Abstract 3029: Dual targeting of ARK5 and CDK4 pathways with ON 123300 as a therapeutic strategy for colorectal carcinoma

Introduction: This study describes the development of a novel dual specificity kinase inhibitor, ON 123300, which exhibits potent activity against colorectal cancers both in vitro and in vivo. While overexpression of Cyclin D1 is closely correlated with the proliferative rate of these tumor cells, metastatic colorectal cancers over-express ARK5, a member of the AMPK family and mediator of AKT activation. In this study, we show that ON 123300, which inhibits both CDK4/6 and ARK5, is a potent inducer of apoptosis of colorectal cancer cells when compared to palbociclib, a highly selective inhibitor of CDK4/6 kinases that does not target ARK5. Results & Conclusions: We examined the effects of palbociclib and ON 123300 on cell cycle progression, modulation of Rb and PI3K/AKT pathways, and induction of apoptosis in multiple colorectal cancer cell lines. Comparative kinase inhibition assays showed that while palbociclib and ON 123300 exhibited equivalent inhibition against CDK4/CDK6, ARK5 activity was inhibited only by ON 123300. When DLD1 and SW480 cells were incubated with increasing concentrations of palbociclib or ON 123300, both compounds were equally efficient in their ability to inhibit phosphorylation of all three members of the Rb family of proteins. However, when the phosphorylation status of proteins associated with the PI3K/AKT pathway was measured by western blot, ON 123300 showed concentration-dependent inhibition of mTOR, AKT, 4EBP1 and S6RB phosphorylation while palbociclib had little or no effect on the phosphorylation of these proteins. Cells treated with palbociclib rapidly accumulated in the G0/G1 stage of the cell cycle with increasing drug concentrations. Although cells treated with ON 123300 also arrested in the G0/G1 phase at lower concentrations (01-0.5 uM), with increasing concentrations of drug there was an accumulation of cells with sub-G1 DNA content, suggesting induction of apoptosis. ON 123300-treated cells showed cleavage of PARP and Caspases (3, 7 and 9) as well as inhibition of FOXO1 phosphorylation, which was not observed in cells treated with palbociclib. Since ARK5 belongs to the AMPK family of kinases, we next examined the effects of ON 123300-mediated ARK5 inhibition on metabolic changes of tumor cells that over-express this gene. Treatment of SW-480 colorectal cancer cells with ON 123300 resulted in an increase in glucose uptake, profound inhibition of glutamine uptake and reduced ATP production. A detailed metabolomic study revealed significant alterations in the levels of metabolites associated with glutamine metabolism. Nude mouse xenograft assays using Colo-205 cells revealed strong inhibition of tumor growth following 100 mg/kg of ON 123300 given QD or QOD, with little evidence of toxicity as measured by change in body weight. Thus, dual inhibition of ARK5 and CDK4 pathways could be an effective therapeutic strategy for the treatment of colorectal cancers. Citation Format: Saikrishna A. Divakar, M.V. Ramana Reddy, Stephen C. Cosenza, Stacey J. Baker, Vinee Purohit, Venugopal Gunda, Pankaj K. Singh, E. Premkumar Reddy. Dual targeting of ARK5 and CDK4 pathways with ON 123300 as a therapeutic strategy for colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3029.