Abstract 4033: A functional genome-wide screen to identify the ER degradation machinery in ER+ breast cancer cells

Abstract

Abstract Targeted protein degradation (TPD) is a new pharmaceutical strategy to hijack the ubiquitin-proteasome system to degrade target proteins. Many TPD drugs are discovered serendipitously, and we are only beginning to harness their potential. Understanding how TPD drugs co-opt the proteolysis pathway is important, as it could allow molecule re-engineering for TPD of new proteins (ie. neo-substrates). A rising example of TPD drugs are the selective estrogen receptor degraders (SERDs) - small molecule antagonists of estrogen receptor alpha (ER) that target ER protein for degradation. SERDs are the next generation of therapy for metastatic ER+ breast cancer. Currently, fulvestrant is the only FDA approved SERD, but fulvestrant is limited by poor pharmacokinetics and resistance conferred by ER mutations (ie. ER-Y537S). An intense pharmaceutical effort has developed five novel SERDs in phase three clinical trials. These novel SERDs have diverse side chains for inducing ER degradation but share advantages including non-steroidal structure, oral bioavailability, and activity against ER-Y537. Based on the diverse molecular properties of SERDs, I hypothesized they utilize different ubiquitin-proteasome pathway genes (such as E3 ligases) for ER degradation. To identify the machinery for SERD-mediated ER degradation, I designed a functional CRISPR/Cas9 screen, with highlights including a genome-wide gRNA library and measurement of endogenous ER protein in ER+ breast cancer cells. Initially focusing on genes required for fulvestrant-mediated ER degradation, we identified the proteolysis pathway as a top hit (q<5x10-6) including central genes in the ubiquitin-proteasome pathway (UBC, PSMA6, and NEDD8) and the ubiquitin-like SUMO pathway (UBC9 and RWDD3). Ubiquitin E3 ligases were also identified, including FBXO45 and VHL, which have been previously implicated as E3 ligases for ER. These hits were validated using inhibitors of ubiquitin-, NEDD8-, and SUMO-activating enzymes. These results demonstrate that multiple proteolysis mechanisms are sufficient for ER protein degradation. Currently, I am applying this screening approach to novel SERDs (such as elacestrant, amnecestrant, and giredestrant) to identify overlapping and unique mechanisms of degradation for wild-type ER and ER-Y537S. Importantly, understanding the mechanisms of these exciting new drugs will allow anticipation of resistance mechanisms in advanced ER+ breast cancer patients. Citation Format: Zachary Sandusky. A functional genome-wide screen to identify the ER degradation machinery in ER+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4033.