Disrupting Mitohormesis As a Novel Therapeutic Strategy for Multiple Myeloma (MM) Including Those with High Risk Disease and Proteosome Inhibitor Resistance

Background: Moderate mitochondrial stress induced by multiple mediators but most notably ROS can lead to activation of persistent mito-protective mechanisms termed "Mitohormesis". As a result of massive protein synthesis, malignant plasma cells (PCs) from MM patients (pts) undergo substantial ER stress but in addition high rates of Ig synthesis contributes to overproduction of ROS. We hypothesized that MM cells exploit mitohormesis to maintain ROS in the hometic zone, thereby increasing mitochondrial fitness to avoid apoptosis. We therefore set out to determine if the processes of mitohormesis are activated in MM and whether unmitigated mitochondrial stress can be exploited as a therapeutic strategy in MM.

Results: Protective stress mechanisms of mitohormesis include the activation of the mitochondrial UPR (UPR ^MT),a mitochondrial-to-nuclear signaling pathway mediated by CHOP and ATF5 that upregulates mitochondrial import proteins, chaperones and proteases to maintain mitochondrial proteastasis. We first demonstrated that UPR ^MT activation occurs with progression from precursor to overt MM. Using a UPR ^MTgene signature derived from published gene-sets we observed upregulation of UPR ^MT genes in single-cell RNA sequencing (scRNA-seq) data generated from PCs derived from Vκ*MYC mice (a transgenic mouse model of MM) spanning the spectrum of the disease. UPR ^MT gene signature scores in PCs from mice increased with disease progression with the highest levels found in late-MM> int-MM> early MM>wild type mice. Similarly, analysis of publicly available gene expression datasets (GSE6477) that includes normal donors, MGUS and newly diagnosed MM (NDMM) revealed higher expression of UPR ^MT genes in the majority of NDMM, weak expression in MGUS and absence in normal PCs. To assess the impact of UPR ^MT expression on pt outcomes we calculated a UPR ^MT index score derived from the median expression of 12 mtUPR classifier genes across the MMRF CoMMpass dataset of NDMM pts. Stratifying pts by UPR ^MT expression score we found that pts in the top quartile had a significantly shorter PFS and OS compared to pts with the lowest quartile weighted score.

Next, we postulated that perturbation of the mitochondrial import protein, Translocase of the Inner Membrane 23 (TIM23) would exaggerate mitochondrial stress as mitochondrial import efficiency is a key regulator of the UPR ^MT. First, we demonstrated that TIM23 complex genes are enriched in pts from the CoMMpass dataset with poor risk (1q gain and PR gene signature) and that shorter PFS and OS is associated with a higher weighted score of TIM23 complex genes. We then demonstrated that genetic (shRNA) knockdown or pharmacologic inhibition of TIM23 with MB-10, a small molecule inhibitor of TIM23 induced apoptosis of MM cell lines and primary pt PCs. Further non-transformed cell lines, CD138 ^- non-MM cells and normal donor hematopoietic progenitor cells were less susceptible to the effects of MB-10. Consistent with activation of the UPR ^MT, treatment of MM cells resulted in increased cytosolic ATF4, CHOP and a shift of ATF5 to the nuclear fraction. Activation of the CHOP-dependent branch of the UPR ^MT resulted in in upregulation of mitochondrial-targeted proteins, cpn10 and ClpP. Interestingly, MB-10 also induced XBP1 splicing demonstrating that inhibition of TIM23 complex can simultaneously activate the IRE1/XBP1 branch of integrated stress response (ISR), This led us to hypothesize that targeting TIM23 as an alternative means of activating the ISR could overcome acquired resistance to proteosome inhibitors (PIs). Indeed, PI-resistant and parental isogenic cell lines were equally susceptible to MB-10 as measured by IC50 values of cell growth. Finally, we demonstrated that doxycycline inducible knockdown of TIM23 in a mouse xenograft model induced tumor regression with significantly small tumor volumes at the end of 17 days of doxycycline treatment compared to tumors expressing an inducible control vector.

Conclusions: These data demonstrate that mitohormesis and UPR ^MT activation is associated with MM progression and worse clinical outcomes. Further we show that disrupting mitochondrial protein import results in unmitigated mitochondrial stress that switches the UPR ^MT from an adaptive cytoprotective to cytotoxic proapoptotic response. Thus, targeting mitochondrial import proteins such as TIM23 may represent novel therapeutic targets for MM.