Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in triple-receptor negative breast cancer

Dean C Singleton,Anne-Lise Dechaume,Euphemia Y Leung,Bruce C Baguley,William P Katt,Pamela M Murray

doi:10.1186/s12885-020-06885-3

Abstract

BackgroundGlutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients.MethodsA panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity.ResultsThe anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness.ConclusionThis study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.

Highlights

Glutamine serves as an important nutrient with many cancer types displaying glutamine dependence
We demonstrate that paracrine secretion of de novo produced pyruvate into the extracellular environment can act as a source of pyruvate and this process can be antagonised using a monocarboxylate transporter 1 (MCT1) inhibitor
Breast cancer cell lines display differences in sensitivity to pharmacological glutaminase inhibition depending on culture medium composition MDA-MB-231 cells grown in Minimum Essential Medium α modification (αMEM) + 5% foetal bovine serum (FBS) were treated with CB-839 and drug sensitivity was assessed 3 days later by IC50 analysis

Summary

Introduction

Glutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Many Triple-receptor Negative Breast Cancer (TNBC) cell lines are dependent on glutamine for growth and viability [5, 6] These cells acquire glutamine and convert it to glutamate in a reaction catalysed by mitochondrial glutaminase – predominantly the GAC splice variant encoded by the GLS gene [5, 6]. The glutamate derived from glutamine has many uses, including glutathione synthesis or further metabolism to α-ketoglutarate (αKG) by glutamate dehydrogenase/aminotransferase-catalysed reactions [1, 4] This αKG contributes to numerous biosynthetic and epigenetic processes or can act as an anaplerotic substrate to replenish tricarboxylic acid (TCA) cycle metabolites that have been exported from the mitochondria for the production of biomass [3]. Once αKG enters the TCA cycle it can support TCA cycle flux either through oxidative decarboxylation or reductive carboxylation [7,8,9,10,11]

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