Sodium valproate to enhance doxorubicin sensitivity in osteosarcoma

B131 Osteosarcoma (OS) remains an incurable and ultimately fatal disease in many patients, and novel forms of therapy are needed. Although current therapies including surgery and adjuvant chemotherapy improve outcome, there have been no major treatment breakthroughs for OS in the last 20 years. The histone deacetylase (HDAC) enzymes are powerful new targets for epigenetic cancer therapy. The acetylation of histones, controlled by multiple histone acetyltransferases and HDACs, is important in governing chromatin structure and thereby can modulate expression of genes associated with cellular proliferation, differentiation and survival. Pharmacologic inhibition of HDAC has multiple anti-tumor effects that stem from the ability to affect cellular processes that are dysregulated in neoplastic cells. Furthermore, the effect of HDAC inhibition on chromatin structure has been shown to increase access of DNA-binding molecules such as transcription factors as well as DNA damaging agents. One available and inexpensive drug with HDAC inhibitory activity is the anticonvulsant sodium valproate(VPA). VPA has been shown to be a broad-spectrum inhibitor of HDAC, blocking the catalytic activity of class I enzymes and inducing proteosomal degradation of some class II enzymes. We hypothesized that VPA can be used to inhibit HDAC and potentiate the anti-tumor effects of doxorubicin (DOX) in OS cells in vitro and in vivo. Canine and human OS cell lines were incubated with and without VPA alone and in combination with DOX, and antiproliferative effects were evaluated using bioreductive and clonogenic assays. Effects on apoptosis were evaluated using TUNEL assay. Immunofluorescence cytochemistry and western analysis were used to evaluate changes in histone H3 acetylation. In vivo efficacy was evaluated in a canine OS xenograft model in athymic mice utilizing single-agent DOX or VPA as well as combination therapy. Treatment with VPA resulted in significant increases in acetylated histone H3. Incubation with VPA alone had modest antiproliferative effects, while pre-incubation with VPA followed by a short-term exposure to DOX resulted in significant chemosensitization. Exposure to VPA also resulted in increased nuclear DOX accumulation and potentiated DOX-induced apoptosis. Mice receiving combination therapy had statistically significant reductions in tumor growth and improvements in overall survival compared to mice receiving either agent alone or placebo. Based on the encouraging in vitro and xenograft data generated, we are currently performing a phase I clinical trial in canine patients with spontaneously occurring tumors, in which dogs scheduled to receive a standard dose of DOX are pre-treated for 48 hours with escalating doses of oral VPA. In conclusion, pretreatment of canine and human OS cells with clinically relevant doses of VPA results in histone hyperacetylation, enhanced nuclear DOX accumulation, and sensitization of these cells to the antiproliferative and proapoptotic effects of DOX in vitro and in vivo. These results justify evaluation of HDAC inhibitor/anthracycline combination therapy in canine and human patients with OS.