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

The AML1 gene (also known as RUNX1) at 21q22 codes for core binding factor (CBF) alpha, which forms a heterodimer with CBF beta that acts as a transcriptional activating factor. CBF is a critical regulator in the generation and differentiation of definitive hematopoietic stem cells and is frequently disrupted in leukemia through chromosome translocations. We cloned a novel AML1 partner gene, PRDX4, in an X;21 translocation in a 74-year-old male patient diagnosed with acute myeloid leukemia-M2. Chromosome analysis detected a t(X;21)(p22;q22) as the sole abnormality in bone marrow samples. The involvement of AML1 was confirmed by fluorescence in situ hybridization studies. Using 3' RACE-PCR, we cloned a fusion between exon 5 of AML1 and exon 2 of PRDX4. RT-PCR confirmed the fusion and detected another fusion between exon 6 of AML1 and exon 2 of PRDX4, indicating alternative splicing of exon 6 of AML1 in the fusion transcripts. PRDX4 is one of six peroxiredoxin-family genes that are highly conserved in eukaryotes and prokaryotes and are ubiquitously expressed. Peroxiredoxin genes exhibit thioredoxin-dependent peroxidase activity and have been implicated in a number of other cellular functions such as cell proliferation and differentiation. PRDX4 plays a regulatory role in the activation of the transcription factor NF-kappaB and is significantly down-regulated in acute promyelocytic leukemia. This is the first example of antioxidant enzyme involvement in a chromosome translocation in leukemia.

Identification of the specific cytogenetic abnormality is one of the critical steps for classification of acute myeloblastic leukemia (AML) which influences the selection of appropriate therapy and provides information about disease prognosis. However at present, the genetic complexity of AML is only partially understood. To obtain a comprehensive, unbiased, quantitative measure, we performed serial analysis of gene expression (SAGE) on CD15 ؉ myeloid progenitor cells from 22 AML patients who had four of the most common translocations, namely t(8;21), t(15;17), t(9;11), and inv(16). The quantitative data provide clear evidence that the major change in all these translocation-carrying leukemias is a decrease in expression of the majority of transcripts compared with normal CD15 ؉ cells. From a total of 1,247,535 SAGE tags, we identified 2,604 transcripts whose expression was significantly altered in these leukemias compared with normal myeloid progenitor cells. The gene ontology of the 1,110 transcripts that matched known genes revealed that each translocation had a uniquely altered profile in various functional categories including regulation of transcription, cell cycle, protein synthesis, and apoptosis. Our global analysis of gene expression of common translocations in AML can focus attention on the function of the genes with altered expression for future biological studies as well as highlight genes͞pathways for more specifically targeted therapy.hematopoietic cell differention ͉ diagnostic microarray T he pathogenesis of acute myeloid leukemia (AML) in many patients is linked to oncogenic fusion proteins, generated as a consequence of chromosome translocations or inversions (1). Many different translocations have been described in AML, the most frequent being the t(9;11), t(15;17), t(8;21), and inv(16), which, taken together with their variants, account for Ϸ20-30% of AML cases (2, 3), although a recent analysis by Mitelman et al. (4) suggests that the proportion may be closer to 10%. These recurring translocations are now the basis for classification of some patients with AML. Despite genetic heterogeneity, there is increasing evidence for some common molecular and biological mechanisms in the genesis of AML. In particular, one of the components of each fusion protein is almost invariably a transcription factor, frequently involved in the regulation of myeloid cell differentiation (5). As a consequence, AML-associated fusion proteins function as aberrant transcriptional regulators with the potential to interfere with the normal processes of myeloid cell differentiation.Genome-wide gene expression profiling is becoming useful for the classification of many types of cancer (6, 7), including AML and acute lymphoblastic leukemia (8-15). Although AML subtypes can be distinguished by oligonucleotide microarrays, the results of analysis of different translocations between laboratories are not always similar. This lack of consistency has probably resulted from the heterogeneous nature of clinical samples (age, se...

The recurring chromosome translocation t(11;16)(q23;p13) is detected in leukemia patients, virtually all of whom have received previous chemotherapy with topoisomerase (topo) II inhibitors. In the t(11;16), 3' CBP, on 16p13, is fused to 5' MLL, on 11q23, resulting in an MLL-CBP fusion gene that plays an important role in leukemogenesis. In this study, we cloned genomic breakpoints of the MLL and CBP genes in the t(11;16) in the SN-1 cell line and in five patients with therapy-related leukemia, all of whom had received topo II inhibitors for previous tumors. In all patients except one, both the genomic MLL-CBP and the reciprocal fusions were cloned. Genomic breakpoints in MLL occurred in the 8.3-kb breakpoint cluster region in all patients, whereas the breakpoints in CBP clustered in an 8.2-kb region of intron 3 in four patients. Genomic breakpoints in MLL occurred in intron 11 near the topo II cleavage site in the SN-1 cell line and in one patient, and they were close to LINE repetitive sequences in two other patients. In the remaining two patients, genomic breakpoints were in intron 9 in Alu repeats. Genomic breakpoints in CBP occurred in and around Alu repeats in one and two patients, respectively. In two patients, the breaks were near LINE repetitive sequences, suggesting that repetitive DNA sequences may play a role. No specific recombination motifs were identified at or near the breakpoint junctions. No topo II cleavage sites were detected in introns 2 and 3 of CBP. However, there were deletions and duplications at the breakpoints in both MLL and CBP and microhomologies or nontemplated nucleotides at most of the genomic fusion junctions, suggesting that a nonhomologous end-joining repair mechanism was involved in the t(11;16).

The t(8;21)(q22;q22) that results in an AML1-ETO fusion gene is frequently detected in de novo and in therapy-related acute myeloid leukemia (AML) patients. Most t-AML patients with t(8;21) have received treatment with topoisomerase (topo) II inhibitors for primary tumors. In our previous study in 31 de novo leukemia patients with t(8;21) genomic breakpoints clustered in each three breakpoint cluster regions (BCRs) in intron 5 in AML1 and in intron 1b in ETO which tended to colocalize with DNA topo II cleavage and DNase I hypersensitive sites, implicating these chromatin structural elements in the mechanisms leading to the t(8;21). In this study, we cloned genomic breakpoints in AML1 and ETO in both derivative chromosomes in six t-AML leukemia patients with t(8;21), all of whom were treated with topo II inhibitors for previous tumors. Genomic breakpoints in AML1 and ETO in t-AML patients cluster in the same BCRs previously identified in de novo patients with t(8;21). Our results are unexpected because in MLL translocations involving 11q23, the location of breakpoints in MLL in de novo and in t-AML patients is different. There were deletions and duplications at the breakpoints in both AML1 and ETO, and microhomologies or non-templated nucleotides occurred at most of the genomic fusion junctions. No specific recombination motifs were identified at or near the breakpoint junctions. Both deletions and duplications were larger in de novo leukemia patients than in t-AML patients. In each 10 de novo patients, the deletions ranged from 5 to 556 bp (251 bp in average) in AML1 and from 6 to 225 bp (88 bp) in ETO, whereas in t-AML patients the deletions in AML1 ranged from 1-111bp (39 bp) in three and from 2-125 bp (32 bp) in ETO in five patients. Duplications in AML1 in seven de novo patients ranged from 1-141 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. Similarly, only very small (2-7 bp) deletions or duplications were detected in MLL and CBP in t-AML patients with t(11;16). Thus, there are some unexplained differences in non-homologous end joining repair in de novo and therapy-related leukemia. We mapped a scaffold attachment region (SAR) in intron 4 of AML1, and two SARs in intron 1b of ETO that are near the BCRs. Moreover, we analyzed the free energy (delta G) that is needed to unwind double strand DNA of the BCR-bearing introns in AML1 and ETO and found that the topo II cleavage sites we identified in both genes have the lowest delta G value suggesting that topo II cleaves DNA at the point of the lowest free energy. Our study suggests that illegitimate recombination between AML1 and ETO in the t(8;21) in both de novo and therapy-related leukemia patients is similar, and that topo II cleavage sites with the lowest free energy provide vulnerable sites for breakage. Non-homologous end joining repair is a likely mechanism involved in the formation of the t(8;21).