Cancer cell culture has been a backbone in cancer research, in which analysis of human cell line mutational profiles often correlates with oncogene addiction and drug sensitivity. We have conducted whole-exome sequence analyses on 33 canine cancer cell lines from 10 cancer types to identify somatic variants that contribute to pathogenesis and therapeutic sensitivity. A total of 66,344 somatic variants were identified. Mutational load ranged from 15.79 to 129.37 per Mb, and 13.2% of variants were located in protein-coding regions (PCR) of 5,085 genes. PCR somatic variants were identified in 232 genes listed in the Cancer Gene Census (COSMIC). Cross-referencing variants with human driving mutations on cBioPortal identified 61 variants as candidate cancer drivers in 30 cell lines. The most frequently mutated cancer driver was TP53 (15 mutations in 12 cell lines). No drivers were identified in three cell lines. We identified 501 non-COSMIC genes with PCR variants that functionally annotate with COSMIC genes. These genes frequently mapped to the KEGG MAPK and PI3K-AKT pathways. We evaluated the cell lines for ERK1/2 and AKT (S473) phosphorylation and sensitivity to the MEK1/2 inhibitor, trametinib. Twelve of the 33 cell lines were trametinibsensitive (IC 50 < 32 nmol/L), all 12 exhibited constitutive or serum-activated ERK1/2 phosphorylation, and 8 carried MAPK pathway cancer driver variants: NF1(2), BRAF(3), N/KRAS(3). This functionally annotated database of canine cell line variants will inform hypothesis-driven preclinical research to support the use of companion animals in clinical trials to test novel combination therapies.

Canine transitional cell carcinoma (TCC) of the bladder accounts for 2% of diagnosed canine cancers. Most TCCs are inoperable and unresponsive to traditional chemotherapy. Median survival time is typically less than a year for all treatments indicating a need for more effective therapies. Recent studies have discovered that approximately 70% of canine TCCs harbor mutations in the proto‐oncogene BRAF, a kinase involved in the mitogen‐activated protein kinase (MAPK) pathway that controls cell proliferation, differentiation, and apoptosis. BRAF mutations are present in several human cancers, most of which exhibit adaptive or intrinsic resistance to BRAF‐targeted inhibitors. The goals of this study were to (1) further characterize the role of mutant BRAF in canine TCC and (2) determine whether inhibition of the MAPK pathway alone or in combination with other gene targets may be an effective therapy for TCC treatment. Analysis of ERK1/2 phosphorylation following serum starvation indicates that TCC cell lines exhibit constitutive MAPK pathway activation independent of their BRAF mutation status. MAPK activity was further quantified using gene expression analysis of ten MAPK target genes, revealing that TCC cell lines have significantly higher MAPK pathway activity compared to other canine cancer cell lines. These data suggest a causative role for MAPK signaling in TCC pathogenesis. To determine whether the MAPK pathway could be a therapeutic target for TCC treatment we assessed the effect of BRAF and MEK inhibition on TCC cell proliferation and ERK1/2 phosphorylation. Four BRAF mutant human cell lines with varying degrees of sensitivity to BRAF‐targeted agents were used to determine the relative sensitivity of canine TCC cell lines. BRAF mutant TCC cell lines were sensitive to BRAF inhibition with the “paradox‐breaking” inhibitor PLX7904 (IC50: 0.2–1.2μM), but not vemurafenib (IC50: 7–21μM). Both BRAF wild type and mutant TCC cell lines were sensitive to MEK inhibition with selumetinib (IC50: 15–420nM) and trametinib (IC50: 0.4–8nM). ERK1/2 phosphorylation decreased after 6‐hour treatments with MAPK inhibitors, but rebounded by 24 hours suggesting the presence of resistance mechanisms. Microarray analysis indicated that the ErbB family of receptors and ligands are up‐regulated in TCC cell lines relative to other canine cancer cell lines (fold‐change > 2, q < 0.05). Combined BRAF and ErbB inhibition synergized in the BRAF mutant Bliley TCC cell line, while combined MEK and ErbB inhibition synergized in both Bliley and BRAF wild type Kinsey cells. These findings suggest that targeting ErbB receptors with MAPK inhibition is a potential therapy for canine TCC treatment. Additionally, our data indicate that canine TCC may serve as a naturally‐occurring model for the study of resistance mechanisms to MAPK inhibition in human cancers. Support or Funding Information Morris Animal Foundation, Shipley University Chair in Comparative Oncology This abstract is from the Experimental Biology 2019 Meeting. There is no full text artic...

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis.Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets.We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

Transitional cell carcinoma (TCC) is the most common bladder cancer in both humans and dogs. The majority of canine TCCs are invasive tumors of intermediate‐ to high‐grade at diagnosis and result in a median survival of less than a year. Approximately 20% of human TCCs are muscle‐invasive tumors, which frequently metastasize and have a poor prognosis. Human and canine TCCs share similarities in risk factors, histopathology, and sites of metastasis. Thus, naturally‐occurring canine TCC has the potential to serve as a model for the assessment of novel therapeutics which may inform treatment modalities in human TCC. In this study, we performed whole exome sequencing of 11 canine TCC tumors and three matched normal tissue samples, identifying 581 variants in known protein coding genes. Protein coding variants showed enrichment of KEGG pathways involved in PI3K‐Akt signaling, axon guidance, focal adhesion, and chemokine signaling. Variants were screened against the COSMIC Cancer Gene Census, identifying 37 variants in 27 cancer‐related genes. BRAF V to E missense mutations were identified in 4/11 samples and later confirmed via Sanger sequencing in 73% of samples (8/11). Other cancer genes mutated in at least 2 samples include LRP1B, CUL3, MSH2, and RNF213. Microarray analysis was utilized to characterize the transcriptome of canine TCC relative to normal bladder. 802 and 1,327 genes were up‐ and down‐regulated in tumor samples, respectively. Up‐regulated genes were enriched for cell cycle, p53 signaling, and metabolic KEGG pathways. Down‐regulated genes include members of focal adhesion, ECM‐receptor interaction, and PI3K‐Akt signaling pathways. Gene set enrichment analysis was performed, identifying four hallmark gene sets enriched in tumors: E2F targets, G2M checkpoint, mitotic spindle, and interferon alpha response. A major advantage of the dog model is the ability to evaluate novel immunotherapies in spontaneous tumors that develop under normal immunosurveilance. However, a basic understanding of the immune landscape of canine cancers, such as TCC, is required. Using immunohistochemical labeling, we quantified CD3+ T cells and MAC387+ myeloid cells in TCC tumors. CD3+ T cell counts were variable, exhibiting a bimodal distribution, ranging from 1 to 415 cells/mm2, suggesting immunologically “hot” and “cold” tumors. MAC387+ cell counts ranged from 3 to 613 cells/mm2 and did not correlate with CD3+ T cell counts. Clustering of tumors using expression of immune‐related genes (GO:0002376) revealed two distinct groups. Group I exhibited a strong type I interferon gene expression signature, showing enrichment of hallmark gene sets for interferon alpha and gamma responses. Group II exhibited enrichment of hallmark gene sets for G2M checkpoint and E2F targets. The type I interferon signature was applied to human muscle‐invasive bladder tumors in the TCGA database and was associated with a better disease‐free survival. Overall, the results of this study establish the utility of canine bladder cancer as a model for identify...

Periostin is a matricellular protein important in regulating bone, tooth, and cardiac development. In pathologic conditions, periostin drives allergic and fibrotic inflammatory diseases and is also overexpressed in certain cancers. Periostin signaling in tumors has been shown to promote angiogenesis, metastasis, and cancer stem cell survival in rodent models, and its overexpression is associated with poor prognosis in human glioblastoma. However, the role of periostin in regulating tumorigenesis of canine cancers has not been evaluated. Given its role in bone development, we sought to evaluate mRNA and protein expression of periostin in canine osteosarcoma (OS) and assess its association with patient outcome. We validated an anti-human periostin antibody cross-reactive to canine periostin via western blot and immunohistochemistry and evaluated periostin expression in microarray data from 49 primary canine OS tumors and 8 normal bone samples. Periostin mRNA was upregulated greater than 40-fold in canine OS tumors compared to normal bone and was significantly correlated with periostin protein expression based on quantitative image analysis. However, neither periostin mRNA nor protein expression were associated with time to metastasis in this cohort. Gene Set Enrichment Analysis demonstrated significant enhancement of pro-tumorigenic pathways including canonical WNT signaling, epithelial-mesenchymal transition, and angiogenesis in periostin-high tumors, while periostin-low tumors demonstrated evidence of heightened antitumor immune responses. Overall, these data identify a novel antibody that can be used as a tool for evaluation of periostin expression in dogs and suggest that investigation of Wnt pathway-targeted drugs in periostin overexpressing canine OS may be a potential therapeutic target.