MLL Translocations Research Articles

Cryptic insertion and translocation or nondividing leukemic cells disclosed by FISH analysis in infant acute leukemia with discrepant molecular and cytogenetic findings.

Of 51 infants with acute leukemia, 13 (25%) had contradictory findings on 11q23/MLL rearrangements that were analyzed by cytogenetic and Southern blot methods: seven had rearranged MLL and normal karyotype, four had rearranged MLL and abnormal karyotype with no 11q23 translocation, and two had germline MLL and 11q23 translocations. Fluorescent in situ hybridization (FISH) analysis using an MLL probe that was performed to elucidate the discrepancy disclosed the presence of normal dividing cells and nondividing leukemic cells in the same bone marrow in five patients, and cryptic insertion or translocation in another five. Subsequent FISH and reverse transcription-polymerase chain reaction analysis identified the MLL-AF10, MLL-AF4, or MLL-AF1q fusions that were produced by the cryptic rearrangements in four of the five patients. In the remaining three patients, the breakpoint of 11q23 translocation was located distal to the MLL locus in one, and the discrepancy was unresolved in two. Thus, FISH should complement cytogenetic analysis when cytogenetic and molecular genetic findings are contradictory in infant leukemia, and when infant leukemia does not show 11q23 translocations or other specific translocations including t(7;12), t(1;22), etc that are recurrently found in infant leukemia.

Proteolytic cleavage of MLL generates a complex of N- and C-terminal fragments that confers protein stability and subnuclear localization.

The mixed-lineage leukemia gene (MLL, ALL1, HRX) encodes a 3,969-amino-acid nuclear protein homologous to Drosophila trithorax and is required to maintain proper Hox gene expression. Chromosome translocations in human leukemia disrupt MLL (11q23), generating chimeric proteins between the N terminus of MLL and multiple translocation partners. Here we report that MLL is normally cleaved at two conserved sites (D/GADD and D/GVDD) and that mutation of these sites abolishes the proteolysis. MLL cleavage generates N-terminal p320 (N320) and C-terminal p180 (C180) fragments, which form a stable complex that localizes to a subnuclear compartment. The FYRN domain of N320 directly interacts with the FYRC and SET domains of C180. Disrupting the interaction between N320 and C180 leads to a marked decrease in the level of N320 and a redistribution of C180 to a diffuse nuclear pattern. These data suggest a model in which a dynamic post-cleavage association confers stability to N320 and correct nuclear sublocalization of the complex, to control the availability of N320 for target genes. This predicts that MLL fusion proteins of leukemia which would lose the ability to complex with C180 have their stability conferred instead by the fusion partners, thus providing one mechanism for altered target gene expression.

Low NAD(P)H:quinone oxidoreductase activity is associated with increased risk of leukemia with MLL translocations in infants and children

An inactivating polymorphism at position 609 in the NAD(P)H:quinone oxidoreductase 1 gene (NQO1 C609T) is associated with an increased risk of adult leukemia. A small British study suggested that NQO1 C609T was associated with an increased risk of infant leukemias with MLL translocations, especially infant acute lymphoblastic leukemia (ALL) with t(4;11). We explored NQO1 C609T as a genetic risk factor in 39 pediatric de novo and 18 pediatric treatment-related leukemias with MLL translocations in the United States. Children with de novo B-lineage ALL without MLL translocations and a calculation of the expected genotype distribution in an ethnically matched population of disease-free subjects served as the comparison groups. Patients with de novo leukemias with MLL translocations were significantly more likely to be heterozygous at NQO1 C609T (odds ratio [OR] = 2.77, 95% confidence intervals [CI] 1.17-6.57; P =.02), and significantly more likely to have low/null NQO1 activity than patients with de novo B-lineage ALL without MLL translocations (OR = 2.47, 95% CI 1.08-5.68; P =.033). They were also significantly more likely to have low/null NQO1 activity than expected in an ethnically matched population of disease-free subjects (OR = 2.50, P =.02). Infants younger than 12 months old at diagnosis of leukemia with t(4;11) were most likely to have low/null NQO1 activity (OR > 10.0). Conversely, the distribution of NQO1 genotypes among patients with treatment-related leukemias with MLL translocations was not statistically different than in the comparison groups. The inactivating NQO1 polymorphism is associated with an increased risk of de novo leukemia with MLL translocations in infants and children.

Molecular Rearrangements and Morphology in Thyroid Cancer

Tumor development within the human thyroid gland provides an attractive experimental model with which to consider the pathogenesis of carcinoma, the most common and clinically significant cancer. Thyroid tumors are readily accessible to morphological and molecular study because their primary treatment is surgical. Investigations of thyroid cancer have expanded our knowledge of carcinoma biology. Thyroid carcinoma is the only adult epithelial malignancy in which specific chromosomal rearrangements have been identified. 1-3 RET and PPARγ rearrangements in thyroid carcinoma create fusion proteins that are hypothesized to play fundamental roles in thyroid oncogenesis. 3,4 The fusion protein pathways are prime targets for new strategies directed at improving thyroid cancer diagnosis and treatment. 5-7

The Leukemia-Associated Gene Mllt1/ENL: Characterization of a Murine Homolog and Demonstration of an Essential Role in Embryonic Development

ABSTRACT MLLT1 (ENL/LTG19) is one of a number of fusion gene partners with the MLL oncogene involved in 11q23 translocations in human leukemia and encodes a transcriptional regulator of unknown function. Leukemias bearing MLL translocations may be myeloid or lymphoid or bear mixed lineage properties; however, those bearing MLL/MLLT1 translocations are predominantly lymphoid, suggesting that MLLT1 may influence the leukemic phenotype. The murine homolog Mllt1 exhibits 86% amino acid sequence identity with the human gene and is broadly expressed in murine tissues and cell lines, with the exception of liver and myeloid cell lines. We have mapped Mllt1 to mouse chromosome 17 band E2 using FISH analysis. The genomic structure and 5′ regulatory sequence of Mllt1 are highly conserved between mouse and human. There is also conservation of the genomic structure, but not the promoter, between MLLT1 and MLLT3/AF9, a homologous gene that is also an MLL translocation partner in human leukemias with a predominant myeloid phenotype. Targeted disruption of Mllt1 in mice leads to embryonic lethality prior to 8.5 dpc. These studies indicate that MLLT1 is involved in essential developmental processes and suggest that expression patterns of MLL fusion partners may influence the lineage of MLL-associated leukemias.

Sensitization by 5‐aza‐2′‐deoxycytidine of leukaemia cells with MLL abnormalities to induction of differentiation by all‐trans retinoic acid and 1α,25‐dihydroxyvitamin D3

Most chromosomal abnormalities associated with breakage at 11q23 in acute leukaemia involve the MLL gene, and the presence of this breakage strongly predicts a poor clinical outcome. We assessed the possibility of differentiation-inducing therapy for acute leukaemias with chromosomal translocations involving 11q23. Among the cell lines with MLL translocations that we examined, KOCL48 and KOPN-1 cells were induced to differentiate into granulocytes by all-trans retinoic acid (ATRA) or into monocytes by 1alpha,25-dihydroxyvitamin D3 (VD3). These cells expressed p16 mRNA before treatment with 5-aza-2'-deoxycytidine (5-AZA), an inhibitor of DNA methylation. On the other hand, differentiation was not induced in SN-1, KOCL33, KOCL51 or KOCL44 cells by ATRA or VD3, and these cells did not express mRNA of this gene. However, these cells were effectively induced to differentiate by ATRA or VD3 in the presence of 5-AZA, and concomitantly exhibited p16 gene expression, suggesting an association between DNA demethylation and restoration of sensitivity to differentiation-inducing activity of ATRA or VD3 in leukaemia cells with MLL abnormalities. Based on these findings, combined treatment with ATRA or VD3 plus 5-AZA may be clinically useful in therapy for acute leukaemia with MLL abnormalities.

Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints.

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.

Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes.

Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts.

Sensitivity of FISH in detection of MLL translocations.

Fluorescence in situ hybridisation (FISH) detection of MLL translocations is now commonplace in cytogenetics laboratories. One of the most widely used probes is the Oncor MLL probe (Oncor, Gaithersburg, MD) that theoretically demonstrates the presence of an MLL rearrangement by a splitting of the FISH signal between the two derivative chromosomes generated by a translocation. Recently, another commercial probe has been made available from Vysis (Vysis, Downers Grove, IL) that uses a dual colour system. We examined material from 29 patients and 4 cell lines, all with recognised MLL translocations by G-banding, that were confirmed using Southern blot analysis of the MLL breakpoint cluster region. Both Oncor and Vysis MLL FISH probes were applied to these cases to compare their performance in detection of the MLL translocations. Thirty of the 33 cases demonstrated a clear splitting of Oncor MLL FISH signal in concordance with the Southern blot analysis and cytogenetics. Three cases failed to demonstrate a split MLL FISH signal. Therefore, we conclude that the Oncor MLL FISH probe has a 9.1% false negative rate, i.e., 90.9% sensitivity in detection of classic MLL translocations. Vysis MLL FISH detected the rearrangement in all 33 cases.

Rapid and sensitive detection of all types of MLL gene translocations with a single FISH probe set.

The MLL gene on chromosome 11 band q23 is frequently involved in chromosome translocations in acute lymphoblastic leukemia and acute myeloid leukemia. The translocation results in the formation of a fusion gene on the derivative 11 chromosome consisting of the 5' part of the MLL gene and the 3' part of another gene; already more than 30 different partner chromosome regions have been described. MLL gene rearrangements are generally correlated with a poor prognosis. Therefore the presence of an 11q23 aberration has direct implications for treatment stratification, making early and rapid detection of utmost importance. In this study, we developed a FISH probe set for detection of MLL gene rearrangements according to strict design criteria. The cosmid probes are derived from the flanking regions of the MLL breakpoint region on chromosome 11 and when used in dual colored FISH experiments give rise to a split of the normally colocalizing (fused) signals in case of a translocation. This split signal was observed in seven out of 10 cases with an 11q23 translocation with various partner chromosomes. In the three other cases, a deletion of the 3' part of the MLL gene, downstream of the breakpoint region was also found. A low false positive value of only 1.7% was obtained for interphase cells in contrast to conventional dual colored FISH where the creation of a fusion signal has cut off values of at least 5-10%. A major advantage of our type of probe set is the application of a single FISH experiment to detect all types of MLL translocations. Moreover, since this cosmid probe set can be used for either interphase or metaphase studies, metaphases are no longer a prerequisite for detecting the presence of an 11q23 translocation. Nevertheless, metaphase FISH with the new probe set is helpful in determining the partner chromosome and therefore may lead to the identification of new partner genes.

Establishment of an inducible expression system of chimeric MLL-LTG9 protein and inhibition of Hox a7, Hox b7 and Hox c9 expression by MLL-LTG9 in 32Dcl3 cells.

The MLL (HRX/ALL-1 gene is frequently disrupted in infantile leukemias and therapy-related leukemias and fused to various translocation partner genes. We previously showed that chimeric MLL proteins localize in the nuclei in a fashion similar to that of MLL protein even if the partner gene encodes a cytoplasmic protein and indicated the importance of the N-terminal portion of MLL common to various MLL translocations. This time we established an inducible expression system for chimeric MLL-LTG9 and truncated N-terminal MLL proteins (MLL-Zf(-)) in 32Dcl3 cells. By utilizing this system, we were able to show inhibition of Hox a7, Hox b7 and Hox c9 genes' expression by induced MLL-LTG9 and MLL-Zf(-). Up-regulation of Hox a7, Hox b7 and Hox c9 was observed when 32Dcl3 cells were cultured with granulocyte colony stimulating factor (G-CSF) in place of interleukin 3 and induction of MLL-LTG9 and MLL-Zf(-) was shown to suppress this upregulation. At the same time, expression of two mammalian Polycomb group genes, M33 and mel-18, which both reportedly affect Hox genes' expression, was not inhibited by MLL-LTG9 and MLL-Zf(-) induction. These results indicate that MLL has an important effect on the expression of at least some Hox genes in hematopoietic cells and suggest that inhibition of the proper expression of Hox genes by chimeric MLL proteins may dysregulate hematopoietic cell differentiation and proliferation, which then can lead to leukemogenesis.

Identification of complex genomic breakpoint junctions in the t(9;11) MLLAF9 fusion gene in acute leukemia

The MLL gene at chromosome 11, band q23, is involved in translocations with as many as 40 different chromosomal bands. Virtually all breakpoints occur within an 8.3 kb BamHI fragment and result in 5` MLL fused to partner genes in a 5′-3′ orientation. The translocation t(9;11)(p22;q23), which results in the fusion of MLL to AF9, is the most common of the 11q23 chromosomal abnormalities observed in de novo acute myeloid leukemia (AML), in therapy related leukemia (t-AML), and rarely in acute lymphoblastic leukemia (ALL). We have studied 24 patients with a t(9;11) and an MLL rearrangement, including 19 patients with AML, four with t-AML, and one with ALL. To understand the mechanisms of this illegitimate recombination, we cloned and sequenced the t(9;11) translocation breakpoint junctions on both derivative chromosomes from one AML patient and from the Mono Mac 6 (MM6) cell line, which was derived from a patient with AML. Two different complex junctions were noted. In the AML patient, both chromosome 11 and 9 breaks were staggered, occurred in Alu DNA sequences, and resulted in a 331 bp duplication. In the MM6 cell line, breaks in chromosomes 11 and 9 were also staggered, but, in contrast to the finding in the AML patient, the breaks did not involve Alu DNA sequences and resulted in a 664 bp deletion at the breakpoints. Using reverse transcriptase (RT-) PCR, we analyzed 11 patient samples, including the two just described, for MLL-AF9 fusions. The fusion occurred in six of seven AML patients, two of two t-AML patients, one patient with ALL, and in the MM6 cell line. Interestingly, all of the breaks within the AF9 gene in AML patients occurred in the central AF9 exon, called Site A by others, whereas in the single ALL patient the breakpoint mapped to a more 3` region of the AF9 gene. Our data, when combined with those of others, suggest that the fusion point within the AF9 gene, and thus the amount of AF9 material included in the MLL-AF9 fusion gene product, may influence the phenotype of the resulting leukemia. This further supports the proposal that the MLL translocation partner genes play a critical role in the leukemogenic process. Genes Chromosomes Cancer 20:185–195, 1997. © 1997 Wiley-Liss, Inc.

Site-specific DNA cleavage within the MLL breakpoint cluster region induced by topoisomerase II inhibitors [see comments

The MLL gene located at 11q23 is frequently disrupted by chromosomal translocation in a wide spectrum of newly diagnosed acute leukemias. Recently, it has become apparent that the MLL gene is very frequently disrupted by chromosomal translocations in patients with secondary leukemias associated with chemotherapeutic regimens incorporating topoisomerase II inhibitors. These secondary leukemias associated with topoisomerase II inhibitors (most commonly teniposide, etoposide, or doxorubicin) have distinct clinical and biologic features which have led to the speculation that they are induced by treatment with topoisomerase II inhibitors. We have identified a site within the MLL breakpoint cluster region (bcr) that is highly sensitive to double-strand DNA cleavage induced by topoisomerase II inhibitors. This finding is quite specific and highly reproducible. Although it was initially discovered in malignant lymphoblasts isolated from a patient receiving multiagent chemotherapy, this site-specific double-strand DNA cleavage can be induced in tissue culture using malignant cell lines as well as peripheral blood from normal individuals. Site-specific cleavage occurs in a significant fraction of cells using a variety of model systems, is both time and dose dependent, and can be induced with either doxorubicin or etoposide. This site-specific cleavage maps to the same region as a consensus topoisomerase II cleavage site within the MLL bcr. These results suggest that site specific cleavage within the MLL bcr induced by topoisomerase II inhibitors may be an early step leading to MLL translocations and secondary leukemia.