The core role of exosomal mir-21-5p/TGF-β1-CXCL12/RANKL axis in bone marrow microenvironment remodeling in multiple myeloma and targeted intervention strategies

Abstract Multiple myeloma (MM) drug resistance and bone disease are closely associated with the remodeling of the bone marrow microenvironment, yet the mechanisms of exosome-mediated communication remain poorly understood. In this study, we utilized a dual-model approach combining patient and mouse data through multi-omics to explore the MM-exosomal miR-21-5p/TGF-β1–CXCL12/RANKL axis and assess its potential for therapeutic intervention. We isolated CD63⁺CD81⁺ exosomes measuring 100–150 nm from 28 newly diagnosed and 17 relapsed/refractory (R/R) MM patients using ultracentrifugation-SEC, yielding a concentration of 2.3 × 10¹¹ particles per mL. miRNA sequencing and LC-MS/MS analysis revealed a greater than tenfold enrichment of miR-21-5p (p < 0.001) and identified TGF-β1 among the top 5% of most abundant proteins (iTRAQ ratio 12.4), with similar profiles confirmed in exosomes from 5TGM1 syngeneic serum. Live-cell imaging using PKH26 demonstrated that over 78% of human or murine mesenchymal stem cells (MSCs) internalized these exosomes within two hours. Dual-luciferase assays indicated that miR-21-5p binds to a specific seed sequence (nucleotides 624-631) in the 3′ untranslated region (UTR) of TGF-β1, leading to a suppression of mRNA and protein expression by 2.8-fold and 3.4-fold, respectively, after 24 hours. This resulted in a 4.1-fold increase in the nuclear accumulation of phosphorylated SMAD2/3, which directed MSCs toward a cancer-associated MSC (CA-MSC) phenotype characterized by α-SMA⁺FAP⁺ markers (GSEA FDR < 0.01). CA-MSCs were found to secrete 5.2-fold more CXCL12 and 6.7-fold more RANKL, which enhanced the chemotaxis of MM cells by 4.5-fold (with anti-CXCL12 treatment reversing 82% of this migration) and increased the number of TRAP⁺ multinucleated osteoclasts from 12 to 89 per well. Micro-CT imaging of calvariae showed a 2.1-fold increase in the bone surface to bone volume ratio compared to controls. Importantly, the administration of a TGF-β1-neutralizing antibody or miR-21-5p antagomiR encapsulated in exosome-mimetic liposomes was able to reverse this pathological condition (p < 0.01). In bortezomib-resistant 5TGM1-BR cells, which have an IC₅₀ of 17.4 nM, the depletion of exosomes or the delivery of antagomiR significantly restored sensitivity, lowering the IC₅₀ to 3.3 nM and increasing apoptosis from 14% to 71% when treated with 5 nM for 48 hours. In a study involving NSG mice with 5TGM1-BR tumors, the combination of bortezomib administered at a dose of 0.5 mg kg⁻¹ twice weekly and MLN4924-antagomiR liposomes at 2 mg kg⁻¹ weekly resulted in a notable reduction of tumor bioluminescence imaging (BLI) flux from 1.1 × 10⁷ to 3.2 × 10⁶ photons s⁻¹, and it also extended the median survival of the mice from 34 to 71 days (p < 0.001). Additionally, there was a significant recovery of trabecular bone volume by 68% (p < 0.01). These findings suggest that multiple myeloma (MM) exosomes play a crucial role in remodeling the bone marrow niche through the miR-21-5p/TGF-β1–CXCL12/RANKL signaling axis, which drives the conversion of cancer-associated mesenchymal stem cells (CA-MSC), osteolysis, and resistance to proteasome inhibitors. Therefore, targeting this exosome-mediated pathway presents a promising strategy for overcoming drug resistance and improving conditions associated with MM-related bone disease.