Increased expression of Hypoxia-Inducible Factor 1α (HIF-1α) in breast tumours is associated with poor survival outcome. HIF-1α promotes phenotypic adaptations including metabolic reorganisation, angiogenesis and increased cell invasiveness. Notably, HIF-1α activity is increased in Triple receptor Negative Breast Cancer (TNBC) compared with other breast cancer subtypes. One reason for this elevated HIF-1α stabilisation in TNBC has been described; intracellular cysteine depletion which leads to impaired prolyl hydroxylase-domain (PHD) enzyme activity. Normally, cystine is acquired in exchange for intracellular glutamate using the xCT antiporter. However, in TNBC the excessive secretion and extracellular accumulation of glutamate impairs cystine exchange. Since the majority of glutamate produced in TNBC is derived from glutaminase-catalysed hydrolysis of glutamine, we hypothesised that glutaminase inhibition using a potent and selective inhibitor, CB-839, would improve glutamate/cystine exchange and reduce HIF-1α levels.

We detected normoxic HIF-1α expression in BT549, Hs578T and SUM159PT TNBC cell cultures. In these cases HIF-1α protein was destabilised upon treatment with CB-839 and the expression of some HIF-1α-target genes was significantly decreased (e.g. CA9, BNIP3, PDK1, LDHA) but not others (e.g. ADM and VEGFA). HIF-1α destabilisation corresponded with increased PHD-dependent hydroxylation, supporting the hypothesis that glutaminase inhibition can improve PHD function. Consistently, WT but not a proline hydroxylation-null mutant HIF-1α protein was depleted by CB-839. We confirmed that CB-839 decreased glutamate secretion but, unexpectedly, HIF-1α levels were not restabilised by addition of excess extracellular glutamate. Thus glutamine metabolism drives normoxic HIF-1α stabilisation via an additional mechanism distinct from perturbation of the glutamate/cystine exchange system.

To elucidate the underlying process we focused on the glutamine-derived metabolite (S)-2-Hydroxyglutarate [(S)-2HG], a competitive inhibitor of 2-Oxoglutarate (2OG)-dependent enzymes, including the PHD enzymes. Glutaminase inhibition decreased the levels of (S)-2HG. Furthermore, growing CB-839-treated cells in culture medium containing cell permeable (S)-2HG, but not (R)-2HG, could restabilise HIF-1α. In addition, increasing 2OG levels in these cells further stabilised HIF-1α, an effect that was blunted by inhibition of lactate dehydrogenase A (LDHA), a promiscuous source of (S)-2HG generated via reduction of 2OG.

Conclusion: CB-839 has demonstrated encouraging clinical responses in patients with heavily pretreated and therapy-resistant metastatic TNBC. Our work demonstrates that high levels of glutamine metabolism provoke HIF-1α stabilisation through the LDHA-catalysed production of (S)-2HG. Assessment of HIF-1α activity may be a useful approach to identify glutamine-addicted tumours and predict responsiveness to glutaminase inhibitor therapy.

Citation Format: Laura Cussonneau, Anne-Lise Dechaume, Pamela M. Murray, Henegama Liyanage, Tet-Woo Lee, Frederik Pruijn, Euphemia Y. Leung, Dean C. Singleton. HIF-1α stabilisation in triple receptor negative breast cancer is sensitive to glutaminase inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3561.