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.     

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.     

Chromatin-related properties of CBP fused to MLL generate a myelodysplastic-like syndrome that evolves into myeloid leukemia

As a result of the recurring translocation t(11;16) (q23;p13.3), MLL (mixed-lineage leukemia) is fused in frame to CBP (CREB binding protein). This translocation has been documented almost exclusively in cases of acute leukemia or myelodysplasia secondary to therapy with drugs that target DNA topo isomerase II. The minimal chimeric protein that is produced fuses MLL to the bromodomain, histone acetyltransferase (HAT) domain, EIA-binding domain and steroid-receptor coactivator binding domains of CBP. We show that transplantation of bone marrow retrovirally transduced with MLL-CBP induces myeloid leukemias in mice that are preceded by a long preleukemic phase similar to the myelodysplastic syndrome (MDS) seen in many t(11;16) patients but unusual for other MLL translocations. Structure-function analysis demonstrated that fusion of both the bromodomain and HAT domain of CBP to the amino portion of MLL is required for full in vitro transformation and is sufficient to induce the leukemic phenotype in vivo. This suggests that the leukemic effect of MLL-CBP results from the fusion of the chromatin association and modifying activities of CBP with the DNA binding activities of MLL.     

Common chromatin structures at breakpoint cluster regions may lead to chromosomal translocations found in chronic and acute leukemias

The t(9;22) BCR/ABL fusion is associated with over 90% of chronic myelogenous and 25% of acute lymphocytic leukemia. Chromosome 11q23 translocations in acute myeloid and lymphoid leukemia cells demonstrate myeloid lymphoid leukemia (MLL) fusions with over 40 gene partners, like AF9 and AF4 on chromosomes 9 and 4, respectively. Therapy-related leukemia is associated with the above gene rearrangements following the treatment with topoisomerase II (topo II) inhibitors. BCR, ABL, MLL, AF9 and AF4 have defined patient breakpoint cluster regions. Chromatin structural elements including topo II and DNase I cleavage sites and scaffold attachment sites have previously been shown to closely associate with the MLL and AF9 breakpoint cluster regions, implicating these elements in non-homologous recombination (NHR). In this report, using cell lines and primary cells, chromatin structural elements were analyzed in BCR, ABL and AF4 and, for comparison, in MLL2, which is a homolog to MLL, but not associated with chromosome translocations. Topo II and DNase I cleavage sites associated with all breakpoint cluster regions, whereas SARs associated with ABL and AF4, but not with BCR. No close breakpoint clustering with the topo II/DNase I sites were observed; however, a statistically significant 5′ or 3′ distribution of patient breakpoints to the topo II DNase I sites was found, implicating DNA repair and exonucleases. Although MLL2 was expressed in all cell lines tested, except for the presence of one DNAse I site in the promoter, no other structural elements were found in MLL2. A NHR model presented demonstrates the importance of chromatin structure in chromosome translocations involved with leukemia.     

The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia.

Friedreich ataxia (FA) is associated with the expansion of a GAA trinucleotide repeat in the first intron of the X25 gene. We found both alleles expanded in 67 FA patients from 48 Italian families. Five patients from three families were compound heterozygotes with expansion on one allele and an isoleucine-->phenylalanine change at position 154 on the other one. We found neither expansions nor point mutations in three patients. The length of FA alleles ranged from 201 to 1,186 repeat units, with no overlap with the normal range, and showed a negatively skewed distribution with a peak between 800 and 1,000 repeats. The FA repeat showed meiotic instability with a median variation of 150 repeats. The lengths of both larger and smaller alleles in each patient inversely correlated with age at onset of the disorder. Smaller alleles showed the best correlation, accounting for approximately 50% of the variation of age at onset. Mean allele length was significantly higher in patients with diabetes and in those with cardiomyopathy.     

Expansion of GAA trinucleotide repeats in mammals

We have previously shown that GAA trinucleotide repeats have undergone significant expansion in the human genome. Here we present the analysis of the length distribution of all 10 nonredundant trinucleotide repeat motifs in 20 complete eukaryotic genomes (6 mammalian, 2 nonmammalian vertebrates, 4 arthropods, 4 fungi, and 1 each of nematode, amoebozoa, alveolate, and plant), which showed that the abundance of large expansions of GAA trinucleotide repeats is specific to mammals. Analysis of human-chimpanzee-gorilla orthologs revealed that loci with large expansions are species-specific and have occurred after divergence from the common ancestor. PCR analysis of human controls revealed large expansions at multiple human (GAA)(30+) loci; nine loci showed expanded alleles containing >65 triplets, analogous to disease-causing expansions in Friedreich ataxia, including two that are in introns of genes of unknown function. The abundance of long GAA trinucleotide repeat tracts in mammalian genomes represents a significant mutation potential and source of interindividual variability.     

G130V, a common FRDA point mutation, appears to have arisen from a common founder

Friedreich ataxia (FRDA) is the most common inherited ataxia. About 98% of mutant alleles have an expansion of a GAA trinucleotide repeat in intron 1 of the affected gene, FRDA. The other 2% are point mutations. Of the 17 point mutations so far described, three appear to be more common. One of these is the G130V mutation in exon 4 of FRDA. G130V, when present with an expanded GAA repeat on the other allele, is associated with an atypical FRDA phenotype. Haplotype analysis was undertaken on the four families who have been described with this mutation. The results suggest a common founder for this mutation. Although marked differences in extragenic marker haplotypes were seen in one family, similar intragenic haplotyping suggests the same mutation founder for this family with the differences explicable by two recombination events.     

Detection of interruptions in the GAA trinucleotide repeat expansion in the FXN gene of Friedreich ataxia

Friedreich ataxia is a neurodegenerative disorder caused by the expansion of a GAA trinucleotide repeat sequence within the first intron of the FXN gene. Interruptions in the GAA repeat may serve to alleviate the inhibitory effects of the GAA expansion on FXN gene expression and to decrease pathogenicity. We have developed a simple and rapid PCR- and restriction enzyme-based assay to assess the purity of GAA repeat sequences.     

Nucleotide-resolution mapping of topoisomerase-mediated and apoptotic DNA strand scissions at or near an MLL translocation hotspot

The emergence of therapy-related acute myeloid leukemia (t-AML) has been associated with DNA topoisomerase II (TOP2)-targeted drug treatments and chromosomal translocations frequently involving the MLL, or ALL-1, gene. Two distinct mechanisms have been implicated as potential triggers of t-AML translocations: TOP2-mediated DNA cleavage and apoptotic higher-order chromatin fragmentation. Assessment of the role of TOP2 in this process has been hampered by a lack of techniques allowing in vivo mapping of TOP2-mediated DNA cleavage at nucleotide resolution in single-copy genes. A novel method, extension ligation-mediated polymerase chain reaction (ELMPCR), was used here for mapping topoisomerase-mediated DNA strand breaks and apoptotic DNA cleavage across a translocation-prone region of MLL in human cells. We report the first genomic map integrating translocation breakpoints and topoisomerase I, TOP2, and apoptotic DNA cleavage sites at nucleotide resolution across an MLL region harboring a t-AML translocation hotspot. This hotspot is flanked by a TOP2 cleavage site and is localized at one extremity of a minor apoptotic cleavage region, where multiple single- and double-strand breaks were induced by caspase-activated apoptotic nucleases. This cleavage pattern was in sharp contrast to that observed approximately 200 bp downstream in the exon 12 region, which displayed much stronger apoptotic cleavage but where no double-strand breaks were detected and no t-AML-associated breakpoints were reported. The localization and remarkable clustering of the t-AML breakpoints cannot be explained simply by the DNA cleavage patterns but might result from potential interactions between TOP2 poisoning, apoptotic DNA cleavage, and DNA repair attempts at specific sites of higher-order chromatin structure in apoptosis-evading cells. ELMPCR provides a new tool for investigating the role of DNA topoisomerases in fundamental genetic processes and translocations associated with cancer treatments involving topoisomerase-targeted drugs.     

ABR, an active BCR-related gene.

The human BCR gene on chromosome 22 is specifically involved in the Philadelphia translocation, t(9;22), a chromosomal rearrangement present in the leukemic cells of patients with chronic myeloid leukemia or acute lymphoblastic leukemia. In most cases, the breakpoints on chromosome 22 are found within a 5.8 kb region of DNA designated the major breakpoint cluster region (Mbcr) of the BCR gene. Hybridization experiments have indicated that the human genome contains BCR gene-related sequences. Here we report the molecular cloning of one of these loci, for which we propose the name ABR. In contrast with the other BCR-related genes studied to date, ABR represents a functionally active gene and contains exons very similar to those found within the Mbcr. Unlike the BCR gene, the ABR gene exhibits great genomic variability caused by two different variable tandem repeat regions located in two introns. All other BCR gene-related sequences isolated so far and the BCR gene itself are located on chromosome 22. In contrast, the ABR gene is located on chromosome 17p.     

The MLLT3 Gene Maps between D9S156 and D9S171 and Contains an Unstable Polymorphic Trinucleotide Repeat

MLLT3, one of the genes shown to be a translocation breakpoint partner for the acute lymphocytic leukemia (MLL) gene, has been mapped to 9p22. We have identified a polymorphic trinucleotide repeat within this gene that shows somatic instability. The inheritance pattern of this polymorphism in recombinant individuals from families previously typed for other chromosome 9 markers indicates that the gene lies in the interval bounded by D9S156 and D9S171.     

The identification of novel small-molecule inhibitors targeting WDR5-MLL1 interaction through fluorescence polarization based high-throughput screening

The protein-protein interaction between WDR5 (WD40 repeat protein 5) and MLL1 (mixed-lineage leukemia 1) is important for maintaining optimal H3K4 methyltransferase activity of MLL1. Dysregulation of MLL1 catalytic function is relevant to mixed-lineage leukemia, and targeting WDR5-MLL1 interaction could be a promising therapeutic strategy for leukemia harboring MLL1 fusion proteins. To date, several peptidomimetic and non-peptidomimetic small-molecule inhibitors targeting WDR5-MLL1 interaction have been reported, yet the discovery walk of new drugs inhibiting MLL1 methytransferase activity is still in its infancy. It’s urgent to find other small-molecule WDR5-MLL1 inhibitors with novel scaffolds. In this study, through fluorescence polarization (FP)-based high throughput screening, several small-molecule inhibitors with potent inhibitory activities in vitro against WDR5-MLL1 interaction were discovered. Nuclear Magnetic Resonance (NMR) assays were carried out to confirm the direct binding between hit compounds and WDR5. Subsequent similarity-based analog searching of the 4 hits led to several inhibitors with better activity, among them, DC_M5_2 displayed highest inhibitory activity with IC₅₀ values of 9.63?±?1.46?µM. Furthermore, a molecular docking study was performed and disclosed the binding modes and interaction mechanisms between two most potent inhibitors and WDR5.     

Altered DNA topoisomerase II in multidrug resistance

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected. We propose that MDR associated with alterations in topo II may have clinical consequences, and our current efforts involve exploiting these biochemical and molecular observations in the development of probes that may be useful to identify such drug resistant cells in the tumors of patients.     

Determination of BCR/ABL translocation in radiation-associated acute myeloid leukemia patients by fluorescence in situ hybridization

We report the results of BCR/ABL translocation analysis on interphase leukemic cells of 33 acute myeloid leukemia (AML) patients by fluorescence in situ hybridization. Of these, there were 13 persons exposed to ionizing radiation due to the Chernobyl accident with radiation-associated AML and 20 patients with spontaneous disease. BCR/ABL translocation which was detected in 4 and I case respectively may play an important role in radiation-induced leukemigenesis.     

MLL1 is regulated by KSHV LANA and is important for virus latency

Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi''s sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-Å crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.