Background Daratumumab (Dara), a first-in-class anti-CD38 monoclonal antibody, is pivotal in multiple myeloma (MM) treatment. Nevertheless, primary and acquired resistance significantly limits its clinical efficacy. While tumor-intrinsic mechanisms (e.g., CD38 downregulation) are well-characterized, interventions targeting these pathways show limited success. Emerging evidence implicates gut dysbiosis in treatment resistance, with Dara-resistant patients exhibiting depleted butyrate-producing bacteria and reduced serum butyrate levels, suggesting a potential gut-bone marrow axis in Dara resistance.

Aims This study aims to: 1) Define the role of gut microbiota-derived butyrate in overcoming Dara resistance; 2) Investigate whether butyrate rescues impaired NK cell cytotoxicity through calcium signaling; and 3) Elucidate the epigenetic mechanism by which butyrate regulates Orai1 expression via HDAC3 inhibition.

Methods We conducted integrated multi-omics analysis on 40 subjects (10 newly diagnosed MM, 10 Dara-sensitive, 10 Dara-resistant , including 16S rRNA sequencing, serum metabolomics, and single-cell RNA sequencing. In vitro functional assays utilized NK-MM co-culture systems with butyrate supplementation, Orai1 inhibitor (MRS1845), and HDAC3 modulators (inhibitor RGFP966/activator ITSA). Calcium flux was measured using Fluo-4 AM, while epigenetic changes were assessed via ATAC-seq and ChIP-qPCR for histone H3 acetylation. Cytotoxicity was quantified by granzyme/perforin release and IFN-γ secretion.

Results

Dara-resistant patients displayed severe gut dysbiosis with a 3.2-fold reduction in butyrate-producing genera (p=0.008) and 60% lower serum butyrate versus sensitive patients (p<0.001), correlating with shorter median PFS (11 vs. 28 months; HR=0.32, p=0.002). NK cells from resistant patients showed functional impairment: 45% reduced granzyme release (p=0.003), 52% lower perforin (p=0.001), and 2.8-fold decreased calcium influx (p<0.001), accompanied by 70% downregulated Orai1 expression (p=0.004). Butyrate (1mM) restored NK cytotoxicity, increasing MM cell lysis by 2.1-fold (p<0.001) and normalizing calcium influx. Mechanistically, butyrate inhibited HDAC3, enhancing H3K9/K27 acetylation at the Orai1 promoter (4.1-fold ChIP enrichment; p=0.005) and chromatin accessibility (3.3-fold; p=0.003), thereby boosting Orai1 transcription (3.5-fold; p=0.002). These effects were abolished by Orai1 blockade (MRS1845) or HDAC3 activation (ITSA), while HDAC3 inhibition (RGFP966) phenocopied butyrate.

Summary/Conclusion Our study identifies a novel “gut microbiota-butyrate-HDAC3-Orai1” axis driving Dara resistance. Butyrate deficiency disrupts NK cell cytotoxicity via suppressed Orai1-mediated calcium signaling, which is rescued by butyrate through HDAC3 inhibition and subsequent epigenetic activation of Orai1. Targeting this axis via butyrate prodrugs, probiotics, or selective HDAC3 inhibitors represents a promising translational strategy to overcome Dara resistance in MM.

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2025
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