Genome-Wide RNAi Screens for Novel Regulators of Acute Myeloid Leukemia

175 Pages·2017·12.88 MB·English

Genome-Wide RNAi Screens for Novel Regulators
of Acute Myeloid Leukemia
Citation
Li, Hubo. 2015. Genome-Wide RNAi Screens for Novel Regulators of Acute Myeloid Leukemia.
Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
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http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226105
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Genome-wide RNAi screens for novel regulators of
acute myeloid leukemia

A dissertation presented
by
Hubo Li

to
The Division of Medical Sciences
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
in the subject of
Biological Chemistry and Molecular Pharmacology

Harvard University
Cambridge, Massachusetts

December 2014
© 2014 – Hubo Li
All rights reserved
Dissertation advisor: Dr. David Pellman Hubo Li

Genome-wide RNAi screens for novel regulators of acute myeloid leukemia

Abstract
Acute myeloid leukemia (AML) is a heterogeneous disease with complex molecular
mechanisms. Recent advent of genomic technologies, such as copy number profiling, whole
genome sequencing, and gene expression profiling has accumulated a plethora of large-scale data
in AML cell lines and patient samples. However, the functional relevance of most genes
identified by these methods has yet to be determined. To systematically characterize the genetic
requirement in AML, we conducted genome-wide shRNA screens in 17 AML cell lines in
parallel with 199 cell lines of other cancer types. We identified over 150 genes that were
required for proliferation specifically by AML, but not other cancer cell lines. We further
interrogated the requirements of primary screen hits in vivo with a secondary screen in a
xenotransplantation model driven by the MLL-AF9 oncogenic fusion. Integrating both of the
RNAi screens and additional gene expression data, we identified transcription factor ZEB2 as a
top candidate for regulating AML proliferation. In human AML cells, ZEB2 inhibition impairs
proliferation and promotes granulocytic differentiation. Mechanistically, we showed that ZEB2
interacts with the CtBP co-repressor complex, and transcriptionally represses genes involved in
cell adhesion and migration. ZEB2’s relevance in AML is further demonstrated by its
overexpression in MLL-rearranged AML, and by the epigenetic silencing of its negative
regulators, miR-200 family microRNAs, in AML. Our results extend the role of ZEB2 beyond
regulating epithelial-mesenchymal transition, and establish ZEB2 as a novel regulator of AML
proliferation and differentiation.
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MicroRNA-like off-target effect is a major caveat of RNAi screens, which often leads to
false positive discoveries. However, systematic analysis of off-target effects in large-scale RNAi
screen data can also lead to the discovery of microRNAs with functional relevance. By analyzing
the off-target effects in our AML screen, we identified several microRNAs as candidate
suppressors for AML proliferation. We show that miR-105, miR-140, miR-501, and miR-532 are
novel regulators of the myeloid oncogene MYB. In particular, miR-105 inhibits AML cell growth
and miR-532 is associated with myeloid differentiation. The combination of the ZEB2 and
microRNA work emphasizes the power of RNAi screens in the exploration of novel cancer
regulators.

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Table of Contents
Title page ………………………………………………………………………………………….i
Copyright page…………………………………………………………………………………….ii
Abstract…………………………………………………………………………………………...iii
Table of contents…………………………………………………………………………………..v
List of figures ………………………………………………………………………………….…vi
List of tables…………………………………………………………………………………….viii
Acknowledgments………………………………………………………………………………..ix
Chapter 1: Introduction……………………………………………………………………………1
Chapter 2: shRNA screens identify the EMT regulator ZEB2 as a novel dependency in acute
myeloid leukemia…………………………………………………………………………….…..18
Chapter 3: Off-target analysis of shRNA screens identifies novel miRNAs regulating MYB and
NRAS…………………………………………………………………………………………….85
Chapter 4: The role of polysomy 21 in DNA repair in isogenic cell lines………………..……115
Chapter 5: Conclusions and future directions…………………………………………………..135
References………………………………………………………………………………………144

v
List of figures
Figure 2-1. shRNA screen identifies genes uniquely required by AML cell lines for 23
proliferation.
Figure 2-2. Top screen hits are required by a broad set of AML cell lines for 27
proliferation.
Figure 2-3. Schematic representation of the in vivo secondary screen. 28
Figure 2-4. The secondary screen has high sensitivity and reproducibility. 30
Figure 2-5. Positive control shRNAs are significantly more depleted than negative 31
control shRNAs in the secondary screen.
Figure 2-6. ZEB2 is a top hit of the shRNA screen. 34
Figure 2-7. ZEB2 knockdown inhibits growth of AML cell lines. 36
Figure 2-8. ZEB2 shRNA inhibits growth of AML cells more than its seed control. 38
Figure 2-9. Partial knockout of ZEB2 by CRISPR-Cas9 system impairs AML cell 40
proliferation.
Figure 2-10. Zeb2 is required by mouse AML cells. 42
Figure 2-11. Partial knockout of ZEB2 by CRISPR-Cas9 impairs mouse AML cell. 43
Figure 2-12. ZEB2 inhibition does not significantly alter cell cycle. 45
Figure 2-13. ZEB2 knockdown leads to CD11b upregulation and CD14 dysregulation. 45
Figure 2-14. ZEB2 knockdown leads to CD11b upregulation at the transcriptional level. 46
Figure 2-15. ZEB2 knockdown induces morphological differentiation in HL-60 cells. 46
Figure 2-16. ZEB2 regulates common transcriptional targets between different AML cell 48
lines.
Figure 2-17. ZEB2 knockdown induces granulocytic differentiation transcriptional 49
program.
Figure 2-18. Endogenous ZEB2 interacts with key components of the CtBP complex in 51
AML cells.
Figure 2-19. ZEB2 is overexpressed in MLL rearranged AML. 53
vi
Figure 2-20. miR-200 family miRNAs have low expression in 56
AML cell lines and patient samples.
Figure 2-21. miR-200 family miRNAs are repressed in AML by promoter methylation. 57
59
Figure 2-22. miR-200 negative regulate AML proliferation.
Figure 2-23. ZEB2 transcriptionally repress genes involved in cell adhesion and 62
migration.
Figure 2-24. ZEB2 binding motif is enriched in the promoters of genes upregulated by 64
ZEB2 knockdown.
Figure 2-25. shRNA screen and gene expression profile suggest different roles between 67
ZEB1 and ZEB2 in AML.
Figure 2-26. ZEB2 represses ZEB1 expression in AML. 68
Figure 2-27. ZEB2 dependency and expression in leukemia and non-leukemia cancer 73
cell lines.
Figure 3-1. Analysis of shRNA off-target effects in AML screens by GESS.  92
Figure 3-2. Analysis of shRNA off-target effects in AML screens by miRkat. 93
Figure 3-3. Validation of MYB-targeting miRNAs. 97
Figure 3-4. miR-105 inhibits AML cell proliferation. 98
Figure 3-5. miR-532 regulates MYB during myeloid differentiation. 101
Figure 3-6. Screen for mutant NRAS dependency in AML is dominated by off-target 104
effect.
Figure 4-1. Verification of trisomy and tetrasomy 21 RPE1 cells. 122
Figure 4-2. Polysomy 21 cells have elevated ROS but normal sensitivity to DNA- 124
damaging agents.
Figure 4-3. Repair of I-SceI induced single DSB by HDR and imprecise NHEJ in 126
trisomy 21 cells.
Figure 4-4. Repair of RAG induced DSBs in trisomy 21 cells. 128

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List of tables
Table 1-1. Functional classification of recurrent genetic alterations in AML 7
Table 2-1. 17 AML cell lines used in the shRNA screen 22
Table 2-2. Top hits of AML screen 26
Table 2-3. 24 hits from secondary screen 33
Table 2-4. shRNA sequences 76
Table 2-5. sgRNA sequences 77
Table 2-6. qPCR primers 80
Table 3-1. Representative miRNAs identified by miRkat in AML screen 95
Table 4-1. Sensitivity of trisomy 21 cells to DNA-damaging agents 119
Table 4-2. Primer sequences 134

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Acknowledgements
First and foremost, I would like to thank my thesis advisor Dr. David Pellman. In my first
year of graduate school, I had a discussion about rotation lab choices with my friend Xiaolei Su,
who was then a third year graduate student in David’s lab. Xiaolei’s advice for choosing a lab
was “the most important factor for getting through graduate school is to have a supportive
mentor”. Coincidentally or not, I joined David’s lab a few months after the discussion, as I knew
he was the supportive mentor I was looking for. Looking back to the past five years, it was
indeed David’s countless support that helped me to get through the difficulties and challenges I
encountered in research. David was incredibly patient and encouraging to me. I came to graduate
school with little research experience, and it took me a long time to learn the basics about
research. My project did not progress much in the first two years and I was sometimes frustrated.
David told me numerous times that he believes I am a talented student with potential, and helped
me think about alternative approaches to overcome the difficulties in my project. He also offered
me tremendous freedom for pursuing the projects I am interested in. In my fourth year, I became
interested in the AML project, but was not sure whether I could work on it given it did not align
with the overall research goals in David’s lab. I still remember what David told me: “Go ahead
and work on this project if you are interested in it, I just want you to be successful.” One of the
many memorable moments was that when I proposed to collaborate with Dr. Benjamin Ebert’s
lab during a meeting, David was fully supportive and immediately picked up the phone to call
Ben to set up the collaboration.
Besides his support and guidance over the years, David is also a role model for me as a
great scientist. The research topics in the lab are very diverse, and I was always impressed by the
breadth of David’s knowledge and the depth of his insights in multiple different areas. What
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