Preclinical development of histone deacetylase inhibitors for the treatment of pediatric acute leukemia

A136 Acute leukemia is the most common malignancy of childhood and despite a good outcome for the majority of patients, a proportion still do very poorly. As such, novel approaches to therapy and new agents are clearly needed. We are investigating the utility of histone deacetylase inhibitors (HDACIs) for treatment of pediatric acute leukemias. Two HDACIs, vorinostat (suberoylanilide hydroxamic acid, SAHA) and SNDX-275 (MS-275), exhibit broad spectrum anti-tumor activity against a panel of 13 pediatric acute lymphoid leukemia (ALL) and 9 acute myeloid leukemia (AML) cell lines representing different lineages and developmental stages, with clinically achievable IC50 values of ~200-900nM. Both agents induce apoptosis in a dose-dependent manner, although vorinostat is more efficient in this regard. In contrast, vorinostat and SNDX-275 have differential affects on cell cycle progression. Treatment with SNDX-275 results in accumulation of cells in G1 phase in all cell lines tested, while treatment with vorinostat causes accumulation of cells in G1 phase in only a subset of cell lines. In addition, at higher concentrations vorinostat induces accumulation in G2/M or S phases, while treatment with higher concentrations of SNDX-275 causes accumulation of cells primarily in G1 phase. Vorinostat and SNDX-275 also mediate distinct affects on differentiation. Treatment with SNDX-275 results in cell line-specific changes in the expression of multiple hematopoietic differentiation markers on both AML and ALL cell lines. In contrast, only minimal changes are observed following treatment with vorinostat. Taken together, these data suggest that the anti-leukemia activities mediated by vorinostat and SNDX-275 occur by distinct mechanisms. Consistent with this, median effect analysis demonstrates a synergistic interaction between vorinostat and SNDX-275 in two ALL cell lines. This interaction is sequence-dependent and is optimal when SNDX-275 is administered first, followed by treatment with vorinostat. Similarly, HDACIs demonstrate a sequence-dependent synergistic interaction with 5-azacytidine (5-Aza), with 5-Aza followed by HDACI being the optimal sequence and administration of an HDACI followed by 5-Aza resulting in an antagonistic interaction. The synergistic interaction between HDACIs and 5-Aza is mediated, at least in part, by a synergistic induction of cell death. Cleavage of Caspase 8 to its active form is detected in lysates from cells treated with HDACIs and 5-Aza, but not samples treated with a single agent, suggesting that activation of an extrinsic apoptotic pathway(s) contributes to the synergistic induction of cell death by these agents. Finally, both vorinostat and SNDX-275 exhibit synergy with drugs representing the topoisomerase II and proteosome inhibitor classes based on median effect analysis. Our data suggest a biologically-based mechanism for the clinical utility of HDACI-based combination therapies in the treatment of acute leukemias and reveal important information regarding the mechanisms of action of these agents, both alone and in combination with other therapies.