The Roles Of Histone Deacetylases In Ara-C Sensitivity In Pediatric Acute Myeloid Leukemia

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. Histone deacetylase (HDAC) inhibitors (HDACIs) are a promising new class of anti-cancer drugs. 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. 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 drug combinations including HDACIs with standard chemotherapy drugs. We previously hypothesized that VPA (valproic acid, HDACI) 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 the combination. 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 are deteminants of ara-C sensitivities, and to select the optimal HDACIs that are most efficacious when combined with ara-C for treating AML.Expression profiles of HDAC1-11 in 4 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 classâ…¡HDACs and the entire classâ… 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 classâ… HDACs seemed to predominate. Treatment of THP-1 cells with structurally-diverse HDACIs [SAHA (a pan-HDACI), VPA (a relatively classâ… selective-HDACI), and MS-275 (a classâ… selective-HDACI)] and enzymatic assays following immunoprecipitation of classâ… HDACs, revealed that inhibition of classâ… HDACs could augment ara-C-induced apoptosis. However, classâ…¡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 THP-1 cells. Microarray analysis revealed down-regulation of insulin-like growth factor-1 (IGF-1) and CDA in HDAC1 and HDAC6 knockdown clones, respectively. These results suggest that HDAC1 and HDAC6 may affect ara-C sensitivity through modulating the expression of IGF-1 and CDA in pediatric AML cells.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.