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

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: Hi-C 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. In situ Hi-C was performed based on the previously described protocol Rao et al., 2014.

致癌基因KRAS的抑制剂在治疗转移性结直肠癌（metastatic colorectal cancer，mCRC）方面颇具应用前景。本研究发现，一种可与RAS激活（ON）构象中的G12D突变共价结合的小分子RAS抑制剂RM-044，在早期肝转移结直肠癌模型中展现出强劲的治疗效果，但在晚期转移性疾病中其治疗活性显著减弱。经RM-044处理的转移瘤经历了快速的状态转换：从与不良预后相关的Emp1阳性转录细胞状态，转变为WNT通路驱动的Lgr5阳性干细胞样状态，该状态可在RAS G12D活性缺失的情况下支持肿瘤生长。这种可塑性转化仅涉及转录因子使用模式的切换，无需广泛的染色质重塑。通过RAS G12D抑制将转移细胞强制转化为Lgr5阳性状态，随后对该细胞群进行基因敲除，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。总体实验设计：本研究采用Hi-C（高通量染色体构象捕获）技术，评估RM-044对AKTP MTO的影响。在细胞接种两天后，分别用二甲基亚砜（DMSO）或RM-044进行处理，并于给药后24小时更换一次药物，48小时后收集AKTP MTO。本研究基于Rao等人2014年发表的实验方案开展原位Hi-C实验。