Abstract A055: NSD2 is a Requisite and Targetable Subunit of the AR/FOXA1 Neo-Enhanceosome Complex in Prostate Cancer Cells

The androgen receptor (AR) is a ligand-responsive transcription factor (TF) that binds as a homodimer at FOXA1-pioneered enhancer elements containing a palindromic DNA motif. Prostate cancer (PCa) is highly dependent on the AR enhanceosome complex, and in castration-resistant disease, this dependency is reinforced through alterations in the AR pathway. This centrally involves extensive rewiring of the AR cistrome to gain de novo binding at enhancer sites (aka neo-enhancers) that activate hyperproliferative and metastatic gene programs. However, requisite subunits of the AR neo-enhanceosome and the genes it uniquely activates remain uncharacterized. Here, we CRISPR-engineered an endogenous AR reporter system by fusing the mCherry coding sequence in-frame with the KLK3/PSA gene and conducted a CRISPR knock-out screen to identify druggable chromatin/epigenetic cofactors of AR. Using a sgRNA library covering >200 druggable transcriptional coregulators, ranked alongside BRD4 and TRIM24/28, we identified NSD2 as a novel AR coactivator. NSD2 is an H3K36 di-methyltransferase that activates gene expression through antagonism of the PRC2/EZH2 complex. In PCa cells, genetic inhibition of NSD2 function significantly attenuated the expression of AR targets without affecting the AR protein level, suggesting that NSD2 regulates either AR’s chromatin assembly or trans-activational competence. Cistromic profiling of AR (ChIPseq) in NSD2-null PCa cells showed complete loss of binding at >40,000 genomic sites (~65% of the cistrome), with HOMER motif analyses of NSD2-dependent AR sites showing a higher recurrence of hexameric 5′-AGAACA-3′ AR half-site juxtaposed to the FOXA1, ETS, or HOXB13 motifs. In contrast, NSD2-independent AR sites, a large fraction of which show increased binding in NSD2-null cells, housed the canonical 15-bp palindromic AR motif with two invertedly-oriented half-sites separately recognized by each half of the AR homodimer. Notably, analyses of primary AR cistromes from human tissues revealed AR loading at half-motifs to be 20-30-fold enriched in the tumor-specific AR enhancer circuitry. In phenotypic assays, we found NSD2-deficient PCa cells to lose cancer hallmarks such as colony formation, invasion, and hyper-proliferation. We further developed a potent NSD2 PROTAC, LLC0150, which showed preferential cytotoxicity in AR/FOXA1+ PCa relative to the AR-negative disease, and other cancer and normal cell lines from 22 distinct lineages. In a pan-cancer screen comprising over 100 cell lines, treatment with LLC0150 triggered apoptotic death in AR/FOXA1+ malignancies and NSD2-mutant acute lymphocytic leukemia, and multiple myeloma. Altogether, we identify NSD2 as a novel AR neo-cofactor that assists oncogenic TFs in loading the AR enhanceosome at degenerate, low-affinity AR elements, thereby wiring its cancer-specific gene programs. We develop a novel NSD2 PROTAC that selectively kills AR/FOXA1-driven prostate and NSD2-altered tumors in preclinical models, positioning NSD2 therapeutics for safety and efficacy evaluation in human clinical trials. Citation Format: Abhijit Parolia, Brijesh K. Verma, Sanjana Eyunni, Sweta Aras, Sandra E. Carson, Eleanor Young, Chandan K. Das, James M. George, Reyaz ur Rasool, Prathibha Gajjala, Mohammed Alhusayan, Erick Mitchell-Velasquez, Lanbo Xiao, Jie Luo, Mustapha Jaber, Xuhong Cao, Fengyun Su, Rui Wang, Lianchao Liu, Zhen Wang, Ke Ding, Arul M. Chinnaiyan, Irfan Asangani. NSD2 is a requisite and targetable subunit of the AR/FOXA1 neo-enhanceosome complex in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A055.