Topoisomerase II and the etiology of chromosomal translocations

Acute leukemias with balanced chromosomal translocations, protean morphologic and immunophenotypic presentations but generally shorter latency and absence of myelodysplasia are recognized as a complication of anti-cancer drugs that behave as topoisomerase II poisons. Translocations affecting the breakpoint cluster region of the MLL gene at chromosome band 11q23 are the most common molecular genetic aberrations in leukemias associated with the topoisomerase II poisons. These agents perturb the cleavage-religation equilibrium of topoisomerase II and increase cleavage complexes. One model suggests that this damages the DNA directly and leads to chromosomal breakage, which may result in untoward DNA recombination in the form of translocations. This review will summarize the evidence for topoisomerase II involvement in the genesis of translocations and extension of the model to acute leukemia in infants characterized by similar MLL translocations.     

Genomic DNA of leukemic patients: target for clinical diagnosis of MLL rearrangements

Genomic DNA is the optimal resource to analyze questions concerning genetic changes that are related to oncogenesis. This article tries to summarize recent efforts to analyze chromosomal changes that trigger the development of human acute myeloid and lymphoblastic leukemias. The aim of this study was to establish an universal method that enables the identification and characterization of chromosomal translocations of the human MLL gene at the genomic nucleotide level. Chromosomal translocations of the MLL gene are the result of illegitimate recombination events in hematopoietic stem or precursor cells, strictly associated with the onset of highly malignant leukemic diseases. The applied technology was able to identify specific fusion alleles that were generated by chromosomal translocations, chromosomal deletions, chromosomal inversions and partial tandem duplications. Moreover, it allowed us to investigate even highly complex genetic changes by applying systematic breakpoint analyses. On the basis of these analyses, patient-specific molecular markers were established that turned out to be a very good source for monitoring minimal residual disease (MRD). MRD analyses control the efficiency and efficacy of current treatment protocols and have become a very sensitive molecular tool to monitor therapeutic success or failure in individual leukemia patients.     

Causes of oncogenic chromosomal translocation

Non-random chromosomal translocations are frequently associated with a variety of cancers, particularly hematologic malignancies and childhood sarcomas. In addition to their diagnostic utility, chromosomal translocations are increasingly being used in the clinic to guide therapeutic decisions. However, the mechanisms that cause these translocations remain poorly understood. Illegitimate V(D)J recombination, class switch recombination, homologous recombination, non-homologous end-joining and genome fragile sites all have potential roles in the production of non-random chromosomal translocations. In addition, mutations in DNA-repair pathways have been implicated in the production of chromosomal translocations in humans, mice and yeast. Although initially surprising, the identification of these same oncogenic chromosomal translocations in peripheral blood from healthy individuals strongly suggests that the translocation is not sufficient to induce malignant transformation, and that complementary mutations are required to produce a frank malignancy.     

组蛋白甲基转移酶MLL1的结构与功能研究进展

组蛋白甲基转移酶MLL1因其基因易位重排所引起的混合系白血病（mixed lineage leukemia）而得名。MLL1蛋白在基因调控、细胞增殖、生长分化等正常生理功能中发挥着重要作用,染色体易位重排所产生的MLL1融合蛋白则与急性白血病的发生发展密切相关。目前人们对MLL1蛋白的结构和功能研究取得了很大的进展,为以MLL1和其相互作用蛋白为靶点的新型MLL白血病药物设计奠定了坚实的基础。     

Mll fusions generated by Cre-loxP-mediated de novo translocations can induce lineage reassignment in tumorigenesis

Chromosomal translocations are primary events in tumorigenesis. Those involving the mixed lineage leukaemia (MLL) gene are found in various guises and it is unclear whether MLL fusions can affect haematopoietic differentiation. We have used a model in which chromosomal translocations are generated in mice de novo by Cre-loxP-mediated recombination (translocator mice) to compare the functionally relevant haematopoietic cell contexts for Mll fusions, namely pluripotent stem cells, semicommitted progenitors or committed cells. Translocations between Mll and Enl or Af9 cause myeloid neoplasias, initiating in pluripotent stem cells or multipotent myeloid progenitors. However, while Mll-Enl translocations can also cause leukaemia from T-cell progenitors, no tumours arose with Mll-Af9 translocations in the T-cell compartment. Furthermore, Mll-Enl translocations in T-cell progenitors can cause lineage reassignment into myeloid tumours. Therefore, a permissive cellular environment is required for oncogenicity of Mll-associated translocations and Mll fusions can influence haematopoietic lineage commitment.     

Chromatin structural elements and chromosomal translocations in leukemia

Recurring chromosome abnormalities are strongly associated with certain subtypes of leukemia, lymphoma and sarcomas. More recently, their potential involvement in carcinomas, i.e. prostate cancer, has been recognized. They are among the most important factors in determining disease prognosis, and in many cases, identification of these chromosome abnormalities is crucial in selecting appropriate treatment protocols. Chromosome translocations are frequently observed in both de novo and therapy-related acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The mechanisms that result in such chromosome translocations in leukemia and other cancers are largely unknown. Genomic breakpoints in all the common chromosome translocations in leukemia, including t(4;11), t(9;11), t(8;21), inv(16), t(15;17), t(12;21), t(1;19) and t(9;22), have been cloned. Genomic breakpoints tend to cluster in certain intronic regions of the relevant genes including MLL, AF4, AF9, AML1, ETO, CBFB, MYHI1, PML, RARA, TEL, E2A, PBX1, BCR and ABL. However, whereas the genomic breakpoints in MLL tend to cluster in the 5' portion of the 8.3 kb breakpoint cluster region (BCR) in de novo and adult patients and in the 3' portion in infant leukemia patients and t-AML patients, those in both the AML1 and ETO genes occur in the same clustered regions in both de novo and t-AML patients. These differences may reflect differences in the mechanisms involved in the formation of the translocations. Specific chromatin structural elements, such as in vivo topoisomerase II (topo II) cleavage sites, DNase I hypersensitive sites and scaffold attachment regions (SARs) have been mapped in the breakpoint regions of the relevant genes. Strong in vivo topo II cleavage sites and DNase I hypersensitive sites often co-localize with each other and also with many of the BCRs in most of these genes, whereas SARs are associated with BCRs in MLL, AF4, AF9, AML1, ETO and ABL, but not in the BCR gene. In addition, the BCRs in MLL, AML1 and ETO have the lowest free energy level for unwinding double strand DNA. Virtually all chromosome translocations in leukemia that have been analyzed to date show no consistent homologous sequences at the breakpoints, whereas a strong non-homologous end joining (NHEJ) repair signature exists at all of these chromosome translocation breakpoint junctions; this includes small deletions and duplications in each breakpoint, and micro-homologies and non-template insertions at genomic junctions of each chromosome translocation. Surprisingly, the size of these deletions and duplications in the same translocation is much larger in de novo leukemia than in therapy-related leukemia. We propose a non-homologous chromosome recombination model as one of the mechanisms that results in chromosome translocations in leukemia. The topo II cleavage sites at open chromatin regions (DNase I hypersensitive sites), SARs or the regions with low energy level are vulnerable to certain genotoxic or other agents and become the initial breakage sites, which are followed by an excision end joining repair process.     

A novel selection system for chromosome translocations in Saccharomyces cerevisiae

Chromosomal translocations are common genetic abnormalities found in both leukemias and solid tumors. While much has been learned about the effects of specific translocations on cell proliferation, much less is known about what causes these chromosome rearrangements. This article describes the development and use of a system that genetically selects for rare translocation events using the yeast Saccharomyces cerevisiae. A translocation YAC was created that contains the breakpoint cluster region from the human MLL gene, a gene frequently involved in translocations in leukemia patients, flanked by positive and negative selection markers. A translocation between the YAC and a yeast chromosome, whose breakpoint falls within the MLL DNA, physically separates the markers and forms the basis for the selection. When RAD52 is deleted, essentially all of the selected and screened cells contain simple translocations. The detectable translocation rates are the same in haploids and diploids, although the mechanisms involved and true translocation rates may be distinct. A unique double-strand break induced within the MLL sequences increases the number of detectable translocation events 100- to 1000-fold. This novel system provides a tractable assay for answering basic mechanistic questions about the development of chromosomal translocations.     

Analysis of the V(D)J recombination efficiency at lymphoid chromosomal translocation breakpoints

Chromosomal translocations and deletions are among the major events that initiate neoplasia. For lymphoid chromosomal translocations, misrecognition by the RAG (recombination activating gene) complex of V(D)J recombination is one contributing factor that has long been proposed. The chromosomal translocations involving LMO2 (t(11;14)(p13;q11)), Ttg-1 (t(11;14)(p15;q11)), and Hox11 (t(10;14)(q24;q11)) are among the clearest examples in which it appears that a D or J segment has synapsed with an adventitious heptamer/nonamer at a gene outside of one of the antigen receptor loci. The interstitial deletion at 1p32 involving SIL (SCL-interrupting locus)/SCL (stem cell leukemia) is a case involving two non-V(D)J sites that have been suggested to be V(D)J recombination mistakes. Here we have used our human extrachromosomal substrate assay to formally test the hypothesis that these regions are V(D)J recombination misrecognition sites and, more importantly, to quantify their efficiency as V(D)J recombination targets within the cell. We find that the LMO2 fragile site functions as a 12-signal at an efficiency that is only 27-fold lower than that of a consensus 12-signal. The Ttg-1 site functions as a 23-signal at an efficiency 530-fold lower than that of a consensus 23-signal. Hox11 failed to undergo recombination as a 12- or 23-signal and was at least 20,000-fold less efficient than consensus signals. SIL has been predicted to function as a 12-signal and SCL as a 23-signal. However, we find that SIL actually functions as a 23-signal. These results provide a formal demonstration that certain chromosomal fragile sites can serve as RAG complex targets, and they determine whether these sites function as 12- versus 23-signals. These results quantify one of the three major factors that determine the frequency of these translocations in T-cell acute lymphocytic leukemia.     

Local gene density predicts the spatial position of genetic loci in the interphase nucleus

Specific chromosomal translocations are hallmarks of many human leukemias. The basis for these translocation events is poorly understood, but it has been assumed that spatial positioning of genes in the nucleus of hematopoietic cells is a contributing factor. Analysis of the nuclear 3D position of the gene MLL, frequently involved in chromosomal translocations and five of its translocation partners (AF4, AF6, AF9, ENL and ELL), and two control loci revealed a characteristic radial distribution pattern in all hematopoietic cells studied. Genes in areas of high local gene density were found positioned towards the nuclear center, whereas genes in regions of low gene density were detected closer to the nuclear periphery. The gene density within a 2 Mbp window was found to be a better predictor for the relative positioning of a genomic locus within the cell nucleus than the gene density of entire chromosomes. Analysis of the position of MLL, AF4, AF6 and AF9 in cell lines carrying chromosomal translocations involving these genes revealed that the position of the normal genes was different from that of the fusion genes, and this was again consistent with the changes in local gene density within a 2 Mbp window. Thus, alterations in gene density directly at translocation junctions could explain the change in the position of affected genes in leukemia cells.     

MLL: How complex does it get?

The mixed lineage leukemia (MLL) gene encodes a very large nuclear protein homologous to Drosophila trithorax (trx). MLL is required for the proper maintenance of HOX gene expression during development and hematopoiesis. The exact regulatory mechanism of HOX gene expression by MLL is poorly understood, but it is believed that MLL functions at the level of chromatin organization. MLL was identified as a common target of chromosomal translocations associated with human acute leukemias. About 50 different MLL fusion partners have been isolated to date, and while similarities exist between groups of partners, there exists no unifying property shared by all the partners. MLL gene rearrangements are found in leukemias with both lymphoid and myeloid phenotypes and are often associated with infant and secondary leukemias. The immature phenotype of the leukemic blasts suggests an important role for MLL in the early stages of hematopoietic development. Mll homozygous mutant mice are embryonic lethal and exhibit deficiencies in yolk sac hematopoiesis. Recently, two different MLL-containing protein complexes have been isolated. These and other gain- and loss-of-function experiments have provided insight into normal MLL function and altered functions of MLL fusion proteins. This article reviews the progress made toward understanding the function of the wild-type MLL protein. While many advances in understanding this multifaceted protein have been made since its discovery, many challenging questions remain to be answered.     

Etoposide and illegitimate DNA double-strand break repair in the generation of MLL translocations: new insights and new questions

Faithful repair of chromosomal double-strand breaks (DSBs) is central to genome integrity and the suppression of genome rearrangements including translocations that are a hallmark of leukemia, lymphoma, and soft-tissue sarcomas [B. Elliott, M. Jasin, Double-strand breaks and translocations in cancer, Cell. Mol. Life Sci. 59 (2002) 373-385; D.C. van Gent, J.H. Hoeijmakers, R. Kanaar, Chromosomal stability and the DNA double-stranded break connection, Nat. Rev. Genet. 2 (2001) 196-206]. Chemotherapy agents that target the essential cellular enzyme topoisomerase II (topo II) are known promoters of DSBs and are associated with therapy-related leukemias. There is a clear clinical association between previous exposure to etoposide and therapy-related acute myeloid leukemia (t-AML) characterized by chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene on chromosome band 11q23 [C.A. Felix, Leukemias related to treatment with DNA topoisomerase II inhibitors, Med. Pediatr. Oncol. 36 (2001) 525-535]. Most MLL rearrangements initiate within a well-characterized 8.3 kb region that contains both putative topo II cleavage recognition sequences and repetitive elements leading to the logical hypothesis that MLL is particularly susceptible to aberrant cleavage and homology-mediated fusion to repetitive elements located on novel chromosome partners. In this review, we will discuss the findings and implications of recent attempts to confirm this hypothesis.     

Molecular basis of the mixed lineage leukemia-menin interaction: implications for targeting mixed lineage leukemias

Chromosomal translocations targeting the mixed lineage leukemia (MLL) gene result in MLL fusion proteins that are found in aggressive human acute leukemias. Disruption of MLL by such translocations leads to overexpression of Hox genes, resulting in a blockage of hematopoietic differentiation that ultimately leads to leukemia. Menin, which directly binds MLL, has been identified as an essential oncogenic co-factor required for the leukemogenic activity of MLL fusion proteins. Here, we characterize the molecular basis of the MLL-menin interaction. Using (13)C-detected NMR experiments, we have mapped the residues within the intrinsically unstructured fragment of MLL that are required for binding to menin. Interestingly, we found that MLL interacts with menin with a nanomolar affinity (K(d) ～ 10 nM) through two motifs, MBM1 and MBM2 (menin binding motifs 1 and 2). These motifs are located within the N-terminal 43-amino acid fragment of MLL, and the MBM1 represents a high affinity binding motif. Using alanine scanning mutagenesis of MBM1, we found that the hydrophobic residues Phe(9), Pro(10), and Pro(13) are most critical for binding. Furthermore, based on exchange-transferred nuclear Overhauser effect measurements, we established that MBM1 binds to menin in an extended conformation. In a series of competition experiments we showed that a peptide corresponding to MBM1 efficiently dissociates the menin-MLL complex. Altogether, our work establishes the molecular basis of the menin interaction with MLL and MLL fusion proteins and provides the necessary foundation for development of small molecule inhibitors targeting this interaction in leukemias with MLL translocations.     

The mll-AF9 gene fusion in mice controls myeloproliferation and specifies acute myeloid leukaemogenesis.

The MLL gene from human chromosome 11q23 is involved in >30 different chromosomal translocations resulting in a plethora of different MLL fusion proteins. Each of these tends to associate with a specific leukaemia type, for example, MLL-AF9 is found mainly in acute myeloid leukaemia. We have studied the role of the Mll-AF9 gene fusion made in mouse embryonic stem cells by an homologous recombination knock-in. Acute leukaemias developed in heterozygous mice carrying this fusion as well as in chimeric mice. As with human chromosomal translocation t(9;11), the majority of cases were acute myeloid leukaemias (AMLs) involving immature myeloblasts, but a minority were acute lymphoblastic leukaemia. The AMLs were preceded by effects on haematopoietic differentiation involving a myeloproliferation resulting in accumulation of Mac-1/Gr-1 double-positive mature myeloid cells in bone marrow as early as 6 days after birth. Therefore, non-malignant expansion of myeloid precursors is the first stage of Mll-AF9-mediated leukaemia followed by accumulation of malignant cells in bone marrow and other tissues. Thus, the late onset of overt tumours suggests that secondary tumorigenic mutations are necessary for malignancy associated with MLL-AF9 gene fusion and that myeloproliferation provides the pool of cells in which such events can occur.     

MLL-SEPTIN gene fusions in hematological malignancies

The mixed lineage leukemia (MLL) locus is involved in more than 60 different rearrangements with a remarkably diverse group of fusion partners in approximately 10% of human leukemias. MLL rearrangements include chromosomal translocations, gene internal duplications, chromosome 11q deletions or inversions and MLL gene insertions into other chromosomes, or vice versa. MLL fusion partners can be classified into four distinct categories: nuclear proteins, cytoplasmatic proteins, histone acetyltransferases and septins. Five different septin genes (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) have been identified as MLL fusion partners, giving rise to chimeric fusion proteins in which the N terminus of MLL is fused, in frame, to almost the entire open reading frame of the septin partner gene. The rearranged alleles result from heterogeneous breaks in distinct introns of both MLL and its septin fusion partner, originating distinct gene fusion variants. MLL-SEPTIN rearrangements have been repeatedly identified in de novo and therapy related myeloid neoplasia in both children and adults, and some clinicopathogenetic associations are being uncovered. The fundamental roles of septins in cytokinesis, membrane remodeling and compartmentalization can provide some clues on how abnormalities in the septin cytoskeleton and MLL deregulation could be involved in the pathogenesis of hematological malignancies.     

Binding to nonmethylated CpG DNA is essential for target recognition, transactivation, and myeloid transformation by an MLL oncoprotein

The MLL gene is a frequent target for leukemia-associated chromosomal translocations that generate dominant-acting chimeric oncoproteins. These invariably contain the amino-terminal 1,400 residues of MLL fused with one of a variety of over 30 distinct nuclear or cytoplasmic partner proteins. Despite the consistent inclusion of the MLL amino-terminal region in leukemia oncoproteins, little is known regarding its molecular contributions to MLL-dependent oncogenesis. Using high-resolution mutagenesis, we identified three MLL domains that are essential for in vitro myeloid transformation via mechanisms that do not compromise subnuclear localization. These include the CXXC/Basic domain and two novel domains of unknown function. Point mutations in the CXXC domain that eliminate myeloid transformation by an MLL fusion protein also abolished recognition and binding of nonmethylated CpG DNA sites in vitro and transactivation in vivo. Our results define a critical role for the CXXC DNA binding domain in MLL-associated oncogenesis, most likely via epigenetic recognition of CpG DNA sites within the regulatory elements of target genes.     

Polymorphisms in the MLL breakpoint cluster region (BCR)

The MLL gene is involved in many chromosomal translocations leading to both acute myeloid and lymphoid leukemia. Some patients treated for primary malignancies with chemotherapeutic agents that inhibit DNA topoisomerase II (topo II) develop treatment-related leukemia (t-AML) caused by MLL gene rearrangement. Whether these patients are unusually susceptible to anti-topo II drugs, or whether this is a random adverse event is unknown. To discover genetic polymorphisms that may predispose patients to t-AML development, we sequenced the 8.3-kb MLL breakpoint cluster region (BCR) from 22 patients who had been treated with topo II inhibitors and who developed t-AML and from 37 patients who did not, and from eight infants and 20 normal individuals. Four polymorphic sites within Alu repetitive elements were identified; three affected the length of poly-A tracts and one altered the size of a trinucleotide repeat. The three poly-A tract polymorphisms occurred with equal frequency in leukemic patients and controls and hence are not predictors of risk. The trinucleotide GAA repeat has three alleles: (GAA)4, (GAA)5, and (GAA)6. The (GAA)6 allele is very rare. The adult t-AML patients are almost exclusively (GAA)4/5 heterozygotes (83%), whereas the normal population is only 55% (GAA)4/5 heterozygotic and is represented equally by (GAA)4 and (GAA)5 homozygotes (20% each). Only certain trends could be established because of the small sample size of these leukemic groups. Whereas adult t-AML patients are more likely to be (GAA)4/5 heterozygotes, this is not statistically significant, and this polymorphism within the MLL BCR has only a suggestive association with t-AML development.     

Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia

Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.