Type II topoisomerases orchestrate proper DNA topology, and they are the targets of anti-cancer drugs that cause treatment-related leukemias with balanced translocations. Here, we develop a high-throughput sequencing technology to define TOP2 cleavage sites at single-base precision, and use the technology to characterize TOP2A cleavage genome-wide in the human K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence preferences, occurs in cleavage cluster regions (CCRs), and is enriched in introns and lincRNA loci. TOP2A CCRs are biased toward the distal regions of gene bodies, and TOP2 poisons cause a proximal shift in their distribution. We find high TOP2A cleavage levels in genes involved in translocations in TOP2 poison-related leukemia. In addition, we find that a large proportion of genes involved in oncogenic translocations overall contain TOP2A CCRs. The TOP2A cleavage of coding and lincRNA genes is independently associated with both length and transcript abundance. Comparisons to ENCODE data reveal distinct TOP2A CCR clusters that overlap with marks of transcription, open chromatin, and enhancers. Our findings implicate TOP2A cleavage as a broad DNA damage mechanism in oncogenic translocations as well as a functional role of TOP2A cleavage in regulating transcription elongation and gene activation.
We prospectively observed a child exposed to intensive multimodality therapy for metastatic neuroblastoma from emergence of a MLL translocation to disease diagnosis. The t(4;11)(p12;q23) was detected in the marrow 17 months after starting treatment following topoisomerase II poisons, alkylating agents, local radiation, hematopoietic stem cell transplantation, anti-GD2 monoclonal antibody with granulocyte macrophagecolony-stimulating factor, and a high cumulative dose of oral etoposide. Reciprocal genomic breakpoint junctions and fusion transcripts joined MLL with FRYL, the Drosophila melanogaster protein homologue of which regulates cell fate. Etoposide metabolites induced topoisomerase II cleavage complexes that could form both breakpoint junctions. Cells harboring the translocation replaced the marrow without clinical evidence of leukemia and differentiation appeared unaffected for 37 months. Subsequent bilineage dysplasia and increased blasts in addition to the translocation fulfilled criteria for MDS. The IntroductionEpipodophyllotoxins, anthracyclines, and other chemotherapeutic topoisomerase II poisons are associated with leukemias with translocations of the MLL gene at chromosome band 11q23. The MLL gene product is a multidomain oncoprotein that functions in a macromolecular protein complex to regulate transcription. [1][2][3] Recently dubbed the "MLL recombinome," the approximately 50 MLL partner genes encode diverse nuclear transcription factors, transcriptional regulatory proteins, and cytoplasmic or cell membrane proteins. 4 Murine experiments have converged upon a model where heterogeneous MLL fusion oncoproteins affect the incidence or phenotype of leukemia by altering Hox expression. 5,6 It has been proposed that MLL translocations fall into a group of mutations that impair differentiation 7 and that cooperating mutations that confer a proliferative and/or survival advantage to hematopoietic progenitors (eg, mutation or overexpression of FLT3 [8][9][10] ) are required for leukemia to occur.Many MLL fusion proteins from the der(11) chromosome immortalize murine hematopoietic progenitor cells in serial replating assays and/or cause leukemia in mice. [11][12][13][14][15][16][17] However, certain MLL translocations with genes encoding some of the cytoplasmic partner proteins (eg, GRAF, FBP17, ABI1, LASP1) are inactive in serial replating assays, which may reflect the requirement for cooperating alterations. 18,19 Although nearly all known MLL translocations, including those inactive in serial replating assays, [18][19][20][21] manifest as leukemia/myelodysplastic syndrome (MDS) in patients; in one patient, the morphologically normal clone harboring an MLL-ARHGEF17 translocation steadily declined over 30 months, and neither leukemia nor MDS occurred. 22 We characterized a chemotherapy-associated MLL translocation with the partner gene FRYL (furry homolog-like; originally called MIFL [MLL Insufficient for Leukemia]), and prospectively followed the clinical course of the affected patient. First MLL-FRYL ...
Introduction: Patients with birth defects have a higher incidence of cancer; however, an association of amniotic band syndrome (vascular disruption syndrome) with congenital leukemia has never been reported. We characterized a case of MLL-rearranged leukemia in a neonate affected with this birth defect. Methods: The MLL translocation was characterized in peripheral AML blasts by karyotype analysis, multicolor FISH, Southern blot analysis and genomic and cDNA panhandle PCR. NQO1 genotype was determined by real-time PCR. Clinical history and results: The infant was born at 38 weeks gestation by C-section for suboccipital encephalocele to a 21 year-old gravida 3 para 2 mother with a history of cigarette, marijuana, cocaine and opiate use, and antidepressant, antipsychotic, barbiturate, caffeine and proton pump inhibitor treatment during pregnancy. Drug screen at delivery was positive for opioids and barbiturates. In addition to the encephalocele, a circular constriction of the right arm, consistent with amniotic band syndrome, and blueberry muffin lesions were noted at delivery. The CBC showed mild thrombocytopenia that resolved the next day. Congenital infection was excluded. The encephalocele was repaired. At 19–20 days of age the infant became septic and hepatosplenomegaly and hyperleukocytosis were observed. The peripheral smear and flow cytomery revealed acute myeloid leukemia with monocytic differentiation (CD45+ population positive for CD4, CD14, CD33, CD38, HLA-DR). Karyotype analysis showed a complex structural abnormality disrupting chromosomes 4, 11 and 19 involving MLL. M-FISH showed insertion of chromosome 11 material into a chromosome 19 and translocation between chromosomes 11q and 4q. The infant received cytosine arabinoside and daunomycin but succumbed to AML, sepsis and multi-organ failure within 4 days. Autopsy showed marrow, viscera, brain and skin infiltration with AML and Chiari type III brain malformation. Southern blot analysis detected two MLL bcr rearrangements. Panhandle PCR demonstrated fusion of MLL intron 6 and intron 1 of ELL from band 19p13.1. Short sequence homologies at the breakpoint junction suggested DNA damage resolution by NHEJ repair. The corresponding transcript joined MLL exon 6 to ELL exon 2. The infant was wild-type at NQO1 C609T. Conclusions: This is the first association of amniotic band syndrome and congenital AML, both of which are rare conditions. Although the cause(s) are unknown, both conditions originate in utero and maternal exposures during pregnancy may be relevant. There was a history of maternal fetal loss, which is a risk factor for leukemia in infants. The NQO1 substrate p-benzoquinone in cigarette smoke is a topoisomerase II poison, but the infant did not harbor the NQO1 variant that predisposes to leukemia. Cocaine is an in utero exposure implicated in amniotic band syndrome. The occurrence of amniotic band syndrome and congenital AML in this infant raises questions about potential host predisposition or gene-environment interactions common to both conditions. Alternatively, both rare conditions may have occurred by chance alone in the setting of the many in utero exposures.
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