A plastic EMP1? to LGR5? cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer [RNA-Seq mouse]
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: RNA sequencing was performed to assess the impact of RM-044 on AKTP MTO and APK CTO. 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. RNA extraction was carried out using the PureLink™ RNA Mini Kit (Life Technologies, 12183025) according to the manufacturer's instructions. Elution was performed with distilled water (Invitrogen, 10977-035), and RNA concentrations were measured using a Nanodrop 1000 spectrophotometer (ThermoFisher). Library preparation was conducted using the RNA-seq Stranded mRNA Library Illumina kit, followed by sequencing on the Illumina NextSeq 2000 P3 platform.