Panhandle and reverse-panhandle PCR enable cloning of der(11) and der(other) genomic breakpoint junctions ofMLLtranslocations and identify complex translocation ofMLL,AF-4, andCDK6

Raffini, Leslie; Slater, Diana J.; Rappaport, Eric; Nigro, Luca Lo; Cheung, Nai‐Kong V.; Biegel, Jaclyn A.; Nowell̀, Peter C.; Lange, Beverly J.; Felix, Carolyn A.

doi:10.1073/pnas.062066799

Cited by 49 publications

(56 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Furthermore, most TOP2A breaks are SSNs (Supplemental Fig. S2), and it is well established that paired SSNs on opposite DNA strands and template-directed polymerization of long overhangs are required to create the KMT2A translocation breakpoint junctions with the several-hundred-base sequence duplications typical of leukemia in infants (Gillert et al 1999;Raffini et al 2002). These findings support a model in which TOP2A cleavage triggers KMT2A translocations in both treatment-related and infant leukemia.…”

Section: Genes Involved In Oncogenic Translocations Show Top2a Ccr Ensupporting

confidence: 66%

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci

Davenport

Urtishak

et al. 2017

Genome Res.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Genes Involved In Oncogenic Translocations Show Top2a Ccr Ensupporting

confidence: 66%

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci

Davenport

Urtishak

et al. 2017

Genome Res.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Several clones had breakpoints that localize to MLL bcr sequences identified at rearrangement junctions in therapy-related leukemias. 32,[46][47][48][49] These data indicate that although etoposideinitiated rearrangements of MLL are frequent, only a small subset of chromosomal translocations occurs in cells that are capable of continued proliferation and, ultimately, leukemogenesis. …”

mentioning

confidence: 97%

Therapy-related acute myeloid leukemia–like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion

Libura

Slater

Felix

et al. 2005

Blood

Self Cite

View full text Add to dashboard Cite

Rearrangements involving the MLL gene on chromosome band 11q23 are a hallmark of therapy-related acute myeloid leukemias following treatment with topoisomerase II poisons including etoposide. Therapy-related and de novo genomic translocation breakpoints cluster within a well-characterized 8.3-kb fragment of MLL. Repair of etoposidestabilized DNA topoisomerase II covalent complexes may initiate MLL rearrangements observed in patients. We used a culture system of primary human hematopoietic CD34 ؉ cells and inverse polymerase chain reaction to characterize the spectrum of stable genomic rearrangements promoted by etoposide exposure originating within an MLL translocation hotspot in therapy-related leukemia. Alterations to the region were observed at a readily detectable frequency in etoposidetreated cells. Illegitimate repair events after minimal repair included MLL tandem duplications and translocations, with minor populations of deletions or insertions. In stably repaired cells that proliferated for 10 to 14 days, the significant majority of illegitimate events were MLL IntroductionGenome integrity is controlled by multiple damage surveillance pathways, cell-cycle checkpoints, and repair mechanisms. [1][2][3] Despite these safeguards, illegitimate repair of chromosomal doublestrand breaks (DSBs), such as those produced by chemotherapy agents that target topoisomerase II (topo II), can promote chromosomal rearrangements and, ultimately, tumorigenesis. Topo II is an essential cellular enzyme that catalyzes changes in DNA topology via its cleavage-religation equilibrium. Topo II-targeted drugs that are poisons of this enzyme stabilize topo II-DNA covalent complexes, most often by decreasing the rate of religation in a dose-dependent manner. Disruption of the cleavage-religation reaction results in accumulation of DSBs, p53 activation, and induction of apoptosis or repair. 4,5 Chromosomal DSBs are potent inducers of recombination, stimulating the exchange of homologous sequences between 2 DNA duplexes 1000-fold 6-8 not only between sister chromatids, but also between homologs, and sequence repeats on heterologous chromosomes. 9-12 DSBs may be repaired by homologous recombination or nonhomologous end joining (NHEJ), and both repair mechanisms have been associated with chromosomal translocations, a hallmark of leukemias, lymphomas, and soft-tissue sarcomas. [13][14][15] There is clear evidence that exposure to chemotherapy or irradiation can result in subsequent development of therapy-related leukemias, which occur in 1% to 15% of patients exposed to DNA-damaging agents in anticancer regimens. 16,17 Exposure to topo II poisons such as etoposide is predominantly associated with therapy-related leukemias characterized by rearrangements of the MLL gene on chromosome band 11q23. 16 MLL spans 100 kb, encodes a 430-kDa protein homologous to the Drosophila trithorax gene, and has important functions in embryogenesis and hematopoiesis. 18,19 Mixed-lineage leukemia (MLL) is a critical transcriptional regulator and numerous tr...

show abstract

“…Both methods have been applied successfully in the past to identify chromosomal breakpoints (15)(16)(17)(18)(19)(20)(21), but they were never optimized for high-throughput analysis or for clinical use.…”

mentioning

confidence: 99%

Diagnostic tool for the identification of MLL rearrangements including unknown partner genes

Meyer

Schneider

Reichel

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

169

157

View full text Add to dashboard Cite

Approximately 50 different chromosomal translocations of the human MLL gene are currently known and associated with high-risk acute leukemia. The large number of different MLL translocation partner genes makes a precise diagnosis a demanding task. After their cytogenetic identification, only the most common MLL translocations are investigated by RT-PCR analyses, whereas infrequent or unknown MLL translocations are excluded from further analyses. Therefore, we aimed at establishing a method that enables the detection of any MLL rearrangement by using genomic DNA isolated from patient biopsy material. This goal was achieved by establishing a universal longdistance inverse-PCR approach that allows the identification of any kind of MLL rearrangement if located within the breakpoint cluster region. This method was applied to biopsy material derived from 40 leukemia patients known to carry MLL abnormalities. Thirty-six patients carried known MLL fusions (34 with der(11) and 2 with reciprocal alleles), whereas 3 patients were found to carry novel MLL fusions to ACACA, SELB, and SMAP1, respectively. One patient carried a genomic fusion between MLL and TIRAP, resulting from an interstitial deletion. Because of this interstitial deletion, portions of the MLL and TIRAP genes were deleted, together with 123 genes located within the 13-Mbp interval between both chromosomal loci. Therefore, this previously undescribed diagnostic tool has been proven successful for analyzing any MLL rearrangement including previously unrecognized partner genes. Furthermore, the determined patientspecific fusion sequences are useful for minimal residual disease monitoring of MLL associated acute leukemias.acute leukemia ͉ MLL translocations ͉ translocation partner genes C hromosomal translocations involving the human MLL gene are recurrently associated with high-risk acute leukemias (1-4). MLL translocations correlate with specific disease subtypes (acute myeloid and acute lymphocytic leukemias), a specific gene expression profile (5, 6), and outcome (favorable or poor), depending on the particular MLL fusion (7). Approximately 50 different MLL translocation partner genes have been identified, suggesting that the human MLL gene is a hot spot for illegitimate recombination events. During illegitimate recombination events, one MLL allele is reciprocally fused with one of the many translocation partner genes. The latter encode nuclear or cytosolic proteins that share only a little sequence homology; however, the fused portion of partner protein sequences is necessary to confer oncogenic potential.The unambiguous identification of these MLL translocations is necessary to support rapid clinical decisions and specific therapy regimens. Current procedures to diagnose MLL rearrangements include cytogenetic analysis, FISH experiments (e.g., split-signal FISH) (8), and specific RT-PCR methods. However, results of RT-PCR analyses are influenced strongly by the quality of the investigated RNA samples. Furthermore, only the most frequent MLL fusions routine...

show abstract

Panhandle and reverse-panhandle PCR enable cloning of der(11) and der(other) genomic breakpoint junctions ofMLLtranslocations and identify complex translocation ofMLL,AF-4, andCDK6

Cited by 49 publications

References 38 publications

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci

Therapy-related acute myeloid leukemia–like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion

Diagnostic tool for the identification of MLL rearrangements including unknown partner genes

Contact Info

Product

Resources

About