RUNX1-ETO induces a type I interferon response which negatively effects t(8;21)-induced increased self-renewal and leukemia development.

The 8;21 translocation is the most common chromosomal aberration occurring in acute myeloid leukemia (AML). This translocation causes expression of the RUNX1-ETO (AML1-ETO) fusion protein, which cooperates with additional mutations in leukemia development. We report here that interferons (IFNs) and IFN-stimulated genes are a group of genes consistently up-regulated by RUNX1-ETO in both human and murine models. RUNX1-ETO-induced up-regulation of IFN-stimulated genes occurs primarily via type I IFN signaling with a requirement for the IFNAR complex. Addition of exogenous IFN in vitro significantly reduces the increase in self-renewal potential induced by both RUNX1-ETO and its leukemogenic splicing isoform RUNX1-ETO9a. Finally, loss of type I IFN signaling via knockout of Ifnar1 significantly accelerates leukemogenesis in a t(8;21) murine model. This demonstrates the role of increased IFN signaling as an important factor inhibiting t(8;21) fusion protein function and leukemia development and supports the use of type I IFNs in the treatment of AML

Cooperation between RUNX1-ETO9a and Novel Transcriptional Partner KLF6 in Upregulation of <i>Alox5</i> in Acute Myeloid Leukemia

<div><p>Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene <i>Alox5</i>, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of <i>Alox5</i> expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα <i>in vitro</i>. However, <i>Alox5</i> is not essential for the induction of leukemia by RUNX1-ETO9a <i>in vivo</i>. Finally, we demonstrate that the upregulation of <i>Alox5</i> by RUNX1-ETO9a occurs via the C₂H₂ zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, <i>KLF6</i> is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.</p></div

Analysis of <i>Alox5</i> promoter regulation by RE9a.

<p>(A) Schematic of <i>Alox5</i> promoter-luciferase reporter with motifs differing from RUNX1 consensus binding site (TGYGGT) indicated. Bases differing from consensus site labeled in lowercase. Transcription factor SP1 binding site also indicated. Numbers represent base pairs relative to transcription start site. (B) Basal regulation of <i>Alox5</i> promoter-luciferase truncations. Indicated reporters were transfected in the absence of RE9a and expression was normalized to <i>Renilla</i> luciferase and the −507 to +146 construct was set to 1. * = <i>p</i><0.01 relative to −507 reporter. (C) Inducible regulation of <i>Alox5</i> promoter-luciferase by RE9a. Indicated reporters were co-transfected with control or RE9a and expression was normalized to <i>Renilla</i> luciferase. Each control (Ctrl) transfection normalized to 1. <i>p</i>-value relative to −507 reporter +RE9a.</p

Regulation of <i>KLF6</i> by RUNX1-ETO and RE9a.

<p>(A) Expression of <i>KLF6</i> in control- (Ctrl) or RE9a-transfected K562 cells. RE9a or empty vector was co-transfected into K562 cells along with a GFP-expressing vector to determine transfection efficiency. <i>KLF6</i> mRNA levels were normalized to <i>GAPDH</i> with Ctrl set to 1, and samples were then normalized to account for transfection rate by percent GFP-expressing cells as determined by flow cytometry. Data show averages and standard deviations of three independent transfections. (B) Expression of <i>KLF6</i> in HL60 [t(8;21)-negative] and SKNO and Kasumi-1 [t(8;21)-positive] cell lines. <i>KLF6</i> mRNA levels were normalized to <i>GAPDH</i> and HL60 was set to 1. Data show averages and standard deviations of 3 independent RNA isolations. (C) Expression of <i>KLF6</i> in control-, RUNX1-ETO- or RUNX1-ETO9a-transduced HL60 cells. Following 2 rounds of retroviral transduction, <i>KLF6</i> levels determined as in (B), with control-transduced cells (Ctrl) set to 1. Data display averages and standard deviations of 3 independent transductions.</p

Upregulation of <i>Alox5</i> in acute myeloid leukemia and by RUNX1-ETO9a.

<p>(A) Normalized log₂ expression of <i>Alox5</i> in control or RE9a-leukemic murine lin⁻c-Kit⁺ bone marrow cells. mRNA transcript levels were normalized to <i>Gapdh</i> and control was set to 1. Data show averages and standard deviations from 3 independent mice each. (B) RE9a regulation of mouse <i>Alox5</i> promoter-luciferase reporter. Numbers indicate base pair relative to transcription start site. Two RUNX1 binding sites (TGTGGT) were either wildtype or mutated to TGTtag to abrogate RE9a binding <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003765#pgen.1003765-Meyers1" target="_blank">[54]</a>. Indicated promoter-luciferase reporter was co-transfected with control (Ctrl) or RE9a plasmid and expression was normalized to <i>Renilla</i> luciferase. Wildtype promoter+control set to 1. (C) RE9a regulation of truncated mouse <i>Alox5</i> promoter-luciferase reporter. Luciferase assay performed as described in (B), with −1783 to +146 promoter+control set to 1.</p

Loss of <i>Alox5</i> does not block RE9a leukemia induction <i>in vivo</i>.

<p>(A) Survival of mice receiving wildtype or <i>Alox5</i>-/- fetal liver cells transduced by control (MigR1) or RE9a retrovirus. Number of mice in each cohort shown at right. WT median survival: 30.71 weeks; <i>Alox5</i>-/- median survival: 29.43 weeks; <i>p</i> = 0.39. (B) Presence of hematopoietic blast cells in tissues of mice transplanted with RE9a-transduced wildtype or <i>Alox5</i>-/- cells. Peripheral blood smears and cytocentrifugation of bone marrow and spleen cells were stained with Wright-Giemsa solutions. (C) Immunophenotype of myeloid progenitor cells in wildtype and <i>Alox5</i>-/- leukemias. Distribution of EGFP⁺Lin⁻Sca-1⁻c-Kit⁺ leukemic cells harvested from spleen shown based on expression of CD34 and Fcγ receptors II/III (FcγRII/III). At least 4 mice analyzed per genotype, with representative distributions shown.</p