Targeting CCR7 ubiquitination to disrupt protumoral macrophages: A NEDD4-1-dependent strategy to reverse bortezomib resistance in myeloma

Abstract Background: Chemotherapy resistance in multiple myeloma (MM) is associated with MM-associated macrophages (mMΦs). Although the E3 ubiquitin ligase NEDD4-1 enhances MM cell sensitivity to bortezomib (Bor), its role in modulating mMΦs to improve therapeutic efficacy remains unclear. Methods: NEDD4-1 expression in mMΦs that differentiated from MM patient bone marrow or healthy donor PBMCs was assessed by immunofluorescence and Western blot. Knockdown and overexpression models were established using siRNA and plasmids. mMΦ polarization phenotypes and pro-inflammatory factor expression (IL-6, CCL5, CXCL10) were analyzed via cytokine arrays, qPCR, and ELISA. MM cell apoptosis and CD206⁺ mMΦs were quantified by flow cytometry. NEDD4-1 substrate screening identified CCR7, validated by co-immunoprecipitation (Co-IP) and mass spectrometry. Ubiquitination of CCR7 Lys342 was examined using mutant plasmids (K342A) and ubiquitination Co-IP assays. Results: NEDD4-1 expression was downregulated in mMΦs following MM cell stimulation. NEDD4-1 overexpression promoted M1 polarization, enhanced secretion of IL-6, CCL5, and CXCL10, and activated the NF-κB pathway. This reprogramming enhanced MM cell sensitivity to Bor, an effect abolished by NF-κB inhibition and absent in the enzyme-dead NEDD4-1-C867S mutant. Mechanistically, NEDD4-1 bound CCR7 and mediated K63-linked polyubiquitination at CCR7-Lys342. The modification impaired CCR7 membrane localization (predominant in M1 mMΦs vs. cytoplasmic in M2) and blocked M2 polarization and suppressed M2 polarization. and suppressed M2 polarization. Functionally, high NEDD4-1 levels suppressed M2 polarization, decreased tumor burden, and enhanced Bor-induced MM cell apoptosis in vivo. Conclusions: NEDD4-1 hinders M2 polarization by mediating K63-linked ubiquitination of CCR7 at Lys342, thereby promoting M1-like mMΦs and enhancing Bor sensitivity in MM. This pathway reveals novel mechanisms of MM drug resistance and identifies potential therapeutic targets.