Leukemia (2014) 28, 1838–1843 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

ORIGINAL ARTICLE The leukemogenicity of Hoxa9 depends on alternative splicing

CR Stadler1,2,7, N Vegi3,7, MA Mulaw3, KE Edmaier3, VPS Rawat3, A Dolnik4, L Bullinger4, B Heilmeier5, L Quintanilla-Fend6, K Spiekermann1,2, W Hiddemann1,2,KDo¨ hner4,HDo¨ hner4, M Feuring-Buske4 and C Buske3

Although the transforming potential of Hox genes is known for a long time, it is not precisely understood to which extent splicing is important for the leukemogenicity of this gene family. To test this for Hoxa9, we compared the leukemogenic potential of the wild-type Hoxa9, which undergoes natural splicing, with a full-length Hoxa9 construct, which was engineered to prevent natural splicing (Hoxa9FLim). Inability to undergo splicing significantly reduced in vivo leukemogenicity compared to Hoxa9-wild-typed. Importantly, Hoxa9FLim could compensate for the reduced oncogenicity by collaborating with the natural splice variant Hoxa9T, as co-expression of Hoxa9T and Hoxa9FLim induced acute myeloid leukemia (AML) after a comparable latency time as wild-type Hoxa9. Hoxa9T on its own induced AML after a similar latency as Hoxa9FLim, despite its inability to bind DNA. These data assign splicing a central task in Hox gene mediated leukemogenesis and suggest an important role of homeodomain-less splice variants in hematological neoplasms.

Leukemia (2014) 28, 1838–1843; doi:10.1038/leu.2014.74

INTRODUCTION In this report we now demonstrate for Hoxa9 that its ability to Alternative splicing is one of the fundamental processes to add for generate splice variants is a prerequisite for its full transforming genomic complexity in the organism and is observed in all potential and that homeodomain-less isoforms such as Hoxa9T species. Recent reports impressively documented that the splicing can act as an oncogene without directly binding to DNA in vivo. machinery can be the target of leukemogenic mutations and that leukemias often are characterized by large-scale alterations in alternative splicing.1 One highly conserved family of transcription MATERIALS AND METHODS factors, which also undergoes alternative splicing and has an Patient samples essential role in stem cell biology and myeloid leukemogenesis, is Bone marrow (BM) samples from 114 adult patients with newly diagnosed the homeobox protein family. Several studies have shown that AML, de novo AML, were analyzed via real-time quantitative PCR. The alternative splicing of Hox genes often affects the DNA-binding diagnosis of AML was performed according to the French–American– domain and is evolutionarily highly conserved, being observed British criteria and the WHO classification. Human studies were approved from flies to men.2,3 One of the best known leukemogenic by the Ethics Committee of Ludwig Maximilians University and Ulm University abided by the tenets of the revised World Medical Association homeobox genes is HOXA9, which is highly expressed in normal Declaration of Helsinki (http://www.wma.net/e/policy/b3.htm). For com- hematopoietic stem cells and downregulated during parative expression analyses, BM samples of healthy donors were differentiation. Knockout models of Hoxa9 have documented purchased (Lonza, Basel, Switzerland). that ordered expression of this homeobox gene is essential for normal myeloid and lymphoid development.4 HOXA9 is overexpressed in the majority of acute myeloid leukemia (AML) Real-time PCR analysis and acute lymphoblastic leukemia patients and its aberrant For quantification of HOXA9 full length and HOXA9T and Hoxa9 full length expression correlates with poor prognosis.5 Several murine and Hoxa9T expression, ‘Assay-by-design’ primers (Applied Biosystems, Carlsbad, CA, USA) were used. Primer efficiencies were measured for these models have recapitulated the leukemogenic potential of Hoxa9: 2 6 probes and were equal for murine (Hoxa9 full-length: R ¼ 0.9859; Hoxa9T: overexpression of Hoxa9 induces stem cell expansion and AML in R2 ¼ 0.9894) and for human samples (HOXA9 full-length: R2 ¼ 0.9859; murine models. DNA binding via their homeodomain is HOXA9T: R2 ¼ 0.9863). Details of the primers are given in the considered to be a ‘condicio sine qua non’ for the Supplementary Table 9. Fold expression was calculated using the DDCT leukemogenicity of homeobox genes7 and shapes gene method after normalizing to TATA-box binding protein. Average fold 8 expression patterns of HOX-transformed cells in vitro. In mice, expression of DCT values were calculated from the fold expression of each an alternatively spliced isoform of Hoxa9, Hoxa9T was identified, individual patient. which lacks the homeodomain.9 Sequence analyses of this transcript showed that this truncated isoform is highly cDNA constructs and retroviral vectors conserved in mouse, chicken and humans. Human HOXA9T is For retroviral gene transfer into primary BM cells, all constructs were cloned generated by splice sites in exon 1 of the HOXA9 complimentary into the modified murine stem cell virus (MSCV) 2.1 vector upstream of DNA (cDNA), resulting in lack of its DNA-binding homeodomain. the enhanced green fluorescent protein (EGFP) or enhanced yellow

1Department of Internal Medicine III, Klinikum Grosshadern, Ludwig-Maximilians-University of Munich, Munich, Germany; 2Clinical Cooperative Group ‘Pathogenesis of Acute Myeloid Leukemia’, Helmholtz Center Munich for Environmental Health, Munich, Germany; 3Institute of Experimental Cancer Research, Comprehensive Cancer Center, University Hospital of Ulm, Ulm, Germany; 4Department of Internal Medicine III, University Hospital Ulm, Ulm, Germany; 5Department of Oncology and Hematology, Hospital Barmherzige Bru¨der, Regensburg, Germany and 6Institute of Pathology, University of Tu¨bingen, Tu¨ bingen, Germany. Correspondence: Professor C Buske, Institute of Experimental Cancer Research, Comprehensive Cancer Center, University Hospital Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany. E-mail: [email protected] 7These authors contributed equally to this work. Received 25 November 2013; revised 3 February 2014; accepted 7 February 2014; accepted article preview online 18 February 2014; advance online publication, 11 March 2014 Leukemogenicity of truncated Hoxa9 CR Stadler et al 1839 fluorescent protein (EYFP) gene and the internal ribosomal entry site.10 secondary antibody goat anti-mouse IgG-horseradish peroxidase (1:2000; Hoxa9WT cDNA (NM_152739.3) was subcloned into MSCV/EGFP, whereas SC-2005). Hoxa9T (kindly provided by Dr I Pellerin, Rennes Cedex, France) was subcloned into the MSCV/EYFP vector.11 The cDNA of Hoxa9FLim was Colocalization assay generated from the full-length murine Hoxa9 cDNA, using the Quik-change Phoenix-eco cells were transfected with the YFP/Hoxa9T (His) plasmid. 32D Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to cells were then transduced (transduction efficacy 63%) with collected the manufacturer’s instructions to disrupt the consensus intron recognition virally conditioned medium by spin infection and were sorted for YFP sites in exon 1 of Hoxa9 by substituting in total five base pairs (two at the positivity. Untransduced 32D cells were used as control. Cells were fixed in donor site, A313C and G351C, and three at the acceptor site, T478A, C479G 4% paraformaldehyde for 10 min at room temperature and thereafter and C486T) without changing the amino-acid sequence to prevent natural treated with 0.1% Triton X-100 for 5 min at room temperature. Blocking splicing. This construct was cloned into MSCV/EGFP vector. The correct was performed in 10% bovine serum albumin þ 5% serum (from the host sequences of the constructs were confirmed by complete nucleotide species of the secondary antibody) for 1 h at room temperature. Cells were sequencing, and expression was proved by real-time quantitative PCR and then incubated with primary antibodies (anti-His for Hoxa9T from Qiagen Western blot analysis. Integration of the provirus into the genome of mice 10 express) at room temperature for 2 h. Isotype control sections were was assessed by bubble-linker PCR as previously described. incubated with respective IgG primary antibodies. Incubation with the secondary antibodies labeled with Alexa Flour (Invitrogen) was performed Colony-forming cell/colony-forming unit-spleen assays and mice for 45 min at room temperature and the nuclei were stained with 4,6- BM transplantation experiments diamidino-2-phenylindole. Tissue sections were then mounted with The transforming potential of the different Hoxa9 constructs was antifade fluorescent or aqueous mounting medium (Invitrogen) and determined by serial replating in colony-forming cell assays as previously protein signals were visualized under the microscope (Carl Zeiss, reported.12 To assess the impact of the different Hoxa9 constructs on Oberkochen, Germany). short-term repopulating stem cells, purified transduced 5-FU-mobilized BM cells were injected into lethally irradiated F1 (B6C3) recipient mice 96 h Microarray analysis after the start of infection (colony-forming unit-spleen (CFU-S) assay). The After 48 h gene exposure, RNA of highly purified EGFP/EYFP-positive recovery of CFU-S cells was quantified by determination of the number of murine 5-FU BM cells expressing EGFP, Hoxa9FLim, Hoxa9T or co- macroscopic colonies on the spleen at day 12 post injection after fixation expressing both Hoxa9FLim and Hoxa9T from three independent in Telleyesniczky’s solution.10 Parental-strain mice were bred and experiments was isolated by the Qiagen RNA easy column kit to perform maintained at the animal facility of the Helmholtz Center Munich or the microarray analyses as previously described.13 Pathway analysis was done animal facility of the University of Ulm. Donors of primary BM cells [(C57BL/ based on Kyoto Encyclopedia of Genes and Genomes pathway database. 6Ly-Pep3b  C3H/HeJ) F1 (PepC3) mice] and recipient mice [(C57BL/ 6J  C3H/HeJ) F1 (B6C3)] were 8–12 weeks old. Primary mouse BM cells Statistical analysis were transduced as previously described.10 To coinfect the Hoxa9FLim with Data were evaluated using the two-tailed Student’s t-test for dependent or the Hoxa9T retroviruses, BM cells were cultured on a 1:1 mixture of independent samples (Microsoft Excel 2007, Microsoft Corp, Redmond, irradiated Hoxa9FLim/EGFP and Hoxa9T/EYFP GP þ E86 cell lines. Retroviral 10 WA, USA). Differences with P-values less than 0.05 were considered infection of primary BM cells was performed as described. statistically significant. Kaplan–Meier plots for survival were analyzed with BM transduction efficiencies for Hoxa9FLim-EGFP, Hoxa9T-EYFP or the PRISM Graph pad software (La Jolla, CA, USA) and the logrank test. Hoxa9FLim-EGFP and Hoxa9FLim-EGFP/Hoxa9T-EYFP co-expression were 14 ± Between-group analysis was carried out using the bioconductor package tested by EGFP and/or EYFP expression (EGFP/EYFP positivity: 38.2 6.5 made4 in R statistical computing environment. Supervised comparison s.e.m. for Hoxa9FLim, 24.7±4.88 s.e.m. for Hoxa9T, 27.9±9.1 s.e.m. for between groups was performed and the top two components were Hoxa9WT). The cotransduction efficiency for Hoxa9FLim Hoxa9T was þ selected for plotting. Circos plots were generated using a web-based 4.1±0.7 s.e.m. FACS-purified transduced BM cells or non-sorted 5 application Circos Table Viewer (Vancouver, BC, Canada). A function within transduced BM cells (if less than 4  10 transduced cells per recipient the limma bioconductor14 package was used to draw the venn diagrams. were available) were injected into the tail vein of 8- to 12-week-old lethally irradiated (800 cGy from a 137Cs g-radiation source) recipient F1 (B6C3) mice. For transplantation of secondary mice, 2.5  105 BM cells of diseased primary animals together with 1x106 normal BM cells of healthy mice were RESULTS tail vein injected into the recipients after 850 cGy irradiation. The animal Splicing of HOXA9, generating the HOXA9T splice variant, is studies were approved by the Ethics Committee of Ludwig Maximilians widespread in patients with AML University Munich and the University of Ulm and abided by the tenets of In an initial step we asked whether HOXA9 splicing is widespread the revised World Medical Association Declaration of Helsinki (http:// in normal and leukemic tissues by evaluating the expression of the www.wma.net/e/policy/b3.htm). HOXA9 full length and the HOXA9 splice variant HOXA9T in highly purified normal human BM hematopoietic subpopulations or Western blot AML cells by real-time PCR normalized to TATA-box binding Protein expression of the Hoxa9 constructs in NIH 3T3 cells was evaluated protein (Figure 1a; Supplementary Figures 1A and B). by Western blotting using standard procedures.10 Hoxa9 isoforms were Co-expression of HOXA9T along with HOXA9 full length could be detected in protein lysates of NIH 3T3 cells, which were infected with virus readily documented in all normal human hematopoietic sub- produced by GP þ E86 packaging cell lines. Hoxa9 protein was populations. There was significantly higher expression of HOXA9 immunoprecipitated with an affinity purified anti-Hoxa9 polyclonal rabbit full-length compared with HOXA9T in the primitive CD34 þ /CD38 À antibody (custom made by Anaspec, Fremont, CA, USA), which recognizes compartment, in CD34 þ /CD38 þ progenitors and in the more an N-terminal peptide of the murine and human Hoxa9 isoforms and À þ Rabbit IgG TrueBlot beads (eBioscience, Inc., San Diego, CA, USA). For differentiated CD34 /CD38 compartment, although relative detection of Hoxa9 constructs in hematopoietic 32D cells, Phoenix ampho expression of the truncated form was higher in more primitive cells were transfected by the CaCl2 method using MSCV-Hoxa9WT/EGFP, cells compared with mature lymphoid and myeloid cells MSCV-EGFP and MSCV-Hoxa9T-HisV5/YFP constructs. Murine hemato- (Supplementary Figure 1C, and Supplementary Table 1). We poietic 32D cells were transfected with virally conditioned medium by extended this analysis to leukemic samples: all AML cell lines spin infection for 45 min at 2500 r.p.m. and incubation on retronectin for tested expressed the truncated isoform together with the full- 18–20 h. Successfully transduced cells were highly purified by GFP/YFP length HOXA9 transcript. In contrast to normal hematopoietic expression before protein isolation. Hoxa9WT/MIG was detected by using stem and progenitor cells, leukemic cells expressed the truncated rabbit polyclonal anti-Hoxa9 (1:1000; Merck Millipore, Darmstadt, Germany; 07–178). Hoxa9T-HisV5/MIY was detected by using the anti-V5-horseradish isoform to almost comparable levels of the full-length transcript, peroxidase antibody (1:2000; Invitrogen, Carlsbad, CA, USA; R961–25). For indicating a relatively high expression of the homeodomain-less Hoxa9WT/MIG a secondary antibody goat anti-rabbit IgG-horseradish HOXA9 protein in relation to the full-length form in AML cells peroxidase (1:2000; SC-2004) was used. The membranes were stripped and (Supplementary Figure 1D). Furthermore, all analyzed 114 adult re-probed with mouse monoclonal anti-b-actin (1:2000; SC-4778) and AML patient samples (Supplementary Table 2) were positive for

& 2014 Macmillan Publishers Limited Leukemia (2014) 1838 – 1843 Leukemogenicity of truncated Hoxa9 CR Stadler et al 1840

Figure 1. (a) Schematic diagram of Hoxa9 transcripts showing the exons and splicing region. The nucleotide positions were included along with the protein sequence. Stop codon (TGA) in the exon 1 results in formation of the truncated Hoxa9T protein. The green boxes denote the exon 1(a, b) and exon 2 of Hoxa9 transcripts. ATG (start codon), TGA (stop codon) (b) Quantification of HOXA9 full length and HOXA9T expression in AML primary patient samples in comparison with normal BM CD34 þ subpopulations and (c) in highly purified leukemic subpopulations of patients with cytogenetically normal-AML by real-time quantitative PCR. All the normal and leukemic samples were normalized with the housekeeping gene TBP. Individual samples are shown as dots, the mean is indicated as a bar. Values are given as log10 fold expression (normalized to TBP). Two samples in the AML1-ETO positive subgroup and three samples in the inv(16) AML cases did not show any expression. HD, homeodomain; MIM, MEIS interacting motif; PIM, PBX interacting motif; TBP, TATA-box binding protein.

both transcripts, irrespective of their genotype (Figure 1b). undergo natural splicing (Hoxa9 full length with introduced Comparable to AML cell lines and again in contrast to normal mutations ¼ FLim), or the wild-type Hoxa9 (Hoxa9WT), which hematopoietic BM cells, HOXA9T expression levels were high in generated both the full length as well as the homeodomain-less relation to HOXA9 full-length expression (with an, for example, Hoxa9T splice variant (Figure 2a; Supplementary Figure 2A). only fourfold higher expression of HOXA9 full-length versus Expression was proven by real-time PCR after testing for primer HOXA9T in the CD34 À NPM1 þ /FLT3 À AML subtype, compared specificity and by Western blotting in NIH 3T3 and hematopoietic with a 29-fold higher expression, respectively, in the normal 32D cells, confirming no gross differences in protein expression of CD34 À /CD38 þ BM cells) (Figure 1b; Supplementary Table 3). the different constructs (Figure 2c; Supplementary Figure 2B). Expression analyses in highly purified leukemic subpopulations Furthermore, it was documented that retrovirally engineered from patients with cytogenetically normal AML (n ¼ 3) documen- expression of Hoxa9FLim did not induce expression of the ted co-expression of HOXA9 full-length and HOXA9T in the most truncated isoform and Hoxa9WT did result in expression of full- primitive CD34 þ leukemic stem cell candidates (Figure 1c). Taken length and truncated transcript (Supplementary Figure 2C). together, these data indicate that the HOXA9T transcript is co- Prevention of splicing grossly reduced the ability of Hoxa9 to expressed with the full-length HOXA9 in human AML bulk cells induce serial replating of clonogenic progenitors (Supplementary including the CD34 þ /CD38 À leukemic subpopulation and that Figure 3). Furthermore, in the BM transplantation model, mice in splicing of HOXA9 is widespread in human AML, suggesting a the Hoxa9FLim (n ¼ 17) group died after a prolonged median functional role of HOXA9 splice variants in leukemogenesis. latency of 220.5 days compared with mice in the Hoxa9WT group, which developed AML after a significantly shorter time of 130 days (n ¼ 16; P ¼ 0.006), indicating that prevention of splicing in Splicing of Hoxa9 is essential for its full leukemogenic potential in the Hoxa9FLim construct substantially reduced its leukemogenic the mouse BM transplantation model potential in vivo (Figures 2d and e; Supplementary Tables 4 and 5). On the basis of these findings, we hypothesized that splicing of We next asked whether expression of the homeodomain-less HOXA9 is functionally relevant. To test this, we retrovirally splice variant Hoxa9T affects the leukemogenicity of full-length overexpressed a full-length Hoxa9 construct, which does not Hoxa9. To test this, Hoxa9T, representing the mouse-truncated

Leukemia (2014) 1838 – 1843 & 2014 Macmillan Publishers Limited Leukemogenicity of truncated Hoxa9 CR Stadler et al 1841

Figure 2. (a) Schematic presentation of the constructs for Hoxa9WT, Hoxa9FLim and Hoxa9T. All the Hoxa9 forms were cloned into the multiple cloning sites of the modified MSCV 2.1 vector upstream of the internal ribosomal entry site (IRES) and the enhanced green or yellow fluorescent protein (EGFP/EYFP) gene. (b) Plasmids of both Hoxa9T and Hoxa9 full length were analyzed by taqman primers for Hoxa9T and Hoxa9 full length, documenting their specificity (c) Retrovirally engineered protein expression of Hoxa9WT and Hoxa9T shown by western blot in NIH 3T3 and 32D transfected cells. (d) Kaplan–Meier survival curves of primary mice transplanted with BM cells of the different experimental arms as indicated. The number of mice and statistical differences are given in the table. (e) Histological analyses of representative leukemic mice from the different experimental arms as indicated. (f) Activity of Hoxa9T, Hoxa9FLim and Hoxa9T/Hoxa9FLim in the CFU-S assay. Average is indicated as bars. HD, homeodomain; HE, hematoxylin and eosin; IP, immunoprecipitation; MIM, Meis1 interaction motif; MPO, myeloperoxidase; PBM, Pbx-binding motif; PI, pseudointron; T, truncation; T, Hoxa9T; WB, western blot; WT, Hoxa9 wildtype.

Hoxa9 isoform with high homology to the human HOXA9T Hoxa9FLim in concert with Hoxa9T critically alters gene expression (Supplementary Figure 2B), was retrovirally expressed in 5-FU- We next tested the impact of the different constructs on gene mobilized BM alone or in combination with Hoxa9FLim. Also for expression of primary murine BM cells: of note, aberrant Hoxa9T, specificity of the primers was determined before expression of the truncated isoform induced a more profound analyzing for the expression of the transcript by real-time PCR. perturbation of gene expression as the full-length Hoxa9 FLim, Expression of the truncated protein was proven by western sharing more probesets with the highly leukemogenic blotting and colocalization studies in 3T3 cells and hematopoietic Hoxa9T/Hoxa9FLim combination than with Hoxa9FLim 32D cells, respectively, confirming presence of Hoxa9T in the (Figure 3a; Supplementary Table 7). The modified principal nucleus and cytoplasm (Figures 2a–c; Supplementary Figures 2A component analysis-based between-group analyses separated and B). Furthermore, we excluded that enforced expression of the the Hoxa9T/Hoxa9FLim double-transduced cells from the single- Hoxa9T construct changed significantly endogenous Hoxa9 transduced Hoxa9T or Hoxa9FLim cells, indicating again the full-length transcript levels (Supplementary Figure 2C). Of note, necessity of Hoxa9FLim to collaborate with Hoxa9T to achieve full-length Hoxa9FLim increased its leukemogenic potential by maximal gene deregulation (Supplementary Figure 4). Compared collaborating with the homeodomain-less splice variant Hoxa9T with Hoxa9FLim, eight genes were uniquely associated with in vivo: in the CFU-S assay, cotransduction of BM cells with Hoxa9T expression, among them the nucleolar RNAs snord116 Hoxa9FLim and Hoxa9T showed an increase in their average implicated in tumorigenesis and in regulation of alternative frequency of short-term repopulating stem cells with 187±28.6 splicing.15,16 Similarly, Flt3 was upregulated in Hoxa9FLim (versus CFU-S (mean±s.e.m.) as compared with 43.8±7.8 CFU-S for EGFP), whereas tumor suppressor genes like Cdh1 were Hoxa9T (Po0.005) alone and 15.2±20 CFU-S for the EFGP control downregulated in all the experimental arms (all versus EGFP). Of (Po0.005) (Figure 2f). Furthermore, Hoxa9FLim strongly collabo- note, Hoxa9T induced differential expression of six pathway rated with Hoxa9T in mice (n ¼ 7), inducing AML after a short categories not deregulated by Hoxa9FLim (Figure 3b, latency of 141 days, similar to mice transplanted with BM Supplementary Table 8). cells overexpressing Hoxa9WT mice, compared with a median latency of 296.5 days for Hoxa9T (n ¼ 14) and 220 days for Hoxa9FLim (Figure 2d). All the leukemic mice analyzed showed anemia, hyperleukocytosis, splenomegaly and high blast DISCUSSION percentages, which were positive for c-kit and Gr-1/Mac-1 In summary, our data illustrate that generation of splice variants (Supplementary Tables 4 and 5) and were histopathologically has a major impact on the biological function of Hox proteins and classified as AML without maturation and multiple-organ infiltra- that deletion of splice sites can substantially weaken the tion (Figure 2e). Sequencing of the retroviral integration sites by transforming potential of Hox genes. Our observation that splice ligation-mediated PCR in leukemic mice did not indicate integra- variants of Hox genes do not act as dominant negatives but on the tion into known oncogenes listed in the RTCGD database (http:// contrary are able to support the leukemogenic potential of their variation.osu.edu/rtcgd/index.html), but into intergenic regions or full-length transcript is highly relevant for our understanding of introns, which might also promote Hoxa9-induced leukemogen- Hox gene function and potential concepts to antagonize the esis (Supplementary Table 6). driving force of genes such as HOXA9 in human AML. The

& 2014 Macmillan Publishers Limited Leukemia (2014) 1838 – 1843 Leukemogenicity of truncated Hoxa9 CR Stadler et al 1842

Figure 3. (a) Venn diagram of microarray analyses documenting expression of probesets in the different experimental arms as indicated. (b) Circos plot showing differentially regulated pathways for the experimental arms as indicated. EGFP, EGFP control; Hoxa9F, Hoxa9FLim; Hoxa9T, truncated Hoxa9.

presented data clearly point to an unexpected role of HOX ACKNOWLEDGEMENTS isoforms, which lack DNA-binding capacity, but keep the This work was supported by a grant of the Deutsche Krebshilfe (German Cancer Aid) N-terminal stretch. These observations are in line with in vitro (Project No 108438 to CB). We want to thank the Core Facility FACS, Ulm University studies documenting activity of Hoxa9T with full-length Hoxa9 in and the team of the animal facility at Ulm University and at the Helmholtz Center the colony-forming assay, giving a first hint on the role of Munich. Hoxa9T.17 All these data emphasize the importance of the N-terminal transactivation domain of Hox proteins: recently it was shown that this N-terminal transactivation domain is absolutely AUTHOR CONTRIBUTIONS 18 essential for the transforming capacity of Hoxa1 and Hoxa9. There CRS, NV, MAM, VPSR, KEE, AD, LB, BH and LQ-F performed research and KS, WH, are several possibilities how Hoxa9T might act despite its loss of the KD, HD and MF-B provided patient samples and analyzed data. CB designed the homeodomain: of note, in our mouse model, endogenous Hoxa9 as research. CRS, NV and CB analyzed the data and wrote the manuscript. well as Meis1 protein are present, allowing Hoxa9T to act via multimeric complexes. It is known that Hox proteins act in complexes and that Hoxa9 binds to other homeobox proteins such as Meis1, which itself is able to bind to another member of the TALE homeobox REFERENCES protein family Pbx1, thus forming trimeric complexes.19 Importantly, 1 Hahn CN, Scott HS. Spliceosome mutations in hematopoietic malignancies. the Meis1-interacting site is conserved in Hoxa9T, thereby enabling 20 Nat Genet 2012; 44: 9–10. formation of complexes with Meis1, although previous results 2 Talavera D, Orozco M, de la Cruz X. 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