B and perifosine tiggers synergistic cytotoxicity in MM cells 9. Since previous studies have shown that bortezomib upregulates activated STAT3 in head and neck squamous cell carcinoma 21, we here similarly examined whether bortezomib enhances p-STAT3 in MM cells. Importantly, we observed that bortezomib upregulated p-STAT3, which is completely abrogated in HDAC3, but not in HDAC1 or HDAC2, knockdown cells (Figure 5C). These results suggest that the synergistic cytotoxicity induced by combined HDAC3 knockdown with bortezomib is mediated, at least in part, by inhibition of STAT3 activity. We similarly evaluated the combination effect of bortezomib with selective HDAC3 inhibitor BG45. Of note, BG45 did not inhibit HDAC6 evidenced by hyperacetylation of tubulin (Supplementary Figure 3A). Consistent with HDAC3 knockdown data, BG45 in a dose-dependent fashion also synergistically enhanced bortezomib-induced cytotoxicity (Figure 5D, Table 2C). We also examined whether dual inhibition of both HDAC3 and HDAC6 was more cytotoxic than either HDAC3 or HDAC6 when combined with bortezomib. As expected, HDAC6 selective inhibitor tubastatin-A further enhanced cytotoxicity induced by combined HDAC3 knockdown with bortezomib (Supplementary Figure 3B). BG45 demonstrate significant anti-MM activities in a murine xenograft model To evaluate the in vivo impact of BG45 alone or in combination with bortezomib, we used the subcutaneous MM.1S xenograft model of human MM in mice. BG45 significantly inhibited MM tumor growth in the treatment versus control group in a dose-dependent fashion. For example, significant differences were observed in control versus BG45 15 mg/kg, control versus BG45 50 mg/kg, and BG45 15 mg/kg versus BG45 50 mg/kg at day 22 (p 0.05, Figure 6A). Moreover, BG45 50 mg/kg in combination with bortezomib further enhanced either single agent activity (p 0.01). Representative images of tumor growth inhibition by BG45 (50 mg/kg) are demonstrated in Figure 6B. These results confirmed that BG45 triggers in vivo anti-MM activities.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptLeukemia. Author manuscript; available in PMC 2014 September 16.Minami et al.PageDiscussionHistone deacetylases regulate the activity of tumor-suppressor genes and oncogenes that play pivotal roles in tumorigenesis 22 and have been investigated in preclinical studies in both solid tumors and hematologic malignancies, including MM 4, 23. However, the clinical utility of these agents is limited due to unfavorable toxicities attendant to non-selective HDAC inhibition.PF-06821497 Indeed, non-selective HDAC inhibitors show different inhibitory profiles of class-I to class-IV DACs 12.Tebuconazole To date, however, the biologic impact of isoform-selective HDAC inhibitors on MM cell growth and/or survival has not yet been elucidated.PMID:23671446 Interestingly, previous studies have shown that selective inhibition of HDAC1, 2 by Merck60 treatment triggers significant growth inhibition in B-cell acute lymphocytic leukemia cells 24. We here observed that MS275 (HDAC1, 2, 3 inhibition) induces significantly greater MM cell growth inhibition than Merck60 (HDAC1, 2 inhibition), and demonstrate the biologic impact of HDAC3 inhibition on MM cell growth and survival in the context of the BM microenvironment using combined genetic and pharmacological probes. We examined the biologic impact of HDAC3 in MM cells using HDAC3 knockdown and HDAC3-selective small molecule inhibitor BG45. Both induce signif.