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Insulin-Like Growth Factor 1 Is a Direct HOXA9 Target Important for Hematopoietic Transformation

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Insulin-Like Growth Factor 1 Is a Direct HOXA9 Target Important for Hematopoietic Transformation Leukemia (2015) 29, 901–908 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu ORIGINAL ARTICLE Insulin-like growth factor 1 is a direct HOXA9 target important for hematopoietic transformation J Steger, E Füller, M-P Garcia-Cuellar, K Hetzner and RK Slany HOX homeobox proteins are key oncogenic drivers in hematopoietic malignancies. Here we demonstrate that HOXA1, HOXA6 and predominantly HOXA9 are able to induce the production of insulin-like growth factor 1 (Igf1). In chromatin immunoprecipitations, HOXA9 bound directly to the putative promoter and a DNase-hypersensitive region in the first intron of the Igf1 gene. Transcription rates of the Igf1 gene paralleled HOXA9 activity. Primary cells transformed by HOXA9 expressed functional Igf1 receptors and activated the protein kinase Akt in response to Igf1 stimulation, suggesting the existence of an autocrine signaling loop. Genomic deletion of the Igf1 gene by Cre-mediated recombination increased sensitivity toward apoptosis after serum starvation. In addition, the leukemogenic potential of Igf1-negative, HOXA9-transformed cells was impaired, leading to a significant delay in disease development on transplantation into recipient animals. Leukemia (2015) 29, 901–908; doi:10.1038/leu.2014.287 INTRODUCTION factor FGF2 (fibroblast growth factor) are regulated by HOX 14,15 HOX homeobox genes are increasingly acknowledged as impor- proteins. Apart from these examples and despite the tant drivers of malignant transformation in the hematopoietic discovery of numerous HOXA9-binding sites across the genome 16 system. In line with their major regulatory role in hematopoietic by chromatin immunoprecipitation-sequencing ChIP-Seq, func- stem and precursor cell populations, HOX transcription needs to tionally characterized HOX targets are scarce. be extinguished for proper differentiation to occur (for detailed Here we identify Igf1 (insulin-like growth factor 1) as direct reviews of this topic see Alharbi et al.,1 Argiropoulos and HOXA9 subordinate gene and we show that Igf1 signaling Humphries2 and Eklund3). Consequently, inappropriate HOX contributes functionally to HOXA9-induced leukemia. The IGF activation has been detected in many hematologic malignancies. family has been widely implicated in the etiology of neoplastic High-level expression mainly of HOXA and HOXB family members disease. Insulin, IGF1 and IGF2 bind to a shared group of insulin/ is a hallmark of different leukemia subtypes like those harboring IGF receptor tyrosine kinases leading to the activation of PI3K MLL (mixed-lineage leukemia) or CALM (clathrin assembly (phosphatidylinositol-4,5-bisphosphate 3-kinase) and subse- lymphoid myeloid leukemia) fusion proteins, in cases with quently AKT (v-akt murine thymoma viral oncogene homolog). elevated CDX (caudal homeobox) expression or as a consequence In non-gluconeogenic tissues, this triggers multiple downstream 4–8 signaling cascades with preferential impact on proliferation and of a mutation in NPM1 (nucleophosmin). Besides, chromosomal 17 loci of the clustered HOX genes are themselves targets of survival (reviewed in Pollak ). In addition, the insulin/IGF system is chromosomal translocations9 and HOX expression can also be intricately involved in general aspects of physiology that show an association with the incidence of malignancies, like body mass endogenously upregulated, particularly in acute myeloid leuke- index and birth weight. Multiple clinical trials investigating mia. Completing the picture of HOX proteins as cancer hot spots, strategies to block insulin/IGF-triggered signaling are under way. in-frame fusions of members of the nucleopore complexes and On the basis of the studies reported here, it is possible that certain HOX sequences have been identified.10 Apart from these clinical leukemia types may also benefit from new therapeutics tested in findings, HOX factors are popular model oncoproteins that cause these clinical experiments. leukemia in experimental animals on co-overexpression with their known cooperating factors Meis (murine ecotropic integration site 1) and/or PBX (pre-B-cell leukemia homeobox). MATERIALS AND METHODS In contrast to the importance of HOX in normal and malignant hematopoiesis,1–3 disproportionately little is known about HOX Animals, cell lines, nucleic acids and data deposition downstream targets in hematopoietic stem and precursor cells. Conditional Igf1 knockout animals with a floxed exon 4 (B6.129(FVB)-Igf1- One of the most well-characterized genes under HOX control tm1Dlr-/J)18 and Mx-Cre (C57BL/6 J-Tg(Mx1-cre)1Cgn/J) mice19 have been codes for the proto-oncoprotein MYB (myeloblastosis) that can described previously. Both strains were obtained from Jackson Labora- function itself as hematopoietic oncogene.11 Recently, also LMO2 tories (Bar Harbor, MA, USA) through Charles River (Sulzfeld, Germany). C57BL/6 mice were bred in house or purchased from Janvier (LeGenest, (lim-only domain 2), a crucial hematopoietic regulator involved in France). Animal procedures have been approved by local and institutional T-ALL, and the guanine nucleotide exchange factor VAV2 animal welfare review boards under license number 54-2532.1-41/11. 12,13 have been identified as HOXA9 targets. In addition, the Primary cells transformed by inducible HOX constructs and cells for anti-apoptotic protein BCL2 (B-cell lymphoma) and the growth transplantation were obtained by retroviral transduction of a c-kit-selected Department of Genetics, University of Erlangen, Erlangen, Germany. Correspondence: Professor RK Slany, Department of Genetics, University of Erlangen, Erwin Rommel Strasse 3, 91058 Erlangen, Germany. E-mail: [email protected] Received 20 June 2014; revised 12 September 2014; accepted 22 September 2014; accepted article preview online 25 September 2014; advance online publication, 21 October 2014 Igf1 in HOX-induced leukemia J Steger et al 902 bone marrow fraction as described.20 Cells were cultivated in RPMI, 10% fetal calf serum and recombinant murine cytokines: 50 ng/ml stem cell factor and 5 ng/ml each of IL3 (interleukin 3), IL6 and granulocyte- TAM) macrophage colony-stimulating factor (eBioscience, SanDiego, CA, USA). − For activation of HOX constructs, 100 nM 4-hydroxytamoxifen (Sigma, Taufkirchen, Germany) was added. The structure and cloning of HOXA1- estrogen receptor (ER), HOXA6-ER and HOXA9-ER have been published.21 For technical reasons, murine Hoxa9 was used, whereas HOXA1 and HOXA6 were of human origin. Because human and mouse HOXA9/Hoxa9 = replicates B are identical in 267/272 amino acids and to avoid orthographical , confusion, we are using the human nomenclature with capital letters A (HOXA9) throughout this manuscript. Primers used for quantitative PCR (qPCR) were as follows: Igf1qPCR: fw-5ʹ-CTGGACCAGAGACCCTTTGCG-3ʹ; ʹ ʹ ʹ rev-5 -GGACGGGGACTTCTGAGTCTTG-3 , Igf1rqPCR: fw-5 -CTGTGGGGGC ABA B TCGTGTTTCTC-3ʹ; rev-5ʹ-GATCACCGTGCAGTTTTCCAGG-3ʹ, ChIP: 1fw-5ʹ-G TGCCTCCCATACTGCTTCCTTG-3ʹ;1rev-5ʹ-CTAGATCGAAAGGCAGCTCTCAG-3ʹ, 2fw-5ʹ-GAGCCAAGAATCGGGAATTCTTTG-3ʹ; 2rev-5ʹ-CGCGGTGAGTCTAAGAG CAGAG-3ʹ,3fw-5ʹ-AGAGAATAAGTCAGAGTGGCTGC-3ʹ;3rev-5ʹ-CTCTGGCCAG CTCCTCTACTG-3ʹ, Xfw-5ʹ-AGGGTTTGCTTCCACCCACTCAC-3ʹ;Xrev-5ʹ-CTGGAC CACCCAGAGCTAAACCA-3ʹ, genotyping: ES1-5ʹ-GTTAAAAGCCTCTCAACT On vs off AAGACAATA-3ʹ, IA6-5ʹ-AAACCACACTGCTCGACATTG-3ʹ,ID3-5ʹ-CACTAAGG AGTCTGTATTTGGACC-3ʹ,normfw:5ʹ-GTGCCTCCCATACTGCTTCCTTG-3ʹ; normrev: 5ʹ-CTAGATCGAAAGGCAGCTCTCAG-3ʹ. Array data have been deposited at ArrayExpress (www.ebi.ac.uk) and can be accessed under E-MEXP-3648 and E-MTAB-2603. TAM) Log2 change Hox on (+TAM) Hox off ( − Isolation of nascent RNA To determine RNA synthesis rates, newly transcribed RNA was labeled by the addition of 100 μM 4-thiouridine (Sigma) directly to the cell culture medium. After 1 h, total RNA was isolated by RNeasy spin column = replicates HOXA1 B purification according to the instructions of the manufacturer (Qiagen, , Hilden, Germany). RNA (100 μg) was biotinylated with 200 μg/ml EZ-Link A HPDP Biotin (Pierce-Thermo, Rockford, IL, USA) in 1 ml of 10 mM Tris-HCl, pH7.4, 1 mM EDTA. Biotinylated nucleic acid was bound to μMACS ABA B streptavidin magnetic beads, washed as recommended by the manufac- ND ND ND ND 0.88 42 797 40 530 17 005 28 228 turer (Miltenyi, Bergisch-Gladbach, Germany) and separated by magnetic force. Elution of retained material was done with 100 mM dithiothreitol. After a final purification on RNeasy columns, nascent RNA was subjected to standard reverse transcription and qPCR. a Enzyme-linked immunosorbent assay and antibodies On vs off Enzyme-linked immunosorbent assay (ELISA) reagents specific for mouse Igf1 were obtained from AssayPro (St Charles, MO, USA) and used exactly according to the instructions of the manufacturer. Assay supernatants were 6 TAM) Log2 change Hox on (+TAM) Hox off ( generated by incubating 2 × 10 cells for 18 h in 1 ml of fresh medium. − Mouse monoclonal anti-Igf1 antibody (MAB791) was purchased from R&D Systems (Minneapolis, MN, USA). Anti-Igf1rβ (111A9), anti-Igf1rβ (Tyr1150- /1151P)(19H7) and anti-Akt(Ser473)(D9E) were monoclonal rabbit anti- bodies, anti-panAkt (4DD4) was of murine origin. These antibodies were developed by Cell Signaling Technologies (Danvers, MA, USA). = replicates HOXA6 B , Igf1r stimulation and serum starvation A Starvation experiments were done in serum-free RPMI1640 supplemented with 0.1% buffered bovine serum albumin (Life Technologies, Darmstadt, Germany) and 25 μg/ml iron-saturated bovine transferrin (Sigma). After 4 h,
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