Inhibition of Histone Deacetylases 1 and 6 Enhances Ara-C-Induced Apoptosis In Pediatric Acute Myeloid Leukemia Cells

Abstract 3275

Acute myeloid leukemia (AML) accounts for one-fourth of acute leukemias in children, but is responsible for more than half of the leukemia deaths in this patient population. Resistance to cytarabine (ara-C)-based chemotherapy is a major cause of treatment failure in this disease. Therefore, new therapies for children with AML are urgently needed. Among the newer agents that have been recently investigated in high-risk AML in adults, histone deacetylase (HDAC) inhibitors [HDACIs, e.g., valproic acid (VPA) and Vorinostat (SAHA)] are particularly notable. The ability of HDACIs to induce cell differentiation, cell cycle arrest, and apoptosis in human leukemic cells, but not in normal cells, has stimulated significant interest in their potential as anti-leukemia agents. Numerous HDACIs have been developed during the last decade and the majority of these are in clinical trials including the novel class I-selective HDACIs, MS-275 and MGCD0103, and pan-HDACIs, LBH-589 and PXD101. Despite the well-characterized molecular and cellular effects of HDACIs, single-agent activity for this class of drugs has been modest. However, the clinical usefulness of HDACIs may be increased through rationally designed combination strategies including HDACIs with standard chemotherapy drugs. We previously hypothesized that VPA synergizes with ara-C, resulting in enhanced antileukemic activity in pediatric AML, by inducing apoptosis. We examined the impact of VPA on ara-C cytotoxicities in a panel of pediatric AML cell lines and diagnostic blast samples from children with de novo AML and demonstrated highly synergistic antileukemic activities of combined ara-C and VPA. This was especially pronounced in samples with t(8;21). Our mechanistic studies revealed that induction of DNA damage and Bim underlay the synergistic antileukemic activities of this drug combination. The present study was designed to identify members of the HDAC family which were deteminants of ara-C sensitivities, and to select the optimal HDACIs that were most efficacious when combined with ara-C for treating AML. Expression profiles of HDACs 1–11 in 4 clinically relevant pediatric AML cell lines (THP-1, Kasumi-1, MV4-11, and CMS) suggested that HDACs 5 and 11 were likely not involved due to marginal or lack of expression. The remaining class II HDACs and the entire class I enzymes could be relevant to HDACI anti-leukemic activities, based on the relationships between HDAC levels and HDACI cytotoxicities and responses to the combined VPA and ara-C, although the impact of class I HDACs seemed to predominate. Treatment of THP-1 cells with structurally-diverse HDACIs [SAHA (a pan-HDACI), VPA (a relatively class I selective-HDACI), and MS-275 (a class I selective-HDACI)] and enzymatic assays following immunoprecipitation of class I HDACs, revealed that inhibition of class I HDACs could augment ara-C-induced apoptosis. However, class II HDACs (e.g., HDAC6) were also implicated since SAHA was also effective. shRNA knockdown of HDACs 1 or 6 resulted in ∼2-fold increased apoptosis induced by ara-C in THP-1 AML cells (p<0.05). This was accompanied by substantially increased expression of Bim (2.3- and 1.4-fold, respectively). Down-regulation of HDAC2 resulted in ∼30% decreased ara-C-induced apoptosis. In contrast, shRNA knockdown of HDACs 3 and 4 had no effects on ara-C-induced apoptosis in THP-1 cells. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced ara-C-induced apoptosis in pediatric AML sublines including THP-1 and Kasumi-1. Our results further establish that HDACs are promising therapeutic targets for treating pediatric AML and identified HDACs 1 and 6 as the most relevant drug targets. Accordingly, treating pediatric AML patients with pan-HDACIs may be more beneficial than HDAC isoform-specific drugs. Based on our results, incorporation of pan-HDACIs (e.g., LBH-589 and PXD101) into ara-C-based clinical trials for treating pediatric AML should be strongly considered.