Antiandrogen treatment endows ZMYDN8 dependent epigenetic reprogramming to orchestrate neuroendocrine tumor transdifferentiation [scRNA-seq]

The transdifferentiation from adenocarcinoma following androgen deprivation therapy (ADT) is thought to be the primary process leading to the development of neuroendocrine prostate cancer (NEPC). However, it remains unclear how lineage factors interact with ADT to endow the lineage transition. Through an integrated analysis of NEPC-based CRISPR-Cas9 screening and scRNA-seq tracking of tumor transitions, we unveil that antiandrogen-induced ZMYND8-dependent epigenetic programming orchestrates the transdifferentiation of NEPC. We demonstrate that the ablation of Zmynd8 restricts NEPC development in Pten/Trp53/Rb1 knockout mouse models. Conversely, ASCL1-induced upregulation of ZMYND8 shapes the neuroendocrine (NE) lineage to confer resistance to AR-targeted therapy. Mechanistically, FOXM1, a key regulator in castration-resistant prostate cancer (CRPC), stabilizes ZMYND8 binding to chromatin regions harboring H3K4me1-K14ac modification and FOXM1 targeting. Antiandrogen treatment frees the SWI/SNF chromatin remodeling complex from AR, enabling its interaction with ZMYND8/FOXM1 to upregulate key NE lineage regulators (e.g., FOXA2, SOX2, and POU3F2), thus inducing transdifferentiation. Having identified ZMYND8's link to adverse disease outcomes in CRPC patients, we develop a small molecule, ZMYND8i-34, designed to selectively inhibit its histone recognition. In pre-clinical models, ZMYND8i-34 treatment effectively blocks NE transdifferentiation and curtails CRPC development. Together, our results highlight the importance of antiandrogen treatment endowing ZMYND8-dependent epigenetic reprogramming to orchestrate lineage fate and suggest that targeting ZMYND8 may hold the potential to restrict NEPC development and overcome treatment resistance. To understand the cell lines changes in ZMYND8-OE condition, we treated cell lines with ZMYND8-OE in low MOI of virus and cultured the mixture of cells with CSS (Charcoal-Stripped FBS). Then, we collected the partially ZMYND8 overexpressed cells at three time points in CSS condition, day0, day3 and day30 and used three time points cells (day0, day3 and day30) for scRNA-seq separately.

雄激素剥夺治疗（androgen deprivation therapy, ADT）后腺癌转分化，被认为是神经内分泌前列腺癌（neuroendocrine prostate cancer, NEPC）发生的核心病理过程。然而，谱系因子如何与ADT相互作用以驱动谱系转变，目前仍未阐明。本研究通过整合基于NEPC的CRISPR-Cas9筛选与肿瘤转变的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）追踪分析，揭示了抗雄激素诱导的、依赖ZMYND8的表观遗传编程可调控NEPC的转分化过程。我们证实，在Pten/Trp53/Rb1敲除小鼠模型中，敲除Zmynd8可显著限制NEPC的发生发展。与之相反，ASCL1诱导的ZMYND8上调会重塑神经内分泌（neuroendocrine, NE）谱系，进而赋予肿瘤对雄激素受体（androgen receptor, AR）靶向治疗的耐药性。从机制上看，去势抵抗性前列腺癌（castration-resistant prostate cancer, CRPC）中的关键调控因子FOXM1，可稳定ZMYND8与携带H3K4me1-K14ac修饰及FOXM1结合位点的染色质区域的结合。抗雄激素治疗会将SWI/SNF染色质重塑复合物从AR复合物中释放，使其与ZMYND8/FOXM1相互作用，从而上调关键NE谱系调控因子（如FOXA2、SOX2及POU3F2），最终诱导肿瘤转分化。我们还发现，在CRPC患者中ZMYND8的异常表达与不良疾病预后显著相关，并据此开发了一种小分子抑制剂ZMYND8i-34，可选择性抑制ZMYND8的组蛋白识别功能。在临床前模型中，ZMYND8i-34治疗可有效阻断NE转分化进程，并抑制CRPC的发展。综上，本研究结果表明，抗雄激素治疗可通过依赖ZMYND8的表观遗传重编程调控细胞谱系命运，提示靶向ZMYND8或可成为限制NEPC发生、克服治疗耐药的潜在策略。为探究ZMYND8过表达（ZMYND8 overexpression, ZMYND8-OE）条件下细胞系的转录组变化，我们以低感染复数（multiplicity of infection, MOI）的病毒感染细胞系以诱导ZMYND8过表达，并将感染后的细胞混合培养于活性炭剥离胎牛血清（Charcoal-Stripped FBS, CSS）培养基中。随后，我们在CSS培养条件下的三个时间点（第0天、第3天及第30天）收集部分过表达ZMYND8的细胞，并分别对这三个时间点的细胞进行scRNA-seq测序。