Identification of therapeutic targets in canine bladder cancer: a translational model for MAPK pathway-targeted and immune-based therapies
Date
2021
Authors
Cronise, Kathryn Elizabeth, author
Duval, Dawn, advisor
Gustafson, Daniel, advisor
DeLuca, Jennifer, committee member
Page, Rodney, committee member
Thamm, Douglas, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Activating mutations in the proto-oncogene BRAF are drivers of oncogenesis in several human cancers, including melanoma, thyroid and colorectal carcinomas, and hairy-cell leukemia. Small molecule inhibitors targeting oncogenic BRAF demonstrate initial efficacy in approximately 50% of BRAF mutant melanoma patients; however, acquired resistance invariably develops. Other individuals, including the majority of colorectal cancer patients, exhibit intrinsic resistance to BRAF inhibitors. Combined inhibition of BRAF and its downstream target MEK improves the rate and duration of patient response, but resistance remains an issue. Thus, more effective and robust therapies are necessary. Transitional cell carcinoma (TCC) is the most common bladder cancer in dogs and humans. In this study, we provide a molecular characterization of 11 canine TCC (cTCC) tumors and identified BRAF mutations in 8 out of 11 samples. All BRAF mutations were valine-to-glutamic acid missense substitutions at amino acid residue 596 of canine BRAF (V596E), analogous to the V600E driving variant in human cancer. Additionally, 22 out of 32 formalin-fixed paraffin embedded samples expressed mutant BRAF, indicating an overall prevalence of 70%. Further analysis identified four tumors, three being BRAF mutant, that exhibited increased expression of immune gene markers and gene signatures associated with complete clinical response to checkpoint inhibition in human bladder cancer. We also found that all TCC tumors overexpress cell cycle, DNA repair, and immune-related genes. The high prevalence of BRAF mutations in cTCC makes targeting BRAF with small molecule inhibitors an attractive therapeutic option. We explored this possibility in vitro and determined that BRAF mutant cTCC cell lines are insensitive to the BRAF inhibitor vemurafenib but are sensitive to the newer, "paradox-breaking" BRAF inhibitor PLX7904. All tested cTCC cell lines were sensitive to the MEK1/2 inhibitors trametinib and selumetinib. A phenomenon observed with single-agent BRAF or MEK inhibition was the reactivation of ERK1/2 within 24 hours post-treatment, suggesting built-in mechanisms of bypassing BRAF and MEK inhibition. We also observed upregulation of genes encoding the ErbB family receptors, EGFR and ERBB2, and the EGFR ligand, EREG, in cTCC cell lines compared to other canine cancer cell lines. Treatment with the pan-ErbB inhibitor sapitinib synergized with BRAF or MEK inhibition in the BRAF mutant Bliley cell line and in the BRAF wild-type Kinsey cell line. Next, we generated trametinib-resistant clonal derivatives of the BRAF mutant Tyler1 cTCC cell line (Tyler1-TramR). Tyler1-TramR cells exhibited trametinib IC50 values over 500 nM and maintained suppression of ERK1/2 phosphorylation for 24 hours following trametinib treatment. This response, combined with the insensitivity of Tyler1-TramR cell lines to the ERK1/2 inhibitor ravoxertinib, suggests that resistance to trametinib is independent of ERK1/2 reactivation. Further analysis of two Tyler1-TramR clones using RNA-Seq identified a loss of epithelial gene markers, while mesenchymal genes and transcription factors controlling the epithelial-to-mesenchymal transition were upregulated. Analysis of basal cellular metabolism using a Seahorse XF analyzer revealed that one of the Tyler1-TramR clones exhibited altered metabolism compared to the parental Tyler1 characterized by decreased basal and maximal oxygen consumption rates, diminished spare respiratory capacity, and decreased glycolytic reserve. Collectively, these results demonstrate that spontaneous, BRAF mutant cTCC can be utilized as a translational model for investigating novel targeted and immune-based therapies that may improve treatment in both canine and human MAPK-driven cancers.
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Subject
canine
BRAF
transitional cell carcinoma