Introduction: Proteasome inhibition (PI) remains a cornerstone of multiple myeloma (MM) therapy. Despite decades of clinical use, the molecular mechanisms underlying the development of PI resistance remain only partially understood. In patients, epigenetic repression of PSMD5 and overexpression of the drug efflux transporter ABCB1 were found to induce PI resistance. However, these mechanisms account only for a subset of patients and appear to be drug-specific. Using proteomic profiling,we recently identified upregulated NAD(P)H:quinone oxidoreductase 1 (NQO1) in bortezomib (BTZ)- as well as in carfilzomib (CFZ)-resistant MM cell line models. NQO1 is a cytoprotective oxidoreductase known to prevent redox cycling and the formation of reactive oxygen species (ROS) by catalyzing two-electron reductions of quinones. While NQO1 overexpression has been associated with chemoresistance in solid tumors, its role in hematologic malignancies, especially multiple myeloma, is largely unexplored. Here, we provide a comprehensive functional and clinical evaluation of NQO1 as a novel mediator of PI resistance, providing a potential therapeutic target in MM to overcome PI resistance.

Methods: Patients were first stratified according to their NQO1 expression levels using the CoMMpass dataset (IA22): the top quartile (Q4) was defined as NQO1-high, and the lower three quartiles (Q1–Q3) as NQO1-low. Progression-free survival (PFS) was then compared between these groups, further stratified by whether first-line treatment regimens contained PIs or were based on non-PI therapies. NQO1-overexpressing MM cell lines (AMO1-NQO1 and RPMI8226-NQO1) were generated using the Sleeping Beauty System, and their drug sensitivity was assessed using the AlamarBlue assay following treatment with PIs (BTZ, CFZ) and immunomodulatory drugs (lenalidomide, pomalidomide). To evaluate whether PI resistance was NQO1-dependent and reversible, cells were co-treated with the irreversible NQO1 inhibitor ES936, followed by reassessment of PI sensitivity.

Results: In the CoMMpass dataset (IA22), high NQO1 expression correlated significantly with inferior PFS in patients (n=544) receiving PI-containing first-line regimens (26.2 vs. 40.6 months, p = 0.0004; HR 1.49, 95% CI: 1.17–1.91). This association remained significant when the analysis was restricted to patients treated with PI-containing but IMiD-sparing regimens (n=157; 16.4 vs. 29.5 months, p = 0.0185). In contrast, no significant correlation was observed in patients receiving non-PI-based regimens (n = 39; 40.4 vs. 32.1 months; p = 0.8093), suggesting that elevated NQO1 levels may confer PI-specific resistance and adversely impact survival following first-line therapy. To functionally validate these clinical observations, we overexpressed NQO1 in AMO1 and RPMI8226 MM cell lines and assessed their drug sensitivity. Overexpression of NQO1 decreased sensitivity to both BTZ and CFZ while increasing IC₅₀ values by ~2.3× and ~1.3× in AMO1 and ~1.5× and ~1.2× in RPMI8226, respectively. Consistent with our clinical observations, IMiD sensitivity remained unaffected in our models. To validate whether acquired PI resistance was NQO1-dependent and reversible, cells were treated with the irreversible NQO1 inhibitor ES936. Co-treatment reduced the CFZ IC₅₀ by ~28% in AMO1 and by ~38% in RPMI8226, restoring PI sensitivity. As ABCB1 overexpression is a known driver of CFZ resistance, we next investigated whether combined inhibition of NQO1 and ABCB1 could further enhance drug sensitivity. In CFZ-resistant AMO1 cells, treatment with the ABCB1 inhibitor tariquidar decreased the CFZ IC₅₀ to 23.2 nM, and co-treatment with ES936 further reduced it to 14.3 nM.

Conclusion: Our findings identify NQO1 as a novel mediator of PI resistance in MM. High NQO1 expression is associated with significantly inferior PFS in PI-treated patients, but not in those receiving non-PI-based regimens, indicating a PI-specific mechanism of resistance. Functional studies confirmed that NQO1 overexpression reduces sensitivity to BTZ and CFZ, while pharmacologic inhibition with ES936 restored drug responsiveness. Furthermore, combined inhibition of NQO1 and ABCB1 synergistically enhanced CFZ sensitivity in resistant cells. Together, these results support NQO1 as both a predictive biomarker and a promising therapeutic target, offering a rationale for the development of combinatorial strategies to overcome PI resistance and improve outcomes in MM.

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2025
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