Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia

<div><p>Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as <i>MN1, UHRF1, HOXB3</i>, and <i>HOXB4</i>, as well as <i>TRIM71</i>, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at <i>HOXB3</i> and <i>HOXB4</i> was associated with increased gene expression of both <i>HOXB</i> genes specific to the mid-risk AML, while increased DNA methylation at <i>DCC</i> distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.</p></div

MiR-27a Functions as a Tumor Suppressor in Acute Leukemia by Regulating 14-3-3θ

<div><p>MicroRNAs (miRs) play major roles in normal hematopoietic differentiation and hematopoietic malignancies. In this work, we report that miR-27a, and its coordinately expressed cluster (miR-23a∼miR-27a∼miR-24-2), was down-regulated in acute leukemia cell lines and primary samples compared to hematopoietic stem-progenitor cells (HSPCs). Decreased miR-23a cluster expression in some acute leukemia cell lines was mediated by c-MYC. Replacement of miR-27a in acute leukemia cell lines inhibited cell growth due, at least in part, to increased cellular apoptosis. We identified a member of the anti-apoptotic 14-3-3 family of proteins, which support cell survival by interacting with and negatively regulating pro-apoptotic proteins such as Bax and Bad, as a target of miR-27a. Specifically, miR-27a regulated 14-3-3θ at both the mRNA and protein levels. These data indicate that miR-27a contributes a tumor suppressor-like activity in acute leukemia cells via regulation of apoptosis, and that miR-27a and 14-3-3θ may be potential therapeutic targets.</p> </div

MiR-27a regulates14-3-3θ via a single binding site in the 3′UTR.

<p>(A)–(C) Expression of mature miR-27a and 14-3-3θ mRNA were measured via microarray. Correlation plots of 14-3-3θ and miR-27a expression in (A) pre-B-ALL (n = 37), (B) T-ALL (n = 26), and (C) AML (n = 39) cell lines and primary samples are shown. Significant inverse correlation was determined via Pearson r (r<<b>−</b>0.30, p<0.05*). (D) The effect of miR-27a on 14-3-3θ protein expression was measured via Western blot; the top panel is a representative experiment, and the bottom panel is a graph of normalized protein expression from 3 independent experiments (±SEM), determined by densitometry analysis (p<0.05*). (E) A schematic showing the predicted miR-27a binding site in the 3′UTR of 14-3-3θ (bottom). HEK293T cells were co-transfected with 14-3-3θL-27a or 14-3-3θL-27a-del (500 ng, white bars) alone and with 25 nM of miR-23a (grey bars) or miR-27a (black bars) (top). Normalized mean Luc expression (±SEM) is represented on the graphs. (F) The 14-3-3θ ORF rescues miR-27a-induced cell death. FUGW and FUGW/miR-27a transduced K562 clones were transfected with control plasmid (black bars) or 2 µg 14-3-3θ ORF (white bars), and stained with PI. FUGW/miR-27a transduced clones expressing the 14-3-3θ ORF had significantly less PI+ cells compared to those clones transfected with control vector. Significant differences were determined by Student's t-test (n≥3, p<0.05*).</p

Effects of c-MYC on expression of the miR-23a cluster.

<p>(A) Knock down of c-MYC expression in P493B cells by doxycycline-treatment. Cells were treated with 0 µg/ml (white bar), 0.1 µg/ml (grey bar) or 0.3 µg/ml (black bar) doxycycline for 48 hr, harvested, and total RNA isolated. Levels of miR-23a cluster member expression were measured via qRT-PCR, using U18 as an endogenous control, and normalized to untreated (0 µg/ml Dox) cells (2^−ΔΔCt = 1). (B) and (C) The effects of pharmacologic inhibition of c-MYC on miR-23a (B) and miR-27a (C) in REH, KOPN8, SUPB15, and K562 cell lines were determined by treatment with 2 doses of 10058-F4 (25 µM [grey bar] and 50 µM [black bar]) or vehicle (white bar) for 48 hr. Fold-expression of mature miR-23a and miR-27a were assessed by qRT-PCR and analyzed as above. Effect of the c-MYC inhibitor on miR-23a and miR-27a expression was determined by comparison of treated cells to the mean expression (±SEM) of vehicle-treated cells Significance was determined by a Student's t-test where p<0.05 (*) indicated significance (n = 2–6 independent experiments). (D) and (E) The effect of siRNA knock-down of endogenous c-MYC on expression of miR-23a (D) and miR-27a (E) in K562, Karpas45, and Molt16 cells were assessed by transfection with 50 nM (grey bar) or 100 nM (black bar) of an siRNA-c-MYC pool or 100 nM of control siRNA (white bar). Expression levels of miR-23a and miR-27a and significance of results were assessed as in (B) and (C). n≥3 independent experiments for all samples.</p

The miR-23a cluster does not regulate 14-3-3ζ, or other 14-3-3 isotypes.

<p>(A) and (B) The effect of the miR-23a cluster on (A) 14-3-3ζ and (B) 14-3-3γ protein expression was measured via Western blot. The top panels are representative blots from one experiment, and the bottom panels are graphs of normalized protein expression from 3 independent experiments (±SEM), determined by densitometry analysis (p<0.05*). (C) The schematics show predicted miR-27a and miR-24 binding sites in the CDS and 3′UTR of 14-3-3ζ (top panel). HEK293T cells were co-transfected with 14-3-3ζL-157, 14-3-3ζL-627, or 14-3-3ζL-1075 (500 ng, white bars) alone and with 25 nM of miR-23a (light grey bars) or miR-24 (dark grey bars); HEK293 cells were also co-transfected with 14-3-3ζL-1266 (500 ng, white bars) alone and with 25 nM of miR-23a (light grey bars) or miR-27a (black bars) (bottom panel). Normalized mean Luc expression (±SEM, n≥3) is represented on the graphs.</p

Enforced expression of miR-27a in pre-B-ALLs and T-ALLs.

<p>(A) Viability of FUGW and FUGW/miR-27a transduced REH (pre-B-ALL) clonal populations was measured at indicated times as above. Statistical differences in viability over time in FUGW and FUGW/miR-27a K562 colonies were determined by a 1-way ANOVA test (p<0.0001*). (B) REH cells were stained with AnnexinV and 7AAD and FACS-analyzed. Each data point is a replicate for the designated clonal population. Significant differences between FUGW and FUGW/miR-27a clones were compared as above. (C) FUGW and FUGW/miR-27a transduced Molt16 (T-ALL) cells were FACS-analyzed for GFP expression and PI staining. GFP expression positively correlated with PI+ cells in FUGW/miR-27a transduced cells (Pearson r = 0.9965, p<0.0001) compared to FUGW control cells which did not demonstrate this correlation. (D) GFP expression was monitored via FACS over time in FUGW and FUGW/miR-27a Molt16 GFP−/GFP+ mixed populations.</p

Enforced expression of miR-27a in AMLs.

<p>(A) Effects on proliferation were demonstrated by plating 10⁵ cells/ml in suspension cultures (Day 0) and counting viable cells (via trypan blue exclusion) on Days 1, 2, and 4 (n = 3 per colony). Growth was plotted as the mean viable cell number ±SD. Colony #'s were arbitrary. (B) MiR-27a expression levels in vehicle- and Dox-treated (0.30 µg/ml) TG-27a transduced K562 cells was determined by qRT-PCR and are represented as mean fold expression (2^−ΔΔCt) ±SEM. (C) Untransduced K562 cells (WT), K562 cells transduced with a control tet-inducible plasmid, and K562 cells transduced with the TG-27a construct were treated with increasing doses of Dox (as indicated). Cell growth of control- or tet-inducible construct-transduced cells was normalized to that of untransduced K562 cells. (D) Viability of FUGW and FUGW/miR-27a transduced stable K562 clones was measured at indicated times after isolation from methylcellulose colonies. Each data point represents 1 clonal population (i.e. cells grown up from 1 colony and followed over time). Statistical differences in viability over time in FUGW and FUGW/miR-27a K562 colonies were determined by a 1-way ANOVA test (p<0.0001*). (E) FUGW and FUGW/miR-27a K562 clonal populations were stained with PI and FACS-analyzed. Each data point on the graph represents percent PI of an individual clonal population. The mean (±SEM) of FUGW clones (n = 4) was compared to FUGW/miR-27a clones (n = 7), and significant differences were assessed via student's t-test (p<0.05*). (F) K562, TF1, and HL60 AML cells were stained with AnnexinV and 7AAD and FACS-analyzed. Each data point represents the % AnnexinV⁺/7AAD⁻ population within an individual clone. Significant differences between FUGW and FUGW/miR-27a clones were compared as above.</p

MiR-23a cluster expression in pre-B-ALL, T-ALL, and AML cell lines and primary samples.

<p>Expression of miR-27a (A), miR-23a (B), and miR-24 (C) was determined via microarray analysis in pre-B-ALL, T-ALL, and AML. (A) Average miR-27a expression (±SEM, p<0.05*) was compared to expression in normal CD34+ HSPCs (5.76±0.35, represented by black line) in pre-B-ALL cell lines (n = 36 [replicates of 9 cell lines]), pre-B-ALL primary samples (n = 16), T-ALL cell lines (n = 23 [replicates of 5 cell lines]), T-ALL primary samples (n = 13), AML cell lines (n = 24 [replicates of 7 cell lines]), and AML primary samples (n = 13). (B) Average miR-23a expression (±SEM, p<0.05*) was compared to normal CD34+ HSPCs (9.76±0.19, represented by black line). (C) Average miR-24 expression (±SEM, p<0.05*) was compared to expression in normal CD34+ HSPCs (10.23±0.14). n values for (B) and (C) were as in (A).</p

Expression levels of mature miR-27a in AML, pre-B-ALL, and T-ALL cell lines and primary samples.

<p>Total RNA was isolated from human AML (A), pre-B-ALL (B), and T-ALL (C) cell lines (black bars) and primary cases (primary patient cases identified with unique numbers, white bars). MiR-27a expression levels were normalized to mean levels in normal human CD34+ HSPCs (2^−ΔΔCt = 0.9756±0.022, black line). A Student's t-test was used to determine the significance of the difference in mean miR-27a expression (±SEM) between each leukemia samples and normal CD34+ HSPCs; significance is indicated as p<0.05 (*). n≥3 independent experiments in all cases except for the 10 primary AMLs which were analyzed in triplicate due to limited sample quantity.</p

MiR-24, but not miR-27a regulates 14-3-3β via sites in the 3′UTR and CDS.

<p>(A)–(C) Expression of mature miR-24 and 14-3-3β mRNA were measured via microarray. Correlation plots of 14-3-3β and miR-24 expression in (A) pre-B-ALL (n = 37), (B) T-ALL (n = 20), and (C) AML (n = 36) cell lines and primary samples are shown. Significant inverse correlation was determined via Pearson r (r<−0.03, p<0.05*). (D) The effect of miR-23a cluster members on endogenous 14-3-3β protein expression was measured via Western blot. The left panel is a representative blot from one experiment, and the right panel is a graph of normalized protein expression from 4 independent experiments (±SEM), determined by densitometry analysis (p<0.05*). (E) A schematic showing predicted miR-27a binding sites in the 3′UTR of 14-3-3β (top). HEK293T cells were co-transfected with 14-3-3βL-1537 or 14-3-3βL-1537-del (middle panel), and 14-3-3βL-2061 or 14-3-3βL-2061-del (bottom panel) (500 ng, white bars) alone and with 25 nM of miR-23a (grey bars) or miR-27a (black bars). Normalized mean Luc expression (±SEM, n≥3) is represented on the graph. (F) A schematic shows predicted miR-24 binding sites in the CDS and 3′UTR of 14-3-3β (top). HEK293T cells were co-transfected with 14-3-3βL-3′UTR (or deletion mutants 1 and 2; middle panel) or 14-3-3βL-CDS (or deletion mutants 1 and 2; bottom panel), (500 ng, white bars) alone and with 25 nM of miR-24 (grey bars) or miR-23a (black bars). Normalized mean Luc expression (±SEM, n≥3) is represented.</p