Heat Shock Factor 1 Regulation of Multiple Myeloma Pathogenesis Open Access

Shah, Shardule (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/rx913q62j?locale=en
Published

Abstract

Abstract

Heat Shock Factor 1 Regulation of Multiple Myeloma Pathogenesis

By

Shardule Pankajkumar Shah

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.

Dissertation queries are welcomed and can be e-mailed to:

Shardule P. Shah, shardule@gmail.com or Lawrence H. Boise, lboise@emory.edu

Table of Contents

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

TABLE OF FIGURES AND TABLES

CHAPTER 1

Table 1: Human Myeloma Cell Lines 23

Table 2: HSF1 Kinases, Their Targets, and Functional Consequences 24

Table 3: HSF1 Inhibitors 25

Figure 1A: HSF1 Post-Translational Modifications 26

Figure 1B: HSF1 Activation Lifecycle 27

Figure 2: Inhibitors of the HSF1-dependent Heat Shock Response 28

CHAPTER 2

Table 1: Patient Sample Clinical Diagnostics 52

Figure 1: Bortezomib Induces HSP Expression in Multiple Myeloma Cells, and

HSF1 Silencing Sensitizes Multiple Myeloma Cells to Bortezomib

Treatment 53

Figure 2: In Combination with Bortezomib Treatment, HSF1 Silencing is More

Effective than HSP Silencing at HSR Downregulation 55

Figure 3: HSF1 is Phosphorylated upon Bortezomib Treatment in Multiple

Myeloma Cells 57

Figure 4: Phosphoproteomics Reveals that HSF1 Serine 326 is a Bortezomib-

Inducible Phosphorylation Site and Serine 303 is a Constitutive

Phosphorylation Site 58

Figure 5: Phospho-specific Antibodies Confirm that HSF1 Serine 326 is a

Bortezomib-Inducible Phosphorylation Site and Serine 303 is a

Constitutive Phosphorylation Site 59

Supplementary Table 1 64

Supplementary Figure 1: HSP or HSF1 Silencing Leads to Robust Knockdown

48h After Transfection 65

CHAPTER 3

Figure 1: Schematic of HSF1 Overexpressors 71

Figure 2: HSF1 Overexpression Sensitizes Myeloma Cells to Bortezomib

Treatment 72

Figure 3: HSF1 Overexpression Inhibits the Bortezomib-inducible HSR 73

Figure 4: HSF1 Overexpression Falsely Activates the HSR and Inhibits the

Bortezomib-inducible HSR 74

CHAPTER 4

Figure 1A: HSF1 Serine 326 is Phosphorylated in the Cytoplasm 83

Figure 1B: Human Kinome Phosphoprotein Microarray Identifies BZ-induced

Targets 83

Figure 2A: TG02 Inhibits Proteasome-Inhibitor Induced HSF1 Serine 326

Phosphorylation and Proteasome Inhibitor-Induced HSR in Myeloma

Cells 84

Figure 2B: TG02 and Proteasome Inhibitor Combination Leads to an Additive

Effect on Apoptosis in MM.1s Cells 84

Figure 2C: TG02 Inhibits Bortezomib-induced HSF1 Serine 326 Phosphorylation

and Bortezomib-induced HSR in H929 Cells and HSF1 Serine 326

Phosphorylation in U266 Cells 84

Figure 2D: TG02 and Bortezomib Combination Leads to an Additive

Effect on Apoptosis in H929 Cells but not U266 Cells 84

Figure 2E: TG02 Inhibits Carfilzomib-Induced HSF1 Serine 326 Phosphorylation

and Bortezomib-Induced HSR in Patient Samples 84

Figure 2F: CDK9 is not Responsible for Bortezomib-Induced Serine 326

Phosphorylation and Its Silencing does not Sensitize Cells to

Bortezomib-Induced Apoptosis 84

Supplemental Table 86

CHAPTER 5

Figure 1: Proteasome Inhibition Skews HSF1-Dependent HSP Upregulation

Toward Large (greater or equal to 40 kDa) HSPs 101

Figure 2: HSF1 Inhibition Can Lead to Dual Inhibition of Glucose

Metabolism 102

Figure 3: HSF1 Phospho-serine 326 Staining is Observed in a Patient Sample

Both Pre- and Post-Oprozomib Treatment 103

Figure 4: A Proposed Schematic for HSF1 Biomarker Studies to Predict

Proteasome Inhibitor Response 104

Figure 5: HSF1 Serine 326 Phosphorylation Occurs in the Cytosol Followed by

HSF1 Nuclear Translocation 105

Figure 6: The Threshold for PI-induced HSF1 Serine 326 Phosphorylation is

Lower than that of HSP Upregulation 106

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files