Synergistic Antileukemic Interactions Between Valproic Acid and Cytarabine: A Potential Means to Improve the Treatment of Pediatric Acute Myeloid Leukemia.

Abstract 2084

Poster Board II-61

Acute myeloid leukemia (AML) accounts for one fourth of acute leukemias in children, but it is responsible for more than half of the leukemia deaths in this patient population. In contrast to the tremendous success in the treatment of acute lymphoblastic leukemia over the last three decades, resulting in a >80% cure rate, improvements in AML therapy have been limited. Resistance to cytarabine (ara-C), the most active drug in the treatment of AML, is a major cause of treatment failure in this disease. Therefore, new therapies for children with AML need to be developed to overcome drug resistance, decrease relapse rate, and reduce short- and long-term adverse effects of treatment. Histone deacetylase inhibitors (HDACIs) possess antitumor activity and are currently being tested in clinical trials for the treatment of a variety of different cancers. Valproic acid (VPA), an FDA-licensed drug for treating both children and adults with epilepsy, also acts as an HDACI and can induce apoptosis in leukemic cells but not normal cells. In this study, we hypothesized that VPA synergizes with ara-C in antileukemic activity by inducing apoptosis in AML cells. To model this concept and to provide the basis for future clinical studies, we examined the effects of VPA on sensitivities to ara-C in 8 AML cell lines derived from patients (4 were children) with different subtypes of AML and in AML blasts collected at the time of diagnosis from 10 children with de novo AML treated at Children's Hospital of Michigan. We demonstrated synergistic antileukemic interactions between ara-C and VPA in all of the AML cell lines and additive to synergistic antileukemic interactions between the two drugs in the patient samples by standard isobolograms and calculation of combination indexes. It is interesting to note that MV4-11 [which harbors t(4;11)] and Kasumi-1 [which harbors t(8;21)] cells were substantially more sensitive to VPA than the other AML cell lines. Analogous to the Kasumi-1 cells, diagnostic blasts from t(8;21) AML cases (n=4) were significantly more sensitive to VPA than blasts from non-t(8;21) AML cases (n=6) (mean VPA IC₅₀ 0.51 mM vs 1.95 mM, p=0.0095) and showed median 53.9-fold increased ara-C sensitivities when combined with VPA at concentrations of 0.5 mM or lower. By contrast, non-t(8;21) AML blasts only showed median 2.1-fold increased ara-C sensitivities when combined with 0.5 mM VPA (p=0.048). In a pilot experiment, treatment of SCID mice with K562 xenograft tumors with combined Palmo-ara-C and VPA resulted in a 31% T/C and a 0.8 gross log cell kill compared to treatments with Palmo-ara-C (67% T/C) or VPA alone (100% T/C), establishing unambiguous in vivo synergy. Real-time RT-PCR analyses revealed changes in transcript levels for hENT1 and cytidine deaminase in Kasumi-1 cells post VPA and ara-C treatment alone or in combination. However, these changes would antagonize ara-C sensitivity in Kasumi-1 cells, suggesting that the effects of VPA or ara-C alone or in combination on expression of genes related to ara-C transport and metabolism do not contribute to the observed synergistic effects in AML cells. Interestingly, ara-C and VPA co-treatment resulted in synergistic induction of apoptosis and S-phase arrest in Kasumi-1 cells determined by flow cytometry analysis with annexin V and PI staining. The synergy between ara-C and VPA in induction of apoptosis in Kasumi-1 cells was accompanied by synergistic activation of caspase-3, induction of both total and acetylated p53 (ac-p53), and release of the active form of Bax determined by caspase-3 assays, co-immunoprecipitation, and Western blotting. Collectively, these results suggest that VPA enhances ara-C sensitivity in Kasumi-1 cells most likely by modulating levels of total and ac-p53 proteins and then release of the active form of Bax to trigger apoptosis. Based on our laboratory results, VPA has been incorporated into a treatment arm for high risk AML patients enrolled in the St. Jude Children's Research Hospital (SJCRH) clinical trial AML08: “A Randomized Trial of Clofarabine Plus Cytarabine Versus Conventional Induction Therapy and of Natural Killer Cell Transplantation Versus Conventional Consolidation Therapy in Patients with Newly Diagnosed Acute Myeloid Leukemia”. The results of our study provide compelling evidence to support the use of VPA in combination with ara-C in clinical trials for treating different risk groups of pediatric AML.