Oncogenes, Fusion Genes and Tumor Suppressor Genes

New insights to the MLL recombinome of acute leukemias

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Abstract

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The 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 MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

Introduction

Chromosomal rearrangements involving the human MLL gene at 11q23 are associated with the development of acute leukemias.1, 2 The presence of certain MLL rearrangements is an independent dismal prognostic factor and patients are usually treated according to high-risk protocols. Therefore, 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,3, 4 fluorescence in situ hybridization (FISH) experiments (for example, MLL split-signal FISH),5, 6, 7 specific reverse transcriptase (RT)–PCR8 or genomic PCR methods.9, 10 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 on the molecular level.11 This includes chromosomal translocations, complex chromosomal rearrangements, gene internal duplications, deletions or inversions on chromosome 11q 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 distinct MLL rearrangements, we analyzed prescreened and unscreened biopsy material of pediatric and adult leukemia patients. Prescreening tests (cytogenetic analysis, FISH, Southern blot, RT–PCR or NG2 positivity) were performed at different European centers and centers located outside Europe, where acute leukemia patients are enrolled in different study groups. Nearly all prescreened MLL rearrangements were successfully analyzed and patient-specific MLL fusion sequences—for minimal residual disease (MRD) monitoring—were obtained. In some centers, no prescreening could be performed. In these cases, a successful identification of MLL rearrangements was in the range of 5–10%.

On the basis of the results obtained in the present (n=346) and previous studies (414 patients were already published in 2005 and 2006),11, 12 64 translocation partner genes (TPGs) and their specific breakpoint regions have now been identified. Additional 35 chromosomal translocations of the human MLL gene were characterized by cytogenetics, however, without any further molecular characterization. In this study, five additional fusion loci were sequenced that do not encode any known gene. Thus, the MLL recombinome presently comprises 104 different fusion sites. In addition, we present a list of 48 ‘reciprocal MLL gene fusions’ that derives from complex rearrangements. These reciprocal MLL gene fusions represent 48 genes fused to the 3′-portion of the MLL gene. They have never been described before as MLL TPG, and thus, represent a novel subclass of reciprocal recombination partners.

Material and methods

Patient material

Biopsy material from acute leukemia patients—diagnosed to bear an MLL rearrangement was used to isolate genomic DNA from bone marrow and/or peripheral blood samples. Genomic DNA was sent to the Diagnostic Center of Acute Leukemia (DCAL) at the Frankfurt University. Patient samples were obtained from study groups (the AMLCG study group, Munich; the GMALL study group, Frankfurt/Main; Polish Pediatric Leukemia and Lymphoma Study Group; Zabrze) or participating diagnostic centers. 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.11, 12 Briefly, 1 μg genomic patient DNA was digested with restriction enzymes and re-ligated 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.

Results

Identification of MLL rearrangements and their distribution in clinical subgroups

To analyze the recombinome of the human MLL gene, we obtained 1018 acute leukemia samples—either prescreened or unscreened—from different centers over a period of 6 years. Successful analysis could be performed for 760 patient samples. Unsuccessful analyses were in the range of 25% and were due to absence of any prescreening (21%), false-positive prescreening experiments (1%, depending on the participating center), limited biopsy material or insufficient quality of genomic DNA (1%), insufficient amount of leukemic blasts (1%) or by intrinsic limitations of the applied method (length of IPCR amplimers >15 kb; noncanonical breakpoints, 1% of all investigated cases).

Within the group of characterized patients (n=760), one adult patient was diagnosed with primary myelofibrosis (PMF) and displayed an MLL translocation involving MLL intron 8 fused to a region at 1p13.1 where no gene is encoded. All other patients (n=759) were classified as pediatric or adult acute leukemia patients. Pediatric leukemia patients (n=384) were diagnosed either as acute lymphoblastic leukemia (ALL, n=237) or acute myeloid leukemia (AML, n=147); adult leukemia patients (n=375) were classified either as ALL (n=246) or AML (n=129), respectively. All MLL rearrangements in these four subgroups are summarized in Table 1. On the basis of the above distribution, about 94% of all (pediatric and adult) ALL patients (n=483) with MLL gene fusions are characterized by the fusion genes MLL·AF4 (66.0%), MLL·ENL (14.9%), MLL·AF9 (8.5%), MLL·AF10 (2.7%) and MLL·AF6 (1.5%), respectively. In pediatric and adult AML patients (n=276) about 77% of all characterized MLL fusion genes were MLL·AF9 (30.4%), MLL·AF10 (14.5%), MLL·ELL (10.9%), MLL·AF6 (10.1%), MLL·ENL (5.4%), MLL·AF17 (2.9%) and MLL·SEPT6 (2.5%), respectively. This is in line with recently published data on the frequency and distribution of different MLL fusion partner genes.13, 14

Table 1 Distribution of MLL fusion in clinical subgroups

Breakpoint distribution according to clinical subtypes

We also investigated the distribution of chromosomal breakpoints within the MLL BCR in the four investigated clinical subgroups (pediatric vs adult leukemia patients; ALL vs AML). For this purpose, we analyzed the chromosomal breakpoints within the MLL gene when recombined to the 9 most frequent TPGs (AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, MLLT6/AF17, ELL, EPS15 and SEPT6) or the other 29 identified recombination partners (ABI1, ACACA, ACTN4, AFF3, ARHGEF17, BCL9L, C2CD3, CASC5, DCP1A, EEFSEC, FLNA, FOXO3, KIAA0284, LAMC3, LOC100128568, MAML2, MLLT11, MYO1F, NEBL, NRIP3, PICALM, SEPT5, SEPT9, SMAP1, TET1, TIRAP/DCPS, TNRC18, UBE4A and VAV1). In Figure 1, all these data are summarized for the four clinical subgroups. On the basis of the 760 analyzed patients, pediatric ALL patients (n=237) have their chromosomal breakpoints within MLL intron 11, whereas adult ALL patients (n=246) recombine more frequently in MLL intron 9. Pediatric (n=147) and adult AML patients (n=129) show a preference for recombination events affecting MLL intron 9. The exception was the MLLT3/AF9 gene in pediatric and adult AML patients that show a preference for recombination events to occur within MLL intron 11. The same was true for SEPT6 in pediatric AML patients, but not in adult AML patients. Therefore, we conclude that pediatric ALL patients are different from all other subgroups concerning their breakpoint distribution within the MLL BCR.

Figure 1
figure1

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.

Novel translocation partner genes

Eleven novel TPGs were discovered: DCP1A (decapping enzyme homologue A), TNRC18 (trinucleotide repeat containing 18), LAMC3 (laminin, γ-3), NEBL (nebulette), NRIP3 (nuclear receptor interacting protein 3), C2CD3 (C2 calcium-dependent domain containing 3), UBE4A (ubiquitination factor E4A/UFD2 homologue), VAV1 (vav 1 guanine nucleotide exchange factor), LOC100128568 (similar to hCG2045263), ACTN4 (actinin, α-4) and FLNA (filamin A, α/actin-binding protein 280).

The DCP1A gene encodes a homologue of the DCP1 decapping enzyme, involved in mRNA degradation.15 The protein localizes in the mRNA processing body (P-body) that regulates degradation and abundance of RNA molecules (RNA decay). For TNRC18, no function is known. LAMC3 is a non-basement membrane-associate filamentous protein that is downregulated or deleted in carcinomas. NEBL makes structural part of the Z-line in cardiac myofibrils and binds to α-actinin.16 NRIP3 is a nuclear receptor interacting protein of unknown function. C2CD3 is a Ca2+-binding protein of unknown function. UBE4A is expressed in skeletal muscle, kidney and liver, and weakly expressed in hematopoietic cells. It has been postulated that UBE4A is involved in cell-cycle control (ubiquitination) and by protecting the cell against environmental stress. UBE4A is frequently mutated and deleted in neuroblastomas.17 The identified MLL·UBE4A fusion was a head-to-head genomic fusion, and thus, created loss of heterozygosity (LOH) for the UBE4A gene. VAV1 is a Dbl-domain containing proto-oncoprotein with GDP/GTP exchange function that is selectively expressed in hematopoietic cells. It interacts with CBL and GRB2 and influences RAC/RHO signaling processes and migration behavior.18 LOC100128568 is a hypothetical protein with unknown function. ACTN4 encodes a non-muscle α-actinin that appears to promote tumor growth and invasiveness.19 Basically, ACTN4 regulates stress fiber formation of the cytoskeleton. Moreover, ACTN4 protein is a regulator of AKT1 localization and of its function;20 ACTN4 is also involved in insulin signaling.21 FLNA encodes a cytoskeletal filamin protein (280 kDa) that interacts and reorganizes the actin cytoskeleton. It is a substrate of granzyme B and different caspases.22 Interestingly, the cleaved C-terminal portion (100 kDa) localizes in the nucleus. This proteolytic fragment is able to bind and regulate androgen receptor.23 Another study has demonstrated colocalization of FLNA and Caveolin 1.24 Caveolin 1 is downregulated in tumor cells as it inhibits anchorage-independent growth, anoikis and invasiveness.25

The MLL recombinome

Within the past 16 years, several genetic aberrations involving the human MLL gene located on chromosome 11 band q23 have been described. Out of 104 TPGs, 64 are now characterized at the molecular level (see Table 2). Forty-four fusion genes have been described by others, whereas 20 TPGs have been identified at the Frankfurt DCAL. Additional 35 genetic loci were identified by cytogenetic experiments but not further characterized (for references see Meyer et al.12). Five MLL rearrangements were identified that did not display a fusion to an annotated gene or open reading frame. These partner loci were 1p31.2 (patient with PMF), 9p13.3, 11q22, 11q23.3 and 21q22, respectively. Several attempts to identify spliced fusion partners in vicinity failed so far (data not shown). Thus, these fusions most likely represent nonfunctional MLL fusions that are, however, associated with acute leukemias, and in one case, with PMF.

Table 2 The MLL recombinome of acute leukemia

Genetic alterations resulting in genetic MLL aberrations

In general, human MLL rearrangements are initiated by a DNA damage situation, which induces DNA repair by the nonhomologous-end-joining DNA repair pathway.26, 27 Genetic recombinations involving the human MLL gene predominantly result in reciprocal chromosomal translocations (see Figure 2, rCTL), involving recurrently the following TPGs: ABI1, AFF1/AF4, CASC5, CREBBP, ELL, EPS15/AF1P, FOXO3, FOXO4, FRYL, GPHN, KIAA0284, MLLT3/AF9, MLLT1/ENL, MLLT4/AF6, MLLT6/AF17, MLLT11/AF1Q, MYO1F, SEPT2, SEPT5, SEPT9, TET1 and TNRC18, respectively. Other TPGs (also from the literature) were identified so far only once (ACTN4, ARHGAP26, ARHGEF17, ASAH3, DAB2IP, DCP1A, EEFSEC, EP300, GAS7, GMPS, LAMC3, LASP1, LPP, MAPRE1, NCKIPSD, NEBL, SEPT11, SH3GL1 and SMAP1).

Figure 2
figure2

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+Δ, 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.

Gene internal partial tandem duplications (PTDs) of specific MLL gene portions (duplication of MLL gene segments coding either for introns 2–9, 2–10, 2–11, 4–9, 4–11 or 3–8) are frequently observed in AML patients.28, 29, 30 MLL PTDs are being discussed to mediate dimerization of the MLL N terminus, a process that seems to be sufficient to mediate leukemogenic transformation.31 We have observed MLL PTDs in three of the four investigated subgroups: 1 patient within the group of pediatric AML, 1 patient within the group of adult ALL and 24 patients within the group of adult AML. This demonstrates that MLL PTDs are predominantly present in adult AML patients, in line with previously published data.32, 33

MLL recombinations involving only chromosome 11 are based on two independent DNA strand breaks that are accompanied either by inversions or deletions on 11p or 11q (Inv, Del). Several recombinations have been characterized that belong to these two groups. MLL gene fusions to C2CD3, MAML2, NRIP3, PICALM and UBE4A are based on the inversion of a chromatin portion of 11p or 11q, leading to reciprocal MLL gene fusions. By contrast, a deletion of chromosome material located telomere to MLL fused the 5′-portion of MLL directly to other gene sequences (ARHGEF12, BCL9L and CBL). A fourth deletion at 11q fused 5′-MLL sequences to the 3′-UTR of the TIRAP gene, which is located several kilobases upstream of the DCPS gene. In that particular case, only transcription of MLL and a subsequent splice process allowed to generate an MLL·DCPS fusion mRNA, encoding a bona fide MLL·DCPS fusion protein (MLL spliced fusion).

Beside reciprocal chromosomal translocations of MLL (rCTL), MLL PTDs and 11p/q rearrangements (Del and Inv), additional genetic rearrangements were identified in the genomic DNA of analyzed leukemia biopsy material. Although the previous rearrangements are based on two independent DNA strand breaks, all other genetic events observed for the MLL gene represent more complex rearrangements with at least three or more DNA double-strand breaks. In these cases, the expected reciprocal MLL fusion gene cannot be detected, because other sequences will be fused to the 3′-portion of the MLL gene.

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

Figure 3
figure3

Overview over reciprocal MLL fusion genes. (a) Identified reciprocal MLL fusion partners fused in-frame (green) to the 3′-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 3′-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 3′-MLL gene segment (MLL exons 10/11/12–37); a total of 17 genes have been identified. The 3′-MLL gene segment is still able to be transcribed from its gene internal promoter element located upstream of MLL exon 12 (see 3′-MLL on the bottom); this results in a shorter version of the MLL protein that still exhibit an H3K4 methyltransferase activity.

The second category are reciprocal chromosomal translocations that are associated with deletions on either of the involved chromosomes (CTL+Δ). Such cases were predominantly identified in complex t(4;11)(q21;q23) translocations.

The third category are chromosomal fragment insertions. This includes the insertion of chromosome 11 material (including portions of the MLL gene) into other chromosomes (Ins1), or vice versa, the insertion of chromosome material (including portions of a TPG) into the BCR of the MLL gene (Ins2). An insertion mechanism is required in those cases where the transcriptional orientation of a given TPG is not identical to the transcriptional orientation of the MLL gene. The MLL gene is transcribed in telomeric direction. TPGs with a transcriptional orientation in direction to the centromere are predominantly recombining with MLL by such insertion mechanisms. These genes are ACACA, AFF3/LAF4, AFF4/AF5Q31, CENPK, CIP29, CREBBP, FLNA, FNBP1, LOC1000128568, MLLT10/AF10, SEPT6, SORBS2/ARGBP2 and VAV1. In these cases (Ins1/2), three independent fusion genes will be generated. In three patients bearing a recombination event between MLL and MLLT10/AF10, more complex rearrangements were identified that cannot be explained by a simple insertion mechanism.

Finally, spliced fusion were observed. Spliced fusions are generated by fusing the 5′-portion of the MLL gene to the upstream region of a TPG. Thus, a functional MLL fusion mRNA can only be generated by a coupled process of transcription and splicing (last MLL exon 5′ to the breakpoint spliced to an exon (≠1) of the partner gene fused to the MLL). Beside the above-mentioned DCPS gene, other genes have been identified that can transcriptionally fuse to 5′-MLL sequences. These were ZFYVE19, but also the MLL fusion partners like, AFF1/AF4, EPS15, MLLT3/AF9, MLLT1/ENL and SEPT5. In case of MLLT1/ENL, about 50% of all recombination events were spliced fusions,34 and for MLL·EPS15 fusions about 30%. Spliced fusions to AFF1/AF4, MLLT3/AF9 and SEPT5 represent very rare events.

All the above-mentioned mechanisms can be combined to generate more complex genetic rearrangements, requiring four or more DNA double-strand breaks.

Reciprocal MLL gene fusions

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

Another 19 gene fusions were out of frame because of recombining noncompatible introns of both involved genes (ARMC3, CACNA1B, CMAH, CRLF1, FXYD6, GRIA4, MMP13, NFkB1, PAN3, PBX1, PPM1G, PARP14, PIWIL4, RPS3, SCGB1D1, SFRS4, TCF12, TNRC6B and TRIP4). This genetic situation represents an LOH situation. CRLF1 is part of a signaling complex that regulates immune responses during fetal development. MMP13 is frequently overexpressed in tumor cells. NFkB1 blocks the apoptotic pathway. PAN3 is a tumor suppressor protein that is also part of the polyA-specific 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 interference. TCF12 is a transcription factor that associates with E2A to steer somatic recombination of T-cell receptor genes. TRIP4 is part of the inflammasome and inhibits the activation of interleukin 1 (IL1) and IL18 by modulating CASP1 activity.

The final group of 3′-MLL fusion represent head-to-head fusion. Thus, the transcriptional orientation of the fused TPG is opposite to the orientation of the MLL gene. In these case, the TPG became disrupted by replacing its promoter region by the 3′-MLL portion. Thus, this genetic situation represents again an LOH situation. Identified fusion partners were ADSS, CACNB2, CUGBP1, DSCAML1 (2 × ), ELF2, FCHSD2, FXYD2, GTDC1, KIAA0999 (2 × ), KIAA1239, MPZL2, NCAM1, NT5C2, SVIL, TMEM135, TUBGCP2 and UNC84A, respectively. Two genes, DSCAML1 and KIAA0999, were identified twice in different leukemia patients, indicating that such reciprocal MLL gene fusions do also show recurrence. CUGBP1 binds to RNA, influences splicing processes and translation efficiency; it also binds to EWS. ELF2 is an ETS transcription factor that is overexpressed under hypoxic conditions; ELF2 is a direct binding partner of AML1/RUNX1. NCAM1 is also known as CD56 and a known tumor suppressor protein. SCIL binds to the actin cytoskeleton, whereas TUBGCP2 binds to microtubuli and regulates centrosome formation. UNC84A is associated to the nuclear lamina and to centrosomes. We have to mention that promoterless 3′-MLL gene segments are per se able to transcribe most of the remaining open reading frame of the MLL gene by a gene internal promoter recently identified upstream of MLL exon 12.36 Therefore, also these arbitrary MLL fusion may still allow to transcribe most of the parts of the MLL coding region, resulting in a shorter MLL protein version (230 kDa) that still exhibits the ability to function as ‘nonspecific’ H3K4 methyltransferase due to the missing N terminus.

Classification of MLL TPGs into functional categories

All TPGs were categorized according to their gene ontology into functional subclasses. They can be classified into cytosolic/membrane proteins and nuclear proteins. According to their functions, they were grouped into extracellular proteins (LAMC3), cell adhesion proteins (LPP, SORBS2) with functions in the organization of focal adhesion plaques, endocytotic proteins (EPS15, FNBP1, PICALM, ZFYVE19, proteins involved in diverse signaling pathways AF6, ARHGEF12, ARHGEF17, ASAH3, C2CD3, DAP2IP, LASP1, SMAP1, TIRAP, VAV1), organization and regulation of cytoskeleton (actin and microtubuli; ABI1, ACTN4, ARHGAP26, FLNA, GPHN, KIAA0284, MAPRE1, MYO1F, NEBL, SH3GL1), translation elongation (EEFSEC), metabolic functions (ACACA, CBL, GMPS, UBE4A) and proapoptotic proteins (MLLT11/AF1Q). The nuclear compartment is subclassified into cell-cycle control and organization of nuclear cytoskeleton during cytokinesis (NCKIPSD, SEPT2, SEPT5, SEPT6, SEPT9, SEPT11), nucleic acid binding (CIP29, TNRC18), RNA decay (DCP1A, DCPS), chromosome association (CENPK, CASC5), chromatin regulation (CREBBP, EP300), transcription factors and regulation of transcription (AF17, BCL9L, FOXO3, FOXO4, FRYL, GAS7, MAML2, NRIP3, TET1) as well as transcriptional elongation factors (AFF1, AFF3, AFF4, AF9, AF10, ELL, ENL). No cellular function is known for the gene product of LOC100128568. All categorized TPGs are summarized in Table 3.

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

Discussion

On the basis of published data and own findings during the analysis of 760 MLL-rearranged leukemia patients, we present an update of the MLL recombinome associated with acute leukemia. All our analyses were performed by using small amounts of genomic DNA. In some cases, we analyzed cDNA from a given patient to validate an MLL spliced fusion, or to investigate alternative splice products generated from an MLL fusion gene. The results of this study allow to draw several conclusions.

We successfully verified that the applied LDI-PCR technique is a valid approach to identify reciprocal MLL gene fusions, 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, we successfully extended our knowledge by the analysis of more complex MLL rearrangements. During the latter analyses, the novel subclass of reciprocal MLL gene fusions was identified and investigated. About 25% represent in-frame fusions that can be readily expressed into reciprocal fusion proteins. All other fusions are associated with an LOH of the identified reciprocal MLL fusion gene, however, still allow to transcribe and express a 5′-truncated MLL protein.

The analysis of 760 MLL fusion alleles led to the discovery of 20 novel TPGs in the past 4 years, of which 9 have already been 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 MLL protein variants.

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

An important translational aspect of this study is the establishment of patient-specific DNA sequences that can be used for monitoring MRD by quantitative PCR techniques. For each of these 760 acute leukemia patients at least one MLL fusion allele was identified and characterized by sequencing. Prospective studies were already initiated and first published data demonstrate that these MRD markers contribute to stratification, improved treatment and outcome of leukemia patients.37

The analysis of the MLL recombinome allows to classify MLL fusion partner genes into functional categories. As summarized in Table 3, genes coding for cytosolic and nuclear proteins are affected by MLL rearrangements. Recurrence of MLL rearrangements was observed in about 44% of all yet identified TPGs. The encoded proteins of these TPGs are part of different cellular processes: EPS15 and PICALM are proteins that are involved in endocytotic processes; AF6, ABI1, GPHN, KIAA0284 and MYO1F are involved in signaling processes and the regulation of the cytoskeleton, whereas MLLT11/AF1Q is a proapoptotic protein; different SEPTINS are involved in the process of cytokinesis, by reorganization and stabilization of the nuclear cytoskeleton; nucleic-acid-binding protein TNRC18 and the chromosome-associated CASC5 protein are both located in the nucleus. The histone acetyltransferase CREBBP modifies histone core particles. Several transcription factors (AF17, FOXO3, FOXO4, FRYL, MAML2 and TET1) influence genetic programs. Finally, the most frequent TPGs in MLL translocations encode nuclear proteins (AFF1/AF4, AFF3/LAF4, AFF4/AF5Q31, MLLT3/AF9, MLLT1/ENL and MLLT10/AF10) that belong to a protein network that transmits DOT1L38 and pTEF-B to promoter-arrested RNA polymerase II, and thus, allows active transcription and elongation.39, 40 pTEF-B phosphorylates the C-terminal domain of RNA polymerase II, whereas DOT1L enables methylation of lysine 79 of histone H3 proteins, a prerequisite for the maintenance of RNA transcription.41 In addition, expression of this MLL·AF4 fusion protein confers a global increase of H3K79 methylation, a potentially novel oncogenic mechanism.42

Certain MLL rearrangements are associated with poor outcome in pediatric and adult acute leukemia. It can be assumed that a systematic analysis of the MLL recombinome will allow to draw conclusions on certain aspects of hematological tumor development.

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Acknowledgements

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

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Correspondence to R Marschalek.

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

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Meyer, C., Kowarz, E., Hofmann, J. et al. New insights to the MLL recombinome of acute leukemias. Leukemia 23, 1490–1499 (2009) doi:10.1038/leu.2009.33

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Keywords

  • MLL
  • translocations partner genes
  • acute leukemia
  • ALL
  • AML

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