Improve therapeutic response through inhibition of endosome machinery in multiple myeloma Free

With advanced therapies, the 5-year relative survival rate for multiple myeloma (MM) is about 58%, yet 15-20% of patients survive less than 3 years; and it remains incurable. As the most common frontline regimens, proteasome inhibitors, such as bortezomib and carfilzomib, offer a good initial response. However, most MM patients relapse and become refractory after treatment. Exploration of new therapeutic targets to overcome MM drug resistance is the prerequisite to improving patients' outcomes.

Using public database with large cohorts of MM patients, we analyzed both bortezomib-based or carfilzomib-based therapy, categorizing patients as responders (those achieving complete to partial response) and non-responders (those with stable or progressive disease). We identified that sodium hydrogen exchanger 6 (NHE6), encoded by SLC9A6 gene, is upregulated in non-responders. Screening of NHE family members using Hanamura dataset showed that MM cells highly expressed NHE6, whileother members expressed low levels. We confirmed the high expression of NHE6 in both patient-derived primary MM cells and human MM cell lines. Compared to their respective parental cells, bortezomib- or carfilzomib-resistant cell lines showed elevated NHE6 expression. Moreover, the expression level of NHE6 was negatively associated with overall survival and progression-free survival in patients retrieved from multiple databases. This colossal evidence suggests a robust correlation of NHE6 elevation in MM cells with non-responsiveness to treatment.

NHE6 is located on endosomal membrane and acts as a transporter that exchanges one monovalent alkali ion for one proton ion across the membrane. NHE6 has been previously implicated in neurological diseases, but its role in malignancies is less clear. To examine the role of NHE6 in MM therapeutic response, we knocked out NHE6 expressions in a panel of MM cell lines (sgSLC9A6). We found sgSLC9A6 MM cells with improved sensitivity and increased apoptotic cell death toward bortezomib or carfilzomib treatment in vitro. Preclinical mouse models showed that sgSLC9A6 MM-bearing mice significantly prolonged survival following bortezomib treatment compared to sgCtrl MM-bearing mice, reaffirming the contribution of NHE6 to drug resistance.

Mechanistically, both bortezomib and carfilzomib treatment induced a strong autophagic flux in sgCtrl MM cells but was significantly reduced in sgSLC9A6 MM cells. Adding autophagy inhibitors sensitized sgCtrl MM cells to bortezomib or carfilzomib-induced apoptosis, which mimics the phenotype observed in sgSLC9A6 cells. Cellular fractionation by ultracentrifugation revealed that NHE6 primarily distributed on early endosomes. Using immunoprecipitation, we uncovered a new mechanism through which NHE6 enhances the GTPase switch of Rab7 by promoting Rab7 recruitment to endosomal membranes, driving the early-to-late endosomal maturation process. This is critical for endosome-autophagosome fusion, leading to amphisome formation and activation of autophagy-mediated protein degradation in MM cells.

The clinical relevance of NHE6-mediated endosomal machinery in proteasome inhibitor-based therapies was further validated through analysis of CoMMpass^SM database. We found that non-responders exhibited significantly higher expressions of both Rab7 and NHE6 compared to responders. The high expression level was associated with poorer overall survival and progress-free survival. Pharmaceutical inhibition of the NHE6-Rab7 axis not only improved the efficacy of bortezomib but also resensitized resistant MM cells to bortezomib treatment in vitro and in vivo.

Our findings not only expand the functional repertoire of NHE6 into malignancy but also suggest potential therapeutic opportunities. NHE6 inhibitor could provide a selective upstream strategy to suppress autophagy at the level of endosomal maturation, contrasting to broader disruption of lysosomal function. Since endosomal machinery also impacts receptor recycling, antigen processing, and lysosomal degradation, NHE6 inhibitors could broadly sensitize MM cells to a range of treatments, such as proteasome inhibitors or targeted therapies like bispecific antibodies and chimeric antigen receptor T-cell therapy. Thus, our work provides an unexplored insight into MM refractoriness and provides compelling evidence supporting targeting endosomal events as a promising therapeutic strategy to overcome drug resistance and advance precision medicine in MM.