NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis [RNA-seq]. NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis [RNA-seq]

Androgen receptor (AR) is a ligand-responsive transcription factor that binds at enhancers to drive terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to drive hyper-proliferative, metastatic, or therapy-resistant phenotypes, the molecular mechanisms of which remain poorly understood. Here we show that the tumor-specific enhancer circuitry of AR is critically reliant on the activity of Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone 3 lysine 36 di-methyltransferase. NSD2 is ectopically expressed in prostate cancer cells and catalytic inhibition of NSD2 impairs AR trans-activation potential through partial off-loading from over 40,000 genomic sites or greater than 65% of its cistrome. The NSD2-dependent AR sites distinctly harbor a chimeric AR-half motif juxtaposed to a FOXA1 element. Similar chimeric motifs of AR are absent at the NSD2-independent AR enhancers and contain the canonical palindromic motifs instead. Meta-analyses of AR cistromes from patient tumors uncovered chimeric AR motifs to exclusively participate in tumor-specific enhancer circuitries, with a minimal role in physiological activity of AR. Accordingly, NSD2 inactivation attenuated hallmark cancer phenotypes that were fully reinstated upon exogenous NSD2 re-expression. Inactivation of NSD2 also engendered increased dependency on its paralog NSD1, which independently maintained AR and MYC hyper-transcriptional programs in cancer cells. Therapeutically exploiting these insights, we developed a dual NSD1/2 PROTAC degrader, called LLC0150, which was preferentially cytotoxic in AR-dependent prostate cancer and synergized with enzalutamide. In a pan-cancer screen, comprising over 120 cell lines from 22 distinct lineages, NSD1/2 co-degradation triggered apoptotic cell death in AR-addicted prostate cancer as well as NSD2-altered hematologic malignancies. Altogether, we identify NSD2 as a novel subunit of the AR neo-enhanceosome that wires prostate cancer gene expression programs, positioning NSD1/2 paralog co-targeting as a novel and potent therapeutic strategy. Overall design: Comparative RNA profiles of prostate cancer cells upon CRISPR or siRNA-mediated inactivation of NSD1, NSD2, and/or NSD3 genes, treatment with the EZH2 inhibitor EPZ6438 (72h at 2uM), or treatment with NSD1/2 PROTAC degrader LLC0150 (2um for 24h).

雄激素受体（Androgen receptor, AR）是一类配体响应型转录因子，可结合增强子以驱动前列腺腔上皮细胞的终末分化。与之相反，在源自此类细胞的肿瘤中，AR的染色质占据模式被广泛重编程，进而驱动过度增殖、转移或治疗耐药的表型，其背后的分子机制仍未被充分阐明。本研究发现，AR的肿瘤特异性增强子环路关键依赖于核受体结合SET结构域蛋白2（Nuclear Receptor Binding SET Domain Protein 2, NSD2）的活性，该蛋白是一种组蛋白3赖氨酸36二甲基转移酶。NSD2在前列腺癌细胞中异位表达，对其进行催化抑制可通过从超过4万个基因组位点（或其顺反组的65%以上）中部分解离，削弱AR的反式激活潜能。依赖NSD2的AR结合位点显著含有一类嵌合型AR半位点基序，该基序与叉头框蛋白A1（FOXA1）元件毗邻排布。而不依赖NSD2的AR增强子区域则不存在此类AR嵌合基序，反而包含经典的回文基序。对患者肿瘤来源的AR顺反组开展荟萃分析后发现，嵌合型AR基序仅参与肿瘤特异性增强子环路，在AR的生理活性中仅发挥极小作用。据此，NSD2失活可减弱典型的癌症表型，且该表型可通过外源性NSD2重新表达完全恢复。NSD2失活还会使癌细胞对其旁系同源基因NSD1的依赖程度升高，而NSD1可独立维持癌细胞中AR与MYC原癌基因（MYC）的高转录程序。基于上述发现开发治疗策略，我们研发了一款靶向NSD1/2的双靶点蛋白水解靶向嵌合体（PROTAC）降解剂LLC0150，该降解剂在AR依赖型前列腺癌细胞中表现出优先细胞毒性，并与恩扎卢胺（enzalutamide）具有协同作用。在一项涵盖22种不同谱系、共120余株细胞系的泛癌症筛选中，同时降解NSD1/2可在AR成瘾型前列腺癌以及NSD2异常的血液系统恶性肿瘤中诱导细胞凋亡。综上，本研究确认NSD2是AR新生增强体（neo-enhanceosome）的新型亚基，可调控前列腺癌的基因表达程序，从而将共靶向NSD1/2旁系同源基因确立为一种全新且高效的治疗策略。实验整体设计：对前列腺癌细胞采用成簇规律间隔短回文重复序列（CRISPR）或小干扰RNA（small interfering RNA, siRNA）介导的方式进行NSD1、NSD2及/或NSD3基因失活处理、用zeste基因增强子同源物2（EZH2）抑制剂EPZ6438处理（2μM浓度孵育72小时），或用NSD1/2双靶点PROTAC降解剂LLC0150处理（2μM浓度孵育24小时），并对各组细胞进行比较转录组RNA谱分析。