Polycomb-Like 2 (Mtf2/Pcl2) Mediated Epigenetic Regulation of Hematopoiesis and Refractory Leukemia

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Polycomb-Like 2 (Mtf2/Pcl2) Mediated Epigenetic Regulation of Hematopoiesis and Refractory Leukemia Polycomb-like 2 (Mtf2/Pcl2) mediated epigenetic regulation of hematopoiesis and refractory leukemia Harinad Maganti This thesis is submitted to the Faculty of Graduate and Postdoctoral Studies as a partial fulfillment of the Ph.D. program in Microbiology and Immunology. Department of Biochemistry Faculty of Medicine University of Ottawa Ottawa, Ontario Canada © Harinad Maganti, Ottawa, Canada, 2018 Abstract The Polycomb Repressive Complex 2 (PRC2) epigenetically regulates gene expression by methylating lysine 27 on histone 3 (H3K27me3). While the role of PRC2 core members during hematopoiesis has been elucidated, the role of PRC2 accessory protein, Mtf2, has not been well characterized outside of mouse embryonic stem cells. To investigate the role of Mtf2 in vivo, we created a gene-targeted knockout mouse model. Using this model, we discovered that Mtf2 was a critical regulator of hematopoiesis and its loss within the hematopoietic cells leads to loss of global H3K27me3 levels at the transcriptional start sites (TSS) therefore leading to the overexpression of multiple signalling networks. These findings presented in the first part of my thesis place Mtf2 as a critical regulator of hematopoiesis and expand the role of Mtf2 beyond a canonical accessory PcG protein. While our murine studies revealed that the loss of Mtf2 did not cause leukemia in mice, our studies of MTF2 in human cells demonstrated that MTF2 deficiency within human Hematopoietic Stem and Progenitor Cells (HSPCs) causes a myelo-proliferative phenotype that is reminiscent of pre-leukemia. Furthermore, when we screened MTF2 expression within leukemic stem cell (LSC) enriched CD34+CD38- cells isolated from primary Acute Myeloid Leukemia (AML) patient samples at diagnosis, we observed that MTF2 is miss-regulated in AML and its loss predicted refractory AML. Using MTF2 knockdown (KD) transcriptomic and ChIP-seq data, we drafted MTF2-PRC2 Gene Regulatory Network (GRN) in human HSPCs and LSC enriched cells. Finally, using the MTF2-PRC2 GRN, we uncovered a direct mechanism by which MTF2 regulates chemoresistance in AML and show that targeting this mechanism via MDM2 inhibitors sensitizes refractory AML to standard induction therapy. These findings presented in second part of my thesis ii demonstrate MTF2 as a novel prognostic marker for refractory AML and provide a novel therapy that helps target MTF2 deficient refractory AML. iii List of Tables Table S1. Genes identified as losing H3K27me3 in Mtf2-/- erythroblasts Table S2. Genes contained in the Mtf2-PRC2 erythroid-specific gene regulatory network Table S3. Genes contained in the Mtf2-PRC2 HSPC-specific gene regulatory network Table S4. Primers used for genotyping, qPCR and ChIP-qPCR analysis Table S5. Clinical characteristics of responders Vs non-responders within local AML cohort Table S6. Clinical characteristics of MTF2 Basal Vs MTF2 Low patients within local AML cohort Table S7. Clinical data of patients belonging to local cohort Table S8. Clinical characteristics of TCGA AML cohort Table S9. Multivariate Overall Survival Analysis using TCGA P53 WT AML patients Table S10. Multivariate Disease Free Survival Analysis using TCGA P53WT AML patients Table S11. Primers used for qPCR and ChIP-qPCR iv List of Figures Figure 1.1. The Polycomb Repressive Complex 2 (PRC2) consists of several core and accessory proteins. Figure 1.2. PRC2 mediated recruitment of PRC1. Figure 1.3. Mtf2 is alternatively spliced into 3 isoforms: Mtf2A, Mtf2B and Mtf2C. Figure 1.4. Pcl2/Mtf2 regulated pluripotency gene regulatory network in mESCs. Figure 1.5. Hematopoietic lineage determination in mice is different to the one observed in the human hematopoietic system. Figure 1.6. MTF2 loss within human HSPCs causes a myeloproliferative phenotype. Figure 2.1. Mtf2-/- mice die at e15.5 due to severe anemia. Figure 2.2. Mtf2 is required for normal erythroid differentiation. Figure 2.3. Mtf2 is required for HSC self-renewal. Figure 2.4. Mtf2 is required for promoter-proximal histone trimethylation of lysine 27. Figure 2.5. Mtf2 regulates Wnt-dependent erythroid maturation. Figure 2.6. Mtf2 regulates multiple signaling nodes in HSPCs. Figure 2.7. Hippo pathway regulation by Taz inhibitors Dasatinib and Pazopanib rescues primitive progenitor cell exhaustion caused by loss of Mtf2. Figure S2.1. Mtf2 expression in the mouse hematopoietic system.. v Figure S2.2. Morphology of wild-type and Mtf2-/- fetal liver and peripheral blood erythroblasts. Figure S2.3. Mtf2 regulates core PRC2 protein levels and H3K27me3 in erythroblasts. Figure S2.4. Genes associated with Mtf2 binding in erythroid cells show little overlap with Mtf2 and PRC2 targets in ESCs. Figure S2.5. Wnt pathway inhibition in Mtf2-deficient erythroblasts increases their ability to differentiate. Figure S2.6. Some erythroid master regulators are targets of Mtf2-PRC2 as seen through loss of H3K27me3. Figure S2.7. Wnt pathway inhibition in Mtf2-deficient HSPCs does not affect the number of LT-CIC colonies observed Figure 3.1. MTF2 deficiency correlates with poor response to standard treatment of care. Figure 3.2. MTF2 knockdown in Lin- CD34+ HSPCs or chemoresponsive leukemic cells, but not EZH2 inhibition alone confers resistance to standard treatment of care. Figure 3.3. MTF2 gene regulatory network (GRN) modulates refractory AML via the MDM2/p53 signaling pathway. vi Figure 3.4. MDM2 inhibitors sensitize MTF2-deficient HSPCs and primary refractory AML samples to standard treatment of care. Figure S3.1. MTF2 deficiency correlates with poor prognosis. MTF2 mRNA expression was analyzed within CD34+CD38- LSC enriched cells from the local patient cohort (n=32) at diagnosis and normalized to MTF2 mRNA expression within CD34+CD38- cells isolated from healthy bone marrow aspirates (n=7). Figure S3.2. MTF2 deficiency correlates with poor response to standard treatment of care. Figure S3.3. Restoring MTF2 levels re-establishes H3K27me3 levels within patient AML CD34+CD38- cells. Figure S3.4. MTF2 deficiency promotes an anti-apoptotic effect and resistance to DNA damage post induction treatment. Figure S3.5. Knocking down MTF2 levels in leukemic Lin- CD34+ cells with basal levels of MTF2 [B-AML] alters their chemoresponsivenes. Figure S3.6. Dual inhibition of EZH1 and EZH2 methyltransferase activity confers chemoresistance in responsive primary AML cells. Figure S3.7. DNA damage response (DDR) is hyperactivated in MTF2-deficient HSPCs. Figure S3.8. MDM2 inhibitors restore p53 levels within MTF2-deficient cells. Figure S3.9. Cell cycle progression and proliferation is normalized when p53 levels are restored. vii Figure S3.10. Rescue of MTF2 within MTF2-deficient refractory patient BM aspirates decreases MDM2 and restores p53 levels. Figure S3.11. Combinatory treatment regimen comprised of an MDM2 inhibitor and induction drugs reverses chemoresistance in MTF2-deficient cells. Figure S3.12. Induction therapy and combination therapy kills chemoresponsive AML cells with basal MTF2 levels in vivo. Figure S3.13. Combination therapy kills MTF2-deficient refractory AML cells in vivo. Figure S3.14. Combination therapy treated PDX cells demonstrate multi-lineage engraftment potential and contribute to event free survival in secondary transplants. viii Tables of Contents Abstract List of Tables List of Figures List of Abbreviations Acknowledgements Chapter 1 - Introduction 1 1.1 Epigenetics and stem cell fate decisions 2 1.2 Transcriptional repression and Polycomb group proteins 2 1.3 The Polycomb Repressive Complex 2 (PRC2) 3 1.4 Polycomb accessory proteins 6 1.4.1 Aebp2 6 1.4.2 Jarid2 7 1.4.3 Polycomblike proteins 8 1.4.3.1 Phf1/Pcl1and Phf19/Pcl3 8 1.4.3.2 Pcl2/Mtf2 9 1.5 Pcl2/Mtf2 is a critical regulator of embryonic stem cell fate 10 1.6 Gene Regulatory Networks (GRNs) 11 1.7 The role of Pcl2/Mtf2 In vivo 12 1.8 Polycomb Response Elements mediated targeting of PRC2 12 1.9 Accessory proteins mediated targeting of PRC2 13 1.10 Hematopoiesis 13 ix 1.10.1 Hematopoiesis during development 14 1.10.2 PRC2 mediated cell cycle regulation of hematopoiesis 16 1.10.3 PRC2 hematopoietic mouse model phenotypes 17 1.11 PRC2 and leukemia 18 1.12 Acute Myeloid Leukemia 18 1.13 Differences between mouse and human hematopoietic cell fate decisions 20 1.14 Rationale and overview 23 1.14.1 Overarching hypothesis 24 1.14.2 Specific Aims 24 Chapter 2 - Mtf2 dynamically regulates Wnt and Hippo signaling to control 25 hematopoiesis Author contributions 26 Abstract 28 Introduction 29 Results 31 Mtf2 null mice die in utero from impaired definitive erythropoiesis 31 Mtf2 is required for erythroid maturation in a cell-intrinsic manner 34 Mtf2 is required for maintenance of the HSC pool 37 Mtf2 regulates core PRC2 member protein abundance in the hematopoietic 39 system x Mtf2 regulates promoter-proximal H3K27me3 in erythroblast 40 Mtf2 regulates Wnt-dependent erythroid maturation 43 Wnt inhibition does not affect HSC activity in Mtf2-deficient cells 47 Mtf2 regulates primitive progenitor cell self-renewal via Hippo signaling 48 Discussion 53 Materials and Methods 56 Generation of Mice and Embryonic Analysis 56 Flow Cytometry 56 Capillary Electrophoresis Immunodetection 58 Lentiviral production of Mtf2 shRNA 58 Lentivirus-mediated Mtf2 knockdown of mouse bone marrow cells 59 Colony Forming Assays and LT-CIC analysis 59 RNA-seq and ChIP-seq 60 Bone Marrow Transplants and Analysis 61 Dual knock down experiments 62 Wnt and Hippo inhibition 63 Methyl-ChIP-qPCR 63 Ex-vivo differentiation of erythroblasts 63 Statistics 64 Study Approval 64
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