BMI1 is a novel, preclinical target in fusion-positive rhabdomyosarcoma Open Access

Shields, Cara (Summer 2021)

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Rhabdomyosarcoma (RMS) is a rare, understudied pediatric cancer. There are two main types of RMS: fusion-negative rhabdomyosarcoma (FN-RMS) and fusion-positive rhabdomyosarcoma (FP-RMS). FP-RMS harbors the PAX3-FOXO1 or PAX7-FOXO1 fusion proteins, which is associated with global epigenetic dysregulation. To restore dysfunctional epigenetic signaling in FP-RMS, we investigated B lymphoma Mo-MLV insertion region 1 (BMI1), a druggable chromatin protein member of Polycomb Repressive Complex 1 as a potential target. We discovered that FP-RMS expresses high levels of BMI1 and that inhibition of BMI1 by chemical or genetic methods leads to a dramatic decrease in FP-RMS proliferation and an increase in cell death. We utilized PTC-028, a small molecule inhibitor of BMI1, to study the effects of BMI1 inhibition in FP-RMS. We found that PTC-028 treatment leads to downregulation of DNA replication and halted cell progression to S phase. To understand how BMI1 regulated oncogenic signaling, we next examined Hippo signaling due to its importance in cancer and previous data linking BMI1 to a YAP, a protein involved in the Hippo pathway. Our data show that BMI1 suppresses Hippo pathway activation through repression of LATS1/2 phosphorylation, therefore keeping growth and proliferation genes “on” in FP-RMS. In addition to this, we explored the differences between FP-RMS cell line responses to BMI1 inhibition. We noted similar and contrasting responses in the downstream transcription of Hippo YAP/TAZ/TEAD targets, revealing intertumor heterogeneity in FP-RMS and possibly different mechanisms of LATS1/2 phosphorylation. We then explored other effects of BMI1 inhibition and discovered that PTC-028 treatment results in a downregulation of metabolic pathways and DNA replication, and an increase in p53 signaling. Overall, we find that BMI1 inhibition is an effective therapy in FP-RMS and should be considered a potential translatable target. 

Table of Contents

Table of Contents

1. Introduction 1

1.1 Overview of cancer 2

1.1.1 History of cancer 2

1.1.2 Hallmarks of cancer 3

1.1.3 Adult and pediatric cancer 4

1.1.4 Pediatric soft-tissue sarcomas 5

1.2 Rhabdomyosarcoma 6

1.2.1 Background 6

1.2.2 Fusion-negative and fusion-positive rhabdomyosarcoma 7

1.3 Epigenetic therapies in cancer 8

1.3.1 What is epigenetics? 8

1.3.2 Overview of epigenetic machinery 10

1.3.3 Current epigenetic therapies 12

1.3.4 Efficacy of epigenetic therapies in rhabdomyosarcoma 13

1.4 Polycomb group complexes 15

1.4.1 Polycomb groups 15

1.4.2 Alternative Polycomb roles 17

1.5 BMI1 18

1.5.1 BMI1 function and expression 18

1.5.2 BMI1 is recruited to DNA breaks and regulates repair 19

1.5.3 BMI1 promotes the epithelial to mesenchymal transition 20

1.5.4 BMI1 inhibition as a novel therapeutic 21

1.6 Scope of this dissertation 22

2. Epigenetic regulator BMI1 promotes alveolar rhabdomyosarcoma proliferation and constitutes a novel therapeutic target 28

Abstract 31

2.1 Introduction 32

2.2 Materials & Methods 35

2.2.1 In silico data 35

2.2.2 Cell culture 35

2.2.3 Plasmids, lentiviral preparation, and transduction 35

2.2.4 siRNA transfection 36

2.2.5 Real-Time PCR and western blots 37

2.2.6 Cell growth assays 37

2.2.7 Flow cytometry 38

2.2.8 In vivo xenograft model 38

2.2.9 Immunohistochemistry 39

2.2.10 Statistical analyses 40

2.3 Results 41

2.3.1 BMI1 is highly expressed in rhabdomyosarcoma 41

2.3.2 Genetic knockdown of BMI1 leads to reduced cellular proliferation in ARMS cells 42

2.3.3 Pharmacologic inhibition of BMI1 decreases cell proliferation in vitro 42

2.3.4 Targeting BMI1 decreases cell cycle progression and increases apoptosis in ARMS 44

2.3.5 Single agent PTC-028 treatment causes tumor growth delay in vivo 45

2.3.6 BMI1 negatively influences Hippo signaling 45

2.4 Discussion 49

2.5 Conclusions 52

3. Differential epigenetic effects of BMI1 inhibitor PTC-028 on two fusion-positive rhabdomyosarcoma cell line models 73

Abstract 75

3.1 Introduction 76

3.2 Materials & Methods 78

3.2.1 Cell culture 78

3.2.2 RNA-seq study design 78

3.2.3 Data analyses and graph production 79

3.3 Results 80

3.3.1 BMI1 inhibition influences global gene expression in FP-RMS 80

3.3.2 Gene ontology and pathway analyses reveal downregulation of DNA replication and activation of cellular differentiation during BMI1 inhibition 81

3.3.3 TEAD-regulated gene expression in Rh30 is more sensitive to BMI1 and Hippo pathway inhibition 83

3.3.4 BMI1 inhibition increases the expression of distinct LATS1/2 kinases in Rh28 and Rh30 84

3.4 Discussion 86

4. Discussion and future directions 102

4.1 Summary of findings in Chapters 2 and 3 103

4.2 DNA damage repair 106

4.3 Intersection of BMI1 with metabolic pathways 108

4.4 Invasion and metastasis 109

4.5 Further modeling of PRC1 and BMI1 in FP-RMS 110

4.6 Combining BMI1 inhibition with other translatable targets 112

4.7 Final conclusions and thoughts 114

References 117

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