Oncogenic MAPK signalling induces epithelial cell state change and therapeutic resistance in vivo [scRNA-Seq]
The colorectal epithelium is a rapidly renewing tissue, with a remarkable capacity to regenerate following injury. In colorectal cancer (CRC), this regenerative capacity can be co-opted to drive epithelial plasticity. Activation of oncogenic MAPK signalling in CRC is common, with mutations frequently found in both KRAS (40-50%) and BRAF (10%), alongside other mechanisms of EGFR pathway upregulation. Nonetheless, inhibition of this pathway is often linked to rapid therapeutic resistance in patients. Given the recent development of KRAS inhibitors and the licensing of BRAF+EGFR inhibitor combinations we have investigated mechanisms of resistance to these agents in complex mouse models of CRC. Here we show that MAPK signalling supports the regenerative/revival stem cell phenotype in vivo, while its inhibition leads to rapid remodelling of both Kras and Braf mutant mouse models to a high Wnt activity canonical stem cell phenotype marked by expression of Lgr5. This results in acute therapeutic resistance in Kras driven, and delayed resistance in Braf driven models. Importantly, where therapeutic MAPK targeting is applied in the presence of a single regenerative epithelial population, such as early metastatic disease, or where plasticity is restrained, for example through Wnt pathway restriction, a marked impact on tumourigenesis is observed. Modelling of an BRAF and RNF43-mutant patient population, clinically super-responders to BRAF/EGFR inhibitor therapy, demonstrates excellent response to therapy due to restricted epithelial plasticity, highlighting the criticality of cellular plasticity in therapeutic response. Together, our data provide clear insight into the mechanisms underpinning resistance to targeting oncogenic MAPK signalling in CRC. Moreover, we demonstrate that strategies that aim to restrict epithelial plasticity or corral into or intervene when tumours are in a regenerative cell fate may improve efficacy of MAPK targeting agents in CRC. Overall design: Mice were induced with a single 70uL injection of 100uM 4-OH tamoxifen into the colonic submucosa. When an established tumour was identified on colonoscope (beginning weekly two weeks post injection), mice were placed on a treatment regime. Mice were treated twice daily with MRTX1133 IP AM and PM (MRTX1133 100uL 7.5mg/kg) or vehicle (100uL10% hydroxpropyl beta cyclodextrin in 50mM citrate buffer ph5) for 4 days and sampled two hours post AM dose on the fourth day. Single cell RNAseq data from colon and SI tumours (submitted previously in GSE280631 & GSE293737 as detailed in GSE307774_reanalysis_samples.xlsx), from mice expressing mutations in combinations of Apc, Braf, Kras, p53 and Rnf43. Genetic alterations were induced with intraperitoneal tamoxifen and tumours were homogenised and digested for sequencing using a Mcllwain Tissue Chopper and the GentleMACS Octo Dissociator in combination with the mouse tumour dissociation kit. Sequencing libraries were prepared using reagents from the 10x Chromium Single-Cell 3' v3 Gel Bead Kit and Library (10x Genomics) according to the manufacturer's protocol and then sequenced on an Illumina Novaseq 6000. Sequence alignment of single-cell data to the mm10 genome was performed using the count tool from the Cell Ranger package version 6.1.2 according to the developer's instructions, generating barcodes, features and matrix output files for each sample. *************************************************************** The table below lists GEO accessions reused/reanalyzed for this study. ***************************************************************