Myeloma Genomics and Microenvironment and immune profiling

Category: Myeloma Genomics and Microenvironment and immune profiling

NEDD4-1-Mediated Ubiquitination of CCR7 Reprograms Macrophage Function and Enhances theraputic Sensitivity in Multiple Myeloma.

(PA-279) NEDD4-1-Mediated Ubiquitination of CCR7 Reprograms Macrophage Function and Enhances theraputic Sensitivity in Multiple Myeloma.

Friday, September 19, 2025

Shunnan Yao

MD
Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University

Introduction: Drug resistance in multiple myeloma (MM) is associated with MM-associated macrophages (mMΦs). Although the E3 ubiquitin ligase NEDD4-1 has been shown to enhance MM cell sensitivity to Bortezomib (Bor), its role in modulating mMΦs functional properties to improve drug efficacy remains unclear.

Methods: Immunofluorescence and Western blot were assessed to detect the expression level of NEDD4-1 in mMΦs differentiated from bone marrow of MM patients and peripheral blood mononuclear cells of healthy donors. Transfection of mMΦs with small interfering RNA and overexpressed plasmids was carried out to construct a knock-down and overexpression model of the NEDD4-1 gene. Expression of pro-inflammatory factor in mMΦs and mMΦs polarization phenotype were assessed by cytokine array, qPCR, and ELISA. The expression of CD206 in mMΦs and apoptosis of MM cells were detected by flow cytometry. The NEDD4-1 substrate protein CCR7 was screened and validated using mass spectrometry-based proteomic analysis and Co-IP. The mutant plasmid of Lys 342 site of CCR7 was transfected into mMΦs, and the degree of its ubiquitination was detected by ubiquitination co

-precipitation assay.

Results: We observed that as M-CSF induced the differentiation of healthy donor PBMCs into macrophages, the expression of NEDD4-1 gradually increased, accompanied by a progressive elevation in the expression of the M2 marker CD206. Then, we established NEDD4-1 knockdown (KD) and overexpression (OE) models using adenovirus vectors in mMΦs, detection revealed that the NEDD4-1-KD group exhibited decreased expression of iNOS, IL-6, CCL5 and CXCL10 along with increased expression of ARG-1, CD206, and IL-10. Additionally, its protective effect on primary MM cells and cell lines was attenuated in co-culture conditions. In contrast, the NEDD4-1-OE group showed the opposite trends. The above evidence suggested that macrophages with low NEDD4-1 expression tend to exhibit M2 polarization phenotype. In vivo, NEDD4-1KD or OE mMΦs in xenograft NCG mice revealed that high NEDD4-1 mediated M1 macrophage polarization to decrease tumor burden. Mechanistically, further analysis revealed that NEDD4-1 bound to CCR7, and mediated K63-linked ubiquitination at the Lys 342 site of CCR7, impairing CCR7 membrane function and suppressing mMΦs transition to the M2 phenotype.

Conclusions: This study demonstrates that NEDD4-1 hindered MΦ polarization toward the M2 type by facilitating K63-linked ubiquitination of CCR7 at Lys342, ultimately enhancing Bor sensitivity in MM cells. These findings provide novel insights into MM drug resistance mechanisms and highlight potential therapeutic targets.