Hypoxia Induces Drug Resistance In Multiple Myeloma
Authors: Muz Barbara , Azab Feda , Puente Pilar De La , Ghobrial Irene M. , Vij Ravi , Azab Abdel Kareem 
Published: October 2019

Abstract

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.