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

Abstract

Background: Glutamine 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. Methods: A 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. Results: The 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.

In Vitro and In Vivo Characterization of MCT1 Inhibitor AZD3965 Confirms Preclinical Safety Compatible with Breast Cancer Treatment

“…Upon AZD3965 treatment, proliferating cancer cells, but not nonmalignant cells, accelerated OCR linked to ATP production, hence their OXPHOS rates. Such increase could primarily result from inhibition of pyruvate export [ 53 ], but discrepancies between cancer and nonmalignant cells rather indicate that selective lactate export inhibition in cancer cells is a main contributor to the switch, notwithstanding a possible contribution of other available oxidative substrates (glucose, glutamine, lipids).…”

Targeting Glutaminolysis: New Perspectives to Understand Cancer Development and Novel Strategies for Potential Target Therapies

“…Noteworthy, reducing cell culture medium nutrients to physiological levels also compromised the sensitivity of lung cancer cells to glutaminase inhibitors (137). Singleton et al found that CB-839 activity was significantly compromised in three dimensional spheroids assay compared with two dimensional monolayer culture in TNBC cells (138) The questions then arise: what is the molecular mechanism of glutaminase inhibition resistance and how could we overcome the therapy resisatnce? Firstly, as the most frequently used glutaminase inhibitors such as BPTES and CB-839 are GLS1 selective, the resistance to glutaminase inhibition may be due to the differential expression of GLS1 and GLS2 in cells, as demonstrated in luminal and basal-like breast cancer cells (78).…”

“…Application of a pan-glutaminase inhibitor 968 suppresses BPTES-resistant breast cancer growth. Importantly, a number of studies have demonstrated that glutaminase inhibition could be rescued by alternative metabolic pathways, such as glycolysis and fatty acid oxidation (FAO) (130,138,142). A combinatorial strategy may help to overcome glutaminase inhibition resistance.…”

Targeting the Proline–Glutamine–Asparagine–Arginine Metabolic Axis in Amino Acid Starvation Cancer Therapy

“…Strictly speaking, Gln starvation therapy by targeting GLS is to block Gln consumption (utilization) rather attacking Gln itself. Treating breast cancer cells with GLS inhibitors decreases downstream metabolites of Glu, including those in the TCA cycle that requires anaplerosis for replenishment [20].…”