HDAC and CDK inhibitor combinations suppress neutrophil activation in myeloma

Abstract

Abstract Neutrophils foster a pro-tumorigenic microenvironment in multiple myeloma (MM) by releasing inflammatory cytokines, immunosuppressive factors, and reactive oxygen species (ROS), contributing to immune evasion and disease progression. Elevated neutrophil-to-lymphocyte ratio serves as a poor prognostic marker, yet neutrophil-targeted therapies remain underexplored. Our prior work identified three novel drug combinations that disrupt both epigenetic regulation and cell cycle control—particularly effective in transcriptionally active, treatment-resistant MM. These combinations also upregulate Smad signaling, indicating potential to reprogram the immune microenvironment and enhance anti-myeloma immunity. The CDKi/HDACi combination of dinaciclib and entinostat was identified as a potent combinatorial therapy, as it showed the greatest synergy in treatment-resistant myeloma subtypes and presented higher survival in a murine Vk*MYC; Nras LSL Q61R/+; IgG1-Cre (VQ) allograft mouse model. We investigated the CDKi/HDACi combination for further preclinical development. We explored the targets, mechanism of action, and efficacy of combined CDK/HDAC inhibition in vitro (cell lines) and in vivo (NSG xenografts and Bcl-xL transgenic spontaneous tumors) and confirmed that the combination leads to MYC suppression, upregulation of tumor suppressors like p16, anddelayed tumor progression. Freshly obtained smoldering myeloma patient bone marrow biopsy cells were utilized to evaluate the efficacy of the combination compared to single-agent treatment. The CDKi/HDACi combination was effective at selectively reducing the viability of human CD138+ MM cells ex vivo.Finally, a genetic signature of combined CDK/HDAC inhibition emerged for MM cells. RNA-seq and gene set enrichment analysis revealed downregulation of DNA replication, protein folding, and, importantly,immune pathways, including those related to leukocyte migration and pro-inflammatory chemokines (e.g., CXCL1, CXCL2, IL1A, IL23A). These changes align with the suppression of inflammatory and neutrophil-recruiting signals, suggesting that CDK/HDAC inhibition may indirectly suppress neutrophil activation and trafficking in the MM microenvironment. To overcome translational barriers associated with dinaciclib and entinostat, we evaluated next-generation agents: KB0742, a selective and orally bioavailable CDK9 inhibitor, in combination with quisinostat or zabadinostat, two potent HDAC inhibitors with more favorable pharmacokinetic and safety profiles. We validated their synergistic action in limiting proliferation and alteration of biomarker expression. Our next steps are to use co-culture conditions of MM cells with neutrophils, to observe the effects of CDKi/HDAi combination treatment by monitoring neutrophil activation markers, including CD11b and ROS production, compared to single agents. We will further investigate pro-inflammatory cytokines and chemokines involved in neutrophil recruitment and activation (e.g., CXCL1, CXCL2, CXCL8, IL1A, IL23A). These transcriptional changes, accompanied by increased re-expression of tumor suppressors (e.g.,p16) and TGFβ/SMAD signaling components, would predict reprogramming of an anti-inflammatory microenvironment by these combination treatments in myeloma. These findings point to a promising but underdeveloped therapeutic avenue whereby suppressing neutrophil-driven inflammation enhances anti-myeloma immunity.