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.
RB1 loss-of-function genomic alterations confer resistance to CDK4/6 inhibitors (CDK4/6i) and are enriched post treatment of CDK4/6i in estrogen receptor-positive (ER+) metastatic breast cancer. ER+/Rb-deficient breast cancer is a rising patient population in need of novel therapeutic strategies. Herein, we used a genome-wide CRISPR screen and identified protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in this refractory breast cancer subtype. sgRNA-induced depletion of PRMT5 arrested growth of MCF-7 and T47D RB1 knockout (RBKO) cells. PRMT5 catalyzes symmetric dimethylation of arginine (SDMA). In RBKO cells carrying doxycycline-inducible shRNA targeting the 3’UTR of PRMT5, rescue with wild-type but not an enzymatically dead mutant of PRMT5 restored cell growth, supporting that PRMT5 methyltrasferase activity is essential for growth of these cells. Gene set enrichment analysis (GSEA) of RNA-seq data revealed significant downregulation of cell cycle-related Hallmark gene signatures in RBKO cells treated with PRMT5 siRNA versus control siRNA. Both gene silencing and pharmacological blockade of PRMT5 with the small molecule inhibitor pemrametostat impeded G1-to-S cell cycle progression in MCF-7 and T47D RBKO cells and in lung, prostate, and triple-negative breast cancer cells with natural RB1 mutations or deletions, suggesting that PRMT5 inhibition can block the G1-to-S transition even in the absence of Rb. To identify the protein interactome of PRMT5 and the mechanism by which it promotes cell cycle progression in Rb-deficient cells, we performed proteomics analysis of Co-IP mass spectrometry and an SDMA post-translational modification scan and pinpointed FUS (fused in sarcoma) as a putative downstream effector of PRMT5. FUS is known to regulate RNA polymerase II (Pol II)-mediated transcription. Inhibition of PRMT5 with pemrametostat significantly reduced SDMA levels on FUS and dissociated FUS from Pol II as evidenced by FUS Co-IP and immunoblot analysis. ChIP-seq analysis revealed that treatment of RBKO cells with pemrametostat derepressed phosphorylation of Ser2 in the C-terminus of Pol II at transcription start sites (TSS) of genes involved in cell cycle progression. In accordance with the abnormal accumulation of pSer2 Pol II at TSS, pemrametostat treatment also resulted in an increased Pol II pausing index and an enrichment of intron retention splicing variants. Finally, therapeutic inhibition of PRMT5 with pemrametostat synergized with fulvestrant (a selective ER degrader) against growth of ER+/Rb-deficient breast cancer cell line- and patient-derived xenografts in mice, suggesting this combination as a novel therapeutic strategy for ER+/Rb-deficient metastatic breast cancers. Citation Format: Chang-Ching Lin, Tsung-Cheng Chang, Yunguan Wang, Yanfeng Zhang, Andrew Lemoff, Yisheng V. Fang, He Zhang, Dan Ye, Isabel Soria-Bretones, Alberto Servetto, Kyung-min Lee, Xuemei Luo, Joseph J. Otto, Hiroaki Akamatsu, David W. Cescon, Lin Xu, Yang Xie, Joshua T. Mendell, Ariella B. Hanker, Carlos L. Arteaga. PRMT5 is an actionable target in CDK4/6 inhibitor-resistant ER+/Rb-deficient breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3934.
CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer acquired resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Using a genome-wide CRISPR screen, we identified protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout (RBKO) breast cancer cells. PRMT5 inhibition blocked cell cycle G1-to-S transition independent of RB, thus arresting growth of RBKO cells. Proteomics analysis uncovered fused in sarcoma (FUS) as a downstream effector of PRMT5. Pharmacological inhibition of PRMT5 resulted in dissociation of FUS from RNA polymerase II (Pol II), Ser2 Pol II hyperphosphorylation, and intron retention in genes that promote DNA synthesis. Treatment with the PRMT5i inhibitor pemrametostat and fulvestrant synergistically inhibited growth of ER+/RB-deficient patient-derived xenografts, suggesting dual ER and PRMT5 blockade as a novel therapeutic strategy to treat ER+/RB-deficient breast cancer.
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