Abstract 975: Functional CRISPR screen towards identifying novel epigenetic co-factors of oncogenic AR-activity

Abstract

Prostate cancer (PCa) is the second leading cause of cancer-related mortality in North American men. In recent years, there has been mounting evidence establishing the centrality of epigenetic mechanisms in PCa initiation and progression. Accordingly, various epigenetic genes have been described to collaborate with the androgen receptor (AR) in enabling its oncogenic transcriptional program and aberrantly restoring its activity in metastatic castration resistant PCa. Concordantly, our laboratory has recently described two epigenetic genes, BRD4 and MLL2, as key co-activators of AR-signaling. Furthermore, we have demonstrated that inhibition of these genes synergistically work with AR antagonists to attenuate PCa progression in preclinical models. Thus, in a setting where all metastatic patients eventually progress to evolve resistance to anti-AR therapies, there exists a dire clinical need to identify novel therapeutic targets. To this end, using CRISPR-Cas9 technology, we have engineered a unique AR-reporter LNCaP model that harbors the mCherry gene directly downstream of the endogenous KLK3/PSA promoter (a canonical AR-target). Notably, validation experiments confirm that the mCherry reporter gene, akin to KLK3, is dynamically regulated by AR activity in response to treatment with dihydrotestosterone (DHT; a physiological AR agonist) or enzalutamide (an AR inhibitor). Furthermore, we have designed a focused, high-depth library of small-guide RNAs (sgRNAs) to target over 200 distinct epigenetic genes, both at transcriptional start sites and functionally-essential enzymatic domains. Overall, this library comprises of roughly 1400 distinct guide-RNAs that include 50 non-targeting negative controls. Using these molecular tools, we plan to perform a marker-based, functional screen that involves the sorting of sgRNA-infected AR-reporter cells into mCherryHIGH and mCherryLOW populations. Notably, we have successfully validated the stated screening design: Starting from a mixed pool of AR-targeting siRNAs or R1881 (a synthetic AR-agonist) treated reporter cells, we sorted them into LOW15% or HIGH15% fractions based on the intensity of mCherry fluorescence. As expected, transcriptomic analyses of these fractions confirmed that AR-signaling was indeed significantly inhibited and enhanced in the LOW15% and HIGH15% fractions, respectively, relative to the unsorted cells. Thus, for our CRISPR screen, we theorize that the genomic interrogation of sgRNAs enriched in the mCherryLOW population will reveal novel epigenetic genes that function as essential co-factors of oncogenic AR-activity. These epigenetic factors can be co-targeted to extort durable therapeutic responses in patients treated with the current line of anti-AR therapies, thus directly addressing an urgent clinical need.Citation Format: Abhijit Parolia, Lanbo Xiao, Josh N. Vo, Marcin Cieslik, Xuhong Cao, Arul M. Chinnaiyan. Functional CRISPR screen towards identifying novel epigenetic co-factors of oncogenic AR-activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 975.