A plastic EMP1? to LGR5? cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer [ATAC-Seq]

Inhibitors of the oncogene KRAS holds promise for treating metastatic CRC (mCRC). Here we show that the activity of a small molecule RAS inhibitor, RM-044, which covalently binds to the G12D mutation in the active (ON) conformation of RAS, demonstrated strong curative effects in CRC models of early liver metastases, but its therapeutic activity was diminished in the advanced metastatic disease. RM-044-treated metastases underwent a fast transition from a poor-prognosis-associated Emp1? transcriptional cell state to a WNT-driven Lgr5? stem cell-like state that supported tumor growth in the absence of RAS G12D activity. This plastic conversion involved a switch in transcription factor usage, and did not require extensive chromatin remodeling. Enforced conversion of metastatic cells to the Lgr5? state via RAS G12D inhibition, followed by genetic ablation of this population, produced strong therapeutic effects. Overall, these findings demonstrate a central role for oncogenic KRAS in governing cellular plasticity in mCRC. Overall design: ATAC-seq was performed to assess the impact of RM-044 on AKTP MTO. Treatments with either DMSO or RM-044 were administered two days post-seeding, and MTOs were harvested 48 hours later, with a treatment refresh at the 24-hour mark. ATAC-seq was performed based on previously described protocols (Buenrostro et al., 2015 and Corces et al., 2017).

致癌基因KRAS的抑制剂有望治疗转移性结直肠癌（metastatic CRC, mCRC）。本研究表明，一种可与活性（ON）构象RAS蛋白的G12D突变位点共价结合的小分子RAS抑制剂RM-044，在早期肝转移性结直肠癌模型中展现出显著的治疗效果，但在晚期转移性疾病中其治疗活性有所减弱。经RM-044处理的转移灶会快速从与不良预后相关的Emp1?转录细胞状态，转变为WNT信号通路驱动的Lgr5?干细胞样状态，该状态可在RAS G12D活性缺失的情况下支持肿瘤生长。这种可塑性转化涉及转录因子使用模式的切换，且无需广泛的染色质重塑。通过抑制RAS G12D活性将转移细胞强制转化为Lgr5?状态，随后对该细胞群进行遗传消融，可产生显著的治疗效果。综上，本研究结果证实致癌KRAS在调控转移性结直肠癌的细胞可塑性中发挥核心作用。实验整体设计：本研究采用ATAC-seq技术评估RM-044对AKTP MTO的影响。具体操作如下：细胞接种两天后，分别用二甲基亚砜（dimethyl sulfoxide, DMSO）或RM-044进行处理，并在处理24小时后更换一次药物，48小时后收集样本。ATAC-seq实验按照已发表的方案（Buenrostro等，2015；Corces等，2017）开展。