Improve proteasome inhibitor response by targeting NHE6-mediated endosomal-autophagic machaniery in multiple myeloma

Multiple myeloma (MM) remains incurable, and resistance to frontline proteasome inhibitors (PIs) is a major barrier to improving patient survival. Identifying new therapeutic targets that overcome PI resistance is therefore essential. Through transcriptomic analysis of PI resistance gene signatures across public datasets, we identified NHE6 ( SLC9A6 ) as a regulator of MM cell survival under treatment-induced stress. NHE6, an endosomal Na⁺/H⁺ exchanger that controls vesicular pH and membrane trafficking, has not previously been implicated in MM. We found that NHE6 is highly expressed in patient-derived primary MM cells and human MM cell lines, with further upregulation in PI-resistant cell lines. Elevated NHE6 expression levels correlated with inferior survival across independent patient cohorts. To examine the role of NHE6 in MM therapeutic response, we knocked out NHE6 expression ( sgSLC9A6 ) in a panel of MM cell lines (ARP-1, MM1S, RPMI8266 and H929). We found that sg SLC9A6 cells exhibited markedly enhanced PI-induced apoptosis in vitro , and significantly reduced tumor burden with prolonged survival in vivo . Since autophagy is a key survival mechanism under PI stress, we inhibited autophagy pharmacologically and found that PI-induced apoptosis was increased in sg Ctrl cells, making them similarly sensitive to sg SLC9A6 cells. These results demonstrate that the enhanced drug response caused by NHE6 loss is autophagy dependent. Consistent with this, sg SLC9A6 cells exhibited higher LC3B and p62 levels than sg Ctrl cells, and autophagy inhibition raised their accumulation in both groups to comparable levels, indicating that NHE6 loss blocks autophagic flux. Cellular fractionation localized NHE6 predominantly to early endosomes. Immunoprecipitation studies revealed that NHE6 promotes Rab7 recruitment and GTP loading, thereby accelerating early-to-late endosomal maturation and facilitating endosome-autophagosome fusion and amphisome formation. Pharmacologic inhibition of Rab7 enhanced bortezomib efficacy, like the effect of NHE6 loss. Analysis of the CoMMpass SM cohort supported the clinical relevance of this pathway: non-responders displayed significantly higher Rab7 and NHE6 levels, which correlated with poorer survival. In summary, our study identifies NHE6 as a regulator of endosomal maturation, contributing to PI resistance in MM. Suppressing NHE6 restores drug sensitivity and prolongs survival in preclinical MM models. Targeting NHE6-mediated endosomal dynamics presents a promising therapeutic strategy for MM patients.