Novel indirubin-3-monoxime derivative I3MV-8b enhances anti-CD38 monoclonal antibody efficacy in multiple myeloma through dual proteasome and HDAC6 inhibition

Abstract

Abstract Background: Anti-CD38 monoclonal antibodies (mAbs) constitute a revolutionary class of targeted immunotherapeutic agents that have garnered significant attention due to their effectiveness in the treatment of multiple myeloma (MM). These antibodies exert their therapeutic effects through binding to CD38 on MM cells and inducing various cytotoxicity mechanisms. Clinically approved anti-CD38 mAbs, daratumumab and isatuximab, have become​ the ​foundation of therapy​ for MM. However, resistance ​develops in over 20% of patients undergoing this treatment. The concurrent administration of anti-CD38 mAbs with other anti-MM drugs has been shown to enhance therapeutic outcomes. We previously identified a novel I3MO derivative, I3MV-8b (compound 8b), which functions as a dual inhibitor of the proteasome and HDAC6, exhibiting strong anti-myeloma activity (Biomark Res. 2025)​. The current study reveals that treatment with compound 8b significantly upregulates CD38 expression on MM cells and restores immune cell function, thereby enhancing the efficacy of anti-CD38 mAbs in combating MM. Method & Results: ​First,​​ in the NK-humanized NSG mouse model, the combined treatment of compound 8b markedly improved the therapeutic efficacy of daratumumab (Dara). Using a co-culture system, we observed that​ compound 8b treatment augmented the efficacy of Dara-mediated NK cell cytotoxicity against MM in vitro. Subsequently, we evaluated the effect of 8b on CD38 expression in MM cell lines and primary patient-derived cells. RNA-seq analysis indicated that compound 8b significantly upregulated CD38 expression in MM cells, a finding confirmed​ by flow cytometry in both MM cell lines and CD138⁺ primary MM cells. Additionally, we investigated the combined administration of I3MO and HDAC6i, the principal pharmacophores of compound 8b, ​finding that this combination better enhanced CD38 expression on MM cells. Mechanistically, the inhibition of HDAC6 by compound 8b ​elevated histone acetylation levels, particularly H3K27 acetylation (H3K27ac). ATAC-seq analysis demonstrated that compound 8b treatment significantly enhanced chromatin accessibility, and H3K27ac ChIP-Seq ​analysis​ confirmed that compound 8b robustly facilitated CD38 transcription. ​Building on our previous investigations demonstrating I3MO's ability to inhibit USP7 and modulate protein stability,​​ we ​show​ that the I3MO group of compound 8b enhances the stabilization of the CD38 protein through USP7 inhibition. ​Furthermore, compound 8b exhibited notable immunomodulatory effects in the C57BL/KaLwRij myeloma mouse model. Multicolor spectral flow cytometry analysis indicated that treatment with compound 8b significantly increased the proportion of natural killer (NK) cells within the tumor immune microenvironment. Functionally, compound 8b augmented IFN-γ production in NK cells and downregulated the expression of the NK cell exhaustion marker TIGIT. These findings were corroborated by in vitro experiments, which showed that compound 8b treatment reduced TIGIT expression on NK cells, resulting in enhancing NK cell cytotoxicity and reducing NK cell fratricide. Conclusion: Our findings suggest that compound 8b enhances CD38 expression and modulates immune cell function, offering a promising strategy to potentiate anti-CD38 mAbs against MM.