Targeting Glycolysis in Multiple Myeloma: Novel Strategies in the Treatment of Proteasome Inhibitor Resistant in Hypoxic Conditions

Introduction: Multiple myeloma (MM) is one of the hematological malignancy and characterized by the clonal expansion of plasma cells in the bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs, however, many patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Hypoxia is an important component of the bone marrow microenvironment. Hypoxia may increase myeloma cell survival. Because cells shift primarily to a glycolytic mode for generation of energy in hypoxic conditions, glycolytic activities can be targeted therapeutically in MM patients. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is responsible for maintaining the cellular levels of fructose-2,6-bisphosphate which is a regulator of glycolysis.

Materials and Methods: In this study, we investigated whether PFKFB was involved in myeloma cells in hypoxia condition. We also investigated whether PFKFB inhibitors could suppress myeloma cells and enhance the sensitivity of myeloma cells to proteasome inhibition.

Results: We first investigated the expression of PFKBP in the myeloma cell lines in hypoxia condition. PFKFB family contains four tissue-specific isoenzymes encoded by four different genes. We found expression of PFKBP3 and PFKBP4 were increased in hypoxia condition. We found gene expression of PFKBP3 and PFKBP4 were involved in myeloma cell lines and myeloma patient samples in hypoxia condition from the public microarray datasets (GSE80140 and GSE80545). In hypoxia condition, expression of hypoxia-inducible factor 1α (HIF1α) was increased and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was activated in myeloma cell lines. Expression of PFKBP3 and PFKBP4 were inhibited by HIF1α inhibitor and p38 MAPK inhibitor treatment. In the hypoxia condition, activity of proteasome inhibitors were reduced compared to normoxia condition. We next investigated whether PFKBP3 inhibitor, PFK158 and PFKBP4 inhibitor, 5MPN could inhibit the proliferation of myeloma cells. We found PFK158 and 5MPN treatment inhibited the growth of myeloma cells in a dose dependent manner in hypoxia condition. Combined treatment of myeloma cells with carfilzomib and PFK158 or 5MPN caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We found proteasomal activity was also reduced by carfilzomib and PFK158 or 5MPN treatment. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased after carfilzomib and PFK158 or 5MPN treatment. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential in cell death, which were induced by carfilzomib and PFK158 or 5MPN on myeloma cells. In the hypoxia condition and inhibitor treatment, glycolytic activities (e.g. glucose and lactate) were changed in myeloma cells.

Conclusion: The PFKBP3 and PFKBP4 are enhanced in hypoxia condition and involved in proteasome inhibitor sensitivity. Our data also suggested that administration of PFKBP3 and PFKBP4 inhibitors may be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in hypoxia condition.