Role of Selective HDAC6 Inhibition On Multiple Myeloma Bone Disease

Abstract 328

In multiple myeloma (MM), deregulated osteoclast (OC)/osteoblast (OB) cross-talk induces osteolytic bone lesions. The HDAC6 selective inhibitor, rocilinostat (ACY-1215), in combination with bortezomib has shown potent anti myeloma activity in preclinical studies, which provided the rationale for a clinical trial that is currently recruiting relapsed/refractory MM patients (NCT01323751). However, while the beneficial role of bortezomib in tumor-related bone disease has been previously described, the effect of HDAC6 inhibition is not known. Evidence suggests a positive effect on bone turnover as pan HDAC inhibitors accelerate OB maturation and suppress OC maturation in vitro.

Here, we evaluated effects of the selective HDAC6 inhibitor rocilinostat (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib in MM bone disease. Rocilinostat (1 μM) alone and in combination with bortezomib (2.5 nM) inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, rocilinostat (1 μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines and in combination with bortezomib inhibited transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS, and NFATC1. Importantly, such an effect was cytokine (RANKL and M-CSF) dependent. Further, rocilinostat, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase (ALP) enzyme activity and alizarin red staining. In addition, we found increased mRNA expression of beta-catenin, osteocalcin, ALP, and RUNX2.

Based on this promising in vitro data, we used the xenograft model of disseminated human MM in SCID mice to study the effect of rocilinostat, alone and in combination with bortezomib, on MM bone disease. MM.1S-GFP-Luc cells were injected intravenously, and MM disease progression was followed by bioluminescence imaging. A significant decrease in tumor burden was observed in mice following three weeks of treatment with rocilinostat, alone or in combination with bortezomib. Isolating serum from control and treated mice, we also observed a significant decrease of TRAPc5b levels, a marker of bone resorption, as well as a significant increase in osteocalcin levels, a marker of bone formation, in the serum of the combination treated cohort. Cells isolated from the calvaria from the combination treated group compared to the control group showed a significant increase in the mRNA expression of ALP, RUNX2, and osterix, as well as a significant decrease in the mRNA expression ratio of RANKL/OPG. To elucidate the role of HDAC6 inhibition on bone turnover, we used HDAC6 knockout mice. Cells were isolated from femurs, tibia, and spine of 2 month-old wild type (WT) and HDAC6 knockout (KO) mice and mRNA expression for osteocalcin, ALP, RUNX2 and osterix was assessed by qPCR. We observed a significant increase in osteocalcin mRNA expression without significant changes in the mRNA expression of ALP, RUNX2 and osterix. Bone marrow stromal cells (BMSCs) differentiated from WT and KO mice were co-cultured with MM murine cell lines and, notably, the proliferative advantage conferred by BMSC isolated from HDAC6 KO mice to MM cell lines was significantly decreased compared to WT BMSCs. These data suggest that a microenviroment lacking HDAC6 reduces MM cell proliferation. Moreover, treatment with rocilinostat (1mM) for 24 h inhibited proliferation of MM cells cocultured with WT BMSCs to levels observed in MM cells cultured with KO BMSC lacking endogenous HDAC6. Finally, the effect of co-treatment with rocilinostat (1μM) and bortezomib (2.5 nM) on proliferation of MM cells co-cultured with WT-BMSC was similar to that observed when bortezomib was added to MM cells in cocultures with KO BMSC.

In conclusion, the in vitro data and the in vivo results from the xenograft models of human MM in SCID mice, as well as data in the HDAC6 KO mice, indicate a potential beneficial role of HDAC6 inhibition on MM-related bone disease. We are currently performing dynamic and static histomorphometric analysis to confirm this effect on bone remodeling at the tissue level. These effects on bone remodeling are an added benefit for MM patients and will be assessed prospectively in our ongoing clinical trial.