CRISPR screening identifies synergy between BET and mTOR inhibitors in cholangiocarcinoma through impaired serine glycine one carbon metabolism [CHIP-seq]

Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify novel drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV-825, from which we identified anti-cancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the two inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key regulators PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single agent inhibition of either BET or mTOR. Our results tied together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease. Compare H3K27ac from each group (vehicle, BETp, mTORi, and BETp + mTORi) of SNU1079 with 48h of drug treatment

胆管癌患者因确诊延迟、预后不良及治疗策略匮乏，临床结局极差。为发掘该疾病的新型药物联合方案，我们依托溴结构域和额外末端结构域（bromodomain and extraterminal domain, BET）蛋白降解靶向嵌合体（PROTAC）降解剂ARV-825开展了全基因组成簇规律间隔短回文重复序列（CRISPR）筛选，从中发现该降解剂与哺乳动物雷帕霉素靶蛋白（mTOR）通路成员的基因敲除联用时可产生抗癌协同效应。该联合效应通过多种药理学BET抑制剂与mTOR抑制剂得到验证，实验同时观察到细胞凋亡水平升高与细胞周期阻滞。在异种移植模型中，联合BET降解与mTOR抑制可诱导肿瘤消退。机制层面，两类抑制剂共同作用于经H3K27ac标记的丝氨酸-甘氨酸-一碳代谢（SGOC）通路的表观遗传抑制，该通路包含关键调控因子PHGDH与PSAT1。仅敲低PSAT1即可重现单独抑制BET或mTOR时的协同效应。本研究将表观遗传调控、代谢过程与凋亡诱导关联为该临床需求未被充分满足的疾病的关键治疗靶点，以供后续探索。对经48小时药物处理的SNU1079细胞的各实验组（溶剂对照组、BET抑制剂组（BETp）、mTOR抑制剂组（mTORi）及BET抑制剂+mTOR抑制剂联合组（BETp + mTORi））的H3K27ac水平进行比较。