Leukemia Patients with Absent AF4.MLL Fusion Allele

Complex MLL rearrangements in t(4;11) leukemia patients with absent AF4 . MLL fusion allele

E Kowarz1, T Burmeister2, L Lo Nigro3, MWJC Jansen4, E Delabesse5, T Klingebiel6, Theo Dingermann1, C Meyer1 and R Marschalek1

1Institute of Pharmaceutical Biology / ZAFES / DCAL, JWG-University Frankfurt, Biocenter, Frankfurt/Main, Germany; 2Charite´, Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Medizinische Klinik III, Hindenburgdamm 30, Berlin, Germany; 3Center of Pediatric Hematology Oncology, University of Catania, Catania, Italy; 4Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; 5Department of Haematology, INSERM U563, Hoˆpital Purpan, Toulouse, France and 6ZKI, Medical Faculty III, JWG-University Frankfurt, Frankfurt/Main, Germany

The human mixed lineage leukemia (MLL) gene is frequently into a rapidly proliferating population of preleukemic cells. involved in genetic rearrangements with more than 55 different Over time, this cell population may acquire secondary muta- translocation partner genes, all associated with acute leukemia. tions finally resulting in a malignant leukemia phenotype.9–11 Reciprocal chromosomal translocations generate two MLL 0 0 Genes found to be affected by the der(11)-encoded MLL fusion fusion alleles, where 5 - and 3 -portions of MLL are fused to 6 12 gene segments of given fusion partners. In case of t(4;11) proteins are specific HOX genes, the MYB gene and certain patients, about 80% of all patients exhibit both reciprocal fusion cyclin kinase inhibitors.13,14 In several studies, direct binding of alleles, MLL . AF4 and AF4 Á MLL, respectively. By contrast, 20% the der(11)-encoded MLL fusion protein to promoter regions of of all t(4;11) patients seem to encode only the MLL Á AF4 fusion þ À the above-mentioned genes has been demonstrated by ChIP allele. Here, we analyzed these ’MLL Á AF4 /AF4 Á MLL ’ patients (chromatin immunoprecipitation) experiments).13,15–17 This in- at the genomic DNA level to unravel their genetic situation. Cryptic translocations and three-way translocations were found dicates that MLL fusion proteins encoded by the der(11) in this group of t(4;11) patients. Reciprocal MLL fusions with chromosome are able to influence directly the transcription of novel translocation partner genes, for example NF-KB1 and specific target genes. RABGAP1L, were identified and actively transcribed in leuke- Transgenic mice or the genetic manipulation of murine mic cells. In other patients, the reciprocal 30-MLL gene segment hematopoietic stem/precursor cells by retroviral transduction was fused out-of-frame to PBX1, ELF2, DSCAML1 and FXYD6. and subsequent transplantation into syngenic mice has demon- The latter rearrangements caused haploinsufficiency of genes that are normally expressed in hematopoietic cells. Finally, strated successfully that the overexpression of certain der(11)- patients were identified that encode only solitary 30-MLL gene encoded MLL fusion proteins is sufficient and necessary for the segments on the reciprocal allele. Based on these data, we onset of acute myeloid leukemia. This concept has been tested propose that all t(4;11) patients exhibit reciprocal MLL alleles, for MLL Á AF9, MLL Á ENL, MLL Á ELL, MLL Á GAS7, MLL Á CBP and but due to the individual recombination events, provide MLL Á AF10. All of them showed malignant transformation of different pathological disease mechanisms. myeloid precursors and developed a leukemic disease in mouse Leukemia (2007) 21, 1232–1238. doi:10.1038/sj.leu.2404686; 6,18–30 published online 5 April 2007 models. By contrast, some der(11)-encoded MLL fusions Keywords: MLL; AF4; acute leukemia; reciprocal MLL alleles (MLL Á ABI1, MLL Á AF4, MLL Á FBP17, MLL Á GRAF and MLL Á LASP1) failed to form immortalized colonies in semi-solid agar or engraftment in recipient mice.31 Thus, their malignant potential could not be investigated. Reasons to explain the failure of these fusion constructs to induce a leukemic disease in Introduction the mouse models are presumably complex, including differences between mouse and human proteins, missing mutations in Chromosomal translocations of the human mixed lineage recipient cells, or simply culture conditions used for the leukemia (MLL) gene are associated with infant, childhood, manipulated hematopoietic murine stem cells. Another possibi- adult and therapy-related acute lymphoblastic or myeloid 1,2 lity could be that these negatively tested MLL fusion proteins leukemias (ALL and AML). The molecular pathology of require a specific ’permissive cell type’ to provide their different MLL translocations has been assigned to the presence 3 malignant potential or their inability to be ’genetically instruc- of the der(11)-allele, which encodes the N-terminal portion of tive’. MLL fused to one of the 55 different translocation partner Á 4,5 An exception is the recently characterized MLL AF4 fusion genes. Molecular analyses revealed a direct link between the protein. By using the inverter mouse model in conjunction with presence of der(11)-encoded MLL fusion proteins and the Á 6–8 cell-type specifically expressed Cre-recombinase, the MLL AF4 deregulation of certain genes of the HOXA cluster. In fusion protein was able to instruct genetically lymphocytic particular, the deregulated transcription of HOXA7 and HOXA9, progenitors and early T-cells to develop only into the B- in conjunction with the upregulation of MEIS1, appears to be lineage.32 Although genetically instructive, the MLL Á AF4 fusion sufficient to immortalize myeloid progenitors by turning them protein was not able to cause an acute leukemia rather than a lymphoma disease phenotype. Correspondence: Professor Dr R Marschalek, Institute of Pharmaceu- Here, we want to draw attention to the possibility that t(4;11) tical Biology / ZAFES, University of Frankfurt, Marie-Curie Str. 9, leukemia is mediated not only by the presence of the MLL Á AF4 60439 Frankfurt/Main, Germany. E-mail: [email protected] fusion protein, but by the presence of both reciprocal Received 6 March 2007; accepted 7 March 2007; published online MLL fusion proteins. It has already been demonstrated that 5 April 2007 the MLL Á AF4 fusion gene alone is not sufficient to form t(4;11) genetics E Kowarz et al 1233 immortalized colonies in semi-solid agar (So et al., Blood 2004; cloning the MLL Á AF4 fusion allele, all subsequent fusion alleles 104: 467 abstract). Both the Mll Á Af4 knock-in and the Mll Á Af4 were cloned step by step by using reciprocal LDI-PCR or LR-PCR inverter mouse developed a B-cell lymphoma with low approaches to identify and clone the proposed AF4 Á X and penetrance (50% developed the B-cell lymphoma after a X Á MLL fusion alleles (X represents a recombined allele). By median time of 540 days, which is more than two-thirds of the applying this systematic approach, the genetic situation in 10 natural lifespan of a mouse).32,33 By contrast, the reciprocal out of 13 MLL Á AF4 þ /AF4 Á MLLÀ patients (P03-159, P03-175, AF4 Á MLL fusion protein was able to growth transform murine P03-178, P03-187, P03-217, P03-231, P04-268, P04-275, P05- fibroblasts in classical focus formation assays.34 Moreover, co- 403, P05-442) was determined. Oligonucleotide sequences will transfected cells expressing both the MLL Á AF4 and AF4 Á MLL be made available upon request. fusion protein were shown to have a selective advantage over single-transfected cells with respect to cell cycling, cell growth, apoptotic behavior and growth transformation.14 These data RNA preparation, cDNA synthesis and RT-PCR demonstrated that both reciprocal MLL fusion proteins in t(4;11) experiments leukemia are syngergistically contributing to the leukemia RNA was isolated from cryopreserved MNC using the RNeasy disease phenotype. Mini Kit (Qiagen Hilden, Germany) according to the manufac- However, about 20% of investigated t(4;11) patients seem to turer’s recommendations. RNA was reverse-transcribed with encode only the der(11) allele. The reciprocal AF4 Á MLL fusion 200 units of M-MLV reverse transcriptase (Invitrogen, Karlsruhe, allele can neither be identified at the RNA nor at the genomic Germany) and 100 pmol random hexamers at 421C for 1 h. RT- DNA level by using direct long-range polymerase chain reaction PCR experiments were performed using standard conditions (PCR) experiments.35,36 This has led to the conclusion that the using oligonucleotides specific for a large variety of genes tested reciprocal AF4 Á MLL fusion allele could not have any biological in this study. 50- and 30-RACE experiments were performed by importance, because otherwise it would be present in all t(4;11) using the GeneRacer Kit (Invitrogen) according to the recom- patients. mendations of the manufacturer. Oligonucleotide sequences The controversial situation between genotypes and functional will be made available upon request. data prompted us to investigate the genetic situation in t(4;11) leukemia patients in more detail. For this purpose we investigated a subgroup of t(4;11) patients, which were Results identified by reverse transcriptase-(RT)PCR analyses as MLL Á AF4 þ /AF4 Á MLLÀ patients. Assuming that the selection process Identification of reciprocal MLL fusion alleles in MLL Á relies only on the presence of a functional der(11) fusion allele, AF4 þ /AF4 Á MLLÀ leukemia patients reciprocal MLL fusion alleles should not be present in any of Acute leukemia patients carrying t(4;11) translocations were these patients. However, if reciprocal MLL fusion alleles have pre-screened by RT-PCR and split-signal FISH experiments.39–42 any importance or contribute to the leukemogenic process, we Genomic DNA of 76 patients were subjected to the recently should be able to identify such reciprocal alleles. developed long-distance inverse (LDI)-PCR method to clone Ten out of 13 investigated MLL Á AF4 þ /AF4 Á MLLÀ patients MLL fusion alleles.38 In all cases, the genomic fusion site were successfully analyzed. All MLL Á AF4 þ /AF4 Á MLLÀ patients between the MLL gene and the AF4 gene was identified. By encode either in-frame reciprocal MLL fusions, out-of-frame using the reciprocal LDI-PCR approach, only 63 patients reciprocal MLL fusions or solitary 30-MLL gene segments. These displayed an AF4 Á MLL fusion allele (83%) while 13 patients 50 truncated MLL gene segments, however, are still able to remained negative (17%). A direct genomic long range-PCR (LR- express the recently identified MLL* transcript and the MLL* PCR) approach,36 testing for unusual rearrangements including protein.37 Therefore, all analyzed MLL Á AF4 þ /AF4 Á MLLÀ pa- larger deletions, inversions or duplications, remained also tients are per se able to express the C-terminal portion of the negative for reciprocal AF4 Á MLL fusion alleles. Therefore, the MLL protein. reciprocal LDI-PCR experiments were modified by using new combinations of restriction enzymes and oligonucleotides, including AF4- and MLL-specific oligonucleotides. This ap- Materials and methods proach led to the identification of reciprocal alleles in 10 out of 13 MLL Á AF4 þ /AF4 Á MLLÀ patients. By using the novel Patient data sequence information, direct genomic PCR experiments were Biopsy material of 76 t(4;11) patients with de novo ALL disease conducted by using oligonucleotides specific for the reciprocal phenotype was obtained within the framework of the I-BFM, alleles in combination with MLL-orAF4-specific oligonucleo- Interfant-99 and GMALL study group. Residual material from tides. All genomic PCR amplimers were sequenced to identify diagnostic procedures was used. Genomic DNA was isolated the precise genomic fusion sites and to reconstruct the complex from bone marrow and/or peripheral blood samples and sent to recombination mechanisms. Thus, the genetic situation in each the Diagnostic Center of Acute Leukemia (DCAL, Frankfurt). of these 10 patients was determined. All recombination events Informed consent was obtained from all patients, parents or disrupted the AF4 and the MLL gene in their breakpoint cluster legal guardians and control individuals. regions. Therefore, we termed the translocated gene segments as 50-MLL (exons1–9/10/11), 30-MLL (exons10/11/12-37),50-AF4 (exons 1–3) and 30-AF4 gene segment (exons4–20) to explain the Long distance inverse PCR experiments unusual gene fusions described below. All DNA samples were treated and analyzed as described.38 Briefly, 1 mg genomic patient DNA was digested with appro- priate restriction enzymes and religated to form DNA circles Reciprocal gene fusions of the MLL and AF4 gene before LDI-PCR analyses. Restriction polymorphic PCR ampli- Six out of 10 MLL Á AF4 þ /AF4 Á MLLÀ patients exhibited mers were isolated from the gel and subjected to DNA sequence reciprocal gene fusions (see Figure 1). Patient P03-159 displayed analysis to obtain the patient-specific fusion sequences. After a 3-way-translocation involving chromosomes 1, 4 and 11,

Leukemia t(4;11) genetics E Kowarz et al 1234

Figure 1 Schematic overview of rearranged alleles in MLL Á AF4 þ /AF4 Á MLLÀ patients. The genomic situation of 10 MLL Á AF4 þ /AF4 Á MLLÀ patients is shown schematically. For each patient, the patient identiﬁer is shown in the lower left corner. In the lower middle, sizes of insertion or deletions are indicated in Mbp. In the lower right corner, one of the four possible recombination mechanism is indicated (3-chrom-TL: chromosomal translocation involving three different chromosomes; ins(4)in11: insertion of chromosome 4 material into chromosome 11; ins(11)in4: insertion of chromosome 11 material into chromosome 4; t(D4;11): a t(4;11) translocation in combination with a deletion on chromosome 4). Derivative chromosomes are shown for each panel on the left. Gene names are given for each allele (AF4D or DMLL: 50-AF4 or 30- MLL gene segments). ‘Head-to-head’ or ‘tail-to-tail’ fusions are indicated by writing the gene names above and below the fusion genes. If two fusion sites are present on a derivative chromosome, the alleles are connected by the half-circles.

respectively. The resulting derivative chromosomes der(1), single exons) may lead to in-frame fusions, however, no mRNA der(4) and der(11) were coding for a PBX1 Á MLL fusion gene, of this patient was available to test this possibility. an AF4 Á PBX1 fusion gene and an MLL Á AF4 fusion gene Patient P03-178 represents a reciprocal translocation in (intron11Hintron3), respectively. The recombination sites within combination with a deletion of chromosome 4 sequences the PBX1 Á MLL (intron1Hinton12) and AF4 Á PBX1 fusion genes (0.1 Mbp). The der(11) chromosome displayed the MLL Á AF4 (intron3Hintron1) disrupted per se the open reading frames. We fusion (intron9Hintron3). The genomic fusion site on the der(4) cannot exclude that alternative splice processes (skipping of chromosome was identiﬁed 20.724 bp downstream of AF4

Leukemia t(4;11) genetics E Kowarz et al 1235

Figure 2 RT-PCR analyses. RT-PCR analysis of patients P03-217, P05-442 and P03-175, respectively. M, DNA size marker (1.5 kb ladder for panel 1 and 2; 1 kb ladder for panel 3); NC, negative water controls. Left panel: transcripts of all three fusion genes (MLL . AF4, AF4 . NFkB1 and NFkB1 . MLL) of patient P03-217. Middle panel: transcripts of all three fusion genes (RABGAP1L . MLL, AF4 . RABGAP1L and MLL . AF4) of patient P05-442. Right panel: for patient P03-175, a ‘spliced fusion’ mRNA transcript between a 50-AF4 gene segment and exon 3 of the adjacent UNQ6975 gene, and the MLL . AF4 fusion transcript was observed. exon1a2. 50-RACE experiments revealed that the AF4 exons1a1 gene segment (intron16, intron12). However, 50-RACE PCR and 1a2 are spliced to MLL exon10. However, the resulting experiments revealed a 36 nucleotide long nucleotide sequence fusion mRNA is out-of-frame. (50-GAAGACGGTTACTGACCCCGGCAATCGTTTTTTTTTT-30) Patient P03-217 carries an insertion of chromosome 4 fused to exon14 of the 30-MLL gene. material (15.5 Mbp) into the MLL gene. This led to two fusion Finally, patient P05-442 displayed a 3-way-translocation sites on the resulting der(11) chromosome. At the centromeric involving chromosomes 1, 4 and 11, respectively. The resulting fusion site the MLL Á AF4 fusion gene (intron10Hintron3) was derivative chromosomes der(1), der(4) and der(11) were coding identified, while the telomeric fusion site encodes an for a RABGAP1L Á MLL fusion gene (intron4Hintron11), an NFkB1 Á MLL fusion gene (intron23Hintron10). On the der(4) AF4 Á RABGAP1L fusion gene (intron3Hintron4) and the chromosome, an AF4 Á NFkB1 fusion gene was identified MLL Á AF4 fusion gene (intron11Hintron3), respectively. All (intron3Hintron23). All three fusion genes represent in-frame three fusion genes represent in-frame fusions and are transcribed fusions and are expressed in the patient (Figure 2a). in the patient (Figure 2b). Patient P03-231 carries an insertion of chromosome 11 material (0.6 Mbp) into the AF4 gene. This led to two fusion Truncated MLL and AF4 genes on derivative alleles sites on the resulting der(4) chromosome. At the centromeric In three out of 10 MLL Á AF4 þ /AF4 Á MLLÀ patients only 30-MLL fusion site, the AF4 gene was fused ‘tail-to-tail’ to the FXYD6 or 50-AF4 gene segments were identified (Figure 1) without any gene (intron3Hintron1), while the telomeric fusion site encodes genomic fusion to another gene. the MLL Á AF4 fusion gene (intron10Hintron3). The transcrip- Patient P03-175 displayed a three-way-translocation invol- tional orientation of the AF4 and the FXYD6 gene is in opposite ving chromosomes 2, 4 and 11, respectively. The resulting direction, and thus, neither an AF4 nor an FXYD6 fusion product der(2) chromosome exhibits only a translocated 50-MLL gene could be produced. The der(11) chromosome exhibits a segment, while the der(4) chromosome exhibits an 50-AF4 gene 30-FXYD6 gene segment fused ‘head-to-head’ with the remain- segment adjacent to the UNQ6975 gene. Interestingly, an in- ing 30-MLL gene segment (intron1Hintron10). frame fusion between AF4 and UNQ6975 (exon3Hexon3) was Patient P04-268 carries an insertion of chromosome 4 identified at the mRNA level (Figure 2c). The der(11) chromo- material (52 Mbp) into the MLL gene. This led to two fusion some encodes the MLL Á AF4 fusion gene (intron9Hintron3). sites on the resulting der(11) chromosome. At the centromeric Patient P03-187 carries an insertion of chromosome 11 fusion site, the MLL Á AF4 fusion gene was identified (intro- material (14.1 Mbp) into the AF4 gene. This led to two genomic n11Hintron3), while the telomeric recombination site encodes fusion sites on the resulting der(4) chromosome. At the the 30-ELF2 gene segment fused ‘head-to-head’ to the 30-MLL centromeric fusion site only a 50-AF4 gene segment was gene segment (intron3Hintron11). On the der(4) chromosome, a identified, while at the telomeric fusion site the MLL Á AF4 ‘tail-to-tail’ fusion between a 30-truncated AF4 gene segment fusion gene was identified (intron9Hintron3). On the der(11) and the 30-truncated ELF2 gene segment was observed chromosome, only solitary 30-MLL gene segment was identified. (intron3Hintron3). Again, the transcriptional orientation of the Finally, patient P04-275 represents a reciprocal translocation in truncated ELF2 gene is in opposite orientation with regard to the combination with a large deletion of chromosome 4 sequences AF4 and MLL gene, and thus, neither a functional ELF2 Á MLL nor (27.2 Mbp). The der(11) chromosome displayed the MLL Á AF4 a functional AF4 Á ELF2 fusion product could be produced. fusion (intron11Hintron3). The genomic fusion site on the der(4) Patient P05-403 carries an insertion of chromosome 11 chromosome involved an area located in the subcentromic area material (1 Mbp) into the AF4 gene. This led to two fusion sites of chromosome 4q and the 30-MLL gene segment. on the resulting der(4) chromosome. At the centromeric fusion site, the 50-AF4 gene segment was fused ‘tail-to-tail’ with the 50-DSCAML1 gene segment (intron3Hintron16), while the Discussion telomeric fusion site encodes the MLL Á AF4 fusion gene (intron12Hintron3). On the der(11) chromosome, the 30- A panel of 76 t(4;11) leukemia patients (proB ALL) was analyzed DSCAML1 gene segment was fused ‘head-to-head’ with the 30-MLL for genomic MLL fusion alleles. For 63 t(4;11) patients

Leukemia t(4;11) genetics E Kowarz et al 1236 both reciprocal MLL fusion alleles were identified and sequenced (83%), while 13 patients did not display the reciprocal AF4 Á MLL fusion allele (17%). These patients were further analyzed by using LDI-PCR and LR-PCR strategies in combination with oligonucleotides specific for the 50-portion of the AF4 gene, the 30-portion of the MLL gene, or subsequently for the identified AF4-orMLL-fused sequences. By using this ‘breakpoint hopping technique’ the genetic rearrangements in 10 out of 13 patients were determined. They all displayed complex rearrangements between chromosome 4, 11 and in some cases involving a third chromosome (Figure 1). For the remaining three patients, the genetic situation could not be determined. We cannot exclude that these three patients also exhibit complex rearrangements, but they remained uncovered (likely owing to technical limitations of the applied LDI-PCR method). The genetic rearrangements in these 10 t(4;11) patients revealed four different recombination mechanisms: (1) three genes of three different chromosomes were involved in three- way-translocations (Figure 1: 3-way-TL); (2) a segment of chromosome 4 – disrupting at least the AF4 gene – was inserted into the breakpoint cluster region of the MLL gene (Figure 1: ins(4)in11); (3) vice versa, a segment of chromosome 11 – disrupting at least the MLL gene – was inserted into the breakpoint cluster region of the AF4 gene (Figure 1: ins(11)in4); (4) the chromosomal translocation t(4;11) was not balanced owing to an additional deletion on chromosome 4 (Figure 1: t(D4;11). Both mechanisms (2) and (3) represent a ‘cut and paste mechanism’ of chromosomal fragments into other chromosomes Figure 3 Principles of complex recombination events in t(4,11) (see Figure 3). patients. ‘Cut and paste’ mechanism observed in t(4;11) translocations. In three patients, a 3-way translocation created three For simplicity, only ‘cut and paste’ insertions into chromosome 4 are independent fusion alleles. This explains why these patients shown. (a) A chromosomal fragment coding for the 50-MLL gene were diagnosed as MLL Á AF4 þ /AF4 Á MLLÀ patients. Three-way- segment becomes inserted into the AF4 gene; an in-frame fusion translocations of the MLL gene were already described in two between MLL and AF4 is being created on the resulting der(4) chromosome. On the der(11) chromosome remains a 50- truncated other studies. One patient with a complex rearrangement of MLL gene segment. (b) A chromosomal fragment coding for a 30-TPG chromosome 4, 7 and 11 carried a reciprocal CDK6 Á MLL fusion (translocation partner gene) and the 50-MLL gene segment becomes allele.43 In a second study, conventional cytogenetics revealed a inserted into the AF4 gene; in these cases, three fusion alleles between three-way translocation involving the MLL, the AF4 and a third the TPG, MLL and AF4 are being created on the resulting der(4) and locus on chromosome 13q34.44 der (11) chromosomes, respectively. Seven out of 10 patients bear only two affected chromosomes. The reciprocal alleles represented genomic fusions between MLL and PBX1, AF4, NFkB1, FXYD6, ELF2, DSCAML1 and (Figure 2c). This represented a ‘spliced fusion’, according to RABGAP1L. None of these genes has ever been described the recently discovered mechanism in t(11;19) patients.45 before as translocation partner gene of MLL. However, only the Spliced fusions can be observed in cases where a 30-truncated NFkB1 Á MLL and the RABGAP1L Á MLL fusion alleles repre- gene is located adjacent to an intact gene with the same sented in-frame gene fusions that were transcribed and able to transcriptional orientation. By transcriptional read-through and be translated into chimeric fusion proteins (Figure 2a and b). All subsequent splicing reactions, exons of both genes can be fused other genomic fusions represented either out-of-frame, ‘head-to- together. For all other leukemia patients no RNA material was head’ or ‘tail-to-tail’ gene fusions owing to the ‘cut and paste available. Therefore, we were not able to test systematically for mechanism’. However, no obvious MLL fusion protein could be other mRNA fusions in the remaining leukemia patients (most of encoded by these rearranged alleles. the patient biopsy material was derived from retrospective In an attempt to identify chimeric transcripts for the latter studies). alleles, 50- and 30-RACE experiments were performed. For the Six patients exhibited either a solitary 30-MLL gene segment ‘head-to-head’ DSCAML1 Á MLL fusion allele of patient P05-403, (P03-175, P03-187, P04-275) or a ‘head-to head’ fusion with a stretch of 36 nucleotides without any potential AUG other genes (P03-231, P04-268, P05-403). It has been recently translational start codon was fused at the RNA level to the 30- demonstrated that 30-MLL alleles are still able to produce a MLL gene segment. However, BLAST searches revealed only shorter MLL transcript, termed MLL*. The MLL* transcript starts similarities to short stretches of DNA derived from different in front of MLL exon 12 and gives rise to an mRNA transcript chromosomes. The short nucleotide stretch was not derived that comprises MLL exon 12 until exon 37,37 which is translated from the DSCAML1 gene, neither form exonic nor from intronic into the MLL* protein (coding for about 2.300 amino-acids of sequences. In patient P03-178 the first two AF4 exons, exons1a1 the MLL C-terminus). Thus, leukemia patients carrying either a and exon1a2, were fused out-of-frame to exon 10 of the MLL solitary 30-MLL gene segment or a ‘head-to head’ fusion with the gene. In patient P03-175, we are able to identify an in-frame 30-MLL gene segment, are per se able to transcribe the MLL* fusion between the AF4 gene (exons 1-3) and exon 3 of the mRNA.37 The expression of the C-terminal portion of MLL has to downstream located UNQ6975 gene on the RNA level be verified in future studies, when leukemia patients with such

Leukemia t(4;11) genetics E Kowarz et al 1237 complex genetic alterations can be analyzed at the RNA and at analysis of genomic breakpoints to identify patients with the genomic DNA level in parallel. complex MLL rearrangements. Hints for potential disease mechanisms in this particular Currently, there are no biological data available that would group of t(4;11) leukemia patients may derive from our allow to draw any conclusion on the biology of these patients. knowledge of the genes that were identified to be involved in Therefore, we would like to invite potential collaborators to the genetic rearrangements. PBX1 has first been identified in analyze this group of MLL Á AF4 þ /AF4 Á MLLÀ leukemia patients t(1;19) translocations, where E2A is fused in-frame to PBX1.46,47 in more detail. Besides the genotypic analysis at the RNA and Either dimeric or trimeric protein complexes of PBX1, MEIS1 genomic DNA level, we suggest to use gene expression profiling and HOXA9 are binding to promoters of leukemia-associated experiments in combination with patient treatment data (treat- genes, for example the FLT3 gene.48 The balance between these ment response, outcome, etc.) to subclassify this t(4;11) patient factors is critical for normal hematopoietic development. The subgroup. This may help to understand differences in leukemo- haploinsufficient situation in case of the non-functional PBX1 genic processes and will allow us to identify interesting targets fusion alleles will presumably cause a shift towards HOXA9/ for the development of novel therapeutic strategies. MEIS1 complexes, which in turn will lead to a developmental arrest of myeloid progenitors. A similar situation can be predicted for the ‘head-to-head’ Acknowledgements fusion between 30-ELF2 and 30-MLL. Wild-type ELF2 protein is directly binding to the runt homology domain of AML1. Binding This study was supported by grants MA 1876/7-1 from the DFG, of ELF2 to AML1 strongly transactivates the AML1 protein N1KR-S12T13 from the BMBF, 2001.061.2 from the Wilhelm- complex.49 Thus, the disruption of one ELF2 allele is presumably Sander-Foundation, and 102362 from the German Cancer compromising AML1 functions due to gene dosage effects, and Foundation to R.M. TB was supported by a grant from the Berliner thus, mimics an AML1 translocation because of the ELF2 Krebsgesellschaft. TB thanks Professor E. Thiel and Professor D. haploinsufficiency (AML1 translocations, for example Hoelzer for their support and Ms. Mara Molkentin for excellent AML1 Á ETO, are converting the AML1 transcriptional activator technical assistance. into a constitutive transcriptional repressor).50 NFkB1 is known to be constitutively overexpressed in various subtypes of ALL51 and AML.52 One of the two NFkB1 References fusion proteins, the NFkB1 Á MLL fusion protein, will potentially interfere with normal cellular functions. This fusion protein 1 Felix CA, Hosler MR, Winick NJ, Masterson M, Wilson AE, Lange encodes exons 1–23 of the NFkB1 gene fused to the C-terminal BJ. ALL-1 gene rearrangements in DNA topoisomerase II inhibitor- exons of the MLL gene. Thus, the DNA-binding domain of related leukemia in children. Blood 1995; 85: 3250–3256. the rel-family transcription factor NFkB1 is fused to the 2 Mitterbauer-Hohendanner G, Mannhalter C. The biological and clinical significance of MLL abnormalities in haematological enzymatic domains of MLL. This may result in ectopic gene malignancies. Eur J Clin Invest 2004; 34 (Suppl 2): 12–24. expression of NFkB1 target genes, The reciprocal AF4 Á NFkB1 3 Rowley JD. The der(11) chromosome contains the critical break- fusion protein is coding only for the first three exons of AF4 and point junction in the 4;11, 9;11, and 11;19 translocations in acute the last exon 24 of the NFkB1 gene. This fusion is highly similar leukemia. Genes Chrom Cancer 1992; 5: 264–266. to the FelC protein. Thus, the AF4 Á NFkB1 fusion protein 4 Meyer C, Schneider B, Jakob S, Strehl S, Attarbaschi A, Schnittger S may have a similar function as the FelC protein, which is able et al. The MLL recombinome of acute leukemias. Leukemia 2006a; 20: 777–784. to bind to SIAH1 and SIAH2, and so, lead to the retention of 5 Meyer C, Kowarz E, Oehm C, Klingebiel T, Dingermann T, the two E3-ligases in the cytoplasm. This protects the AF4 full- Marschalek R. Genomic DNA of leukemia patients: target for length protein that is localized in the nucleus against clinical diagnosis of MLL rearrangements. Biotechnol J 2006b; 1: proteasomal degradation.34 656–663. The RABGAP1L protein is a GTPase activating protein that is 6 Ayton PM, Cleary ML. Molecular mechanisms of leukemogenesis specifically expressed in hematopoietic cells. Both DCSAML1 mediated by MLL fusion proteins. Oncogene 2001; 20: 5695– 5707. and FXYD6 are also expressed in hematopoietic cells, however, 7 Hess JL. Mechanisms of transformation by MLL. Crit Rev Eukaryot their biological function is yet unknown. Gene Expr 2004; 14: 235–254. In conclusion, the analysis of reciprocal alleles in 8 Horton SJ, Grier DG, McGonigle GJ, Thompson A, Morrow M, De MLL Á AF4 þ /AF4 Á MLLÀ patients revealed several novelties: Silva I et al. Continuous MLL-ENL expression is necessary to (1) complex genetic rearrangements are able to masque the establish a ‘Hox Code’ and maintain immortalization of hemato- presence of reciprocal MLL alleles; (2) completely novel genes – poietic progenitor cells. Cancer Res 2005; 65: 9245–9252. 9 Rozovskaia T, Feinstein E, Mor O, Foa R, Blechman J, Nakamura T not yet identified as MLL translocation partner genes – were et al. Upregulation of Meis1 and HoxA9 in acute lymphocytic 0 identified as reciprocal fusion partners for the 3 -portion of the leukemias with the t(4;11) abnormality. Oncogene 2001; 20: MLL gene (e.g. NFkB1 and RABGAP1L); (3) several genes 874–878. expressed in hematopoietic cells were disrupted owing to the 10 Ayton PM, Cleary ML. Transformation of myeloid progenitors by ‘cut and paste’ recombination mechanism (e.g. PBX1, FXYD6, MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev ELF2, DSCAML1); (4) solitary 30-MLL gene segments – able to 2003; 17: 2298–2307. 11 Zeisig BB, Milne T, Garcia-Cuellar MP, Schreiner S, Martin ME, express the C-terminal portion of the MLL protein – may still Fuchs U et al. Hoxa9 and Meis1 are key targets for MLL-ENL- remain on one of the resulting derivative chromosomes. The mediated cellular immortalization. Mol Cell Biol 2004; 24: results of this study extends our current knowledge on molecular 617–628. mechanism involving the human MLL gene in acute leukemia 12 Hess JL, Bittner CB, Zeisig DT, Bach C, Fuchs U, Borkhardt A et al. patients, especially for t(4;11) leukemia. In most cases, a ‘cut c-Myb is an essential downstream target for homeobox-mediated and paste’ recombination mechanism makes it nearly impos- transformation of hematopoietic cells. Blood 2006; 108: 297–304. 13 Xia ZB, Popovic R, Chen J, Theisler C, Stuart T, Santillan DA et al. sible to identify the reciprocal allele by standard diagnostic The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase procedures. Therefore, any future analysis should involve inhibitor CDKN1B (p27kip1) expression. Proc Natl Acad Sci USA cytogenetic and RT-PCR experiments in combination with the 2005; 102: 14028–14033.

Leukemia t(4;11) genetics E Kowarz et al 1238 14 Gaussmann A, Wenger T, Eberle I, Bursen A, Bracharz S, Herr I is consistently detected in t(4;11)(q21;q23)-containing acute et al. The combined effects of the two reciprocal t(4;11) fusion lymphoblastic leukemia. Blood 1994; 83: 330–335. proteins, MLL Á AF4 and AF4 Á MLL, confer resistance to apoptosis, 36 Reichel M, Gillert E, Angermuller S, Hensel JP, Heidel F, Lode M cell cycling capacity and growth transformation. Oncogene 2006, et al. Biased distribution of chromosomal breakpoints involving the [E pub ahead of print]. MLL gene in infants versus children and adults with t(4;11) ALL. 15 Milne TA, Hughes CM, Lloyd R, Yang Z, Rozenblatt-Rosen O, Dou Oncogene 2001; 20: 2900–2907. Y et al. Menin and MLL cooperatively regulate expression of 37 Scharf S, Zech J, Bursen A, Schraets D, Oliver PL, Kliem S et al. cyclin-dependent kinase inhibitors. Proc Natl Acad Sci USA Transcription linked to recombination: a gene-internal promoter 2005a; 102: 749–754. coincides with the recombination hot spot II of the human MLL 16 Milne TA, Dou Y, Martin ME, Brock HW, Roeder RG, Hess JL. MLL gene. Oncogene 2007; 26: 1361–1371. associates specifically with a subset of transcriptionally active 38 Meyer C, Schneider B, Reichel M, Angermueller S, Strehl S, target genes. Proc Natl Acad Sci USA 2005b; 102: 14765–14770. Schnittger S et al. Diagnostic tool for the identification of MLL 17 Milne TA, Martin ME, Brock HW, Slany RK, Hess JL. Leukemo- rearrangements including unknown partner genes. Proc Natl Acad genic MLL fusion proteins bind across a broad region of the Hox a9 Sci USA 2005; 102: 449–454. locus, promoting transcription and multiple histone modifications. 39 van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Cancer Res 2005c; 65: 11367–11374. Saglio G et al. Standardized RT-PCR analysis of fusion gene 18 Corral J, Lavenir I, Impey H, Warren AJ, Forster A, Larson TA et al. transcripts from chromosome aberrations in acute leukemia for An Mll-AF9 fusion gene made by homologous recombination detection of minimal residual disease. Report of the BIOMED-1 causes acute leukemia in chimeric mice: a method to create fusion Concerted Action: investigation of minimal residual disease in oncogenes. Cell 1996; 85: 853–861. acute leukemia. Leukemia 1999; 13: 1901–1928. 19 Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and 40 van der Burg M, Beverloo HB, Langerak AW, Wijsman J, van leukemic transformation of a myelomonocytic precursor by Drunen E, Slater R et al. Rapid and sensitive detection of all types retrovirally transduced HRX-ENL. EMBO J 1997; 16: 4226–4237. of MLL gene translocations with a single FISH probe set. Leukemia 20 Slany RK, Lavau C, Cleary ML. The oncogenic capacity of HRX- 1999; 13: 2107–2113. ENL requires the transcriptional transactivation activity of ENL and 41 Andersson A, Ho¨glund M, Johansson B, Lassen C, Billstro¨mR, the DNA binding motifs of HRX. Mol Cell Biol 1998; 18: 122–129. Garwicz S et al. Paired multiplex reverse-transcriptase polymerase 21 Dobson CL, Warren AJ, Pannell R, Forster A, Lavenir I, Corral J chain reaction (PMRT-PCR) analysis as a rapid and accurate et al. The Mll-AF9 gene fusion in mice controls myeloproliferation diagnostic tool for the detection of MLL fusion genes in and specifies acute myeloid leukaemogenesis. EMBO J 1999; 18: hematologic malignancies. Leukemia 2001; 15: 1293–1300. 3564–3574. 42 van der Burg M, Poulsen TS, Hunger SP, Beverloo HB, Smit EM, 22 DiMartino JF, Miller T, Ayton PM, Landewe T, Hess JL, Cleary ML Vang-Nielsen K et al. Split-signal FISH for detection of chromo- et al. A carboxy-terminal domain of ELL is required and sufficient some aberrations in acute lymphoblastic leukemia. Leukemia for immortalization of myeloid progenitors by MLL-ELL. Blood 2004; 18: 895–908. 2000; 96: 3887–3893. 43 Raffini LJ, Slater DJ, Rappaport EF, Nigro LL, Cheung NK, Biegel JA 23 Lavau C, Du C, Thirman M, Zeleznik-Le N. Chromatin-related et al. Panhandle and reverse-panhandle PCR enable cloning of properties of CBP fused to MLL generate a myelodysplastic-like der(11) and der(other) genomic breakpoint junctions of MLL syndrome that evolves into myeloid leukemia. EMBO J 2000a; 19: translocations and identify complex translocation of MLL, AF-4, 4655–4664. and CDK6. Proc Natl Acad Sci USA 2002; 99: 4568–4573. 24 Lavau C, Luo RT, Du C, Thirman MJ. Retrovirus-mediated gene 44 Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, transfer of MLL-ELL transforms primary myeloid progenitors and Mugneret F et al. Ten novel 11q23 chromosomal partner causes acute myeloid leukemias in mice. Proc Natl Acad Sci USA sites. European 11q23 Workshop participants. Leukemia 1998; 2000b; 97: 10984–10989. 12: 811–822. 25 DiMartino JF, Ayton PM, Chen EH, Naftzger CC, Young BD, Cleary 45 Meyer C, Burmeister T, Strehl S, Schneider B, Hubert D, Zach O ML. The AF10 leucine zipper is required for leukemic transformation et al. Spliced MLL fusions: a novel mechanism to generate of myeloid progenitors by MLL-AF10. Blood 2002; 99: 3780–3785. functional chimaeric MLL Á MLLT1 transcripts in t(11;19) 26 Eguchi M, Eguchi-Ishimae M, Greaves M. The small oligomeriza- (q23;p13.3) leukaemia. Leukemia 2007; 21: 588–590. tion domain of gephyrin converts MLL to an oncogene. Blood 46 Hunger SP, Galili N, Carroll AJ, Crist WM, Link MP, Cleary ML. 2004; 103: 3876–3882. The t(1;19)(q23;p13) results in consistent fusion of E2A and PBX1 27 Liu H, Chen B, Xiong H, Huang QH, Zhang QH, Wang ZG et al. coding sequences in acute lymphoblastic leukemias. Blood 1991; Functional contribution of EEN to leukemogenic transformation by 77: 687–693. MLL-EEN fusion protein. Oncogene 2004; 23: 3385–3394. 47 Kamps MP, Look AT, Baltimore D. The human t(1;19) translocation 28 So CW, Karsunky H, Wong P, Weissman IL, Cleary ML. Leukemic in pre-B ALL produces multiple nuclear E2A-Pbx1 fusion transformation of hematopoietic progenitors by MLL-GAS7 in the proteins with differing transforming potentials. Genes Dev 1991; absence of Hoxa7 or Hoxa9. Blood 2004a; 103: 3192–3199. 5: 358–368. 29 So CW, Cleary ML. Dimerization: a versatile switch for oncogen- 48 Wang GG, Pasillas MP, Kamps MP. Persistent transactivation by esis. Blood 2004; 104: 919–922. meis1 replaces hox function in myeloid leukemogenesis models: 30 Wang J, Iwasaki H, Krivtsov A, Febbo PG, Thorner AR, Ernst P evidence for co-occupancy of meis1-pbx and hox-pbx complexes et al. Conditional MLL-CBP targets GMP and models therapy- on promoters of leukemia-associated genes. Mol Cell Biol 2006; related myeloproliferative disease. EMBO J 2005; 24: 368–381. 26: 3902–3916. 31 Strehl S, Borkhardt A, Slany R, Fuchs UE, Konig M, Haas OA. The 49 Cho JY, Akbarali Y, Zerbini LF, Gu X, Boltax J, Wang Y et al. human LASP1 gene is fused to MLL in an acute myeloid leukemia Isoforms of the Ets transcription factor NERF/ELF-2 physically with t(11;17)(q23;q21). Oncogene 2003; 22: 157–160. interact with AML1 and mediate opposing effects on AML1- 32 Metzler M, Forster A, Pannell R, Arends MJ, Daser A, Lobato MN mediated transcription of the B cell-specific blk gene. J Biol Chem et al. A conditional model of MLL-AF4 B-cell tumourigenesis using 2004; 279: 19512–19522. invertor technology. Oncogene 2006; 25: 3093–3103. 50 Wildonger J, Mann RS. The t(8;21) translocation converts AML1 33 Chen W, Li Q, Hudson WA, Kumar A, Kirchhof N, Kersey JH. A into a constitutive transcriptional repressor. Development 2005; murine Mll-AF4 knock-in model results in lymphoid and myeloid 132: 2263–2272. deregulation and hematologic malignancy. Blood 2006; 108: 669– 51 Kordes U, Krappmann D, Heissmeyer V, Ludwig WD, Scheidereit 677. C. Transcription factor NF-kappaB is constitutively activated in 34 Bursen A, Moritz S, Gaussmann A, Moritz S, Dingermann T, acute lymphoblastic leukemia cells. Leukemia 2000; 14: Marschalek R. Interaction of AF4 wild-type and AF4.MLL fusion 399–402. protein with SIAH proteins: indication for t(4;11) pathobiology? 52 Guzman ML, Neering SJ, Upchurch D, Grimes B, Howard DS, Oncogene 2004; 23: 6237–6249. Rizzieri DA et al. Nuclear factor-kappaB is constitutively activated 35 Downing JR, Head DR, Raimondi SC, Carroll AJ, Curcio-Brint AM, in primitive human acute myelogenous leukemia cells. Blood Motroni TA et al. The der(11)-encoded MLL/AF-4 fusion transcript 2001; 98: 2301–2307.

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