A plastic EMP1? to LGR5? cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer [scRNA-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: FACS-sorted Epcam+/CD45- epithelial cells were concentrated by centrifugation (500g for 7 min at 4ºC) and counted. Cells were partitioned into Gel Bead In Emulsions (GEMs) with a target of 5.000 cells per sample using the Chromium Controller system (10X Genomics). cDNA sequencing libraries were prepared using the Chromium Single Cell 3' Kit v3 (PN-1000075). cDNA was amplified, quality checked, and quantified on an Agilent Bioanalyzer High Sensitivity chip (Agilent Technologies), then used for Gene Expression (GEX) library preparation (PN-220103 Chromiumi7 Sample Index Plate). Final libraries were indexed by PCR and verified for size and concentration. Sequencing was performed on an Illumina NovaSeq 6000 to obtain approximately 40.000 reads per cell.