The Novel Subtype-Selective Histone Deacetylase (HDAC) Inhibitor, Chidamide, Exerts Dual Anti-Myeloma and Bone Protective Effect in Vitro and In Vivo

Abstract

Background and Purpose: Despite the advance in multiple myeloma treatment, drug resistance is still inevitable and the prognosis of most patients remains poor. Chidamide, a new subtype-selective histone deacetylase (HDAC) inhibitor, which is produced independently in China, is designed to inhibit the activity of HDAC 1, 2, 3 and 10. In the present work, we investigated the effect of chidamide on multiple myeloma and its associated osteolytic bone disease with different models.Experiment Design: In vitro cytotoxicity of chidamide was tested on myeloma cell lines (MM.1s, CAG, ARP-1, 8226, U266, H929, LP-1, ANBL-6, OPM2) with CCK-8 and flow cytometry. Synergistic combinations with standard drugs in 8226 and CAG cell lines were analyzed by compusyn software. The effect on osteoclast formation was assessed in osteoclastic cultures. The mechanisms of chidamide were explored by western blot and immunofluorescence. Murine models were employed for in vivo works, IVIS in vivo imaging system, ELISA (human Ig lambda, CTX-I, PINP) and micro-CT were used to surveillance the tumor burden and examine the bone destruction.Results: The cytotoxic effect of chidamide on myeloma cells is due to induction of cell apoptosis and cell cycle disruption (G1 phase arrest) in a dose dependent manner (IC50 varies from 2 to 5 uM in different cell lines) which mediated by increasing Cleaved Caspase-3, Caspase-7, Caspase-8, Caspase-9, p21 and p27, decreasing C-Myc, Mcl-1, Bcl-xL, CDK4 and CDK6. SensitiveSOCS3/JAK2/STAT3 cell signaling was involved in regulating these proteins by increasing the expression of SOCS3, reducing the expression of phospho-JAK2 and phospho-STAT3, thus inhibiting the proliferation and inducing cell cycle disruption. After treating with chidamide, the expression levels of HDAC1, 2, 3, 10 were not changed significantly, which indicated chidamide suppressed the function of these targets while not affected their expression. Notably, chidamide exerted synergistic anti-myeloma effect with dexamethasone, carfilzomib or pomalidomide (combination index < 1). Also, chidamide showed effective cytotoxicity in the co-culture of myeloma cells with bone marrow stromal cells and osteoclasts. Importantly, chidamide suppressed in vitro osteoclast formation and resorption, inhibited the expression of the key molecules (Cathepsin K, c-fos, NFATc1) and de-phosphorylation of p-AKT and p-JNK, and disrupted the F-acting ring formation. Finally, chidamide significantly reduced the tumor volume in the subcutaneous xenografts models, also the level of monoclonal protein Ig lambda secreted by myeloma cells decreased in disseminated myeloma murine models after treatment. Moreover, chidamide not only prevented tumor-associated bone loss in the disseminated murine model which was partially related with reducing the tumor burden but also had a direct bone protective effect on RANKL-induced rapid bone loss in non-tumor bearing mouse model with serum PINP (bone formation) and trabecular numbers increased while serum CTX-I (bone resorption) and trabecular space diminished compared with the vehicle group.Conclusion: Our work demonstrates dual anti-myeloma and bone protective effect of chidamide in vitro and in vivo, and shows its synergistic anti-myeloma effect with dexamethasone, carfilzomib and pomalidomide. These findings strongly suggest the potential clinical use of this drug in multiple myeloma in the near future. DisclosuresNo relevant conflicts of interest to declare.