Molecular Subtyping Combined with Biological Pathway Analyses to Study Regorafenib Response in Clinically Relevant Mouse Models of Colorectal Cancer.

ABSTRACT

PURPOSE: Regorafenib (REG) is approved for the treatment of metastatic colorectal cancer, but has modest survival benefit and associated toxicities. Robust predictive/early response biomarkers to aid patient stratification are outstanding. We have exploited biological pathway analyses in a patient-derived xenograft (PDX) trial to study REG response mechanisms and elucidate putative biomarkers. EXPERIMENTAL DESIGN: Molecularly subtyped PDXs were annotated for REG response. Subtyping was based on gene expression (CMS, consensus molecular subtype) and copy-number alteration (CNA). Baseline tumor vascularization, apoptosis, and proliferation signatures were studied to identify predictive biomarkers within subtypes. Phospho-proteomic analysis was used to identify novel classifiers. Supervised RNA sequencing analysis was performed on PDXs that progressed, or did not progress, following REG treatment. RESULTS: Improved REG response was observed in CMS4, although intra-subtype response was variable. Tumor vascularity did not correlate with outcome. In CMS4 tumors, reduced proliferation and higher sensitivity to apoptosis at baseline correlated with response. Reverse phase protein array (RPPA) analysis revealed 4 phospho-proteomic clusters, one of which was enriched with non-progressor models. A classification decision tree trained on RPPA- and CMS-based assignments discriminated non-progressors from progressors with 92% overall accuracy (97% sensitivity, 67% specificity). Supervised RNA sequencing revealed that higher basal EPHA2 expression is associated with REG resistance. CONCLUSIONS: Subtype classification systems represent canonical "termini a quo" (starting points) to support REG biomarker identification, and provide a platform to identify resistance mechanisms and novel contexts of vulnerability. Incorporating functional characterization of biological systems may optimize the biomarker identification process for multitargeted kinase inhibitors.