The Role of Notch Signaling in the Pathogenesis of Acute Promyelocytic Leukemia

224 Pages·2016·5.16 MB·English

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All Theses and Dissertations (ETDs)
1-1-2012
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Nicole Grieselhuber
Washington University in St. Louis
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Grieselhuber, Nicole, "The Role of Notch Signaling in the Pathogenesis of Acute Promyelocytic Leukemia"
(2012). All Theses and Dissertations (ETDs). 579.
https://openscholarship.wustl.edu/etd/579
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WASHINGTON UNIVERSITY IN ST. LOUIS

Division of Biology and Biomedical Sciences

Immunology

Dissertation Examination Committee:
Timothy Ley, Chair
Daniel Link
Jeffrey Milbrandt
Barry Sleckman
Michael Tomasson
Matthew Walter

The Role of Notch Signaling in the Pathogenesis
of Acute Promyelocytic Leukemia

by

Nicole Renée Grieselhuber

A dissertation presented to the
Graduate School of Arts and Sciences
of Washington University in
partial fulfillment of the
requirements for the degree
of Doctor of Philosophy

May 2012

Saint Louis, Missouri
copyright by

Nicole Renée Grieselhuber

2012
ABSTRACT OF THE DISSERTATION

The Role of Notch Signaling in the Pathogenesis of Acute Promyelocytic Leukemia

by

Nicole Renée Grieselhuber

Doctor of Philosophy in Biology and Biomedical Sciences
(Immunology)

Washington University in St. Louis, 2012

Professor Timothy Ley, Chairperson

The t(15;17) translocation is found in nearly 98% of acute promyelocytic
leukemia (APL, FAB subtype M3) cases and results in the fusion of the promyelocytic
leukemia (PML) gene with the retinoic acid receptor alpha (RARA) gene. The fusion
product, PML-RARA, encodes a functionally altered transcription factor that is the
initiating event in APL. To better understand the transcriptional changes associated with
APL pathogenesis, we compared the gene expression profiles of APL samples to those of
other acute myeloid leukemia FAB subtypes and of enriched normal human
promyelocytes. We identified a signature of genes that are specifically dysregulated in
APL relative to other AML subtypes and normal promyelocytes. We found that most
dysregulated genes are not direct targets of PML-RARA, but are rather distal events in
pathogenesis. In contrast, the APL signature was enriched in leukemia cells derived from
a mouse model of APL, demonstrating that common leukemogenic pathways exist in
mouse and human cells.
We then observed that human APL overexpresses the Notch ligand Jagged-1
(JAG1) compared to other AML and normal promyelocytes. Unlike many APL signature
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genes, overexpression of JAG1 is also found in human APL cell lines and in murine APL.
We hypothesized that Notch signaling, which has known roles in proliferation and
survival, may be important in leukemogenesis. Inhibition of Notch signaling by
pharmacological and genetic approaches resulted in a loss of serial replating by marrow
cells from young non-leukemic mCG-PML-RARA animals. In contrast, colony
formation by wildtype marrow is unaffected by Notch inhibition, suggesting that PML-
RARA expressing cells are uniquely dependent upon Notch signaling for increased self
renewal. Growth of primary murine APL cells in vitro was variably reduced by
pharmacological inhibition of Notch signaling (6/9 samples), demonstrating that while
Notch signaling is required for early events in leukemogenesis, in some cases it is
dispensible for the fully transformed tumor. However, inhibition of Notch signaling in
four tumor samples tested did not result in reduced tumor burdens in vivo. In conclusion,
we have demonstrated a previously unappreciated role for the Notch signaling pathway in
the development of acute promyelocytic leukemia.

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ACKNOWLEDGEMENTS

During the past 5 years, I have had the privilege of undertaking graduate studies
at the Washington University School of Medicine. I have received much help along the
way, and hope to convey my appreciation in these few pages. My first thanks go to my
advisor, Dr. Timothy Ley. As a mentor, Dr. Ley has created a collegial and intellectually
stimulating environment in which science is done with rigor, and ideas, information, and
expertise are freely shared amongst colleagues. I am especially appreciative of the
opportunity to forge a new path of research within the Ley laboratory; it was not always
easy but I have learned much from it. The bar has been set very high indeed in looking
for post-doctoral research opportunities and perhaps in establishing my own research
laboratory someday. I am also grateful to have had a committee composed of talented
and wise scientists to guide me. Dr. Daniel Link deserves special recognition for
agreeing to serve as the chair of my committee. Like Dr. Ley, he is a wonderful role
model of a physician scientist, and I have sincerely enjoyed our discussions. Drs. Jeffry
Milbrandt, Barry Sleckman, Michael Tomasson, and Matthew Walter have also provided
excellent advice during my journey. I additionally want to thank Dr. James Hsieh, who
was a valued member of my committee until his recent departure from the university; I
wish him all the best when he begins his new appointment at the Memorial Sloan-
Kettering Cancer Center.
The Ley laboratory is composed of many talented individuals with whom I have
had the pleasure of working during years in graduate school. In particular, I thank John
Welch, Lukas Wartman and Sheng Cai for helpful discussions and Jackie Payton for her
invaluable expertise in data analysis. Additional thanks go to Mieke Hoock, Dan George
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and Nick Protopsaltis for their excellent animal husbandry assistance and Erin Wehmeyer
for technical assistance. The core facilities of the Siteman Cancer Center, including the
High Speed Cell Sorter Core, Bioinformatics Core, Laboratory of Clinical Genomics,
Molecular Imaging Core and Tissue Procurement Core, which were instrumental in
carrying out my research, and I thank their directors and staff for their skill and
dedication. At various times during my graduate school career, the National Institutes of
Health, the Barnes-Jewish Hospital Foundation and the Medical Scientist Training
Program provided financial support for either my stipend or research endeavors.
I also thank everyone in the section of Stem Cell Biology for making the sixth
floor of the Southwest Tower a warm and friendly place in which to work. I will
especially miss Adam Greenbaum, Kilannin Krysiak, Ghada Kunter and Maria Trissal,
and have fond memories of several people whose graduations preceded mine, including
Jennifer Cain, Kyle Eash, David Grenda and Julie O’Neal. I appreciate the Washington
University Medical Scientist Training Program for skillfully handling all the details
inherent in pursuing two degrees, leaving students free to concentrate on their studies.
Finally, I must acknowledge a group of people who are, and will remain,
anonymous to me, but without whom none of my research would be possible: the AML
patients of Washington University who agreed to participate in our tumor banking
program. On what was surely one of the worst days of their lives, these patients and their
families decided to contribute to science, and for that I offer my sincerest thanks and
gratitude.
On a personal level, I have made many friends and happy memories during my
years at Washington University. My classmates are some of the brightest, most hard-
v
working and kindest individuals I have met, and I look forward to seeing what paths we
all take in the coming years. And although it is perhaps not traditional, I thank my 4-
legged friends Missy, Pixie, Belle, Nora and Heidi, for providing the quiet equine and
feline companionship that kept me sane and balanced. Outside of my work in the
laboratory, nothing has taught me more about persistence, flexibility and patience than
my feisty little chestnut mare Pixie. I am definitely a better scientist and a better person
for being so challenged.
My path through graduate school would have been much more difficult without
my family to cheer me on. I thank my sisters, Leslie and Teresa, and my aunt, Doris
Emich, for their love, laughter and support over the years, and am sincerely glad they can
join me in this momentous occasion. Although age and poor health prevent my
grandparents, Otto and Esther Emich, from making the journey to St. Louis to see my
thesis defense, I know that they are with me in my heart, and in the lessons they have
taught me. I also wish to thank them for having the foresight to buy me savings bonds
for my birthdays when I was young and probably would have rather received another toy.
All of my various proposals, grants, updates, data analyses and presentations over these
past 5 years have been crafted on the computer I bought with those “boring” savings
bonds!
Finally, my greatest thanks go to my parents, Rene and Caroline, who have been
my biggest cheerleaders. I thank my father for introducing me to science with PBS
documentaries, for his steadfast belief in me and for his dedicated care of my beloved
first horse Missy in her retirement. I thank my mother for her patience and
encouragement when I had moments of doubt, for her sensible advice and for teaching
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me to cook (despite my initial lack of interest), which became not just a means of
survival but a stress relieving hobby. Mom and Dad, I offer you my heartfelt
appreciation for your love and support and dedicate this thesis to you.
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TABLE OF CONTENTS

Abstract of the Dissertation ii

Acknowledgements iv

List of Figures xiii

List of Tables xv

Chapter 1 – Introduction 2

1.1. Acute Myeloid Lekemia and Acute Promyelocytic Leukemia 3
1.2. Identification of the PML-RARA fusion protein 3
1.3. Treatment of APL with all trans-retinoic acid (ATRA) 5
1.4. Mouse models of APL 6
1.5. Cellular effects of PML-RARA expression 9
1.6. Normal RARA functions 11
1.6.1. Protein structure of RARA 11
1.6.2. RARA and hematopoeisis 13
1.7. Normal PML functions 14
1.8. DNA binding properties of PML-RARA 16
1.8.1. PML-RARA as a dominant negative RARA 16
1.8.2. PML-RARA specific consensus sites 18
1.9. Protein-protein interactions of PML-RARA 21
1.9.1. Interactions which Produce gene repression 21
1.9.2. Interactions which produce gene activation 23
1.9.3. Interactions with transcription factors 24
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