A plastic EMP1⁺ to LGR5⁺ cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer

Most patients with metastatic colorectal cancer (mCRC) ultimately succumb to the disease due to a lack of effective therapeutic options. A new generation of inhibitors targeting the oncogene KRAS holds promise for treating mCRC and are currently under clinical investigation. In this study, we explore the activity of a small molecule KRAS inhibitor, RM-044, which covalently binds to the G12D mutation in the active (ON) conformation of KRAS. We found that despite robust inhibition of the KRAS signaling pathway, RM-044 exhibited limited therapeutic efficacy in patient-derived models of mCRC. Instead, liver metastases treated with RM-044 transitioned from a transcriptionally poor-prognosis-associated Emp1⁺ cell state to a WNT-driven Lgr5⁺ stem cell-like state that supported tumor growth even in the absence of KRAS-G12D activity. This plastic conversion occurred within hours of treatment, involved a switch in transcription factor usage, and did not require extensive chromatin remodeling. To explore vulnerabilities of RM-044–treated metastases, we engineered mCRC models to express the diphtheria toxin receptor under control of the Lgr5 locus. Enforced conversion of CRC cells to the Lgr5⁺ state via KRAS-G12D inhibition, followed by targeted ablation of this population using diphtheria toxin, produced strong therapeutic effects in the liver metastatic context. CUT&RUN 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. CUT&RUN was performed in biological duplicates based on previously described protocols (Skene & Henikoff, 2017) with minor modifications.