Abstract 383: In vitro effects of PI3KmTOR inhibition in canine hemangiosarcoma

While extremely rare in humans, hemangiosarcoma (HSA) accounts for nearly 2 of canine neoplasia, and is characterized by both aggressive local growthinvasion and a high rate of metastasis. Both canine and human HSA exhibit sustained aberrant PI3KAktmTOR pathway signaling. The purpose of this study was to examine the in vitro effects of a novel dual PI3KmTOR inhibitor, VDC-597, in canine HSA cells. Three canine HSA cell lines (DEN-HSA, CIN-HSA, and SB-HSA) were employed in multiple in vitro assays. Western analysis evaluated activation (phosphorylation) of key downstream pro-survival proteins in the PI3KmTOR pathway. Changes in tumor cell growthapoptosis were assessed using both bioreductive assays (Alamar Blue) and in vitro live-cell imaging (IncuCyte), in the presence and absence of doxorubicin, a standard-of-care cytotoxic drug for canine and human sarcomas, as well as in the presence and absence of U-0126, an inhibitor of the MEK pathway. Migration was assessed using both Boyden chamber and scratch assays, via live-cell imaging (IncuCyte). Matrigel invasion was assessed using traditional Boyden chambers. Finally, ELISA was utilized to quantify relative expression of vascular endothelial growth factor (VEGF). VDC-597 suppressed activation of both Akt and 4eBP1 in canine HSA cells in a dose- and time-dependent fashion, with an IC50 of approximately 0.3 uM, a concentration predicted to be clinically achievable based on preliminary early-phase canine and human studies. VDC-597 dose-dependently reduced proliferation, migration, invasion, and VEGF expression in HSA cells, while promoting tumor cell apoptosis. VDC-597 demonstrated additive antiproliferative effects when combined with doxorubicin and U-0126. Together, these results suggest that inhibitors of the PI3KAktmTOR pathway may act against multiple components of the neoplastic process, including proliferationapoptosis, chemosensitivity, invasionmigration and angiogenesis, and justify the evaluation of PI3KmTOR inhibitors in canine, and eventually human, HSA. Experiments to examine the effect of VDC-597 in reducing tumor burden and metastasis in a rodent model are ongoing.