Early epigenetic changes and DNA damage do not predict clinical response in an overlapping schedule of 5-azacytidine and entinostat in patients with myeloid malignancies

Abstract

Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15(INK4B), CDH-1, DAPK-1, and SOCS-1) were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34(+) population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage-associated variant histone gamma-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination "epigenetic" therapy. This trial is registered with http://www.clinicaltrials.gov under NCT00101179.

Figure 1

Methylation status of candidate TSGs in CD34⁻ cells from bone marrow DNA. Promoter methylation of p15^INK4B, CDH-1, DAPK-1, and SOCS-1 was monitored by MSP in 29 patients before treatment (day 0), on days 15 to 16, and on days 28 to 29 of the first cycle of therapy. Red color indicates methylated status, green color indicates unmethylated status, and white color indicates sample was unavailable. No statistically significant association between reversal of candidate TSG methylation status and clinical response to combination therapy with 5AC and entinostat was detected. CR indicates complete response; PR, partial response; HI, hematologic improvement; and NR, no response.

Figure 2

Clinical response is not uniformly associated with p15^INK4B methylation reversal. (A) Methylation reversal in clinical responders. DNA BSS of 27 CpG sites in the promoter region of p15^INK4B in 2 clinical responders (patients 10 and 16). Patient 16 showed reversal of methylation at days 15 and 29 (left panel), whereas patient 10 (right panel) did not show methylation reversal by BSS. (B) Reversal of methylation of p15^INK4B in clinical nonresponders. DNA BSS of 27 CpG sites in the promoter region of p15^INK4B on days 0, 15, and 29 of therapy showed methylation reversal in both clinical responder and nonresponder (patients 7 and 3, respectively). Patient 7 showed methylation reversal at day 29 only. ∘ indicates unmethylated CpGs and • indicates methylated CpGs. The solid line labeled MSP product marks the start and end position of the CG sites of the MSP amplicon. The numbers below the horizontal bar describe the position of the CpGs relative to the transcription start site (+1). %M indicates percentage methylation calculated by dividing the number of • by the number of ∘ (270) and multiplied by 100.

Figure 3

p15^INK4B promoter methylation in a complete responder (patient 4). (A) Genomic DNA BSS of 10 clones at 3 different time points (days 0, 15, and 29) during the first cycle of treatment for patient 4. The numbers below the horizontal bar describe the position of the CpGs relative to the transcription start site (+1). Notations as per Figure 2. (B) MSP gel for the DNA from CD34⁻ and enriched CD34⁺ cells at 3 different time points day 0 (d0), days 15 to 16 (d15), and days 28 to 29 (d29) of therapy. U indicates unmethylated lane; and M, methylated lane. In vitro methylated (IVD) DNA was used as a positive control for methylated status, HCT 116 double knockout (DKO) for DNMT1 and DNMT3b was used as a positive control for unmethylated status, and water (H₂O) was used as a negative control for the PCR reaction.

Figure 4

Methylation status and gene expression of candidate TSGs in purified CD34⁺ hematopoietic progenitors. (A) Promoter methylation of p15^INK4B and SOCS-1 was monitored by MSP in CD34⁺ cells from 30 patients before treatment (day 0) and on days 15 and 29 of the first cycle of therapy. There is no association of reversal of candidate TSG methylation status with clinical response to combination therapy with 5AC and entinostat. Red color indicates methylated status, green color indicates unmethylated status, and white color indicates sample was unavailable. Notations as per Figure 1. (B) Plots (log scale) of normalized gene expression in evaluable patients before and after 1 cycle of 5AC/entinostat therapy in TSG genes tested for methylation status as well as 2 other genes (RASSF1 and CEBPA) frequently hypermethylated in MDS. Green indicates clinical responder; and red, clinical nonresponder. (C) Hierarchical clustering of individual patients (n = 12) at day 0 and day 15.

Figure 5

Treatment with 5AC and entinostat induces transient and reversible global DNA hypomethylation. LINE-1 methylation reversal was monitored by DNA pyrosequencing in 5 patients from different 5AC dose cohorts on days 0, 15, and 29 during the first cycle of treatment. The bar graph demonstrates the average of LINE-1 methylation from the 5 patients at the designated time points. The values represent the mean of duplicate runs ± SD. * indicates significant difference from day 0 at P ≤ .015.

Figure 6

Changes in H3/H4 acetylation and the DNA damage marker γ-H2AX in peripheral blood (PB) during therapy with 5AC and entinostat. (A) Western blotting of H3/H4 acetylation results from 3 representative patient samples (patients 10, 11, and 24) during the first cycle of treatment. H2A was used as a loading control. AcH3 and AcH4 indicate acetylated H3 and acetylated H4, respectively. Note that day-3 samples were procured before any administration of the HDAC inhibitor. (B) Three-dimensional representation of interaction between doses of 5AC and entinostat (denoted MS) administered and median maximal H4 acetylation in peripheral blood mononuclear cells. The median values of the intensity index of acetylated H4 after normalization with nonacetylated H2A (loading control) are shown. The maximal increase in H4 acetylation was observed with the sequential administration of 5AC (40 and 50 mg/m²) and entinostat (6 and 8 mg). (C) DNA damage induction in both clinical responders and nonresponders during the first cycle of treatment. Western blotting showing up-regulation of γ-H2AX in 2 clinical responders (patients 4 and 17) and 2 clinical nonresponders (patients 6 and 21). Actin was used as a loading control. Day-15 (empty lane) and day-29 samples were not available for patients 17 and 21, respectively. Day-4, -11, and -12 samples were not available for patient 6. (D) Three-dimensional representation of relationship between doses of 5AC and entinostat (denoted MS) administered and median maximal γ-H2AX induction in peripheral blood mononuclear cells. The median values of the intensity index of γ-H2AX after normalization with actin (loading control) are shown.