Changes in Promoter Methylation and Gene Expression in Patients with MDS and MDS-AML Treated with 5-Azacitidine and Sodium Phenylbutyrate.

Despite its clinical activity in myelodysplastic syndrome (MDS), the relationship between the inhibition of DNA methyltransferase 1 (DNMT) by 5-azacitidine (5AC) and hematologic improvement has not been well-explored. Optimal in vitro re-expression of genes silenced through promoter methylation requires sequential exposure to a DNMT inhibitor (i) and an histone deacetylase (HDAC)i. In a dose/schedule exploration study, patients with MDS (including MDS-AML) were treated with various regimens of 5AC SQ, followed by a 7 day infusion of the HDACi sodium phenylbutyrate (PB). Patients received 5AC at the following doses: 50 mg/m²/day * 10 doses (cohort A, n = 8), 50 mg/m²/day * 14 doses (cohort B, n = 3), and 25 mg/m²/day * 14 doses (cohort C, n = 5). Patients received a minimum of 4 cycles (q28 days). Clinical responses were graded according to IWG criteria. Changes in promoter methylation of p15^INK4B and E-Cadherin (E-CAD), the most commonly methylated genes in MDS and AML, were monitored using a real time-PCR modification of methylation specific-PCR (rtMSP). Changes in gene expression were monitored using real time rtPCR. Dose-limiting hematologic toxicity (myelosuppression > 14 days) occurred in 2/3 patients in cohort B. The other two dose schedules were well-tolerated. Clinical responses developed in 4/8 patients in cohort A (3 CR, 1 PR), 2/3 patients in cohort B (1 hematologic improvement (HI)-P, N, major, 1 HI-P-major), and 3/3 currently evaluable patients in cohort C (2 HI-N-major, 1 HI-P, major). p15 was methylated in 10/10 evaluable samples pre-treatment; E-CAD was methylated in 5/7. Treatment with 5AC decreased p15 methylation in 50% of patients studied. p15 expression increased in patients in whom methylation was decreased; this included 3/4 clinical responders studied. In several cases, reversal of methylation was confirmed by bisulfite sequencing (BSQ) of serial samples. BSQ suggested that 5AC treatment led to gradual demethylation of the clone, rather than replacement of a methylated clone with a normal unmethylated clone. Surprisingly, treatment with 5AC increased global acetylation of histones H3 and/or H4 (Western analysis) in 7/8 patients in cohort A, and 2/2 evaluable patients in cohort B, (cohort C data pending). Acetylation was further increased following PB administration in 4/8 patients in cohort A and 1/2 in cohort B. These data confirm for the first time that clinical administration of 5AC leads to substantial reversal of promoter methylation associated with gene re-expression. Administration of 5AC is also associated with induction of global histone acetylation; the mechanism underlying histone acetylation in response to 5AC is unclear. While administration of 5AC and PB is associated with re-expression of p15, the relative contributions of the DNMT and HDAC inhibitors cannot be determined from the present study. Gene methylation data obtained using rtMSP correlates well with BSQ. The use of this semi-quantitative technique to monitor larger studies of 5AC with and without HDAC inhibitors will facilitate determination of the relationship between reversal of promoter methylation of p15 and other genes and clinical response to DNMTis.