RAS(ON) multi-selective inhibition remodels cancer-associated fibroblast subtypes and extracellular matrix in pancreatic cancer

Oncogenic KRAS mutations occur in >90% of pancreatic ductal adenocarcinomas (PDACs) and are the principal drivers of malignant transformation and tumor progression. Sustained KRAS signaling in malignant epithelial cells orchestrates a network of paracrine interactions that define the cellular and extracellular composition of the tumor microenvironment (TME). In the TME, cancer-associated fibroblasts (CAFs) are particularly responsive to KRAS-dependent paracrine signals, including Sonic Hedgehog (SHH), a ligand that is overexpressed and secreted from malignant epithelial cells, thereby driving the proliferation of cancer-associated myofibroblasts. RMC-7977 is a RAS(ON) multi-selective inhibitor that inhibits GTP-bound RAS proteins, including wild type and mutant variants of KRAS, NRAS, and HRAS. It showed broad anti-tumor activity in PDAC preclinical models, extending overall survival of the KrasLSL.G12D/+ ;Trp53LSL.R172H/+ ;Pdx1-cretg/+ (KPC) mouse model by more than 3-fold. The KPC mouse model recapitulates the desmoplasia observed in patients, allowing the assessment of KRAS inhibition on stromal composition and function. To study the impact of RAS inhibition in PDAC, we did single cell RNA sequencing (scRNA-seq) on KPC tumors treated with RMC-7977 at multiple timepoints. After a week of RMC-7977 treatment, the dominant CAF subtype in KPC tumors shifted from myofibroblastic CAFs (myCAFs) to inflammatory CAFs (iCAFs). This observation was validated by co-immunofluorescence on tumor sections, which indicated that (PDPN+ , alphaSMA+ ) myCAFs showed decreased abundance and proliferation whereas (PDPN+ , IL6+ ) iCAF abundance increased. Similar effects were observed ex vivo in human and murine PDAC explants. Mechanistic studies showed that this phenotype was mediated by a downregulation of SHH ligand expression in response to RAS inhibition. We also used the scRNA-seq data to assess the contribution of CAF subtypes to the generation, regulation, and degradation of the extracellular matrix (ECM) in PDAC. We found that myCAFs and iCAFs have both overlapping and distinct roles in the regulation of the ECM and that these were differentially impacted by RMC-7977 treatment. Thus, pan-RAS-GTP inhibition indirectly remodeled ECM composition both by impact CAF subtype distribution and by altering matrix composition and regulation by CAFs. Accordingly, nanoindentation measurements on KPC tumors and human explants confirmed a significant decrease in modulus (a measurement for tumor stiffness) after RMC-7977 treatment. In summary, we report that RMC-7977 switches the myCAF-dominant CAF landscape towards an iCAF-enriched TME through the KRAS-SHH axis and this alters TME composition and reduces tumor stiffness through ECM remodeling. Future studies will examine the impact of these finding on drug delivery, tumor invasiveness, tumor differentiation state.