In vivo molecular implications of the combination of the hypo-methylating agent 5-aza-2’-deoxycytidine and the histone deacetylase inhibitor valproic acid in patients with leukemia: Results of a phase I/II study
Authors: Blanca Sanchez-Gonzalez, Hui Yang, Marta Cuadros, Claritza Santos-Malave, Koyu Hoshino, Hagop Kantarjian, Jean-Pierre Issa, Guillermo Garcia-Manero
Journal: Cancer Research (2005)

3979  Epigenetic silencing can be reversed in vitro using agents with hypomethylating or histone deacetylase inhibitory (HDI) activity. The combination of 5-aza-2’-deoxycytidine (DAC), a potent hypomethylating agent, and valproic acid (VPA), an antiepileptic drug with HDI activity, has synergistic antileukemia activity in vitro. We have developed a phase I/II study of this combination in patients (pts) with leukemia. In this study, a fixed low dose of DAC (15 mg/m2 IV daily x 10) was combined with VPA. In the phase I portion of the study, three doses of VPA have been studied: 20, 35 and 50 mg/kg orally daily x 10 concomitant with DAC. The phase II portion uses the combination at its MTD level targeting a response rate of 30%. Responses are defined as complete remissions (CR) or hematological improvements (=CR without platelet recovery). The study stops if 0/10 pts, ≤ 3/20, or ≤ 5/30 respond. Forty-two pts with relapsed/refractory acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) and 9 untreated elderly pts with AML/MDS (total 51) have been registered. VPA doses up to 50 mg/kg orally daily have been combined with DAC with no excess toxicity. At these doses, 9 out of 28 evaluable pts responded. Three out of 4 untreated elderly pts responded to the therapy. So far no relapse has occurred (range 1 to 7+ months). Blood or bone marrow has been obtained on days 0,1,5,10,21 and 28 of each cycle. Free and bound VPA levels are available in 43 pts. Higher levels of both free and bound VPA were observed in responders than in non-responders (36 mg/L vs. 21; 127 ug/ml vs 104 respectively, p=0.02). Histone H3 and H4 acetylation was measured using Western blots in 38 pts. At VPA doses of 20 or 35 mg/kg, 1 out 11 pts (9%) had evidence of histone acetylation compared to 11 out 28 pts (37%) at the 50 mg/kg dose level (p=0.002). No association has been yet observed between histone acetylation and VPA blood levels or response. Subsequently, we evaluated the effects of the combination on global DNA methylation using a bisulfite-based pyrosequencing assay of LINE methylation. In the first 30 pts evaluated, a gradual median decline of 7% (+/− 5%) in LINE methylation is observed by day 10 of treatment. LINE methylation levels are restored to baseline by day 28. No DNMT1 trapping was observed in 10 patients evaluated. Finally, p21 induction was evaluated sequentially during therapy using a real-time PCR assay in 30 patients. An increase in more than 2 fold expression was observed in 20% of patients of 30 evaluated. So far no association between LINE hypomethylation or p21 induction and response has been observed. In summary, the combination of DAC and VPA is safe and has significant activity in patients with AML and MDS. In vivo, it induces histone acetylation, LINE hypomethylation and p21 induction in a significant fraction of patients. The study is ongoing.