Targeting the glutamine metabolic pathway enhances sensitivity to BH3 mimetics in different malignancies

The development of chemoresistance is a major obstacle facing several promising therapies in cancer. Resistance to anti-cancer agents occur via different mechanisms, including failure to undergo apoptosis. Apoptosis is a sophisticated cell death signalling pathway that is mainly regulated by the BCL-2 family of proteins, which can be anti- or pro-apoptotic. The anti-apoptotic members, such as BCL-2, BCL-XL, MCL-1, BCL-w and BCL-2A1, are highly expressed in many cancers. These proteins antagonise apoptosis by binding and inhibiting pro-apoptotic members of the BCL-2 family. The balance among the different anti- and pro-apoptotic members dictates the fate of the cell towards survival or death. Small molecule inhibitors of the anti-apoptotic BCL-2 family members called BH3 mimetics have been developed in the last few years. BH3 mimetics that specifically inhibit BCL-2 (ABT-199), BCL-XL (A-1331852) and MCL-1 (A-1210477 and S63845) demonstrate enormous potential to improve treatment in a wide variety of cancers. Of these, ABT-199 has been used successfully in several haematological cancers but resistance to ABT-199 is starting to emerge. Since all BH3 mimetics share a similar mechanism of action, it is likely that resistance will also emerge for other BH3 mimetics. Therefore, the aims of this study were to (1) generate simple resistance models to the different BH3 mimetics, to mimic the rapid chemoresistance observed in the clinic, (2) identify novel combination therapies to overcome such chemoresistance, (3) characterise the underlying mechanisms by which the combination therapies could overcome chemoresistance and (4) extend these observations to other malignancies. In the first results chapter, data presented discussed four different models of resistance to the different BH3 mimetics in three haematological cancer cell lines. In all these models, resistance was attributed neither to consistent changes in expression levels of the anti-apoptotic proteins nor to interactions among different pro- and anti-apoptotic BCL-2 family members. However, resistance to a particular BH3 mimetic was overcome by exposing cells to BH3 mimetics targeting the other anti-apoptotic members, suggesting redundant functions of multiple BCL-2 family members in regulating apoptosis. Since targeting multiple members of the BCL-2 family members together could be limited due to potential toxicity, other ways to tackle this issue were explored in the second results chapter. Targeting the uptake of glutamine as well as its downstream metabolic pathways, either by genetic knockdown or pharmacological inhibitors, overcame resistance to BH3 mimetic-mediated apoptosis. Interestingly, the enzyme that regulates glutamine metabolism, glutaminase (GLS) interacted exclusively with the critical pro-survival BCL-2 family member in all cell lines. For instance, GLS interacted with BCL-2 in MAVER-1 (BCL-2 dependent cell line), with BCL-XL in K562 (BCL-XL dependent cell line) and interacted with MCL-1 in H929 (MCL-1 dependent cell line), suggesting that GLS played a crucial role in apoptosis and potentially, chemoresistance. The third results chapter is aimed at further exploring the downstream signalling pathway of glutamine metabolism for their roles in overcoming resistance to BH3 mimetics. Targeting the different downstream pathways namely: reductive carboxylation, lipogenesis and cholesterogenesis also overcame resistance to BH3 mimetics. Furthermore, the findings highlighted the possibility that repurposing widely used drugs, such as statins, to target intermediary metabolism could improve the efficacy of BH3 mimetic therapy in haematological malignancies. In the final chapter, the validity of these findings in other malignancies was tested by extending these observations in cell lines derived from head and neck squamous cell carcinoma (HNSCC). Targeting GLS reduced the clonogenic potential of a panel of HNSCC cell lines, thus demonstrating a promising combination that could potentially improve therapy in HNSCC. Moreover, GLS expression assessed in tissue microarrays containing tumour cores from hundreds of HNSCC patients revealed that expression levels of GLS was high in the tumour core of oral cavity cancer patients and could be a prognostic marker in HNSCC. Overall this study demonstrates that resistance to BH3 mimetics in haematological malignancies could be overcome by targeting distinct enzymes regulating intermediary metabolism. Moreover, this study demonstrates that targeting glutamine metabolism could be a promising therapeutic strategy in head and neck cancer