Metabolomic Profiling Identifies Mechanisms Regulating Hypoxia-Induced Drug Resistance In Multiple Myeloma

Multiple Myeloma (MM) is the second most prevalent hematological malignancy and remains incurable, with a median survival of 3-7 years. However, despite the success of the new treatments, most patients still succumb to their disease. In about 20-25% of high-risk patients, MM progresses rapidly and does not respond to conventional therapies leading to rapid extramedullary disease and demise of these patients. One such regulator of dissemination and drug resistance is the dynamic process of oxygen deprivation or hypoxia. A number of studies show that hypoxia promotes neo-angiogenesis, cancer progression, epithelial-mesenchymal transition (EMT), acquisition of metastasis potential and stem-cell features, as well as resistance to therapy by activating adaptive transcriptional programs. Targeting hypoxia, and the metabolic pathways regulated by hypoxia in the tumor cells, could lead to novel opportunities for cancer therapy. Rapidly proliferating hypoxic cancer cells undergo a “metabolic switch” to anaerobic glycolysis. This altered energy metabolism has been shown to be associated with activated oncogenes and mutant tumor suppressors, which are more prevalent in patients with high-risk MM. We aimed 1) to examine the role of HIF-1a and HIF-2ain regulating drug resistance in vitro and in vivo and 2) to identify specific hypoxia-regulated genes and regulators of energy metabolism leading drug resistance in MM.

The effect of hypoxia was analyzed in different MM cell lines (MM1S, RPMI8226, U266 and H929) in basal conditions and after the treatment with bortezomib, dexamethasone or melphalan. The cytotoxicity was analyzed by means of MTT assay. Apoptosis studies were performed by flow cytometry. Gene expression profile of MM1S cells treated with bortezomib was compared in normoxia vs hypoxia using D-chip and GSEA softwares. Genes with expression changes greater or lower than 2 fold in either direction were selected. HIF1A and HIF2A knockdowns were performed in MM1S using lentiviral vectors. For metabolite collection, samples were re-suspended using HPLC grade water for mass spectrometry and analyzed using a 5500 QTRAP hybrid triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu). A total of 254 endogenous water soluble metabolites were analyzed.

We observed that MM cell lines were resistant to bortezomib and melphalan in hypoxic conditions (12 hours at 0.5% of oxygen levels) compared to normoxic conditions. At transcriptional and protein level. cells treated with bortezomib in hypoxic conditions affected a large number of genes/proteins involved in cell cycle such us p21, p53 and p57, cell death and glucose metabolism. However, cell cycle arrest was not responsible for the resistance of MM cells to bortezomib that was observed in hypoxic conditions. Therefore, we investigated mechanisms that are mediated by hypoxia and can regulate drug resistance. HIF1A knockdown restored the effect of bortezomib in MM1S and increased the percentage of apoptosis in cells treated with bortezomib under hypoxic conditions. To further explore the role of hypoxia in the regulation of tumor metabolism downstream of HIF1A, metabolomic studies were performed to characterize metabolic alterations following bortezomib treatment in hypoxic and normoxic conditions. This analysis revealed that hypoxic tumor cells treated with or without bortezomib show significant metabolic changes involving multiple pathways, the most significant of which are intermediates in glucose metabolism such us glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate and phosphoenolpyruvate. We also observed a decrease in measured tricarboxylic acid cycle (TCA) cycle intermediates (citrate, fumarate and malate) after hypoxia exposure and a significant increase of LDHA levels. We assessed the metabolic response to several drugs and shRNAs targeting different glycolytic enzymes (HK2, PFKBP3, PFKBP4 and LDHA). Of these, the most significant changes were observed with LDHA knockdown where these overcame resistance to bortezomib in hypoxic conditions.

Hypoxic conditions are essential for drug resistance and glucose utilization. These data provide new therapeutic targets and associated biomarkers for the treatment of Multiple Myeloma.

Onyx: Membership on an entity’s Board of Directors or advisory committees; BMS: Membership on an entity’s Board of Directors or advisory committees; BMS: Research Funding; Sanofi: Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees.