Altered Runx1 subnuclear targeting enhances myeloid cell proliferation and blocks differentiation by activating a miR-24/MKP-7/MAPK network

Abstract

Disruption of Runx1/AML1 subnuclear localization, either by a single amino acid substitution or by a chromosomal translocation [e.g., t(8;21)], is linked to the etiology of acute myeloid leukemia (AML). Here, we show that this defect induces a select set of micro-RNAs (miR) in myeloid progenitor cells and AML patients with t(8;21). Both Runx1 and the t(8;21)-encoded AML1-ETO occupy the miR-24-23-27 locus and reciprocally control miR-24 transcription. miR-24 directly downregulates mitogen-activated protein kinase (MAPK) phosphatase-7 and enhances phosphorylation of both c-jun-NH(2)-kinase and p38 kinases. Expression of miR-24 stimulates myeloid cell growth, renders proliferation independent of interleukin-3, and blocks granulocytic differentiation. Thus, compromised Runx1 function induces a miR-dependent mechanism that, through MAPK signaling, enhances myeloid proliferation but blocks differentiation--key steps that contribute to leukemia.

Figure 1. Genome-wide micro-RNA profile reveals Runx1 subnuclear targeting dependent miR regulation

Total cellular RNA from murine myeloid progenitor 32D cells, stably expressing normal Runx1 (WT) or a subnuclear targeting defective mutant (mSTD), or from human AML patients (Pt1-4) and AML patient derived Kasumi-1 cells were subjected to genome wide micro RNA analysis. (A) The data were obtained as log2 of the control (Control lane for 32D cells, and CD34+ normal myeloid cells for human samples) and represented as hierarchical clusters using web-based dChIP analysis. A group of 10 miRs that were upregulated upon defective subnuclear targeting of Runx1 by a single amino acid substitution (mSTD in 32D cell samples) or by chromosomal translocation (Kasumi-1 and Pt1-4 samples). (B) Total cellular RNA from the indicated 32D stable cell lines was subjected to either quantitative RT-PCR for all the 10 miRs in the selected group (B)or to Northern blot analysis (C)using probes for either miR-24-23-27 cluster or for U6 RNA (as a loading control). The intensities of the bands were semi-quantified by using Image J software (http://rsbweb.nih.gov/ij/). (D) Genomic location and sequence analysis of the miR 24-23-27 cluster. The cluster is present on Chromosome 19 and have numerous Runx binding sites (vertical lines). The horizontal bars represent the region amplified in ChIP assay, while the black arrows indicate the direction of gene transcription.

Figure 2. Runx1 and AML1-ETO transcriptionally regulate miR-24-23-27 cluster

(A) AML patient derived Kasumi-1 cells that express both Runx1 and AML1-ETO, B-cell lymphoma Jurkat cells that express only Runx1, or cervical carcinoma HeLa cells that do not express either protein were subjected to ChIP assay using antibodies specific for Runx1 (light gray bar) or AML1-ETO (white bar). Immuno-precipitated DNA was subjected to qPCR using locus specific primers and the results were represented as bar graphs of % input. IgG was used as a negative control for the immunoprecipitation. (B) K562 cells were infected with retroviruses carrying either Runx1WT, Runx1 mSTD or AML1-ETO cDNAs. Total cellular RNA was isolated from cells 48 hours after the infection and was subjected to qPCR using primers specific for miR 24-23-27 transcript. Bar graphs represent transcript levels in various experimental groups relative to empty vector (EV). (C) Kasumi-1 cells were transfected with siRNA against Runx1 or AML1-ETO. Cells were subjected to total cellular RNA isolation and qPCR 48 hours post-transfection. Data were represented as bar graphs of transcript levels relative to mock transfected cells.

Figure 3. MKP-7 is a direct target of miR-24

(A) A sequence analysis of the 3′ UTR of MKP-7of several organisms revealed the presence of seed sequence for miR-24 (gray box). (B) The 3′ UTR of MKP-7was cloned downstream of a luciferase gene and used in reporter promoter assays. K562 cells were transfected with the reporter plasmid and indicated miRs or anti-miRs. Luciferase activity in the cell lysates expressing indicated combinations of plasmid and miRs or anti-miRs was assessed using a luminometer.

Figure 4. miR-24 expression increases MAPK phosphorylation in hematopoietic cells

Human erythroleukemia K562 cells were transfected with the indicated miRs for 48 hours and total cellular proteins were subjected to western blot analysis. K562 cells transfected with the indicated miRs were subjected to western blot analysis. Specific antibodies were used to detect MKP-7. Cdk2 levels were assessed for protein loading control (A). Phospho-specific antibodies were used to detect activation of JNK, p38 (B) and AKT kinases (C). Antibodies against total JNK and AKT kinases were used for protein loading control

Figure 5. miR-24 expression alters cell cycle profile of hematopoietic cells and modifies their proliferative potential

(A) Live cell count of K562 cells transfected with indicated miRs was carried out using Tryphan blue staining and is represented here as number of cells in each group for 4 days post-transfection. (B) Myeloid progenitor 32D cells were transfected with the indicated miRs in the presence (gray line) or absence (black line) of IL3. Live cells were counted every day for 3 days post transfection. A representative from 2 independent experiments have been shown.

Figure 6. miR-24 blocks granulocytic differentiation of myeloid progenitor 32D cells

32D cells were transfected with the indicated miRs and subjected to granulocytic differentiation by replacing IL-3 with G-CSF in the growth medium 24 hours post-transfection. Cells were allowed to differentiate for an additional 48 hours and harvested for Giemsa-Wright staining (A). Nuclear lobulation, a hallmark of granulocytic differentiation, was scored in all the experimental groups and represented as % cells with segmented nuclei (light gray bars) or with blast nuclei (dark bars) (B). (C) Total cellular RNA was subjected to RT-qPCR to assess the expression of granulocyte differentiation markers. Primers specific for mRNAs of early (Granzyme-B) as well as late (CD11b and MPO) granulocyte markers were used. Bar graphs represent mRNA expression of each of the markers in each experimental set relative to that in mock-transfected cells. Expression of GAPDH in each sample was used as internal control. Only Granzyme B and CD11b showed statistically significant down-regulation (p=0.05, indicated by an asterisk), while MPO was not changed significantly (marked as non-significant; NS) (D) Murine myeloid 32D cells were subjected to granulocytic differentiation by replacing IL-3 with G-CSF and cells were harvested for RNA isolation at Days 0, 3, and 6 of differentiation. Actively proliferating cells were included as a control. Total cellular RNA was isolated and subjected to qRT-PCR using primers specific for Runx1 or miR 23-24-27 transcripts. The expression of the transcripts was normalized with GAPDH internal control.