Abstract C042: Inhibition of Dihydroorotate Dehydrogenase (DHODH) by RP7214 attenuates growth and promotes differentiation of AML cell lines

Abstract

Abstract Background: Dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme in pyrimidine synthesis, is overexpressed in certain cancers; blockade of which impedes 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 single-agent activity of RP7214 on AML cell differentiation and growth. Methods: Viability of representative AML cell lines (U937, HL-60, THP-1, and KG-1) following incubation with RP7214 for 72 h as well as rescue in the presence of 300 µM Uridine was determined in an MTT-based assay. For differentiation experiments, THP-1 cells were incubated with RP7214 in complete media for 72 h. RNA was isolated, reverse transcribed to cDNA, and real-time PCR was performed to analyze CD11b gene expression. In addition, cell surface expression of CD11b in THP-1 cells incubated with RP7214 and the effect-reversal in presence of Uridine was evaluated by flow cytometry. Apoptosis and cell cycle were determined following incubation with RP7214 for 72 and 48 h respectively. Results: RP7214 inhibited AML cell proliferation in all the four cell lines tested with GI50 ranging from 2.4 to 7.6 µM. Addition of 300 µM Uridine caused a rightward shift with GI50 >10 µM. RT-PCR analysis indicated an 80-fold increase in mRNA expression of the differentiation marker CD11b in THP-1 cells treated with 3 µM RP7214. Similarly, expression of the CD11b marker on THP-1 cell surface was enhanced by 40% following incubation with 5 µM compound; effect of which was reduced to 15% in the presence of 300 µM Uridine. RP7214 dose-dependently induced apoptosis (30-50% at 3 µM) and arrested cycle progression in the “S-phase” (40-45% at 3 µM) in U937 and THP-1 cells. Conclusions: Results demonstrate the utility of RP7214 as a potent DHODH inhibitor in AML acting via suppression of cellular pyrimidine pools and promoting differentiation to fully mature cells. The compound is currently being evaluated in IND-enabling tolerability studies with Phase-1 trial in AML expected to commence in 2020. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Swaroop Vakkalanka. Inhibition of Dihydroorotate Dehydrogenase (DHODH) by RP7214 attenuates growth and promotes differentiation of AML cell lines [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 C042. doi:10.1158/1535-7163.TARG-19-C042