Targeting Redox Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma

Proteasome inhibitors (PIs) like bortezomib (Btz) and carfilzomib (Crflz) induce an oxidative stress response in Multiple Myeloma (MM) cells. Oxidative stress is a key effector pathway in PI-induced cell death, and altered redox signaling has been implicated in the acquisition of PI resistance. The potential of redox as a therapeutic target/pathway for PI resistant MM has not been realized due to the absence of a precise molecular targeted strategy that exploits redox signaling in a way that attacks PI resistant cells while sparing normal cells. Therefore, we set out in this study to characterize redox adaptations that contribute to PI resistance in MM, and to use drug screening platforms to identify specific redox-targeted small molecules that restore PI sensitivity. Using multiple isogenic pairs of PI sensitive and resistant MM cell lines, we found that resistant cells exist under high basal levels of reactive oxygen species (ROS) and oxidation of protein thiols (i.e., oxidative damage). Resistant cells induce significantly higher relative levels of ROS following PI treatment, but exhibit no further increase in oxidative damage. By comparison, their PI sensitive counterparts have relatively low levels of basal and PI-induced ROS levels, but undergo significantly higher levels of oxidative damage following PI treatment. These findings demonstrate that PI resistance is associated with alterations in redox balance; they further suggest that PI resistant cells have acquired adaptations that allow them to survive under high basal levels of oxidative stress, and that provide protection from PI-induced oxidative damage. We also identified significant changes in cellular bioenergetics that are typical of PI resistant cells. Generally, PI resistant cells appear to be more metabolically efficient, relying on mitochondrial respiration as their primary source of ATP production. Specifically, PI resistant cells have higher basal oxygen consumption rates (OCR), expanded respiratory capacity, increased NAD(P)H levels and pyruvate dehydrogenase (PDH) activity, and nearly absent activation of the AMP kinase energy stress signaling pathway. Thus, the acquisition of PI resistance is associated with significant changes in redox balance as well as in cellular bioenergetics. Given these findings, we next used a cell-based drug screening method to screen for redox-targeted small molecules capable of restoring PI sensitivity to resistant cells. We screened a compound collection of known pro- and anti-oxidant small molecules with wide-ranging mechanisms of action. From this screen we identified compound E61, which demonstrated strong synergy with multiple PIs, including Btz, Crflz, ixazomib, and oprozomib. E61 induced an oxidative stress response characterized by a burst of ROS generation and oxidation of protein thiols, and synergistically enhanced the PI-induced oxidative stress response in resistant cells. The synergistic cytotoxic response to E61 and PI co-treatment was dependent on ROS, and was evident across several models of PI resistance, representing cells of diverse genetic backgrounds. While E61 enhanced PI-induced cell death in resistant MM cells, its effects were protective in normal cell types, including peripheral blood mononuclear cells (PMBCs) and lymphocytes from normal human donors. These findings suggest that compound E61 will have a wide therapeutic index in combination with PI therapy in preclinical mouse models of MM, a hypothesis that we are currently testing. All together, our findings identify specific redox and bioenergetics changes that are acquired by PI resistant MM cells. Furthermore, our work offers a novel redox-targeted small molecule, E61, to be used in combination with PI-based therapeutic regimens in refractory MM.