The ROR?/SREBP2 pathway is a master regulator of cholesterol metabolism and serves as potential therapeutic target in t(4;11) leukemia

Dysregulated cholesterol homeostasis promotes tumorigenesis and progression. Therefore, metabolic reprogramming constitutes a new hallmark of cancer. However, until today, only few therapeutic approaches exist to target this pathway due to the often-observed negative feedback induced by agents like statins leading to controversially increased cholesterol synthesis upon inhibition. Sterol regulatory element-binding proteins (SREBPs) are key transcription factors regulating the synthesis of cholesterol and fatty acids. Since SREBP2 is difficult to target, we performed pharmacological inhibition of retinoic acid receptor (RAR)-related orphan receptor gamma (ROR?), which acts upstream of SREBP2 and serves as master regulator of the cholesterol metabolism. This resulted in an inactivated cholesterol-related gene program with significant downregulation of cholesterol biosynthesis. Strikingly, these effects were more pronounced than the effects of fatostatin, a direct SREBP2 inhibitor. Upon ROR? inhibition, RNA sequencing showed strongly increased cholesterol efflux genes leading to leukemic cell death and cell cycle changes in a dose- and time-dependent manner. Combinatorial treatment of t(4;11) cells with the ROR? inhibitor showed additive effects with cytarabine and even strong anti-leukemia synergism with atorvastatin by circumventing the statin-induced feedback. Our results suggest a novel therapeutic strategy to inhibit tumor-specific cholesterol metabolism for the treatment of t(4;11) leukemia. Overall design: human cord blood-derived CD34+ cells were modified using CRISPR/Cas9 to carry an endogenous t(4;11) translocation. RNA sequencing was performed using TruSeq mRNA Stranded Sequencing (Illumina) from three donors in technical duplicates, comparing XY018-treated and untreated cells.

胆固醇稳态失调可促进肿瘤发生与进展，因此代谢重编程已成为癌症的新型标志特征。然而时至今日，针对该代谢通路的治疗手段仍寥寥无几，原因在于他汀类药物等常可诱导负反馈效应，反而会在抑制过程中引发胆固醇合成的异常升高。固醇调节元件结合蛋白（Sterol regulatory element-binding proteins, SREBPs）是调控胆固醇与脂肪酸合成的关键转录因子。由于SREBP2难以直接靶向，本研究对视黄酸受体（RAR）相关孤儿受体γ（RORγ）进行了药理学抑制——该受体位于SREBP2上游，是胆固醇代谢的核心调控因子。这一干预可使胆固醇相关基因程序失活，并显著下调胆固醇生物合成通路相关基因的表达。值得注意的是，其调控效果相较于直接靶向SREBP2的抑制剂fatostatin更为显著。在抑制RORγ后，RNA测序结果显示胆固醇外流相关基因的表达显著上调，进而以剂量依赖和时间依赖的方式诱导白血病细胞死亡，并引发细胞周期改变。将RORγ抑制剂与t(4;11)型白血病细胞联合处理后，其与阿糖胞苷（cytarabine）联用可产生相加效应，而与阿托伐他汀（atorvastatin）联用则可通过规避他汀类药物诱导的负反馈，展现出强劲的抗白血病协同作用。本研究结果表明，靶向肿瘤特异性胆固醇代谢的新型治疗策略，可用于治疗t(4;11)型白血病。
实验整体设计：采用CRISPR/Cas9技术对人脐带血来源的CD34+细胞进行基因编辑，使其携带内源性t(4;11)染色体易位。本研究使用TruSeq链特异性mRNA测序（Illumina）平台，对3名供体来源的细胞进行技术重复测序，每组设置XY018处理组与未处理对照组并进行对比。