Characterization and therapeutic targeting of SPOP missense mutation-mediated oncogenesis 公开
Doyle, Sean (Summer 2021)
Published
Abstract
SPOP is an adaptor subunit of Cullin3-RING E3 ubiquitin ligase complexes that plays a key role in maintenance of cellular homeostasis through its interactions with other proteins. Missense mutations in SPOP’s substrate binding cleft alter SPOP’s ability to bind to other proteins and frequently drive prostate cancer development. However, our knowledge of the SPOP interactome and our understanding of how SPOP mutations influence SPOP function remain limited. In this dissertation, the characterized SPOP interactome is expanded through SPOP-focused, high-throughput protein-protein interaction (PPI) screens to generate new hypotheses for SPOP-mediated biology. We further evaluate how recurrent, prostate adenocarcinoma-associated missense mutations in SPOP alter SPOP PPIs. Recurrent SPOP missense mutations reduce interaction with several cancer-associated proteins, but recurrent SPOP F133L and F133V mutations are also observed to enhance or induce interaction with another set of proteins, including oncogenic transcription factor c-Jun. This dissertation further characterizes SPOP F133L- and F133V-mutation-induced interactions with c-Jun. Mechanistically, SPOP F133L and F133V mutants bind to c-Jun through the mutated SPOP MATH domain and serve to enhance c-Jun protein stability and transcriptional activity. Overall, these data reveal new differential protein-protein interaction connectivity for SPOP point variants, and suggest unique mechanisms of oncogenesis for recurrent SPOP F133L/V mutants through mutation-induced gain-of-interaction with oncogenic transcription factor c-Jun.
Therapeutic approaches that selectively target SPOP mutation-driven oncogenesis are currently limited. Because recurrent, prostate cancer-associated SPOP missense mutations have been characterized to promote prostate tumorigenesis in part through loss of SPOP interaction with chromatin reader BRD4, restoration of mutant SPOP interactions with BRD4 may represent a therapeutic strategy to reverse SPOP missense mutation-driven tumorigenesis. This dissertation describes the development and pilot implementation of a time-resolved, fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening assay to identify small molecule inducers of SPOP missense mutant F133V interaction with BRD4.
Overall, this dissertation explores novel mechanisms of SPOP mutation-mediated oncogenesis that occur through SPOP PPI dysregulation and describes a strategy to rescue mutant SPOP function through restoration of SPOP PPIs. These studies suggest several novel cellular signaling axes mediated by SPOP and nominate potential therapeutic strategies that may be developed to treat patients with SPOP mutation-driven prostate tumors.
Table of Contents
Chapter 1: Introduction…………………………………………………………………..1
1.1 Overview………………………………………………………………………..….......2
1.2 Cancer………………………………………………………………..………………....2
1.3 Protein-Protein interaction network mapping to identify therapeutic opportunities
for cancer……………….…………………………………………………………..3
1.4 E3 ubiquitin ligases are essential mediators of cell biology that function through
selective protein-protein interactions………...…………………………………….5
1.4.1 Dysregulation of E3 ligase-protein interactions promotes oncogenesis…...…6
1.5 SPOP: An E3 ubiquitin ligase subunit frequently altered in cancer……………………9
1.5.1 SPOP discovery and initial characterization………………………………….9
1.5.2 SPOP structure and determinants of SPOP-protein interactions…….……….10
1.5.3 SPOP mechanistic function and biological roles……………………………..11
1.5.4 Mechanisms of SPOP regulation……………………………………………..13
1.5.5 Mechanisms of SPOP dysregulation…….……………………………………15
1.5.6 SPOP is the most frequently mutated gene in primary prostate
adenocarcinoma………………………………………….…………………17
1.5.7 Current therapeutic strategies targeting SPOP-driven tumorigenesis…...……18
1.6 Dissertation scope………………...…………………………………………………….22
Chapter 2: Analysis of differential wild-type and mutant SPOP protein-protein
interactions reveals SPOP F133L/V mutation-induced gain of interaction that
promotes c-Jun protein stability and transcriptional activity………….………………30
2.1 Introduction…………………………..…………………………………………………31
2.2 Experimental Procedures………….……………………………………………………33
2.3 Results…………………………………………………………………………………..39
2.4 Discussion………………………………………………………………………………45
Chapter 3: Development of an ultra-high-throughput screening assay to identify
small molecule inducers of SPOP F133V-BRD4 protein-protein
interactions...............................................................................................................73
3.1 Introduction……………………………………………………………………………..74
3.2 Experimental Procedures……………….………………………………………………75
3.3 Results…..………………………………………………………………………………80
3.4 Discussion……………...……………………………………………………………….83
Chapter 4: Discussion and future directions…………….………………………………94
4.1 Identification of novel SPOP-binding proteins: functional implications…….…………95
4.2 SPOP point variants feature unique protein-protein interactomes that suggest novel
mechanisms of SPOP-mediated biology, oncogenesis, and therapeutic
vulnerability………………………………………………………..………………101
4.3 Unique structural determinants of SPOP-c-Jun interactions………..………………….105
4.4 SPOP-mediated stabilization of c-Jun as a potential mechanism of SPOP-mediated
oncogenesis……………………………………………………..…………………..108
4.5 Small molecule restorers of SPOP protein-protein interactions as a strategy to
reverse SPOP mutation-driven prostate adenocarcinoma……………….………….111
4.6 Limitations……………………………………………………………………………....112
4.7 Conclusions and future directions………………………………………………………113
References……………………...…………………………………………………………..129
Appendix…………..……………………………………………………………………….159
A1 Patient-derived prostate cancer cell line models for cellular characteristics of
oncogenic transformation driven by SPOP variant……………...………………….160
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