Abstract P5-17-09: A genome-wide CRISPR screen identifies PRMT5 as a novel therapeutic target in ER+/RB1-deficient breast cancer
Authors: Lin Chang-Ching , Chang Tsung-Cheng , Servetto Alberto , Lee Kyung-min , Zhang He , Wang Yunguan , Ye Dan , Chatterjee Sumanta , Sudhan Dhivya R , Akamatsu Hiroaki , Xie Yang , Mendell Joshua T , Hanker Ariella B , Arteaga Carlos L 
Published: March 2022

Abstract
Background: CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with advanced estrogen receptor-positive (ER+) breast cancer. However, this benefit is transient as virtually all these tumors eventually develop drug resistance and recur. Clinical studies have reported an association of RB1 loss-of-function genomic alterations with acquired resistance to CDK4/6i. Given the enrichment of RB1 alterations post CDK4/6i treatment, ER+/RB1-deficient breast cancer will become a rising patient population in need of discovery of novel treatment strategies. In this study, we sought to identify actionable vulnerabilities for this refractory breast cancer subtype using a genome-wide CRISPR screen. Methods: RB1 was knocked out in ER+ MCF-7 and T47D breast cancer cells using CRISPR-Cas9; complete gene knockout was confirmed by PCR-based genotyping, Sanger sequencing, and immunoblot analysis. Isogenic RB1 knockout (RBKO) and wild-type (WT) T47D cells were used for the genome-wide CRISPR screen. MAGeCKFlute was used to identify differentially essential genes in T47D RBKO vs WT cells; Gene Ontology (GO) analysis was used to prioritize hits. MCF-7 and T47D RBKO cells were used for validating and studying the function of the identified genes. Results: Knockout of RB1 in MCF-7 and T47D cells increased IC50 of abemaciclib, palbociclib, and ribociclib 10-200 fold compared to WT cells. RNA-seq analysis showed upregulation of E2F target gene expression in RBKO vs WT cells. The CRISPR screen revealed that CCND1 and CDK4 lost their essentiality in T47D RBKO cells, suggesting that loss of RB1 uncouples the CDK4/Cyclin D1 complex from E2F-regulated transcription. GO analysis of the top 50 differentially essential hits of RBKO vs WT cells showed an enrichment of protein arginine methyltransferase activity, primarily PRMT5, which post-translationally mono-methylates and symmetrically di-methylates protein arginine. In agreement with this finding, PRMT5 knockout by three individual sgRNAs resulted in more potent growth inhibition of MCF-7 and T47D RBKO cells than WT cells. Further, transfection of PRMT5 siRNA or treatment with the PRMT5 small molecule inhibitor GSK3326595 - currently in clinical trials - resulted in G1 arrest of MCF-7 and T47D RBKO cells as assayed by propidium iodide staining but did not induce caspase 3/7 or PARP cleavage (apoptosis). RNA-seq of PRMT5 siRNA vs control siRNA in MCF-7 and T47D RBKO cells exhibited significant downregulation of E2F Hallmark gene signature, further suggesting PRMT5 inhibition as a strategy to suppress E2F-regulated gene expression when cells lose Rb. The CRISPR screen also revealed that transcription factors that drive ER signaling, such as FOXA1, GATA3, MYC, SPDEF, and ESR1 (the gene encoding ERα), were commonly essential in both T47D WT and RBKO cells. Estrogen deprivation or treatment with fulvestrant inhibited estrogen responsive element (ERE) luciferase reporter activity, expression of putative E2F target genes, and proliferation of both WT and RBKO cells, suggesting that ER+ cells still rely on ERα irrespective of RB1 status. Treatment of MCF-7 and T47D RBKO cells with fulvestrant and GSK3326595 resulted in more potent growth inhibition than each drug alone, suggesting a novel approach to treat ER+/RB1-deficient breast cancer. We are currently testing the antitumor activity of fulvestrant plus GSK3326595 against RBKO xenografts as well as the requirement of arginine methyltransferase activity associated with PRMT5 for growth of ER+/RB1-deficient breast cancer cells. Conclusion: PRMT5 is essential for proliferation of ER+/RB1-deficient breast cancer cells. Targeting PRMT5 in combination with anti-estrogens is a novel and testable strategy to suppress E2F-regulated cell cycle progression of this CDK4/6 inhibitor-resistant breast cancer subtype.
Citation Format: Chang-Ching Lin, Tsung-Cheng Chang, Alberto Servetto, Kyung-min Lee, He Zhang, Yunguan Wang, Dan Ye, Sumanta Chatterjee, Dhivya R Sudhan, Hiroaki Akamatsu, Yang Xie, Joshua T Mendell, Ariella B Hanker, Carlos L Arteaga. A genome-wide CRISPR screen identifies PRMT5 as a novel therapeutic target in ER+/RB1-deficient breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-09.