Abstract C113: The monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 triggers MCT4-dependent lactate accumulation and blocks pyruvate-lactate exchange in human cancer cells

Abstract

Abstract Background: Monocarboxylate transporters (MCTs) are key modulators of lactate homeostasis and represent promising metabolic targets for molecular cancer therapeutics. The MCT1 inhibitor AZD3965 is now in clinical trial and understanding the impact of this drug on tumour cell metabolism may enable the discovery of pharmacodynamic (PD) biomarkers of target inhibition that will support the clinical development of such agents. Since MCT1 mediates the bidirectional transport of lactate and other monocarboxylates including pyruvate, here we use NMR spectroscopy to investigate the effect of AZD3965 on a) intracellular lactate levels and b) hyperpolarized 13C-pyruvate-lactate exchange, as biomarkers for MCT1 inhibition in human cancer cells with varying MCT4 expression (predictive of resistance to AZD3965). Materials and Methods: Human Raji (MCT4-) and Hut78 (MCT4 low (+)) lymphoma as well as HT29 (MCT4 high (+++)) colon carcinoma cells were treated with either 5nM or 500nM AZD3965 for 24h and levels of intracellular lactate determined by 1H NMR of cell extracts. Raji cells were also exposed to additional AZD3965 concentrations spanning 1nM-500nM. For 13C-pyruvate-lactate exchange studies, Raji cells were treated with either 5nM or 25nM AZD3965 for 24h. Cells were then incubated at 37°C in FBS-free medium and dynamic 13C NMR spectra acquired for 4 minutes with 2s intervals immediately after the addition of 10mM hyperpolarised [1-13C]pyruvic acid and 10mM unlabelled lactate. The ratio of the area under the curve for the summed lactate and pyruvate signals (LacAUC/PyrAUC) was determined to estimate pyruvate-lactate exchange. Data represent mean±SE. Results: 24h exposure to a low concentration of AZD3965 (5nM) led to increased intracellular lactate in MCT4- Raji and MCT4+ Hut78 human lymphoma cells to 2.65-fold and 10-fold respectively (p = ≤0.02) while the effect in MCT4+++ HT29 human colon carcinoma cells was insignificant (167±31% of controls, p = 0.1). Exposure to a high concentration of AZD3965 (500nM) increased intracellular lactate accumulation in HT29 cells albeit to a lesser degree than in Raji and Hut78 cells (4-fold (0.02) vs. 14 to 15-fold (p≤0.01) in the lymphoma lines). This effect is consistent with the expected blockade of lactate release in cells with low or no MCT4 expression following MCT1 inhibition. Lactate build-up in Raji cells was AZD3965 concentration-dependent being observed with as little as 1nM (up 1.7-fold), reaching a maximum at 25nM (12-fold) and plateauing thereafter. Analysis of the hyperpolarized 13C NMR data showed a significant decrease in LacAUC/PyrAUC to 31±6% in 5nM and 19±2% in 25nM AZD3965-treated Raji cells relative to controls (p<0.001). These results are consistent with MCT1-mediated transport of 13C-pyruvate being a rate limiting step in the 13C NMR-observed pyruvate-lactate exchange. Conclusions: Our data show that AZD3965 triggers intracellular lactate accumulation in a concentration- and MCT4 expression-dependent manner and inhibits 13C-pyruvate-lactate exchange (via blockade of 13C-pyruvate uptake). Intracellular lactate and hyperpolarized 13C-pyruvate-lactate exchange measurements are translatable to in vivo imaging studies and are therefore promising non-invasive metabolic biomarkers for AZD3965 and potentially other MCT1 inhibitors. Citation Format: Mounia Beloueche-Babari, Slawomir Wantuch, Markella Koniordou, Harry G. Parkes, Vaitha Arunan, Thomas R. Eykyn, Paul D. Smith, Martin O. Leach. The monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 triggers MCT4-dependent lactate accumulation and blocks pyruvate-lactate exchange in human cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C113.