Inhibiting both Class IIb HDACs is superior to isoform-specific inhibition for improved antitumor immune response.

Class IIb histone deacetylases, including HDAC6 and HDAC10, have garnered significant interest due to their non-canonical structure, cytoplasmic localization, and their role in immunomodulatory antitumor responses. It is reported that HDAC6 inhibitors, Tubastatin A and Nexturastat A (NextA), inhibit both HDAC6 and HDAC10 isoforms with varying selectivity. In addition, HDAC6 genetic knockout models failed to recapitulate the effects observed with highly selective and isoform-specific HDAC6is, suggesting potential off-target effects. Therefore, it is crucial to discriminate the effect of inhibiting specific isoforms of Class IIb HDACs. We previously reported that dual HDAC6/10 inhibitors significantly suppressed tumor-promoting M2-like macrophage (Mφ) function and phenotype in vitro and in vivo. In this study, we report novel HDAC inhibitors, ITF3756 and ITF6475, referred to as ITF3 and ITF6, respectively, and their effects on macrophage function and phenotype using murine bone marrow-derived Mφs (BMDMs), murine RAW264.7 cells, human PBMC-isolated monocyte-derived Mφs, and Thp1 monocyte-derived human Mφs. We performed several assays, including cytotoxicity, HDAC inhibition profiling, gene expression analysis by qPCR, flow cytometry analysis of phenotypic markers, tumor cell phagocytosis, antigen cross-presentation, and T-cell activation assays to comprehensively characterize the effect of dual HDAC6/10 inhibition vs HDAC6-specific inhibition. As reference molecules, we also used NextA (dual HDAC6/10) and CPD11352 (CPD) (HDAC6-specific) inhibitors to compare the effect on Mφs. Finally, we demonstrated the therapeutic effect of ITF3 in combination with radiation therapy to suppress prostate cancer in an immunocompetent Myc-Cap murine model. The data indicated that both cytotoxicity and HDAC inhibitory activity of ITF3 were similar to NextA, and ITF6 was similar to CPD on both murine and human Mφs. Gene expression analysis of phenotypic markers by qPCR and protein levels by immunoblot indicated that ITF3 significantly suppressed M2-like Mφs, whereas ITF6 did not. In contrast, neither ITF3 nor ITF6 affected M1 Mφs. Furthermore, murine BMDM-derived M1-like Mφs treated with ITF3 and ITF6 significantly increased tumor cell phagocytosis, SIINFEKL antigen cross-presentation, and SIINFEKL-mediated activation of OT-1 CD8 T-cells. These results mirrored the effects observed in murine and human macrophages treated with NextA and CPD. Finally, a combination of ITF3 with radiation therapy significantly suppressed Myc-Cap prostate cancer tumor growth while inducing an immune-stimulatory effect, as evidenced by increased infiltration of antitumor M1 Mφs and CD8 effector T cells. Overall, we demonstrated that dual HDAC6/10 inhibition is superior to HDAC6 inhibition alone for instigating an immunomodulatory antitumor immune response in prostate cancer therapy.