AbstractBackgroundMultiple myeloma (MM) is a hematopoietic malignancy for which proteasome inhibitors have become available in recent years. However, many patients develop resistance to these drugs during treatment. Therefore, it is important to elucidate the mechanisms underlying resistance acquisition by proteasome inhibitors. Side population (SP) cells, which have a high drug efflux capacity and hypoxic responses in the microenvironment have both provided important insights into drug resistance in MM; however, little is known about the characteristics of SP cells in hypoxic microenvironments.MethodsWe performed cDNA microarray analysis for SP and non‐SP obtained from RPMI‐8226 and KMS‐11 cell lines cultured for 48 h in normoxic and hypoxic conditions (1% O2). Genes specifically upregulated in hypoxic SP were examined.ResultsOur comprehensive gene expression analysis identifiedHMOX1,BACH2, andDUX4as protein‐coding genes that are specifically highly expressed in SP cells under hypoxic conditions. We have shown thatHMOX1/heme oxygenase‐1 (HMOX1/HO‐1) is induced by hypoxia‐inducible reactive oxygen species (ROS) and reduces ROS levels. Furthermore, we found that HMOX1 contributes to hypoxia‐induced resistance to proteasome inhibitors in vitro and in vivo. Excessive ROS levels synergistically enhance bortezomib sensitivity. In clinical datasets,HMOX1had a strong and significantly positive correlation withMAFBbut notMAF. Interestingly, hypoxic stimulation increasedMAFB/MafBexpression in myeloma cells; in addition, the knockdown of MAFB under hypoxic conditions suppressed HMOX1 expression.ConclusionThese results suggest that the hypoxia‐ROS‐HMOX1 axis and hypoxia‐induced MafB may be important mechanisms of proteasome inhibitor resistance in hypoxic microenvironments.