Constitutive IRF8 Expression Inhibits AML by Activation of Repressed Immune Response Signaling

ORIGINAL ARTICLE Constitutive IRF8 expression inhibits AML by activation of repressed immune response signaling

A Sharma1,7,HYun1,7, N Jyotsana1, A Chaturvedi1, A Schwarzer2, E Yung3, CK Lai3, F Kuchenbauer4, B Argiropoulos5,KGo¨ rlich1, A Ganser1, RK Humphries3,6 and M Heuser1

Myeloid differentiation is blocked in acute myeloid leukemia (AML), but the molecular mechanisms are not well characterized. Meningioma 1 (MN1) is overexpressed in AML patients and confers resistance to all-trans retinoic acid-induced differentiation. To understand the role of MN1 as a transcriptional regulator in myeloid differentiation, we fused transcriptional activation (VP16) or repression (M33) domains with MN1 and characterized these cells in vivo. Transcriptional activation of MN1 target genes induced myeloproliferative disease with long latency and differentiation potential to mature neutrophils. A large proportion of differentially expressed genes between leukemic MN1 and differentiation-permissive MN1VP16 cells belonged to the immune response pathway like interferon-response factor (Irf) 8 and Ccl9. As MN1 is a cofactor of MEIS1 and retinoic acid receptor alpha (RARA), we compared chromatin occupancy between these genes. Immune response genes that were upregulated in MN1VP16 cells were co-targeted by MN1 and MEIS1, but not RARA, suggesting that myeloid differentiation is blocked through transcriptional repression of shared target genes of MN1 and MEIS1. Constitutive expression of Irf8 or its target gene Ccl9 identiﬁed these genes as potent inhibitors of murine and human leukemias in vivo. Our data show that MN1 prevents activation of the immune response pathway, and suggest restoration of IRF8 signaling as therapeutic target in AML.

Leukemia (2015) 29, 157–168; doi:10.1038/leu.2014.162

INTRODUCTION healthy volunteers.11 MN1 expression above the median The inability of acute myeloid leukemia (AML) cells to differentiate expression has been identified as an independent prognostic into phagocytically active macrophages and neutrophils is a key factor in patients with AML with normal cytogenetics, associated characteristic of this disease placing patients at high risk of with shorter relapse-free survival, overall survival and resistance to infectious complications. Several transcription factors that regulate ATRA-induced differentiation.5,10,12–15 Whether MN1 expression is myeloid differentiation are mutated or dysregulated in AML cells, required for AML cell survival has been investigated in a study leading to a block in differentiation.1,2 Although the differentiation using the THP1 AML cell line wherein MN1 downregulation via block can be overcome effectively by all-trans retinoic acid (ATRA) RNA interference impaired proliferation and significantly in acute promyelocytic leukemia driven by the PML–RARA decreased clonogenic activity.16 It is still not completely known (promyelocytic leukemia–retinoic acid receptor alpha) fusion in which proportion of AML patients MN1 has a similar critical 3,4 gene, other AML subtypes are resistant to ATRA. A pathogenetic role as shown in the study of THP1 cells. paradigmatic model of ATRA-resistant AML is the meningioma 1 The role of MN1 as one of the most potent oncogenes in murine (MN1) model, which induces AML in mice on constitutive und human leukemogenesis is well established. MN1 overexpre- expression and a 3000-fold resistance to ATRA in preleukemic ssion alone or in cooperation with inv(16)-generated CBFb–MYH11 cells.5 MN1 was identified as a gene disrupted by balanced fusion gene has been shown to rapidly induce leukemia in mice translocation t(12;22)(p13;q11) in patients with myeloid with high blast counts.5,11 Although MN1 overexpression leads to malignancies.6 Additional evidence of a pathogenetic role of repression of the myeloid transcription factors CEBPa and PU.1,5 MN1 upregulation came from gene expression profiling studies, the mechanisms how MN1 blocks myeloid differentiation are not which identified MN1 as part of a gene signature associated with well understood. Moreover, MN1 is able to expand normal human treatment-resistant AML.7 Subsequently, other gene expressing CD34 þ cord blood cells, suggesting that it has leukemogenic profiling studies revealed that high MN1 expression is present in potential in human cells as well.17 MN1 possesses the ability to AML patients with inv(16) or EVI1 overexpression.8,9 The levels of transform single common myeloid progenitor cells to MN1 expression vary in a wide range in cytogenetically normal immortalized leukemic stem cells, showing that common AML patients with a significantly higher median expression of myeloid progenitors are one source of cells of origin in MN1- MN1 in AML patients compared with peripheral blood induced AML.18 Our previous data suggested that MN1 enhances mononuclear cells in healthy volunteers.10 MN1 expression in the transcriptional activity of MEIS1 and HOXA9 as a leukemic blasts of patients with inv(16) AML was found to be transcriptional cofactor.18 MN1 is colocalized with MEIS1 at a 17–112-fold higher as compared with that of bone marrow of large proportion of MEIS1-binding sites, and MEIS1 with its

1Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; 2Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 3Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada; 4Department of Internal Medicine III, University Hospital Medical Center, Ulm, Germany; 5Department of Medical Genetics, HSC, University of Calgary, Calgary, Alberta, Canada and 6Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada. Correspondence: Dr M Heuser, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany, E-mail: [email protected] 7These authors contributed equally to this work. Received 21 August 2013; revised 28 April 2014; accepted 5 May 2014; accepted article preview online 20 May 2014; advance online publication, 24 June 2014 Irf8 inhibits MN1 leukemia A Sharma et al 158 cofactor HOXA9 is essential for MN1 leukemogenesis.18 MEIS1 and Mice were kept in pathogen-free conditions at the central animal HOXA9 themselves are well-established drivers of self-renewal, laboratory of Hannover Medical School. All animal experiments were capable of leukemia induction on overexpression.19 The quest for approved by the Lower Saxony state office for consumer protection, potential cooperative partners of several oncogenic fusion genes Oldenburg, Germany. (NUP98–HOXD13,20 CALM–AF10,21 MLL–AF922 and MLL–ENL16)or AML1 mutations23 has led to the identification of Mn1 as a Bone marrow transplantation and monitoring of mice common target of insertional mutagenesis, thereby promoting Transduced bone marrow cells from C57BL/6 J mice were injected into the leukemogenesis. More recently, a hematopoietic stem cell gene tail vein of lethally irradiated syngeneic recipient mice that were exposed therapy trial has confirmed that aberrant MN1 activation due to to a single dose of 800 cGy total-body irradiation accompanied by a life- g-retroviral insertion in a patient has promoted the development of sparing dose of 1 Â 105 freshly isolated bone marrow cells from syngenic null AML.24 mice. In the case of NOD/SCID/IL2rg (NSG) mice, a dose of 300 cGy total- ICSBP (interferon consensus sequence-binding protein) also body irradiation was used without helper cells. known as interferon-response factor (Irf8) is a transcription factor of the interferon regulatory factor (IRF) family and has a decisive Clonogenic progenitor assay role for differentiation of macrophages derived from granulo- Colony-forming cell units were assayed in methylcellulose (Methocult cyte-macrophage progenitors.2,25–27 Both heterozygous and M3234; StemCell Technologies Inc., Vancouver, BC, Canada) as described in homozygous losses of Irf8 deregulate hematopoiesis and induce supplementary materials. a syndrome similar to human chronic myelogenous leukemia in 28 mice; moreover, this syndrome can be further accelerated by Cell cycle analysis in vitro overexpression of Meis1 or Meis3.29 Loss of Irf8 has also been For in vitro labeling, 10 mM of 5-bromodeoxyuridine (BrdU) was added to shown to be a collaborating event in NUP98-TOP1-induced 0.5 million cells for 6 h. Cell cycle analysis was performed according to the 30 murine leukemia. Patients with chronic myelogenous leukemia manufacturers’ protocol (BD Pharmingen Cat. no. 557892, Heidelberg, have low levels of IRF8 in BCR-ABL-expressing cells, and chronic Germany). myelogenous leukemia therapy with interferon alpha or gamma 31–33 restores IRF8 levels. Forced expression of Irf8 inhibits BCR- Immunoblotting and enzyme-linked immunosorbent assay ABL-induced myeloproliferative disorder.34,35 Re-expression of For western blotting, cell lysates were prepared and immunoblotting was chemokine (C–C motif) ligand 6 (Ccl6) and chemokine (C–C performed using monoclonal mouse anti-Irf8 antibody (ICSBP (C-19): motif) ligand 9 (Ccl9) immune response genes, downstream of Irf8, sc-6058) from Santa Cruz Biotechnology Inc. (Heidelberg, Germany), and abolishes the ability of BCR-ABL-transformed cells to cause monoclonal mouse anti--actin (AC-15, Sigma-Aldrich, Seelze, Germany). leukemia in a syngeneic mouse leukemia model by inducing a T-cell-mediated response against antigens preferentially H O measurement expressed on BCR-ABL-transformed cells.36 In addition, stable 2 2 and conditional expression of IRF8 in BCR-ABL-transformed 32D The measurement of hydrogen peroxide was carried out using the Amplex cells inhibited BCR-ABL-mediated leukemogenesis in vivo, and red hydrogen peroxide/peroxidase assay kit as described by the manufacturer (Molecular Probes, Invitrogen, Darmstadt, Germany). overrodes BCR-ABL-induced inhibition of differentiation by repression of Bcl-2, a major antiapoptotic target of BCR-ABL.37 In patients with therapy-related AML, gene expression profiling Gene expression profiling of CD34 þ hematopoietic stem/progenitor cells revealed that Gene expression profiling of MN1VP16 bone marrow cells that were patients with loss of chromosome 5 or deletion of 5q, in addition transplanted and collected from mice was performed on Affymetrix to upregulation of growth-promoting genes, had no expression of GeneChip Mouse 430 2.0 arrays (Affymetrix, High Wycombe, UK). Gene 38 expression profiles of MN1 and Gr1 þ /CD11b þ bone marrow cells have IRF8. The IRF8 promoter has also been found hypermethylated in 18 39 been described previously. Data were analyzed using R and patients with myelodysplastic syndrome and AML. Recently, it Bioconductor. was shown that altered innate immune signaling has an important role in the pathogenesis of myelodysplastic syndrome.40,41 However, whether forced expression of Irf8 or its downstream Quantitative reverse-transcriptase PCR target genes would mimic similar antileukemic effects in AML as in Quantitative reverse-transcriptase PCR (qRT-PCR) was done as previously described using SYBR green (Qiagen, Hilden, Germany) on the StepOne- chronic myelogenous leukemia is presently unknown. 43 We employed the MN1-induced AML model to investigate Plus Real-Time PCR system (Applied Biosystems, Darmstadt, Germany). Primer sequences are provided in Supplementary Table S1. mechanisms underlying the severe differentiation block in AML cells, and found that innate immunity signaling is severely impaired in MN1-mediated leukemogenesis, whereas activation ChIP-seq data processing and bioinformatic analysis of the repressed immune response pathway inhibited MN1 Chromatin immunoprecipitation (ChIP)-seq on MN1 and MEIS1 was leukemia and human AML in vivo. performed as described previously.18 RARA ChIP-seq data were obtained from the GEO database under accession number GSM1047939.44 Relative distance of peak maxima between ChIP-seq data sets was calculated with MATERIALS AND METHODS ChIP-Cor module provided by the Swiss Institute of Bioinformatics. Intersection of MN1 chromatin peaks with MEIS1 or RARA peaks was More detailed information on Materials and Methods can be found in the performed using Table Browser from UCSC genome browser. Genome Supplementary Information. features and motif finding of overlapping peaks were generated by CEAS module from Cistrome/Galaxy.45 Target gene annotation and gene Retroviral vectors and vector production ontology analysis were performed with GPAT46 and DAVID bioinformatic 47 Retroviral vectors pSF91-IRESeGFP, pSF91-MN1-IRESeGFP, pSF91-MN1VP16- resource. IRESeGFP and MSCV-IRES-YFP have been described previously.5,18,42 The human MN1 gene was subcloned into EcoRI sites in the MSCV-IRES-YFP Gene set enrichment analysis vector from pSF91-hMN1-IRES-eGFP as described previously.5,18 Genome-wide gene expression data from MN1, MN1VP16 and Gr1 þ /CD11b þ cells were used for gene set enrichment analysis (GSEA). Cell lines and culture conditions The Broad Institute GSEA software package was employed48 for GSEA Mouse cell lines were generated from 6- to 8-week-old female C57BL/6 J using gene ontology gene sets from the Molecular Signatures Database mice, which were purchased from Charles River, Sulzfeld, Germany. (http://www.broad.mit.edu/gsea/msigdb/).

Leukemia (2015) 157 – 168 & 2015 Macmillan Publishers Limited Irf8 inhibits MN1 leukemia A Sharma et al 159 ChIP-PCR Immune response signaling is inhibited in MN1 leukemic cells but Chromatin immunoprecipitates of MN1- and MEIS1-transduced cells were activated in MN1VP16 cells. prepared as previously reported.18 Immunoprecipitated samples and input To identify the gene signature that determines the differentiation- samples were quantified for selected target genes and control regions by permissive capacity of MN1VP16 cells, gene expression profiles of qPCR using SYBR green and the StepOnePlus Real-Time PCR system. The MN1 and MN1VP16 cells from mouse bone marrow at 4 weeks enrichment of targets and controls was calculated as percent of input DNA À DCT after transplantation and Gr1 þ /CD11b þ bone marrow cells from using 2 (DCT refers to CTtarget-CTinput) methods. Primer sequences are provided in Supplementary Table S1. normal mice were compared by unsupervised hierarchical clustering. In this analysis, MN1VP16 cells clustered more closely with Gr1 þ /CD11b þ cells (Figure 2a). The most differentially Statistical analysis expressed genes between MN1 and MN1VP16 cells included a Pairwise comparisons were performed using Student’s t-test for contin- number of immune response-related genes, which are upregu- uous variables and using w2-test for categorical variables. The two-sided lated in MN1VP16 cells (Figure 2b and Supplementary Table S3). level of significance was set at Po0.05. Comparison of survival curves was GSEA showed that 38% of the 60 most differentially expressed performed using the log-rank test. Statistical analyses were performed gene sets belonged to immune response and immune regulation using Microsoft Excel (Microsoft, Munich, Germany) and GraphPad Prism 5 pathways, and 6% were related to hypoxia and oxygen (GraphPad Software, La Jolla, CA, USA). Leukemia-initiating cell frequencies metabolism (Figure 2c, Supplementary Table S4). Enrichment were calculated with the L-Calc software (StemCell Technologies). plots for ‘immune effector process’ and ‘cytokine activity’ showed good enrichment in MN1VP16 cells compared with MN1 cells (Figure 2d). One of the most upregulated genes in MN1VP16 cells RESULTS compared with MN1 cells was Irf8, which is a well-studied MN1-mediated transcriptional activation releases the regulator of monocytic differentiation.2,25–27 Differential expression differentiation block in AML of several immune response-related genes was confirmed by To study whether the transcription cofactor MN1 primarily quantitative RT-PCR, whereas Meis2 was one of the most activates or represses gene transcription, a transcriptional activa- downregulated genes in MN1VP16 compared with MN1 cells in tion domain (VP16) or a transcriptional repression domain contrast to Meis1, which was not differentially expressed between (repression domain of M33/CBX2) was fused to the C-terminus MN1VP16 and MN1 cells (Figure 2e). We next investigated whether of MN1. Full-length MN1, MN1VP16 and MN1M33 were trans- the activated immune response pathway resulted in increased duced into mouse bone marrow cells, and transgene-positive cells phagocytic activity of MN1VP16 cells. Indeed, stimulation with were evaluated in colony-forming cell assays. MN1 as well as lipopolysaccharide induced significantly higher levels of hydrogen MN1M33 and MN1VP16 immortalized cells in vitro (data not peroxide in MN1VP16 compared with MN1 cells (Figure 2f). Thus, shown), and could be replated in colony-forming cell assays immune response pathways were significantly upregulated in producing high numbers of colonies (Figure 1a). MN1VP16 cells MN1VP16 cells compared with MN1 cells, resulting in increased expressed more myeloid markers and less c-Kit in vitro than MN1 phagocytic activity on lipopolysaccharide stimulation. and MN1M33 cells (Figure 1b). Supplementary Figures S1A–C Employing a detailed structure–function analysis of MN1 (to be show the blast-like morphology and immunophenotype of MN1 reported elsewhere), we found that the C terminus of MN1 is cells and negativity of the mast cell marker IgER. In vivo, MN1 and critical for the myeloid differentiation block and ATRA resistance, MN1M33 cells induced a rapid leukemia with the same short whereas loss of the 206 most C-terminal amino acids (MN1DC) latency (median 35.5 and 39 days, respectively), whereas mice restored ATRA sensitivity (Supplementary Figure S2). Unsupervised receiving MN1VP16 cells died after a significantly longer latency hierarchical clustering of gene expression profiling data of MN1, (median 146 days, Figure 1c). At death, MN1, MN1M33 and MN1VP16, MN1DC and normal Gr1 þ /CD11b þ cells demonstrated MN1VP16 mice had high white blood cell counts (Figure 1d) and that MN1VP16 and MN1DC cells cluster together with differentiated low red blood cell counts in peripheral blood (Figure 1e). All MN1, normal Gr1 þ /CD11b þ cells (Supplementary Figure S3). Validation MN1M33 and MN1VP16 mice showed splenomegaly (Figure 1f), by RT-PCR showed that Ccl9 and Irf8 were also upregulated and leukemic bone marrow cells were transplantable in secondary in MN1DC cells as in MN1VP16 compared with MN1 cells animals (Figure 1g). As in primary transplantation, MN1VP16 mice (Supplementary Figure S4A). As an indicator of the phagocytic survived significantly longer after secondary transplantation than activity of MN1DC, we measured H2O2 levels and found significantly MN1 or MN1M33 mice. Morphology of MN1 and MN1M33 bone higher levels of H2O2 in MN1DC cells similar to MN1VP16 cells when marrow cells at death proved the diagnosis of AML with very high compared with MN1 cells (Supplementary Figure S4B). Thus, both blast counts (Figure 1h). In contrast, the bone marrow of MN1VP16 functional (MN1VP16) and structural (MN1DC) variants of MN1 mice showed almost exclusively neutrophils, demonstrating that induce a gene expression signature of differentiated myeloid cells, MN1VP16 induced a myeloproliferative disease in mice with full suggesting that transcriptional activation and the C terminus of differentiation potential (Figure 1h). We compared cell cycle MN1 are required to block myeloid differentiation. distribution in MN1 and MN1VP16 cells and found significantly more MN1VP16 cells in G0/G1 phases and fewer cells in S/G2/M phases compared with MN1 cells, consistent with reduced Immune response genes are directly regulated by MN1 through proliferation of MN1VP16 cells (Figure 1i). To investigate whether MN1/MEIS1 but not MN1/RARA co-occupied chromatin targets the fusion of M33 to MN1 had any biologic effect, we performed As previously reported by Van Wely et al.,49 MN1 localizes to limiting dilution transplantation to determine the leukemic stem retinoic acid response elements at the chromatin level. Moreover, cell frequency of MN1 and MN1M33 cells. The stem cell frequency as MN1 is associated with a block in myeloid differentiation and of MN1M33 cells was 24-fold higher compared with MN1 cells, induces resistance against ATRA, the ligand of RARA, we indicating increased self-renewal in MN1M33 cells (Figure 1j and hypothesized that MN1 represses RARA target genes. To test Supplementary Table S2). In summary, transcriptional activation of this hypothesis, we first identified common chromatin targets of MN1 target genes by VP16 released the myeloid differentiation MN1 and RARA and of MN1 and MEIS1 as a control. First, ChIP-seq block and attenuated cycling of these cells, whereas transcrip- data sets of MN1, MEIS1 and RARA were compared with each tional repression of MN1 cells by M33 enhanced the leukemogenic other. As shown before, MN1 and MEIS1 peaks overlapped to a activity of MN1 by increasing the leukemia stem cell frequency. high degree (26 207 out of 37 610 MEIS1 peaks (69.68%) were These data suggest that MN1 blocks myeloid differentiation by centered within ±250 bp of MN1 peak maxima, Figures 3a and c). transcriptional repression of its target genes. Similarly, RARA peaks were enriched at MN1 peaks (8347 out of

& 2015 Macmillan Publishers Limited Leukemia (2015) 157 – 168 Irf8 inhibits MN1 leukemia A Sharma et al 160

Figure 1. MN1-mediated transcriptional activation releases the differentiation block in AML. (a) Cumulative colony-forming cell (CFC) output of bone marrow cells transduced with control, MN1, MN1M33 or MN1VP16 over six rounds of platings (mean±s.e.m., n ¼ 4). (b) Immunophenotype of GFP þ MN1, MN1M33 and MN1VP16 cells in vitro (mean±s.e.m., n ¼ 4). (c) Survival of mice receiving transplants of bone marrow cells transduced with the indicated constructs (log-rank test). (d) White blood cell count in peripheral blood of mice at the time of killing (mean±s.e.m. of the indicated number of mice). (e) Red blood cell count in peripheral blood of mice at the time of killing (mean±s.e.m. of the indicated number of mice). (f) Spleen weight of mice at the time of killing (mean±s.e.m. of the indicated number of mice). (g) Survival of secondary recipient mice that received bone marrow cells from primary recipients collected at time of killing (log-rank test). (h) Morphology of bone marrow cytospins from primary moribund MN1, MN1M33 and MN1VP16 mice (more than 90% of bone marrow cells were GFP positive at the time of death). (i) Cell cycle analysis of MN1 and MN1VP16 cells in vitro using BrdU (mean±s.e.m., n ¼ 4) (j) Leukemia stem cell frequency in MN1- and MN1M33-transduced bone marrow cell cultures determined by limiting dilution transplantation assay (proportion of LSCs, n ¼ 3 mice per dose group, four cell doses, see also Supplementary Table S2). *Po0.05; **Po0.01; ns, not signiﬁcant.

23 404 RARA peaks (35.66%) were centered within ±250 bp of were most frequently located in the promoter or 50-untranslated MN1 peak maxima, Figures 3b and c). A high proportion of MEIS1 region of genes, whereas shared MN1/MEIS1 peaks without RARA and RARA peaks overlapped with MN1, respectively, but not with peak were most frequently located in the intronic or intergenic each other (Figure 3c and Supplementary Table S5). We further region (Figure 3d). One of the most enriched transcription factor- evaluated the two peak sets MN1/MEIS1 without RARA and binding motifs for shared MN1/RARA peaks without MEIS1 peak MN1/RARA without MEIS1 and found a very distinct distribution was the AP2alpha motif, whereas the MEIS1 motif was highly within the genome. Shared MN1/RARA peaks without MEIS1 peak enriched for MN1/MEIS1 peaks without RARA peak (Figure 3e).

Leukemia (2015) 157 – 168 & 2015 Macmillan Publishers Limited Irf8 inhibits MN1 leukemia A Sharma et al 161

Figure 2. Immune response signaling is inhibited in MN1 leukemic cells but activated in MN1VP16 cells. (a) Unsupervised hierarchical clustering of gene expression proﬁles from MN1 and MN1VP16 bone marrow cells from leukemic mice and Gr1 þ /CD11b þ bone marrow cells from normal mice. (b) Heatmap from unsupervised hierarchical clustering showing the top 50 differentially expressed genes in cells transduced with MN1 or MN1VP16. * indicates genes related to immune response signaling. (c) Graphical representation of enriched categories of gene ontology terms for MN1VP16 cells compared with MN1 cells based on GSEA from gene chip arrays. The top 60 gene sets upregulated in MN1VP16 were selected for the analysis. (d) Enrichment plot for the gene sets ‘immune effector process’ and ‘cytokine activity’ comparing MN1VP16 and MN1 cells. (e) RT-PCR validation of differentially expressed genes between MN1VP16 and MN1 cells (mean±s.e.m., n ¼ 3). (f)Hydrogenperoxidelevels in lipopolysaccharide-treated cells (1 mg/ml) measured by absorbance of MN1 and MN1VP16 cells that were incubated with Amplex red reaction mixture for 12 h (mean±s.e.m., n ¼ 4). a.u., arbitrary units; NES, normalized enrichment score. **Po0.01.

We next identiﬁed genes that were differentially expressed in MN1 detail. Genes upregulated in MN1VP16 vs MN1 cells that were and MN1VP16 cells and were targeted by either MN1/RARA directly targeted by MN1 and MEIS1, but not RARA, were without MEIS1 or MN1/MEIS1 without RARA. Interestingly, many highly enriched for immune response function (Figure 3f genes of the immune response pathway were regulated by and Supplementary Table S6). ‘Regulation of immune process’, MN1/MEIS1, but not by MN1/RARA. Functional annotation analysis ‘immune response’, ‘lysosome’ and ‘leukocyte/lymphocyte activation’ was performed with these gene lists to investigate the association were among the ﬁve most enriched gene sets of this gene list. of target gene ontology with corresponding regulators in more However, genes downregulated in MN1VP16 vs MN1 and targeted

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Figure 3. Immune response genes are directly regulated by MN1 through MN1/MEIS1 but not MN1/RARA co-occupied chromatin targets. (a) Histogram showing the distance between peak maxima of MN1 peaks to their closest MEIS1 peak, indicating that MN1-binding sites are highly enriched at MEIS1-binding sites. (b) Histogram showing the distance between peak maxima of MN1 peaks to their closest RARA peak, indicating that MN1-binding sites are enriched at RARA-binding sites. (c) Venn diagram of unique and overlapping chromatin peaks of MN1, MEIS1 and RARA ChIP-seq libraries. (d) Distribution of peaks in promoter, intragenic and intergenic regions that are shared by MN1 and MEIS1, but not RARA (white bars), and peaks that are shared by MN1 and RARA, but not MEIS1 (black bars). (e) Transcription factor motif enriched at shared binding sites of MN1 and MEIS1, but not RARA, showing the consensus motif of MEIS1 (top). Transcription factor motif enriched at shared binding sites of MN1 and RARA, but not MEIS1, showing the motif of AP2alpha (bottom). (f) Gene ontology terms of upregulated (upper panel) and downregulated (lower panel) genes between MN1VP16 and MN1 cells that are directly targeted by MN1 and MEIS1 but not RARA. (g) Gene ontology terms of upregulated (upper panel) and downregulated (lower panel) genes between MN1VP16 and MN1 cells that are directly targeted by MN1 and RARA but not MEIS1.

by MN1 and MEIS1, or genes differentially expressed in MN1VP16 vs Re-expression of Irf8 or its target gene Ccl9 increases reactive MN1 and targeted by MN1 and RARA but not MEIS1 did not oxygen species in MN1-transformed cells in vitro show a clear enrichment of immune response-related gene sets Irf8 and its target gene Ccl9 were among the most upregulated (Figures 3f and g). These data show that the immune response genes in MN1VP16 cells (Supplementary Table S3) and were pathway, which is repressed in MN1 leukemic cells but upregulated directly targeted by MN1 and MEIS1 (Figure 4a and in MN1VP16 cells, is co-regulated by MN1 and MEIS1, but not RARA. Supplementary Table S6). Chromatin binding of MN1 and MEIS1

Leukemia (2015) 157 – 168 & 2015 Macmillan Publishers Limited Irf8 inhibits MN1 leukemia A Sharma et al 163

Figure 4. Re-expression of Irf8 or its target gene Ccl9 increases reactive oxygen species in MN1-transformed cells in vitro.(a) UCSC browser visualization of chromatin peaks of MN1-, MEIS1- and IgG-control immunoprecipitations at the Ccl9 gene locus. (b) Validation of selected chromatin peaks shared by MN1 and MEIS1, and of control regions (chromatin sites not targeted by either MN1 or MEIS1). Chromatin immunoprecipitates of MN1 or MEIS1 from MN1 or MEIS1 þ ND13 leukemic cells, respectively, were quantified by qPCR and compared with input DNA (n ¼ 4, mean±s.e.m.). (c) Western blot showing protein expression of Irf8 in MN1 cells transduced with control or Irf8 vectors. (d) Enzyme-linked immunosorbent assay for Ccl9 in MN1 cells transduced with control or Ccl9 vector (Ccl9 protein concentration (pg/ml), mean±s.e.m., n ¼ 4). (e) Cell cycle analysis of MN1 þ CTL, MN1 þ Ccl9 and MN1 þ Irf8 cells in vitro using BrdU (mean±s.e.m., n ¼ 4, two repeats from two independently transduced cell lines). (f) Morphology of in vitro-cultured cells—cytospins of MN1 þ CTL, MN1 þ Ccl9 and MN1 þ Irf8. (g) Hydrogen peroxide levels in lipopolysaccharide-treated cells (1 mg/ml) measured by absorbance of MN1 þ CTL, MN1 þ Ccl9 and MN1 þ Irf8 cells that were incubated with Amplex red reaction mixture for 12 h (mean±s.e.m., n ¼ 4, two repeats from two independently transduced cell lines). a.u., arbitrary units. *Po0.05; **Po0.01; ns, not significant. to regulatory regions of Irf8 and Ccl9 was confirmed by ChIP-PCR target genes of MN1, which can mimic, on re-expression, the (Figure 4b). We hypothesized that re-expression of genes of the characteristics of differentiation-permissive MN1VP16 cells. immune response pathway in MN1 cells will alter MN1 leukemic cell properties. Irf8 or its target gene Ccl9 or a control vector was transduced in MN1 cells and their expression was confirmed by Irf8 and Ccl9 inhibit MN1-induced leukemia in mice western blot and enzyme-linked immunosorbent assay, respec- The antileukemic potential of Irf8 and Ccl9 was assessed in MN1 tively, and by RT-PCR (Figures 4c and d, and Supplementary Figure leukemic cells by constitutive expression of Irf8, Ccl9 or a control S5A and B). Overexpression of Irf8 led to reduced cell cycling, as vector in MN1 cells and transplantation of 1 Â 105 sorted, double- more cells were observed in G0/G1 phase and less cells were in positive cells in lethally irradiated mice. Interestingly, engraftment S/G2/M phases compared with MN1 þ CTL cells (Figure 4e). of Ccl9- or Irf8-transduced cells was significantly lower at 4 weeks MN1 þ Ccl9 and MN1 þ Irf8 cells showed distinct morphologic (Figure 5a). At 8 weeks, engraftment of MN1 þ Ccl9 cells increased, characteristics in vitro. MN1 þ Ccl9 cells showed a high rate of whereas it stayed constant for MN1 þ Irf8 cells (Figure 5a). Ccl9 vesiculation at their surface, and MN1 þ Irf8 cells were highly prolonged the time until disease onset and Irf8 prevented granulated (Figure 4f). Quantitation of hydrogen peroxide levels as leukemia in 6 of 10 mice (Figure 5b). At 4 weeks, white blood a measure of phagocytic activity was significantly elevated in cell counts were lower in MN1 þ Irf8 and MN1 þ Ccl9 mice than in MN1 þ Irf8 and to a lesser extent in MN1 þ Ccl9 cells (Figure 4g), MN1 þ CTL mice (although not significantly, Figure 5c), hemo- suggesting that Irf8 indeed is one of the key repressed globin levels and platelet counts were higher in MN1 þ Irf8 and

& 2015 Macmillan Publishers Limited Leukemia (2015) 157 – 168 Irf8 inhibits MN1 leukemia A Sharma et al 164

Figure 5. Irf8 and Ccl9 inhibit MN1-induced leukemia in mice. (a) Engraftment of double-transduced cells in peripheral blood of mice 4 and 8 weeks after transplantation (MN1-YFP and CTL/Ccl9/Irf8-GFP double positivity shown by flow cytometry). Mice received transplants of cells transduced with the indicated genes (mean±s.e.m. of the indicated number of mice, two independent repeats from two independently transduced cell lines). (b) Survival of mice receiving transplants of bone marrow cells transduced with the indicated constructs (log-rank test). (c–e) White blood cell count (c), hemoglobin (d) and platelet count (e) in peripheral blood of mice at 4 weeks. Mice received transplants of cells transduced with the indicated genes (mean±s.e.m. of the indicated number of mice). (f) Spleen weight of mice at the time of killing that received transplants of cells transduced with the indicated genes (mean±s.e.m. of the indicated number of mice). (g) Morphology of bone marrow cytospins from moribund MN1 þ CTL, MN1 þ Ccl9 and MN1 þ Irf8 mice. (h) Survival of mice receiving transplants of bone marrow cells transduced with the indicated constructs (log-rank test). (i) Survival of immunodeficient NOD-SCID-IL2rg null mice receiving transplants of bone marrow cells transduced with the indicated constructs (log-rank test). *Po0.05; **Po0.01; ns, not significant.

Leukemia (2015) 157 – 168 & 2015 Macmillan Publishers Limited Irf8 inhibits MN1 leukemia A Sharma et al 165 MN1 þ Ccl9 mice compared with MN1 þ CTL mice (Figures 5d and e) least in part cell autonomously in the MN1 model (Figure 5j). and spleen weight was significantly lower in MN1 þ Irf8 mice Importantly, five out of six mice transplanted with MN1 þ Irf8 in than in MN1 þ CTL mice (Figure 5f). Differential blood counts did NSG mice died from MN1 leukemia that had lost Irf8 expression not demonstrate any significant differences between MN1 þ CTL, (Supplementary Figure S7). In summary, re-expression of Irf8 or its MN1 þ Ccl9 and MN1 þ Irf8 mice (Supplementary Figure S6). Bone target gene Ccl9 inhibits MN1 leukemia, suggesting that activation marrow morphology of moribund mice showed a high blast count of the immune response pathway in leukemic cells can override for MN1 þ CTL and MN1 þ Ccl9 mice, but o20% blasts and many proliferation and survival signals in AML cells. neutrophils for MN1 þ Irf8 (Figure 5g). We also found Klf4 and Ccl5 In addition, we studied the role of IRF8 in AML xenograft genes upregulated in MN1VP16 cells, but their re-expression in models. IRF8 or a control vector was transduced in the MN1 cells did not significantly prolong survival of mice (Figure 5h) human AML cell lines OCI-AML3 and U937, and was further A previous study showed that re-expression of Irf8 in BCR-ABL- transplanted subcutaneously in NSG mice. After 15 days of transformed cells induced a T-cell response that prevented transplantation, tumor volumes were monitored every 5 days. outgrowth of leukemic cells in vivo, suggesting that the effect Overexpression of IRF8 in both AML cell lines led to significantly was non-cell autonomous.36 To evaluate whether Irf8 and Ccl9 act reduced tumor volumes in mice at all time points and at death cell autonomously or mainly attract immune effector cells, we compared with vector control (Figure 6a–f). Thus, overexpression transplanted MN1 þ Irf8 or MN1 þ Ccl9 cells in immunodeficient of IRF8 in AML xenograft models too possesses antitumor NOD-SCID-IL2rg null (NSG) mice. Expression of Irf8 or Ccl9 again activity, which further advocates its role for potential therapeutic prolonged leukemic onset, suggesting that these genes act at strategies.

Figure 6. IRF8 overexpression inhibits human AML in vivo.(a) Tumor volumes from OCI-AML3 cells transduced with control or IRF8 that were inoculated subcutaneously in both flanks of NSG mice. Tumor volumes were measured at the indicated time points after inoculation (mean±s.e.m., number of mice n ¼ 5; number of tumors n ¼ 10). (b) Tumor volumes from OCI-AML3 cells transduced with control or IRF8 that were inoculated subcutaneously in both flanks of NSG mice at death (mean±s.e.m., number of mice n ¼ 5; number of tumors n ¼ 10). (c) Macroscopic pictures of explanted tumors at the time of killing of NSG mice inoculated subcutaneously with OCI-AML3 cells transduced with control or IRF8 (scale bar 1 cm). (d) Tumor volumes from U937 cells transduced with control or IRF8 that were inoculated subcutaneously in both flanks of NSG mice. Tumor volumes were measured at the indicated time points after inoculation (mean±s.e.m., number of mice n ¼ 5 for control and n ¼ 10 for IRF8; number of tumors n ¼ 10 for control and n ¼ 20 for IRF8). (e) Tumor volumes from U937 cells transduced with control or IRF8 that were inoculated subcutaneously in both flanks of NSG mice at death (mean±s.e.m., number of mice n ¼ 5 for control and n ¼ 10 for IRF8; number of tumors n ¼ 10 for control and n ¼ 20 for IRF8). (f) Macroscopic pictures of explanted tumors at the time of killing of NSG mice inoculated subcutaneously with U937 cells transduced with control or IRF8 (scale bar 1 cm). *Po0.05; **Po0.01; ns, not significant.

& 2015 Macmillan Publishers Limited Leukemia (2015) 157 – 168 Irf8 inhibits MN1 leukemia A Sharma et al 166 DISCUSSION Further evidence for a role of MN1/MEIS1 target genes in MN1 induces an aggressive form of myeloid leukemia that is myeloid differentiation recently came from a study by Ficara 51 phenotypically arrested at the myeloid progenitor stage. We et al., showing rapid myeloid differentiation of hematopoietic found that fusion of the transcriptional activation domain VP16 stem cells on loss of PBX1, the dimerization partner of MEIS1. with MN1 dissociated the self-renewal from the differentiation- Oxygen metabolism was another functional category significantly inhibiting function, allowing full maturation to neutrophils in vivo. enriched in MN1VP16 vs MN1 cells, and reactive oxygen species The ability to block differentiation was found to be correlated with responsible for phagocytic function were found to be increased in repression of immune response pathways by MN1. On fusion of MN1VP16, MN1 þ Ccl9 and MN1 þ Irf8 cells. Recent studies have MN1 with VP16 or by deletion of the C terminus of MN1, repressed shown that MEIS1 is required to scavenge reactive oxygen species 52,53 immune response pathways were reactivated and the cells in hematopoietic stem cells and its expression is 18 phenotypically and functionally differentiated to neutrophils. downregulated during myeloid differentiation. In the present Interestingly, regulatory chromatin regions of genes of the study, we found that Meis2 expression was strongly immune response pathway were co-occupied by MN1 and MEIS1, downregulated in MN1VP16 cells, suggesting that low levels of and not RARA, providing a novel mechanism of differentiation Meis2 allow myeloid differentiation and induction of reactive blockage in AML cells with high MN1 expression. To further oxygen species. elucidate and mechanistically prove the role of immune response Irf8 was one of the most upregulated genes in differentiation- signaling in differentiation, we re-expressed two of the most permissive MN1VP16 cells and prolonged or prevented outgrowth downregulated genes in MN1 cells, Irf8 and Ccl9. Overexpression of MN1 leukemias. Constitutive expression of Irf8 (and Ccl9)in of Ccl9 and Irf8 genes significantly inhibited leukemic outgrowth, MN1 cells was 10-fold higher than endogenous expression of Irf8 prolonged disease latency or even prevented disease onset (Irf8) in MN1VP16 cells. Thus, our experiments may partly overestimate in vivo both in murine and human AML models. Re-expression of the effect of Irf8. However, 44 immune response-related genes these genes also showed increased phagocytic activity of were significantly upregulated in MN1VP16 cells compared with leukemic cells in vitro. These results demonstrate an antileukemic MN1 cells, which corresponds to 25% of all significantly effect of immune response signaling in leukemic cells (Figure 7). upregulated genes. Thus, even if Irf8 cannot induce differentiation MN1 confers resistance to ATRA-induced differentiation in vitro alone in MN1VP16 cells, it is an important component as it is and induces AML in mice.5 MN1 colocalizes with the nuclear sufficient to inhibit MN1 leukemia at high expression levels. A hormone receptor RARA at chromatin49 and while ATRA resistance previous study found that viral integrations in the MN1 locus of has been linked to transcriptional repression of RARA target Irf8-knockout mice accelerated leukemic onset,29 suggesting genes,50 we hypothesized that transcriptional activation of RARA contrasting roles of MN1 and Irf8. In a different model, Irf8 by target genes might allow differentiation of MN1 cells. Indeed, itself and via its downstream signaling has prevented leukemic MN1VP16 cells possessing transcriptional activator function outgrowth of BCR-ABL leukemia.36 This study identified Ccl6 and induced a myeloproliferative disease with longer latency and full Ccl9 as downstream targets of Irf8, which could delay leukemic differentiation potential. Differentiated myeloid cells are onset of BCR-ABL-positive cells on combined overexpression. characterized by their phagocytic activity; hence, it was not Interestingly, knockdown of Ccl6 or Ccl9 in Irf8-expressing BCR- unanticipated to find genes related to immune response most ABL-positive cells restored the leukemic phenotype of BCR-ABL differentially expressed in differentiation-permissive MN1VP16 or cells, suggesting that the antileukemic effects of Irf8 are mediated MN1DC cells. We have previously shown that a large proportion of by Ccl6 and Ccl9.36 These authors also showed that the MN1 chromatin peaks are co-occupied by MEIS1, suggesting a antileukemic effect of Irf8 was non-cell autonomous, because no similar function of these transcriptional regulators. Surprisingly, antileukemic effect of Irf8 was observed in Rag2-knockout mice. genes of the immune response pathway were co-occupied by However, in the present study, when Irf8 was expressed in MN1 MEIS1 and MN1, not with RARA, suggesting a novel role of MN1 cells and transplanted in immunodeficient NOD-SCID-IL2rg null and MEIS1 in suppressed innate immunity signaling. More general mice, Irf8 still exhibited an antileukemic effect, as it did in human these data suggest that myeloid differentiation is blocked in AML AML cells transplanted in NSG mice, suggesting that in our model through transcription factors like MN1 and MEIS1 and less through Irf8 acts at least in part cell autonomous. RAR signalling, thereby explaining the low activity of all-trans In summary, we identify immune response signaling as a retinoic acid in non-APL AML. repressed pathway in MN1-induced AML, which is co-regulated by

Figure 7. Summary highlighting the inhibitory effect of MN1 on immune response signaling and the antileukemic effect of activated IRF8 immune response signaling.

Leukemia (2015) 157 – 168 & 2015 Macmillan Publishers Limited Irf8 inhibits MN1 leukemia A Sharma et al 167 MN1 and MEIS1. Activation of the repressed immune signaling via acute myeloid leukemia: a cancer and leukemia group B study. J Clin Oncol 2009; re-activation of Irf8 or Ccl9 delays or prevents murine and human 27: 3198–3204. leukemia in vivo. We propose to activate immune signalling in 14 Schwind S, Marcucci G, Kohlschmidt J, Radmacher MD, Mrozek K, Maharry K et al. AML cells as a therapeutic strategy, for example by targeting MN1 Low expression of MN1 associates with better treatment response in older or MEIS1/2, which could be more effective than activating retinoic patients with de novo cytogenetically normal acute myeloid leukemia. Blood 2011; acid response signalling in these cells. 118: 4188–4198. 15 Miller BG, Stamatoyannopoulos JA. Integrative meta-analysis of differential gene expression in acute myeloid leukemia. PLoS One 2010; 5: e9466. CONFLICT OF INTEREST 16 Liu T, Jankovic D, Brault L, Ehret S, Baty F, Stavropoulou V et al. Functional characterization of high levels of meningioma 1 as collaborating oncogene in The authors declare no conflict of interest. acute leukemia. Leukemia 2010; 24: 601–612. 17 Kandilci A, Grosveld GC. Reintroduction of CEBPA in MN1-overexpressing hematopoietic cells prevents their hyperproliferation and restores myeloid ACKNOWLEDGEMENTS differentiation. Blood 2009; 114: 1596–1606. We thank Dr AM Baru and PD Dr Michael Morgan for discussions and Renate 18 Heuser M, Yun H, Berg T, Yung E, Argiropoulos B, Kuchenbauer F et al. Cell of Schottmann and Martin Wichmann for technical help. We also thank Dr Matthias origin in AML: susceptibility to MN1-induced transformation is regulated by the Ballmaier and the staff of the Cell Sorting Core Facility of Hannover Medical School MEIS1/AbdB-like HOX protein complex. Cancer Cell 2011; 20: 39–52. for their excellent service (supported in part by the Braukmann-Wittenberg-Herz- 19 Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM, Sauvageau G. Stiftung). This study was supported by DFG grants HE 5240/5-1, grants DJCLS H09/1f, Hoxa9 transforms primary bone marrow cells through specific collaboration with R 10/22, SP 12/02 and R13/14 from Deutsche-Jose´-Carreras Leuka¨mie-Stiftung e.V., Meis1a but not Pbx1b. EMBO J 1998; 17: 3714–3725. grants 109003, 110284, 110292 and 111267 from Deutsche Krebshilfe and the 20 Slape C, Hartung H, Lin YW, Bies J, Wolff L, Aplan PD. Retroviral insertional German Federal Ministry of Education and Research grant 01EO0802 (IFB-Tx). mutagenesis identifies genes that collaborate with NUP98-HOXD13 during leukemic transformation. Cancer Res 2007; 67: 5148–5155. 21 Caudell D, Harper DP, Novak RL, Pierce RM, Slape C, Wolff L et al. Retroviral AUTHOR CONTRIBUTIONS insertional mutagenesis identifies Zeb2 activation as a novel leukemogenic A Sharma and MH designed the research; A Sharma, HY, NJ, AC, A Schwarzer, collaborating event in CALM-AF10 transgenic mice. Blood 2010; 115: 1194–1203. 22 Bergerson RJ, Collier LS, Sarver AL, Been RA, Lugthart S, Diers MD et al. EY, CKL, FK, BA, KG and MH performed the research; and A Sharma, HY, NJ, AC, An insertional mutagenesis screen identifies genes that cooperate with Mll-AF9 in A Schwarzer, EY, CKL, FK, BA, KG, AG, RKH and MH analyzed the data. A Sharma, a murine leukemogenesis model. Blood 2012; 119: 4512–4523. HY and MH wrote the manuscript. All authors read and agreed to the final 23 Watanabe-Okochi N, Kitaura J, Ono R, Harada H, Harada Y, Komeno Y et al. AML1 version of the manuscript. mutations induced MDS and MDS/AML in a mouse BMT model. Blood 2008; 111: 4297–4308. 24 Braun CJ, Boztug K, Paruzynski A, Witzel M, Schwarzer A, Rothe M et al. Gene REFERENCES therapy for wiskott-Aldrich syndrome--long-term efficacy and genotoxicity. Sci 1 Ley TJ. Genomic and epigenomic landscapes of adult de novo acute myeloid Transl Med 2014; 6: 227ra233. leukemia. N Engl J Med 2013; 369: 98–98. 25 Li L, Jin H, Xu J, Shi Y, Wen Z. Irf8 regulates macrophage versus neutrophil fate 2 Rosenbauer F, Tenen DG. Transcription factors in myeloid development: during zebrafish primitive myelopoiesis. Blood 2010; 117: 1359–1369. balancing differentiation with transformation. Nat Rev Immunol 2007; 7: 26 Kurotaki D, Osato N, Nishiyama A, Yamamoto M, Ban T, Sato H et al. Essential role 105–117. of the IRF8-KLF4 transcription factor cascade in murine monocyte differentiation. 3 Milligan DW, Wheatley K, Littlewood T, Craig JI, Burnett AK. Fludarabine and Blood 2013; 121: 1839–1849. cytosine are less effective than standard ADE chemotherapy in high-risk acute 27 Tamura T, Nagamura-Inoue T, Shmeltzer Z, Kuwata T, Ozato K. ICSBP directs myeloid leukemia, and addition of G-CSF and ATRA are not beneficial: results of bipotential myeloid progenitor cells to differentiate into mature macrophages. the MRC AML-HR randomized trial. Blood 2006; 107: 4614–4622. Immunity 2000; 13: 155–165. 4 Lo-Coco F, Avvisati G, Vignetti M, Breccia M, Gallo E, Rambaldi A et al. Front-line 28 Holtschke T, Lohler J, Kanno Y, Fehr T, Giese N, Rosenbauer F et al. Immuno- treatment of acute promyelocytic leukemia with AIDA induction followed by risk- deficiency and chronic myelogenous leukemia-like syndrome in mice with adapted consolidation for adults younger than 61 years: results of the AIDA-2000 a targeted mutation of the ICSBP gene. Cell 1996; 87: 307–317. trial of the GIMEMA Group. Blood 2010; 116: 3171–3179. 29 Hara T, Schwieger M, Kazama R, Okamoto S, Minehata K, Ziegler M et al. 5 Heuser M, Argiropoulos B, Kuchenbauer F, Yung E, Piper J, Fung S et al. MN1 Acceleration of chronic myeloproliferation by enforced expression of Meis1 or overexpression induces acute myeloid leukemia in mice and predicts ATRA Meis3 in Icsbp-deficient bone marrow cells. Oncogene 2008; 27: 3865–3869. resistance in patients with AML. Blood 2007; 110: 1639–1647. 30 Gurevich RM, Rosten PM, Schwieger M, Stocking C, Humphries RK. Retroviral 6 Buijs A, Sherr S, van Baal S, van Bezouw S, van der Plas D, Geurts van Kessel A et al. integration site analysis identifies ICSBP as a collaborating tumor suppressor gene Translocation (12;22) (p13;q11) in myeloproliferative disorders results in fusion of in NUP98-TOP1-induced leukemia. Exp Hematol 2006; 34: 1192–1201. the ETS-like TEL gene on 12p13 to the MN1 gene on 22q11. Oncogene 1995; 10: 31 Schmidt M, Hochhaus A, Nitsche A, Hehlmann R, Neubauer A. Expression of 1511–1519. nuclear transcription factor interferon consensus sequence binding protein 7 Heuser M, Wingen LU, Steinemann D, Cario G, von Neuhoff N, Tauscher M et al. in chronic myeloid leukemia correlates with pretreatment risk features and Gene-expression profiles and their association with drug resistance in adult acute cytogenetic response to interferon-alpha. Blood 2001; 97: 3648–3650. myeloid leukemia. Haematologica 2005; 90: 1484–1492. 32 Schmidt M, Nagel S, Proba J, Thiede C, Ritter M, Waring JF et al. Lack of interferon 8 Ross ME, Mahfouz R, Onciu M, Liu HC, Zhou X, Song G et al. Gene expression consensus sequence binding protein (ICSBP) transcripts in human myeloid profiling of pediatric acute myelogenous leukemia. Blood 2004; 104: 3679–3687. leukemias. Blood 1998; 91: 22–29. 9 Valk PJ, Verhaak RG, Beijen MA, Erpelinck CA, Barjesteh van Waalwijk van Doorn- 33 Niederle N, Kloke O, Wandl UB, Becher R, Moritz T, Opalka B. Long-term treatment Khosrovani S, Boer JM et al. Prognostically useful gene-expression profiles in of chronic myelogenous leukemia with different interferons: results from three acute myeloid leukemia. N Engl J Med 2004; 350: 1617–1628. studies. Leuk Lymphoma 1993; 9: 111–119. 10 Heuser M, Beutel G, Krauter J, Dohner K, von Neuhoff N, Schlegelberger B et al. 34 Hao SX, Ren R. Expression of interferon consensus sequence binding protein High meningioma 1 (MN1) expression as a predictor for poor outcome in acute (ICSBP) is downregulated in Bcr-Abl-induced murine chronic myelogenous myeloid leukemia with normal cytogenetics. Blood 2006; 108: 3898–3905. leukemia-like disease, and forced coexpression of ICSBP inhibits Bcr-Abl-induced 11 Carella C, Bonten J, Sirma S, Kranenburg TA, Terranova S, Klein-Geltink R et al. myeloproliferative disorder. Mol Cell Biol 2000; 20: 1149–1161. MN1 overexpression is an important step in the development of inv(16) AML. 35 Deng M, Daley GQ. Expression of interferon consensus sequence binding protein Leukemia 2007; 21: 1679–1690. induces potent immunity against BCR/ABL-induced leukemia. Blood 2001; 97: 12 Haferlach C, Kern W, Schindela S, Kohlmann A, Alpermann T, Schnittger S et al. 3491–3497. Gene expression of BAALC, CDKN1B, ERG, and MN1 adds independent prognostic 36 Nardi V, Naveiras O, Azam M, Daley GQ. ICSBP-mediated immune protection information to cytogenetics and molecular mutations in adult acute myeloid against BCR-ABL-induced leukemia requires the CCL6 and CCL9 chemokines. leukemia. Genes Chromosomes Cancer 2012; 51: 257–265. Blood 2009; 113: 3813–3820. 13 Langer C, Marcucci G, Holland KB, Radmacher MD, Maharry K, Paschka P et al. 37 Burchert A, Cai D, Hofbauer LC, Samuelsson MK, Slater EP, Duyster J et al. Prognostic importance of MN1 transcript levels, and biologic insights from MN1- Interferon consensus sequence binding protein (ICSBP; IRF-8) antagonizes associated gene and microRNA expression signatures in cytogenetically normal BCR/ABL and down-regulates bcl-2. Blood 2004; 103: 3480–3489.

& 2015 Macmillan Publishers Limited Leukemia (2015) 157 – 168 Irf8 inhibits MN1 leukemia A Sharma et al 168 38 Qian Z, Fernald AA, Godley LA, Larson RA, Le Beau MM. Expression proﬁling 45 Ji X, Li W, Song J, Wei L, Liu XS. CEAS: cis-regulatory element annotation system. of CD34 þ hematopoietic stem/ progenitor cells reveals distinct subtypes of Nucleic Acids Res 2006; 34(Web Server issue): W551–W554. therapy-related acute myeloid leukemia. Proc Natl Acad Sci USA 2002; 99: 46 Krebs A, Frontini M, Tora L. GPAT: retrieval of genomic annotation from large 14925–14930. genomic position datasets. BMC Bioinformatics 2008; 9: 533. 39 Otto N, Manukjan G, Gohring G, Hofmann W, Scherer R, Luna JC et al. ICSBP 47 Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis promoter methylation in myelodysplastic syndromes and acute myeloid of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009; 4: leukaemia. Leukemia 2011; 25: 1202–1207. 44–57. 40 Starczynowski DT, Karsan A. Deregulation of innate immune signaling in 48 Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA et al. myelodysplastic syndromes is associated with deletion of chromosome arm 5q. Gene set enrichment analysis: a knowledge-based approach for interpreting Cell Cycle 2010; 9: 855–856. genome-wide expression proﬁles. Proc Natl Acad Sci USA 2005; 102: 15545–15550. 41 Wei Y, Chen R, Dimicoli S, Bueso-Ramos C, Neuberg D, Pierce S et al. Global 49 van Wely KH, Molijn AC, Buijs A, Meester-Smoor MA, Aarnoudse AJ, Hellemons A H3K4me3 genome mapping reveals alterations of innate immunity signaling and et al. The MN1 oncoprotein synergizes with coactivators RAC3 and p300 in overexpression of JMJD3 in human myelodysplastic syndrome CD34 þ cells. RAR-RXR-mediated transcription. Oncogene 2003; 22: 699–709. Leukemia 2013; 27: 2177–2186. 50 Wang ZY, Chen Z. Acute promyelocytic leukemia: from highly fatal to highly 42 Schambach A, Wodrich H, Hildinger M, Bohne J, Krausslich HG, Baum C. Context curable. Blood 2008; 111: 2505–2515. dependence of different modules for posttranscriptional enhancement of gene 51 Ficara F, Crisafulli L, Lin C, Iwasaki M, Smith KS, Zammataro L et al. Pbx1 restrains expression from retroviral vectors. Mol Ther 2000; 2: 435–445. myeloid maturation while preserving lymphoid potential in hematopoietic 43 Heuser M, Yap DB, Leung M, de Algara TR, Tafech A, McKinney S et al. progenitors. J Cell Sci 2013; 126(Pt 14): 3181–3191. Loss of MLL5 results in pleiotropic hematopoietic defects, reduced neutrophil 52 Kocabas F, Zheng J, Thet S, Copeland NG, Jenkins NA, DeBerardinis RJ et al. Meis1 immune function, and extreme sensitivity to DNA demethylation. Blood 2009; regulates the metabolic phenotype and oxidant defense of hematopoietic stem 113: 1432–1443. cells. Blood 2012; 120: 4963–4972. 44 Hu G, Cui K, Northrup D, Liu C, Wang C, Tang Q et al. H2 A.Z facilitates access of 53 Unnisa Z, Clark JP, Roychoudhury J, Thomas E, Tessarollo L, Copeland NG et al. active and repressive complexes to chromatin in embryonic stem cell self-renewal Meis1 preserves hematopoietic stem cells in mice by limiting oxidative stress. and differentiation. Cell Stem Cell 2012; 12: 180–192. Blood 2012; 120: 4973–4981.

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