New Insights to the MLL Recombinome of Acute Leukemias

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 Cave´10, E Clappier10, JJM van Dongen11, BV Balgobind12, MM van den Heuvel-Eibrink12, HB Beverloo13, R Panzer-Gru¨mayer14, A Teigler-Schlegel15, J Harbott15, E Kjeldsen16, S Schnittger17, U Koehl18, B Gruhn19, O Heidenreich20, LC Chan21, SF Yip21, M Krzywinski22, C Eckert23,AMo¨ricke24, M Schrappe24, CN Alonso25, BW Scha¨fer26, 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 Me´decine et des Sciences de la Sante´, Laboratoire d’Histologie, Embryologie et Cytogeńe´tique, Universite´ de Bretagne Occidentale, Brest, France; 7INSERM-U613, Brest, France; 8CHU Purpan, Laboratoire d’He´matologie, Toulouse, France; 9Pediatric Hematology- Oncology Program, Research Center, Instituto Nacional de Cancer Rio de Janeiro, Rio de Janeiro, Brazil; 10De´partement de Geńe´tique, 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 Cancer Research Institute, Vienna, Austria; 15Department of Pediatric Hematology and Oncology, Children’s University Hospital, Giessen, Germany; 16Cancercytogenetics Laboratory, Aarhus University Hospital, Aarhus, Denmark; 17MLL Munich Leukemia Laboratory, 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, Newcastle University, Newcastle upon Tyne, UK; 21Department of Pathology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China; 22Canada’s Michael Smith Genome Sciences Center, Vancouver, British Columbia, Canada; 23Department of Pediatric Oncology and Hematology, Charite´ Medical University Berlin, CVK, Berlin, Germany; 24Department of Paediatrics, University of Schleswig-Holstein, Kiel, Germany; 25Servcio de Hemato-Oncologıá, Hospital Nacional de Pediatrıá Prof Dr JP Garrahan, Buenos Aires, Argentina; 26Department of Oncology, University Children’s Hospital, Zurich, Switzerland; 27Clinic for Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hanover Medical School, Hanover, Germany; 28Division of Pediatrics, Cell Therapy Section, University of Texas MD Anderson Cancer Center, Houston, TX, USA; 29Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 30Department of Paediatrics and Oncohematology, University of Padua, Padua, Italy; 31Sydney Children’s Hospital, Children’s Cancer Institute, Sydney, New South Wales, Australia; 32Department of Pediatric Hemato-Oncology, The Chaim Sheba Medical Center, Tel Aviv, Israel; 33The Cancer Research Center, Tel Aviv, Israel; 34Sackler Medical School Tel Aviv University, Tel Aviv, Israel; 35Center of Pediatric Hematology Oncology, University of Catania, Catania, Italy; 36Regional Children Hospital 1, Pediatric Oncology and Hematology Center, Research Institute of Medical Cell Technologies, Ekaterinburg, Russia; 37Department of Pediatric Hematology and Oncology, 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 are on the molecular level. Nine TPGs seem to be predominantly associated with high-risk pediatric, adult and therapy-asso- involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/ ciated acute leukemias. These patients need to be identified, AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, treated appropriately and minimal residual disease was MLLT6/AF17 and SEPT6, respectively. Moreover, we describe monitored by quantitative PCR techniques. Genomic DNA was for the first time the genetic network of reciprocal MLL gene isolated from individual acute leukemia patients to identify and fusions deriving from complex rearrangements. characterize chromosomal rearrangements involving the Leukemia (2009) 23, 1490–1499; doi:10.1038/leu.2009.33; human MLL gene. A total of 760 MLL-rearranged biopsy published online 5 March 2009 samples obtained from 384 pediatric and 376 adult leukemia Keywords: MLL; translocations partner genes; acute leukemia; patients were characterized at the molecular level. The ALL; AML distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different Introduction MLL rearrangements of which 64 TPGs are now characterized Chromosomal rearrangements involving the human MLL gene at Correspondence: Professor Dr R Marschalek, Diagnostic Center of 11q23 are associated with the development of acute leuke- Acute Leukemia, Institute of Pharmaceutical Biology, ZAFES, Uni- mias.1,2 The presence of certain MLL rearrangements is an versity of Frankfurt, Max-von-Laue-Str. 9, Frankfurt/Main D-60438, independent dismal prognostic factor and patients are usually Germany. E-mail: [email protected] treated according to high-risk protocols. Therefore, the identi- Received 9 December 2008; revised 15 January 2009; accepted 28 fication of MLL gene fusions is necessary for rapid clinical January 2009; published online 5 March 2009 decisions resulting in specific therapy regimens. Current The MLL recombinome C Meyer et al 1491 procedures to identify MLL rearrangements include cytogenetic from the gel and subjected to DNA sequence analyses to obtain analysis,3,4 fluorescence in situ hybridization (FISH) experi- the patient-specific fusion sequences. ments (for example, MLL split-signal FISH),5–7 specific reverse transcriptase (RT)–PCR8 or genomic PCR methods.9,10 This repertoire of technologies was recently extended by a Results long-distance inverse PCR (LDI-PCR) method that uses small amounts of genomic DNA to determine any type of MLL gene Identification of MLL rearrangements and their rearrangement on the molecular level.11 This includes chromo- distribution in clinical subgroups somal translocations, complex chromosomal rearrangements, To analyze the recombinome of the human MLL gene, we gene internal duplications, deletions or inversions on chromo- obtained 1018 acute leukemia samplesFeither prescreened or some 11q and MLL gene insertions into other chromosomes, or unscreenedFfrom different centers over a period of 6 years. vice versa, the insertion of chromatin material into the MLL Successful analysis could be performed for 760 patient samples. gene. Unsuccessful analyses were in the range of 25% and were due To gain insight into the frequency of distinct MLL rearrange- to absence of any prescreening (21%), false-positive prescreen- ments, we analyzed prescreened and unscreened biopsy ing experiments (B1%, depending on the participating center), material of pediatric and adult leukemia patients. Prescreening limited biopsy material or insufficient quality of genomic DNA tests (cytogenetic analysis, FISH, Southern blot, RT–PCR or NG2 (1%), insufficient amount of leukemic blasts (1%) or by intrinsic positivity) were performed at different European centers and limitations of the applied method (length of IPCR amplimers centers located outside Europe, where acute leukemia patients 415 kb; noncanonical breakpoints, B1% of all investigated are enrolled in different study groups. Nearly all prescreened cases). MLL rearrangements were successfully analyzed and patient- Within the group of characterized patients (n ¼ 760), one specific MLL fusion sequencesFfor minimal residual disease adult patient was diagnosed with primary myelofibrosis (PMF) (MRD) monitoringFwere obtained. In some centers, no and displayed an MLL translocation involving MLL intron 8 prescreening could be performed. In these cases, a successful fused to a region at 1p13.1 where no gene is encoded. All other identification of MLL rearrangements was in the range of patients (n ¼ 759) were classified as pediatric or adult acute 5–10%. leukemia patients. Pediatric leukemia patients (n ¼ 384) were On the basis of the results obtained in the present (n ¼ 346) diagnosed either as acute lymphoblastic leukemia (ALL, and previous studies (414 patients were already published in n ¼ 237) or acute myeloid leukemia (AML, n ¼ 147); adult 2005 and 2006),11,12 64 translocation partner genes (TPGs) and leukemia patients (n ¼ 375) were classified either as ALL their specific breakpoint regions have now been identified. (n ¼ 246) or AML (n ¼ 129), respectively. All MLL rearrange- Additional 35 chromosomal translocations of the human MLL ments in these four subgroups are summarized in Table 1. On gene were characterized by cytogenetics, however, without any the basis of the above distribution, about 94% of all (pediatric further molecular characterization. In this study, five additional and adult) ALL patients (n ¼ 483) with MLL gene fusions are fusion loci were sequenced that do not encode any known gene. characterized by the fusion genes MLL Á AF4 (B66.0%), MLL Á Thus, the MLL recombinome presently comprises 104 different ENL (B14.9%), MLL Á AF9 (B8.5%), MLL Á AF10 (B2.7%) and fusion sites. In addition, we present a list of 48 ‘reciprocal MLL MLL Á AF6 (B1.5%), respectively. In pediatric and adult gene fusions’ that derives from complex rearrangements. These AML patients (n ¼ 276) about 77% of all characterized MLL reciprocal MLL gene fusions represent 48 genes fused to the fusion genes were MLL Á AF9 (B30.4%), MLL Á AF10 (B14.5%), 30-portion of the MLL gene. They have never been described MLL Á ELL (B10.9%), MLL Á AF6 (B10.1%), MLL Á ENL (B5.4%), before as MLL TPG, and thus, represent a novel subclass of MLL Á AF17 (B2.9%) and MLL Á SEPT6 (B2.5%), respec- reciprocal recombination partners. tively. This is in line with recently published data on the frequency and distribution of different MLL fusion partner genes.13,14 Material and methods Breakpoint distribution according to clinical subtypes Patient material We also investigated the distribution of chromosomal break- Biopsy material from acute leukemia patientsFdiagnosed to points within the MLL BCR in the four investigated clinical bear an MLL rearrangement was used to isolate genomic DNA subgroups (pediatric vs adult leukemia patients; ALL vs AML). from bone marrow and/or peripheral blood samples. Genomic For this purpose, we analyzed the chromosomal breakpoints DNA was sent to the Diagnostic Center of Acute Leukemia within the MLL gene when recombined to the 9 most frequent (DCAL) at the Frankfurt University. Patient samples were TPGs (AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, obtained from study groups (the AMLCG study group, Munich; MLLT4/AF6, MLLT6/AF17, ELL, EPS15 and SEPT6) or the other the GMALL study group, Frankfurt/Main; Polish Pediatric 29 identified recombination partners (ABI1, ACACA, ACTN4, Leukemia and Lymphoma Study Group; Zabrze) or participating AFF3, ARHGEF17, BCL9L, C2CD3, CASC5, DCP1A, EEFSEC, diagnostic centers. Informed consent was obtained from all FLNA, FOXO3, KIAA0284, LAMC3, LOC100128568, MAML2, patients or patients’ parents/legal guardians and control MLLT11, MYO1F, NEBL, NRIP3, PICALM, SEPT5, SEPT9, individuals. SMAP1, TET1, TIRAP/DCPS, TNRC18, UBE4A and VAV1). In Figure 1, all these data are summarized for the four clinical subgroups. On the basis of the 760 analyzed patients, pediatric Long-distance inverse PCR experiments ALL patients (n ¼ 237) have their chromosomal breakpoints All DNA samples were treated and analyzed as described.11,12 within MLL intron 11, whereas adult ALL patients (n ¼ 246) Briefly, 1 mg genomic patient DNA was digested with restriction recombine more frequently in MLL intron 9. Pediatric (n ¼ 147) enzymes and re-ligated to form DNA circles before LDI-PCR and adult AML patients (n ¼ 129) show a preference for analyses. Restriction polymorphic PCR amplimers were isolated recombination events affecting MLL intron 9. The exception

Leukemia The MLL recombinome C Meyer et al 1492 Table 1 Distribution of MLL fusion in clinical subgroups E4A/UFD2 homologue), VAV1 (vav 1 guanine nucleotide exchange factor), LOC100128568 (similar to hCG2045263), TPG Pediatric Adult Total ACTN4 (actinin, a-4) and FLNA (filamin A, a/actin-binding protein 280). All AML Sum All AML Sum The DCP1A gene encodes a homologue of the DCP1 15 AFF1/AF4 109 1 110 210 1 211 321 decapping enzyme, involved in mRNA degradation. The MLLT3/AF9 37 47 84 4 37 41 125 protein localizes in the mRNA processing body (P-body) that MLLT1/ENL 4955423103387 regulates degradation and abundance of RNA molecules (RNA MLLT10/AF10 12 34 46 1 6 7 53 decay). For TNRC18, no function is known. LAMC3 is a non- MLLT4/AF6 6 11 17 1 17 18 35 basement membrane-associate filamentous protein that is F ELL 17 17 1 13 14 31 downregulated or deleted in carcinomas. NEBL makes structural EPS15/AF1P 7512F 1113 a 16 MLLT6/AF17 F 22F 66 8part of the Z-line in cardiac myofibrils and binds to -actinin. SEPT6 F 66F 11 7NRIP3 is a nuclear receptor interacting protein of unknown function. C2CD3 is a Ca2 þ -binding protein of unknown MLLT11 178FFF 8 function. UBE4A is expressed in skeletal muscle, kidney and SEPT9 F 11F 33 4liver, and weakly expressed in hematopoietic cells. It has been FFF TET1/LCX 2133postulated that UBE4A is involved in cell-cycle control AFF3/LAF4 2 F 2 FFF 2 CASC5/AF15Q14 F 11F 11 2(ubiquitination) and by protecting the cell against environmental FOXO3/AF6Q21 2 F 2 FFF 2 stress. UBE4A is frequently mutated and deleted in neuro- 17 KIAA0284 FFFF 22 2blastomas. The identified MLL Á UBE4A fusion was a head-to- MAML2 2 F 2 FFF 2 head genomic fusion, and thus, created loss of heterozygosity PICALM/CALM 1 F 1 F 11 2(LOH) for the UBE4A gene. VAV1 is a Dbl-domain containing F F SEPT5/CDCREL 1 1 11 2proto-oncoprotein with GDP/GTP exchange function that is TNRC18/KIAA1856 2 F 2 FFF 2 ABI1 F 11FFF 1 selectively expressed in hematopoietic cells. It interacts with ACACA F 11FFF 1 CBL and GRB2 and influences RAC/RHO signaling processes 18 ACTN4 FFF 1 F 11and migration behavior. LOC100128568 is a hypothetical ARHGEF17 F 11FFF 1 protein with unknown function. ACTN4 encodes a non-muscle BCL9 L 1 F 1 FFF 1 a-actinin that appears to promote tumor growth and invasive- FFFF C2CD3 11 1ness.19 Basically, ACTN4 regulates stress fiber formation of the DCP1A 1 F 1 FFF 1 F FFF cytoskeleton. Moreover, ACTN4 protein is a regulator of AKT1 EEFSEC/SELB 1 1 1 20 FLNA FFFF 11 1localization and of its function; ACTN4 is also involved in 21 LAMC3 F 11FFF 1 insulin signaling. FLNA encodes a cytoskeletal filamin protein LOC100128568 F 11FFF 1 (280 kDa) that interacts and reorganizes the actin cytoskeleton. MYO1F F 11FFF 1 It is a substrate of granzyme B and different caspases.22 F FFF NEBL 11 1 Interestingly, the cleaved C-terminal portion (100 kDa) localizes NRIP3 F 11FFF 1 FFFF in the nucleus. This proteolytic fragment is able to bind and SMAP1 11 1 23 TIRAP (DCPS) FFFF 11 1regulate androgen receptor. Another study has demonstrated 24 UBE4A 1 F 11colocalization of FLNA and Caveolin 1. Caveolin 1 is VAV1 F 11FFF 1 downregulated in tumor cells as it inhibits anchorage-indepen- MLL PTD F 111242526dent growth, anoikis and invasiveness.25 9p13.3 1 F 1 FFF 1 11q12 1 F 1 FFF 1 11q23 FFF 1 F 11The MLL recombinome 21q22 1 F 1 FFF 1 Within the past 16 years, several genetic aberrations involving Total 237 147 384 246 129 375 759 the human MLL gene located on chromosome 11 band q23 have Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute been described. Out of 104 TPGs, 64 are now characterized at myeloid leukemia; TPG, translocation partner gene. the molecular level (see Table 2). Forty-four fusion genes have All identified MLL fusions were classified by pediatric vs adult patients been described by others, whereas 20 TPGs have been and disease phenotype (ALL vs AML). Total numbers are indicated for identified at the Frankfurt DCAL. Additional 35 genetic loci all clinical subgroups and sorted by frequency. were identified by cytogenetic experiments but not further characterized (for references see Meyer et al.12). Five MLL was the MLLT3/AF9 gene in pediatric and adult AML patients rearrangements were identified that did not display a fusion to that show a preference for recombination events to occur within an annotated gene or open reading frame. These partner loci MLL intron 11. The same was true for SEPT6 in pediatric AML were 1p31.2 (patient with PMF), 9p13.3, 11q22, 11q23.3 and patients, but not in adult AML patients. Therefore, we conclude 21q22, respectively. Several attempts to identify spliced fusion that pediatric ALL patients are different from all other subgroups partners in vicinity failed so far (data not shown). Thus, these concerning their breakpoint distribution within the MLL BCR. fusions most likely represent nonfunctional MLL fusions that are, however, associated with acute leukemias, and in one case, with PMF. Novel translocation partner genes Eleven novel TPGs were discovered: DCP1A (decapping enzyme homologue A), TNRC18 (trinucleotide repeat contain- Genetic alterations resulting in genetic MLL aberrations ing 18), LAMC3 (laminin, g-3), NEBL (nebulette), NRIP3 In general, human MLL rearrangements are initiated by a (nuclear receptor interacting protein 3), C2CD3 (C2 calcium- DNA damage situation, which induces DNA repair by the dependent domain containing 3), UBE4A (ubiquitination factor nonhomologous-end-joining DNA repair pathway.26,27 Genetic

Leukemia The MLL recombinome C Meyer et al 1493

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 are localized in the MLL BCR (exons 10, 11 and 12). Breakpoint distributions are shown for pediatric acute lymphoblastic leukemia (ALL), pediatric acute myeloid leukemia (AML), adult ALL and adult AML patients. Breakpoints are shown only for the nine most frequent translocation partner genes (TPGs) and ‘all other’ chromosomal rearrangements identiﬁed in the respective subgroups. Sizes of all MLL introns and the sum of MLL exons 10–12 are given below in base pairs.

recombinations involving the human MLL gene predominantly MLL recombinations involving only chromosome 11 are result in reciprocal chromosomal translocations (see Figure 2, based on two independent DNA strand breaks that are rCTL), involving recurrently the following TPGs: ABI1, AFF1/ accompanied either by inversions or deletions on 11p or 11q AF4, CASC5, CREBBP, ELL, EPS15/AF1P, FOXO3, FOXO4, (Inv, Del). Several recombinations have been characterized that FRYL, GPHN, KIAA0284, MLLT3/AF9, MLLT1/ENL, MLLT4/ belong to these two groups. MLL gene fusions to C2CD3, AF6, MLLT6/AF17, MLLT11/AF1Q, MYO1F, SEPT2, SEPT5, MAML2, NRIP3, PICALM and UBE4A are based on the inversion SEPT9, TET1 and TNRC18, respectively. Other TPGs (also from of a chromatin portion of 11p or 11q, leading to reciprocal MLL the literature) were identified so far only once (ACTN4, gene fusions. By contrast, a deletion of chromosome material ARHGAP26, ARHGEF17, ASAH3, DAB2IP, DCP1A, EEFSEC, located telomere to MLL fused the 50-portion of MLL directly to EP300, GAS7, GMPS, LAMC3, LASP1, LPP, MAPRE1, NCKIPSD, other gene sequences (ARHGEF12, BCL9L and CBL). A fourth NEBL, SEPT11, SH3GL1 and SMAP1). deletion at 11q fused 50-MLL sequences to the 30-UTR of the Gene internal partial tandem duplications (PTDs) of specific TIRAP gene, which is located several kilobases upstream of the MLL gene portions (duplication of MLL gene segments coding DCPS gene. In that particular case, only transcription of MLL either for introns 2–9, 2–10, 2–11, 4–9, 4–11 or 3–8) are and a subsequent splice process allowed to generate an frequently observed in AML patients.28–30 MLL PTDs are being MLL Á DCPS fusion mRNA, encoding a bona fide MLL Á DCPS discussed to mediate dimerization of the MLL N terminus, a fusion protein (MLL spliced fusion). process that seems to be sufficient to mediate leukemogenic Beside reciprocal chromosomal translocations of MLL (rCTL), transformation.31 We have observed MLL PTDs in three of the MLL PTDs and 11p/q rearrangements (Del and Inv), additional four investigated subgroups: 1 patient within the group of genetic rearrangements were identified in the genomic DNA of pediatric AML, 1 patient within the group of adult ALL and 24 analyzed leukemia biopsy material. Although the previous patients within the group of adult AML. This demonstrates that rearrangements are based on two independent DNA strand MLL PTDs are predominantly present in adult AML patients, in breaks, all other genetic events observed for the MLL gene line with previously published data.32,33 represent more complex rearrangements with at least three or

Leukemia The MLL recombinome C Meyer et al 1494 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, CML 2 t(1;11)(q21;q23) 1q21 MLLT11/AF1Q Tse et al. (1995) AML 3 t(2;11)(q11.2Bq12;q23) 2q11.2Bq12 AFF3/LAF4 von Bergh et al. (2003) ALL 4 t(2;11)(q37;q23) 2q37 SEPT2 Cerveira et al. (2006) AML, t-MDS, t-AML 5 t(3;11)(p21;q23) 3p21 NCKIPSD/AF3P21 Sano et al. (2000) t-AML 6 t(3;11)(p21.3;q23) 3p21.3 DCP1A This manuscriptb ALL 7 t(3;11)(q21.3;q23) 3q21.3 EEFSEC/SELB Meyer et al (2005)b ALL 8 t(3;11)(q24;q23) 3q24 GMPS Pegram et al. (2000) t-AML 9 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-AML 11 t(4;11)(q21.1;q23) 4q21.1 SEPT11/FLJ10849 Kojima et al. (2004) CML 12 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) AML 14 complex abnormalities 5q12.3 CENPK/FKSG14 Taki et al. (1996) AML 15 ins(5;11)(q31;q13q23) 5q31 AFF4/AF5Q31 Taki et al. (1999) ALL 16 t(5;11)(q31;q23) 5q31 ARHGAP26/GRAF Borkhardt et al. (2000) JMML 17 t(6;11)(q12B13;q23) 6q12Bq13 SMAP1 Meyer et al (2005)b AML 18 t(6;11)(q21;q23) 6q21 FOXO3/AF6Q21 Hillion et al. (1997) t-AML 19 t(6;11)(q27;q23) 6q27 MLLT4/AF6 Prasad et al. (1993) AML, t-AML, ALL 20 t(7;11)(p22.1;q23) 7p22.1 TNRC18/KIAA1856 This manuscriptb ALL 21 t(9;11)(p22;q23) 9p22 MLLT3/AF9 Nakamura et al. (1993) AML, t-AML, ALL 22 t(9;11)(q33.1Bq33.3;q23); 9q33.1Bq33.3 DAB2IP/AF9Q34 von Bergh et al. (2004) AML 23 ins(11;9)(q23;q34)inv(11)(q13q23) 9q34 FNBP1/FBP17 Fuchs et al. (2001) AML 24 t(9;11)(q31Bq34;q23) 9q31Bq34 LAMC3 This manuscriptb t-AML 25 t(10;11)(p11.2;q23) 10p11.2 ABI1 Taki et al. (1998) AML 26 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 AML 28 t(10;11)(q21;q23) 10q21 TET1/LCX Ono et al. (2002) AML 29 inv(11)(p15.3q23) 11p15.3 NRIP3 Balgobind et al, submitteda AML 30 t(11;11)(q13.4;q23) 11q13.4 ARHGEF17 Teuffel et al. (2005)b AML 31 inv(11)(q13.4q23) 11q13.4 C2CD3/DKFZP586P0123 This manuscriptb AML 32 inv(11)(q14q23) 11q14 PICALM/CALM Wechsler et al. (2003) AML 33 inv(11)(q21q23) 11q21 MAML2 Meyer et al (2006)b t-T-ALL, t-AML 34 t(11;15)(q23q;q21)inv(11)(q23q23) 11q23 UBE4A This manuscriptb MDS 35 del(11)(q23q23.3) 11q23.3 ARHGEF12/LARG Kourlas et al. (2000) AML 36 del(11)(q23q23.3) 11q23.3 CBL Fu et al. (2003) AML 37 del(11)(q23q23.3) 11q23.3 BCL9 L Meyer et al (2006)b ALL 38 del(11)(q23q24.2) 11q24.2 TIRAP Meyer et al (2006)b AML 39 del(11)(q23q24.2) 11q24.2 DCPS Meyer et al (2005)b AML 40 t(11;12)(q23;q13.2) 12q13.2 CIP29 Hashii et al. (2004) AML 41 t(11;14)(q23.3;q23.3) 14q23.3 GPHN Kuwada et al. (2001) AML, t-AML 42 t(11;14)(q32.33;q32.33) 14q32.33 KIAA0284 Burmeister et al (2008)b AML 43 t(11;15)(q23;q14) 15q14 CASC5/AF15Q14 Hayette et al. (2000) AML, ALL 44 t(11;15)(q23;q14) 15q14 ZFYVE19/MPFYVE Chinwalla et al. (2003) AML 45 t(11;16)(q23;p13.3) 16p13.3 CREBBP/CBP Taki et al. (1997) t-MDS, t-AML, t-ALL 46 t(11;17)(q23;p13.1) 17p13.1 GAS7 Megonigal et al. (2000) t-AML 47 ins(11;17)(q23;q21) 17q21 ACACA Meyer et al (2005)b AML 48 t(11;17)(q23;q21) 17q21 MLLT6/AF17 Prasad et al. (1994) AML 49 t(11;17)(q23;q11Bq21.3) 17q11Bq21.3 LASP1 Strehl et al. (2003) AML 50 t(11;17)(q23;q25) 17q25 SEPT9/AF17Q25 Osaka et al. (1999) t-AML, AML 51 t(11;19)(q23;p13.1) 19p13.1 ELL Thirman et al. (1994) AML, t-AML 52 t(11;19)(q23;p13) 19p13.3 SH3GL1/EEN So et al. (1997) AML 53 ins(11;19)(q23;p13.2) 19p13.2 VAV1 This manuscriptb AML 54 t(11;19)(q23;p13.3) 19p13.3 MLLT1/ENL Tkachuk et al. (1992) ALL, AML, t-AL 55 t(11;19)(q23;p13.3) 19p13.3 ASAH3/ACER1 Lo Nigro et al. (2002) ALL 56 t(2;11;19)(p23.3;q23;p13.3) 19p13.3 LOC100128568 This manuscriptb AML 57 t(11;19)(q23;p13.3Bp13.2) 19p13.3Bp13.2 MYO1F Lo Nigro et al. (2002) AML 58 t(11;19)(q23;q13) 19q13 ACTN4 This manuscriptb ALL 59 t(11;20)(q23;q11) 20q11 MAPRE1 Fu et al. (2005) ALL 60 t(11;22)(q23;q11.21) 22q11.21 SEPT5/CDCREL Megonigal et al. (1998) AML, T-ALL 61 t(11;22)(q23;q13.2) 22q13.2 EP300/P300 Ida et al. (1997) t-AML 62 t(X;11)(q13.1;q23) Xq13.1 FOXO4/AFX Parry et al. (1994) ALL, AML 63 ins(X;11)(q24;q23) Xq24 SEPT6 Borkhardt et al. (2001) AML 64 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, myelodysplastic syndrome; 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.

Leukemia The MLL recombinome C Meyer et al 1495

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) or 11q 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 genes belong 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 recombination process. Green vertical bars, MLL; red, orange, blue and pink vertical bars, partner genes involved in recombination events; derivative 11 chromosomes 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; CTL þ D, chromosomal translocation 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. more DNA double-strand breaks. In these cases, the expected ACACA, AFF3/LAF4, AFF4/AF5Q31, CENPK, CIP29, CREBBP, reciprocal MLL fusion gene cannot be detected, because other FLNA, FNBP1, LOC1000128568, MLLT10/AF10, SEPT6, sequences will be fused to the 30-portion of the MLL gene. SORBS2/ARGBP2 and VAV1. In these cases (Ins1/2), three The first class of complex MLL rearrangements are three-way independent fusion genes will be generated. In three patients chromosomal translocations (3W-CTL) involving three indepen- bearing a recombination event between MLL and MLLT10/ dent chromosomes and resulting in three different fusion genes. AF10, more complex rearrangements were identified that The most frequent involved genes in 3W-CTLs were AFF1/AF4, cannot be explained by a simple insertion mechanism. MLLT3/AF9, MLLT1/ENL, MLLT11/AF1Q and ELL in combina- Finally, spliced fusion were observed. Spliced fusions are tion with partner genes shown in Figure 3. generated by fusing the 50-portion of the MLL gene to the The second category are reciprocal chromosomal transloca- upstream region of a TPG. Thus, a functional MLL fusion mRNA tions that are associated with deletions on either of the involved can only be generated by a coupled process of transcription and chromosomes (CTL þ D). Such cases were predominantly splicing (last MLL exon 50 to the breakpoint spliced to an exon identified in complex t(4;11)(q21;q23) translocations. (a1) of the partner gene fused to the MLL). Beside the above- The third category are chromosomal fragment insertions. This mentioned DCPS gene, other genes have been identified that includes the insertion of chromosome 11 material (including can transcriptionally fuse to 50-MLL sequences. These were portions of the MLL gene) into other chromosomes (Ins1), or vice ZFYVE19, but also the MLL fusion partners like, AFF1/AF4, versa, the insertion of chromosome material (including portions EPS15, MLLT3/AF9, MLLT1/ENL and SEPT5. In case of MLLT1/ of a TPG) into the BCR of the MLL gene (Ins2). An insertion ENL, about 50% of all recombination events were spliced mechanism is required in those cases where the transcriptional fusions,34 and for MLL Á EPS15 fusions about 30%. Spliced orientation of a given TPG is not identical to the transcriptional fusions to AFF1/AF4, MLLT3/AF9 and SEPT5 represent very rare orientation of the MLL gene. The MLL gene is transcribed events. in telomeric direction. TPGs with a transcriptional orientation in All the above-mentioned mechanisms can be combined to direction to the centromere are predominantly recombining generate more complex genetic rearrangements, requiring four with MLL by such insertion mechanisms. These genes are or more DNA double-strand breaks.

Leukemia The MLL recombinome C Meyer et al 1496

Figure 3 Overview over reciprocal MLL fusion genes. (a) Identified reciprocal MLL fusion partners fused in-frame (green) to the 30-MLL gene segment (MLL exons 10/11/12–37); a total of 12 genes were identified. CDK6 has been described in the literature. (b) Identified reciprocal MLL fusion 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 has been described in the literature. (c) Identified reciprocal MLL fusion partners fused head-to-head (orange) to the 30-MLL gene segment (MLL exons 10/11/12–37); a total of 17 genes have been identified. The 30-MLL gene segment is still able to be transcribed from its gene internal promoter element 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 H3K4 methyltransferase activity.

Reciprocal MLL gene fusions 12 out of these 48 reciprocal fusions represent bona fide gene Analyses of complex MLL rearrangements (3W-CTL, CTL þ D, fusions between the given TPG and MLL introns 9–12 (ADARB2, Ins1, Ins2 and cCTLs) allowed to identify a new class of MLL APBB1IP, ATG16L2, CDK6, FLJ46266, GPSN2, MEF2C, recombination events that provide new insights into the MYO18A, NKAIN2, RABGAP1L, RNF115 and UVRAG). complex spectrum of MLL rearrangements. By using a systema- ADARB2 is an RNA-editing enzyme that desaminates A tic breakpoint analysis approach, we identified 3–5 fusion nucleotides. Overexpressed APBB1IP results in cell adhesion alleles in these patients, of which only one of these alleles and is predominantly expressed in myeloid cells. ATG16L2 is represented the reciprocal MLL fusion allele. Most of these a protein that promotes autophagy. CDK6 is a cell-cycle- reciprocal MLL fusion were not able to produce fusion proteins, dependent kinase that associates with cyclin D during because the recombination occurred either in noncompatible G1 phase. MEF2C resembles a transcription factor that enhances introns or represented head-to-head gene fusions. As summarized c-jun-mediated transcriptional processes; the MEF2C gene was in Figure 3, 48 reciprocal MLL fusion have been identified. Only found to be overexpressed in MLL-rearranged leukemias. The

Leukemia The MLL recombinome C Meyer et al 1497 NKAIN2 gene resembles a genetic hotspot that is frequently organization and regulation of cytoskeleton (actin and micro- recombined in T-cell lymphomas. RABGAP1L regulates the tubuli; ABI1, ACTN4, ARHGAP26, FLNA, GPHN, KIAA0284, cytoskeleton, whereas UVRAG is a tumor suppressor protein MAPRE1, MYO1F, NEBL, SH3GL1), translation elongation that promotes autophagy. It was defined as tumor suppressor (EEFSEC), metabolic functions (ACACA, CBL, GMPS, UBE4A) protein, because overexpression suppresses proliferation and and proapoptotic proteins (MLLT11/AF1Q). The nuclear com- tumorigenicity. partment is subclassified into cell-cycle control and organiza- Another 19 gene fusions were out of frame because of tion of nuclear cytoskeleton during cytokinesis (NCKIPSD, recombining noncompatible introns of both involved genes SEPT2, SEPT5, SEPT6, SEPT9, SEPT11), nucleic acid binding (ARMC3, CACNA1B, CMAH, CRLF1, FXYD6, GRIA4, MMP13, (CIP29, TNRC18), RNA decay (DCP1A, DCPS), chromosome NFkB1, PAN3, PBX1, PPM1G, PARP14, PIWIL4, RPS3, association (CENPK, CASC5), chromatin regulation (CREBBP, SCGB1D1, SFRS4, TCF12, TNRC6B and TRIP4). This genetic EP300), transcription factors and regulation of transcription situation represents an LOH situation. CRLF1 is part of a (AF17, BCL9L, FOXO3, FOXO4, FRYL, GAS7, MAML2, NRIP3, signaling complex that regulates immune responses during fetal TET1) as well as transcriptional elongation factors (AFF1, AFF3, development. MMP13 is frequently overexpressed in tumor AFF4, AF9, AF10, ELL, ENL). No cellular function is known for cells. NFkB1 blocks the apoptotic pathway. PAN3 is a tumor the gene product of LOC100128568. All categorized TPGs are suppressor protein that is also part of the polyA-specific summarized in Table 3. ribonuclease complex. PBX1 is a transcription factor that interacts with MEIS1 and HOXA proteins to steer developmental processes. PBX1 is already well known as fusion partner of the E2A gene in a subset of ALL cases.35 PIWIL4 is involved in the maintenance and self-renewal of stem cells, as well as in RNA Table 3 Overview on the cellular function of all yet known TPGs interference. TCF12 is a transcription factor that associates with E2A to steer somatic recombination of T-cell receptor genes. A. Cell surface/membrane proteins (n ¼ 1) TRIP4 is part of the inflammasome and inhibits the activation of LAMC3 interleukin 1 (IL1) and IL18 by modulating CASP1 activity. The final group of 30-MLL fusion represent head-to-head B. Cytosolic proteins (n ¼ 33) fusion. Thus, the transcriptional orientation of the fused TPG is Cell adhesion LPP, SORBS2 opposite to the orientation of the MLL gene. In these case, the Endocytosis TPG became disrupted by replacing its promoter region by the EPS15, FNBP1, PICALM, ZFYVE19 0 3 -MLL portion. Thus, this genetic situation represents again an Signaling and regulation of signaling LOH situation. Identified fusion partners were ADSS, CACNB2, AF6, ARHGEF12, ARHGEF17, ASAH3, C2CD3, DAP2IP, LASP1, CUGBP1, DSCAML1 (2 Â ), ELF2, FCHSD2, FXYD2, GTDC1, SMAP1, KIAA0999 (2 Â ), KIAA1239, MPZL2, NCAM1, NT5C2, SVIL, TIRAP, VAV1 Cytoskeleton organization/signaling TMEM135, TUBGCP2 and UNC84A, respectively. Two genes, ABI1, ACTN4, ARHGAP26, FLNA, GPHN, KIAA0284, MAPRE1, DSCAML1 and KIAA0999, were identified twice in different MYO1F, leukemia patients, indicating that such reciprocal MLL gene NEBL, SH3GL1 fusions do also show recurrence. CUGBP1 binds to RNA, Translation elongation influences splicing processes and translation efficiency; it also EEFSEC binds to EWS. ELF2 is an ETS transcription factor that is Metabolism overexpressed under hypoxic conditions; ELF2 is a direct ACACA, CBL, GMPS, UBE4A Mitochondrial membrane binding partner of AML1/RUNX1. NCAM1 is also known as AF1Q CD56 and a known tumor suppressor protein. SCIL binds to the actin cytoskeleton, whereas TUBGCP2 binds to microtubuli and C. Nuclear proteins (n ¼ 30) regulates centrosome formation. UNC84A is associated to the Cell cycle, cytokinesis, organization of nuclear cytoskeleton nuclear lamina and to centrosomes. We have to mention that NCKIPSD, SEPT2, SEPT5, SEPT6, SEPT9, SEPT11 promoterless 30-MLL gene segments are per se able to transcribe Nucleic acid binding CIP29, TNRC18 most of the remaining open reading frame of the MLL gene by a Chromosome associated gene internal promoter recently identified upstream of MLL exon CENPK, CASC5 12.36 Therefore, also these arbitrary MLL fusion may still allow RNA decay metabolism to transcribe most of the parts of the MLL coding region, DCP1A, DCPS resulting in a shorter MLL protein version (230 kDa) that still Histone acetylation exhibits the ability to function as ‘nonspecific’ H3K4 methyl- CREBBP, EP300 Transcription and regulation of transcription transferase due to the missing N terminus. AF17, BCL9L, FOXO3, FOXO4, FRYL, GAS7, MAML2, NRIP3, TET1 Transcriptional elongation Classification of MLL TPGs into functional categories AFF1, AFF3, AFF4, AF9, AF10, ELL, ENL All TPGs were categorized according to their gene ontology into functional subclasses. They can be classified into cytosolic/ D. Not classified (n ¼ 1) LOC100128568 membrane proteins and nuclear proteins. According to their functions, they were grouped into extracellular proteins All yet characterized TPGs (n ¼ 64) encode proteins that have distinct (LAMC3), cell adhesion proteins (LPP, SORBS2) with functions functions within a living cell. The TPG-encoded proteins were classified according to their cellular localization and into functional groups. One in the organization of focal adhesion plaques, endocytotic protein could not be classified due to the absence of any knowledge proteins (EPS15, FNBP1, PICALM, ZFYVE19, proteins involved about potential function(s). All gene names marked in italics are in diverse signaling pathways AF6, ARHGEF12, ARHGEF17, recurrently involved in MLL rearrangements (based on this study and ASAH3, C2CD3, DAP2IP, LASP1, SMAP1, TIRAP, VAV1), published data), whereas all others have been identified only once.

Leukemia The MLL recombinome C Meyer et al 1498 Discussion nuclear proteins (AFF1/AF4, AFF3/LAF4, AFF4/AF5Q31, MLLT3/ AF9, MLLT1/ENL and MLLT10/AF10) that belong to a protein On the basis of published data and own findings during the network that transmits DOT1L38 and pTEF-B to promoter- analysis of 760 MLL-rearranged leukemia patients, we present arrested RNA polymerase II, and thus, allows active transcrip- an update of the MLL recombinome associated with acute tion and elongation.39,40 pTEF-B phosphorylates the C-terminal leukemia. All our analyses were performed by using small domain of RNA polymerase II, whereas DOT1L enables amounts of genomic DNA. In some cases, we analyzed cDNA methylation of lysine 79 of histone H3 proteins, a prerequisite from a given patient to validate an MLL spliced fusion, or to for the maintenance of RNA transcription.41 In addition, investigate alternative splice products generated from an MLL expression of this MLL Á AF4 fusion protein confers a global fusion gene. The results of this study allow to draw several increase of H3K79 methylation, a potentially novel oncogenic conclusions. mechanism.42 We successfully verified that the applied LDI-PCR technique Certain MLL rearrangements are associated with poor out- is a valid approach to identify reciprocal MLL gene fusions, MLL come in pediatric and adult acute leukemia. It can be assumed gene internal duplications, chromosome 11 inversions, chro- that a systematic analysis of the MLL recombinome will allow to mosomal 11 deletions and the insertion of chromosome 11 draw conclusions on certain aspects of hematological tumor material into other chromosomes, or vice versa, the insertion of development. chromatin material of other chromosomes into the MLL gene. Moreover, we successfully extended our knowledge by the analysis of more complex MLL rearrangements. During the latter Acknowledgements analyses, the novel subclass of reciprocal MLL gene fusions was identified and investigated. About 25% represent in-frame This work was made possible by and conducted within the fusions that can be readily expressed into reciprocal fusion framework of the International BFM Study Group. This study was proteins. All other fusions are associated with an LOH of the supported by grant 107819 from the Deutsche Krebshilfe to RM, identified reciprocal MLL fusion gene, however, still allow to TD and TK; supported by grant R06/22 from the German Jose´ transcribe and express a 50-truncated MLL protein. Carreras Leukemia foundation to TB and supported by grant 2 The analysis of 760 MLL fusion alleles led to the discovery of P054 095 30 from the Polish Ministry of Science and Higher 20 novel TPGs in the past 4 years, of which 9 have already been Education to TS. published.11,12 These are more than 30% of all identified MLL fusion partner genes so far. Moreover, these novel MLL gene fusions provide a rich source for future analyses of oncogenic References MLL protein variants. A total of 64 TPGs are now characterized at the molecular 1 Pui CH, Gaynon PS, Boyett JM, Chessells JM, Baruchel A, Kamps level (see Supplementary Figure 1). According to our data, the W et al. Outcome of treatment in childhood acute lymphoblastic most frequent TPGs in acute leukemias are AFF1/AF4, MLLT3/ leukaemia with rearrangements of the 11q23 chromosomal region. AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, Lancet 2002; 359: 1909–1915. MLLT6/AF17 and SEPT6. Noteworthy, different clinical subtypes 2 Pui CH, Chessells JM, Camitta B, Baruchel A, Biondi A, Boyett JM (pediatric vs adult leukemia patients, ALL vs AML) displayed et al. Clinical heterogeneity in childhood acute lymphoblastic different percentages for these nine TPGs (see Table 1). leukemia with 11q23 rearrangements. Leukemia 2003; 17: 700–706. An important translational aspect of this study is the 3 Johansson B, Moorman AV, Secker-Walker LM. Derivative establishment of patient-specific DNA sequences that can be chromosomes of 11q23-translocations in hematologic malignan- used for monitoring MRD by quantitative PCR techniques. For cies. European 11q23 Workshop participants. Leukemia 1998; 12: each of these 760 acute leukemia patients at least one MLL 828–833. fusion allele was identified and characterized by sequencing. 4 Heerema NA, Sather HN, Ge J, Arthur DC, Hilden JM, Trigg ME Prospective studies were already initiated and first published et al. Cytogenetic studies of infant acute lymphoblastic leukemia: poor prognosis of infants with t(4;11)Fa report of the Children’s data demonstrate that these MRD markers contribute to Cancer Group. Leukemia 1999; 13: 679–686. stratification, improved treatment and outcome of leukemia 5 van der Burg M, Beverloo HB, Langerak AW, Wijsman J, patients.37 van Drunen E, Slater R et al. Rapid and sensitive detection of all The analysis of the MLL recombinome allows to classify MLL types of MLL gene translocations with a single FISH probe set. fusion partner genes into functional categories. As summarized Leukemia 1999; 13: 2107–2113. in Table 3, genes coding for cytosolic and nuclear proteins are 6 van der Burg M, Poulsen TS, Hunger SP, Beverloo HB, Smit EM, Vang-Nielsen K et al. Split-signal FISH for detection of chromo- affected by MLL rearrangements. Recurrence of MLL rearrange- some aberrations in acute lymphoblastic leukemia. Leukemia ments was observed in about 44% of all yet identified TPGs. The 2004; 18: 895–908. encoded proteins of these TPGs are part of different cellular 7 Harrison CJ, Moorman AV, Barber KE, Broadfield ZJ, Cheung KL, processes: EPS15 and PICALM are proteins that are involved in Harris RL et al. Interphase molecular cytogenetic screening for endocytotic processes; AF6, ABI1, GPHN, KIAA0284 and chromosomal abnormalities of prognostic significance in child- MYO1F are involved in signaling processes and the regulation hood acute lymphoblastic leukaemia: a UK Cancer Cytogenetics Group Study. Br J Haematol 2005; 129: 520–530. of the cytoskeleton, whereas MLLT11/AF1Q is a proapoptotic 8 van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, protein; different SEPTINS are involved in the process of Saglio G et al. Standardized RT–PCR analysis of fusion gene cytokinesis, by reorganization and stabilization of the nuclear transcripts from chromosome aberrations in acute leukemia for cytoskeleton; nucleic-acid-binding protein TNRC18 and the detection of minimal residual disease. Report of the BIOMED-1 chromosome-associated CASC5 protein are both located in the Concerted Action: investigation of minimal residual disease in nucleus. The histone acetyltransferase CREBBP modifies histone acute leukemia. Leukemia 1999; 13: 1901–1928. 9 Reichel M, Gillert E, Angermu¨ller S, Hensel JP, Heidel F, Lode M core particles. Several transcription factors (AF17, FOXO3, et al. Biased distribution of chromosomal breakpoints involving the FOXO4, FRYL, MAML2 and TET1) influence genetic programs. MLL gene in infants versus children and adults with t(4;11) ALL. Finally, the most frequent TPGs in MLL translocations encode Oncogene 2001; 20: 2900–2907.

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