A Pancreas Organoids-based Screen Identifies a Specific KRAS Mutation Inhibitor that Attenuating Cholesterol Biosynthesis Pathway in Pancreatic Cancer

we reported a high-content drug screening platform of a series of pancreas organoids which contain common mutations found in human pancreatic cancer, wide type (WT), KC (KRASG12D), KPC (KRASG12D; Tp53R172H), KSC (KRASG12D; SMAD4-/-) and KPSC (KRASG12D; Tp53R172H and SMAD4-/-). Based on this platform, we screened about 6000 compounds and identified Perhexiline, which can inhibit the growth of pancreas organoids with KRAS mutation in vitro and in vivo. Single cell RNA-seq data shows that the cholesterol synthesis pathway is activated specifically caused by KRAS in mutated organoid lines. The key cholesterol synthesis regulator of SREBP2 and its downstream genes are activated in KRAS mutated organoids compared with WT. Interestingly, Perhexiline can reverse the metabolism changes in pancreas organoids with KRAS through downregulating SREBP2 at transcription level. Finally, we further validated targeting SREBP2 can attenuate tumor growth significantly with chemical inhibitor and genetic perturbation. These findings boost further understanding of KRAS in PDAC and provide a valuable resource for targeting KRAS therapeutics development. Five kinds of pancreas organoids, wide type (WT), KC (KRASG12D), KPC (KRASG12D; Tp53R172H), KSC (KRASG12D; SMAD4-/-) and KPSC (KRASG12D; Tp53R172H and SMAD4-/-) were treated with or without perhexiline. Ten samples were barcoded by 10 CMOs, respectively. And then, 10 samples were pooled for library preparation and sequence.

本研究报道了一套基于一系列携带人类胰腺癌常见突变的胰腺类器官的高内涵药物筛选平台，所涉类器官基因型包括野生型（Wild Type, WT）、KC（KRASG12D）、KPC（KRASG12D; Tp53R172H）、KSC（KRASG12D; SMAD4基因敲除，SMAD4-/-）及KPSC（KRASG12D联合Tp53R172H与SMAD4基因敲除，SMAD4-/-）。依托该平台，本研究筛选了约6000种化合物，最终鉴定出哌克昔林（Perhexiline），其可在体内外抑制携带KRAS突变的胰腺类器官的生长。单细胞RNA测序（single cell RNA-seq）数据显示，胆固醇合成通路在KRAS突变的类器官系中被特异性激活。与野生型类器官相比，胆固醇合成关键调控因子固醇调节元件结合蛋白2（SREBP2）及其下游基因在KRAS突变类器官中均被激活。值得注意的是，哌克昔林可通过在转录水平下调SREBP2，逆转KRAS突变胰腺类器官的代谢异常。本研究进一步通过化学抑制剂与基因扰动实验验证，靶向SREBP2可显著抑制肿瘤生长。本研究成果加深了学界对KRAS在胰腺导管腺癌（PDAC）中作用的理解，并为靶向KRAS的治疗药物开发提供了宝贵的研究资源。本研究将上述5种胰腺类器官分别施以哌克昔林处理或空白对照，10个样本分别通过10个CMO进行条码标记，随后将10个样本混合以完成文库制备与测序。