ORIGINAL ARTICLE

New insights to the MLL recombinome of acute leukemias

C Meyer1, E Kowarz1, J Hofmann1, A Renneville2,3, J Zuna4, J Trka4, R Ben Abdelali5, E Macintyre5, E De Braekeleer6,7,M De Braekeleer6,7, E Delabesse8, MP de Oliveira9, H Cave10, E Clappier10, JJM van Dongen11, BV Balgobind12,MM van den Heuvel-Eibrink12, HB Beverloo13, R Panzer-Grumayer14, A Teigler-Schlegel15, J Harbott15, E Kjeldsen16,S Schnittger17, U Koehl18, B Gruhn19, O Heidenreich20, LC Chan21, SF Yip21, M Krzywinski22, C Eckert23, A Moricke24,M Schrappe24, CN Alonso25, BW Schafer26, J Krauter27, DA Lee28, U zur Stadt29, G Te Kronnie30, R Sutton31, S Izraeli32,33,34,L Trakhtenbrot32,33,34, L Lo Nigro35, G Tsaur36, L Fechina36, T Szczepanski37, S Strehl14, D Ilencikova38, M Molkentin39,T Burmeister39, T Dingermann1, T Klingebiel18 and R Marschalek1

1Diagnostic Center of Acute Leukemia, Institute of Pharmaceutical Biology, ZAFES, University of Frankfurt, Frankfurt/Main,Germany; 2Department of Hematology, Biology and Pathology Center, CHU of Lille, Lille, France; 3INSERM, U-837, Team 3,Lille, France; 4CLIP, Department of Paediatric Haematology/Oncology, Second Faculty of Medicine, Charles University Prague,Prague, Czech Republic; 5Biological Hematology, AP-HP Necker, Universite Paris Descartes, Paris, France; 6Faculte de Medecineet des Sciences de la Sante, Laboratoire d’Histologie, Embryologie et Cytogenetique, Universite de Bretagne Occidentale, Brest,France; 7INSERM-U613, Brest, France; 8CHU Purpan, Laboratoire d’Hematologie, Toulouse, France; 9Pediatric Hematology-Oncology Program, Research Center, Instituto Nacional de Cancer Rio de Janeiro, Rio de Janeiro, Brazil; 10Departement deGenetique, Hopital Robert Debre, Paris, France; 11Department of Immunology, Erasmus MC, Sophia Children’s Hospital,Rotterdam, The Netherlands; 12Department of Pediatric Oncology/Hematology, Erasmus MC, Sophia Children’s Hospital,Rotterdam, The Netherlands; 13Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands; 14Children’s CancerResearch Institute, Vienna, Austria; 15Department of Pediatric Hematology and Oncology, Children’s University Hospital,Giessen, Germany; 16Cancercytogenetics Laboratory, Aarhus University Hospital, Aarhus, Denmark; 17MLL Munich LeukemiaLaboratory, Munich, Germany; 18Department of Pediatric Hematology and Oncology, University of Frankfurt, Frankfurt/Main,Germany; 19Department of Pediatrics, University of Jena, Jena, Germany; 20Northern Institute for Cancer Research, NewcastleUniversity, Newcastle upon Tyne, UK; 21Department of Pathology, Queen Mary Hospital, The University of Hong Kong, HongKong, China; 22Canada’s Michael Smith Genome Sciences Center, Vancouver, British Columbia, Canada; 23Department ofPediatric Oncology and Hematology, Charite Medical University Berlin, CVK, Berlin, Germany; 24Department of Paediatrics,University of Schleswig-Holstein, Kiel, Germany; 25Servcio de Hemato-Oncologıa, Hospital Nacional de Pediatrıa Prof Dr JPGarrahan, Buenos Aires, Argentina; 26Department of Oncology, University Children’s Hospital, Zurich, Switzerland; 27Clinic forHematology, Hemostasis, Oncology and Stem Cell Transplantation, Hanover Medical School, Hanover, Germany; 28Division ofPediatrics, Cell Therapy Section, University of Texas MD Anderson Cancer Center, Houston, TX, USA; 29Department of PediatricHematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 30Department of Paediatricsand Oncohematology, University of Padua, Padua, Italy; 31Sydney Children’s Hospital, Children’s Cancer Institute, Sydney, NewSouth Wales, Australia; 32Department of Pediatric Hemato-Oncology, The Chaim Sheba Medical Center, Tel Aviv, Israel; 33TheCancer Research Center, Tel Aviv, Israel; 34Sackler Medical School Tel Aviv University, Tel Aviv, Israel; 35Center of PediatricHematology Oncology, University of Catania, Catania, Italy; 36Regional Children Hospital 1, Pediatric Oncology and HematologyCenter, Research Institute of Medical Cell Technologies, Ekaterinburg, Russia; 37Department of Pediatric Hematology andOncology, Medical University of Silesia, Zabrze, Poland; 38Department of Clinical Genetics, National Cancer Hospital,Bratislava, Slovakia and 39Medical Faculty III, CBF, Charite Medical University Berlin, Berlin, Germany

Chromosomal rearrangements of the human MLL gene areassociated with high-risk pediatric, adult and therapy-asso-ciated acute leukemias. These patients need to be identified,treated appropriately and minimal residual disease wasmonitored by quantitative PCR techniques. Genomic DNA wasisolated from individual acute leukemia patients to identify andcharacterize chromosomal rearrangements involving thehuman MLL gene. A total of 760 MLL-rearranged biopsysamples obtained from 384 pediatric and 376 adult leukemiapatients were characterized at the molecular level. Thedistribution of MLL breakpoints for clinical subtypes (acutelymphoblastic leukemia, acute myeloid leukemia, pediatric andadult) and fused translocation partner genes (TPGs) will bepresented, including novel MLL fusion genes. Combined dataof our study and recently published data revealed 104 differentMLL rearrangements of which 64 TPGs are now characterized

on the molecular level. Nine TPGs seem to be predominantlyinvolved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P,MLLT6/AF17 and SEPT6, respectively. Moreover, we describefor the first time the genetic network of reciprocal MLL genefusions deriving from complex rearrangements.Leukemia (2009) 23, 1490–1499; doi:10.1038/leu.2009.33;published online 5 March 2009Keywords: MLL; translocations partner genes; acute leukemia;ALL; AML

Introduction

Chromosomal rearrangements involving the human MLL gene at11q23 are associated with the development of acute leuke-mias.1,2 The presence of certain MLL rearrangements is anindependent dismal prognostic factor and patients are usuallytreated according to high-risk protocols. Therefore, the identi-fication of MLL gene fusions is necessary for rapid clinicaldecisions resulting in specific therapy regimens. Current

Received 9 December 2008; revised 15 January 2009; accepted 28January 2009; published online 5 March 2009

Correspondence: Professor Dr R Marschalek, Diagnostic Center ofAcute Leukemia, Institute of Pharmaceutical Biology, ZAFES, Uni-versity of Frankfurt, Max-von-Laue-Str. 9, Frankfurt/Main D-60438,Germany.E-mail: Rolf.Marschalek@em.uni-frankfurt.de

Leukemia (2009) 23, 1490–1499& 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00

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procedures to identify MLL rearrangements include cytogeneticanalysis,3,4 fluorescence in situ hybridization (FISH) experi-ments (for example, MLL split-signal FISH),5–7 specific reversetranscriptase (RT)–PCR8 or genomic PCR methods.9,10 Thisrepertoire of technologies was recently extended by along-distance inverse PCR (LDI-PCR) method that uses smallamounts of genomic DNA to determine any type of MLL generearrangement on the molecular level.11 This includes chromo-somal translocations, complex chromosomal rearrangements,gene internal duplications, deletions or inversions on chromo-some 11q and MLL gene insertions into other chromosomes, orvice versa, the insertion of chromatin material into the MLLgene.

To gain insight into the frequency of distinct MLL rearrange-ments, we analyzed prescreened and unscreened biopsymaterial of pediatric and adult leukemia patients. Prescreeningtests (cytogenetic analysis, FISH, Southern blot, RT–PCR or NG2positivity) were performed at different European centers andcenters located outside Europe, where acute leukemia patientsare enrolled in different study groups. Nearly all prescreenedMLL rearrangements were successfully analyzed and patient-specific MLL fusion sequencesFfor minimal residual disease(MRD) monitoringFwere obtained. In some centers, noprescreening could be performed. In these cases, a successfulidentification of MLL rearrangements was in the range of5–10%.

On the basis of the results obtained in the present (n¼ 346)and previous studies (414 patients were already published in2005 and 2006),11,12 64 translocation partner genes (TPGs) andtheir specific breakpoint regions have now been identified.Additional 35 chromosomal translocations of the human MLLgene were characterized by cytogenetics, however, without anyfurther molecular characterization. In this study, five additionalfusion loci were sequenced that do not encode any known gene.Thus, the MLL recombinome presently comprises 104 differentfusion sites. In addition, we present a list of 48 ‘reciprocal MLLgene fusions’ that derives from complex rearrangements. Thesereciprocal MLL gene fusions represent 48 genes fused to the30-portion of the MLL gene. They have never been describedbefore as MLL TPG, and thus, represent a novel subclass ofreciprocal recombination partners.

Material and methods

Patient materialBiopsy material from acute leukemia patientsFdiagnosed tobear an MLL rearrangement was used to isolate genomic DNAfrom bone marrow and/or peripheral blood samples. GenomicDNA was sent to the Diagnostic Center of Acute Leukemia(DCAL) at the Frankfurt University. Patient samples wereobtained from study groups (the AMLCG study group, Munich;the GMALL study group, Frankfurt/Main; Polish PediatricLeukemia and Lymphoma Study Group; Zabrze) or participatingdiagnostic centers. Informed consent was obtained from allpatients or patients’ parents/legal guardians and controlindividuals.

Long-distance inverse PCR experimentsAll DNA samples were treated and analyzed as described.11,12

Briefly, 1mg genomic patient DNA was digested with restrictionenzymes and re-ligated to form DNA circles before LDI-PCRanalyses. Restriction polymorphic PCR amplimers were isolated

from the gel and subjected to DNA sequence analyses to obtainthe patient-specific fusion sequences.

Results

Identification of MLL rearrangements and theirdistribution in clinical subgroupsTo analyze the recombinome of the human MLL gene, weobtained 1018 acute leukemia samplesFeither prescreened orunscreenedFfrom different centers over a period of 6 years.Successful analysis could be performed for 760 patient samples.Unsuccessful analyses were in the range of 25% and were dueto absence of any prescreening (21%), false-positive prescreen-ing experiments (B1%, depending on the participating center),limited biopsy material or insufficient quality of genomic DNA(1%), insufficient amount of leukemic blasts (1%) or by intrinsiclimitations of the applied method (length of IPCR amplimers415 kb; noncanonical breakpoints, B1% of all investigatedcases).

Within the group of characterized patients (n¼ 760), oneadult patient was diagnosed with primary myelofibrosis (PMF)and displayed an MLL translocation involving MLL intron 8fused to a region at 1p13.1 where no gene is encoded. All otherpatients (n¼ 759) were classified as pediatric or adult acuteleukemia patients. Pediatric leukemia patients (n¼ 384) werediagnosed either as acute lymphoblastic leukemia (ALL,n¼ 237) or acute myeloid leukemia (AML, n¼ 147); adultleukemia patients (n¼ 375) were classified either as ALL(n¼ 246) or AML (n¼ 129), respectively. All MLL rearrange-ments in these four subgroups are summarized in Table 1. Onthe basis of the above distribution, about 94% of all (pediatricand adult) ALL patients (n¼ 483) with MLL gene fusions arecharacterized by the fusion genes MLL �AF4 (B66.0%), MLL �ENL (B14.9%), MLL �AF9 (B8.5%), MLL �AF10 (B2.7%) andMLL �AF6 (B1.5%), respectively. In pediatric and adultAML patients (n¼ 276) about 77% of all characterized MLLfusion genes were MLL �AF9 (B30.4%), MLL �AF10 (B14.5%),MLL � ELL (B10.9%), MLL �AF6 (B10.1%), MLL � ENL (B5.4%),MLL �AF17 (B2.9%) and MLL � SEPT6 (B2.5%), respec-tively. This is in line with recently published data on thefrequency and distribution of different MLL fusion partnergenes.13,14

Breakpoint distribution according to clinical subtypesWe also investigated the distribution of chromosomal break-points within the MLL BCR in the four investigated clinicalsubgroups (pediatric vs adult leukemia patients; ALL vs AML).For this purpose, we analyzed the chromosomal breakpointswithin the MLL gene when recombined to the 9 most frequentTPGs (AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10,MLLT4/AF6, MLLT6/AF17, ELL, EPS15 and SEPT6) or the other29 identified recombination partners (ABI1, ACACA, ACTN4,AFF3, ARHGEF17, BCL9L, C2CD3, CASC5, DCP1A, EEFSEC,FLNA, FOXO3, KIAA0284, LAMC3, LOC100128568, MAML2,MLLT11, MYO1F, NEBL, NRIP3, PICALM, SEPT5, SEPT9,SMAP1, TET1, TIRAP/DCPS, TNRC18, UBE4A and VAV1). InFigure 1, all these data are summarized for the four clinicalsubgroups. On the basis of the 760 analyzed patients, pediatricALL patients (n¼ 237) have their chromosomal breakpointswithin MLL intron 11, whereas adult ALL patients (n¼ 246)recombine more frequently in MLL intron 9. Pediatric (n¼ 147)and adult AML patients (n¼ 129) show a preference forrecombination events affecting MLL intron 9. The exception

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was the MLLT3/AF9 gene in pediatric and adult AML patientsthat show a preference for recombination events to occur withinMLL intron 11. The same was true for SEPT6 in pediatric AMLpatients, but not in adult AML patients. Therefore, we concludethat pediatric ALL patients are different from all other subgroupsconcerning their breakpoint distribution within the MLL BCR.

Novel translocation partner genesEleven novel TPGs were discovered: DCP1A (decappingenzyme homologue A), TNRC18 (trinucleotide repeat contain-ing 18), LAMC3 (laminin, g-3), NEBL (nebulette), NRIP3(nuclear receptor interacting protein 3), C2CD3 (C2 calcium-dependent domain containing 3), UBE4A (ubiquitination factor

E4A/UFD2 homologue), VAV1 (vav 1 guanine nucleotideexchange factor), LOC100128568 (similar to hCG2045263),ACTN4 (actinin, a-4) and FLNA (filamin A, a/actin-bindingprotein 280).

The DCP1A gene encodes a homologue of the DCP1decapping enzyme, involved in mRNA degradation.15 Theprotein localizes in the mRNA processing body (P-body) thatregulates degradation and abundance of RNA molecules (RNAdecay). For TNRC18, no function is known. LAMC3 is a non-basement membrane-associate filamentous protein that isdownregulated or deleted in carcinomas. NEBL makes structuralpart of the Z-line in cardiac myofibrils and binds to a-actinin.16

NRIP3 is a nuclear receptor interacting protein of unknownfunction. C2CD3 is a Ca2þ -binding protein of unknownfunction. UBE4A is expressed in skeletal muscle, kidney andliver, and weakly expressed in hematopoietic cells. It has beenpostulated that UBE4A is involved in cell-cycle control(ubiquitination) and by protecting the cell against environmentalstress. UBE4A is frequently mutated and deleted in neuro-blastomas.17 The identified MLL �UBE4A fusion was a head-to-head genomic fusion, and thus, created loss of heterozygosity(LOH) for the UBE4A gene. VAV1 is a Dbl-domain containingproto-oncoprotein with GDP/GTP exchange function that isselectively expressed in hematopoietic cells. It interacts withCBL and GRB2 and influences RAC/RHO signaling processesand migration behavior.18 LOC100128568 is a hypotheticalprotein with unknown function. ACTN4 encodes a non-musclea-actinin that appears to promote tumor growth and invasive-ness.19 Basically, ACTN4 regulates stress fiber formation of thecytoskeleton. Moreover, ACTN4 protein is a regulator of AKT1localization and of its function;20 ACTN4 is also involved ininsulin signaling.21 FLNA encodes a cytoskeletal filamin protein(280 kDa) that interacts and reorganizes the actin cytoskeleton.It is a substrate of granzyme B and different caspases.22

Interestingly, the cleaved C-terminal portion (100 kDa) localizesin the nucleus. This proteolytic fragment is able to bind andregulate androgen receptor.23 Another study has demonstratedcolocalization of FLNA and Caveolin 1.24 Caveolin 1 isdownregulated in tumor cells as it inhibits anchorage-indepen-dent growth, anoikis and invasiveness.25

The MLL recombinomeWithin the past 16 years, several genetic aberrations involvingthe human MLL gene located on chromosome 11 band q23 havebeen described. Out of 104 TPGs, 64 are now characterized atthe molecular level (see Table 2). Forty-four fusion genes havebeen described by others, whereas 20 TPGs have beenidentified at the Frankfurt DCAL. Additional 35 genetic lociwere identified by cytogenetic experiments but not furthercharacterized (for references see Meyer et al.12). Five MLLrearrangements were identified that did not display a fusion toan annotated gene or open reading frame. These partner lociwere 1p31.2 (patient with PMF), 9p13.3, 11q22, 11q23.3 and21q22, respectively. Several attempts to identify spliced fusionpartners in vicinity failed so far (data not shown). Thus, thesefusions most likely represent nonfunctional MLL fusions that are,however, associated with acute leukemias, and in one case,with PMF.

Genetic alterations resulting in genetic MLL aberrationsIn general, human MLL rearrangements are initiated by aDNA damage situation, which induces DNA repair by thenonhomologous-end-joining DNA repair pathway.26,27 Genetic

Table 1 Distribution of MLL fusion in clinical subgroups

TPG Pediatric Adult Total

All AML Sum All AML Sum

AFF1/AF4 109 1 110 210 1 211 321MLLT3/AF9 37 47 84 4 37 41 125MLLT1/ENL 49 5 54 23 10 33 87MLLT10/AF10 12 34 46 1 6 7 53MLLT4/AF6 6 11 17 1 17 18 35ELL F 17 17 1 13 14 31EPS15/AF1P 7 5 12 F 1 1 13MLLT6/AF17 F 2 2 F 6 6 8SEPT6 F 6 6 F 1 1 7

MLLT11 1 7 8 F F F 8SEPT9 F 1 1 F 3 3 4TET1/LCX F F F 2 1 3 3AFF3/LAF4 2 F 2 F F F 2CASC5/AF15Q14 F 1 1 F 1 1 2FOXO3/AF6Q21 2 F 2 F F F 2KIAA0284 F F F F 2 2 2MAML2 2 F 2 F F F 2PICALM/CALM 1 F 1 F 1 1 2SEPT5/CDCREL 1 F 1 F 1 1 2TNRC18/KIAA1856 2 F 2 F F F 2ABI1 F 1 1 F F F 1ACACA F 1 1 F F F 1ACTN4 F F F 1 F 1 1ARHGEF17 F 1 1 F F F 1BCL9 L 1 F 1 F F F 1C2CD3 F F F F 1 1 1DCP1A 1 F 1 F F F 1EEFSEC/SELB 1 F 1 F F F 1FLNA F F F F 1 1 1LAMC3 F 1 1 F F F 1LOC100128568 F 1 1 F F F 1MYO1F F 1 1 F F F 1NEBL F 1 1 F F F 1NRIP3 F 1 1 F F F 1SMAP1 F F F F 1 1 1TIRAP (DCPS) F F F F 1 1 1UBE4A 1 F 1 1VAV1 F 1 1 F F F 1MLL PTD F 1 1 1 24 25 26

9p13.3 1 F 1 F F F 111q12 1 F 1 F F F 111q23 F F F 1 F 1 121q22 1 F 1 F F F 1Total 237 147 384 246 129 375 759

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acutemyeloid leukemia; TPG, translocation partner gene.All identified MLL fusions were classified by pediatric vs adult patientsand disease phenotype (ALL vs AML). Total numbers are indicated forall clinical subgroups and sorted by frequency.

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recombinations involving the human MLL gene predominantlyresult in reciprocal chromosomal translocations (see Figure 2,rCTL), involving recurrently the following TPGs: ABI1, AFF1/AF4, CASC5, CREBBP, ELL, EPS15/AF1P, FOXO3, FOXO4,FRYL, GPHN, KIAA0284, MLLT3/AF9, MLLT1/ENL, MLLT4/AF6, MLLT6/AF17, MLLT11/AF1Q, MYO1F, SEPT2, SEPT5,SEPT9, TET1 and TNRC18, respectively. Other TPGs (also fromthe literature) were identified so far only once (ACTN4,ARHGAP26, ARHGEF17, ASAH3, DAB2IP, DCP1A, EEFSEC,EP300, GAS7, GMPS, LAMC3, LASP1, LPP, MAPRE1, NCKIPSD,NEBL, SEPT11, SH3GL1 and SMAP1).

Gene internal partial tandem duplications (PTDs) of specificMLL gene portions (duplication of MLL gene segments codingeither for introns 2–9, 2–10, 2–11, 4–9, 4–11 or 3–8) arefrequently observed in AML patients.28–30 MLL PTDs are beingdiscussed to mediate dimerization of the MLL N terminus, aprocess that seems to be sufficient to mediate leukemogenictransformation.31 We have observed MLL PTDs in three of thefour investigated subgroups: 1 patient within the group ofpediatric AML, 1 patient within the group of adult ALL and 24patients within the group of adult AML. This demonstrates thatMLL PTDs are predominantly present in adult AML patients, inline with previously published data.32,33

MLL recombinations involving only chromosome 11 arebased on two independent DNA strand breaks that areaccompanied either by inversions or deletions on 11p or 11q(Inv, Del). Several recombinations have been characterized thatbelong to these two groups. MLL gene fusions to C2CD3,MAML2, NRIP3, PICALM and UBE4A are based on the inversionof a chromatin portion of 11p or 11q, leading to reciprocal MLLgene fusions. By contrast, a deletion of chromosome materiallocated telomere to MLL fused the 50-portion of MLL directly toother gene sequences (ARHGEF12, BCL9L and CBL). A fourthdeletion at 11q fused 50-MLL sequences to the 30-UTR of theTIRAP gene, which is located several kilobases upstream of theDCPS gene. In that particular case, only transcription of MLLand a subsequent splice process allowed to generate anMLL �DCPS fusion mRNA, encoding a bona fide MLL �DCPSfusion protein (MLL spliced fusion).

Beside reciprocal chromosomal translocations of MLL (rCTL),MLL PTDs and 11p/q rearrangements (Del and Inv), additionalgenetic rearrangements were identified in the genomic DNA ofanalyzed leukemia biopsy material. Although the previousrearrangements are based on two independent DNA strandbreaks, all other genetic events observed for the MLL generepresent more complex rearrangements with at least three or

Figure 1 Breakpoint distribution within the human MLL gene. The distribution of breakpoints within the MLL breakpoint cluster region is shown.Breakpoints were categorized by their occurrence within MLL introns 7–12 or by recombination events that occurred within exons that arelocalized in the MLL BCR (exons 10, 11 and 12). Breakpoint distributions are shown for pediatric acute lymphoblastic leukemia (ALL), pediatricacute myeloid leukemia (AML), adult ALL and adult AML patients. Breakpoints are shown only for the nine most frequent translocation partnergenes (TPGs) and ‘all other’ chromosomal rearrangements identified in the respective subgroups. Sizes of all MLL introns and the sum of MLL exons10–12 are given below in base pairs.

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Table 2 The MLL recombinome of acute leukemia

No. Cytogenetic abnormality Breakpoint Partner gene Referencea Leukemia type

1 t(1;11)(p32;q23) 1p32 EPS15/AF1P Bernard et al. (1994) ALL, AML, CML2 t(1;11)(q21;q23) 1q21 MLLT11/AF1Q Tse et al. (1995) AML3 t(2;11)(q11.2Bq12;q23) 2q11.2Bq12 AFF3/LAF4 von Bergh et al. (2003) ALL4 t(2;11)(q37;q23) 2q37 SEPT2 Cerveira et al. (2006) AML, t-MDS, t-AML5 t(3;11)(p21;q23) 3p21 NCKIPSD/AF3P21 Sano et al. (2000) t-AML6 t(3;11)(p21.3;q23) 3p21.3 DCP1A This manuscriptb ALL7 t(3;11)(q21.3;q23) 3q21.3 EEFSEC/SELB Meyer et al (2005)b ALL8 t(3;11)(q24;q23) 3q24 GMPS Pegram et al. (2000) t-AML9 t(3;11)(q27Bq28;q23) 3q27Bq28 LPP Daheron et al. (2001) t-AML

10 t(4;11)(p12;q23) 4p12 FRYL Hayette et al. (2006) t-ALL, t-AML11 t(4;11)(q21.1;q23) 4q21.1 SEPT11/FLJ10849 Kojima et al. (2004) CML12 t(4;11)(q21;q23) 4q21 AFF1/AF4 Gu et al. (1992) ALL, t-ALL,(AML)13 t(4;11)(q35.1;q23) 4q35.1 SORBS2/ARGBP2 Pession et al. (2006) AML14 complex abnormalities 5q12.3 CENPK/FKSG14 Taki et al. (1996) AML15 ins(5;11)(q31;q13q23) 5q31 AFF4/AF5Q31 Taki et al. (1999) ALL16 t(5;11)(q31;q23) 5q31 ARHGAP26/GRAF Borkhardt et al. (2000) JMML17 t(6;11)(q12B13;q23) 6q12Bq13 SMAP1 Meyer et al (2005)b AML18 t(6;11)(q21;q23) 6q21 FOXO3/AF6Q21 Hillion et al. (1997) t-AML19 t(6;11)(q27;q23) 6q27 MLLT4/AF6 Prasad et al. (1993) AML, t-AML, ALL20 t(7;11)(p22.1;q23) 7p22.1 TNRC18/KIAA1856 This manuscriptb ALL21 t(9;11)(p22;q23) 9p22 MLLT3/AF9 Nakamura et al. (1993) AML, t-AML, ALL22 t(9;11)(q33.1Bq33.3;q23); 9q33.1Bq33.3 DAB2IP/AF9Q34 von Bergh et al. (2004) AML23 ins(11;9)(q23;q34)inv(11)(q13q23) 9q34 FNBP1/FBP17 Fuchs et al. (2001) AML24 t(9;11)(q31Bq34;q23) 9q31Bq34 LAMC3 This manuscriptb t-AML25 t(10;11)(p11.2;q23) 10p11.2 ABI1 Taki et al. (1998) AML26 ins(10;11)(p12;q23q13) 10p12 MLLT10/AF10 Chaplin et al. (1995) AML, t-AML, (ALL)27 ins(10;11)(p12;q23) 10p12 NEBL This manuscriptb AML28 t(10;11)(q21;q23) 10q21 TET1/LCX Ono et al. (2002) AML29 inv(11)(p15.3q23) 11p15.3 NRIP3 Balgobind et al, submitteda AML30 t(11;11)(q13.4;q23) 11q13.4 ARHGEF17 Teuffel et al. (2005)b AML31 inv(11)(q13.4q23) 11q13.4 C2CD3/DKFZP586P0123 This manuscriptb AML32 inv(11)(q14q23) 11q14 PICALM/CALM Wechsler et al. (2003) AML33 inv(11)(q21q23) 11q21 MAML2 Meyer et al (2006)b t-T-ALL, t-AML34 t(11;15)(q23q;q21)inv(11)(q23q23) 11q23 UBE4A This manuscriptb MDS35 del(11)(q23q23.3) 11q23.3 ARHGEF12/LARG Kourlas et al. (2000) AML36 del(11)(q23q23.3) 11q23.3 CBL Fu et al. (2003) AML37 del(11)(q23q23.3) 11q23.3 BCL9 L Meyer et al (2006)b ALL38 del(11)(q23q24.2) 11q24.2 TIRAP Meyer et al (2006)b AML39 del(11)(q23q24.2) 11q24.2 DCPS Meyer et al (2005)b AML40 t(11;12)(q23;q13.2) 12q13.2 CIP29 Hashii et al. (2004) AML41 t(11;14)(q23.3;q23.3) 14q23.3 GPHN Kuwada et al. (2001) AML, t-AML42 t(11;14)(q32.33;q32.33) 14q32.33 KIAA0284 Burmeister et al (2008)b AML43 t(11;15)(q23;q14) 15q14 CASC5/AF15Q14 Hayette et al. (2000) AML, ALL44 t(11;15)(q23;q14) 15q14 ZFYVE19/MPFYVE Chinwalla et al. (2003) AML45 t(11;16)(q23;p13.3) 16p13.3 CREBBP/CBP Taki et al. (1997) t-MDS, t-AML, t-ALL46 t(11;17)(q23;p13.1) 17p13.1 GAS7 Megonigal et al. (2000) t-AML47 ins(11;17)(q23;q21) 17q21 ACACA Meyer et al (2005)b AML48 t(11;17)(q23;q21) 17q21 MLLT6/AF17 Prasad et al. (1994) AML49 t(11;17)(q23;q11Bq21.3) 17q11Bq21.3 LASP1 Strehl et al. (2003) AML50 t(11;17)(q23;q25) 17q25 SEPT9/AF17Q25 Osaka et al. (1999) t-AML, AML51 t(11;19)(q23;p13.1) 19p13.1 ELL Thirman et al. (1994) AML, t-AML52 t(11;19)(q23;p13) 19p13.3 SH3GL1/EEN So et al. (1997) AML53 ins(11;19)(q23;p13.2) 19p13.2 VAV1 This manuscriptb AML54 t(11;19)(q23;p13.3) 19p13.3 MLLT1/ENL Tkachuk et al. (1992) ALL, AML, t-AL55 t(11;19)(q23;p13.3) 19p13.3 ASAH3/ACER1 Lo Nigro et al. (2002) ALL56 t(2;11;19)(p23.3;q23;p13.3) 19p13.3 LOC100128568 This manuscriptb AML57 t(11;19)(q23;p13.3Bp13.2) 19p13.3Bp13.2 MYO1F Lo Nigro et al. (2002) AML58 t(11;19)(q23;q13) 19q13 ACTN4 This manuscriptb ALL59 t(11;20)(q23;q11) 20q11 MAPRE1 Fu et al. (2005) ALL60 t(11;22)(q23;q11.21) 22q11.21 SEPT5/CDCREL Megonigal et al. (1998) AML, T-ALL61 t(11;22)(q23;q13.2) 22q13.2 EP300/P300 Ida et al. (1997) t-AML62 t(X;11)(q13.1;q23) Xq13.1 FOXO4/AFX Parry et al. (1994) ALL, AML63 ins(X;11)(q24;q23) Xq24 SEPT6 Borkhardt et al. (2001) AML64 ins(11;X)(q23;q28q13.1) Xq28 FLNA This manuscriptb AML

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; MDS, myelodysplasticsyndrome; TPG, translocation partner gene.List of cytogenetic localizations of all yet characterized TPGs, references of first description and observed leukemia disease phenotypes.aBibliographic data of all references are summarized in supplementary data.bDCAL publications.

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more DNA double-strand breaks. In these cases, the expectedreciprocal MLL fusion gene cannot be detected, because othersequences will be fused to the 30-portion of the MLL gene.

The first class of complex MLL rearrangements are three-waychromosomal translocations (3W-CTL) involving three indepen-dent chromosomes and resulting in three different fusion genes.The most frequent involved genes in 3W-CTLs were AFF1/AF4,MLLT3/AF9, MLLT1/ENL, MLLT11/AF1Q and ELL in combina-tion with partner genes shown in Figure 3.

The second category are reciprocal chromosomal transloca-tions that are associated with deletions on either of the involvedchromosomes (CTLþD). Such cases were predominantlyidentified in complex t(4;11)(q21;q23) translocations.

The third category are chromosomal fragment insertions. Thisincludes the insertion of chromosome 11 material (includingportions of the MLL gene) into other chromosomes (Ins1), or viceversa, the insertion of chromosome material (including portionsof a TPG) into the BCR of the MLL gene (Ins2). An insertionmechanism is required in those cases where the transcriptionalorientation of a given TPG is not identical to the transcriptionalorientation of the MLL gene. The MLL gene is transcribedin telomeric direction. TPGs with a transcriptional orientation indirection to the centromere are predominantly recombiningwith MLL by such insertion mechanisms. These genes are

ACACA, AFF3/LAF4, AFF4/AF5Q31, CENPK, CIP29, CREBBP,FLNA, FNBP1, LOC1000128568, MLLT10/AF10, SEPT6,SORBS2/ARGBP2 and VAV1. In these cases (Ins1/2), threeindependent fusion genes will be generated. In three patientsbearing a recombination event between MLL and MLLT10/AF10, more complex rearrangements were identified thatcannot be explained by a simple insertion mechanism.

Finally, spliced fusion were observed. Spliced fusions aregenerated by fusing the 50-portion of the MLL gene to theupstream region of a TPG. Thus, a functional MLL fusion mRNAcan only be generated by a coupled process of transcription andsplicing (last MLL exon 50 to the breakpoint spliced to an exon(a1) of the partner gene fused to the MLL). Beside the above-mentioned DCPS gene, other genes have been identified thatcan transcriptionally fuse to 50-MLL sequences. These wereZFYVE19, but also the MLL fusion partners like, AFF1/AF4,EPS15, MLLT3/AF9, MLLT1/ENL and SEPT5. In case of MLLT1/ENL, about 50% of all recombination events were splicedfusions,34 and for MLL � EPS15 fusions about 30%. Splicedfusions to AFF1/AF4, MLLT3/AF9 and SEPT5 represent very rareevents.

All the above-mentioned mechanisms can be combined togenerate more complex genetic rearrangements, requiring fouror more DNA double-strand breaks.

Figure 2 Overview of all known MLL rearrangements. Genes are listed according to their transcriptional orientation on their chromosomes.(a) Genes transcribing into telomeric direction are categorized either by reciprocal chromosomal translocation (rCTL), spliced fusion (Spl) or11q deletions (Del). A total of 46 genes belong to this group. Gray gene names: genes listed also under ‘reciprocal chromosomal translocations’.(b) Genes transcribing into centromeric direction are categorized either by insertions (Ins1 and Ins2) or inversions at 11p/q (Inv). A total of 18 genesbelong to this group. Bottom: all identified recombination events, arranged according to the number of DNA double-strand breaks (DSBs)necessary to explain the recombination event. Green, chromosome 11; red and orange, partner chromosomes involved in the recombinationprocess. Green vertical bars, MLL; red, orange, blue and pink vertical bars, partner genes involved in recombination events; derivative 11chromosomes are always depicted by ‘Der’. Black and white horizontal lines, recombination sites on wild-type and derivative chromosomes;rCTL, reciprocal chromosomal translocation; Del/Inv, deletion/inversion; 3W-CTL, three-way chromosomal translocation; CTLþD, chromosomaltranslocation including deletion(s); Ins1, chromosomal fragment including portions of the MLL gene is inserted into a partner chromosome; Ins2,chromosomal fragment including portions of a partner gene is inserted into the MLL gene; cCTL, complex chromosomal translocations.

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Reciprocal MLL gene fusionsAnalyses of complex MLL rearrangements (3W-CTL, CTLþD,Ins1, Ins2 and cCTLs) allowed to identify a new class of MLLrecombination events that provide new insights into thecomplex spectrum of MLL rearrangements. By using a systema-tic breakpoint analysis approach, we identified 3–5 fusionalleles in these patients, of which only one of these allelesrepresented the reciprocal MLL fusion allele. Most of thesereciprocal MLL fusion were not able to produce fusion proteins,because the recombination occurred either in noncompatibleintrons or represented head-to-head gene fusions. As summarizedin Figure 3, 48 reciprocal MLL fusion have been identified. Only

12 out of these 48 reciprocal fusions represent bona fide genefusions between the given TPG and MLL introns 9–12 (ADARB2,APBB1IP, ATG16L2, CDK6, FLJ46266, GPSN2, MEF2C,MYO18A, NKAIN2, RABGAP1L, RNF115 and UVRAG).ADARB2 is an RNA-editing enzyme that desaminates Anucleotides. Overexpressed APBB1IP results in cell adhesionand is predominantly expressed in myeloid cells. ATG16L2 isa protein that promotes autophagy. CDK6 is a cell-cycle-dependent kinase that associates with cyclin D duringG1 phase. MEF2C resembles a transcription factor that enhancesc-jun-mediated transcriptional processes; the MEF2C gene wasfound to be overexpressed in MLL-rearranged leukemias. The

Figure 3 Overview over reciprocal MLL fusion genes. (a) Identified reciprocal MLL fusion partners fused in-frame (green) to the 30-MLL genesegment (MLL exons 10/11/12–37); a total of 12 genes were identified. CDK6 has been described in the literature. (b) Identified reciprocal MLLfusion partners fused out-of-frame (red) to the 30-MLL gene segment (MLL exons 10/11/12–37); a total of 19 genes have been identified. ARMC3 hasbeen described in the literature. (c) Identified reciprocal MLL fusion partners fused head-to-head (orange) to the 30-MLL gene segment (MLL exons10/11/12–37); a total of 17 genes have been identified. The 30-MLL gene segment is still able to be transcribed from its gene internal promoterelement located upstream of MLL exon 12 (see 30-MLL on the bottom); this results in a shorter version of the MLL protein that still exhibit an H3K4methyltransferase activity.

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NKAIN2 gene resembles a genetic hotspot that is frequentlyrecombined in T-cell lymphomas. RABGAP1L regulates thecytoskeleton, whereas UVRAG is a tumor suppressor proteinthat promotes autophagy. It was defined as tumor suppressorprotein, because overexpression suppresses proliferation andtumorigenicity.

Another 19 gene fusions were out of frame because ofrecombining noncompatible introns of both involved genes(ARMC3, CACNA1B, CMAH, CRLF1, FXYD6, GRIA4, MMP13,NFkB1, PAN3, PBX1, PPM1G, PARP14, PIWIL4, RPS3,SCGB1D1, SFRS4, TCF12, TNRC6B and TRIP4). This geneticsituation represents an LOH situation. CRLF1 is part of asignaling complex that regulates immune responses during fetaldevelopment. MMP13 is frequently overexpressed in tumorcells. NFkB1 blocks the apoptotic pathway. PAN3 is a tumorsuppressor protein that is also part of the polyA-specificribonuclease complex. PBX1 is a transcription factor thatinteracts with MEIS1 and HOXA proteins to steer developmentalprocesses. PBX1 is already well known as fusion partner of theE2A gene in a subset of ALL cases.35 PIWIL4 is involved in themaintenance and self-renewal of stem cells, as well as in RNAinterference. TCF12 is a transcription factor that associates withE2A to steer somatic recombination of T-cell receptor genes.TRIP4 is part of the inflammasome and inhibits the activation ofinterleukin 1 (IL1) and IL18 by modulating CASP1 activity.

The final group of 30-MLL fusion represent head-to-headfusion. Thus, the transcriptional orientation of the fused TPG isopposite to the orientation of the MLL gene. In these case, theTPG became disrupted by replacing its promoter region by the30-MLL portion. Thus, this genetic situation represents again anLOH situation. Identified fusion partners were ADSS, CACNB2,CUGBP1, DSCAML1 (2� ), ELF2, FCHSD2, FXYD2, GTDC1,KIAA0999 (2� ), KIAA1239, MPZL2, NCAM1, NT5C2, SVIL,TMEM135, TUBGCP2 and UNC84A, respectively. Two genes,DSCAML1 and KIAA0999, were identified twice in differentleukemia patients, indicating that such reciprocal MLL genefusions do also show recurrence. CUGBP1 binds to RNA,influences splicing processes and translation efficiency; it alsobinds to EWS. ELF2 is an ETS transcription factor that isoverexpressed under hypoxic conditions; ELF2 is a directbinding partner of AML1/RUNX1. NCAM1 is also known asCD56 and a known tumor suppressor protein. SCIL binds to theactin cytoskeleton, whereas TUBGCP2 binds to microtubuli andregulates centrosome formation. UNC84A is associated to thenuclear lamina and to centrosomes. We have to mention thatpromoterless 30-MLL gene segments are per se able to transcribemost of the remaining open reading frame of the MLL gene by agene internal promoter recently identified upstream of MLL exon12.36 Therefore, also these arbitrary MLL fusion may still allowto transcribe most of the parts of the MLL coding region,resulting in a shorter MLL protein version (230 kDa) that stillexhibits the ability to function as ‘nonspecific’ H3K4 methyl-transferase due to the missing N terminus.

Classification of MLL TPGs into functional categoriesAll TPGs were categorized according to their gene ontology intofunctional subclasses. They can be classified into cytosolic/membrane proteins and nuclear proteins. According to theirfunctions, they were grouped into extracellular proteins(LAMC3), cell adhesion proteins (LPP, SORBS2) with functionsin the organization of focal adhesion plaques, endocytoticproteins (EPS15, FNBP1, PICALM, ZFYVE19, proteins involvedin diverse signaling pathways AF6, ARHGEF12, ARHGEF17,ASAH3, C2CD3, DAP2IP, LASP1, SMAP1, TIRAP, VAV1),

organization and regulation of cytoskeleton (actin and micro-tubuli; ABI1, ACTN4, ARHGAP26, FLNA, GPHN, KIAA0284,MAPRE1, MYO1F, NEBL, SH3GL1), translation elongation(EEFSEC), metabolic functions (ACACA, CBL, GMPS, UBE4A)and proapoptotic proteins (MLLT11/AF1Q). The nuclear com-partment is subclassified into cell-cycle control and organiza-tion of nuclear cytoskeleton during cytokinesis (NCKIPSD,SEPT2, SEPT5, SEPT6, SEPT9, SEPT11), nucleic acid binding(CIP29, TNRC18), RNA decay (DCP1A, DCPS), chromosomeassociation (CENPK, CASC5), chromatin regulation (CREBBP,EP300), transcription factors and regulation of transcription(AF17, BCL9L, FOXO3, FOXO4, FRYL, GAS7, MAML2, NRIP3,TET1) as well as transcriptional elongation factors (AFF1, AFF3,AFF4, AF9, AF10, ELL, ENL). No cellular function is known forthe gene product of LOC100128568. All categorized TPGs aresummarized in Table 3.

Table 3 Overview on the cellular function of all yet known TPGs

A. Cell surface/membrane proteins (n¼ 1)LAMC3

B. Cytosolic proteins (n¼33)Cell adhesion

LPP, SORBS2Endocytosis

EPS15, FNBP1, PICALM, ZFYVE19Signaling and regulation of signaling

AF6, ARHGEF12, ARHGEF17, ASAH3, C2CD3, DAP2IP, LASP1,SMAP1,

TIRAP, VAV1Cytoskeleton organization/signaling

ABI1, ACTN4, ARHGAP26, FLNA, GPHN, KIAA0284, MAPRE1,MYO1F,

NEBL, SH3GL1Translation elongation

EEFSECMetabolism

ACACA, CBL, GMPS, UBE4AMitochondrial membrane

AF1Q

C. Nuclear proteins (n¼30)Cell cycle, cytokinesis, organization of nuclear cytoskeleton

NCKIPSD, SEPT2, SEPT5, SEPT6, SEPT9, SEPT11Nucleic acid binding

CIP29, TNRC18Chromosome associated

CENPK, CASC5RNA decay metabolism

DCP1A, DCPSHistone acetylation

CREBBP, EP300Transcription and regulation of transcription

AF17, BCL9L, FOXO3, FOXO4, FRYL, GAS7, MAML2, NRIP3,TET1

Transcriptional elongationAFF1, AFF3, AFF4, AF9, AF10, ELL, ENL

D. Not classified (n¼ 1)LOC100128568

All yet characterized TPGs (n¼64) encode proteins that have distinctfunctions within a living cell. The TPG-encoded proteins were classifiedaccording to their cellular localization and into functional groups. Oneprotein could not be classified due to the absence of any knowledgeabout potential function(s). All gene names marked in italics arerecurrently involved in MLL rearrangements (based on this study andpublished data), whereas all others have been identified only once.

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Discussion

On the basis of published data and own findings during theanalysis of 760 MLL-rearranged leukemia patients, we presentan update of the MLL recombinome associated with acuteleukemia. All our analyses were performed by using smallamounts of genomic DNA. In some cases, we analyzed cDNAfrom a given patient to validate an MLL spliced fusion, or toinvestigate alternative splice products generated from an MLLfusion gene. The results of this study allow to draw severalconclusions.

We successfully verified that the applied LDI-PCR techniqueis a valid approach to identify reciprocal MLL gene fusions, MLLgene internal duplications, chromosome 11 inversions, chro-mosomal 11 deletions and the insertion of chromosome 11material into other chromosomes, or vice versa, the insertion ofchromatin material of other chromosomes into the MLL gene.Moreover, we successfully extended our knowledge by theanalysis of more complex MLL rearrangements. During the latteranalyses, the novel subclass of reciprocal MLL gene fusions wasidentified and investigated. About 25% represent in-framefusions that can be readily expressed into reciprocal fusionproteins. All other fusions are associated with an LOH of theidentified reciprocal MLL fusion gene, however, still allow totranscribe and express a 50-truncated MLL protein.

The analysis of 760 MLL fusion alleles led to the discovery of20 novel TPGs in the past 4 years, of which 9 have already beenpublished.11,12 These are more than 30% of all identified MLLfusion partner genes so far. Moreover, these novel MLL genefusions provide a rich source for future analyses of oncogenicMLL protein variants.

A total of 64 TPGs are now characterized at the molecularlevel (see Supplementary Figure 1). According to our data, themost frequent TPGs in acute leukemias are AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P,MLLT6/AF17 and SEPT6. Noteworthy, different clinical subtypes(pediatric vs adult leukemia patients, ALL vs AML) displayeddifferent percentages for these nine TPGs (see Table 1).

An important translational aspect of this study is theestablishment of patient-specific DNA sequences that can beused for monitoring MRD by quantitative PCR techniques. Foreach of these 760 acute leukemia patients at least one MLLfusion allele was identified and characterized by sequencing.Prospective studies were already initiated and first publisheddata demonstrate that these MRD markers contribute tostratification, improved treatment and outcome of leukemiapatients.37

The analysis of the MLL recombinome allows to classify MLLfusion partner genes into functional categories. As summarizedin Table 3, genes coding for cytosolic and nuclear proteins areaffected by MLL rearrangements. Recurrence of MLL rearrange-ments was observed in about 44% of all yet identified TPGs. Theencoded proteins of these TPGs are part of different cellularprocesses: EPS15 and PICALM are proteins that are involved inendocytotic processes; AF6, ABI1, GPHN, KIAA0284 andMYO1F are involved in signaling processes and the regulationof the cytoskeleton, whereas MLLT11/AF1Q is a proapoptoticprotein; different SEPTINS are involved in the process ofcytokinesis, by reorganization and stabilization of the nuclearcytoskeleton; nucleic-acid-binding protein TNRC18 and thechromosome-associated CASC5 protein are both located in thenucleus. The histone acetyltransferase CREBBP modifies histonecore particles. Several transcription factors (AF17, FOXO3,FOXO4, FRYL, MAML2 and TET1) influence genetic programs.Finally, the most frequent TPGs in MLL translocations encode

nuclear proteins (AFF1/AF4, AFF3/LAF4, AFF4/AF5Q31, MLLT3/AF9, MLLT1/ENL and MLLT10/AF10) that belong to a proteinnetwork that transmits DOT1L38 and pTEF-B to promoter-arrested RNA polymerase II, and thus, allows active transcrip-tion and elongation.39,40 pTEF-B phosphorylates the C-terminaldomain of RNA polymerase II, whereas DOT1L enablesmethylation of lysine 79 of histone H3 proteins, a prerequisitefor the maintenance of RNA transcription.41 In addition,expression of this MLL �AF4 fusion protein confers a globalincrease of H3K79 methylation, a potentially novel oncogenicmechanism.42

Certain MLL rearrangements are associated with poor out-come in pediatric and adult acute leukemia. It can be assumedthat a systematic analysis of the MLL recombinome will allow todraw conclusions on certain aspects of hematological tumordevelopment.

Acknowledgements

This work was made possible by and conducted within theframework of the International BFM Study Group. This study wassupported by grant 107819 from the Deutsche Krebshilfe to RM,TD and TK; supported by grant R06/22 from the German JoseCarreras Leukemia foundation to TB and supported by grant 2P054 095 30 from the Polish Ministry of Science and HigherEducation to TS.

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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