Publications by authors named "Genta Nagae"

53 Publications

Accumulation of Molecular Aberrations Distinctive to Hepatocellular Carcinoma Progression.

Cancer Res 2020 09 8;80(18):3810-3819. Epub 2020 Jul 8.

Genome Science Division, RCAST, University of Tokyo, Tokyo, Japan.

Cancer develops through the accumulation of genetic and epigenetic aberrations. To identify sequential molecular alterations that occur during the development of hepatocellular carcinoma (HCC), we compared 52 early and 108 overt HCC samples by genome sequencing. Gene mutations in the p53/RB1 pathway, WNT pathway, MLL protein family, SWI/SNF complexes, and AKT/PI3K pathway were common in HCC. In the early phase of all entities, was the most frequently upregulated gene owing to diverse mechanisms. Despite frequent somatic mutations in driver genes, including and , early HCC was a separate molecular entity from overt HCC, as each had a distinct expression profile. Notably, WNT target genes were not activated in early HCC regardless of mutation status because β-catenin did not translocate into the nucleus due to the E-cadherin/β-catenin complex at the membrane. Conversely, WNT targets were definitively upregulated in overt HCC, with mutation associated with downregulation of and hypomethylation of CpG islands in target genes. Similarly, cell-cycle genes downstream of the p53/RB pathway were upregulated only in overt HCC, with or gene mutations associated with chromosomal deletion of 4q or 16q. HCC was epigenetically distinguished into four subclasses: normal-like methylation, global-hypomethylation (favorable prognosis), stem-like methylation (poor prognosis), and CpG island methylation. These methylation statuses were globally maintained through HCC progression. Collectively, these data show that as HCC progresses, additional molecular events exclusive of driver gene mutations cooperatively contribute to transcriptional activation of downstream targets according to methylation status. SIGNIFICANCE: In addition to driver gene mutations in the WNT and p53 pathways, further molecular events are required for aberrant transcriptional activation of these pathways as HCC progresses.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0225DOI Listing
September 2020

A novel sensitive detection method for DNA methylation in circulating free DNA of pancreatic cancer.

PLoS One 2020 10;15(6):e0233782. Epub 2020 Jun 10.

Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Despite recent advances in clinical treatment, pancreatic cancer remains a highly lethal malignancy. In order to improve the survival rate of patients with pancreatic cancer, the development of non-invasive diagnostic methods using effective biomarkers is urgently needed. Here, we developed a highly sensitive method to detect DNA methylation in cell-free (cf)DNA samples based on the enrichment of methyl-CpG binding (MBD) protein coupled with a digital PCR method (MBD-ddPCR). Five DNA methylation markers for the diagnosis of pancreatic cancer were identified through DNA methylation microarray analysis in 37 pancreatic cancers. The sensitivity and specificity of the five markers were validated in another independent cohort of pancreatic cancers (100% and 100%, respectively; n = 46) as well as in The Cancer Genome Atlas data set (96% and 90%, respectively; n = 137). MBD-ddPCR analysis revealed that DNA methylation in at least one of the five markers was detected in 23 (49%) samples of cfDNA from 47 patients with pancreatic cancer. Further, a combination of DNA methylation markers and the KRAS mutation status improved the diagnostic capability of this method (sensitivity and specificity, 68% and 86%, respectively). Genome-wide MBD-sequencing analysis in cancer tissues and corresponding cfDNA revealed that more than 80% of methylated regions were overlapping; DNA methylation profiles of cancerous tissues and cfDNA significantly correlated with each other (R = 0.97). Our data indicate that newly developed MBD-ddPCR is a sensitive method to detect cfDNA methylation and that using five marker genes plus KRAS mutations may be useful for the detection of pancreatic cancers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233782PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286528PMC
August 2020

Defined lifestyle and germline factors predispose Asian populations to gastric cancer.

Sci Adv 2020 May 6;6(19):eaav9778. Epub 2020 May 6.

Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Tokyo, Japan.

Germline and environmental effects on the development of gastric cancers (GC) and their ethnic differences have been poorly understood. Here, we performed genomic-scale trans-ethnic analysis of 531 GCs (319 Asian and 212 non-Asians). There was one distinct GC subclass with clear alcohol-associated mutation signature and strong Asian specificity, almost all of which were attributable to alcohol intake behavior, smoking habit, and Asian-specific defective allele. Alcohol-related GCs have low mutation burden and characteristic immunological profiles. In addition, we found frequent (7.4%) germline variants among Japanese GCs, most of which were attributed to a few recurrent single-nucleotide variants shared by Japanese and Koreans, suggesting the existence of common ancestral events among East Asians. Specifically, approximately one-fifth of diffuse-type GCs were attributable to the combination of alcohol intake and defective allele or to variants. These results revealed uncharacterized impacts of germline variants and lifestyles in the high incidence areas.
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http://dx.doi.org/10.1126/sciadv.aav9778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202881PMC
May 2020

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Nat Commun 2020 03 6;11(1):1222. Epub 2020 Mar 6.

Division of Cancer Cell Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.
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http://dx.doi.org/10.1038/s41467-020-15006-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060239PMC
March 2020

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells.

Epigenetics Chromatin 2019 12 19;12(1):77. Epub 2019 Dec 19.

Japan Agency for Medical Research and Development (AMED-CREST), AMED, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan.

Background: Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood.

Results: To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing.

Conclusions: This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.
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http://dx.doi.org/10.1186/s13072-019-0319-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921469PMC
December 2019

Spatial and temporal expansion of intrahepatic metastasis by molecularly-defined clonality in multiple liver cancers.

Cancer Sci 2020 Feb 11;111(2):601-609. Epub 2020 Jan 11.

Genome Science Division, RCAST, University of Tokyo, Tokyo, Japan.

Multiple hepatocellular carcinoma (HCC) is divided into two categories: intrahepatic metastasis (IM), which is a true relapse of HCC, and multicentric origin (MO), which is a second primary tumor. Clinical diagnosis of multiple HCC is usually made based on tumor location and/or time to recurrence; however, it is often difficult to distinguish the two types of multiple HCC. Using 41 matched pairs of multiple HCC specimens, we confirmed the accuracy of clinical diagnoses using exome sequence data and investigated the importance of discriminating the type of multiple HCC. Genomic analysis revealed that 18 (43.9%) patients diagnosed as having genomic IM had common mutations in a pair of HCC tumors with the main tumor of these patients being more progressive compared to those with genomic MO. The accuracy of clinical diagnosis based on lobe (Definition 1) and segment (Definition 2) were 68.3% and 78.0%, respectively. Intriguingly, recurrence ≥2 years after initial surgery for 3 patients was IM. The survival of patients with clinical IM was significantly shorter than for those with clinical MO based on both Definition 1 (P = 0.045) and Definition 2 (P = 0.043). However, mean survival was not different between the patients with genomic IM and those with MO (P = 0.364). Taken together, genomic analysis elucidated that liver cancer may spread more extensively and more slowly than previously thought. In addition, distinguishing multiple HCC as IM or MC may have provided biological information but was not of clinical importance with respect to patient prognosis.
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http://dx.doi.org/10.1111/cas.14282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004543PMC
February 2020

Impact of AAV2 and Hepatitis B Virus Integration Into Genome on Development of Hepatocellular Carcinoma in Patients with Prior Hepatitis B Virus Infection.

Clin Cancer Res 2019 10 18;25(20):6217-6227. Epub 2019 Jul 18.

Genome Science Division, RCAST, University of Tokyo, Tokyo, Japan.

Purpose: Hepatitis B viral (HBV) DNA is frequently integrated into the genomes of hepatocellular carcinoma (HCC) in patients with chronic HBV infection (chronic HBV, hereafter), whereas the frequency of HBV integration in patients after the disappearance of HBV (prior HBV, hereafter) has yet to be determined. This study aimed to detect integration of HBV and adeno-associated virus type 2 (AAV2) into the human genome as a possible oncogenic event.

Experimental Design: Virome capture sequencing was performed, using HCC and liver samples obtained from 243 patients, including 73 with prior HBV without hepatitis C viral (HCV) infection and 81 with chronic HBV.

Results: Clonal HBV integration events were identified in 11 (15.0%) cases of prior HBV without HCV and 61 (75.3%) cases of chronic HBV ( < 0.001). Several driver genes were commonly targeted by HBV, leading to transcriptional activation of these genes; [four (5.4%) vs. 15 (18.5%)], [two (2.7%) vs. five (6.1%)], [zero vs. one (1.2%)], [zero vs. one (1.2%)]. Conversely, and were, respectively, targeted by AAV2 only in prior HBV. In liver samples, HBV genome recurrently integrated into fibrosis-related genes , and in chronic HBV. There was not history of alcohol abuse and 3 patients with a history of nucleoside analogue treatment for HBV in 8 prior HBV with driver gene integration.

Conclusions: Despite the seroclearance of hepatitis B surface antigen, HBV or AAV2 integration in prior HBV was not rare; therefore, such patients are at risk of developing HCC.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-4041DOI Listing
October 2019

Publisher Correction: DNA demethylation is associated with malignant progression of lower-grade gliomas.

Sci Rep 2019 May 23;9(1):7935. Epub 2019 May 23.

Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-019-43790-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531430PMC
May 2019

Reduced Neoantigen Expression Revealed by Longitudinal Multiomics as a Possible Immune Evasion Mechanism in Glioma.

Cancer Immunol Res 2019 07 14;7(7):1148-1161. Epub 2019 May 14.

Department of Immunotherapeutics, The University of Tokyo Hospital, Tokyo, Japan.

Immune-based therapies have shown limited efficacy in glioma thus far. This might be at least in part due to insufficient numbers of neoantigens, thought to be targets of immune attack. In addition, we hypothesized that dynamic genetic and epigenetic tumor evolution in gliomas might also affect the mutation/neoantigen landscape and contribute to treatment resistance through immune evasion. Here, we investigated changes in the neoantigen landscape and immunologic features during glioma progression using exome and RNA-seq of paired primary and recurrent tumor samples obtained from 25 WHO grade II-IV glioma patients (glioblastoma, IDH-wild-type, = 8; grade II-III astrocytoma, IDH-mutant, = 9; and grade II-III oligodendroglioma, IDH-mutant, 1p/19q-codeleted, = 8). The number of missense mutations, predicted neoantigens, or expressed neoantigens was not significantly different between primary and recurrent tumors. However, we found that in individual patients the ratio of expressed neoantigens to predicted neoantigens, designated the "neoantigen expression ratio," decreased significantly at recurrence ( = 0.003). This phenomenon was particularly pronounced for "high-affinity," "clonal," and "passenger gene-derived" neoantigens. Gene expression and IHC analyses suggested that the decreased neoantigen expression ratio was associated with intact antigen presentation machinery, increased tumor-infiltrating immune cells, and ongoing immune responses. Our findings imply that decreased expression of highly immunogenic neoantigens, possibly due to persistent immune selection pressure, might be one of the immune evasion mechanisms along with tumor clonal evolution in some gliomas.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0599DOI Listing
July 2019

DNA demethylation is associated with malignant progression of lower-grade gliomas.

Sci Rep 2019 02 13;9(1):1903. Epub 2019 Feb 13.

Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

To elucidate the mechanisms of malignant progression of lower-grade glioma, molecular profiling using methylation array, whole-exome sequencing, and RNA sequencing was performed for 122, 36 and 31 gliomas, respectively. This cohort included 24 matched pairs of initial lower-grade gliomas and recurrent tumors, most of which showed malignant progression. Nearly half of IDH-mutant glioblastomas that had progressed from lower-grade gliomas exhibited characteristic partial DNA demethylation in previously methylated genomic regions of their corresponding initial tumors, which had the glioma CpG island methylator phenotype (G-CIMP). In these glioblastomas, cell cycle-related genes, RB and PI3K-AKT pathway genes were frequently altered. Notably, late-replicating domain was significantly enriched in the demethylated regions that were mostly located in non-regulatory regions, suggesting that the loss of DNA methylation during malignant transformation may involve mainly passive demethylation due to a delay in maintenance of methylation during accelerated cell division. Nonetheless, a limited number of genes including IGF2BP3, which potentially drives cell proliferation, were presumed to be upregulated due to demethylation of their promoter. Our data indicated that demethylation of the G-CIMP profile found in a subset of recurrent gliomas reflects accelerated cell divisions accompanied by malignant transformation. Oncogenic genes activated by such epigenetic change represent potential therapeutic targets.
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http://dx.doi.org/10.1038/s41598-019-38510-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374451PMC
February 2019

Differential regulation of CpG island methylation within divergent and unidirectional promoters in colorectal cancer.

Cancer Sci 2019 Mar 2;110(3):1096-1104. Epub 2019 Mar 2.

Division of Cellular Signaling, National Cancer Center Research Institute, Tokyo, Japan.

The silencing of tumor suppressor genes by promoter CpG island (CGI) methylation is an important cause of oncogenesis. Silencing of MLH1 and BRCA1, two examples of oncogenic events, results from promoter CGI methylation. Interestingly, both MLH1 and BRCA1 have a divergent promoter, from which another gene on the opposite strand is also transcribed. Although studies have shown that divergent transcription is an important factor in transcriptional regulation, little is known about its implication in aberrant promoter methylation in cancer. In this study, we analyzed the methylation status of CGI in divergent promoters using a recently enriched transcriptome database. We measured the extent of CGI methylation in 119 colorectal cancer (CRC) clinical samples (65 microsatellite instability high [MSI-H] CRC with CGI methylator phenotype, 28 MSI-H CRC without CGI methylator phenotype and 26 microsatellite stable CRC) and 21 normal colorectal tissues using Infinium MethylationEPIC BeadChip. We found that CGI within divergent promoters are less frequently methylated than CGI within unidirectional promoters in normal cells. In the genome of CRC cells, CGI within unidirectional promoters are more vulnerable to aberrant methylation than CGI within divergent promoters. In addition, we identified three DNA sequence motifs that correlate with methylated CGI. We also showed that methylated CGI are associated with genes whose expression is low in normal cells. Thus, we here provide fundamental observations regarding the methylation of divergent promoters that are essential for the understanding of carcinogenesis and development of cancer prevention strategies.
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http://dx.doi.org/10.1111/cas.13937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398885PMC
March 2019

Transduced caudal-type homeobox (CDX) 2/CDX1 can induce growth inhibition on CDX-deficient gastric cancer by rapid intestinal differentiation.

Cancer Sci 2018 Dec 26;109(12):3853-3864. Epub 2018 Oct 26.

Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Intestinal metaplasia induced by ectopic expression of caudal-type homeobox (CDX)2 and/or CDX1 (CDX) is frequently observed around gastric cancer (GC). Abnormal expression of CDX is also observed in GC and suggests that inappropriate gastrointestinal differentiation plays essential roles in gastric tumorigenesis, but their roles on tumorigenesis remain unelucidated. Publicly available databases show that GC patients with higher CDX expression have significantly better clinical outcomes. We introduced CDX2 and CDX1 genes separately into GC-originated MKN7 and TMK1 cells deficient in CDX. Marked suppression of cell growth and dramatic morphological change into spindle-shaped flat form were observed along with induction of intestinal marker genes. G0-G1 growth arrest was accompanied by changed expression of cell cycle-related genes but not with apoptosis or senescence. Microarray analyses additionally showed decreased expression of gastric marker genes and increased expression of stemness-associated genes. Hierarchical clustering of 111 GC tissues and 21 non-cancerous gastric tissues by selected 18 signature genes based on our transcriptome analyses clearly categorized the 132 tissues into non-cancer, "CDX signature"-positive GC, and "CDX signature"-negative GC. Gene set enrichment analysis indicated that "CDX signature"-positive GC has lower malignant features. Immunohistochemistry of 89 GC specimens showed that 50.6% were CDX2-deficient, 66.3% were CDX1-deficient, and 44.9% were concomitant CDX2/CDX1-deficient, suggesting that potentially targetable GC cases by induced intestinal differentiation are quite common. In conclusion, exogenous expression of CDX2/CDX1 can lead to efficient growth inhibition of CDX-deficient GC cells. It is based on rapidly induced intestinal differentiation, which may be a future therapeutic strategy.
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http://dx.doi.org/10.1111/cas.13821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272106PMC
December 2018

Fusion Kinases Identified by Genomic Analyses of Sporadic Microsatellite Instability-High Colorectal Cancers.

Clin Cancer Res 2019 01 2;25(1):378-389. Epub 2018 Oct 2.

Department of Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Purpose: Colorectal cancers with microsatellite instability-high (MSI-H) status, due to mismatch repair deficiency, are associated with poor patient outcomes after relapse. We aimed to identify novel therapeutic targets for them.

Experimental Design: We performed MSI analyses of over 2,800 surgically resected colorectal tumors obtained from consecutive patients treated in Japan from 1998 through June 2016. Whole-exome sequencing, transcriptome sequencing, and methylation analyses were performed on 149 of 162 tumors showing MSI in BAT25 and BAT26 loci. We analyzed patient survival times using Bonferroni-adjusted log-rank tests.

Results: Sporadic MSI-H colorectal cancers with promoter methylation of (called MM) had a clinicopathological profile that was distinct from that of colorectal cancers of patients with germline mutations (Lynch syndrome, LS-associated) or somatic, Lynch-like mutations in mismatch repair genes. MM tumors had more insertions and deletions and more recurrent mutations in and than LS-associated or Lynch-like MSI-H tumors. Eleven fusion kinases were exclusively detected in MM MSI-H colorectal cancers lacking oncogenic / missense mutations and were associated with worse post-relapse prognosis. We developed a simple method to identify MM tumors and applied it to a validation cohort of 28 MSI-H colorectal cancers, identifying 16 MM tumors and 2 fusion kinases.

Conclusions: We discovered that fusion kinases are frequently observed among sporadic MM MSI-H colorectal cancers. The new method to identify MM tumors enables us to straightforwardly group MSI-H patients into candidates of LS or fusion kinase carriers.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1574DOI Listing
January 2019

Integrated molecular profiling of juvenile myelomonocytic leukemia.

Blood 2018 04 2;131(14):1576-1586. Epub 2018 Feb 2.

Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Juvenile myelomonocytic leukemia (JMML), a rare and aggressive myelodysplastic/myeloproliferative neoplasm that occurs in infants and during early childhood, is characterized by excessive myelomonocytic cell proliferation. More than 80% of patients harbor germ line and somatic mutations in RAS pathway genes (eg, , , , , and ), and previous studies have identified several biomarkers associated with poor prognosis. However, the molecular pathogenesis of 10% to 20% of patients and the relationships among these biomarkers have not been well defined. To address these issues, we performed an integrated molecular analysis of samples from 150 JMML patients. RNA-sequencing identified tyrosine kinase fusions (, and ) in 3 of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed fusion-positive JMML cell proliferation in vitro. Therefore, we administered crizotinib to a chemotherapy-resistant patient with the fusion who subsequently achieved complete molecular remission. In addition, crizotinib also suppressed proliferation of JMML cells with canonical RAS pathway mutations. Genome-wide methylation analysis identified a hypermethylation profile resembling that of acute myeloid leukemia (AML), which correlated significantly with genetic markers with poor outcomes such as gene mutations, 2 or more genetic mutations, an AML-type expression profile, and expression. In summary, we identified recurrent activated fusions in JMML patients without canonical RAS pathway gene mutations and revealed the relationships among biomarkers for JMML. Crizotinib is a promising candidate drug for the treatment of JMML, particularly in patients with fusions.
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http://dx.doi.org/10.1182/blood-2017-07-798157DOI Listing
April 2018

An Excess of CYP24A1, Lack of CaSR, and a Novel lncRNA Near the PTH Gene Characterize an Ectopic PTH-Producing Tumor.

J Endocr Soc 2017 Jun 3;1(6):691-711. Epub 2017 May 3.

Department of Biochemistry, Teikyo University School of Medicine, Tokyo 173-0003, Japan.

Thus far, only 23 cases of the ectopic production of parathyroid hormone (PTH) have been reported. We have characterized the genome-wide transcription profile of an ectopic PTH-producing tumor originating from a retroperitoneal histiocytoma. We found that the calcium-sensing receptor (CaSR) was barely expressed in the tumor. Lack of CaSR, a crucial braking apparatus in the presence of both intraparathyroid and, probably, serendipitous PTH expression, might contribute strongly to the establishment and maintenance of the ectopic transcriptional activation of the PTH gene in nonparathyroid cells. Along with candidate drivers with a crucial frameshift mutation or copy number variation at specific chromosomal areas obtained from whole exome sequencing, we identified robust tumor-specific cytochrome P450 family 24 subfamily A member 1 (CYP24A1) overproduction, which was not observed in other non-PTH-expressing retroperitoneal histiocytoma and parathyroid adenoma samples. We then found a 2.5-kb noncoding RNA in the PTH 3'-downstream region that was exclusively present in the parathyroid adenoma and our tumor. Such a co-occurrence might act as another driver of ectopic PTH-producing tumorigenesis; both might release the control of PTH gene expression by shutting down the other branches of the safety system ( CaSR and the vitamin D3-vitamin D receptor axis).
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http://dx.doi.org/10.1210/js.2017-00063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686629PMC
June 2017

Aberrant DNA methylation of pregnane X receptor underlies metabolic gene alterations in the diabetic kidney.

Am J Physiol Renal Physiol 2018 04 6;314(4):F551-F560. Epub 2017 Dec 6.

Division of Clinical Epigenetics, The University of Tokyo , Tokyo , Japan.

Epigenetic abnormalities have been suggested to mediate metabolic memory observed in diabetic complications. We have shown that epigenetic alterations may induce persistent phenotypic changes in the proximal tubules of the diabetic kidneys. In this study, we show that pregnane X receptor (PXR), a xenobiotic nuclear receptor, is epigenetically altered and upregulated and may have a possible function in the diabetic kidney. PXR has been shown to play a critical role in metabolic changes in obesity and diabetes; however, its distribution and function in the kidney are unknown. In the normal kidney, Pxr was selectively expressed in the proximal tubular cells with demethylation in the promoter DNA. In db/db mice, significant increases in Pxr mRNA, further demethylation of DNA, and stimulatory histone marks in the promoter were observed. Epigenetic changes are likely to play a causative role in PXR induction, since a DNA methyltransferase inhibitor increased PXR mRNA in cultured human proximal tubular cells. Administration of a PXR agonist increased mRNA levels of solute carrier organic anion transporter family member 2B1 ( Slco2b1), a xenobiotic transporter; response gene to complement 32 ( Rgc32), a molecule known to exert fibrotic effects in the kidney; and phosphoenolpyruvate carboxykinase 1 ( Pck1), a gluconeogenic enzyme in the kidney. The expressions of these genes were inhibited by PXR small interfering RNA in cultured proximal tubular cells. Increased mRNA levels of Slco2b1, Rgc32, and Pck1 were also observed in the kidney of db/db mice. These data indicate that PXR is upregulated in the diabetic kidney with aberrant epigenetic modifications and may modulate the course of diabetic kidney disease through the activation of these genes.
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http://dx.doi.org/10.1152/ajprenal.00390.2017DOI Listing
April 2018

Distinct molecular profile of diffuse cerebellar gliomas.

Acta Neuropathol 2017 12 29;134(6):941-956. Epub 2017 Aug 29.

Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Recent studies have demonstrated that tumor-driving alterations are often different among gliomas that originated from different brain regions and have underscored the importance of analyzing molecular characteristics of gliomas stratified by brain region. Therefore, to elucidate molecular characteristics of diffuse cerebellar gliomas (DCGs), 27 adult, mostly glioblastoma cases were analyzed. Comprehensive analysis using whole-exome sequencing, RNA sequencing, and Infinium methylation array (n = 17) demonstrated their distinct molecular profile compared to gliomas in other brain regions. Frequent mutations in chromatin-modifier genes were identified including, noticeably, a truncating mutation in SETD2 (n = 4), which resulted in loss of H3K36 trimethylation and was mutually exclusive with H3F3A K27M mutation (n = 3), suggesting that epigenetic dysregulation may lead to DCG tumorigenesis. Alterations that cause loss of p53 function including TP53 mutation (n = 9), PPM1D mutation (n = 2), and a novel type of PPM1D fusion (n = 1), were also frequent. On the other hand, mutations and copy number changes commonly observed in cerebral gliomas were infrequent. DNA methylation profile analysis demonstrated that all DCGs except for those with H3F3A mutations were categorized in the "RTK I (PDGFRA)" group, and those DCGs had a gene expression signature that was highly associated with PDGFRA. Furthermore, compared with the data of 315 gliomas derived from different brain regions, promoter methylation of transcription factors genes associated with glial development showed a characteristic pattern presumably reflecting their tumor origin. Notably, SOX10, a key transcription factor associated with oligodendroglial differentiation and PDGFRA regulation, was up-regulated in both DCG and H3 K27M-mutant diffuse midline glioma, suggesting their developmental and biological commonality. In contrast, SOX10 was silenced by promoter methylation in most cerebral gliomas. These findings may suggest potential tailored targeted therapy for gliomas according to their brain region, in addition to providing molecular clues to identify the region-related cellular origin of DCGs.
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http://dx.doi.org/10.1007/s00401-017-1771-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663812PMC
December 2017

DNA-friendly Cu(ii)/TEMPO-catalyzed 5-hydroxymethylcytosine-specific oxidation.

Chem Commun (Camb) 2017 May;53(42):5756-5759

Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

The mixture of copper(ii) perchlorate and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) mildly and specifically oxidized 5-hydroxymethylcytosine to yield 5-formylcytosine because of its allyl alcohol specificity. The genomic DNA oxidized at 5-hydroxymethylcytosine by the copper(ii)/TEMPO oxidation was efficiently analyzed using various practical methods: DNA cleavage by piperidine, specific functionalization by hydrazone formation, and genome-wide high-resolution microarray.
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http://dx.doi.org/10.1039/c7cc02814hDOI Listing
May 2017

Genetic and epigenetic stability of oligodendrogliomas at recurrence.

Acta Neuropathol Commun 2017 03 7;5(1):18. Epub 2017 Mar 7.

Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.

Among diffuse gliomas, oligodendrogliomas show relatively better prognosis, respond well to radiotherapy and chemotherapy, and seldom progress to very aggressive tumors. To elucidate the genetic and epigenetic background for such behavior and tumor evolution during tumor relapse, we comparatively analyzed 12 pairs of primary and recurrent oligodendrogliomas with 1p/19q-codeletion. Initial treatment for these patients was mostly chemotherapy alone. Temozolomide was used for 3, and procarbazine, nimustine and vincristine (PAV chemotherapy) were used for 7 patients. World Health Organization histological grade at recurrence was mostly stable; it was increased in 2, the same in 9, and decreased in 1 cases. Whole-exome sequencing demonstrated that the rate of shared mutation between the primary and recurrent tumors was relatively low, ranging from 3.2-57.9% (average, 33.3%), indicating a branched evolutionary pattern. The trunk alterations that existed throughout the course were restricted to IDH1 mutation, 1p/19q-codeletion, and TERT promoter mutation, and mutation of the known candidate tumor suppressor genes CIC and FUBP1 were not consistently observed between primary and recurrent tumors. Multiple sampling from different regions within a tumor showed marked intratumoral heterogeneity. Notably, in general, the number of mutations was not significantly different after recurrence, remaining under 100, and no hypermutator phenotype was observed. FUBP1 mutation, loss of chr. 9p21, and TCF12 mutation were among a few recurrent de novo alterations that were found at recurrence, indicating that these events were clonally selected at recurrence but were not enough to enhance malignancy. Genome-wide methylation status, measured by Illumina 450 K arrays, was stable between recurrence and the primary tumor. In summary, although oligodendroglioma displays marked mutational heterogeneity, histological malignant transformation accompanying events such as considerable increase in mutation number and epigenetic profile change were not observed at recurrence, indicating that noticeable temporal and spatial genetic heterogeneity in oligodendrogliomas does not result in rapid tumor progression.
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http://dx.doi.org/10.1186/s40478-017-0422-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339990PMC
March 2017

Base-Resolution Analysis of 5-Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate.

J Am Chem Soc 2016 11 24;138(43):14178-14181. Epub 2016 Oct 24.

Department of Chemistry and Biotechnology, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

5-Hydroxymethylcytosine (C) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring C by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of C, trihydroxylated thymine (T), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that C sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of C in double-stranded DNA. Furthermore, Illumina sequencing analysis of C-containing synthetic genome fragments enabled us to identify simultaneously the positions of C in base resolution. This bisulfite-free simple C detection technique could facilitate the acquisition of epigenomic information.
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http://dx.doi.org/10.1021/jacs.6b06428DOI Listing
November 2016

Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer.

Nat Genet 2016 05 11;48(5):500-9. Epub 2016 Apr 11.

Laboratory for Genome Sequencing Analysis, RIKEN Center for Integrative Medical Sciences, Tokyo, Japan.

Liver cancer, which is most often associated with virus infection, is prevalent worldwide, and its underlying etiology and genomic structure are heterogeneous. Here we provide a whole-genome landscape of somatic alterations in 300 liver cancers from Japanese individuals. Our comprehensive analysis identified point mutations, structural variations (STVs), and virus integrations, in noncoding and coding regions. We discovered mutational signatures related to liver carcinogenesis and recurrently mutated coding and noncoding regions, such as long intergenic noncoding RNA genes (NEAT1 and MALAT1), promoters, CTCF-binding sites, and regulatory regions. STV analysis found a significant association with replication timing and identified known (CDKN2A, CCND1, APC, and TERT) and new (ASH1L, NCOR1, and MACROD2) cancer-related genes that were recurrently affected by STVs, leading to altered expression. These results emphasize the value of whole-genome sequencing analysis in discovering cancer driver mutations and understanding comprehensive molecular profiles of liver cancer, especially with regard to STVs and noncoding mutations.
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http://dx.doi.org/10.1038/ng.3547DOI Listing
May 2016

Integrated Multiregional Analysis Proposing a New Model of Colorectal Cancer Evolution.

PLoS Genet 2016 Feb 18;12(2):e1005778. Epub 2016 Feb 18.

Department of Surgery, Kyushu University Beppu Hospital, Beppu, Japan.

Understanding intratumor heterogeneity is clinically important because it could cause therapeutic failure by fostering evolutionary adaptation. To this end, we profiled the genome and epigenome in multiple regions within each of nine colorectal tumors. Extensive intertumor heterogeneity is observed, from which we inferred the evolutionary history of the tumors. First, clonally shared alterations appeared, in which C>T transitions at CpG site and CpG island hypermethylation were relatively enriched. Correlation between mutation counts and patients' ages suggests that the early-acquired alterations resulted from aging. In the late phase, a parental clone was branched into numerous subclones. Known driver alterations were observed frequently in the early-acquired alterations, but rarely in the late-acquired alterations. Consistently, our computational simulation of the branching evolution suggests that extensive intratumor heterogeneity could be generated by neutral evolution. Collectively, we propose a new model of colorectal cancer evolution, which is useful for understanding and confronting this heterogeneous disease.
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http://dx.doi.org/10.1371/journal.pgen.1005778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758664PMC
February 2016

Prognostic significance of CpG island methylator phenotype in surgically resected small cell lung carcinoma.

Cancer Sci 2016 Mar 19;107(3):320-5. Epub 2016 Feb 19.

Division of Pathology, The Cancer Institute, Department of Pathology, The Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan.

Methylation is closely involved in the development of various carcinomas. However, few datasets are available for small cell lung cancer (SCLC) due to the scarcity of fresh tumor samples. The aim of the present study is to clarify relationships between clinicopathological features and results of the comprehensive genome-wide methylation profile of SCLC. We investigated the genome-wide DNA methylation status of 28 tumor and 13 normal lung tissues, and gene expression profiling of 25 SCLC tissues. Following unsupervised hierarchical clustering and non-negative matrix factorization, gene ontology analysis was performed. Clustering of SCLC led to the important identification of a CpG island methylator phenotype (CIMP) of the tumor, with a significantly poorer prognosis (P = 0.002). Multivariate analyses revealed that postoperative chemotherapy and non-CIMP were significantly good prognostic factors. Ontology analyses suggested that the extrinsic apoptosis pathway was suppressed, including TNFRSF1A, TNFRSF10A and TRADD in CIMP tumors. Here we revealed that CIMP was an important prognostic factor for resected SCLC. Delineation of this phenotype may also be useful for the development of novel apoptosis-related chemotherapeutic agents for treatment of the aggressive tumor.
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http://dx.doi.org/10.1111/cas.12876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814245PMC
March 2016

Integrated molecular analysis of adult T cell leukemia/lymphoma.

Nat Genet 2015 Nov 5;47(11):1304-15. Epub 2015 Oct 5.

Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Adult T cell leukemia/lymphoma (ATL) is a peripheral T cell neoplasm of largely unknown genetic basis, associated with human T cell leukemia virus type-1 (HTLV-1) infection. Here we describe an integrated molecular study in which we performed whole-genome, exome, transcriptome and targeted resequencing, as well as array-based copy number and methylation analyses, in a total of 426 ATL cases. The identified alterations overlap significantly with the HTLV-1 Tax interactome and are highly enriched for T cell receptor-NF-κB signaling, T cell trafficking and other T cell-related pathways as well as immunosurveillance. Other notable features include a predominance of activating mutations (in PLCG1, PRKCB, CARD11, VAV1, IRF4, FYN, CCR4 and CCR7) and gene fusions (CTLA4-CD28 and ICOS-CD28). We also discovered frequent intragenic deletions involving IKZF2, CARD11 and TP73 and mutations in GATA3, HNRNPA2B1, GPR183, CSNK2A1, CSNK2B and CSNK1A1. Our findings not only provide unique insights into key molecules in T cell signaling but will also guide the development of new diagnostics and therapeutics in this intractable tumor.
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http://dx.doi.org/10.1038/ng.3415DOI Listing
November 2015

Integrated genetic and epigenetic analysis defines novel molecular subgroups in rhabdomyosarcoma.

Nat Commun 2015 Jul 3;6:7557. Epub 2015 Jul 3.

Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Here we studied 60 RMSs using whole-exome/-transcriptome sequencing, copy number (CN) and DNA methylome analyses to unravel the genetic/epigenetic basis of RMS. On the basis of methylation patterns, RMS is clustered into four distinct subtypes, which exhibits remarkable correlation with mutation/CN profiles, histological phenotypes and clinical behaviours. A1 and A2 subtypes, especially A1, largely correspond to alveolar histology with frequent PAX3/7 fusions and alterations in cell cycle regulators. In contrast, mostly showing embryonal histology, both E1 and E2 subtypes are characterized by high frequency of CN alterations and/or allelic imbalances, FGFR4/RAS/AKT pathway mutations and PTEN mutations/methylation and in E2, also by p53 inactivation. Despite the better prognosis of embryonal RMS, patients in the E2 are likely to have a poor prognosis. Our results highlight the close relationships of the methylation status and gene mutations with the biological behaviour in RMS.
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http://dx.doi.org/10.1038/ncomms8557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506514PMC
July 2015

Age-Dependent Decrease of DNA Hydroxymethylation in Human T Cells.

J Clin Exp Hematop 2015 ;55(1):1-6

Department of Hematology, Graduate School of Comprehensive Human Science, University of Tsukuba.

Hydroxymethylcytosine (hmC) is a natural nucleobase, which is converted from methylcytosine (mC) by tet methylcytosine dioxygenase (TET) family (TET1-3) enzymes. Decrease of genomic hmC is postulated to confer a risk for myeloid-lineage as well as T-cell neoplasms, based on the fact that loss-of-function mutations in the TET2 gene were frequently identified in these diseases. The relationship between hmC and aging remains to be elucidated. Here, we demonstrated that hmC content decreased with age in the peripheral blood T cells of 53 human volunteers. We further identified that the mRNA expression levels of TET1 and TET3 decreased with age, while those of TET2 were not influenced by age. The genomic hmC content was correlated with the mRNA expression level of TET3, but not those of TET1 and TET2. Our study suggests the presence of new epigenetic regulatory mechanisms in aging T cells.
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http://dx.doi.org/10.3960/jslrt.55.1DOI Listing
March 2016

BRCC3 mutations in myeloid neoplasms.

Haematologica 2015 Aug 22;100(8):1051-7. Epub 2015 May 22.

Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA Department of Pathology and Tumor Biology, Kyoto University, Japan

Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3 mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84-4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25-11.0). Clinically, BRCC3 mutations were also related to higher age (P=0.01), although prognosis was not affected. Knockdown of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.
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http://dx.doi.org/10.3324/haematol.2014.111989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004421PMC
August 2015

Trans-ancestry mutational landscape of hepatocellular carcinoma genomes.

Nat Genet 2014 Dec 2;46(12):1267-73. Epub 2014 Nov 2.

1] Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan. [2] Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Diverse epidemiological factors are associated with hepatocellular carcinoma (HCC) prevalence in different populations. However, the global landscape of the genetic changes in HCC genomes underpinning different epidemiological and ancestral backgrounds still remains uncharted. Here a collection of data from 503 liver cancer genomes from different populations uncovered 30 candidate driver genes and 11 core pathway modules. Furthermore, a collaboration of two large-scale cancer genome projects comparatively analyzed the trans-ancestry substitution signatures in 608 liver cancer cases and identified unique mutational signatures that predominantly contribute to Asian cases. This work elucidates previously unexplored ancestry-associated mutational processes in HCC development. A combination of hotspot TERT promoter mutation, TERT focal amplification and viral genome integration occurs in more than 68% of cases, implicating TERT as a central and ancestry-independent node of hepatocarcinogenesis. Newly identified alterations in genes encoding metabolic enzymes, chromatin remodelers and a high proportion of mTOR pathway activations offer potential therapeutic and diagnostic opportunities.
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http://dx.doi.org/10.1038/ng.3126DOI Listing
December 2014