Abstract 4875: Addition of RP10107, a novel and potent glutaminase inhibitor, accentuates 5-FU activity in lung cancer cell lines in vitro
Authors: Viswanadha Srikant , Eleswarapu Satyanarayana , Nyayapathy Seeta , Vakkalanka Swaroop 
Published: August 2018

Abstract
Background: Drug resistance remains a significant limitation to the clinical use of 5-Fluorouracil (5-FU) in lung cancer. With cancer cell metabolism emerging as a critical regulator of tumor progression, combining 5-FU with an inhibitor of the metabolic machinery represents a potential therapeutic strategy to prevent lung cancer progression. Cancer cell metabolism is reprogrammed wherein glutamine utilization is increased via elevation of glutaminase activity thereby generating the necessary substrates required for eventual ATP synthesis and energy production. RP10107 is a novel, potent, and selective glutaminase (GLS-1) inhibitor that demonstrated high potency against mouse (IC50=21.2 nM), rat (IC50=18.2 nM) and human (IC50=26.4 nM) enzymes with selectivity over GLS-2 (&gt;380-fold). The objective of this study was to evaluate the effect of a combination of 5-FU and RP10107 in lung cancer cells.
Methods: Glutamate concentrations in lung cancer cell lines (A549, NCI-H460, NCI-H1975, NCI-H441, and NCI-H2170) following treatment with RP10107 was estimated by LC-MS/MS. Synergism with 5-FU was determined using different concentrations of the compounds in a 5 x 5 grid. Synergism, additivity, or antagonism was calculated based on the BLISS score. Apoptosis was determined by Annexin V/7AAD staining using a MUSE® Annexin V and dead cell assay kit (Millipore) while the effect on cell cycle was estimated using Guava Cell Cycle Reagent (Millipore). Expression of caspase 3/7, phosphor-p53 (S15), and Bcl-2 were determined by Western Blotting.
Results: Treatment of lung cancer cell lines with RP10107 resulted in an increased ratio of glutamine to glutamate with a doubling noticed between 100-300 nM. The change in glutamine utilization was accompanied by a dose-dependent inhibition in cell growth with half-maximal inhibitions ranging from 1.3 to 9.0 μM. Synergism or additivity between RP10107 and 5-FU for anti-proliferative activity was noticed in all the cell lines tested with effect being most pronounced in NCI-H460 and NCI-H441. Additionally, incubation with the combination (1-5 μM RP10107 + 3-5 μM 5-FU) for 48-72 h caused a G2/M or S-phase arrest with a corresponding increase in the percent of apoptotic cells (1-5 to 2.5-fold) compared to the individual agents. Combination of RP10107 and 5-FU increased expression of phospho-p53 expression in all cell lines tested while it led to 40-75% reduction in caspase 3, caspase 7, and Bcl-2 in NCI-H2170 and NCI-H460.
Conclusions: Addition of RP10107, a potent glutaminase inhibitor, potentiated 5-FU activity in lung cancer cell lines. Findings provide a rationale for use of the combination in future clinical trials involving lung cancer patients thereby providing a safer and more effective alternative to currently available therapy.
Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Seeta Nyayapathy, Swaroop Vakkalanka. Addition of RP10107, a novel and potent glutaminase inhibitor, accentuates 5-FU activity in lung cancer cell lines in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4875.