Therapeutic Effects Of The Novel Subtype-selective Histone Deacetylase(HDAC)Inhibitor Chidamide On Myeloma-associated Bone Disease

BackgroundMultiple myeloma(MM)is an incurable plasma malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow(BM),the secretion of high levels of monoclonal immunoglobulins and osteolytic bone lesions.Myeloma cells interact with different cell types,such as osteoclasts(OCs)and mesenchymal stromal cells,in the BM microenvironment,which supports their growth,drug resistance and the development of bone disease through cell-cell adhesion and the release of growth factors,such as interleukin-6(IL-6)and vascular endothelial growth factor(VEGF).Osteolytic bone lesions,which result from increased bone resorption by OCs and reduced osteoblastic bone formation,are the most common complication of MM and often decrease the quality of life and survival time of patients with MM.New active drugs,including proteasome inhibitors and immunomodulatory agents,have improved the outcomes of patients with MM and alleviated bone damage.However,disease progression is still inevitable.Therefore,the identification of new targets and development of new drugs that focus on MM cells and their BM microenvironment are crucial to the development of more effective treatments.Chidamide is a novel oral histone deacetylase inhibitor(HDACi)designed to inhibit the activity of HDAC1,2,3 and 10 that is produced independently in China and is now undergoing I phase clinical trials in America and Japan.Chidamide has been shown to induce cell apoptosis and cell cycle arrest in a variety of cancers,such as lung cancer,pancreatic cancer,leukemia and lymphoma.Moreover,HDAC3 was recently shown to be overexpressed in MM and was a key factor that propelled myeloma cell proliferation.Additionally,HDAC1 and HDAC3 are important positive regulators of osteoclastogenesis.Based on these data,we hypothesize that chidamide may exert dual anti-myeloma and bone-protective effects.Objects:In the present work,we investigated the dual anti-myeloma and bone-protective effects on myeloma cells and preclinical models.To the best of our knowledge,this compound is the first HDACi that was independently developed in China and shows both anti-myeloma and anti-osteolytic bone disease activity in vitro and in vivo,supporting the future clinical application of this drug as a treatment for MM.Methods: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.The effect on osteoclast formation was assessed inosteoclastic 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)mediated by increasing Caspase-3,Caspase-7,Caspase-8,Caspase-9,p21 and p27,decreasing C-Myc,Mcl-1,Bcl-xL,CDK4 and CDK6.SOCS3/JAK2/STAT3 cell signaling was involved in regulating these proteins by increasing the expression of SOCS3,reducing the expression of 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 indicatedchidamide suppressed the function of these targets while not affected their expression.Also,chidamide showed effective cytotoxicity in the co-culture of myeloma cells with BMSC or OC.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 burden insubcutaneous and disseminated myeloma murine models.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.Conclusions:Our work demonstrates dual anti-myeloma and bone protective effect of chidamide in vitro and in vivo.These findings strongly suggest the potential clinical use of this drug in multiple myeloma in the near future.