Investigating MGMT expression as a resistance mechanism to temozolomide in canine hemangiosarcoma cell lines

Hemangiosarcoma (HSA) is the most common splenic cancer diagnosed in dogs and despite treatment with surgery and adjuvant doxorubicin chemotherapy, long term prognosis remains poor. Attempting to improve outcomes, several clinical studies have assessed combination chemotherapy protocols; however, none appear vastly superior and are generally more toxic compared to single-agent doxorubicin. Temozolomide (TMZ), an oral chemotherapy drug, has favorable safety data available when combined with doxorubicin in dogs. TMZ exerts anticancer efficacy by methylating nucleotide bases. Overexpression of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein, is a major determinant of TMZ resistance. This study investigates canine HSA cell line sensitivity to TMZ and assesses for correlation with MGMT expression. If such correlation exists, MGMT expression may be explored as a biomarker to stratify patients that may benefit from the combination of doxorubicin and TMZ. MGMT expressions were investigated in five canine HSA and one canine endothelial cell lines by qPCR and western blot. Cytotoxicity assays (± an MGMT inhibitor, O 6 -benzylguanine) were performed to assess the sensitivity of canine HSA cell lines to TMZ. MGMT was variably expressed by the canine HSA cell lines and expression patterns were comparable between gene and protein assessments. Overall, canine HSA cell lines are considered resistant to TMZ, and their resistance did not correlate with MGMT expression levels. This in vitro investigation demonstrates that MGMT status cannot predict sensitivity to TMZ and therefore suggests canine HSA cells have an MGMT-independent resistance mechanism. These in vitro data do not support the clinical exploration of TMZ in combination with doxorubicin for treating dogs with HSA.