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Introduction Multiple Myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) and overproduction of immunoglobulin. The interaction of MM cells with the BM microenvironment was shown to play an important role in the drug resistance in MM. Hypoxia was shown to develop in the BM niche during progression of MM and to play a major role in the dissemination of MM cells to the new BM niches. In this study, we tested the effect of hypoxia on the proliferation, cells cycle, apoptosis and induction of drug resistance in MM cells. Methods MM cell lines were exposed to normoxia (21% O2) or hypoxia (1% O2) and the effect of hypoxia on cell survival (by MTT assay), apoptosis (by annexin-PI staining and analysis with flow cytometry) and cell cycle (by cell fixation and permeabilization, RNA degradation and DNA staining with PI, and analysis with flow cytometry) was tested. Moreover, the effect of hypoxia on the expression of PI3K pathway-associated proteins (p-PI3K, p-AKT and p-mTOR), cell cycle proteins (cyclin-D, cyclin-E, p-Rb and p27) and apoptosis proteins (cleaved PARP, caspase-3, Bcl-2, Bcl-Xl and Mcl-1) were studied. We tested the effect of hypoxia on the sensitivity of MM cells to different therapeutic agents using MTT assay after the MM cells were treated with increasing concentrations of bortezomib, carfilzomib and melphalan. Finally, we examined the effect of a HIF inhibitor on the sensitivity of MM cells to therapy. Results Hypoxia decreased the proliferation of MM cells and reduced phosphorylation of p-PI3K, p-AKT and p-mTOR. Similarly, MM cells exhibited G1 cell cycle arrest, decreased expression of cell cycle-associated proteins including cyclin-D3, cyclin-E, p-Rb, and increase in cell cycle inhibitory protein p27. However, hypoxia did not alter the apoptosis of MM cells, where neither apoptosis was detected in MM cells due to hypoxia using annexin-PI staining, nor pro-apoptosis proteins were activated due to hypoxia as shown by the unchanged levels of cleaved PARP, caspase-3, Bcl-2, Bcl-Xl, and Mcl-1 proteins. Moreover, we found that hypoxic cells displayed less sensitivity to bortezomib and carfilzomib, but it did not induce any change in melphalan activity. Treatment with carfilzomib or bortezomib (5nM for 48hrs) showed decreased survival of about 50% of normoxic cells, while no effect of the drugs was observed on survival in hypoxia. The same trends where observed in higher and lower concentrations of the drugs. Moreover, we found that treatment with a HIF inhibitor could partially rescue the sensitivity of MM cells to bortezomib and carfilzomib. Conclusions We report that hypoxia decreases the proliferation and cell cycle of MM cells without signs of apoptosis, and that hypoxia induces drug resistance to bortezomib and carfilzomib, an effect which was partially reversed by a HIF inhibitor. This data suggests the hypoxia signaling as a therapeutic target for sensitization of MM cells to therapy, and suggests the use of HIF inhibitors in combination with other drugs as a novel therapeutic strategy for treatment of MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

INTRODUCTION: Hypoxia and T-helper cell 1 (Th1) cytokine-driven inflammation are key features of rheumatoid arthritis (RA) and contribute to disease pathogenesis by promoting angiogenesis. The objective of our study was to characterise the angiogenic gene signature of RA fibroblast-like synoviocytes (FLS) in response to hypoxia, as well as Th1 and T-helper cell 2 (Th2) cytokines, and in particular to dissect out effects of combined hypoxia and cytokines on hypoxia inducible transcription factors (HIFs) and angiogenesis. METHODS: Human angiogenesis PCR arrays were used to screen cDNA from RA FLS exposed to hypoxia (1% oxygen) or dimethyloxalylglycine, which stabilises HIFs. The involvement of HIF isoforms in generating the angiogenic signature of RA FLS stimulated with hypoxia and/or cytokines was investigated using a DNA-binding assay and RNA interference. The angiogenic potential of conditioned media from hypoxia-treated and/or cytokine-treated RA FLS was measured using an in vitro endothelial-based assay. RESULTS: Expression of 12 angiogenic genes was significantly altered in RA FLS exposed to hypoxia, and seven of these were changed by dimethyloxalylglycine, including ephrin A3 (EFNA3), vascular endothelial growth factor (VEGF), adipokines angiopoietin-like (ANGPTL)-4 and leptin. These four proangiogenic genes were dependent on HIF-1 in hypoxia to various degrees: EFNA3 >ANGPTL-4 >VEGF >leptin. The Th1 cytokines TNFα and IL-1β induced HIF-1 but not HIF-2 transcription as well as activity, and this effect was additive with hypoxia. In contrast, Th2 cytokines had no effect on HIFs. IL-1β synergised with hypoxia to upregulate EFNA3 and VEGF in a HIF-1-dependent fashion but, despite strongly inducing HIF-1, TNFα suppressed adipokine expression and had minimal effect on EFNA3. Supernatants from RA FLS subjected to hypoxia and TNFα induced fewer endothelial tubules than those from FLS subjected to TNFα or hypoxia alone, despite high VEGF protein levels. The Th2 cytokine IL-4 strongly induced ANGPTL-4 and angiogenesis by normoxic FLS and synergised with hypoxia to induce further proangiogenic activity. CONCLUSION: The present work demonstrates that Th1 cytokines in combination with hypoxia are not sufficient to induce angiogenic activity by RA FLS despite HIF-1 activation and VEGF production. In contrast, Th2 cytokines induce angiogenic activity in normoxia and hypoxia, despite their inability to activate HIFs, highlighting the complex relationships between hypoxia, angiogenesis and inflammation in RA.