Chromatin structural elements and chromosomal translocations in leukemia

d n a r e p a i r 5 ( 2 0 0 6 ) 1282–1297

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Chromatin structural elements and chromosomal translocations in leukemia Yanming Zhang, Janet D. Rowley ∗ Section of Hematology/Oncology, Department of Medicine, University of Chicago, 5841 S. Maryland Ave., Chicago, IL, United States

a r t i c l e

i n f o

a b s t r a c t

Article history:

Recurring chromosome abnormalities are strongly associated with certain subtypes of

Published on line 7 August 2006

leukemia, lymphoma and sarcomas. More recently, their potential involvement in carcinomas, i.e. prostate cancer, has been recognized. They are among the most important

Keywords:

factors in determining disease prognosis, and in many cases, identification of these chro-

Non-homologous end joining

mosome abnormalities is crucial in selecting appropriate treatment protocols. Chromosome

Topoisomerase II

translocations are frequently observed in both de novo and therapy-related acute myeloid

Leukemia

leukemia (AML) and myelodysplastic syndromes (MDS). The mechanisms that result in such

Chromosomal translocations

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 therapyrelated leukemia. We propose a non-homologous chromosome recombination model as

∗

Corresponding author. Tel.: +1 773 702 6117; fax: +1 773 702 3002. E-mail address: [email protected] (J.D. Rowley).

1568-7864/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.dnarep.2006.05.020

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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. © 2006 Published by Elsevier B.V.

1. Overview of recurring chromosome translocations in leukemia and lymphoma Since the discovery of the Philadelphia chromosome in 1960 which later was recognized as a balanced t(9;22) [1,2], the hallmark of chronic myeloid leukemia (CML), many recurring chromosome abnormalities have been identified by cytogenetic analysis in various human leukemia, lymphoma and other cancers [3–5]. In the past decade, using molecular cytogenetic techniques, i.e. fluorescence in situ hybridization (FISH), spectral karyotyping (SKY or M-FISH) and comparative genomic hybridization, more chromosome abnormalities in hematological diseases and solid tumors have been identified (Table 1) [6,7]. These recurring chromosome abnormalities are acquired somatic mutations present in the malignant cells. They are strongly associated with particular histological and immunological subtypes of leukemia and lymphoma as well as sarcoma and recently, epithelial cancers [8,9]. They are among the most important factors in determining disease prognosis, and in many cases, detection of these chromosome abnormalities is crucial not only in diagnosis and sub-classification but also in selecting appropriate treatment protocols [10]. Identification of these chromosome aberrations greatly facilitates cloning of the relevant genes that are involved in tumorigenesis, in particular in leukemia and lymphoma, and the resultant altered proteins form the basis for tumor specific therapy targeted at the genetic mutation, such as the recent success of treatment with Gleevec in CML and acute lymphoblastic leukemia (ALL) with t(9;22) [11]. Because of their specific association with distinct subtypes of leukemia, certain chromosome translocations and inversions have recently been incorporated in the WHO classification as the criteria for sub-classification of acute myeloid leukemia (AML), including the t(8;21), t(15;17), inv(16) or 11q23 rearrangements, regardless of the morphology or percent blast cells [8]. Most chromosome translocations in leukemia and lymphoma arise in hematopoietic stem cells and are reciprocal, stable and balanced at the cytogenetic level. In childhood leukemia, translocations may arise before birth, during fetal hematopoiesis [12]. Many of the recurring chromosome translocations in various B- and T-cell leukemia and lymphoma, such as t(8;14), t(11;14) and t(14;18), result in the juxtaposition of the immunoglobulin genes (IGH, IGK and IGL) or T-cell receptor (TCR) genes with certain oncogenes that leads to an increased expression of oncogenes or anti-apoptotic genes but the protein produced is normal [13,14]. In contrast, the majority of common chromosome translocations in myeloid leukemia, such as the t(8;21) and t(15;17), result in a novel chimeric fusion gene with novel functions that interferes with myeloid cell maturation [15]. Recently, specific gene rearrangements and chromosome translocations have also been identified in certain chronic myeloproliferative dis-

eases, such as polycythemia vera, essential thrombocythemia. These chromosome translocations mostly involve the tyrosine kinase genes, such as PDGFRB, FGFR1 and JAK2 (Table 1) [16–18]. The function of the novel fusion genes may involve tyrosine kinases, such as ABL, transcription factors (AML1), growth factors (IL3) or their receptors (TAN1). According to their function or the targeted genes, chromosome translocations in leukemia may be grouped into the seven subsets indicated in Table 1. Chromosome translocations are frequently observed not only in de novo acute leukemia but also in therapy-related AML (t-AML) and myelodysplastic syndromes (MDS), which may develop in patients who received chemotherapy and/or radiation treatment for previous cancers [19,20]. Therapyrelated leukemia is a major later complication of successful therapy for a prior cancer or other disease, occurring in between 2% and 10% of patients depending on the treatment. More than 90% of t-AML/MDS patients show clonal chromosome abnormalities, with abnormalities involving chromosomes 5 and/or 7 in 70% and balanced chromosome translocations that frequently involve MLL at 11q23 and AML1 at 21q22 in 10% of patients [19–22]. t(9;11) is detected in 48% of t-AML involving MLL and the t(4;11) in 9% of all t-ALL [23]. Translocations in t-AML involving the AML1 gene are most commonly t(8;21) (35%) and t(3;21) (13%) [24]. Two types of t-AML/MDS have been identified each with unique features. One is related to previous treatment with alkylating agents and/or radiation, is usually preceded by MDS and is characterized by deletion of the long arm of chromosomes 5 and/or 7 or loss of one whole chromosome 7; the other type is strongly associated with previous treatment with topoisomerase II (topo II) inhibitors and also with balanced chromosome translocations involving MLL or AML1 [19,22,25]. In addition, the NUP98 gene at 11p15 is involved in some rarely recurring chromosome translocations including t(7;11), t(2;11) and t(11;20), and fused with various homeobox genes, usually HOXA9, HOXD13 and PMX1 (Table 1). The NUP98 is strongly associated with t-AML/MDS with prior exposure to topo II inhibitors [26–29]. t-AML/MDS provides a unique opportunity to study the etiology of leukemia and the possible mechanisms, especially damage to DNA, that results in certain chromosome abnormalities. In this review, we will focus entirely on recurring chromosome translocations in human leukemia, particularly AML for which there are adequate data about the genomic location of the breakpoints and especially those subsets that are also frequently involved in t-AML/MDS.

2. Genomic analysis of recurring chromosome translocation breakpoints in leukemia The MLL gene (also called ALL1 or HRX) is one of the few genes that is involved with multiple translocation partners

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Table 1 – Recurring chromosome translocations in leukemia and the involved genes Translocations

Genes involved

(1) MLL associated translocations t(X;11)(q13;q23) AFX1/MLL t(X;11)(q22;q23) Septin2/MLL t(1;11)(p32;q23) AF1p/MLL t(1;11)(q21;q23) AF1q/MLL t(2;11)(q11;q23) AFF3(LAF4)/MLL t(3;11)(p21;q23) AF3p21 NCKIPSD/MLL t(3;11)(q21;q23) EEFSEC(SELB)/MLL t(3;11)(q25;q23) GMPS/MLL t(3;11)(q28;q23) LPP/MLL t(4;11)(p12;q23) AF4p12/MLL t(4;11)(q21;q23) AF4/MLL t(4;11)(q21;q23) SEPT 11/MLL t(5;11)(q31;q23) GRAF/MLL ins(5;11)(q31;q13q23) AF5q31/MLL t(6;11)(q12;q23) SMAP1/MLL t(6;11)(q21;q23) AF6q21/MLL t(6;11)(q27;q23) AF6/MLL t(8;16)(p11;p13) MOZ/CBP inv(8)(p11q13) MOZ/TIF2 t(8;22)(p11;q13) MOZ/p300 t(9;11)(p22;q23) AF9/MLL t(9;11)(q34;q23) AF9q34/MLL t(10;11)(p12;q23) AF10/MLL t(10;11)(p11.2;q23) ABI1/MLL t(10;11)(p12;q14) AF10/CALM1 t(10;11)(q21;q23) CXXC6(TET1)/MLL t(11;11)(q21;q23) MLL/PICALM t(11;11)(q23;q23) MLL/CBL t(11;11)(q23;q23) MLL/ARHGEF12 t(11;11)(q23;q24) MLL/TIRAP t(11;12)(q23;q13) MLL/CIP29 t(11;14)(q23;q24) MLL/GPHN t(11;15)(q23;q14) MLL/CASC5 (AF15q14) t(11;15)(q23;q14) MLL/MPFYVE t(11;16)(q23;p13.3) MLL/CBP t(11;17)(q23;p13) t(11;17)(q23;q21) t(11;17)(q23;q21) t(11;17)(q23;q21) t(11;17)(q23;q21) t(11;17)(q23;q25) t(11;19)(q23;p13.1) t(11;19)(q23;p13.3) t(11;19)(q23;p13.3) t(11;20)(q23;q11) t(11;22)(q23;q11) t(11;22)(q23;q13)

MLL/GAS7 MLL/AF17 MLL/RARa MLL/ACACA MLL/LASP1 MLL/MSF1 MLL/ELL MLL/ENL MLL/EEN MLL/MAPRE1 (EB1) MLL/hCDCrel MLL/p300

(2) CBF (AML1/CBFA and CBFB) and TEL/ETV6-associated translocations/inversion t(X;21)(p22;q22) PRDX4/AML1 t(3;21)(q26;q22) EVI1/MDS1/EAP/AML1

t(8;21)(q22;q22) t(8;21)(q23;q22) t(8;21)(q24;q22) t(16;21)(q24;q22) t(19;21)(q13;q22) t(12;21)(p12;q22)

ETO/AML1 FOG2/AML1 TRPS1/AML1 MTG16/AML1 AMP19/AML1 TEL/AML1

Associated diseases AML AML ALL AML ALL t-AML/ALL ALL t-AML AML t-ALL pro B ALL aCML AML/ALL ALL AML AML AML AML/t-AML AML AML AML/ALL AML AML AML AML, T-ALL AML AML AML AML ANLL AML AML/AUL AML/ALL T-ALL AML/ALL/tCMML t-AML AML AML AML AML t-AML AML AML/ALL AML ALL AML AML

AML tAML/CMLACC/BC AML MDS ALL/AML t-AML t-AML ALL

Table 1 (Continued ) Translocations

Genes involved

t(21;21)(q11;q22) inv(16)/t(16;16)(p13;q22) t(1;12)(p36;p13) t(1;12)(q21;p13) t(1;12)(q25;p13) t(3;12)(q26;p13) t(4;12)(q11;p13) t(5;12)(q31;p13) t(5;12)(q33;p13) t(6;12)(q23;p13) t(7;12)(q36;p13) t(9;12)(p24;p13) t(9;12)(q22;p13) t(9;12)(q34;p13) t(12;13)(p13;q12) t(12;13)(p13;q14) t(12;15)(p13;q25)

UPS25/AML1 MYH11/CBFB MDS2/TEL ARNT/TEL ARG/TEL MDS1/EVI/TEL BTL/TEL ACS2/TEL PDGFRB/TEL STL/TEL HLXB9/TEL JAK2/TEL SYK/TEL ABL/TEL TEL/CDX2 TEL/TTL TEL/NTRK3

t(12;17)(p13;p12) t(12;21)(p13;q11) t(12;16)(q13;p11) t(16;21)(p11;q22)

TEL/PER1 TEL/MN1 CHOP/TLS/FUS TLS/FUS/ERG

Associated diseases MDS AML-M4 CML/MDS AML AML MPD AML AML CMML ALL AML ALL, aCML MDS CMML AML ALL AML, fibrosarcoma AML AML AML AML, MLS

(3) RARA associated translocations t(15;17)(q22;q21) PML/RARA t(5;17)(q32;q21) NPM/RARA t(11;17)(q23;q21) PLZF/RARA t(11;17)(q13;q21) NUMA/RARA der(17) STAT5/RARA t(3;5)(q25;q35) MLF1/NPM

APL AML AML AML AML AML/MDS

(4) E2A associated translocations t(1;19)(q23;p13) PBX1/E2A t(17;19)(q23;p13) HLF/E2A

B-ALL B-ALL

(5) Tyrosine kinase associated translocations del(4)(q12q12) FIP1L1/PDGFRA t(4;22)(q12;q11) PDGFRA/BCR t(1;5)(q23;q33) Myomegalin/PDGFRB t(5;7)(q33;q11.2) PDGFRB/HIP1 t(5;10)(q33;q21) PDGFRB/H4 t(5;12)(q33;p13) PDGFRB/TEL t(5;14)(q33;q32) PDGFRB/CAV14 t(5;14)(q33;q24) PDGFRB/NIN t(5;15)(q33;q15) PDGFRB/TP53BP1 t(5;17)(q33;p13) PDGFRB/RAB5 t(5;17)(q33;p11.2) PDGFRB/HCMOGT t(6;8)(q27;p11) FOP/FGFR1 t(7;8)(q32;p11) TRIM24/FGFR1 t(8;9)(p12;q33) FGFR1/CEP110 t(8;13)(p11;q12) FGFR1/ZNF198 t(8;17)(p11;q11) FGFR1/MYO18A MPD t(8;22)(p11;q11) FGFR1/BCR t(8;19)(p11;q13.3) FGFR1/LOC113386 t(9;22)(q34;q11) ABL/BCR t(9;12)(q34;p13) ABL/TEL JAK2/BCR t(9;22)(p24;q11) t(8;9)(p22;p24) PCM1/JAK2

MPD CMML MPD CMML AML MPD MPD CMML JMML MPD MPD MPD MPD MPD MPD MPD CML, ALL CML like CMML MPD

(6) NUP98/NUP214 associated translocations t(1;11)(q23;p15) PMX1/NUP98 t(2;11)(q31;p15) HOXD13/NUP98 t(4;11)(q21;p15) RAP1GDS1/NUP98 t(5;11)(q35;p15) NSD/NUP98 t(7;11)(p15;p15) HOXA9/NUP98 t(9;11)(p22;p15) LEDGF/NUP98

AML t-AML T-ALL AML AML AML

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Table 1 (Continued ) Translocations inv(11)(p15q22) t(11;20)(p15;q11) t(6;9)(p23;q34) Normal karyotype

Genes involved NUP98/DDX10 NUP98/TOP1 DEK/NUP214(CAN) SET/NUP214(CAN)

Associated diseases t-AML t-MDS AML AML

(7) Immunoglobulin (IG) or TCR gene related translocations t(8;14)(q24;q32) IGH/c-MYC ALL (Burkitt) t(2;8)(p12;q32) IGK/c-MYC ALL (Burkitt) t(8;22)(q24;q11) IGL/c-MYC ALL (Burkitt) t(5;14)(q31;q32) IL3/IGH pre B ALL t(7;14)(q21;q32) IGH/CDK6 B-CLL t(14;19)(q32;p13) IGH/BCL-3 B-CLL t(1;14)(p32;q11) TAL1/SCL/TCRA T-ALL t(9;14)(p21;q11) p16/p19/TCRA T-ALL t(10;14)(q24;q11) HOX11/TCRA T-ALL t(11;14)(p15;q11) LMO1/TCRA T-ALL t(11;14)(p13;q11) LMO2/TCRA T-ALL TCRA/BHLHB1 T-ALL t(14;21)(q11;q22) t(7;9)(q35;q34) TCRB/TAL2 T-ALL t(7;10)(q35;q24) TCRB/HOX11 T-ALL t(7;11)(q35;p13) TCRB/LMO2 T-ALL t(7;19)(q35;p13) TCRB/LYL1 T-ALL AML: acute myeloid leukemia; APL: acute promyelocytic leukemia; ALL: acute lymphoblastic leukemia; Pre-B ALL: precursor B ALL; AUL: acute undifferentisl leukemia; CML: chronic myeloid leukemia; ACC: accelerated phase; BC: blast crisis; aCML: atypical CML; CLL: chronic lymphocytic leukemia; MDS: myelodysplastic syndrome; MPD: myeloproliferative disease; CMML: chronic myelomonocytic leukemia; JMML: juvenile myelomonocytic leukemia; T-: T cell; B-: B cell; t-: therapy-related. Boldface represents common chromosome translocations in leukemia.

in leukemia. The MLL involvement is found in approximately 15% of patients with AML and ALL [5,22]. More than 70 MLL translocations have been reported in de novo and therapyrelated AML or ALL, and more than 50 of the MLL partner genes have been cloned (Table 1). The most common MLL translocations in AML are t(9;11)(p22;q23) resulting in a fusion of the MLL and AF9 genes and t(11;19)(q23;p13.1) leading to a fusion of MLL and ELL. Among the most frequent MLL translocations in ALL are the t(4;11)(q21;q23) that results in a fusion of MLL and AF4 and is noted in more than 80% of infant patients and 10% of childhood and adult patients, and the t(11;19)(q23;p 13.3) that results in a fusion of MLL and ENL [30,31]. Generally, patients with an MLL rearrangement, except for the t(9;11), have a very poor prognosis. The amino terminus of the MLL protein contains three AT-hook DNA-binding domains, a transcriptional repression domain, and a region of homology to mammalian DNA methyltransferases, whereas the carboxyterminus contains a plant homeobox domain (PHD) with three zinc-binding C4HC3 motifs and the SET [Su(var)3–9 enhancer of zeste and trithorax] domain. The SET domain is the region most conserved with the Drosophila trithorax (trx) protein, and it is required to maintain normal embryonic and adult development of Drosophila [32].

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The t(8;21)(q22;q22) was identified in 1973 as the first recurring chromosome translocation in acute leukaemia [33], and is associated primarily with the M2 subtype (30% of AML-M2 and about 8–10% of all AML patients) [4]. It is the most common translocation in AML and is characterized by a good response to therapy and a prolonged disease-free survival. The t(8;21) translocation results in a fusion between the AML1 (also called RUNX1, CBFA2) gene at 21q22 and the ETO (MTG8) gene at 8q22 [34,35]. The AML1 gene is also involved in other recurrent chromosome translocations in myeloid leukemia, such as t(3;21) and t(16;21) that occur more frequently in t-AML/MDS than in de novo AML as well as in the t(12;21), the most common aberration in childhood pre B-ALL (Table 1) [5]. So far, the AML1 gene has been identified in more than 31 chromosome translocations in leukemia, and 12 partner genes have been cloned [7]. They result in a novel chimeric gene between AML1 and its partners that plays an important role in leukemogenesis. In the t(8;21), the 5
part of the AML1 gene is fused with nearly the entire ETO gene at 8q22, resulting in an inframe AML1-ETO fusion located on 8q22 [34,35]. AML1 is a transcription factor and a critical regulator of hematopoietic cell development; it contains the Runt DNA binding domain at the N-terminus and a transactivation domain at the Cterminus [36]. In most of these chromosome translocations involving the AML1 gene, the chimeric fusions have a dominant negative effect over the normal AML1 gene through the aberrant recruitment of an N-CoR repressor complex [37]. Several mouse model studies with knock-in strategy have shown that the AML1-ETO gene results in death during midgestion from central nervous system hemorrhage and impaired hematopoiesis in the fetal liver [38,39]. AML1 heterodimerizes with the core binding factor beta (CBFB) that does not bind to DNA directly, but rather enhances the affinity of AML1 binding to the core enhancer sequence (TGTGG) leading to the expression of its target genes including IL3, GMCSF, CSF1R, MOP and TCR [36]. The inv(16)(p13q22) or t(16;16)(p13;q22), one of the most frequent chromosome abnormalities in AML and specifically associated with AML-M4 with abnormal eosiophils [40], results in the fusion of the CBFB gene at 16q22 and the MYH11 gene at 16q13 [41]. Mouse model studies also showed that the CBFBMYH11 results in a block of definitive hematopoieisis during embryogenesis and embryonic lethal hemorrhage, similar to the phenotype observed in the AML1-ETO knock-in mice [42]. These studies showed that CBF has a critical regulatory role in normal hematopoiesis. Both the t(8;21) and inv(16) result in the abnormal repression of the CBF target genes, although the leukemia cells with each translocation have distinctive different morphologies. The t(15;17) is the hallmark of acute promyelocytic leukemia (APL), which accounts for 10–15% of young de novo AML patients. The t(15;17) results in a fusion between PML at 15q22 and RARA at 17q21 [43,44]. RARA is a member of the steroid hormone receptor superfamily and mediates the effect of retinoic acid at specific response elements. APL is characterized by unusual sensitivity to differentiation by retinoids, such as ATRA [45]. In addition to the involvement in t(15;17), the RARA gene is also involved in five other chromosome translocations and rearrangements including t(11;17) and t(5;17) (Table 1). The t(11;17) results in a fusion of RARA

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and PLZF and is associated with resistance to ATRA treatment [46].

3. Genomic breakpoints cluster in certain intronic regions of the relevant genes of chromosome translocations in de novo and therapy-related leukemia patients To understand the mechanisms of the formation of chromosome translocations in de novo and therapy-related leukemia, we and others have characterized genomic breakpoints of most common recurring chromosome translocations in terms of chromatin structural features and DNA sequence changes at or around genomic junctions. Comparison of genomic breakpoints in these genes in de novo and t-AML/MDS patients could help to clarify potential mechanisms of breakage and translocations. Recent reports of cloning of genomic breakpoints of t(4;11), t(9;11), t(8;21), inv(16), t(15;17), t(11;16), t(12;21), t(1;19) and t(9;22) have identified several common features [47–69]. In most of these chromosome translocations, the genomic breakpoints are distributed not throughout the whole gene, but rather cluster in certain intronic regions. Certain BCRs have been identified in these genes, and some distribution bias of the locations of breakpoints were noted, such as in t(4;11) among infant and childhood and adult leukemia patients and in t(9;11), t(8;21) and t(15;17) among de novo and t-AML patients [50,55,56,59,68]. Virtually all chromosome translocations in leukemia that have been analyzed at present show no homologous sequences at the breakpoints. However, genomic breakpoint cluster regions in the relevant genes often co-localize with chromatin structural elements, i.e. in vivo topo II cleavage sites, DNase I hypersensitive sites and scaffold attachment regions (SARs), implying that they have a role in the formation of leukemia-associated chromosome translocations [55,68–71]. MLL associated chromosome translocations are among those studied in most detail. In MLL translocations, almost all of the breakpoints in MLL occur in an 8.3 kb Bam HI fragment known as the breakpoint cluster region (BCR), encompassing exons 8–14. The genomic breakpoints in MLL in infant leukemia, namely t(4;11), and in t-AML tend to cluster in the 3
portion of the BCR, near exon 12, whereas those in childhood and adult patients with de novo AML tend to occur in the 5
portion of the BCR between exons 9 and 10 [50,68,72]. In AF4, the breakpoints are dispersed through out a 50 kb region from exon 3 to exon 6 [47–51]. Two weak cluster regions were identified, a smaller centromeric region of about 17 kb at the 5
portion of intron 3 and a large telomeric region of 31 kb extending from the 3
portion of intron 3 to exon 6 [50]. For non-infant patients, genomic breakpoints distribute equally in the entire breakpoint cluster regions of the AF4, whereas in infant patients genomic breakpoints occur with a weak bias towards the centromeric region [48,50]. About 9% of patients with t-ALL had a t(4;11) but no genomic breakpoint information has been reported. The t(9;11) that results in a fusion between MLL and AF9 is a recurring chromosomal translocation in de novo AML and is one of the most common recurring chromosome translocations detected in 48% of t-AML patients with MLL involvement.

AF9 is greater than 100 kb and contains 10 exons. Genomic analysis identified two BCRs in AF9 in t(9;11) patients, i.e. BCR1 in intron 4 and BCR2 spanning introns 7 and 8 [53,54]. Using Southern blot analysis, Strissel et al. [68] mapped genomic breakpoints in MLL in six de novo AML patients and three tAML patients all with a t(9;11). All three t-AML patients have breakpoints in the 3
portion of the BCR near exon 12, whereas four of six de novo patients have breakpoints in the 5
portion of the BCR. In a series of 24 patients with t(9;11), including two t-AML patients, Gill-Super et al. [52] defined genomic breakpoints in MLL in 10 patients. Six patients had breakpoints in the 5
portion and four patients including those two t-AML patients had breakpoints in the 3
portion of the BCR. In AF9, the breakpoints seemed to cluster in a large region BCR1 (previously called site A). In one patient, genomic breakpoints occurred in Alu repeats in both MLL and AF9, whereas in the cell line Mono Mac 6 which was derived from a de novo AML patient, breakpoints were not near Alu repeats. In four t-AML patients with t(9;11), Atlas et al. [53] were able to define the genomic breakpoints in both MLL and AF9. All of genomic breakpoints in MLL were in the 8.3 kb BCRs with two very close to exon 12. In AF9, genomic breakpoints in four patients distributed through out intron 4. In a series of 11 childhood leukemia patients with t(9;11) including three AML, five t-AML, three ALL and two cell lines (THP-1 and Mono Mac 6), Langer et al. [54] again found all breakpoints in MLL in the 8.3 kb BCR region. Genomic breakpoints occur about equally in the 5
and 3
portions in de novo AML patients, whereas all five t-AML patients had genomic breakpoints in the 3
portion of the BCR in a 6 bp hot spot. This breakage hot spot is inducible by topo II inhibitors or apoptotic triggers in vitro [71,73]. Two of these five t-AML patients had received daunorubicin (a topo II inhibitor) during treatment of their primary malignant disease. In AF-9, genomic breakpoints in four leukemia patients including 2 tAML patients and two cell lines were located in BCR1, whereas in two other de novo patients breaks occurred in BCR2. Moreover, in five patients including three t-AML patients, the AF9 breakpoints were found outside the previously described BCRs within the centromeric region of intron 4 and even within intron 3 in one case. In summary, it is evident that in MLL translocations in de novo and t-AML patients, genomic breakpoints are located in the 8.3 kb BCR of MLL with the tendency to occur in the 3
portion in infant leukemia patients and in t-AML patients [50,68]. In MLL partner genes, including AF4 and AF9, there is only a weak bias in the distribution of genomic breakpoints between de novo and t-AML patients. In AF9, more t-AML patients with t(9;11) have breakpoints in BCR1. The difference in the distribution of genomic breakpoints in the MLL gene in de novo and t-AML as well as between infant and older children leukemia patients could be due to different mechanisms in the formation of these translocations. The AML1 gene is about 260 kb in size and contains nine exons. In the t(8;21), we cloned the AML1/ETO and the reciprocal ETO/AML1 genomic fusion in 35 patients with de novo AML [55 and Zhang et al., unpublished data]. The genomic breakpoints in AML1 in all of these patients are located in intron 5, clustering in three BCRs (Fig. 1) [55]. These results were confirmed by studies of others. In 16 childhood leukemia patients and in two leukemia cell lines with a t(8;21), Xiao et al. [56]

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Fig. 1 – Colocalization of in vivo topo II cleavage sites and DNase I hypersensitive sites with genomic breakpoint clusters at lowest free energy (
G) level in AML1 in de novo and t-AML patients with t(8;21). The vertical boxes represent exons. The green and blue arrowheads at the bottom represent in vivo topo II cleavage sites and DNase I hypersensitive sites in introns 5 and 7a. The yellow and arrowheads above the enlarged intron 5 indicate the cloned genomic breakpoints in de novo and therapy-related t(8;21) patients, whereas the breakpoints that were mapped by Southern blot analysis but not cloned yet are represented as brown arrowheads. The genomic breakpoints in intron 7a of AML1 in four t-AML with t(3;21) are indicated by arrows. At the bottom, the free energy (
G, kcal/mol) level from intron 5 through intron 7a was calculated with the WEB-THERMODYN [105] program. Three BCRs of genomic breakpoints in t(8;21) patients were defined in intron 5, and some of these BCRs colocalize with in vivo topo II cleavage sites and DNase I hypersensitive sites. Genomic breakpoints in AML1 in four t(3;21) patients cluster in the 3 portion of intron 7a and colocalize with several in vivo topo II cleavage sites and DNase I hypersensitive sites. BCR1 in intron 5 is located at the region with the lowest free energy level.

mapped genomic breakpoints in AML1 in nine patients to the 3
part of intron 5 that covers BCR-3 identified in our study. Moreover, in a study of 21 patients with t(8;21), Shimizu et al. [74] mapped genomic breakpoints in AML1 in 8 and 10 patients in the middle and the 3
part of intron 5, respectively, which overlap with BCR-1 and BCR-3. In our study, the clustering of genomic breakpoints in ETO in t(8;21) is even stronger (Fig. 2). Except for a few patients with breakpoints in intron 1a, the breakpoints in ETO in 24 patients clustered in three BCRs in intron 1b. Tighe and Calabi [75] identified two BCRs in intron 1b and one BCR in intron 1a in 18 patients with t(8;21). These two BCRs in intron 1b may overlap BCR-I and BCR-II, respectively. In the study of t(8;21) by Xiao et al. [56], a large BCR was defined spanning the middle and the 3
portion of intron 1b, which covers our BCR-II and BCR-III. Thus, genomic breakpoints in both AML1 and ETO in patients with t(8;21) tend to occur in defined BCRs. Genomic breakpoints in AML1 and ETO

in 6 t-AML patients, all of whom were treated with topo II inhibitors, cluster in the same BCRs previously identified in de novo patients with t(8;21) [55, Zhang et al. unpublished data]. This suggests that the same mechanism could be involved in the formation of t(8;21) in t-AML, which accounts for 35% of tAML involving AML1. Nevertheless, this observation needs to be confirmed in a larger series of t-AML patients with t(8;21). The t(3;21)(q26;q22) that results in a chimeric fusion between AML1 and MDS1/EVI1 also is a recurring chromosomal translocation and is often observed in CML in the accelerated/blast phase (ACC/BC) and in t-AML/MDS but is rarely detected in de novo AML patients [76]. It accounts for 13% of t-AML patient with AML1 involvement [24]. EVI1 spans over 100 kb and contains 12 exons with 10 coding exons. MDS1 covers more than 234 kb and contains four exons. Based on partial genomic sequence, MDS1 is located about 60 kb telomeric to EVI1. Using Southern blot analysis and FISH techniques, we

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Fig. 2 – Colocalization of in vivo topo II cleavage sites and DNase I hypersensitive sites with genomic breakpoint clusters at lowest free energy (
G) level in ETO in de novo and t-AML patients with t(8;21). The vertical boxes represent exons. The green and blue arrowheads at the bottom represent in vivo topo II cleavage sites and DNase I hypersensitive sites in introns 1b. The yellow and arrowheads above the enlarged intron 1b indicate the cloned genomic breakpoints in de novo and therapy-related t(8;21) patients, whereas the breakpoints that were mapped by Southern blot analysis but not cloned yet are represented as brown arrowheads. At the bottom, the free energy (
G, kcal/mol) level of introns 1b and 1a was calculated with the WEB-THERMODYN [105] program. Three BCRs of genomic breakpoints in t(8;21) patients were defined in intron 1b, and two of these BCRs colocalize with in vivo topo II cleavage sites and DNase I hypersensitive sites. The BCRs in intron 1b are located at the region with the low free energy level.

mapped genomic breakpoints of AML1 in 10 t(3;21) patients, including 4 patients with CML-ACC/BC, 5 patients with tMDS/AML and 2 patients with de novo MDS/AML [Zhang et al. unpublished observation]. Genomic breakpoints occurred in introns 5 and 7a in four patients each (Fig. 1). Genomic breakpoints in AML1 in the SKH-1 cell line with a t(3;21) and in two leukemia patients with t(5;21) and t(12;21) were also mapped in introns 5 and 7a, respectively [77,78]. The genomic breakpoints in intron 5 in these t(3;21) patients overlap with BCR2 and BCR3 that were defined in the t(8;21). In one t(3;21) patient, we used inverse PCR and cloned the genomic breakpoints in intron 7a of AML1 and in intron 1 of MDS1 (Fig. 1) [Zhang et al. unpublished data]. Moreover, using FISH we also mapped the genomic breakpoint in intron 1 of MDS1 in another t(3;21) patient. Thus, it is possible that the genomic breakpoints in the MDS1/EVI1 clustered in certain regions of intron 1 of MDS1 in t(3;21) patients. In the inv(16)/t(16;16), genomic breakpoints are tightly clustered in both CBFB and MYH11. In a series of 24 de novo patients with inv(16), van der Reijden et al. [57] found all

genomic breakpoints in CBFB are located in a 5 kb intron 5. This intron is rich in repetitive sequences including Alu, LINE1 and MIRs. The genomic breakpoints mapped about equally in the 5
and 3
portions; 8 and 3 breakpoints occur in the Alu and LINE-1/MIR repeats, respectively. In MYH11, 21 of 24 breakpoints were located in a 370 bp intron 30. Three genomic breakpoints occur in the Alu repeats and three another breaks are located adjacent to a V(D)J recombinase signal sequence (heptamer and nonamer sequences), possibly implicating either the Alu repeats or a V(D)J mediated recombination in the inv(16). In 37 de novo leukemia patients with t(15;17), Reiter et al. [58] cloned genomic breakpoints in both the PML and RARA genes. Genomic breakpoints in PML are located within three BCRs in introns 3 (1.5 kb), 5 (0.4 kb) and 6 (1 kb) of the PML gene on chromosome 15, whereas genomic breakpoints occur in three significant microcluster regions (from 137 to 545 bp) in intron 2 of RARA on chromosome 17, indicating sequenceassociated or structural factors may play a role in the formation of the t(15;17). There was no correlation of the location

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of the breakpoints of PML with those of RARA [58]. In a recent paper by Mistry et al. [59], genomic breakpoints in all 35 de novo APL patients were equally distributed throughout the entire 1 kb intron 6 of PML, whereas genomic breakpoints in four of six t-APL patients who received mitoxantrone for primary tumors clustered tightly in an 8-bp region in the middle of intron 6. Genomic breakpoints in PML in 7 t-APL patients who did not receive topo II inhibitors but mainly radiotherapy were located throughout the entire intron 6 as in de novo APL patients. In both de novo and therapy-related APL patients, genomic breakpoints in RARA were dispersed through intron 2 [58,59]. Thus, as in AML1 and ETO in the t(8;21) andAF9 in the t(9;11), genomic breakpoints in both PML and RARA genes in the t(15;17) are located in the same intronic regions in de novo and t-APL patients; however, there is more clustering in some hot-spot regions in t-APL patients. The discussion thus far has been concentrated on genomic breakpoint in AML. Similar data are available for a few breakpoints in B-ALL with a t(12;21) or t(1;19). About 25% of children with pre B ALL have the t(12;21) that results in a fusion between AML1 at 21q22 and TEL at 12p12. This translocation is associated with a good prognosis and is generally undetectable with conventional cytogenetic analysis. In contrast to other AML1 associated translocations, AML1 with a break in intron 1 or 2 forms the 3
part of the fusion gene in the t(12;21). In a series of 20 ALL patients with t(12;21), Wiemels et al. [61] found tight clustering of genomic breakpoints in intron 5 of TEL and in introns 1 and 2 in AML1. Two strong microclusters in TEL were identified in the middle and 3
portion of intron 5. Interestingly, there is a highly repetitive purine/pyrimidine repeat region [62] and a 738 bp deletion polymorphism [63,79] just 5
of the middle BCR. These unstable regions undergo expansion and contraction and may be a hotspot that is prone for DNA recombination events, such as insertions or translocations [63]. Genomic breakpoints in the TEL gene in 9 patients with t(12;21) were mapped at these regions [61–63]. In AML1, four highly significant microclusters were mapped in introns 1 and 2. One cluster is in the 5
portion of intron 1, close to exon 1, whereas the other three clusters are in the 3
portion of intron 1 with two overlapping with exons 2 and 3 [61]. In the t(1;19) that occurs in about 5% of pre-B ALL in children and adult patients and results in a fusion between E2A and PBX1, Wiemels et al. [64] identified a 5 bp BCR in intron 13 of the E2A gene that contained the genomic breakpoints in 16 of 24 patients with t(1;19). In PBX1, two BCRs were mapped in intron 1; one (8 kb) is located very close to exon 2 and the other one of 21 kb is in the middle in intron 1. Thus, as in the t(12;21), genomic breakpoints in both E2A and PBX1 in the t(1;19) are clustered tightly in certain intronic regions. The t(9;22) that results in a fusion of BCR and ABL is associated with 100% of CML by definition and about 25% adult ALL and is characterized with a favorable response to Gleevec treatment in most patients [8,11]. In a majority of CML patients with t(9;22) and in some ALL patients with t(9;22), genomic breakpoints in the BCR gene at 22q11 are located in a 5.8 kb region called the major breakpoint cluster region (M-BCR) between exons 11 and 16 and, whereas ALL breakpoints are mainly located in a larger 35 kb minor breakpoint region (mBCR) towards the 3
end of intron 1 [65–67]. Furthermore,

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three BCRs have been described in introns 1b and 1a of the ABL gene [67]. In some CML patients, genomic breakpoints in both BCR and ABL were defined at or near Alu repeats, implicating these repeats in the formation of the t(9;22) [65–67]. We have already indicated that there are two broad groups of t-AML/MDS, those associated with alkylating agents and radiation and the second associated with prior treatment with topo II inhibitors [21,22]. The cytogenetic abnormalities associated with the former group are primarily deletions of chromosome 5 and/or 7, whereas balanced chromosome translocations, especially involving MLL and AML1 are more frequently associated with the latter treatment [19,21]. A number of investigators have questioned whether particular drugs are preferentially associated with any specific translocations [80,81]. To obtain more accurate data, a Workshop on recurring chromosome abnormalities and prior treatment in therapyrelated leukemia and MDS patients was held, and data on 511 patients were evaluated in detail regarding chromosome aberrations and prior treatment history in each individual patients [22]. One hundred and sixty two and 79 patients had involvement of 11q23 and 21q22, respectively [23,24]. It was difficult to correlate specific translocations with particular drugs, because more than half of the patients (278 patients) had received both radiation and chemotherapy treatment, and for the 169 patients who received chemotherapy, the majority received both alkylating agents and topo II inhibitors. However, careful subset analysis revealed that patients with chromosome translocations involving MLL and AML1 were more likely to have received topo II inhibitors than those with inv(16), t(15;17) or other groups. Too few patients received only one type of topo II inhibitor to subdivide the cytogenetic groups further, so the questions remain unanswered. As discussed earlier, it is worth noting that in the analysis of t-APL patients with t(15;17) and with prior exposure to mitoxantrone treatment for primary cancers [59], an 8 bp breakpoint hot spot region in the PML gene was identified, suggesting that there may be a specific vulnerability of certain chromosome regions to particular drugs.

4. Chromatin structural elements including in vivo topo II cleavage sites, DNase I hypersensitive sites, SARs and lowest free energy level sites colocalize with genomic breakpoints of chromosome translocation in leukemia Several chromatin structural elements including in vivo topo II DNA cleavage sites, DNase I hypersensitive sites and SARs have been characterized with respect to their possible involvement in chromosome translocations in leukemia [55,69–73,82,83]. Topo II is a primary chromosome scaffold protein and is essential for chromosome condensation, transcription and replication as well as for apoptosis [84–86]. It binds preferentially to the SARs [87,88]. Human topo II cleavage sites have been mapped by many investigators using in vitro topo II cleavage assays on naked DNA, or in nuclei and in vivo topo II cleavage assay in living cells. In in vitro topo II cleavage assays, naked DNA is incubated with human topo II␣ in the pres-

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ence of ATP and topo II inhibitors [89]. There are a great many such sites in the genes that we are considering in this paper. Our concern is based on the notion that chromatin structure, instead of DNA nucleotide sequence, may be the primary determination of topo II cleavage sites in vivo [90–92]; thus, these in vitro topo II cleavage sites are not specific and do not necessarily accurately reflect topo II cleavage of DNA in a living cell. In vivo topo II cleavage sites are determined selectively by the inhibitors used in the assay [93]. Many DNase I hypersensitive sites are associated with transcriptional regulatory DNA elements and SARs at gene boundaries or within genes; some of these sites colocalize with SARs and/or topo II sites at breakpoint cluster regions in IFN, MLL, AF9, AF4, AML1, ETO, BCR and ABL [55,69,82,83,94,95]. AT-rich DNA SARs usually define the attachment sites of interphase and metaphase chromatin loops in the DNA scaffold-loop model of chromosomes [87] and are presumed to facilitate the entry of transcription, replication or chromosome condensation protein factors to target sequences. They represent regions of DNA fragility due to the DNA unwinding property of some of these proteins [95]. Thus, these chromatin structural elements appear to interact in vivo in maintaining chromosome structure and function. Agents that inhibit topo II, such as the epipodophyllotoxins, e.g. VP16, and Doxorubicin (Dox) that are used frequently in chemotherapy as well as natural bioflavonoids found in certain foods and dietary supplements, can trap topo II in a DNA-cleavable complex and result in DNA double strand breaks [82,83,96,97]. Position-specific trapping sites of topo II by some intercalators have been clarified in suppression of topo II mediated DNA cleavage [97]. Topo II appears to recognize DNA structure rather than a DNA specific sequence. In previous studies by us and others, these in vivo topo II DNA cleavage and DNase I hypersensitive sites have been identified near the genomic breakpoints in the genes frequently involved in leukemia, such as MLL, AF9, AF4, AML, ETO, BCR and ABL [30,55,69,82,83,98–100]. In the MLL gene, we and others identified a single strong in vivo topo II cleavage site near exon 12 that co-localizes with a DNase I hypersensitive site [69–71]. Moreover, we also defined S1 nuclease and Mung bean cleavage sites that specifically recognize and cleave single-strand regions in super-coiled DNA at the same regions. Both DNase I hypersensitive sites and S1/Mung bean nuclease cleavage sites represent open and accessible regions of DNA as defined primarily by double strand DNA susceptibility to DNase I enzyme cleavage and single stranded DNA susceptibility to the S1/Mung bean enzymes. As indicated above, genomic breakpoints in t-AML patients and infant leukemia patients withMLL translocations occur mainly in this region. In the t(11;16) which is a rare recurring chromosome translocation and is detected virtually only in t-AML/MDS, we mapped genomic breakpoints of MLL in four of six patients to the 3
portion of the BCR with two breakpoints very close to this topo II cleavage site [60]. We defined two SARs in MLL, one mapped just 5
of the BCR and the other which includes exons 12 and 13 in the 3
portion of the BCR region. The 3
SAR region overlaps the topo II cleavage and DNase I hypersensitive sites near exon 12 [69]. In AF9, we identified one strong topo II cleavage site that colocalizes with a DNase I hypersensitive site near exon 8 and

three weaker centromeric topo II cleavage sites [82]. Two SARs were identified which both were located centromeric to the topo II and DNase I hypersensitive sites and bordered both patient BCRs; SAR1 was located in intron 4, whereas SAR2 encompassed parts of exons 6–7 [69,82]. In AF4, we identified a strong topo II cleavage site and a strong colocalizing topo II/DNase I hypersensitive site in introns 3 just 5
to exon 4 within a strong 6 kb SAR [99]. Two strong colocalizing topo II/DNase I hypersensitive sites and two weak topo II cleavage sites were identified in introns 5 and 6, all located within or near a 0.8 kb SAR. Notably, although t(4;11) is primarily observed in de novo infant leukemia, these specific chromatin sites are located in or very near to the BCR in AF4. Thus, these specific chromatin structural elements clearly colocalize with genomic breakpoint clusters in MLL and its partner genes in leukemia, in particular in t-AML and infant leukemia patients. In the t(8;21), three and seven in vivo topo II DNA cleavage sites were identified in introns 5 and 7a of AML1, respectively, and two topo II cleavage sites were identified in intron 1b of ETO (Fig. 1) [55]. Notably, these topo II DNA cleavage sites in AML1 colocalize with genomic BCRs in leukemia patients. Topo II sites A and C are located within BCR-2 and BCR-3, respectively, whereas topo II site B is within a 0.2 kb region surrounded by the breakpoints of two patients. However, we were not able to identify topo II or DNase I hypersensitive sites at or near BCR1 in AML1. A stronger correlation of topo II DNA cleavage sites with genomic breakpoints was observed in intron 1b of ETO (Fig. 2). Topo II site A is located at the same region as the 1.9 kb BCR-I, whereas topo II site B is within the 2.5 kb BCR-II (Fig. 2). We have also identified five and two DNase I hypersensitive sites in AML1 and ETO, respectively, all colocalizing with all strong in vivo topo II sites in both genes. Recently, Iarovaia et al. [94] showed that the BCRs we identified in AML1 and ETO contain SAR elements and were preferentially associated with the nuclear matrix in proliferating HEL cells. They further mapped the SARs to a 0.9 kb region of BCR3 in AML1 and to a 1.2 kb region of BCR-II of the ETO gene. Thus, topo II cleavage sites, DNase I hypersensitive sites and SARs all are implicated in the formation of the t(8;21). The correlation of breakpoints and these chromatin structural elements is also revealed in the t(3;21). Seven topo II DNA cleavage sites are clustered in the 3
portion of intron 7a of AML1 (Fig. 1). In our ongoing study of t(3;21) patients, genomic breakpoints in AML1 in 4 t-AML patients mapped in the regions containing seven topo II sites including sites D-G and I and J in intron 7a (Fig. 1). Genomic breakpoints in AML1 in t(5;21) and t(12;21) were also located in the same regions in intron 7a [78]. Thus, genomic breakpoints in AML1 in t(3;21) and in these less common translocations may also be clustered in and thus correlated with topo II cleavage sites. Similarly, in MLL and AF9 in the t(9;11), and in BCR in the t(9;22) in CML, all DNase I hypersensitive sites colocalized only with strong in vivo topo II sites [69,70,82,99]. These results support previous proposals that topo II preferentially cleaves at specific open chromatin sites that correlate with stronger topo II DNA cleavage sites. Importantly, using an in vitro topo II cleavage assay, Mistry et al. [59] examined the intron regions of the PML and RARA genes that encompass the relevant translocation breakpoints in t(15;17) patients. An 8 bp breakpoint hot spot region was

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identified in the middle of intron 6 of the PML gene in four tAPL patients with prior treatment with mitoxanthrone. An in vitro topo II cleavage site was mapped in this 8 bp region, and it is markedly enhanced by VP16 and mitoxantrone. This in vitro topo II cleavage site is a preferred site of cleavage by topo II in the presence of mitoxanthrone, and it remains detectable after heating, indicating stability of the cleavage complexes. Similarly, several mitoxanthrone-induced in vitro topo II cleavage sites in the RARA gene were mapped at or proximal to genomic breakpoints of t-APL patients. Moreover, in one patient with tAPL with prior exposure to VP16 and doxorubicin, in vitro topo II cleavage sites were identified at the breakpoints in both the PML and RARA genes [59]. Thus, genomic breakpoints in both the PML and RARA genes in t(15;17) are correlated with in vitro topo II cleavage sites. As indicated earlier, genomic breakpoints in both BCR and ABL in t(9;22) leukemia patients cluster in certain introns. In a few CML patients, these breakpoints appear to occur at or near Alu repeats in both genes. A detail analysis of both BCR and ABL genes by Jeffs et al. [67] showed that Alu repeats are the most abundant class of repeats in both genes, but they occupy fewer sites than previously estimated and they are distributed non-randomly through both genes. A significant lack of Alu elements was observed across the M-BCR and m-BCR of the BCR gene as well as the 25 kb region showing a high frequency of breakages in the ABL gene. Thus, it seems also possible that genomic breakpoints are correlated by chance with Alu repeats in BCR and ABL because of the high density of Alu in these two genes. In our recent chromatin structural analysis of BCR and ABL [99], we identified a strong colocalizing topo II/DNase I hypersensitive site in the middle of intron 13 and a single strong DNase I hypersensitive site in the 3
portion of intron 8 of the BCR gene. Notably, the colocalzing topo II/DNase I hypersensitive site in intron 13 is located within the M-BCR that contained most of genomic breakpoints in CML. In the ABL gene, we identified a colocalizing topo II/DNase I hypersensitive site and a single DNase I hypersensitive site in the middle of intron 1a as well as three colocalizing topo II/DNase I hypersensitive sites in intron 4. We mapped a 5.1 kb SAR that overlaps with the colocalizing topo II/DNase I hypersensitive site in intron 1a of the ABL gene. No SAR was mapped in the BCR gene. Thus, chromatin structural elements including in vivo topo II cleavage sites, DNase I hypersensitive sites colocalize with genomic breakpoints in both BCR and ABL. In ABL, a SAR is probably implicated as well [99]. However, not all BCRs identified to date in the relevant genes involved in leukemia-related chromosome translocations colocatize with topo II cleavage sites, as demonstrated in the AML1 and CBP genes [55,60]. Thus, other chromatin structural properties probably are also involved in determining the location of chromosome breakage. One of these properties may be the free energy level that is required for unwinding sequences in supercoiled double stranded DNA [101–105]. Using the WEB-THERMODYN developed by Huang and Kowalski [102], we analyzed the free energy (
G) level of the BCRbearing introns in AML1 and ETO and found that some of the BCRs we identified in both AML1 and ETO have low
G values (Figs. 1 and 2). Two BCRs in intron 5 of AML1 and two BCRs in intron 1b of ETO that colocalize with topo II cleavage sites and DNase I hypersensitive sites have a low free

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energy level compared with other regions of the same intron, whereas BCR1 in intron 5 of AML1 that contains no in vivo topo II sites or DNase I hypersensitive sites has the lowest level. We also analyzed the 8.3 kb BCR of MLL and found similar results, namely, that the location of topo II cleavage site, DNase I hypersensitive site, S1/Mung bean sites and SARs near exon 12 is at the lowest level of the free energy (
G). Our studies suggest that in vivo topo II cleavage sites and the regions with the lowest free energy provide vulnerable sites for breakage which is subsequently repaired using a non-homologous end joining (NHEJ). Nuclear topography and the spatial positioning of genes in the nucleus of hematopoietic cells have been presumed to be contributing factors in the formation of recurring chromosome translocations in leukemia and lymphoma [106–109]. Spatial proximity of chromosomal loci that participate in rearrangement is required as a precondition for the formation of a chromosome translocation [110]. The MYC, BCL2 and BCL6 and IGH genes are non-randomly positioned in three-dimensioned space toward the nuclear interior in normal cells. This local proximity is the consequence of higher-order genomic structure rather than a property of individual genes. The degree of physical proximity between two gene loci before the translocations events actually correlated with the frequency of certain chromosome translocations observed in lymphoma [106]. The ABL and BCR loci that are involved in the t(9;22) are close to each other in CD34+ bone marrow cells [108]. The RET and H4 genes are involved in the inv(10) by illegitimate recombination which is frequently observed in radiation-associated papillary thyroid cancer. The RET and H4 loci were found to be closer to one another in the nuclei of thyroid cells than in those of lymphocytes [110]. Our recent analysis of the nuclear 3D position of the MLL gene, and five of its common partners (AF4, AF6, AF9, ENL and ELL) revealed a characteristic radial distribution pattern in all hematopoietic cells studied. Genes in chromosomal areas of high local gene density were positioned towards the nuclear center, whereas genes in regions of low gene density were detected closer to the nuclear periphery [107]. 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 genes in normal cells was different from that of the fusion genes. Thus, alterations in gene density directly at translocation junctions could explain the change in the position of affected genes in leukemia cells.

5. Non-homologous end joining repair signatures at genomic breakpoints and junctions in chromosome translocations Cloning both reciprocal genomic junctions in various chromosome translocations in leukemia has enabled investigators to characterize the specific sequence changes at the breakpoints and genomic junctions. Several studies have shown that almost all chromosome translocations observed in leukemia are unbalanced at the genomic level. There are small deletions and duplications at the breakpoints in many translocations. However, comparison of genomic fusion in de novo and therapy-related leukemia with t(8;21), t(15;17),

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t(8;16) and t(9;11) as well as t(4;11) and t(12;21) shows that the size of both deletions and duplications is larger in de novo leukemia patients than in t-AML/MDS patients [54,58–60]. Whether these differences in the extent of chromosome unbalance reflect some unexplained differences in NHEJ repair in de novo and therapy-related leukemia is unknown. No specific recombination motifs have been identified in the myeloid leukemias but small (2–5 bp) micro-homologies or non-templated nucleotides exist at or near most of the breakpoint junctions characterized so far. These features, in particular micro-homologies at genomic breakpoints, are all consistent with an NHEJ repair mechanism in religation of these DNA double strand breaks in chromosome translocations [47–64,68–71,82]. In t(4;11) patients who are primarily de novo infant patients with ALL, duplications and deletions in MLL ranged from 7 to 324 bp (average 95 bp) and 5–1014 bp (average 253 bp), respectively [48,51]. In AF4, duplications and deletions were between 35 and 274 bp (average 140 bp) and between 23 and 169 bp (average 169 bp), respectively [48,51]. In three de novo t(9;11) patients, duplications of 38 and 103 bp and a deletion of 75 bp in MLL and deletions of 6, 16 and 105 bp in AF9 were detected, whereas in four t-AML patients with t(9;11), deletions or duplications of 2–4 bp in either or both MLL and AF9 were detected [54]. Gill-Super et al. [52] cloned genomic breakpoints of the t(9;11) in the Mono Mac 6 cell line and found a deletion of 499 bp in the MLL gene and a deletion of a 165 bp in the AF9 gene. In four t-AML patients with t(11;16), we found 2–3 bp microhomologies at all genomic junctions and a perfect junction between MLL and CBP in two patients and only 2–7 bp deletions or duplications in two other patients [60]. Whereas in the SN-1 cell line, which was derived from a 2-year-old TALL patient with no history of previous tumor or treatment, we identified an insertion between the CBP and MLL of 79 bp derived from a chromosome 17 sequence [60]. In the t(8;16) that involves CBP and MOZ, Panagopoulos et al. [111] identified a small non-template insertion (up to 10 bp) at the genomic junctions and small deletions (1–162 bp) in both CBP and MOZ in two t-AML patients and a duplication of 465 bp in CBP in one of two de novo leukemia patients. In 31 de novo patients with t(8;21) [55, Zhang et al. unpublished data], the deletions ranged from 5 to 556 bp (251 bp on average) in AML1 and from 6 to 225 bp (88 bp) in ETO, whereas in six t-AML patients with t(8;21), the deletions in AML1 ranged from 1 to 111 bp (39 bp) in three and from 2 to 125 bp (32 bp) in ETO in five patients. Duplications in AML1 in seven de novo t(8;21) patients ranged from 1 to 141 bp (76 bp) and in ETO in eight patients from 51 to 355 bp (185 bp); in contrast, duplications in AML1 were 1, 2 and 1085 bp in three t-AML patients and only one t-AML patient had a duplication (1 bp) in ETO. In 29 de novo APL with t(15;17) [58], only two patients had perfectly balanced reciprocal translocations. Duplications ranging from 2 to 109 bp with a median of 5 bp were observed in either or both PML and RARA genes in 16 patients, whereas deletions from 1 to 351 bp (median 5 bp) were detected in 14 patients. In contrast, in all four mitoxanthrone treatment-related APL patients with t(15;17), the breakpoint junctions had no duplications or deletions in either gene [59].

6. A model of non-homologous chromosome translocation (NHCT) as one of the mechanisms resulting in the formation of chromosome translocations in leukemia For understanding the mechanisms leading to chromosome translocations in leukemia, genomic breakpoints in many patients with various translocations have been cloned, and several models have been proposed based in some studies on only a few patient samples. For example, translin binding sites at or near the breakpoint junctions have been found in t(8;14), t(9;11) and t(14;18) [53,112]. Illegitimate V(D)J recombination and class switch recombination with site-specific DNA cleavage at heptamer/nonamer signal sequences were identified near genomic breakpoints in lymphoid malignancies including leukemia and lymphoma [63,113,114]. ␹-Like signal sequences were found to be associated with several chromosome translocations in leukemia and lymphoma [115,116]. Alu and long interspersed nuclear elements (LINEs) have been proposed to play a role in the formation of BCR-ABL in the t(9;22) and in the partial tandem duplication of the MLL gene in AML as well as in non-leukemic fusions in solid tumors, such as EWS-FLI1 [65,117–119]. However, in most patients these recombination-related motif sequences were not identified at or near the genomic junctions. Based on our results and those of others in studies in t(8;21), t(15;17) and t(9;11), we propose a non-homologous chromosome translocation model of a common mechanism for both de novo and t-AML leukemia [55,69,82,99]. We propose that topo II normally functions in vivo at these sites to monitor the helicity of DNA and that the genomic breakpoint cluster regions are evolutionarily conserved chromatin regions essential for replication, transcription, condensation and apoptosis. DNA damage and repair at these sites, which could lead to chromosome translocations, are a rare occurrence, but they perhaps could also be due to the natural topo II inhibitors, such as some bioflavonoids, which we recently proposed as a potential mechanism for de novo infant leukemia involving MLL [120]. Other DNA damaging agents, such as pesticides and organic chemicals or apoptotic nucleases could also act at these topo II sites either by inhibiting topo II religation or by cleaving open chromatin, thus playing a role in the generation of translocations [121,122]. In addition, certain chromosome regions with a lower free energy level that is required for unwinding double DNA strands are also prone to break upon exposure to these agents. In most chromosome translocations including t(8;21), t(9;11) and t(15;17), many DNA breakpoints do not colocalize exactly with, but map close to or at a variable distance from the in vivo topo II cleavage sites. It is possible that DNA cleavage is induced at these topo II cleavage sites, followed by complex DNA repair mechanisms including 3
–5
exonucleolytic processing which is associated with deletions and duplications at the breaks as a result of the repair machinery as well as microhomologies and non-templated insertions at genomic junctions. Obviously, for cellular transformation to occur, the chromosome translocations formed by this model of colocalization of specific chromatin structural elements and genomic breakpoint cluster regions must lead to a fusion

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oncoprotein that promotes a growth advantage in the cell. [10]

7.

Conclusions

Recurring chromosome translocations are the hallmarks of leukemia and lymphoma. They are strongly associated with certain types of clinical and pathologic entities and are of prognostic significance. Based on our current knowledge, several different mechanisms are probably involved in mediating the formation of these chromosome translocations. As proposed in this paper, several chromatin structural elements including topo II cleavage sites, DNase I hypersensitive sites, SARs are very likely associated with preferentially breakage sites after exposure to damage including topo II inhibitors. Understanding such mechanisms is clinically important as new anticancer drugs could be developed with less inhibition of topo II and thus potentially a decreased induction of therapyrelated leukemia. For further understanding of the mechanisms resulting in chromosome translocations, we need to know whether and how such chromatin structural elements relate to certain recurring translocation translocations, and whether and how the open chromatin structural elements that colocalize with breakpoint cluster regions may be active sites for cellular processes that promote chromosome translocations.

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Acknowledgement Supported in part by the National Institute of Health/National Cancer Institute (grant CA84405 to JDR).

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