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.

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Mechanisms of MLL gene rearrangement: site-specific DNA cleavage within the breakpoint cluster region is independent of chromosomal context

Human Molecular Genetics ◽

10.1093/hmg/10.22.2481 ◽

2001 ◽

Vol 10 (22) ◽

pp. 2481-2491 ◽

Cited By ~ 18

Author(s):

M. Stanulla

Keyword(s):

Dna Cleavage ◽

Gene Rearrangement ◽

Site Specific ◽

Breakpoint Cluster Region ◽

Cluster Region ◽

Mll Gene ◽

Mll Gene Rearrangement

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DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.4070 ◽

1997 ◽

Vol 17 (7) ◽

pp. 4070-4079 ◽

Cited By ~ 130

Author(s):

M Stanulla ◽

J Wang ◽

D S Chervinsky ◽

S Thandla ◽

P D Aplan

Keyword(s):

Dna Cleavage ◽

Cellular Response ◽

Topoisomerase Ii ◽

Apoptotic Cell ◽

Chemotherapeutic Agents ◽

Higher Order ◽

Site Specific ◽

Breakpoint Cluster Region ◽

Cluster Region ◽

Topo Ii

A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML.

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Comparison of DNA cleavage induced by etoposide and doxorubicin in two human small-cell lung cancer lines with different sensitivities to topoisomerase II inhibitors

Lung Cancer ◽

10.1016/0169-5002(90)90227-d ◽

1990 ◽

Vol 6 (3-4) ◽

pp. 139-140

Keyword(s):

Lung Cancer ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Dna Cleavage ◽

Topoisomerase Ii ◽

Small Cell ◽

Small Cell Lung ◽

Topoisomerase Ii Inhibitors

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Site-Specific DNA Cleavage byChlorellaVirus Topoisomerase II†

Biochemistry ◽

10.1021/bi025802g ◽

2002 ◽

Vol 41 (39) ◽

pp. 11761-11769 ◽

Cited By ~ 11

Author(s):

John M. Fortune ◽

Jennifer S. Dickey ◽

Oleg V. Lavrukhin ◽

James L. Van Etten ◽

R. Stephen Lloyd ◽

...

Keyword(s):

Dna Cleavage ◽

Topoisomerase Ii ◽

Site Specific

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Conformational properties of topoisomerase II inhibitors and sequence specificity of DNA cleavage

Journal of Molecular Recognition ◽

10.1002/jmr.300070312 ◽

1994 ◽

Vol 7 (3) ◽

pp. 227-231 ◽

Cited By ~ 5

Author(s):

M. Palumbo ◽

M. Mabilia ◽

A. Pozzan ◽

G. Capranico ◽

S. Tinelli ◽

...

Keyword(s):

Dna Cleavage ◽

Topoisomerase Ii ◽

Sequence Specificity ◽

Topoisomerase Ii Inhibitors ◽

Conformational Properties

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Specific Histone Patterns and Acetylase/Deacetylase Activity at the Breakpoint-Cluster Region of the Human MLL Gene

Cancer Research ◽

10.1158/0008-5472.can-03-1126 ◽

2004 ◽

Vol 64 (8) ◽

pp. 2656-2662 ◽

Cited By ~ 14

Author(s):

Andriy Khobta ◽

Carmelo Carlo-Stella ◽

Giovanni Capranico

Keyword(s):

Breakpoint Cluster Region ◽

Cluster Region ◽

Mll Gene

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Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia: correlation with scaffold attachment regions and topoisomerase II consensus binding sites

Blood ◽

10.1182/blood.v87.5.1912.1912 ◽

1996 ◽

Vol 87 (5) ◽

pp. 1912-1922 ◽

Cited By ~ 167

Author(s):

PL Broeker ◽

HG Super ◽

MJ Thirman ◽

H Pomykala ◽

Y Yonebayashi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Topoisomerase Ii ◽

De Novo ◽

Lymphoblastic Leukemia ◽

Breakpoint Cluster Region ◽

Cluster Region ◽

Topo Ii ◽

Acute Myeloid

Abstract A major unresolved question for 11q23 translocations involving MLL is the chromosomal mechanism(s) leading to these translocations. We have mapped breakpoints within the 8.3-kb BamHI breakpoint cluster region in 31 patients with acute lymphoblastic leukemia and acute myeloid leukemia (AML) de novo and in 8 t-AML patients. In 23 of 31 leukemia de novo patients, MLL breakpoints mapped to the centromeric half (4.57 kb) of the breakpoint cluster region, whereas those in eight de novo patients mapped to the telomeric half (3.87 kb). In contrast, only two t-AML breakpoints mapped in the centromeric half, whereas six mapped in the telomeric half. The difference in distribution of the leukemia de novo breakpoints is statistically significant (P = .02). A similar difference in distribution of breakpoints between de novo patients and t-AML patients has been reported by others. We identified a low- or weak-affinity scaffold attachment region (SAR) mapping just centromeric to the breakpoint cluster region, and a high-affinity SAR mapping within the telomeric half of the breakpoint cluster region. Using high stringency criteria to define in vitro vertebrate topoisomerase II (topo II) consensus sites, one topo II site mapped adjacent to the telomeric SAR, whereas six mapped within the SAR. Therefore, 74% of leukemia de novo and 25% of t-AML breakpoints map to the centromeric half of the breakpoint cluster region map between the two SARs; in contrast, 26% of the leukemia de novo and 75% of the t-AML patient breakpoints map to the telomeric half of the breakpoint cluster region that contains both the telomeric SAR and the topo II sites. Thus, the chromatin structure of the MLL breakpoint cluster region may be important in determining the distribution of the breakpoints. The data suggest that the mechanism(s) leading to translocations may differ in leukemia de novo and in t-AML.

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Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia: correlation with scaffold attachment regions and topoisomerase II consensus binding sites

Blood ◽

10.1182/blood.v87.5.1912.bloodjournal8751912 ◽

1996 ◽

Vol 87 (5) ◽

pp. 1912-1922 ◽

Cited By ~ 6

Author(s):

PL Broeker ◽

HG Super ◽

MJ Thirman ◽

H Pomykala ◽

Y Yonebayashi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Topoisomerase Ii ◽

De Novo ◽

Lymphoblastic Leukemia ◽

Breakpoint Cluster Region ◽

Cluster Region ◽

Topo Ii ◽

Acute Myeloid

A major unresolved question for 11q23 translocations involving MLL is the chromosomal mechanism(s) leading to these translocations. We have mapped breakpoints within the 8.3-kb BamHI breakpoint cluster region in 31 patients with acute lymphoblastic leukemia and acute myeloid leukemia (AML) de novo and in 8 t-AML patients. In 23 of 31 leukemia de novo patients, MLL breakpoints mapped to the centromeric half (4.57 kb) of the breakpoint cluster region, whereas those in eight de novo patients mapped to the telomeric half (3.87 kb). In contrast, only two t-AML breakpoints mapped in the centromeric half, whereas six mapped in the telomeric half. The difference in distribution of the leukemia de novo breakpoints is statistically significant (P = .02). A similar difference in distribution of breakpoints between de novo patients and t-AML patients has been reported by others. We identified a low- or weak-affinity scaffold attachment region (SAR) mapping just centromeric to the breakpoint cluster region, and a high-affinity SAR mapping within the telomeric half of the breakpoint cluster region. Using high stringency criteria to define in vitro vertebrate topoisomerase II (topo II) consensus sites, one topo II site mapped adjacent to the telomeric SAR, whereas six mapped within the SAR. Therefore, 74% of leukemia de novo and 25% of t-AML breakpoints map to the centromeric half of the breakpoint cluster region map between the two SARs; in contrast, 26% of the leukemia de novo and 75% of the t-AML patient breakpoints map to the telomeric half of the breakpoint cluster region that contains both the telomeric SAR and the topo II sites. Thus, the chromatin structure of the MLL breakpoint cluster region may be important in determining the distribution of the breakpoints. The data suggest that the mechanism(s) leading to translocations may differ in leukemia de novo and in t-AML.

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MLL-USP2: An Underestimated New Entity of MLL-Rearranged Leukemia Identified By NGS Analysis

Blood ◽

10.1182/blood-2018-99-112594 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3920-3920 ◽

Cited By ~ 1

Author(s):

Claus Meyer ◽

Bruno Lopes ◽

Aurélie Caye-Eude ◽

Hélène Cavé ◽

Chloé Arfeuille ◽

...

Keyword(s):

High Risk ◽

Acute Leukemia ◽

Diagnostic Methods ◽

Patient Specific ◽

Fish Analysis ◽

Acute Leukemias ◽

Breakpoint Cluster Region ◽

Cluster Region ◽

Mll Gene ◽

Partner Gene

Abstract Chromosomal rearrangements of the MLL gene are responsible for 5-10% of all acute leukemias, biphenotypic leukemias and myelodysplastic syndromes. The large number of known MLL fusions (>80) renders a precise diagnosis a demanding task. Even though all MLL rearrangements are associated with high-risk acute leukemia, the outcome (poor or very poor) is influenced by the partner gene. The applied diagnostic methods (LDI-PCR and multiplex PCR) allows the identification of MLL fusion genes at the nucleotide level, providing important information on the genetics of leukemia patients, and patient-specific biomarkers. These biomarkers are used for monitoring of minimal residual disease in acute leukemia patients during and after therapy. Thus, the identification of MLL gene fusions is necessary for rapid clinical decisions to determine the best therapy regimen. We have developed a customized NGS panel for MLL diagnostics to utilize state of the art technology at DCAL. With this new tool, the whole MLL gene is analyzed in contrast to the LDI-PCR where only the main MLL breakpoint cluster region (BCR-1) is covered. The first results of the NGS analysis of 84 patients identified MLL breakpoints located outside the main BCR-1 of MLL. Furthermore, a novel MLL partner gene USP2 was identified in 16 patients. All MLL-USP2 positive patients had a breakpoint located outside BCR-1 and within a newly defined breakpoint cluster region BCR-2. The BCR-2 site was also used in 2 other patients with MLL-AFF1 and one patient with MLL-MLLT3. These findings reveal USP2 as a new entity for MLL rearrangements affecting indifferently children aged 3 months to 10 years old (mean 30 months) with no gender bias (M/F=1.3). Interestingly, only 5/16 affected children were below 1 year of age at diagnosis and thus treated according to the Interfant trial. Clinical presentation as well as outcome associated with this new entity deserves further investigation to define whether those patients should be allocated, as other MLL-rearranged ones, in high-risk treatment groups. More MLL patients should also be analyzed to get a better idea of the frequency of breakpoints within BCR-2, especially the frequency of MLL-USP2 fusions. Indeed, standard FISH analysis and CGH array do not permit reliable detection of this fusion, explaining why they remained undetected so far. The biology of this novel MLL rearrangement also deserves further investigation, considering that USP2 is the only MLL partner fused exclusively to BCR-2. Disclosures No relevant conflicts of interest to declare.

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