Abstract 324: Investigating the dependence of canine bladder transitional cell carcinoma on activated mutant BRAF
Authors: Cronise Kathryn , Hernandez Belen , Gustafson Daniel L. , Duval Dawn L. 
Published: July 2017

Transitional cell carcinoma (TCC) accounts for approximately 2% of diagnosed malignancies in canines. The majority of canine TCCs are invasive, intermediate to high grade tumors sharing similarities with human muscle invasive TCCs in risk factors, histology, sites of metastasis, and therapeutic response to single agents. Whole exome sequencing of canine TCC tumors was performed to identify somatic mutations in known cancer driver and repressor genes that could potentially contribute to canine TCC pathogenesis. A valine to glutamic acid mutation in BRAF homologous to the activating V600E mutation identified in human melanoma, colorectal and thyroid cancers was identified in 70% of sequenced tumors. Sensitivity to the BRAFV600E inhibitor Vemurafenib was tested in three BRAF mutant canine TCC cell lines (Bliley, Tyler1 and Tyler2) and two BRAF wild type canine TCC cell lines (Angus1 and Kinsey). All five canine TCC cell lines exhibited IC50s greater than 10μM, with BRAF mutant cell lines being slightly more sensitive. These sensitivity ranges are similar to those of some mutant human colorectal cancer cell lines, indicating that additional mechanisms may contribute to Vemurafenib resistance. Western blot analysis was performed to measure relative abundance of pERK, a downstream target of BRAF, in canine TCCs in response to serum starvation. All TCC cell lines showed sustained pERK expression in the absence of serum, indicating constitutive activation of the MAPK pathway. The five canine TCC lines were treated with 15μM Vemurafenib for 6 and 24 hours and their lysates were analyzed for pERK protein expression. pERK abundance was decreased in only the BRAF mutant cell lines after 6 hours of treatment. However, this decrease was less pronounced after 24 hours, suggesting that resistance mechanisms are bypassing BRAF to activate ERK. Sequence analysis of an additional panel of formalin-fixed paraffin embedded canine TCCs also revealed a mutation in RanBP2 in 31% of samples. Strikingly, the RanBP2 mutation appeared to be mutually exclusive to BRAF V to E mutant tumors with only two of the analyzed samples carrying both mutations. Significant tumor heterogeneity was implicated due to low level mutant amplification in these samples. It has been reported that loss of RanBP2 is synthetic lethal in BRAF V600E mutant colorectal cancer. Since RanBP2 forms complexes with CRM1 at the nuclear pore complex for nuclear export and at the kinetochore during mitosis, canine TCC cell lines were treated with KPT-185, a CRM1 inhibitor. BRAF mutant TCC cell lines had IC50 values ranging from 45nM to 65nM and were approximately ten-fold more sensitive than wild type cell lines. Overall, this data indicates that the pathogenesis of canine TCC likely depends on driving factors in addition to constitutive BRAF signaling, but Vemurafenib resistant BRAF mutant tumors can be targeted through inhibition of the nucleopore complex.

Citation Format: Kathryn Cronise, Belen Hernandez, Daniel L. Gustafson, Dawn L. Duval. Investigating the dependence of canine bladder transitional cell carcinoma on activated mutant BRAF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 324. doi:10.1158/1538-7445.AM2017-324