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

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: PDOs were cultured in 70% cold BME/medium drops and complete organoid CRC tumor medium (CTM) containing AdvancedDMEM/F12, Hepes, Glutamax, B27, Nicotinamide 10mM, N-Acetyl- cystein 1.25nM, 50 ng/ml EGF, R-spondin1 1ug/ml, Noggin 100 ng/ml, SB202190 10 uM, Gastrin 10nM, A83-01 500nM, ProstaglandinE2 2.5 uM and Normocin 100 ug/ml. For RM-044 experiments, 7-day grown organoids were disaggregated with TrypLE™, counted and plated in 70% BME at a density of 750 cell/µl for P26 and PDO131 and 250 cell/µl for C31M in complete CTM with rock inhibitor Y27632 10uM. After three days, media was changed to CTM without rock inhibitor and cells were treated with RM-044 100nM or DMSO with a daily media change. Samples for RNA-sequencing were collected at day 4 after treatment and growth curves were measured during 10 days from treatment start.