Abstract C044: RP7214, a small molecule inhibitor of dihydroorotate dehydrogenase (DHODH), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML

Abstract

Abstract Background: Interventions that promote differentiation of immature blasts have been shown to be a promising therapeutic strategy for the treatment of AML. Inhibition of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme in pyrimidine synthesis, promotes AML cell differentiation besides impeding tumor cell proliferation via cell cycle arrest. RP7214 is a novel and potent DHODH inhibitor that inhibited DHODH activity in enzyme and PHA induced HWB/PBMC proliferation with IC50 and EC50 values of 7.8 & 2.5/0.60 nM respectively. The objective of this study was to evaluate the effect of combining RP7214 with Gilteritinib, a small molecule inhibitor of FLT-3, or cytarabine in preclinical models of AML. Methods: Cell growth following incubation with RP7214 (1 μM), Gilteritinib (0.25-1.5 µM), or a combination of both agents was determined in the AML lines, THP-1 and U-937. Apoptosis was evaluated following incubation of cell lines with compounds for 72 h, subsequent staining with Annexin-V-PE and 7-AAD, and analysis by flow cytometry. For cell cycle, cells were incubated with compounds for 72 h, fixed with 70% ethanol, incubated at 4°C for 3 h, and stained with Propidium Iodide prior to analysis by flow cytometry. AKT phosphorylation was determined by Western blotting. In vivo efficacy was evaluated in a MV-4-11 mouse xenograft model. Results: In the AML cell lines (THP-1 and U-937), addition of RP7214 potentiated Gilteritinib activity manifested by a significant (P<0.05) growth inhibition when compared to the activity of the individual agents. Similarly, the combination (3 µM RP7214 + 0.75 µM Gilteritinib) led to a 50% increase in the number of apoptotic cells besides increasing cells in the G2/M phase by 31%. Combining RP7214 (3 µM) and Gilteritinib (0.1 µM) reduced AKT phosphorylation by 54% when compared to Gilteritinib in the THP-1 cell line. In the MV-4-11 mouse xenograft model, RP7214 when administered orally at 30 mg/kg/BID for 21 days demonstrated significant (P<0.001) anti-tumor activity both as a single agent and in combination with Cytarabine with tumor growth inhibitions of 37 and 73 %, respectively. No adverse events or body weight changes were observed throughout the study period. Conclusions: Inhibition of DHODH by RP7214 represents a unique therapeutic strategy in AML that accentuates the effect of approved and standard of care drugs such as Gilteritinib and Cytarabine. The compound is currently being tested in Phase-1 enabling studies with human trials expected to commence in 2020. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Kumar V. Penmetsa, Swaroop Vakkalanka. RP7214, a small molecule inhibitor of dihydroorotate dehydrogenase (DHODH), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C044. doi:10.1158/1535-7163.TARG-19-C044