A Novel Indirubin-3-Monoxime Derivative Enhance Immune Cell Mediated Cytotoxicity in Multiple Myeloma
Authors: Zhang Xiaoyu , Sun Xiyue , Liu Lanting , Yu Zhen , Qiu Lu-Gui , Hao Mu 
Published: December 2024

Introduction: Immunotherapy, including monoclonal antibodies (e.g., daratumumab), chimeric antigen receptor T (CAR-T) cell therapy and immune checkpoints inhibitors, has assumed a more prominent role in the treatment of multiple myeloma (MM). Despite these advancements, immunotherapy is frequently limited by immune-suppressive mechanisms within the tumor-bone marrow microenvironment. Consequently, aimed at remodeling the immune microenvironment and mitigating immune escape has become a promising strategy to bolster immune surveillance and prevent relapse. Our prior research reports that Indirubin-3'-oxime (I3MO), a traditional herb-derived compound, shows promise as a new antitumor agent for improving MM prognosis (eBioMedicine 2021). Herein we synthesized a novel derivative of I3MO, compound 8b, with the aim of augmenting therapeutic efficacy and investigating its ability of enhancing immune cell-mediated cytotoxicity in MM treatment.

Methods: The therapeutic effects of compound 8b were evaluated using in vitro co-culture systems of MM cell and human Natural Killer (NK) cells, followed by validation in an NK cell-humanized mouse. RNA-seq analysis was employed to explore the signaling pathways enriched by compound 8b in MM cells to identify key signaling cascades.

Results: Compound 8b demonstrated superior cytotoxic activity compared to I3MO against MM cell lines. Importantly, we observed that low doses of compound 8b (1µM, non-cytotoxic) significantly enhanced daratumumab-mediated NK cell cytotoxicity in co-culture model. The same finding was demonstrated in NK-humanized murine model. Thus, we hypothesize that compound 8b treatment enhances the sensitivity of MM cells to NK cell-mediated cytotoxicity. RNA-seq data revealed that treatment with 8b was involved in lipid regulation pathways, particularly fatty acid metabolism and cholesterol synthesis. Lipid metabolic regulation of compound 8b were examined using Lipid Droplet Dyes and BODIPY™ FL C16. Mechanistically, Small Molecule Pull-Down analysis revealed that I3MO directly binds to STAT3, inhibiting its phosphorylation at Y705 and Y727. HDAC6 inhibition sites in compound 8b effectively suppressed the JAK/STAT3 pathway, providing a molecular rationale for the enhanced efficacy of compound 8b. Furthermore, inhibition of STAT3 phosphorylation significantly reduced transcription of the downstream PIM1 kinase, critical for lipid metabolism reprogramming in various tumor cells. Notably, alterations in lipid metabolism were associated with increased lipid accumulation in tumor cell membranes (known as lipid order), affecting their susceptibility to perforin-mediated cytotoxicity. Using membrane-permeable fluorescent probes Laurdan and negative stain electron microscopy of cell membranes, our study showed that PIM1 overexpression increased membrane lipid accumulation and reduced perforin induced pore formation in membranes. These findings indicate that compound 8b inhibits lipid droplet synthesis and membrane lipid order in MM cell through PIM1 kinase downregulation, thereby heightening MM cell sensitivity to NK cell or cytotoxic T lymphocyte-mediated cytotoxicity. Additionally, immunoprecipitation and mass spectrometry analysis confirmed the interaction of PIM1 with SND1 protein and regulation of downstream SREBF2 expression, implicating cholesterol metabolism in MM cells.

Conclusion:Our study suggest that compound 8b enhances the sensitivity of MM cells to immunotherapy by targeting the STAT3/PIM1/SND1 signaling axis, thereby inducing reprogramming of lipid metabolism. These findings provide critical insights into the immunomodulatory properties of compound 8b and its potential clinical implications in MM treatment.