Characterizing a Canine Urothelial Carcinoma Model of Acquired MEK1/2 Inhibitor Resistance

Abstract

Activating BRAF mutations are drivers of oncogenesis in several human cancers and in over 70% of canine urothelial carcinomas (cUC). Selective BRAF inhibitors are effective alone and in combination with MEK inhibitors in approximately half of BRAF mutant melanoma cases, but the majority of these patients ultimately develop resistance. Thus, intrinsic and acquired resistance to MAP kinase pathway targeted agents remains a challenge requiring the development of novel therapeutic strategies. Using canine urothelial carcinoma as a spontaneous cancer model to explore treatment of BRAF mutant cancers and acquired drug resistance, we sought to identify determinants of MEK1/2 inhibitor sensitivity and resistance. We applied the human‐derived MAPK Pathway Activity Score (MPAS), a predictor of sensitivity to MEK inhibition in human cancers based on expression levels of 10 downstream targets, to a panel of 32 canine cancer cell lines and found that MPAS correlates with trametinib sensitivity in this canine dataset. MPAS genes with significant (p<0.05) Pearson correlations to sensitivity were CCND1, DUSP6, ETV4, ETV5, and SPRY2. We also identified 30 non‐MPAS genes whose expression levels are significantly correlated with trametinib sensitivity, suggesting their role in de novo sensitivity to trametinib. Each of the 5 urothelial carcinoma lines had high MPAS scores and trametinib IC50 values <10nM. CRISPR‐Cas9 knockouts in two canine urothelial carcinoma lines also identified MEK1 as a critical dependency. To investigate mechanisms of acquired resistance, we generated trametinib‐resistant (TramR) clonal derivatives of the BRAF mutant Tyler1 canine urothelial carcinoma cell line (Tyler1‐TramR), with trametinib IC50 values greater than 500 nM versus 1 nM in parental Tyler1. Tyler1‐TramR cell lines exhibited sustained inhibition of ERK1/2 phosphorylation when treated with trametinib and were also resistant to pharmacologic inhibition of ERK1/2. Collectively, these data suggest an ERK‐independent mechanism of resistance. Gene expression analysis of two Tyler1‐TramR clones identified dramatic downregulation of epithelial markers accompanied by an increase in expression of mesenchymal genes and transcription factors that regulate the epithelial‐to‐mesenchymal transition (EMT). One Tyler1‐TramR clone exhibited metabolic alterations including decreased basal and maximal oxygen consumption rates, diminished spare respiratory capacity, and reduced glycolytic capacity. Overall, the findings in this study suggest that the mechanisms of acquired and intrinsic MEK inhibitor resistance are conserved between man and dog, further elucidating the translational value of cUC as a model to optimize therapies for Ras/Raf/MAP kinase driven cancers.