ERG Transcriptional Networks in Primary Acute Leukemia Cells Implicate a Role for ERG in Deregulated Kinase Signaling

<div><p>High expression of the E26 transforming sequence related gene (ERG) is associated with poor prognosis in a subgroup of leukemia patients with acute myeloid (AML) and acute T-lymphoblastic leukemia (T-ALL). In a previous study we proposed that <em>ERG</em> overexpression may deregulate several signaling cascades in acute leukemia. Herein, we further expand those studies by identifying a consensus of biological targets in primary blasts of newly diagnosed acute leukemia patients. Our findings of chromatin immunoprecipitation-on-chip of primary samples revealed 48 significantly enriched single genes including <em>DAAM1</em> and <em>NUMB</em>. Significantly enriched signaling pathways included WNT/β-catenin, p53, and PI3K/AKT with <em>ERG</em> overexpression inducing dephosphorylation of AKT(Ser473) relative to non <em>ERG</em> expressing K562 cells. Cell based <em>ERG</em> overexpression studies also revealed drug resistance to multi-kinase inhibitor, BAY 43-9006 (Sorafenib) and to the tyrosine kinase inhibitor TKI258. Thus in primary leukemic cells, ERG may contribute to the dysregulation of kinase signaling, which results in resistance to kinase inhibitors.</p> </div

The significant genes derived from 7 ChIP-chip microarray data are displayed with the use of MultiExperiment Viewer graphical interface.

<p>The fold enrichment for 48 genes shared by at least 3 of ChIP-chips was uploaded to MultiExperiment Viewer to obtain a heat map representing the gene overlap of all ChIP-chips. Significantly enriched genes are >1.25 fold change with respect to the IgG control. The overlap of 48 unique genes shows that AML D, T-ALL and nBM share a greater number of enriched target genes. AML A-E are ordered with respect to <i>ERG</i> mRNA expression and genes are ranked according AML D P-Values.</p

ERG induced dephosphorylation of AKT(Ser473).

<p>A histogram overlay of ERG overexpressing cells in K562 and Jurkat cells (red tinted peaks) stained for intracellular pan AKT (left side histograms) and phosphorylated AKT(Ser473, right side histograms). Non ERG expressing cells are represented by an untinted black lined peak. The histograms represent two relative AKT levels in duplicate experiments of two K562 Tet-on ERG clones. Jurkat transient transfections of pDom-empty and pDom-ERG were carried out in duplicate.</p

ChIP-chip enriched genes.

<p>Displayed are 8 ChIP-chip samples with the <i>ERG</i> mRNA expression measured by quantitative real-time PCR as well as the number of significantly enriched single genes, identified by ChIP-chip using BRB Array Tools (fold enrichment >1.25, p<0.05), for primary AML samples (AML A through AML E), for T-ALL (T-ALL), a normal bone marrow sample (nBM) and the cell line HL60.</p

<i>ERG</i> overexpression induced resistance to multi-kinase inhibitors. A)

<p>K562 cells transduced with the inducible <i>ERG</i> expression vector (induced indicated as +DOX and uninduced indicated as –DOX) were treated with 10 µM Sorafenib in 5 parallel wells. Following 48 hours, cell proliferation was measured with WST-1 at 450 nm absorbance. Jurkat cells were transiently transfected with <i>ERG</i> expression vector, pDom-<i>ERG</i>, and the control vector pDom-empty. Twenty-four hours after seeding transiently transfected cells in 5 parallel wells, Sorafenib was added to a final concentration of 10 µM. Cell proliferation was measured 48 hours after drug addition. Cell proliferation was measured with WST-1 reagent at the 450 nm absorbance. Cell proliferation was measured with WST-1 in Jurkat cells transiently transfected with <i>ERG</i> expression vector pDom-<i>ERG</i> and the control vector pDom-empty. Twenty-four hours following transient transfection of pDom-<i>ERG</i> and pDom-empty, TKI258 was added to a final concentration of 1 µM. Cell proliferation was analyzed as described above. Bar graphs display the average of 5 experiments. Statistical significance was analyzed using Wilcoxon rang sum test. Asterisk indicates statistically significant results. *: P<0.05. <b>B and C)</b> ERG (+Dox and −Dox) K562 cells were treated with 10 µM Sorafenib or 10 µM TKI258 for 72 hours in order to determine the effects of drug induced apoptosis by Annexin V-FITC detection. This was conducted in two independent <i>ERG</i> inducible K562 Tet-on clones and one of two independent experiments is represented by dot plots.</p

Biological pathways enriched in primary leukemia ChIP-chip analyses.

<p>Displayed are the biological pathways significantly enriched in at least three of seven primary leukemia samples by ChIP-chip. The bars represent the negative logarithm function of Fisher’s P-value (P<0.05).</p

Scheme of ChIP-chip hybridization per sample.

<p>For each primary sample four hybridizations were carried out. IgG-enriched DNA was paired with input-DNA and ERG-enriched with input-DNA. DNA-mixtures were hybridized to a promoter chip array. Thus, genes identified as enriched in the IgG/input hybridizations (hybridization 1 and 2) were treated as unspecifically enriched genes and those identified in the ERG/input hybridizations (hybridization 3 and 4) were regarded as ERG-enriched genes. Finally, only those genes that were enriched in hybridization 3 and 4 only were regarded as putative ERG target genes.</p

Additional file 1: Table S1. of Inhibition of IGF1-R overcomes IGFBP7-induced chemotherapy resistance in T-ALL

Probe sets in the IGF1-R signatures that are over-expressed in the high IGF1-R group. Table S2. Probe sets in the IGF1-R signatures that are under-expressed in the high IGF1-R group. (PDF 209 kb

Promoter ChIP enrichment of <i>DAAM1</i> and <i>NUMB</i> verified by quantitative real-time PCR analysis.

<p><b>A and B).</b> Quantitative PCRs with primers designed to include the conserved ETS binding sequence in the proximal promoter of <i>DAAM1</i> and <i>NUMB</i> were applied. The <i>DAAM1</i> and <i>NUMB</i> promoters were enriched 1.5-fold over IgG control in six ChIPs and in five ChIPs, respectively (one of two representative experiments is shown).</p

Kaplan Meier analysis of overall survival in adult ETP-ALL patients receiving chemotherapy only (without alloSCT) or undergoing alloSCT.

<p>P-value was calculated by the Log-Rank test. Abbreviations: alloSCT, allogeneic stem cell transplantation.</p