Heat Shock Factor 1 Regulation of Multiple Myeloma Pathogenesis Pubblico

Abstract

Multiple myeloma is a plasma cell malignancy with estimated 30,330 new cases and the cause of 12,650 deaths in 2016 in the United States. Proteasome inhibitors have dramatically improved patient outcome but there is no functional cure. The proteasome inhibitors bortezomib and carfilzomib work in part because they exploit the plasma cell backbone of a myeloma cell. Myeloma cells upregulate the heat shock response in order to protect themselves from bortezomib-induced apoptosis. In chapter two, we show that knockdown of the master regulator of the heat shock response, Heat Shock Factor 1 (HSF1), sensitizes myeloma cells to bortezomib-induced apoptosis. HSF1 knockdown results in a greater additive effect on apoptosis than simultaneous knockdown of multiple HSF1-mediated heat shock proteins. We show HSF1 phosphorylation upon bortezomib treatment and that HSF1 serine 326 phosphorylation is an activating post-translational modification and also detail novel HSF1 post-translational modifications. Chapter four details that cell lines stably overexpressing wildtype HSF1 or a serine-to-alanine or serine-to-glutamate mutation at amino acid position 326, all result in downregulation of the bortezomib-induced heat shock response and increased bortezomib-induced apoptosis. Identification of kinases responsible for HSF1 phosphorylation may inform an HSF1 indirect inhibition strategy. In chapter five, we show that the kinase is cytosolic and classify candidate kinases responsible for serine 326 phosphorylation. Also, we show a novel mechanism of action for the multi-kinase inhibitor TG02: inhibition of serine 326 phosphorylation and the proteasome inhibitor-induced heat shock response. We also demonstrate that a TG02 and bortezomib or carfilzomib combination leads to an additive effect on apoptosis in myeloma cells. Our data indicate that the kinase responsible for serine 326 phosphorylation is a cytosolic TG02 target which has likely not yet been elucidated. Our studies show how myeloma cells hijack the HSF1-mediated heat shock response in order to avoid proteasome inhibitor-induced apoptosis. We also demonstrate that inhibition of serine 326 phosphorylation is a novel TG02 mechanism. Ultimately, our work could improve the efficacy of myeloma therapeutic strategies, and can also be broadened to additional malignancies for which proteasome inhibition is a frontline therapy, such as mantle cell lymphoma. Additionally, HSF1 inhibition strategies could inform therapeutic strategies for malignancies which activate HSF1 for apoptosis evasion, such as breast cancer, prostate cancer, and chronic lymphocytic leukemia.

Table of Contents

I. INTRODUCTION 1

A. Abstract 2

B. Introduction 2

C. Proteasome Inhibition 3

D. The Heat Shock Response and Heat Shock Proteins 7

E. Heat Shock Factor 1 9

F. Regulation of HSF1 by Post-translational Modifications 11

G. HSF1 Inhibition in Cancer Treatment 16

H. Conclusions 22

I. Acknowledgments 22

J. Tables and Figures 23

K. Statement of Problem 29

II. HSF1-MEDIATED REGULATION OF BORTEZOMIB-INDUCED

HEAT SHOCK RESPONSE IN MULTIPLE

MYELOMA 32

A. Abstract 33

B. Introduction 34

C. Results 36

D. Discussion 42

E. Materials and Methods 46

F. Acknowledgments 51

G. Tables and Figures 52

H. Supplementary Material 61

III. HSF1 OVEREXPRESSION AND PROTEASOME INHIBITOR

STUDIES IN MULTIPLE MYELOMA 66

A. Introduction 66

B. Hypothesis 66

C. Materials and Methods 66

D. Results 67

E. Discussion 69

F. Figures 71

IV. TG02 REGULATION OF PROTEASOME INHIBITORINDUCED

HSF1 ACTIVATION IN MULTIPLE MYELOMA 75

A. Materials and Methods 80

B. Figures 83

C. Supplemental Table 86

V. DISCUSSION 87

A. Implications from Bortezomib-Induction of Heat Shock Factor 1 Serine

326 Phosphorylation Studies 87

1. Characterization of the MPH 87

2. The Role of the MPH and HSF1 Activation in Combination

Therapy 88

3. Non-HSR HSF1 Functionality in MM 90

4. Non-pS326 HSF1 PI-induced Phosphorylation in MM 91

5. Acetylation Regulation of HSF1 Activation 92

6. pS326 as a MM Biomarker 93

7. Summary 94

B. Implications from TG02 and Proteasome Inhibitor Studies 96

1. The PI-pS326 Kinase is Cytosolic 96

2. TG02 Sensitizes MM Cell Lines and Patient Samples to

Proteasome Inhibition and Inhibits PI-pS326 97

3. Have We Moved Closer to Identifying the PI-pS326 Kinase? 98

4. Early Detection of TG02-Mediated PI-pS326 Inhibition in MM.1s

Cells 100

5. Summary 100

6. Figures 101

VI. REFERENCES 107

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Health Sciences, Immunology Health Sciences, Oncology
Parola chiave	HSP Multiple myeloma Bortezomib Heat Shock Factor 1 Heat Shock Protein HSF1
Committee Chair / Thesis Advisor	Boise, Lawrence, Emory University
Committee Members	Selvaraj, Periasamy, Emory University Iwakoshi, Neal N., Emory University Jacob, Joshy, Emory University Wilkinson, Keith D, Emory University

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