NAMPT in macrophages promotes multiple myeloma drug resistance through mitochondrial metabolic reprogramming and modulates immunosuppression via the SIRT1/STAT3/SPP1 axis.

Abstract Introduction: Macrophages (MΦs) are key components of the multiple myeloma (MM) tumor microenvironment (TME) and contribute to drug resistance, but the mechanisms underlying their crosstalk with MM cells and other immune cells are yet to be elucidated. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme responsible for NAD+ biosynthesis, has been reported to promote tumorigenesis, drug resistance and immunosuppression. Previously, we identified a subgroup of MΦs with high expression of NAMPT through single-cell RNA sequencing (scRNA-seq) of MM bone marrow. Consequently, unraveling the regulation of NAMPT in MΦs is essential for developing potent therapeutic strategies against MM. Methods: To characterize macrophage subpopulations, we performed scRNA-seq of MM bone marrow samples obtained from newly diagnosed MM (NDMM) and relapsed/refractory MM (RRMM) patients. PBMC were incubated for 7 days in medium with M-CSF to become normal MΦs. MΦs were cultured for 72 hours with myeloma-conditioning medium to generate myloma-associated MΦs (mMΦs). We suppressed NAMPT in MΦs through siRNA-mediated knockdown (KD) and FK866 inhibition. Then functional rescue was achieved by supplementing downstream metabolite NMN. Using co-culture models, we assessed the impact of mMΦs on the drug resistance of MM and regulation of CD8+T cells cytotoxic activity, as well as the downstream signaling mechanism through in vitro and in vivo experiments. Results: We found that NAMPT knockdown and FK866 inhibition contributed to a pro-inflammatory phenotype shift in MΦs, with the upregulation of CD80, CD86, IL1b, IL6 and downregulation of CD163, CD206(MRC1). Co-culture experiments demonstrated that mMΦs protected H929 and JJN3 cells from bortezomib-induced apoptosis at high concentrations. Intratumoral injection of NAMPT-silenced mMΦs significantly impaired tumor progression and resensitized NSG mice xenograft models to bortezomib treatment. NMN supplementation reversed the above phenomenon. These results indicated that NAMPT's metabolic properties govern its regulatory role in macrophage function. Next, we confirmed that NAMPT maintained mitochondrial homeostasis by preserving NAD+ pools and redox balance in mMΦs, NAMPT impairment triggered mitochondrial dysfunction, evidenced by reduced ATP production and OCR, collapsed membrane potential and ultrastructural abnormalities including cristae loss and whorled morphology.We further investigated the regulation effects on T cells by studying the interaction between mMΦs and CD8+T cells. mMΦs co-cultured with CD8+ T cells induced the expression of exhaustion markers LAG3, and TIM3, and suppressed the expression of granzyme B (GZMB) and interferon-γ (IFN-γ) in T cells. To elucidate the underlying mechanism, we conducted comparative transcriptome analysis between NAMPT-KD and control mMΦs, identifying SPP1 as a pivotal downstream effector. SPP1, highly expressed in MΦs, interacts with integrin α4/β1 complex to modulate T cell activity. SPP1-deficient mMΦs significantlyenhanced CD8+T cells proliferation and cytotoxic activity while reducing the exhaustion. Chip-qPCR and Luciferase activity assays indicated STAT3 binding to specific promoter motifs of SPP1 in mMΦs. Given that STAT3 acetylation has been reported to modulate its DNA-binding affinity, we proposed that NAMPT facilitates SPP1 gene transcription through STAT3 acetylation. This hypothesis was strengthened by downregulating the expression of SIRT1, an NAD+-dependent deacetylase. SIRT1 knockdown increased acetyl-STAT3 and decreased SPP1 expression, enhancing CD8+T cell proliferation and cytotoxicity while alleviating exhaustion in co-culture system. Importantly, NAMPT inhibition impaired SIRT1 activity through NAD+ depletion, thereby establishing the NAMPT-NAD+-SIRT1-STAT3-SPP1 signaling axis which modulates mMΦs-induced CD8+ T cell dysfunction. Conclusions：This study established NAMPThighmMΦs as a pivotal regulator in the immunosuppressive myeloma niche, orchestrating both drug resistance and T cell dysfunction through NAD+-SIRT1-STAT3-SPP1 axis. Downregulating NAMPT simultaneously disrupted MM cells survival pathways and reversed immunosuppression, which offers a promising approach to overcome drug resistance in RRMM patients. The mechanistic insights into macrophage-T cell crosstalk further support developing NAMPT inhibitors as microenvironment-modulating agents for combination immunotherapy in MM.