Shunpei Ishikawa

Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4–ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.

Downregulation of MicroRNA-200 in EBV-Associated Gastric Carcinoma

EBV-associated gastric carcinoma is a distinct gastric carcinoma subtype with characteristic morphologic features similar to those of cells that undergo epithelial-to-mesenchymal transition. The effect of microRNA abnormalities in carcinogenesis was investigated by measuring the expression of the epithelial-to-mesenchymal transition-related microRNAs, miR-200a and miR-200b, in 36 surgically resected gastric carcinomas using quantitative reverse transcription-PCR analysis. MiR-200 family expression was decreased in EBV-associated gastric carcinoma, as compared with that in EBV-negative carcinoma. Downregulation of the miR-200 family was found in gastric carcinoma cell lines infected with recombinant EBV (MKN74-EBV, MKN7-EBV, and NUGC3-EBV), accompanied by the loss of cell adhesion, reduction of E-cadherin expression, and upregulation of ZEB1 and ZEB2. E-cadherin expression was partially restored by transfection of EBV-infected cells with miR-200 family precursors. Reverse transcription-PCR analysis of primary precursors of miR-200 (pri-miR-200) revealed that the transcription of pri-miR-200 was decreased in EBV-infected cells. Transfection of MKN74 cells with BARF0, EBNA1, and LMP2A resulted in a decrease of pri-miR-200, whereas transfection with EBV-encoded small RNA (EBER) did not. These four latent genes contributed to the downregulation of the mature miR-200 family and the subsequent upregulation of ZEB1/ZEB2, resulting in the reduction of E-cadherin expression. These findings indicate that all the latency type I genes have a synergetic effect on the downregulation of the miR-200 family that leads to reduced E-cadherin expression, which is a crucial step in the carcinogenesis of EBV-associated gastric carcinoma.

Mechanisms of Genomic Instabilities Underlying Two Common Fragile-Site-Associated Loci, PARK2 and DMD, in Germ Cell and Cancer Cell Lines

Common fragile sites (CFSs) are specific chromosome regions that exhibit an increased frequency of breaks when cells are exposed to a DNA-replication inhibitor such as aphidicolin. PARK2 and DMD, the causative genes for autosomal-recessive juvenile Parkinsonism and Duchenne and Becker muscular dystrophy, respectively, are two very large genes that are located within aphidicolin-induced CFSs. Gross rearrangements within these two genes are frequently observed as the causative mutations for these diseases, and similar alterations within the large fragile sites that surround these genes are frequently observed in cancer cells. To elucidate the molecular mechanisms underlying this fragility, we performed a custom-designed high-density comparative genomic hybridization analysis to determine the junction sequences of approximately 500 breakpoints in germ cell lines and cancer cell lines involving PARK2 or DMD. The sequence signatures where these breakpoints occur share some similar features both in germ cell lines and in cancer cell lines. Detailed analyses of these structures revealed that microhomologies are predominantly involved in rearrangement processes. Furthermore, breakpoint-clustering regions coincide with the latest-replicating region and with large nuclear-lamina-associated domains and are flanked by the highest-flexibility peaks and R/G band boundaries, suggesting that factors affecting replication timing collectively contribute to the vulnerability for rearrangement in both germ cell and somatic cell lines.

DHTP Ligands for the Highly Ortho-Selective, Palladium-Catalyzed Cross-Coupling of Dihaloarenes with Grignard Reagents: A Conformational Approach for Catalyst Improvement†

The site-selective cross-coupling of dihaloarenes is a useful method for synthesizing substituted monohaloarenes, which are an important class of compounds that are commonly employed as drug frameworks and synthetic intermediates. To achieve their efficient site-selective cross-coupling, two main problems must be addressed: First, the difficulty in differentiating two reactive sites, particularly when the desired coupling position is sterically and electronically unfavorable, and second, the difficulty in suppressing undesired doubly cross-coupled products. We recently developed the site-selective, palladiumcatalyzed cross-coupling of dibromobenzenes with Grignard reagents. The cross-coupling occurred site-selectively at the positions ortho to the hydroxy or amino groups on the substrate. In most cases, the reactions occurred at sterically and electronically unfavorable sites. The key to this system was the use of hydroxy-substituted terphenylphosphine ligands (1 or 2 ; Scheme 1). We assume that these phosphines form bimetallic palladium/magnesium species in the presence of palladium and Grignard reagents, and that the OMgX moiety acts as a binding site for the substrate (which also exists as the magnesium salt), and holds the ortho halo group close to the palladium center (Scheme 1). In this mechanism, oxidative addition to the palladium atom occurs preferentially at the positions ortho to the OMg group of the substrate. Whilst the ortho selectivity for substrates that have a strongly electron-donating substituent is unique, and cannot be achieved using other phosphine ligands, the selectivities were often modest and the formation of doubly cross-coupled products was a severe problem in many cases. Therefore, improvement of the catalysts was necessary to expand the applicability of this ortho-selective cross-coupling procedure. Herein, we present dihydroxyterphenylphosphine (DHTP) ligands 3–6 that improved the palladium-catalyzed orthoselective cross-coupling of dihaloarenes remarkably and expanded the scope of the reaction. The design of DHTPs was based on the following ideas. The assumed catalytic species formed from 1 or 2 is conformationally rigid owing to the para-terphenyl framework but retains flexibility in rotation of the C C single bonds. As shown in Scheme 2a, the conformation in which the palladium and the magnesium oxido moieties are located proximal to each other is in equilibrium with that in which they are on opposing sides of the terphenyl group. In the latter conformation, the cooperative effect of the palladium and magnesium oxido groups cannot work (Scheme 1). Conversely, when DHTPs are used, there is always a magnesium oxido group on the same face of the terphenyl structure as the palladium atom, even if C C bond rotation occurs (Scheme 2b). Therefore, cooperation between the palladium and magnesium oxido moieties would be more effective when DHTP ligands are used, thus affording higher selectivities in the ortho-selective cross-coupling reaction. The magnesium oxido moiety of the catalytic species also has conformational flexibility (Scheme 2c). Although it was expected that controlling the spatial arrangement of the magnesium atom should affect the catalytic performance, it was unknown how that would affect the ortho-selective crosscoupling. To control the position of the magnesium atom, we introduced two types of substituents at the position ortho to Scheme 1. Structures of terphenylphosphines (1 and 2 ; top left), dihydroxyterphenylphosphines (3–6; top right), and a mechanism for the ortho-selectivity in the site-selective cross-coupling of dibromophenol with a Grignard reagent.

Abstract 4602: Recurrent pathway mutations of multiple components of cohesin complex in myeloid neoplasms.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. So, we analyzed 50 paired tumor-normal samples of myeloid neoplasms using whole exome sequencing, among which we identified recurrent mutations involving STAG2, a core cohesin component, and two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex which is composed of four core subunits (SMC1, SMC3, RAD21 and STAG proteins), and is engaged in cohesion of sister chromatids, DNA repair and transcriptional regulation. To extend the findings in the whole-exome analysis, an additional 534 primary samples of various myeloid neoplasms was examined for mutations and deletions in a total of 9 components of the cohesin complexes, using high-throughput sequencing and SNP arrays. In total, mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205), in a mutually exclusive manner. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles revealed that majority of the cohesin mutations existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we examined several myeloid leukemia cell lines with or without cohesin mutations for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of several components of cohesin was significantly reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we introduced the wild-type RAD21 allele into RAD21-mutated cell lines (Kasumi-1), which effectively suppressed the proliferation of Kasumi-1, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, about half of cohesin-mutated cases in our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Of note, the number of mutations determined by our whole exome analysis was significantly higher in cohesin-mutated cases compared to non-mutated cases. Since cohesin participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Citation Format: Ayana Kon, Lee-Yung Shih, Masashi Minamino, Masashi Sanada, Yuichi Shiraishi, Yasunobu Nagata, Kenichi Yoshida, Yusuke Okuno, Masashige Bando, Shunpei Ishikawa, Aiko Sato-Otsubo, Genta Nagae, Aiko Nishimoto, Claudia Haferlach, Daniel Nowak, Yusuke Sato, Tamara Alpermann, Teppei Shimamura, Hiroko Tanaka, Kenichi Chiba, Ryo Yamamoto, Tomoyuki Yamaguchi, Makoto Otsu, Naoshi Obara, Mamiko Sakata-Yanagimoto, Tsuyoshi Nakamaki, Ken Ishiyama, Florian Nolte, Wolf-Karsten Hofmann, Shuichi Miyawaki, Shigeru Chiba, Hiraku Mori, Hiromitsu Nakauchi, H. Phillip Koeffler, Hiroyuki Aburatani, Torsten Haferlach, Katsuhiko Shirahige, Satoru Miyano, Seishi Ogawa. Recurrent pathway mutations of multiple components of cohesin complex in myeloid neoplasms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4602. doi:10.1158/1538-7445.AM2013-4602