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.

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. Cell cycle and apoptosis studies were performed by flow cytometry. Proteomic changes induced after treatment were analyzed under normoxic and hypoxic conditions by western-blotting. Gene expression profile of MM1S cells treated with bortezomib was compared in normoxia vs hypoxia using D-chip software. 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 hypoxic conditions (12 hours at 0.7% of oxygen levels) suppressed the effect of melphalan and more significantly the effect of bortezomib. At the transcriptional level and protein level, we observed that cells treated with bortezomib in hypoxic conditions affected a large number of genes/proteins involved in cell cycle, cell death, glucose metabolism and the Wnt signaling pathway. Hypoxia blocked cell cycle progression, which was accompanied by p21, p53 and p57 up-regulation. In addition, apoptosis pathways were inhibited after exposure to hypoxia including inactivation of caspases 3, 8 and 9 and PARP cleavage. HIF1A and HIF2A knockdowns restore 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, metabolomic studies were performed to characterize metabolic alterations following bortezomib treatment. This analysis revealed that hypoxic tumor cells treated with bortezomib show significant metabolic changes involving multiple pathways, the most significant of which are intermediates in glucose and, sucrose metabolism. Bortezomib treatment under hypoxic conditions was accompanied by a significant decrease in UDP-D-glucose, UDP-D-glucuronate, and glutathione disulfide.

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.

Ghobrial:Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other.