Reprogramming the Immune Phenotype of Rb-deficient Prostate Cancer Cells via BET Inhibition Open Access

Abstract

While prostate cancer has been shown to be amenable to some immunotherapeutic strategies, it has remained largely unresponsive to immune checkpoint blockade that has revolutionized the treatment of many other malignancies. A potential factor contributing to this lack of response is the absence of a T-cell gene signature in most prostate cancer patients, which can predict responsiveness to immunotherapies. We’ve identified that these immunologically ‘cold’, non-inflamed tumors are enriched for Rb loss, suggesting that targeting the consequences of Rb loss may be a rational approach for promoting an immunologically hot tumor microenvironment and enhanced responsiveness to checkpoint blockade. BET inhibitors target the BET family of chromatin readers such as BRD4, which acts in the Rb pathway to stimulate E2F-mediated transcriptional activity in the absence of Rb. In this study we use BI894999, a novel, clinically relevant BET inhibitor to target Rb-deficient prostate cancer. We hypothesize that Rb-deficiency in prostate cancer is associated with an immunologically cold tumor microenvironment, and targeting this pathway with BET inhibition will convert immunologically cold tumors into hot tumors. We found that Rb-deficient tumor cells have increased susceptibility to BETi-mediated cell death compared with wild-type cells. Examination of checkpoint ligand expression indicated that while Rb loss led to increased PD-L1 expression, BET inhibition reduced this expression in a dose-dependent fashion. There were also noticeable changes in chemokine expression following Rb loss and BETi treatment, indicating that BETi is capable of altering the immune phenotype. These data lend support to the idea that Rb loss is a potential biomarker of an immunologically cold tumor microenvironment, while also potentially identifying the use of BETi in promoting an immunologically hot phenotype that may enhance efficacy of checkpoint blockade in patients with Rb-deficient malignancies. 

Table of Contents

Introduction

            Overview of prostate cancer………………………….………………………….………….........……………….……….1

            Effectiveness of checkpoint blockade treatment on prostate cancer………………...................……………..2

            Rb loss as a potential biomarker indicating sensitivity to BET inhibition……………................……………4

            Significance of epigenetic mechanisms as a therapeutic target in prostate cancer…….....................……5

            BET proteins: a viable epigenetic target………………………….………………………….……………..........…….6

            BET inhibition………………………….………………………….………………………….……………………..…………8

            Objectives of this study: targeting the Rb pathway via BET inhibition……………………….................…..10

Methods

            Cell culture………………………….………………………….………………………….………………………………......11

            Generation of CRISPR knockouts and transfection of E. coli stocks…………………………....................….12

            E. coli plasmid purification………………………….………………………….………………………......….………….13

            Transfection of Myc-CaP cells………………………….………………………….……………………......…………...14

            Fluorescence-activated cell sorting (FACS) and expansion of clonal lines………………...............…..…...14

            In vitrotreatment of BI894999 or JQ-1 to Myc-CaP cells………………………….……………..............……....15

            In vivotreatment of BI894999 or JQ-1 to FVB mice……………………………………………….............……..…15

            Analysis of tumor-infiltrating lymphocytes/splenocytes via flow cytometry………….…….....................17

            Myc-Cap protein purification and bicinchoninc assay (BCA)………………………….…...................……....19

            Western blot………………………….………………………….………………………….………………….…..………...19

            Chemokine array………………………….………………………….………………………….……………..…..………..20

            Flow cytometry………………………….………………………….………………………….……………….…..………..21

            MTT cell proliferation assay………………………….………………………….………………………….……....…...23

            RNA purification………………………….………………………….………………………………………..………….…23

            qRT-PCR………………………….………………………….………………………….………………………….……….…24

Results

            Generation of Rb-deficient prostate cancer cell lines………………………….………………............………...25

            BET inhibition can pharmacologically inhibit BET function in vitro…………………………............……....25

            Rb-deficient Myc-Cap cells have increased susceptibility to BETi-mediated cell lysis……...................26

            Rb-deficiency increases checkpoint ligand expression that can be modulated by BETi….....................26

            BETi alters chemokine expression………………………….………………………….………………………..…......28

            BETi increases immune infiltration into the tumor microenvironment……………………................……28

            Generation of Rb-deficient MC38 and CDKN2A-deficient KPC cell lines…………………..……...............29

Discussion

            Rb deficiency promotes an immunosuppressive phenotype in Myc-CaP cells……..................……….…30

            BETi prostate cancer tumor cell intrinsic effects……..........…………………….…………………………….….31

            BETi appears to reprogram Myc-CaP cells through alterations of checkpoint ligand expression…...…32

            BETi alters chemokine expression of Myc-CaP cells………………………….………………..........…………...33

            BETi shapes the tumor immune landscape in vivo……………….……………………...........……..……………34

            Rational for combinatorial strategies with BETi in mCRPC………………………..........………….………….35

            Identification of malignancies that are susceptible to BETi………………………….............……...……..…37

            Conclusion………………………….………………………….………………………….…………………………..……..38

References…………….……………………………….……………………………….……………………………….………..…..40

Figures

Figure 1…………….……………………….……………………………….……………………………….………………47

Figure 2…………….……………………………….……………………………….……………………………….………48

Figure 3…………….……………………………….……………………………….……………………………….……….49

Figure 4…………….……………………………….……………………………….……………………………….……….50

Figure 5…………….……………………………….……………………………….……………………………….……….51

Figure 6…………….……………………………….……………………………….……………………………….……….52

Figure 7…………….……………………………….……………………………….……………………………….……….53

Figure 8…………….……………………………….……………………………….……………………………….……….55

Figure 9…………….……………………………….……………………………….………………………………………..56

Table 1…………….……………………………….……………………………….…………………………………………57

Table 2…………….……………………………….……………………………….…………………………………………57

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Biology, Cell Health Sciences, Oncology
Keyword	cytotoxicity BET chemokine cancer Rb prostate checkpoint
Committee Chair / Thesis Advisor	Brian Olson, Emory University
Committee Members	Sarah Fankhauser, Emory University Gregory Lesinski, Emory University Gregg Orloff, Emory University

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