Androgen receptor (AR) is a ligand-responsive transcription factor that drives terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to activate malignant phenotypes, the molecular mechanisms of which remain unknown. Here, we show that tumor-specific AR enhancers are critically reliant on H3K36 dimethyltransferase activity of NSD2. NSD2 expression is abnormally induced in prostate cancer, where its inactivation impairs AR transactivation potential by disrupting over 65% of its cistrome. NSD2-dependent AR sites distinctively harbor the chimeric FOXA1:AR half-motif, which exclusively comprise tumor-specific AR enhancer circuitries defined from patient specimens. NSD2 inactivation also engenders increased dependency on the NSD1 paralog, and a dual NSD1/2 PROTAC degrader is preferentially cytotoxic in AR-dependent prostate cancer models. Altogether, we characterize NSD2 as an essential AR neo-enhanceosome subunit that enables its oncogenic activity, and position NSD1/2 as viable co-targets in advanced prostate cancer. CRISPR screen identifies coactivators of the androgen receptor (AR) complex, including NSD2. NSD2 contributes to AR cistrome reprogramming during prostate cancer progression, and its degradation via a novel PROTAC reduces prostate cancer cell viability in vitro.

Abstract Androgen receptor (AR) signaling is critical for survival of prostate cancer (PCa) cells, thus making androgen deprivation therapy (ADT; e.g., castration) as the mainstay for treatment. Notably, oncogenic functions of AR rely on chromatin-binding regulatory proteins, which includes a pioneer transcription factor called FOXA1. FOXA1 de-compacts chromatin to enable AR’s binding to the DNA and expression of it's target genes. Recently, our lab found FOXA1 alterations to recur within three distinct structural classes in over 35% of metastatic castration resistant PCa (mCRPC) in Caucasian men. Subsequent studies found FOXA1 mutations to be prevalent in over 40% of primary PCa in Chinese men, positioning FOXA1 as a principal oncogene in this disease. Yet, hitherto, no studies have defined the pathobiology of FOXA1 alterations in mouse models. Here, we have developed the first-in-field transgenic mice with conditional overexpression of FOXA1 mutants in the prostate luminal epithelia. We found truncal FOXA1 Class1 mutants to initiate luminal hyperplasia in a monogenic model, or hyperproliferative prostate adenocarcinoma in a compound Trp53-deficient genetic background. Mechanistically, Class1 mutants concurrently upregulate the AR and mTORC1/2 pathways to drive transformation, with this being the first report of FOXA1-driven PCa formation in mice. In contrast, FOXA1 Class2 mutants—which are acquired in mCRPC—do not drive prostate luminal transformation. Instead, single-cell multi-omics (RNA+ATAC) profiling of Class2-mutant mouse prostate tissue uncovered extensive transcriptional remodeling of epithelial cells into a luminal stem-like cell fate, leading to a dramatic 15-20-fold expansion of the progenitor population vs control tissue. These luminal stem cells are similar to the Club/Hillock cells detected in the human prostate that have been implicated in driving resistance to ADT. Consistently, we found Class2-mutant prostates to show minimal atrophy upon castration, with immunohistological assessment uncovering a higher density of Ki67+ luminal epithelial cells relative to wild type and Class1 tissues. Class2-mutant organoids also showed higher subcutaneous grafting ability in mice in limiting dilution assays. Mechanistically, we found the cistromically-dominant Class2 mutants to pioneer over 40,000 neo-enhancer elements harboring motifs of stemness-associated transcription factors, which in turn instruct the ADT-resistant luminal progenitor gene program. Altogether, findings from our mouse models uncover FOXA1’s versatility as a driver oncogene that, depending on the mutation type, either activates enhancer-wired luminal tumorigenesis (Class1-initiating event) or therapy resistance-associated stemness (Class2-promoting alteration) gene programs in the mouse prostate tissue. Citation Format: Sanjana Eyunni, Abhijit Parolia, Eleanor Young, James Matthew George, Rahul Mannan, Sandra E. Carson, Yuping Zhang, Jean Tien, Mustapha Jaber, Jie Luo, Matthew Pang, Rohit Mehra, Xuhong Cao, Fengyun Su, Rui Wang, Marcin Cieslik, Dong Kee Lee, Jianming Xu, Arul M. Chinnaiyan. FOXA1 alterations distinctively drive prostate tumorigenesis or therapy resistance in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1433.

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.

The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers.

Metastatic neuroendocrine prostate cancer (NEPC) is a highly aggressive disease, whose incidence is rising. Long noncoding RNAs (lncRNAs) represent a large family of disease‐ and tissue‐specific transcripts, most of which are still functionally uncharacterized. Thus, we set out to identify the highly conserved lncRNAs that play a central role in NEPC pathogenesis. To this end, we performed transcriptomic analyses of donor‐matched patient‐derived xenograft models (PDXs) with immunohistologic features of prostate adenocarcinoma (AR+/PSA+) or NEPC (AR−/SYN+/CHGA+) and through differential expression analyses identified lncRNAs that were upregulated upon neuroendocrine transdifferentiation. These genes were prioritized for functional assessment based on the level of conservation in vertebrates. Here, LINC00261 emerged as the top gene with over 3229‐fold upregulation in NEPC. Consistently, LINC00261 expression was significantly upregulated in NEPC specimens in multiple patient cohorts. Knockdown of LINC00261 in PC‐3 cells dramatically attenuated its proliferative and metastatic abilities, which are explained by parallel downregulation of CBX2 and FOXA2 through distinct molecular mechanisms. In the cell cytoplasm, LINC00261 binds to and sequesters miR‐8485 from targeting the CBX2 mRNA, while inside the nucleus, LINC00261 functions as a transcriptional scaffold to induce SMAD‐driven expression of the FOXA2 gene. For the first time, these results demonstrate hyperactivation of the LINC00261‐CBX2‐FOXA2 axes in NEPC to drive proliferation and metastasis, and that LINC00261 may be utilized as a therapeutic target and a biomarker for this incurable disease.