Misao Ohki

The t(8;21) translocation in acute myeloid leukemia results in production of an AML1-MTG8 fusion transcript.

The t(8;21) translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia. It has been shown that the t(8;21) breakpoints on chromosome 21 cluster within a single specific intron of the AML1 gene, which is highly homologous to the Drosophila segmentation gene runt. Here we report that this translocation juxtaposes the AML1 gene with a novel gene, named MTG8, on chromosome 8, resulting in the synthesis of an AML1‐MTG8 fusion transcript. The fusion protein predicted by the AML1‐MTG8 transcript consists of the runt homology region of AML1 and the most part of MTG8, which contains putative zinc finger DNA binding motifs and proline‐rich regions constituting a characteristic feature of transcription factors. The MTG8 gene is not expressed in normal hematopoietic cells, whereas AML1 is expressed at high levels. Our results indicate that the production of chimeric AML1‐MTG8 protein, probably a chimeric transcription factor, may contribute to myeloid leukemogenesis.

Interaction and functional cooperation of the leukemia‐associated factors AML1 and p300 in myeloid cell differentiation

The AML1 transcription factor and the transcriptional coactivators p300 and CBP are the targets of chromosome translocations associated with acute myeloid leukemia and myelodysplastic syndrome. In the t(8;21) translocation, the AML1 (CBFA2/PEBP2αB) gene becomes fused to the MTG8 (ETO) gene. We previously found that the terminal differentiation step leading to mature neutrophils in response to granulocyte colony‐stimulating factor (G‐CSF) was inhibited by the ectopic expression of the AML1–MTG8 fusion protein in L‐G murine myeloid progenitor cells. We show here that overexpression of normal AML1 proteins reverses this inhibition and restores the competence to differentiate. Immunoprecipitation analysis shows that p300 and CREB‐binding protein (CBP) interact with AML1. The C‐terminal region of AML1 is responsible for the induction of cell differentiation and for the interaction with p300. Overexpression of p300 stimulates AML1‐dependent transcription and the induction of cell differentiation. These results suggest that p300 plays critical roles in AML1‐dependent transcription during the differentiation of myeloid cells. Thus, AML1 and its associated factors p300 and CBFβ, all of which are targets of chromosomal rearrangements in human leukemia, function cooperatively in the differentiation of myeloid cells.

Expression profiling in ovarian clear cell carcinoma: Identification of hepatocyte nuclear factor-1β as a molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma

Of all of the epithelial ovarian cancers, clear cell carcinoma (CCC) of the ovary has the worst prognosis. We applied the oligonucleotide array technique to identify genes generally involved in CCC. Of the approximately 12,600 genes that were analyzed, 28 were expressed significantly differently between four CCC and seven non-CCC cell lines. Among 16 up-regulated genes in CCC, we further investigated a transcription factor, hepatocyte nuclear factor-1 beta (HNF-1 beta). We validated up-regulation of HNF-1 beta in CCC in terms of both mRNA and protein level using real-time quantitative reverse transcriptase-polymerase chain reaction and immunoblotting. Immunohistochemical analysis of 83 surgically resected ovarian cancers showed that almost all CCC specimens (21 of 22 cases) had nuclear staining for HNF-1 beta, whereas most non-CCC specimens (60 of 61 cases) showed no immunostaining or only focal and faint staining in the nucleus. Furthermore, we investigated the significance of HNF-1 beta expression in CCC using RNA interference. The reduction of HNF-1 beta expression by RNA interference induced apoptotic cell death in ovarian CCC cells, which was confirmed by terminal dUTP nick-end labeling and fluorescence-activated cell-sorting analyses. Our results suggest that HNF-1 beta is not only an excellent CCC-specific molecular marker but also a molecular target for therapy of ovarian CCC.

Activation of AML1-mediated transcription by MOZ and inhibition by the MOZ–CBP fusion protein

The AML1–CBFβ transcription factor complex is the most frequent target of specific chromosome translocations in human leukemia. The MOZ gene, which encodes a histone acetyltransferase (HAT), is also involved in some leukemia‐associated translocations. We report here that MOZ is part of the AML1 complex and strongly stimulates AML1‐mediated transcription. The stimulation of AML1‐mediated transcription is independent of the inherent HAT activity of MOZ. Rather, a potent transactivation domain within MOZ appears to be essential for stimulation of AML1‐mediated transcription. MOZ, as well as CBP and MOZ–CBP, can acetylate AML1 in vitro. The amount of AML1–MOZ complex increases during the differentiation of M1 myeloid cells into monocytes/macrophages, suggesting that the AML1–MOZ complex might play a role in cell differentiation. On the other hand, the MOZ–CBP fusion protein, which is created by the t(8;16) translocation associated with acute monocytic leukemia, inhibits AML1‐mediated transcription and differentiation of M1 cells. These results suggest that MOZ–CBP might induce leukemia by antagonizing the function of the AML1 complex.

The AML1-MTG8 Leukemic Fusion Protein Forms a Complex with a Novel Member of the MTG8(ETO/CDR) Family, MTGR1

ABSTRACT The AML1-CBFβ transcription factor complex is essential for the definitive hematopoiesis of all lineages and is the most frequent target of chromosomal rearrangements in human leukemia. In the t(8;21) translocation associated with acute myeloid leukemia (AML), theAML1(CBFA2/PEBP2αB) gene is juxtaposed to theMTG8(ETO/CDR) gene. We show here that the resultant AML1-MTG8 gene product specifically and strongly interacts with an 85-kDa phosphoprotein. Molecular cloning of cDNA indicated that the AML1-MTG8-binding protein (MTGR1) is highly related to MTG8 and similar to Drosophila Nervy. Comparison of amino acid sequences among MTGR1, MTG8, and Nervy revealed four evolutionarily conserved regions (NHR1 to NHR4). Ectopic expression of AML1-MTG8 in L-G murine myeloid progenitor cells inhibits differentiation to mature neutrophils and induces cell proliferation in response to granulocyte colony-stimulating factor (G-CSF). Analysis with C-terminal deletion mutants of AML1-MTG8 indicated that the region of 51 residues (488 to 538), which contains NHR2, is essential for the induction of G-CSF-dependent cell proliferation. Immunoprecipitation analysis indicates that this region is required for AML1-MTG8 to form a stable complex with MTGR1. Overexpression of MTGR1 stimulates AML1-MTG8 to induce G-CSF-dependent proliferation of L-G cells and to interfere with AML1-dependent transcription. These results suggest that AML1-MTG8 could function as a complex with MTGR1 and that the complex might be important in promoting leukemogenesis.

Overexpression of KIT in chromophobe renal cell carcinoma

We analysed gene-expression profiles in 15 surgical specimens of conventional, papillary, and chromophobe renal cell carcinomas (RCCs) using high-density oligonucleotide arrays. From about 12 000 genes targeted by the array, 67 were upregulated specifically in each histological type of RCC. The oncogene KIT was one of the genes whose expression was upregulated specifically in chromophobe RCCs. Immunohistochemical analysis demonstrated the KIT gene product on the cell membrane of chromophobe RCC in all cases, although it was not detected in conventional RCCs or non-neoplastic kidneys except for weak staining in the cytoplasm of renal tubules. These results suggest that each histological subtype of RCC has a unique gene-expression profile, and in particular indicates for the first time that KIT could be a useful marker for chromophobe RCC. As overexpression of KIT might be involved in tumor growth, KIT could be a new therapeutic target in this special type of RCC.

Fusion of MOZ and p300 histone acetyltransferases in acute monocytic leukemia with a t(8;22)(p11;q13) chromosome translocation.

Histone acetyltransferase p300 functions as a transcriptional co-activator which interacts with a number of transcription factors. Monocytic leukemia zinc finger protein (MOZ) has histone acetyltransferase activity. We report the fusion of the MOZ gene to the p300 gene in acute myeloid leukemia with translocation t(8;22)(p11;q13). FISH and Southern blot analyses showed the rearrangement of the MOZ and p300 genes. We determined the genomic structure of the p300 and the MOZ genes and the breakpoints of the translocation. Analysis of fusion transcripts indicated that the zinc finger and acetyltransferase domains of MOZ are fused to a largely intact p300. These results suggest that MOZ-p300, which has two acetyltransferase domains, could be involved in leukemogenesis through aberrant regulation of histone acetylation.

Genome‐wide array‐based comparative genomic hybridization analysis of pancreatic adenocarcinoma: Identification of genetic indicators that predict patient outcome

We analyzed the subchromosomal numerical aberrations of 44 surgically resected pancreatic adenocarcinomas by array‐based comparative genomic hybridization. The aberration profile ranged widely between cases, suggesting the presence of multiple or complementary mechanisms of evolution in pancreatic cancer, and was associated with lymph node metastasis and venous or serosal invasion. A large number of small loci, previously uncharacterized in pancreatic cancer, showed non‐random loss or gain. Frequent losses at 1p36, 4p16, 7q36, 9q34, 11p15, 11q13, 14q32‐33, 16p13, 17p11‐13, 17q11‐25, 18q21‐tel, 19p13, 21q22 and 22q11‐12, and gains at 1q25, 2p16, 2q21‐37, 3q25, 5p14, 5q11‐13, 7q21, 7p22, 8p22, 8q21‐23, 10q21, 12p13, 13q22, 15q13‐22 and 18q11 were identified. Sixteen loci were amplified recurrently. We identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Gain of LUNX, HCK, E2F1 and DNMT3b at 20q11, loss of p73 at 1p36 and gain of PPM1D at 17q23 independently predicted patient outcome. Expression profiling of amplified genes identified Smurf1 and TRRAP at 7q22.1, BCAS1 at 20q13.2‐3, and VCL at 10q22.1 as potential novel oncogenes. Our results contribute to a complete description of genomic structural aberrations and the identification of potential therapeutic targets and genetic indicators that predict patient outcome in pancreatic adenocarcinoma. (Cancer Sci 2007; 98: 392–400)

Association of amino acid substitution polymorphisms in DNA repair genes TP53, POLI, REV1 and LIG4 with lung cancer risk.

Single nucleotide polymorphisms (SNPs) were searched for in 36 genes involved in diverse DNA repair pathways, and 50 nonsynonymous (associated with amino acid changes) SNPs identified were assessed for associations with lung cancer risk by a case‐control study consisting of 752 adenocarcinoma cases, 250 squamous cell carcinoma cases and 685 controls. An SNP, Arg72Pro, of the TP53 gene encoding a DNA damage response protein showed the strongest association with squamous cell carcinoma risk (OR Pro/Pro vs. Arg/Arg = 2.2), while 2 other SNPs, Phe257Ser of the REV gene encoding a translesion DNA polymerase and Ile658Val of the LIG4 gene encoding a DNA double‐strand break repair protein, also showed associations (OR Ser/Ser vs. Phe/Phe = 2.0 and OR Ile/Val vs. Ile/Ile = 0.4, respectively). An SNP, Thr706Ala, in the POLI gene encoding another translesion DNA polymerase was associated with adenocarcinoma and squamous cell carcinoma risk, particularly in individuals of ages < 61 years (OR Ala/Ala + Ala/Thr vs. Thr/Thr = 1.5 and 2.4, respectively). POLI is the human counterpart of PolI, a strong candidate for the Par2 (pulmonary adenoma resistance 2) gene responsible for adenoma/adenocarcinoma susceptibility in mice. The present results suggest that these 4 SNPs function as genetic factors underlying lung cancer susceptibility by modulating activities to maintain the genome integrity of each individual.

Genetic Classification of Lung Adenocarcinoma Based on Array-Based Comparative Genomic Hybridization Analysis: Its Association with Clinicopathologic Features

The array-based comparative genomic hybridization using microarrayed bacterial artificial chromosome clones allows high-resolution analysis of genome-wide copy number changes in tumors. To analyze the genetic alterations of primary lung adenocarcinoma in a high-throughput way, we used laser-capture microdissection of cancer cells and array comparative genomic hybridization focusing on 800 chromosomal loci containing cancer-related genes. We identified a large number of chromosomal numerical alterations, including frequent amplifications on 7p12, 11q13, 12q14-15, and 17q21, and two homozygous deletions on 9p21 and one on 8p23. Unsupervised hierarchical clustering analysis of multiple alterations revealed three subgroups of lung adenocarcinoma that were characterized by the accumulation of distinct genetic alterations and associated with smoking history and gender. The mutation status of the epidermal growth factor receptor (EGFR) gene was significantly associated with specific genetic alterations and supervised clustering analysis based on EGFR gene mutations elucidated a subgroup including all EGFR gene mutated tumors, which showed significantly shorter disease-free survival. Our results suggest that there exist multiple molecular carcinogenesis pathways in lung adenocarcinoma that may associate with smoking habits and gender, and that genetic cancer profiling will reveal previously uncharacterized genetic heterogeneity of cancer and be beneficial in estimating patient prognosis and discovering novel cancer-related genes including therapeutic targets.