same trend in hair as
skin. This discrepancy may be a result of differential expression contribution of hair structures
that remain in the skin biopsy and are void in plucked hair (Pan, Lin et al., 2004). The germ
capsule, sebaceous gland, arrector pili muscles and dermal papilla are example structures that
may contribute to these expression differences. We did not examine the plucked follicle under a
microscope but do believe that we are capturing the highly proliferative epithelial sheath
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45
surrounding the hair bulb. The phase associations were derived from a compilation of data from
different sources and assay platforms. Published material included multiple rodent species, yet
no distinction was made between them when observing phase-associated genes (Ishimatsu-Tsuji,
Moro et al., 2005; Umeda-Ikawa, Shimokawa et al., 2009). Only directionality of expression
change and not magnitude were used to quantify hair-cycle phases associated with gene
expression changes herein. Analysis was performed based on directionality due to the inability
to standardize the multiple sources of data utilized to report expression changes. If a method of
normalization and inclusion of magnitude changes could have been implemented, perhaps a
stronger relationship could have been established between tissue types.
4.4 Gamma-secretase inhibitor perturbs normal hair cycle gene expression patterns
After confirming that the majority of hair cycle genes showed a similar expression trend
in hair follicles as skin biopsies, we investigated the effect of GSI in hair follicles. It was
possible that the gamma-secretase inhibitor altered hair cycle gene expression patterns due to the
Notch pathway involvement in hair development and cycling (Pan, Lin et al., 2004). A
qualitative analysis showed that the inhibitor most strongly affected hair cycle genes during
anagen phase (corresponding to day 3-12) and less so at day 25 (corresponding to telogen) by
comparing hair cycle gene expression of vehicle to GSI exposed hair follicles. The early stages
of anagen phase have the highest rates of proliferation in the connective tissue sheath and
presumably would be highly regulated by a chemotherapeutic that is slowing proliferation in
target and ancillary tissues. No change was noted during day 19 of the regressing catagen phase
at which time apoptosis rates are the highest (Tobin, Gunin et al., 2003).
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46
Studies using transgenic mice have shown that Notch signaling is essential for
maintenance of many structures of the hair follicle including, but not limited to the inner root
sheath, outer root sheath and hair shaft. Deletion of gamma-secretase in post-natal skin has led
to an absence of sebocytes and hair follicle conversion into epithelial cysts due to dysregulated
epithelial proliferation (Pan, Lin et al., 2004; Watt, Estrach et al., 2008). Loss and gain of
function Notch mutants have shown similar structural phenotypes indicating that the level of
Notch signaling plays an important role in proper hair follicle development (Aubin-Houzelstein,
G. 2011).
While the GSI is intended to completely ablate downstream signaling, it is unknown what
level of inhibition was reached in the hair at the time of tissue collection occurring 8 hours post
dosing. Morphological analysis was not applied to the plucked hairs, but it can be inferred that
retarded structural development could contribute to the gene expression patterns observed.
Additionally, there is a wide array of evidence in transgenic mice with inducible deletions of
Notch signaling components, which implicate Notch in controlling the hair cycle clock and
transitions between phases (Aubin-Houzelstein, G. 2011). Taken together, it is probable that
GSI treated hair cannot be utilized for hair cycle phase identification evidenced by altered hair
cycle gene expression patterns in GSI exposed follicles compared to vehicle exposed follicles.
On the other hand, the role that Notch signaling plays in hair follicle development may enhance
the biomarker response to GSI, making hair follicle a stronger and potentially more reliable
surrogate tissue in GSI treated animals.
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4.5 Refined Notch Signature Scores allows hair follicles to be used as a surrogate tissue
across all presumed phases of the hair growth cycle
One objective of this study was to understand whether the phase of the hair cycle during
which samples are collected affect the validity of hair follicles as a robust surrogate tissue for
tumor. The strength of the correlation between hair follicle and tumor response was measured by
the treatment effect on the various Notch scores at different times during the hair cycle. Each
Notch score was a composite of Notch signaling pathway genes, with each score reflecting
specific tissue responsive genes. It was hypothesized that the anagen phase would prove most
effective and reliable since the proliferation rate peaks during this time.
Contrary to the hypothesis of peak modulation in anagen phase, significant Notch scores
were observed across all phases of the hair cycle. The Notch Scores are a read-out of Notch
signaling abundance, while the treatment effects demonstrate how strongly the GSI has down-
regulated Notch signaling targets at each timepoint. The Notch scores that showed the highest
rates of significance across time were Refined Notch Signature Score 1 and Refined Notch
Signature Score 2, which were comprised of genes shown to be highly responsive in the tissue
types selected. These scores, compared to the Original Notch Signature Score, were selected to
obtain larger magnitude Notch signaling difference between vehicle and treated samples. Notch
signaling plays a major role in hair development and cycle control (Aubin-Houzelstein, G. 2011),
which we infer would alter hair cycle gene expression. Notch receptors are present within the
hair follicle and at its base, the site of active proliferation (Watt, Estrach et al., 2008). Notch
expression has been shown to appear during times of terminal differentiation (Aubin-
Houzelstein, G. 2011),(Watt, Estrach et al., 2008). Also, Notch1 deletion in the epithelium of a
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conditional gene targeted mouse that induces inactivation of critical Notch signaling components
results in a truncated anagen period along with premature catagen entry in the first hair cycle
(Aubin-Houzelstein, G. 2011). Anagen re-entry can also be delayed in addition to dysregulated
proliferation and apoptosis rates during hair cycle transitions (Lin, Kao et al.). Additionally,
gamma-secretase deficient mice have improper development of skin appendages like sebaceous
glands that support the hair follicle (Pan, Lin et al., 2004). Therefore, we infer that the significant
shift in GSI induced Notch expression occurs across all phases of the hair cycle and leads to
modulated hair cycle gene expression (Table 8). Our data is in agreement with this inference
based on examination of individual hair cycle genes during multiple points in the hair cycle
(Figure 17). Taken together, we believe the hair cycle may be altered in some way that does not
allow for a direct correlation of phase to surrogate efficacy with a Notch-dependent GSI. The
treatment effect is time matched but may not be phase matched due to GSI effects on hair cycle
phase. Therefore, we cannot conclude whether phase alters the hair's ability to be an effective
surrogate. As the data shows, use of a Notch Signature Score that is highly responsive in the
surrogate tissue sampled, will allow for hair sampling across all presumed phases of the hair
cycle.
Conclusions
In conclusion, we were able to examine gene expression patterns in the multiple surrogate
tissues and found distinct differences in hair cycle gene expression patterns within hair and hair-
containing skin. Differences between skin and hair may be related to sebaceous gland structural
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49
defects of the hair follicle as a result of exposure to GSI, which may lead to perturbed expression
patterns if such structures were a source of a phase specific genes (Pan, Lin et al., 2004). The
knowledge of the tissue specific hair cycle patterns is an important tool that can be used during
experimental design of pre-clinical models in which surrogate tissues will be collected. We have
shown that it is difficult to interpret hair follicle surrogacy effectiveness during Notch dependent
normal hair cycle phases using gene expression analysis due to the Notch targeting GSI
treatment. Introduction of this Notch targeting compound led to a skewing of normal hair cycle
gene expression patterns though it was not determined whether hair cycling patterns were
altered. Despite this, we have demonstrated that plucked mouse hair follicle can be used to
measure GSI response in TALL-1 xenografts using a highly responsive de novo Refined Notch
Signature Score 2 across all presumed phases of the hair cycle. Additionally, this depilation
model can be applied more broadly in xenograft studies to assess numerous other compounds
that do not alter hair follicle development as strongly as a Notch targeted inhibitor.
Future Directions
This study provided a strong foundation for hair follicles as a surrogate tissue for tumor.
While we were able to replicate published hair cycle gene expression patterns in skin with a
limited number of assays, it could be advantageous to look at a wider range of gene expression
assays. The inclusion of additional, more frequent, time points could also allow us to capture the
many sub-phases of anagen and catagen. Extending the duration of the study would capture
telogen phase more thoroughly. Inclusion of histological data would be pertinent to verifying the
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proposed structural abnormalities and determining which structures remained embedded in the
skin after hair is plucked. This will also allow visualization of hair cycle gene expression
patterns in embedded structures, which may explain the discrepancy of some hair cycle genes
between skin biopsies and plucked hair. Lastly, the use of a different chemotherapeutic that does
not modify hair follicle structures or hair cycle transitions would allow us to tease out the initial
hypothesis of whether hair cycle phase greatly alters the hair's effectiveness as a surrogate tissue.
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Figures
Figure 1: Key Players of Notch Signaling pathway. The Notch pathway is highly conserved and normally functions to determine cell fates and regulate pattern formation. Dysfunction of this cascade has been implicated in a variety of human diseases. Notch signaling is mediated by cell to cell contact in which Notch genes encode transmembrane bound receptors that are activated by two distinct families of transmembrane bound ligands. Four receptors (Notch 1-4) and five ligands (Delta-like 1,3 and 4, and Jagged 1 and 2) are found in mammals. The receptors differ in the number of EGF-like repeats and the length of the intracellular domain.
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Figure 2: Mechanism of Notch Signaling. Notch signaling activation is the result of ligand-receptor interaction between adjacent cells resulting in induction of two proteolytic cleavages. A metalloprotease is responsible for the first cleavage, which severs the Notch receptor protein extracellularly to release the ligand binding domain which continues to interact with the ligand, DSL (Delta, Serrate, Lag-2). The ligand-expressing cell endocytoses the ligand-Notch extracellular domain complex. The portion of the receptor that remains on the Notch-expressing cell is further cleaved by a γ(gamma)-secretase enzyme inside the inner leaflet of the cell membrane of that cell. The intracellular domain of the Notch protein (ICN) is released and moves to the nucleus where it regulates gene expression by activating transcription factor, CSL (CBF1/RBP-Jk, Suppressor of Hairless, LAG-1). Use of a γ -secretase inhibitor (GSI) blocks Notch signaling by preventing the second proteolytic cleavage of the Notch protein.
©2005 by American Society of Clinical Oncology
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Figure 3: Plucked hair transcription PD biomarker (Notch Signature score) successfully used in normal healthy volunteers. Dose- and time-dependent modulation of Notch Signature score by gamma-secretase inhibitor, MK-072, is driven by a subset of 5 genes.
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Figure 4: Hair Follicle Structure. The hair follicle structure is essentially conserved throughout mammalian species aside modifications for certain specialized functions. It is a separate entity within the skin and its production is initiated by epidermal cell differentiation and results in a keratinized hair fiber. Matrix cells are epidermal derived cells found close to the dermal papilla and are the dividing cells that contribute to the growth of the hair follicle. Moving outwards, cortex cells make up the hair fiber and are derived from cells produced in the center of the hair follicle. Surrounding the cortex is the inner root sheath produced by matrix cells and is made up of several layers (cuticle, Huxley’s, Henle’s). The outer root sheath is continuous with the epidermis and its bulge region containing stem cells and is the site of arrector pili muscle attachment.
http://www.dolcera.com/wiki/images/Hairbasics.jpg
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Figure 5: Hair cycle growth phases. The hair cycle can be divided into three main phases: a long growing phase (anagen), a brief apoptosis driven regressing phase (catagen) and a short relatively quiescent phase (telogen). At the end of the resting phase, the hair falls out (exogen) and a new hair starts growing in the follicle, beginning the cycle again.
Cotsarelis G J. Clinical Inv. Vol. 116:19-22 (2006)
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Figure 6: Timeline of cell injections and phase induction. TALL1 cell lines were injected into thirteen mice at days (-32,-29, -26, -20, -14, -7) corresponding to six unique groups of mice (expected final n =10 due to tumor take rate). Each group was monitored for 28 days and then dosed with 100 mpk of MK-003 or vehicle. At 8 hours post dosing, primary tumor tissue and surrogate hair, whiskers, skin were collected. Depilation occurred at day 0 inducing synchronization of hair growth in order to allow for subsequent collection of tissues at presumed hair cycle phases.
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Figure 7: Average PK concentrations of MK-003 dosed groups. The time points represent unique days post depilation over the course of the study during which groups of mice (n=5) were dosed with 100 mpk of MK-003 eight hours prior to tissue collection. There is no statistically significant difference in the average MK-003 concentration in whole blood between time points. One-way ANOVA and Bonferronni’s Multiple Test Correction showed no significant difference amongst time points.
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Figure 8: Treatment effect at multiple times post depilation across potential surrogate tissues. Treatment effect is calculated from the difference in Original Notch Signature Scores between vehicle and MK-003 dosed mice. Variation in treatment effect is noted in all tissues most strongly in whisker. Dark line indicates average while dashed line is + 1 standard deviation.
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R2 = 0.07
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Figure 9: Correlations of treatment effect by Original Notch Signature Score in multiple tissue combinations. The change in Original Notch Signature Score between time matched vehicle and dosed mice for each tissue type is plotted to determine the correlation strength between pairs of tissues. The Pearson correlation is not significant using p< 0.05 between any two tissue types.
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Figure 10: Comparison of Original Notch Signature Score and Refined Notch Signature Score 1 to measure treatment effects. All samples were evaluated by use of both scores. Refined Notch Signature Score reduced variation in treatment effect at various times post depilation within tumor, hair, whiskers and increased in skin.
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Figure 11: Poorly and highly responsive genes to MK-003 across all potential surrogate tissue types. ATOH1, HMGCS, HEY1 and NRARP expression changes across tumor, skin, hair and whiskers in response to MK-003 were noted by qPCR. Genes are separated by panels and treatment is color coded (MK-003 – pink, Vehicle - blue). Responsiveness was determined by treatment effect separation based on log2fold. ATOH1 and HMGCS1 were poorly responsive to MK-003 while HEY1 and NRARP were highly responsive to MK-003.
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Hair RNNS1 Skin RNNS1
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Figure 12: Refined Notch Signature Score 1 resulted in improved correlation of scores between surrogate and target tissue. The correlation values of all surrogate tissues are statistically significant with p < 0.01. Hair has the highest R-squared value indicating its consistent strength as a surrogate tissue.
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Figure 13: Refined Notch Signature Score 2 differs from Refined Notch Signature Score 1 by inclusion of HEY1 and exclusion of HES1. Score make-up was determined by responsiveness of tissues of interest to MK-003. Responsiveness was evaluated by separation of vehicle and MK-003 dosed samples by log2fold. HES1 is a poorly responsive genes to MK-003 in hair and tumor compared to HEY1.
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Figure 14: Refined Notch Signature Score 2 shows enhanced treatment effect read-out in hair compared to tumor in all times post depilation. The Refined Notch Signature Score 2 is composed of genes highly responsive in hair and tumor.
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Figure 15: Examples of genes demonstrating poor agreement of gene expression directionality between skin biopsy and hair follicle. Agreement scores were determined by similar expression directionality between hair follicle (green arrows) and skin biopsy (blue arrows) for each time interval. The 25% agreement score for Eif5 is attributed to the expression directionality between 19 and 25 days post depilation. Jun was the only gene assessed to have a 0% agreement score highlighting the similarity in gene expression directionality of hair follicle to skin biopsy.
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Figure 16: Examples of genes demonstrating high agreement of gene expression directionality between skin biopsy and hair follicle. The magnitude of gene expression directionality was not considered in calculation of agreement scores resulting in a 100% agreement score for Mt4 and Cryba4. Seven of the thirty hair cycle genes assessed had a 100% agreement score.
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Figure 17: Significance of disrupted hair cycle gene expression patterns by MK-003 was initially determined by three-way ANOVA. KCNE1(a) and CCL7(e) are significantly modulated by gamma-secretase inhibitor while CAR2(b),CRYBA4 (c), KRT25(d) are not. P < 0.05
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Figure 18: Presumed phase does not greatly alter significance of Refined Notch Signature 1 and 2 scores in hair follicle. The advantage of further refining the Original Notch Signature Score (ONSS) is evidenced by the increasing number of times when significant treatment effects occur. The Original Notch Signature Score, Refined Notch Signature Score 1 (RNSS 1) and Refined Notch Signature Score 2 (RNSS 2) showed 2, 4 and 5 significant treatment effects not limited to any particular phase.
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Tables Table 1: Hair Cycle Genes Gene Symbol Gene Title Applied Biosystems
Human Assay Identification
Applied Biosystems Mouse Assay Identification
Ankrd1 ankyrin repeat domain 1 (cardiac muscle) Hs00173317_m1* Mm00496512_m1*
Atf3 activating transcription factor 3 Hs00231069_m1* Mm00476032_m1*
Birc5
Baculoviral inhibitor of apoptosis repeat containing 5 NA Mm00599749_m1*
Car2 carbonic anhydrase 2 Hs00163869_m1* Mm00501572_m1* Car6 carbonic anhydrase 6 NA Mm00486722_m1*
CCL7
Chemokine(C-C motif) ligand 7/small inducible cytokine A7/monocyte chemotactic protein 3 Hs00171147_m1* Mm00443113_m1*
CDCA3 cell division cycle associated 3 Hs00229905_m1* Mm00496601_m1*
CDK5RAP2
CDK5 regulatory subunit associated protein 2 Hs00217403_m1* Mm00524401_m1*
Cryba4 crystallin, beta A4 Hs00608089_m1* Mm00517516_m1* Ctse cathepsin E Hs00157213_m1* Mm00456010_m1*
Eif5 eukaryotic translation initiation factor 5 Hs01028811_g1* Mm00730998_s1*
Igf1 insulin-like growth factor 1 Hs01547656_m1* Mm00439560_m1
Igfbp3 insulin-like growth factor binding protein 3 Hs00181211_m1* Mm00515156_m1*
Igfbp5 insulin-like growth factor binding protein 5 Hs00181213_m1* Mm00516037_m1*
Jun Jun oncogene NA Mm00495062_s1*
Kcne1
potassium voltage-gated channel, Isk-related subfamily, member 1 Hs00897540_s1* Mm01215533_m1*
Kifc1 kinesin family member C1 Hs00292601_m1* Mm03011781_m1*
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Krt25 Keratin 25 Hs00698974_m1 Mm00840837_m1*
Krtap3-3 keratin associated protein 3-3 Hs00953462_s1* Mm04208593_s1
LEF lymphoid enhancer binding factor 1 NA Mm01310389_m1
Lnc2 Lipocalin 2 Hs01008571_m1* Mm01324470_m1*
Mki67
antigen identified by monoclonal antibody Ki-67 NA Mm01278617_m1*
MMP11 matrix metallopeptidase 11 Hs00968295_m1* Mm00485048_m1*
Mt4 metallothionein 4 Hs00262914_m1* Mm00485227_m1*
S100a9 S100 calcium binding protein A9/calgranulin Hs00610058_m1* Mm00656925_m1*
SHCBP1 Shc SH2-domain binding protein 1 Hs00226915_m1* Mm00488184_m1*
Slfn4 Schafen 4 NA Mm01298330_m1* spnb2 spectrin beta 2 NA Mm00486365_m1*
Sprr2a small proline-rich protein 2A Hs03046643_s1* Mm00845122_s1
Thbs1 thrombospondin 1 Hs00962908_m1* Mm01335418_m1
TIMP1 Tissue inhibitor of metalloproteinase 1 Hs00171558_m1* Mm00441818_m1*
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Table 2: Notch Signature Genes Gene Symbol Gene Title Applied Biosystems
Human Assay Identification
Applied Biosystems Mouse Assay Identification
ADAM19 A Disintegrin And Metalloproteinase 19 Hs00224960_m1 Mm00477337_m1
ATOH