Inhibition of Oxidative Phosphorylation and Glycolysis Reduces Viability in Multiple Myeloma By Affecting mTOR-Mediated Protein Synthesis

Introduction

Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Currently, MM patients are treated with combinations of multiple drugs, although drug resistance eventually occurs. Therefore, drug re-tasking may offer readily available, safe and affordable treatment options for relapsed/refractory MM patients. Metabolic changes, initiated by the hypoxic bone marrow environment, lead to both cancer cell survival and drug resistance. In this study, we aimed to evaluate the synergistic activity of monocarboxylate transporter (MCT) inhibition by syrosingopine with metformin on MM viability and survival.

Material and methods

Correlation between expression and survival was investigated using the MMRF cohort. In vitro, multiple MM cell lines (OPM-2, LP-1, RPMI-8226, JJN3, ANBL-6) were used, as well as the stromal cell line HS-5 and freshly isolated CD138+ cells from primary patients samples. A tracer study, whereby RPMI-8226 cells were supplemented with ¹³C-glucose, was performed to measure glucose-to-lactate conversion in normoxia and hypoxia (1% O₂). MCT expression was measured on protein level by western blot. MCT1 and MCT4 were inhibited by syrosingopine, while metformin was used to inhibit complex I of the electron transport chain. Effects on viability were measured by CellTiter Glo, lactate concentration by Lactate Glo, apoptosis by annexin V/7-AAD staining and proliferation by BrdU uptake. Effects on glycolysis and oxidative phosphorylation were measured by the Seahorse Glycolytic Rate assay. Pathways were investigated by western blot. All in vitro experiments were performed in hypoxic conditions.

Results and discussion

High MCT1 or MCT4 expression correlates with a significant lower overall survival. MCT4 expression is also significantly upregulated in MM cells from relapse/refractory patients compared to matched newly-diagnosed patients. Glucose-to-lactate conversion was increased in RPMI-8226 cells in hypoxia, confirming the Warburg effect in MM. JJN3 cells contained the highest expression of MCT1, while RPMI-8226 showed the highest expression of MCT4 alongside the HS-5 cells. Both MCT1 and MCT4 protein expression were increased after 48h of hypoxia.

MCT1/MCT4 dual targeting by syrosingopine increased intracellular lactate levels and decreased viability and proliferation in LP-1 and RPMI-8226. Apoptotic rates were increased.

As syrosingopine only increased cell death at higher doses, we investigated possible new combination strategies. Syrosingopine inhibits lactate transport, causing a feedback loop which limits glycolysis. As metformin inhibits complex I of the electron transport chain, blocking oxidative phosphorylation, we investigated whether targeting both crucial metabolic processes could increase apoptotic cell death in MM. Indeed, metformin increased syrosingopine-mediated cell death in MM cell lines. Seahorse analysis confirmed that metformin decreased oxygen consumption rate (OCR, indicator for oxidative phosphorylation) and syrosingopine decreased extracellular acidification rate (ECAR, indicator for glycolysis). Combination therapy reduced OCR and ECAR, blocking both crucial metabolic processes. Pathway investigation revealed a decrease in mTOR, p-p70S6K, p-S6, p-4EBP1 and p-eIF4E when MM cells were treated with both syrosingopine and metformin. Finally, the combination treatment also successfully decreased viability in CD138+ cells from primary patient samples compared to both single agents.

Conclusion

Syrosingopine reduces MM viability and proliferation while also increasing apoptotic cell death of MM cells. Metformin enhances syrosingopine-mediated cell death in both MM cell lines and primary patient samples. Moreover, combination treatment inhibits the crucial metabolic processes glycolysis and oxidative phosphorylation. A decrease in the mTOR-mediated protein synthesis pathway was also observed for the combination treatment. In conclusion, this combination strategy consisting of syrosingopine and metformin could form a new therapeutic approach to block MM progression and survival.

Inge Oudaert and Arne Van der Vreken contributed equally to this work.

De Veirman:Active Biotech: Research Funding.

Syrosingopine is a antihypertensive agent and MAO inhibitor that also inhibits MCT1/4. Syrosingopine is not approved by the FDA as a MCT1/4 inhibitor. Metformin is an anti-diabetic drug that also inhibits complex I of the electron transport chain. Metformin is not approved by the FDA as a complex I inhibitor.