Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.
Chromosomal rearrangements involving the human MLL gene are recurrently associated with the disease phenotype of acute leukemias.1, 2 For instance, the presence of MLL rearrangements is an independent dismal prognostic factor in infant anti-lymphoblastic factor (ALL), whereas childhood ALL patients with MLL · AF4 fusions are usually treated according to high-risk protocols. Thus, the identification of MLL gene fusions is necessary for rapid clinical decisions resulting in specific therapy regimens. Current procedures to identify MLL rearrangements include cytogenetic analysis, fluorescence in situ hybridization (FISH) experiments (e.g. MLL split-signal FISH), specific reverse transcriptase-PCR (RT-PCR) and genomic PCR methods. This repertoire of technologies was recently extended by a long-distance inverse PCR (LDI-PCR) method that uses small amounts of genomic DNA to determine any type of MLL gene rearrangement at the molecular level.3 This includes chromosomal translocations, gene internal duplications, chromosome 11q deletions or inversions, and MLL gene insertions into other chromosomes, or vice versa, the insertion of chromatin material into the MLL gene.
To gain insight into the frequency of MLL rearrangements, unscreened and prescreened pediatric and adult leukemia patients were analyzed. Prescreening tests (cytogenetic analysis, FISH, Southern blot, RT-PCR or NG2-positivity) were performed at different European centers, where acute leukemia patients are enrolled in different study groups (Interfant-99, AMLCG, GMALL). With the exception of a few patients, all prescreened MLL rearrangements were successfully analyzed and patient-specific MLL fusion sequences were obtained. Data obtained from the literature and results obtained in this study are summarized in a color map where all 51 translocation partner genes (TPGs) and their specific breakpoint regions have been assigned. The applied color code will enable all investigators to identify compatible intron–intron fusions between the MLL gene and all yet characterized TPGs. Moreover, we provide a complete list of 87 MLL rearrangements of which 36 still await molecular characterization.
Materials and methods
Genomic DNA was isolated from bone marrrow and/or peripheral blood samples of all patients and sent to our center. Patient samples were obtained from the Interfant-99 study group (Rotterdam, The Netherlands), the AMLCG study group (Munich, Germany) and the GMALL study group (Berlin, Germany). Informed consent was obtained from all patients or patients' parents/legal guardians and control individuals.
Long-distance inverse PCR experiments
All DNA samples were treated and analyzed as described.3 Briefly, 1 μg genomic patient DNA was digested with restriction enzymes and religated to form DNA circles before LDI-PCR analyses. Restriction polymorphic PCR amplimers were isolated from the gel and subjected to DNA sequence analyses to obtain the patient-specific fusion sequences.
Identification of MLL rearrangements
Acute leukemia patients carrying MLL rearrangements are not exceeding a total of 800 cases per year in Europe (about 300 pediatric and about 500 adult leukemia patients). To analyze the recombinome of the human MLL gene, 414 acute leukemia samples from different European centers were analyzed over a period of 24 months (272 pediatric and 142 adult patients). One Hundred and seventy-six out of 414 leukemia samples were not prescreened for MLL aberrations or were classified as ‘MLL-negative’ cases. A total of seven MLL rearrangements are identified in the group of not prescreened pediatric leukemia patients, which corresponds to the expected frequency of 5–10%. By contrast, 238 patients with acute leukemia were prescreened for MLL rearrangements (see Table 1a). These ‘MLL-positive’ leukemias correspond to 107 pediatric and 131 adult acute leukemia patients. From these patients, 206 were successfully analyzed, whereas for 32 acute leukemia patients, no MLL rearrangement was detected (false-positive prescreening: 3/32; prescreened only by NG2-positivity: 9/32; poor DNA quality: 3/32; failure to produce a positive result: 17/32). The latter failures (17/32) might be explained either by false-positive prescreenings or by the intrinsic limitation of the applied method. which allows the identification of MLL rearrangements only when they occurred within the breakpoint cluster region of the MLL gene. However, chromosomal breakpoints have already been mapped outside the breakpoint region.4
The most frequent rearrangements were t(4;11)(q21;q23) involving the MLLT2 (AF4) gene, t(9;11)(p22;q23) involving the MLLT3 (AF9) gene, t(11;19)(q23;p13.3) involving the MLLT1 (ENL) gene, t(10;11)(p12;q23) involving the MLLT10 (AF10) gene and t(6;11)(q27;q23) involving the MLLT4 (AF6) gene, respectively (see Table 1b). These translocations account for about 85% of all investigated leukemia samples. All identified TPGs in pediatric and adult leukemia patients are summarized in Table 1b.
Novel translocation partner genes
A total of seven new TPGs were discovered: ACACA (acetyl-CoA carboxylase alpha), SELB (elongation factor for selenocysteine), SMAP1 (glycoprotein involved in erythropoietic development), TIRAP (TLR4 adaptor protein), DCPS (mRNA decapping enzyme), BCL9L and ARHGEF17. With the exception of BCL9L and ARHGEF17, all of them have been reported in a technical paper by the authors.3 The BCL9L gene encodes a structural constituent of ribosomes (with similarity to the Drosophila legless gene) and is located on chromosome 11q23.5 The fusion between MLL and BCL9L deletes a chromosomal area of about 300 kb and fused MLL intron 8 tail-to-tail with the 3′-non-translated region (3′-NTR) of BCL9L. Therefore, no functional fusion protein can be produced. The ARHGEF17 gene is located on chromosome 11q13 and encodes a protein of 1510 amino acids (164 kDa) that belongs to the family of Rho guanine nucleotide exchange factors involved in signaling pathways.6 The in-frame fusion between MLL intron 12 and ARHGEF17 intron 1 occurred on both fusion alleles and involved two homologous chromosomes 11.
Different possibilities to cause genetic MLL aberrations
In general, human MLL rearrangements are initiated by DNA cleavage or a DNA damage situation, which induces DNA repair via the non-homologous end joining (NHEJ) DNA repair pathway.7, 8 Although the MLL gene is predominantly involved in reciprocal chromosomal translocations (Figure 1a), other genetic rearrangements were observed in this study or were described in the literature. Gene-internal partial tandem duplications (PTD) of specific MLL gene portions (duplication of introns 2–9, 2–11, 4–9, 4–11 or 3–8; Figure 1b) are frequently observed in AML patients.9, 10, 11 MLL PTD are being discussed to mediate dimerization of the MLL N-terminus, a process that seems to be sufficient to mediate leukemogenic transformation.12 The third possibility is deletions on the long arm of chromosome 11, which affect only portions of the MLL gene, but always lead to functional MLL fusion genes (TPG · MLL or MLL · TPG, depending on whether the deletion involves genes located centromeric or telomeric relative to the MLL gene; Figure 1c). In the latter case, only the derivative(11) chromosome is created (e.g. ARHGEF12). A fourth mechanism is the inversion of a chromosome 11 segment, represented by the MLL · PICALM fusion (Figure 1c). A more complicated picture emerges when chromosome 11 material (including portions of the MLL gene) is inserted into other chromosomes, or vice versa, the insertion of chromosome material (including portions of a TPG) into the breakpoint cluster region of the MLL gene (e.g. MLLT10; Figure 1d). A combination of both events leads to reciprocal insertions. In rare cases, the described insertion mechanism involves the MLL gene and two different TPGs, a mechanism that leads to complex three-partner translocations. The insertion of chromatin fragments is necessary because some TPGs are not transcribed in the telomeric direction, as the MLL gene. Therefore, a ‘simple’ reciprocal translocation would lead to a head-to-head or tail-to-tail fusion of the MLL gene with these TPGs. All recombination events – or any combination thereof – are sufficient to explain all yet known MLL rearrangements. The different genetic mechanisms convert the MLL protein into an oncogenic derivative, necessary for the onset of a preleukemic or leukemic clone, and subsequently, to initiate acute leukemia.
MLL rearrangements and the ‘intron code’
On the basis of results and data obtained from the literature, a total of 51 TPGs are now characterized at the molecular level. These 51 TPGs are summarized in Figure 2, where all MLL fusion genes are shown schematically with their specific exon/intron structure. In addition, all introns were artificially color-coded: colorless introns are named ‘type 0’ introns, whereas red and blue introns are ‘type 1’ and ‘type 2’ introns. A ‘type 0’ intron disrupts a given open reading frame exactly between two triplet codons (triplet–intron–triplet), whereas ‘type 1’ and ‘type 2’ introns disrupt the three nucleotides of a given codon either by 1-intron-2 or 2-intron-1, respectively (see also Marschalek et al.13). Chromosomal rearrangements within the MLL gene occur mostly in ‘type 0’ introns (between MLL exons 9 and 14) and a corresponding ‘type 0’ intron of a recombination partner gene. Figure 2 allows also to identify ‘compatible’ gene fusions, as it predicts MLL fusions at the mRNA level in cases where introns of different colors were recombined (e.g. MLL fusions with RPS3, MLLT6 or MSF). In these rare cases, alternative splice events are fusing compatible exons – flanked by identical intron color – to guarantee functional fusions. To this end, Figure 2 is helpful to design appropriate RT-PCR experiments.
Arbitrary MLL fusions and ‘spliced fusions’
In very rare cases, the MLL gene is fused to TPGs without producing functional products. In one patient who carried a reciprocal translocation between the MLL and the SELB gene, no detectable fusion mRNA species from the two reciprocal fusion products MLL · SELB and SELB · MLL were detected.3 A second unusual observation was identified in a patient with an MLL · TIRAP fusion resulting from an interstitial deletion. The involved TIRAP intron 7 was located within the 3′-untranslated region of the TIRAP gene (TIRAP–11q24; Figure 2). Indeed, no MLL-TIRAP fusion mRNA could be identified. However, a gene located about 5 kb telomeric of TIRAP, the DCPS gene, was used for splicing to generate an in-frame MLL fusion.3 This ‘spliced fusion’ exists only at the RNA level, and thus, establishes another mechanism by which a functional MLL fusion can be generated. A similar situation was found for the MLL · BCL9L fusion mentioned above. As the MLL gene was fused tail-to-tail with the 3′-NTR of BCL9L, no functional fusion mRNA could be encoded by the genomic fusion of both genes. Experimental attempts to identify spliced fusions with telomer located genes of BCL9L (FOXR1, etc.) are still ongoing.
Several conclusions can be drawn from this study. Genomic DNA turned out to be a good source to identify different MLL rearrangements. The applied method allowed the identification of chromosomal translocations, MLL gene-internal duplications, chromosome 11 inversions, chromosomal 11 deletions and the insertion of chromosome 11 material into other chromosomes, or vice versa, the insertion of chromatin material of other chromosomes into the MLL gene. Moreover, it allowed to identify unknown TPGs and/or complex rearrangements that cannot be analyzed by the resolution of cytogenetic or FISH analyses. The successful analysis of more than 200 MLL fusion alleles led to the discovery of new TPGs and a large variety of different mechanisms that fuse the MLL gene to different TPGs or MLL-spliced fusions in order to create oncogenic protein variants. Within the past years, 87 genetic aberrations involving the human MLL gene located on chromosome 11 band q23 have been described (see Table 2 in Supplementary Information). Fifty-one TPGs are now characterized on the molecular level; however, 36 genetic loci were identified only by cytogenetic experiments and still await their molecular characterization.
According to our data, the most frequent TPGs in MLL-mediated acute leukemias are MLLT2 (AF4), MLLT3 (AF9), MLLT1 (ENL), MLLT10 (AF10) and MLLT4 (AF6), which are responsible for about 85% of all investigated rearrangements (see Table 1b, pediatric and adult patients). A second diagnostic center at the University of Erlangen-Nuremberg has investigated only pediatric leukemia patients prescreened for t(4;11), t(9;11) and t(11;19) translocations. They have successfully analyzed 33 t(4;11), 20 t(9;11) and 16 t(11;19) leukemia patients in parallel to our study (see Table 1b, marked by asterisk) by using different genomic PCR approaches.14, 15 Upon taking these data together, the five different chromosomal translocations mentioned above account for about 90% of all MLL-positive leukemias. Routine diagnostic methods based on RT-PCR might be restricted to these few TPGs in order to identify the vast majority of MLL gene rearrangements.
An important result of this study is the establishment of patient-specific DNA sequences that can now be used for monitoring of minimal residual disease (MRD) by quantitative PCR techniques. In general, chromosomal fusion alleles represent the most reliable markers for MRD studies and have several advantages over surrogate markers such as IgH or T-cell receptor rearrangements (as they may represent tumor subpopulations). Owing to the fact that a given MLL fusion allele is genetically stable and a mono-allelic marker for each tumor cell, a more reliable quantification and tracing of residual tumor cells becomes possible. For each of these 213 acute leukemia patients, at least one MLL fusion allele was identified and characterized by sequencing. A first prospective study was already initiated and verified the reliability of these genomic markers for MRD monitoring.16 Therefore, the use of these MRD markers will contribute to stratification, improved treatment and outcome of leukemia patients.
The analysis of the MLL recombinome allows to classify MLL fusion partner genes into functional categories. Interestingly, the most frequent TPGs in MLL translocations encode nuclear proteins (AF4, AF9, ENL and AF10) that were recently identified to belong to the same nuclear protein network (see Figure 3). This may indicate that TPGs are not randomly chosen, rather functionally selected. Briefly, AF4 and AF9 colocalize at distinct nuclear foci,17 and the disruption of the AF4/AF9 protein interaction induces apoptosis in t(4;11) cells.18 By contrast, the AF4 protein seems to be required for growth and differentiation of lymphocytic progenitors.19 ENL was shown to interact directly with the AF4 and AF10 proteins,20 and in addition, AF10 binds to hDOT1L.21 The hDOT1L protein is a non-SET-domain protein that is able to mediate the methylation of lysine 79 of histone H3 proteins. H3K79me seems to be a prerequisite for elongation of RNA polymerase II. Both the MLL and hDOT1L protein guarantee a site-specific histone methylation pattern that play a major role in transcriptional initiation, elongation and, subsequently, maintenance of transcription.22, 23, 24 The limiting factor of this complex seems to be the AF4 protein, as it has a very low abundance in mammalian cells owing to its interaction with the two E3 ubiquitin ligases SIAH1 and SIAH2.25 On taking together, disruption of either component of this protein network might compromise important cellular functions.
Although most MLL translocations are associated with poor outcome in infant and childhood acute leukemia, it can be assumed that a systematic analysis of the MLL recombinome will allow to draw conclusions on certain aspects of hemato-malignant transformation processes. The analyses of different MLL fusion partner genes may help to categorize those for (1) their subcellular localization, (2) their cellular function, (3) specific protein domain structures (e.g. dimerization domains) and, finally, (4) their ability to interact with other proteins. These classifications have to be supplemented by functional studies attempting to demonstrate the oncogenicity of different MLL fusions in the future by retroviral transduction of hematopoietic stem/precursor cells. At the end, this will help to classify the large variety of different MLL translocations into different risk groups, and thus will lead to a better stratification and treatment of leukemia patients.
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This work was made possible by and conducted within the framework of the International BFM Study Group. We thank Reinald Repp, Thorsten Langer, Jörn-D Beck, Markus Metzler und Thomas Leis for providing and sharing unpublished information of their ongoing study (Grant 2002.032.1 from the Wilhelm Sander Foundation) and Michael Karas for critical comments. This study is supported by Grant GEN-AU Child, GZ 200.071/3-VI/2a/2002 to OAH and Grant 2001.061.1 from the Wilhelm-Sander-Foundation to RM, TD and TK.
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