Impact of MLL5 expression on decitabine efficacy and DNA methylation in acute myeloid leukemia

Abstract

Hypomethylating agents are widely used in patients with myelodysplastic syndromes and unfit patients with acute myeloid leukemia. However, it is not well understood why only some patients respond to hypomethylating agents. We found previously that the effect of decitabine on hematopoietic stem cell viability differed between Mll5 wild-type and null cells. We, therefore, investigated the role of MLL5 expression levels on outcome of acute myeloid leukemia patients who were treated with decitabine. MLL5 above the median expression level predicted longer overall survival independent of DNMT3A mutation status in bivariate analysis (median overall survival for high vs. low MLL5 expression 292 vs. 167 days; P=0.026). In patients who received three or more courses decitabine, high MLL5 expression and wild-type DNMT3A independently predicted improved overall survival (median overall survival for high vs. low MLL5 expression 468 vs. 243 days; P=0.012). In transformed murine cells, loss of Mll5 was associated with resistance to low-dose decitabine, less global DNA methylation in promoter regions, and reduced DNA demethylation upon decitabine treatment. Together, these data support our clinical observation of improved outcome in decitabine-treated patients who express MLL5 at high levels, and suggest a mechanistic role of MLL5 in the regulation of DNA methylation.

Figure 1.

Prognostic impact of MLL5 expression in AML patients treated with decitabine (DAC). (A to C) Overall survival (OS) of all patients treated with DAC (irrespective of treatment courses) (A), OS of patients who received 1–2 courses of DAC (B), and OS of patients who received 3 or more courses of DAC (C), according to high versus low MLL5 expression levels.

Figure 2.

Immortalized Mll5 wild-type mouse bone marrow cells are more sensitive to decitabine (DAC) treatment than Mll5 null cells. (A) Cell viability upon low- or high-dose DAC treatment relative to DMSO solvent control (CTL) treatment. 1 × 10⁵ cells were plated in duplicate (mean ± SEM, n=5). (B) DAC-induced cell differentiation by immunophenotyping CD11b expression (mean ± SEM, n=3). (C) Colony yield from CFC assays performed with DAC-treated cells in comparison to CTL-treated cells (mean ± SEM, n=3). (D) Morphology of representative colonies from 3 independent CFC assays. Scale bars represent 100μm. (E) Cell cycle profiles upon CTL or DAC treatment (mean, n=3). (F) Frequency of apoptotic cells represented by PI negative and Annexin-V positive staining upon DAC treatment and normalized to CTL treatment (mean ± SEM, n=3). n.s.: not significant; *P < 0.05; **P < 0.01.

Figure 3.

Leukemic Mll5 wild-type cells exhibit higher global DNA methylation levels in promoter regions compared to Mll5 null cells. (A) Cell viability upon low-, medium- or high-dose DAC treatment relative to CTL treatment. 2 × 10⁴ cells were plated in duplicate (mean ± SEM, n=3). (B) DAC-induced cell differentiation by immunophenotyping Gr-1/CD11b expression (mean ± SEM, n=3). (C) Promoter DNA-methylation represented by the number of methylated array probes at different methylation-defining cut offs of MeDIP-chip assay. The log₂ ratio of immunoprecipitated DNA compared to input DNA at 0.5 was chosen as the threshold to discriminate methylated from non-methylated probes for downstream analyses. (D) Percentage of methylated and non-methylated probes in Mll5 wild-type and null cells. (E) Differential methylation of selected gene promoters based on MeDIP-chip log₂ ratio difference (Δlog₂ ratio). (F) Validation of differentially methylated gene promoters by MeDIP-PCR. Methylation difference was calculated as log₂ -transformed ratio of enrichment of methylated DNA of Mll5 wild-type compared to null cells (log₂ ratio) (mean ± SEM, n=3).

Figure 4.

Leukemic Mll5 wild-type cells are more responsive to DAC-induced promoter demethylation compared to Mll5 null cells. (A) Promoter DNA-methylation analysis of cells upon 20nM DAC exposure. (B) Percentage of DAC-responsive probes in Mll5 wildtype versus null cells. Methylation values of each probe were compared between untreated and 20nM DAC-treated cells. Among all the methylated probes (log₂ ratio > 0.5) in untreated sample, those probes with a decrease of the log₂ ratio of more than 0.2 (Δlog₂ ratio > 0.2) upon DAC treatment were defined as DAC-responsive probes. (C) Functional annotation of genes that were differentially demethylated upon DAC treatment in Mll5 wild-type cells. Mll5 wild-type specific DAC-responsive probes were matched to their closest gene if 2 or more probes were DAC responsive. **P < 0.01.

Figure 5.

High MLL5 expression is associated with increased DNA methylation in promoter regions in patients with CBF-AML and CN-AML. (A and B) Volcano plot of methylation difference of MLL5 high-expressing CBF-AML patients (n=18) (A) and CN-AML (n=51) (B) patients versus CD34⁺ normal bone marrows (NBM) (n=8). Hyper- or hypomethylated probe sets were marked by red or blue circles, respectively. (C) Number and percentage of hyper- or hypomethylated probe sets in patients versus CD34⁺ NBM. (D and E) Heatmap with supervised hierarchical clustering showing the 50 most differentially methylated probe sets of CN-AML patients with high MLL5 (n=51) (D) and low MLL5 (n=51) (E) expression versus CD34⁺ NBM (n=8). Black bars indicate the hypermethylated probes in patients compared to CD34⁺ NBM.