Defined cellular reprogramming of androgen receptor-active prostate cancer to neuroendocrine prostate cancer [CUTnRUN]

Neuroendocrine prostate cancer (NEPC) arises primarily through neuroendocrine transdifferentiation (NEtD) as an adaptive mechanism of therapeutic resistance. Models to define the functional effects of putative drivers of this process on androgen receptor (AR) signaling and NE cancer lineage programs are lacking. We adapted a genetically defined strategy from the field of cellular reprogramming to directly convert AR-active prostate cancer (ARPC) to AR-independent NEPC using candidate factors. We delineated critical roles of the pioneer factors ASCL1 and NeuroD1 in NEtD and uncovered their abilities to silence AR expression and signaling by remodeling chromatin at the somatically acquired AR enhancer and global AR binding sites with enhancer activity. We also elucidated the dynamic temporal changes in the transcriptomic and epigenomic landscapes of cells undergoing acute lineage conversion from ARPC to NEPC which should inform future therapeutic development. Further, we distinguished the activities of ASCL1 and NeuroD1 from the inactivation of RE-1 silencing transcription factor (REST), a master suppressor of a major neuronal gene program, in establishing a NEPC lineage state and in modulating the expression of genes associated with major histocompatibility complex class I (MHC I) antigen processing and presentation. Overall design: CUT&RUN was performed in reprogrammed C4-2B cells for ASCL1, H3K4me3, H3K4me1, H3K27Ac. Experiments performed in triplicates with an IgG control.

神经内分泌前列腺癌（Neuroendocrine prostate cancer, NEPC）主要通过神经内分泌转分化（neuroendocrine transdifferentiation, NEtD）产生，作为肿瘤细胞对抗治疗的适应性抵抗机制。目前尚无能够解析该过程中潜在驱动因子对雄激素受体（androgen receptor, AR）信号通路及神经内分泌癌症谱系程序的功能影响的相关模型。我们借鉴细胞重编程领域的遗传定向策略，通过候选因子将具有雄激素受体活性的前列腺癌细胞系C4-2B重编程为雄激素受体非依赖型NEPC。本研究明确了先锋转录因子ASCL1与NeuroD1在NEtD中的关键作用，并揭示二者可通过在体细胞获得的AR增强子及全局AR结合位点（带有增强子活性）重塑染色质，进而沉默AR的表达与信号通路。我们还解析了从雄激素受体活性型前列腺癌（AR-active prostate cancer, ARPC）向NEPC发生急性谱系转换的细胞的转录组与表观基因组景观的动态时序变化，该结果可为未来的治疗开发提供参考。此外，我们区分了ASCL1与NeuroD1的活性，以及RE-1沉默转录因子（RE-1 silencing transcription factor, REST）——一类主要神经元基因程序的核心抑制因子——的失活，在建立NEPC谱系状态及调控主要组织相容性复合体I类（major histocompatibility complex class I, MHC I）抗原加工与呈递相关基因表达中的不同作用。

整体实验设计：采用CUT&RUN技术对重编程后的C4-2B细胞进行检测，靶标包括ASCL1、H3K4me3、H3K4me1及H3K27Ac；所有实验均设置3次生物学重复，并以IgG作为阴性对照。