Details of a Researcher - Kataoka, Keisuke

Papers - Kataoka, Keisuke

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PD-L1 expression on tumor or stromal cells of nodal cytotoxic T-cell lymphoma: A clinicopathological study of 50 cases

Yamashita, D., Shimada, K., Kohno, K., Kogure, Y., Kataoka, K., Takahara, T., Suzuki, Y., Satou, A., Sakakibara, A., Nakamura, S., Asano, N. and Kato, S.

Pathol Int 2020.05

ISSN 1440-1827

Inhibitors of programmed cell-death 1 (PD-1) and programmed cell-death ligand 1 (PD-L1) have revolutionized cancer therapy. Nodal cytotoxic T-cell lymphoma (CTL) is characterized by a poorer prognosis compared to nodal non-CTLs. Here we investigated PD-L1 expression in 50 nodal CTL patients, with and without EBV association (25 of each). We identified seven patients (14%) with neoplastic PD-L1 (nPD-L1) expression on tumor cells, including three males and four females, with a median age of 66 years. One of the seven cases was TCRalphabeta type, three were TCRgammadelta type and three were TCR-silent type. Six of the seven cases exhibited a lethal clinical course despite multi-agent chemotherapy, of whom four patients died within one year of diagnosis. Morphological findings were uniform, with six cases showing centroblastoid appearance. Among nPD-L1(+) cases, two of three examined had structural variations of PD-L1 disrupting 3'-UTR region. Notably, all of the TCRgammadelta-type nodal CTL cases showed nPD-L1 or miPD-L1 positivity (3 and 10 cases, respectively). TCRgammadelta-type cases comprised 42% of nPD-L1(+) cases (P = 0.043 vs. PD-L1(-) ), and 35% of miPD-L1(+) cases (P = 0.037 vs. PD-L1(-) ). The results indicate that PD-L1(+) nodal CTL cases, especially of the TCRgammadelta type, are potential candidates for anti-PD-1/PD-L1 therapies.

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LUBAC accelerates B-cell lymphomagenesis by conferring B cells resistance to genotoxic stress

Jo, T., Nishikori, M., Kogure, Y., Arima, H., Sasaki, K., Sasaki, Y., Nakagawa, T., Iwai, F., Momose, S., Shiraishi, A., Kiyonari, H., Kagaya, N., Onuki, T., Shin-Ya, K., Yoshida, M., Kataoka, K., Ogawa, S., Iwai, K. and Takaori-Kondo, A.

Blood 2020.04

ISSN 1528-0020

Linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-kB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL), and expression of HOIP which parallels LUBAC activity is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-kB activation, and the analysis of human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-kB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-kB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicates that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage-induced cell death, and is a suitable therapeutic target for B-cell lymphomas.

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Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes

Ochi, Y., Kon, A., Sakata, T., Nakagawa, M. M., Nakazawa, N., Kakuta, M., Kataoka, K., Koseki, H., Nakayama, M., Morishita, D., Tsuruyama, T., Saiki, R., Yoda, A., Okuda, R., Yoshizato, T., Yoshida, K., Shiozawa, Y., Nannya, Y., Kotani, S., Kogure, Y., Kakiuchi, N., Nishimura, T., Makishima, H., Malcovati, L., Yokoyama, A., Takeuchi, K., Sugihara, E., Sato, T. A., Sanada, M., Takaori-Kondo, A., Cazzola, M., Kengaku, M., Miyano, S., Shirahige, K., Suzuki, H. I. and Ogawa, S.

Cancer Discov 2020.04

ISSN 2159-8290

STAG2 encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including RUNX1. However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in STAG2-cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency.

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TET2 haploinsufficiency alters reprogramming into induced pluripotent stem cells

Secardin, L., Limia, C. E. G., di Stefano, A., Bonamino, M. H., Saliba, J., Kataoka, K., Rehen, S. K., Raslova, H., Marty, C., Ogawa, S., Vainchenker, W., Monte-Mor, Bdcr and Plo, I.

Stem Cell Res 44 101755 2020.03

ISSN 1876-7753

The discovery of the Ten-Eleven Translocation (TET) protein family was initiated by the identification of the MLL partner TET1, and of mutations in the TET2 gene in hematological malignancies including myeloproliferative neoplasms (MPN). TET1, 2 and 3 proteins hydroxylate 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and further oxidize 5-hmC into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Previous studies highlight the involvement of TET proteins in somatic cells reprogramming into induced pluripotent stem cells (iPSC), particularly Tet1 and 2 in mouse and TET1 in human. Here, we asked whether endogenous TET2 knockdown also displays this function. Using different shRNA against TET2, we provide evidence that TET2 strongly decreases the reprogramming of human hematopoietic progenitor cells into iPSC. Importantly, using 2 MPN patients, we observed that TET2 mutations affecting catalytic domain allowed iPSC generation. Instead, using another TET2 and TET3-mutated patient, we could only reprogram IPSC with TET3 mutation alone, suggesting that the type of TET2 mutation and/or the cooperation with TET3 mutations may alter the reprogramming activity. Altogether, this work highlights the importance of endogenous TET in the reprogramming process of human hematopoietic progenitors.

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Blockade of EGFR improves responsiveness to PD-1 blockade in EGFR-mutated non-small cell lung cancer

Sugiyama, E., Togashi, Y., Takeuchi, Y., Shinya, S., Tada, Y., Kataoka, K., Tane, K., Sato, E., Ishii, G., Goto, K., Shintani, Y., Okumura, M., Tsuboi, M. and Nishikawa, H.

Sci Immunol 5 ( 43 ) 2020.01

ISSN 2470-9468

The clinical efficacy of anti-PD-1 (programmed cell death-1) monoclonal antibody (mAb) against cancers with oncogenic driver gene mutations, which often harbor a low tumor mutation burden, is variable, suggesting different contributions of each driver mutation to immune responses. Here, we investigated the immunological phenotypes in the tumor microenvironment (TME) of epidermal growth factor receptor (EGFR)-mutated lung adenocarcinomas, for which anti-PD-1 mAb is largely ineffective. Whereas EGFR-mutated lung adenocarcinomas had a noninflamed TME, CD4(+) effector regulatory T cells, which are generally present in the inflamed TME, showed high infiltration. The EGFR signal activated cJun/cJun N-terminal kinase and reduced interferon regulatory factor-1; the former increased CCL22, which recruits CD4(+) regulatory T cells, and the latter decreased CXCL10 and CCL5, which induce CD8(+) T cell infiltration. The EGFR inhibitor erlotinib decreased CD4(+) effector regulatory T cells infiltration in the TME and in combination with anti-PD-1 mAb showed better antitumor effects than either treatment alone. Our results suggest that EGFR inhibitors when used in conjunction with anti-PD-1 mAb could increase the efficacy of immunotherapy in lung adenocarcinomas.

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TP53 and PTEN mutations were shared in concurrent germ cell tumor and acute megakaryoblastic leukemia

Akizuki, K., Sekine, M., Kogure, Y., Kameda, T., Shide, K., Koya, J., Kamiunten, A., Kubuki, Y., Tahira, Y., Hidaka, T., Kiwaki, T., Tanaka, H., Sato, Y., Kataoka, H., Kataoka, K. and Shimoda, K.

BMC Cancer 20 ( 1 ) 5 2020.01

ISSN 1471-2407

BACKGROUND: The occurrence of a mediastinal germ cell tumor (GCT) and hematological malignancy in the same patient is very rare. Due to its rarity, there have been only two reports of the concurrent cases undergoing detailed genetic analysis with whole-exome sequencing (WES), and the possible clonal relationship between the both tumors remained not fully elucidated. METHODS: We performed whole-exome sequencing analysis of mediastinal GCT and acute myeloid leukemia (AML) samples obtained from one young Japanese male adult patient with concurrent both tumors, and investigated the possible clonal relationship between them. RESULTS: Sixteen somatic mutations were detected in the mediastinal GCT sample and 18 somatic mutations in the AML sample. Mutations in nine genes, including TP53 and PTEN both known as tumor suppressor genes, were shared in both tumors. CONCLUSIONS: All in our case and in the previous two cases with concurrent mediastinal GCT and AML undergoing with whole-exome sequencing analysis, TP53 and PTEN mutations were commonly shared in both tumors. These data not only suggest that these tumors share a common founding clone, but also indicate that associated mediastinal GCT and AML harboring TP53 and PTEN mutations represent a unique biological entity.

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Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis

Kakiuchi, N., Yoshida, K., Uchino, M., Kihara, T., Akaki, K., Inoue, Y., Kawada, K., Nagayama, S., Yokoyama, A., Yamamoto, S., Matsuura, M., Horimatsu, T., Hirano, T., Goto, N., Takeuchi, Y., Ochi, Y., Shiozawa, Y., Kogure, Y., Watatani, Y., Fujii, Y., Kim, S. K., Kon, A., Kataoka, K., Yoshizato, T., Nakagawa, M. M., Yoda, A., Nanya, Y., Makishima, H., Shiraishi, Y., Chiba, K., Tanaka, H., Sanada, M., Sugihara, E., Sato, T. A., Maruyama, T., Miyoshi, H., Taketo, M. M., Oishi, J., Inagaki, R., Ueda, Y., Okamoto, S., Okajima, H., Sakai, Y., Sakurai, T., Haga, H., Hirota, S., Ikeuchi, H., Nakase, H., Marusawa, H., Chiba, T., Takeuchi, O., Miyano, S., Seno, H. and Ogawa, S.

Nature 577 ( 7789 ) 260 - 265 2020.01

ISSN 1476-4687

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer(1-3). However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.

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Molecular heterogeneity in peripheral T-cell lymphoma, not otherwise specified revealed by comprehensive genetic profiling

Watatani, Y., Sato, Y., Miyoshi, H., Sakamoto, K., Nishida, K., Gion, Y., Nagata, Y., Shiraishi, Y., Chiba, K., Tanaka, H., Zhao, L., Ochi, Y., Takeuchi, Y., Takeda, J., Ueno, H., Kogure, Y., Shiozawa, Y., Kakiuchi, N., Yoshizato, T., Nakagawa, M. M., Nanya, Y., Yoshida, K., Makishima, H., Sanada, M., Sakata-Yanagimoto, M., Chiba, S., Matsuoka, R., Noguchi, M., Hiramoto, N., Ishikawa, T., Kitagawa, J., Nakamura, N., Tsurumi, H., Miyazaki, T., Kito, Y., Miyano, S., Shimoda, K., Takeuchi, K., Ohshima, K., Yoshino, T., Ogawa, S. and Kataoka, K.

Leukemia 33 ( 12 ) 2867 - 2883 2019.12

ISSN 1476-5551

Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) is a diagnosis of exclusion, being the most common entity in mature T-cell neoplasms, and its molecular pathogenesis remains significantly understudied. Here, combining whole-exome and targeted-capture sequencing, gene-expression profiling, and immunohistochemical analysis of tumor samples from 133 cases, we have delineated the entire landscape of somatic alterations, and discovered frequently affected driver pathways in PTCL, NOS, with and without a T-follicular helper (TFH) cell phenotype. In addition to previously reported mutational targets, we identified a number of novel recurrently altered genes, such as KMT2C, SETD1B, YTHDF2, and PDCD1. We integrated these genetic drivers using hierarchical clustering and identified a previously undescribed molecular subtype characterized by TP53 and/or CDKN2A mutations and deletions in non-TFH PTCL, NOS. This subtype exhibited different prognosis and unique genetic features associated with extensive chromosomal instability, which preferentially affected molecules involved in immune escape and transcriptional regulation, such as HLA-A/B and IKZF2. Taken together, our findings provide novel insights into the molecular pathogenesis of PTCL, NOS by highlighting their genetic heterogeneity. These results should help to devise a novel molecular classification of PTCLs and to exploit a new therapeutic strategy for this group of aggressive malignancies.

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Integrated genetic and epigenetic analysis revealed heterogeneity of acute lymphoblastic leukemia in Down syndrome

Kubota, Y., Uryu, K., Ito, T., Seki, M., Kawai, T., Isobe, T., Kumagai, T., Toki, T., Yoshida, K., Suzuki, H., Kataoka, K., Shiraishi, Y., Chiba, K., Tanaka, H., Ohki, K., Kiyokawa, N., Kagawa, J., Miyano, S., Oka, A., Hayashi, Y., Ogawa, S., Terui, K., Sato, A., Hata, K., Ito, E. and Takita, J.

Cancer Sci 110 ( 10 ) 3358 - 3367 2019.10

ISSN 1349-7006

Children with Down syndrome (DS) are at a 20-fold increased risk for acute lymphoblastic leukemia (ALL). Compared to children with ALL and no DS (non-DS-ALL), those with DS and ALL (DS-ALL) harbor uncommon genetic alterations, suggesting DS-ALL could have distinct biological features. Recent studies have implicated several genes on chromosome 21 in DS-ALL, but the precise mechanisms predisposing children with DS to ALL remain unknown. Our integrated genetic/epigenetic analysis revealed that DS-ALL was highly heterogeneous with many subtypes. Although each subtype had genetic/epigenetic profiles similar to those found in non-DS-ALL, the subtype distribution differed significantly between groups. The Philadelphia chromosome-like subtype, a high-risk B-cell lineage variant relatively rare among the entire pediatric ALL population, was the most common form in DS-ALL. Hypermethylation of RUNX1 on chromosome 21 was also found in DS-ALL, but not non-DS-ALL. RUNX1 is essential for differentiation of blood cells, especially B cells; thus, hypermethylation of the RUNX1 promoter in B-cell precursors might be associated with increased incidence of B-cell precursor ALL in DS patients.

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PAK Kinase Inhibition Has Therapeutic Activity in Novel Preclinical Models of Adult T-Cell Leukemia/Lymphoma

Chung, E. Y., Mai, Y., Shah, U. A., Wei, Y., Ishida, E., Kataoka, K., Ren, X., Pradhan, K., Bartholdy, B., Wei, X., Zou, Y., Zhang, J., Ogawa, S., Steidl, U., Zang, X., Verma, A., Janakiram, M. and Ye, B. H.

Clin Cancer Res 25 ( 12 ) 3589 - 3601 2019.06

ISSN 1078-0432

PURPOSE: To evaluate therapeutic activity of PAK inhibition in ATLL and to characterize the role of PAK isoforms in cell proliferation, survival, and adhesion of ATLL cells in preclinical models. EXPERIMENTAL DESIGN: Frequency and prognostic impact of PAK2 amplification were evaluated in an ATLL cohort of 370 cases. Novel long-term cultures and in vivo xenograft models were developed using primary ATLL cells from North American patients. Two PAK inhibitors were used to block PAK kinase activity pharmacologically. siRNA-based gene silencing approach was used to genetically knockdown (KD) PAK1 and PAK2 in ATLL cell lines. RESULTS: PAK1/2/4 are the three most abundantly expressed PAK family members in ATLL. PAK2 amplifications are seen in 24% of ATLLs and are associated with worse prognosis in a large patient cohort. The pan-PAK inhibitor PF-3758309 (PF) has strong in vitro and in vivo activity in a variety of ATLL preclinical models. These activities of PF are likely attributed to its ability to target several PAK isoforms simultaneously because genetic silencing of either PAK1 or PAK2 produced more modest effects. PAK2 plays a major role in CADM1-mediated stromal interaction, which is an important step in systemic dissemination of the disease. This finding is consistent with the observation that PAK2 amplification is more frequent in aggressive ATLLs and correlates with inferior outcome. CONCLUSIONS: PAK2, a gene frequently amplified in ATLL, facilitates CADM1-mediated stromal interaction and promotes survival of ATLL cells. Taken together, PAK inhibition may hold significant promise as a targeted therapy for aggressive ATLLs.

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Decreased RORC expression and downstream signaling in HTLV-1-associated adult T-cell lymphoma/leukemia uncovers an antiproliferative IL17 link: A potential target for immunotherapy?

Subramanian, K., Dierckx, T., Khouri, R., Menezes, S. M., Kagdi, H., Taylor, G. P., Farre, L., Bittencourt, A., Kataoka, K., Ogawa, S. and Van Weyenbergh, J.

Int J Cancer 144 ( 7 ) 1664 - 1675 2019.04

ISSN 1097-0215

Retinoic acid-related drugs have shown promising pre-clinical activity in Adult T-cell Leukemia/Lymphoma, but RORC signaling has not been explored. Therefore, we investigated transcriptome-wide interactions of the RORC pathway in HTLV-1 and ATL, using our own and publicly available gene expression data for ATL and other leukemias. Gene expression data from ATL patients were analyzed using WGCNA to determine gene modules and their correlation to clinical and molecular data. Both PBMCs and CD4(+) T-Cells exhibited decreased RORC expression in four different ATL cohorts. A small subset of RORC(hi) ATL patients was identified with significantly lower pathognomonic CADM1 and HBZ levels but similar levels of other ATL markers (CD4/CD25/CCR4), hinting at a less aggressive ATL subtype. An age-dependent decrease in RORC expression was found in HTLV-1-infected individuals, but not in healthy controls, suggesting an early molecular event predisposing to leukemogenesis. Genes upstream of RORC signaling were members of a proliferative gene module (containing proliferation markers PCNA/Ki67), whereas downstream members clustered in an anti-proliferative gene module. IL17C transcripts showed the strongest negative correlation to PCNA in both ATL cohorts, which was replicated in two large cohorts of T- and B-cell acute lymphoid leukemia (ALL). Finally, IL17C expression in purified CD4 + CCR4 + CD26-CD7- "ATL-like" cells from HTLV-1-infected individuals and ATL patients was negatively correlated with clonality, underscoring a possible antileukemic/antiproliferative role. In conclusion, decreased RORC expression and downstream signaling might represent an early event in ATL pathogenesis. An antiproliferative IL17C/PCNA link is shared between ATL, T-ALL and B-ALL, suggesting (immuno)therapeutic benefit of boosting RORC/IL17 signaling.

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Identification of enhancer of mRNA decapping 4 as a novel fusion partner of MLL in acute myeloid leukemia

Becker, H., Greve, G., Kataoka, K., Mallm, J. P., Duque-Afonso, J., Ma, T., Niemoller, C., Pantic, M., Duyster, J., Cleary, M. L., Schuler, J., Rippe, K., Ogawa, S. and Lubbert, M.

Blood Adv 3 ( 5 ) 761 - 765 2019.03

ISSN 2473-9537

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Frequent structural variations involving programmed death ligands in Epstein-Barr virus-associated lymphomas

Kataoka, K., Miyoshi, H., Sakata, S., Dobashi, A., Couronne, L., Kogure, Y., Sato, Y., Nishida, K., Gion, Y., Shiraishi, Y., Tanaka, H., Chiba, K., Watatani, Y., Kakiuchi, N., Shiozawa, Y., Yoshizato, T., Yoshida, K., Makishima, H., Sanada, M., Onozawa, M., Teshima, T., Yoshiki, Y., Ishida, T., Suzuki, K., Shimada, K., Tomita, A., Kato, M., Ota, Y., Izutsu, K., Demachi-Okamura, A., Akatsuka, Y., Miyano, S., Yoshino, T., Gaulard, P., Hermine, O., Takeuchi, K., Ohshima, K. and Ogawa, S.

Leukemia 2019.01

ISSN 1476-5551

Viral infection induces potent cellular immunity and activated intracellular signaling, which may dictate the driver events involved in immune escape and clonal selection of virus-associated cancers, including Epstein-Barr virus (EBV)-positive lymphomas. Here, we thoroughly interrogated PD-L1/PD-L2-involving somatic aberrations in 384 samples from various lymphoma subtypes using high-throughput sequencing, particularly focusing on virus-associated lymphomas. A high frequency of PD-L1/PD-L2-involving genetic aberrations was observed in EBV-positive lymphomas [33 (22%) of 148 cases], including extranodal NK/T-cell lymphoma (ENKTL, 23%), aggressive NK-cell leukemia (57%), systemic EBV-positive T-cell lymphoproliferative disorder (17%) as well as EBV-positive diffuse large B-cell lymphoma (DLBCL, 19%) and peripheral T-cell lymphoma-not otherwise specified (15%). Predominantly causing a truncation of the 3'-untranslated region, these alterations represented the most prevalent somatic lesions in ENKTL. By contrast, the frequency was much lower in EBV-negative lymphomas regardless of histology type [12 (5%) of 236 cases]. Besides PD-L1/PD-L2 alterations, EBV-positive DLBCL exhibited a genetic profile distinct from EBV-negative one, characterized by frequent TET2 and DNMT3A mutations and the paucity of CD79B, MYD88, CDKN2A, and FAS alterations. Our findings illustrate unique genetic features of EBV-associated lymphomas, also suggesting a potential role of detecting PD-L1/PD-L2-involving lesions for these lymphomas to be effectively targeted by immune checkpoint blockade.

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Yokoyama, A., Kakiuchi, N., Yoshizato, T., Nannya, Y., Suzuki, H., Takeuchi, Y., Shiozawa, Y., Sato, Y., Aoki, K., Kim, S. K., Fujii, Y., Yoshida, K., Kataoka, K., Nakagawa, M. M., Inoue, Y., Hirano, T., Shiraishi, Y., Chiba, K., Tanaka, H., Sanada, M., Nishikawa, Y., Amanuma, Y., Ohashi, S., Aoyama, I., Horimatsu, T., Miyamoto, S., Tsunoda, S., Sakai, Y., Narahara, M., Brown, J. B., Sato, Y., Sawada, G., Mimori, K., Minamiguchi, S., Haga, H., Seno, H., Miyano, S., Makishima, H., Muto, M. and Ogawa, S.

Nature 565 ( 7739 ) 312 - 317 2019.01

ISSN 1476-4687

Clonal expansion in aged normal tissues has been implicated in the development of cancer. However, the chronology and risk dependence of the expansion are poorly understood. Here we intensively sequence 682 micro-scale oesophageal samples and show, in physiologically normal oesophageal epithelia, the progressive age-related expansion of clones that carry mutations in driver genes (predominantly NOTCH1), which is substantially accelerated by alcohol consumption and by smoking. Driver-mutated clones emerge multifocally from early childhood and increase their number and size with ageing, and ultimately replace almost the entire oesophageal epithelium in the extremely elderly. Compared with mutations in oesophageal cancer, there is a marked overrepresentation of NOTCH1 and PPM1D mutations in physiologically normal oesophageal epithelia; these mutations can be acquired before late adolescence (as early as early infancy) and significantly increase in number with heavy smoking and drinking. The remodelling of the oesophageal epithelium by driver-mutated clones is an inevitable consequence of normal ageing, which-depending on lifestyle risks-may affect cancer development.

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More accurate prognostic prediction in diffuse large B-cell lymphoma: beyond cell-of-origin

Koya, J. and Kataoka, K.

Ann Oncol 29 ( 12 ) 2284 - 2286 2018.12

ISSN 1569-8041

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Using patient-derived iPSCs to develop humanized mouse models for chronic myelomonocytic leukemia and therapeutic drug identification, including liposomal clodronate

Taoka, K., Arai, S., Kataoka, K., Hosoi, M., Miyauchi, M., Yamazaki, S., Honda, A., Aixinjueluo, W., Kobayashi, T., Kumano, K., Yoshimi, A., Otsu, M., Niwa, A., Nakahata, T., Nakauchi, H. and Kurokawa, M.

Sci Rep 8 ( 1 ) 15855 2018.10

ISSN 2045-2322

Chronic myelomonocytic leukemia (CMML) is an entity of myelodysplastic syndrome/myeloproliferative neoplasm. Although CMML can be cured with allogeneic stem cell transplantation, its prognosis is generally very poor due to the limited efficacy of chemotherapy and to the patient's age, which is usually not eligible for transplantation. Comprehensive analysis of CMML pathophysiology and the development of therapeutic agents have been limited partly due to the lack of cell lines in CMML and the limited developments of mouse models. After successfully establishing patient's derived disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with CMML, we utilized these CMML-iPSCs to achieve hematopoietic re-differentiation in vitro, created a humanized CMML mouse model via teratomas, and developed a drug-testing system. The clinical characteristics of CMML were recapitulated following hematopoietic re-differentiation in vitro and a humanized CMML mouse model in vivo. The drug-testing system using CMML-iPSCs identified a MEK inhibitor, a Ras inhibitor, and liposomal clodronate as potential drugs for treating CMML. Clodronate is a drug commonly used as a bisphosphonate for osteoporosis. In this study, the liposomalization of clodronate enhanced its effectiveness in these assays, suggesting that this variation of clodronate may be adopted as a repositioned drug for CMML therapy.

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North American ATLL has a distinct mutational and transcriptional profile and responds to epigenetic therapies

Shah, U. A., Chung, E. Y., Giricz, O., Pradhan, K., Kataoka, K., Gordon-Mitchell, S., Bhagat, T. D., Mai, Y., Wei, Y., Ishida, E., Choudhary, G. S., Joseph, A., Rice, R., Gitego, N., Parrish, C., Bartenstein, M., Goel, S., Mantzaris, I., Shastri, A., Derman, O., Binder, A., Gritsman, K., Kornblum, N., Braunschweig, I., Bhagat, C., Hall, J., Graber, A., Ratner, L., Wang, Y., Ogawa, S., Verma, A., Ye, B. H. and Janakiram, M.

Blood 132 ( 14 ) 1507 - 1518 2018.10

ISSN 1528-0020

Adult T-cell leukemia lymphoma (ATLL) is a rare T cell neoplasm that is endemic in Japanese, Caribbean, and Latin American populations. Most North American ATLL patients are of Caribbean descent and are characterized by high rates of chemo-refractory disease and worse prognosis compared with Japanese ATLL. To determine genomic differences between these 2 cohorts, we performed targeted exon sequencing on 30 North American ATLL patients and compared the results with the Japanese ATLL cases. Although the frequency of TP53 mutations was comparable, the mutation frequency in epigenetic and histone modifying genes (57%) was significantly higher, whereas the mutation frequency in JAK/STAT and T-cell receptor/NF-kappaB pathway genes was significantly lower. The most common type of epigenetic mutation is that affecting EP300 (20%). As a category, epigenetic mutations were associated with adverse prognosis. Dissimilarities with the Japanese cases were also revealed by RNA sequencing analysis of 9 primary patient samples. ATLL samples with a mutated EP300 gene have decreased total and acetyl p53 protein and a transcriptional signature reminiscent of p53-mutated cancers. Most importantly, decitabine has highly selective single-agent activity in the EP300-mutated ATLL samples, suggesting that decitabine treatment induces a synthetic lethal phenotype in EP300-mutated ATLL cells. In conclusion, we demonstrate that North American ATLL has a distinct genomic landscape that is characterized by frequent epigenetic mutations that are targetable preclinically with DNA methyltransferase inhibitors.

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Molecular pathogenesis of disease progression in MLL-rearranged AML

Kotani, S., Yoda, A., Kon, A., Kataoka, K., Ochi, Y., Shiozawa, Y., Hirsch, C., Takeda, J., Ueno, H., Yoshizato, T., Yoshida, K., Nakagawa, M. M., Nannya, Y., Kakiuchi, N., Yamauchi, T., Aoki, K., Shiraishi, Y., Miyano, S., Maeda, T., Maciejewski, J. P., Takaori-Kondo, A., Ogawa, S. and Makishima, H.

Leukemia 2018.09

ISSN 1476-5551

Leukemic relapse is frequently accompanied by progressively aggressive clinical course. To understand the molecular mechanism of leukemic relapse, MLL/AF9-transformed mouse leukemia cells were serially transplanted in C57BL/6 mice (N = 96) by mimicking repeated recurrences, where mutations were monitored by exome sequencing (N = 42). The onset of leukemia was progressively promoted with advanced transplants, during which increasing numbers of somatic mutations were acquired (P < 0.005). Among these, mutations in Ptpn11 (p.G60R) and Braf (p.V637E) corresponded to those identified in human MLL-AML, while recurrent mutations affecting Msn (p.R295C) were observed only in mouse but not in human MLL-AML. Another mutated gene of interest was Gnb2 which was reported to be recurrently mutated in various hematological neoplasms. Gnb2 mutations (p.G77R) were significantly increased in clone size (P = 0.007) and associated with earlier leukemia onset (P = 0.011). GNB2 transcripts were significantly upregulated in human MLL-AML compared to MLL-negative AML (P < 0.05), which was supported by significantly increased Gnb2 transcript induced by MLL/AF9 overexpression (P < 0.001). In in vivo model, both mutation and overexpression of GNB2 caused leukemogenesis, and downregulation of GNB2 expression reduced proliferative potential and survival benefit, suggesting a driver role of GNB2. In conclusion, alterations of driver genes over time may play an important role in the progression of MLL-AML.

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Aberrant splicing and defective mRNA production induced by somatic spliceosome mutations in myelodysplasia

Shiozawa, Y., Malcovati, L., Galli, A., Sato-Otsubo, A., Kataoka, K., Sato, Y., Watatani, Y., Suzuki, H., Yoshizato, T., Yoshida, K., Sanada, M., Makishima, H., Shiraishi, Y., Chiba, K., Hellstrom-Lindberg, E., Miyano, S., Ogawa, S. and Cazzola, M.

Nat Commun 9 ( 1 ) 3649 2018.09

ISSN 2041-1723

Spliceosome mutations are frequently found in myelodysplasia. Splicing alterations induced by these mutations, their precise targets, and the effect at the transcript level have not been fully elucidated. Here we report transcriptomic analyses of 265 bone marrow samples from myelodysplasia patients, followed by a validation using CRISPR/Cas9-mediated gene editing and an assessment of nonsense-mediated decay susceptibility. Small but widespread reduction of intron-retaining isoforms is the most frequent splicing alteration in SF3B1-mutated samples. SF3B1 mutation is also associated with 3' splice site alterations, leading to the most pronounced reduction of canonical transcripts. Target genes include tumor suppressors and genes of mitochondrial iron metabolism or heme biosynthesis. Alternative exon usage is predominant in SRSF2- and U2AF1-mutated samples. Usage of an EZH2 cryptic exon harboring a premature termination codon is increased in both SRSF2- and U2AF1-mutated samples. Our study reveals a landscape of splicing alterations and precise targets of various spliceosome mutations.

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De Novo Mutations Activating Germline TP53 in an Inherited Bone-Marrow-Failure Syndrome

Toki, T., Yoshida, K., Wang, R., Nakamura, S., Maekawa, T., Goi, K., Katoh, M. C., Mizuno, S., Sugiyama, F., Kanezaki, R., Uechi, T., Nakajima, Y., Sato, Y., Okuno, Y., Sato-Otsubo, A., Shiozawa, Y., Kataoka, K., Shiraishi, Y., Sanada, M., Chiba, K., Tanaka, H., Terui, K., Sato, T., Kamio, T., Sakaguchi, H., Ohga, S., Kuramitsu, M., Hamaguchi, I., Ohara, A., Kanno, H., Miyano, S., Kojima, S., Ishiguro, A., Sugita, K., Kenmochi, N., Takahashi, S., Eto, K., Ogawa, S. and Ito, E.

Am J Hum Genet 103 ( 3 ) 440 - 447 2018.09

ISSN 1537-6605

Inherited bone-marrow-failure syndromes (IBMFSs) include heterogeneous genetic disorders characterized by bone-marrow failure, congenital anomalies, and an increased risk of malignancy. Many lines of evidence have suggested that p53 activation might be central to the pathogenesis of IBMFSs, including Diamond-Blackfan anemia (DBA) and dyskeratosis congenita (DC). However, the exact role of p53 activation in each clinical feature remains unknown. Here, we report unique de novo TP53 germline variants found in two individuals with an IBMFS accompanied by hypogammaglobulinemia, growth retardation, and microcephaly mimicking DBA and DC. TP53 is a tumor-suppressor gene most frequently mutated in human cancers, and occasional germline variants occur in Li-Fraumeni cancer-predisposition syndrome. Most of these mutations affect the core DNA-binding domain, leading to compromised transcriptional activities. In contrast, the variants found in the two individuals studied here caused the same truncation of the protein, resulting in the loss of 32 residues from the C-terminal domain (CTD). Unexpectedly, the p53 mutant had augmented transcriptional activities, an observation not previously described in humans. When we expressed this mutant in zebrafish and human-induced pluripotent stem cells, we observed impaired erythrocyte production. These findings together with close similarities to published knock-in mouse models of TP53 lacking the CTD demonstrate that the CTD-truncation mutations of TP53 cause IBMFS, providing important insights into the previously postulated connection between p53 and IBMFSs.

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PD-1 Inhibitor Therapy in Adult T-Cell Leukemia-Lymphoma

Kataoka, K. and Ogawa, S.

N Engl J Med 379 ( 7 ) 696 2018.08

ISSN 1533-4406

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RNA fusions involving CD28 are rare in peripheral T-cell lymphomas and concentrate mainly in those derived from follicular helper T cells

Vallois, D., Dupuy, A., Lemonnier, F., Allen, G., Missiaglia, E., Fataccioli, V., Ortonne, N., Clavert, A., Delarue, R., Rousselet, M. C., Fabiani, B., Llamas-Gutierrez, F., Ogawa, S., Thome, M., Ko, Y. H., Kataoka, K., Gaulard, P. and de Leval, L.

Haematologica 103 ( 8 ) e360 - e363 2018.08

ISSN 1592-8721

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A comprehensive characterization of cis-acting splicing-associated variants in human cancer

Shiraishi, Y., Kataoka, K., Chiba, K., Okada, A., Kogure, Y., Tanaka, H., Ogawa, S. and Miyano, S.

Genome Res 28 ( 8 ) 1111 - 1125 2018.08

ISSN 1549-5469

Although many driver mutations are thought to promote carcinogenesis via abnormal splicing, the landscape of splicing-associated variants (SAVs) remains unknown due to the complexity of splicing abnormalities. Here, we developed a statistical framework to systematically identify SAVs disrupting or newly creating splice site motifs and applied it to matched whole-exome and transcriptome sequencing data from 8976 samples across 31 cancer types, generating a catalog of 14,438 SAVs. Such a large collection of SAVs enabled us to characterize their genomic features, underlying mutational processes, and influence on cancer driver genes. In fact, approximately 50% of SAVs identified were those disrupting noncanonical splice sites (non-GT-AG dinucleotides), including the third and fifth intronic bases of donor sites, or newly creating splice sites. Mutation signature analysis revealed that tobacco smoking is more strongly associated with SAVs, whereas ultraviolet exposure has less impact. SAVs showed remarkable enrichment of cancer-related genes, and as many as 14.7% of samples harbored at least one SAVs affecting them, particularly in tumor suppressors. In addition to intron retention, whose association with tumor suppressor inactivation has been previously reported, exon skipping and alternative splice site usage caused by SAVs frequently affected tumor suppressors. Finally, we described high-resolution distributions of SAVs along the gene and their splicing outcomes in commonly disrupted genes, including TP53, PIK3R1, GATA3, and CDKN2A, which offers genetic clues for understanding their functional properties. Collectively, our findings delineate a comprehensive portrait of SAVs, novel insights into transcriptional de-regulation in cancer.

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ADAM8 Is an Antigen of Tyrosine Kinase Inhibitor-Resistant Chronic Myeloid Leukemia Cells Identified by Patient-Derived Induced Pluripotent Stem Cells

Miyauchi, M., Koya, J., Arai, S., Yamazaki, S., Honda, A., Kataoka, K., Yoshimi, A., Taoka, K., Kumano, K. and Kurokawa, M.

Stem Cell Reports 10 ( 3 ) 1115 - 1130 2018.03

ISSN 2213-6711

Properties of cancer stem cells involved in drug resistance and relapse have significant effects on clinical outcome. Although tyrosine kinase inhibitors (TKIs) have dramatically improved survival of patients with chronic myeloid leukemia (CML), TKIs have not fully cured CML due to TKI-resistant CML stem cells. Moreover, relapse after discontinuation of TKIs has not been predicted in CML patients with the best TKI response. In our study, a model of CML stem cells derived from CML induced pluripotent stem cells identified ADAM8 as an antigen of TKI-resistant CML cells. The inhibition of expression or metalloproteinase activity of ADAM8 restored TKI sensitivity in primary samples. In addition, residual CML cells in patients with optimal TKI response were concentrated in the ADAM8+ population. Our study demonstrates that ADAM8 is a marker of residual CML cells even in patients with optimal TKI response and would be a predictor of relapse and a therapeutic target of TKI-resistant CML cells.

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A novel genetic and morphologic phenotype of ARID2-mediated myelodysplasia

Sakai, H., Hosono, N., Nakazawa, H., Przychodzen, B., Polprasert, C., Carraway, H. E., Sekeres, M. A., Radivoyevitch, T., Yoshida, K., Sanada, M., Yoshizato, T., Kataoka, K., Nakagawa, M. M., Ueno, H., Nannya, Y., Kon, A., Shiozawa, Y., Takeda, J., Shiraishi, Y., Chiba, K., Miyano, S., Singh, J., Padgett, R. A., Ogawa, S., Maciejewski, J. P. and Makishima, H.

Leukemia 32 ( 3 ) 839 - 843 2018.03

ISSN 1476-5551

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Integrated Molecular Characterization of the Lethal Pediatric Cancer Pancreatoblastoma

Isobe, T., Seki, M., Yoshida, K., Sekiguchi, M., Shiozawa, Y., Shiraishi, Y., Kimura, S., Yoshida, M., Inoue, Y., Yokoyama, A., Kakiuchi, N., Suzuki, H., Kataoka, K., Sato, Y., Kawai, T., Chiba, K., Tanaka, H., Shimamura, T., Kato, M., Iguchi, A., Hama, A., Taguchi, T., Akiyama, M., Fujimura, J., Inoue, A., Ito, T., Deguchi, T., Kiyotani, C., Iehara, T., Hosoi, H., Oka, A., Sanada, M., Tanaka, Y., Hata, K., Miyano, S., Ogawa, S. and Takita, J.

Cancer Res 78 ( 4 ) 865 - 876 2018.02

ISSN 1538-7445

Pancreatoblastoma is a rare pediatric pancreatic malignancy for which the molecular pathogenesis is not understood. In this study, we report the findings of an integrated multiomics study of whole-exome and RNA sequencing as well as genome-wide copy number and methylation analyses of ten pancreatoblastoma cases. The pancreatoblastoma genome was characterized by a high frequency of aberrant activation of the Wnt signaling pathway, either via somatic mutations of CTNNB1 (90%) and copy-neutral loss of heterozygosity (CN-LOH) of APC (10%). In addition, imprinting dysregulation of IGF2 as a consequence of CN-LOH (80%), gain of paternal allele (10%), and gain of methylation (10%) was universally detected. At the transcriptome level, pancreatoblastoma exhibited an expression profile characteristic of early pancreas progenitor-like cells along with upregulation of the R-spondin/LGR5/RNF43 module. Our results offer a comprehensive description of the molecular basis for pancreatoblastoma and highlight rational therapeutic targets for its treatment.Significance: Molecular genetic analysis of a rare untreatable pediatric tumor reveals Wnt/IGF2 aberrations and features of early pancreas progenitor-like cells, suggesting cellular origins and rational strategies for therapeutic targeting. Cancer Res; 78(4); 865-76. (c)2017 AACR.

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Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice

Kon, A., Yamazaki, S., Nannya, Y., Kataoka, K., Ota, Y., Nakagawa, M. M., Yoshida, K., Shiozawa, Y., Morita, M., Yoshizato, T., Sanada, M., Nakayama, M., Koseki, H., Nakauchi, H. and Ogawa, S.

Blood 131 ( 6 ) 621 - 635 2018.02

ISSN 1528-0020

Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre-mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2-mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2-mutated mice. RNA sequencing of the Srsf2-mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r, Fyn, Gnas, Nsd1, Hnrnpa2b1, and Trp53bp1 Among the mutant Srsf2-associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults.

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Prognostic relevance of integrated genetic profiling in adult T-cell leukemia/lymphoma

Kataoka, K., Iwanaga, M., Yasunaga, J. I., Nagata, Y., Kitanaka, A., Kameda, T., Yoshimitsu, M., Shiraishi, Y., Sato-Otsubo, A., Sanada, M., Chiba, K., Tanaka, H., Ochi, Y., Aoki, K., Suzuki, H., Shiozawa, Y., Yoshizato, T., Sato, Y., Yoshida, K., Nosaka, K., Hishizawa, M., Itonaga, H., Imaizumi, Y., Munakata, W., Shide, K., Kubuki, Y., Hidaka, T., Nakamaki, T., Ishiyama, K., Miyawaki, S., Ishii, R., Nureki, O., Tobinai, K., Miyazaki, Y., Takaori-Kondo, A., Shibata, T., Miyano, S., Ishitsuka, K., Utsunomiya, A., Shimoda, K., Matsuoka, M., Watanabe, T. and Ogawa, S.

Blood 131 ( 2 ) 215 - 225 2018.01

ISSN 1528-0020

Adult T-cell leukemia/lymphoma (ATL) is a heterogeneous group of peripheral T-cell malignancies characterized by human T-cell leukemia virus type-1 infection, whose genetic profile has recently been fully investigated. However, it is still poorly understood how these alterations affect clinical features and prognosis. We investigated the effects of genetic alterations commonly found in ATL on disease phenotypes and clinical outcomes, based on genotyping data obtained from 414 and 463 ATL patients using targeted-capture sequencing and single nucleotide polymorphism array karyotyping, respectively. Aggressive (acute/lymphoma) subtypes were associated with an increased burden of genetic and epigenetic alterations, higher frequencies of TP53 and IRF4 mutations, and many copy number alterations (CNAs), including PD-L1 amplifications and CDKN2A deletions, compared with indolent (chronic/smoldering) subtypes. By contrast, STAT3 mutations were more characteristic of indolent ATL. Higher numbers of somatic mutations and CNAs significantly correlated with worse survival. In a multivariate analysis incorporating both clinical factors and genetic alterations, the Japan Clinical Oncology Group prognostic index high-risk, older age, PRKCB mutations, and PD-L1 amplifications were independent poor prognostic factors in aggressive ATL. In indolent ATL, IRF4 mutations, PD-L1 amplifications, and CDKN2A deletions were significantly associated with shorter survival, although the chronic subtype with unfavorable clinical factors was only marginally significant. Thus, somatic alterations characterizing aggressive diseases predict worse prognosis in indolent ATL, among which PD-L1 amplifications are a strong genetic predictor in both aggressive and indolent ATL. ATL subtypes are further classified into molecularly distinct subsets with different prognosis. Genetic profiling might contribute to improved prognostication and management of ATL patients.

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Prognostic relevance of genetic alterations in diffuse lower-grade gliomas

Aoki, K., Nakamura, H., Suzuki, H., Matsuo, K., Kataoka, K., Shimamura, T., Motomura, K., Ohka, F., Shiina, S., Yamamoto, T., Nagata, Y., Yoshizato, T., Mizoguchi, M., Abe, T., Momii, Y., Muragaki, Y., Watanabe, R., Ito, I., Sanada, M., Yajima, H., Morita, N., Takeuchi, I., Miyano, S., Wakabayashi, T., Ogawa, S. and Natsume, A.

Neuro Oncol 20 ( 1 ) 66 - 77 2018.01

ISSN 1523-5866

Background: Diffuse lower-grade gliomas (LGGs) are genetically classified into 3 distinct subtypes based on isocitrate dehydrogenase (IDH) mutation status and codeletion of chromosome 1p and 19q (1p/19q). However, the subtype-specific effects of additional genetic lesions on survival are largely unknown. Methods: Using Cox proportional hazards regression modeling, we investigated the subtype-specific effects of genetic alterations and clinicopathological factors on survival in each LGG subtype, in a Japanese cohort of LGG cases fully genotyped for driver mutations and copy number variations associated with LGGs (n = 308). The results were validated using a dataset from 414 LGG cases available from The Cancer Genome Atlas (TCGA). Results: In Oligodendroglioma, IDH-mutant and 1p/19q codeleted, NOTCH1 mutations (P = 0.0041) and incomplete resection (P = 0.0019) were significantly associated with shorter survival. In Astrocytoma, IDH-mutant, PIK3R1 mutations (P = 0.0014) and altered retinoblastoma pathway genes (RB1, CDKN2A, and CDK4) (P = 0.013) were independent predictors of poor survival. In IDH-wildtype LGGs, co-occurrence of 7p gain, 10q loss, mutation in the TERT promoter (P = 0.024), and grade III histology (P < 0.0001) independently predicted poor survival. IDH-wildtype LGGs without any of these factors were diagnosed at a younger age (P = 0.042), and were less likely to have genetic lesions characteristic of glioblastoma, in comparison with other IDH-wildtype LGGs, suggesting that they likely represented biologically different subtypes. These results were largely confirmed in the cohort of TCGA. Conclusions: Subtype-specific genetic lesions can be used to stratify patients within each LGG subtype. enabling better prognostication and management.

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Identification of the genetic and clinical characteristics of neuroblastomas using genome-wide analysis

Uryu, K., Nishimura, R., Kataoka, K., Sato, Y., Nakazawa, A., Suzuki, H., Yoshida, K., Seki, M., Hiwatari, M., Isobe, T., Shiraishi, Y., Chiba, K., Tanaka, H., Miyano, S., Koh, K., Hanada, R., Oka, A., Hayashi, Y., Ohira, M., Kamijo, T., Nagase, H., Takimoto, T., Tajiri, T., Nakagawara, A., Ogawa, S. and Takita, J.

Oncotarget 8 ( 64 ) 107513 - 107529 2017.12

ISSN 1949-2553

To provide better insight into the genetic signatures of neuroblastomas, we analyzed 500 neuroblastomas (included specimens from JNBSG) using targeted-deep sequencing for 10 neuroblastoma-related genes and SNP arrays analysis. ALK expression was evaluated using immunohistochemical analysis in 259 samples. Based on genetic alterations, the following 6 subgroups were identified: groups A (ALK abnormalities), B (other gene mutations), C (MYCN amplification), D (11q loss of heterozygosity [LOH]), E (at least 1 copy number variants), and F (no genetic changes). Groups A to D showed advanced disease and poor prognosis, whereas groups E and F showed excellent prognosis. Intriguingly, in group A, MYCN amplification was not a significant prognostic marker, while high ALK expression was a relevant indicator for prognosis (P = 0.033). Notably, the co-existence of MYCN amplification and 1p LOH, and the co-deletion of 3p and 11q were significant predictors of relapse (P = 0.043 and P = 0.040). Additionally, 6q/8p LOH and 17q gain were promising indicators of survival in patients older than 5 years, and 1p, 4p, and 11q LOH potentially contributed to outcome prediction in the intermediate-risk group. Our genetic overview clarifies the clinical impact of genetic signatures and aids in the better understanding of genetic basis of neuroblastoma.

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A Cryptic NUP214-ABL1 Fusion in B-cell Precursor Acute Lymphoblastic Leukemia

Tsujimoto, S. I., Nakano, Y., Osumi, T., Okada, K., Ouchi-Uchiyama, M., Kataoka, K., Fujii, Y., Ohki, K., Seki, M., Tamagawa, N., Takita, J., Ogawa, S., Kiyokawa, N., Hara, J. and Kato, M.

J Pediatr Hematol Oncol 2017.12

ISSN 1536-3678

Fluorescent in situ hybridization (FISH) analysis is the standard methods for screening ABL1 fusions, which is recurrently translocated in pediatric acute lymphoblastic leukemia (ALL), and potentially targetable by kinase inhibitors. Here we demonstrated a case of B-cell precursor ALL with NUP214-ABL1 fusion, which break-apart FISH assay for ABL1 failed to detect. The cryptic fusion was generated by small duplication from ABL1 to NUP214, which was detected by copy number analysis using genomic microarray and confirmed by PCR. In the context of precision medicine, we should establish how to screen targetable abnormalities for minimizing risk of false-negative.

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Loss of DNA Damage Response in Neuroblastoma and Utility of a PARP Inhibitor

Takagi, M., Yoshida, M., Nemoto, Y., Tamaichi, H., Tsuchida, R., Seki, M., Uryu, K., Nishii, R., Miyamoto, S., Saito, M., Hanada, R., Kaneko, H., Miyano, S., Kataoka, K., Yoshida, K., Ohira, M., Hayashi, Y., Nakagawara, A., Ogawa, S., Mizutani, S. and Takita, J.

J Natl Cancer Inst 109 ( 11 ) 2017.11

ISSN 1460-2105

Background: Neuroblastoma (NB) is the most common solid tumor found in children, and deletions within the 11q region are observed in 11% to 48% of these tumors. Notably, such tumors are associated with poor prognosis; however, little is known regarding the molecular targets located in 11q. Methods: Genomic alterations of ATM , DNA damage response (DDR)-associated genes located in 11q ( MRE11A, H2AFX , and CHEK1 ), and BRCA1, BARD1, CHEK2, MDM2 , and TP53 were investigated in 45 NB-derived cell lines and 237 fresh tumor samples. PARP (poly [ADP-ribose] polymerase) inhibitor sensitivity of NB was investigated in in vitro and invivo xenograft models. All statistical tests were two-sided. Results: Among 237 fresh tumor samples, ATM, MRE11A, H2AFX , and/or CHEK1 loss or imbalance in 11q was detected in 20.7% of NBs, 89.8% of which were stage III or IV. An additional 7.2% contained ATM rare single nucleotide variants (SNVs). Rare SNVs in DDR-associated genes other than ATM were detected in 26.4% and were mutually exclusive. Overall, samples with SNVs and/or copy number alterations in these genes accounted for 48.4%. ATM-defective cells are known to exhibit dysfunctions in homologous recombination repair, suggesting a potential for synthetic lethality by PARP inhibition. Indeed, 83.3% NB-derived cell lines exhibited sensitivity to PARP inhibition. In addition, NB growth was markedly attenuated in the xenograft group receiving PARP inhibitors (sham-treated vs olaprib-treated group; mean [SD] tumor volume of sham-treated vs olaprib-treated groups = 7377 [1451] m 3 vs 298 [312] m 3 , P = .001, n = 4). Conclusions: Genomic alterations of DDR-associated genes including ATM, which regulates homologous recombination repair, were observed in almost half of NBs, suggesting that synthetic lethality could be induced by treatment with a PARP inhibitor. Indeed, DDR-defective NB cell lines were sensitive to PARP inhibitors. Thus, PARP inhibitors represent candidate NB therapeutics.

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Genetic alterations in adult T-cell leukemia/lymphoma

Kogure, Y. and Kataoka, K.

Cancer Sci 108 ( 9 ) 1719 - 1725 2017.09

ISSN 1349-7006

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm with a dismal prognosis. It is caused by human T-cell leukemia virus type-1 (HTLV-1) retrovirus. A long latency period from HTLV-1 infection to ATL onset suggests that not only HTLV-1 proteins, such as Tax and HBZ, but also additional genetic and/or epigenetic events are required for ATL development. Although many studies have demonstrated the biological functions of viral genes, alterations of cellular genes associated with ATL have not been fully investigated. Recently, a large-scale integrated genetic analysis revealed the entire landscape of somatic aberrations in ATL. This neoplasm is characterized by frequent gain-of-function alterations in components of the T-cell receptor/NF-kappaB signaling pathway, including activating mutations in the PLCG1, PRKCB, CARD11 and VAV1 genes, and CTLA4-CD28 and ICOS-CD28 fusions. Importantly, molecules associated with immune surveillance, such as HLA-A/B, CD58 and FAS, are affected recurrently. Among them, one notable lesion occurs as frequent structural variations that truncate the PD-L1 3'-untranslated region, leading to its overexpression. Other genetic targets include transcription factors (IRF4, IKZF2, and GATA3) and chemokine receptors (CCR4, CCR7 and GPR183), which are functionally relevant in normal T cells. A substantial proportion of ATL cases show widespread accumulation of repressive epigenetic changes, such as trimethylation of histone H3 lysine 27 and DNA hypermethylation of CpG islands, which coordinately modulate multiple pathways, including Cys2-His2 zinc finger genes involved in silencing retroelements. Here we review the current understanding of the genetic/epigenetic aberrations in ATL, focusing on their relevance in its molecular pathogenesis.

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Recurrent SPI1 (PU.1) fusions in high-risk pediatric T cell acute lymphoblastic leukemia

Seki, M., Kimura, S., Isobe, T., Yoshida, K., Ueno, H., Nakajima-Takagi, Y., Wang, C., Lin, L., Kon, A., Suzuki, H., Shiozawa, Y., Kataoka, K., Fujii, Y., Shiraishi, Y., Chiba, K., Tanaka, H., Shimamura, T., Masuda, K., Kawamoto, H., Ohki, K., Kato, M., Arakawa, Y., Koh, K., Hanada, R., Moritake, H., Akiyama, M., Kobayashi, R., Deguchi, T., Hashii, Y., Imamura, T., Sato, A., Kiyokawa, N., Oka, A., Hayashi, Y., Takagi, M., Manabe, A., Ohara, A., Horibe, K., Sanada, M., Iwama, A., Mano, H., Miyano, S., Ogawa, S. and Takita, J.

Nat Genet 49 ( 8 ) 1274 - 1281 2017.08

ISSN 1546-1718

The outcome of treatment-refractory and/or relapsed pediatric T cell acute lymphoblastic leukemia (T-ALL) is extremely poor, and the genetic basis for this is not well understood. Here we report comprehensive profiling of 121 cases of pediatric T-ALL using transcriptome and/or targeted capture sequencing, through which we identified new recurrent gene fusions involving SPI1 (STMN1-SPI1 and TCF7-SPI1). Cases positive for fusions involving SPI1 (encoding PU.1), accounting for 3.9% (7/181) of the examined pediatric T-ALL cases, showed a double-negative (DN; CD4(-)CD8(-)) or CD8(+) single-positive (SP) phenotype and had uniformly poor overall survival. These cases represent a subset of pediatric T-ALL distinguishable from the known T-ALL subsets in terms of expression of genes involved in T cell precommitment, establishment of T cell identity, and post-beta-selection maturation and with respect to mutational profile. PU.1 fusion proteins retained transcriptional activity and, when constitutively expressed in mouse stem/progenitor cells, induced cell proliferation and resulted in a maturation block. Our findings highlight a unique role of SPI1 fusions in high-risk pediatric T-ALL.

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Loss of p53 induces leukemic transformation in a murine model of Jak2 V617F-driven polycythemia vera

Tsuruta-Kishino, T., Koya, J., Kataoka, K., Narukawa, K., Sumitomo, Y., Kobayashi, H., Sato, T. and Kurokawa, M.

Oncogene 36 ( 23 ) 3300 - 3311 2017.06

ISSN 1476-5594

As leukemic transformation of myeloproliferative neoplasms (MPNs) worsens the clinical outcome, reducing the inherent risk of the critical event in MPN cases could be beneficial. Among genetic alterations concerning the transformation, the frequent one is TP53 mutation. Here we show that retroviral overexpression of Jak2 V617F mutant into wild-type p53 murine bone marrow cells induced polycythemia vera (PV) in the recipient mice, whereas Jak2 V617F-transduced p53-null mice developed lethal leukemia after the preceding PV phase. The leukemic mice had severe anemia, hepatosplenomegaly, pulmonary hemorrhage and expansion of dysplastic erythroid progenitors. Primitive leukemia cells (c-kit(+)Sca1(+)Lin(-) (KSL) and CD34(-)CD16/32(-)c-kit(+)Sca1(-)Lin(-) (megakaryocyte-erythroid progenitor; MEP)) and erythroid progenitors (CD71(+)) from Jak2 V617F-transduced p53-null leukemic mice had leukemia-initiating capacity, however, myeloid differentiated populations (Mac-1(+)) could not recapitulate the disease. Interestingly, recipients transplanted with CD71(+) cells rapidly developed erythroid leukemia, which was in sharp contrast to leukemic KSL cells to cause lethal leukemia after the polycythemic state. The leukemic CD71(+) cells were more sensitive to INCB18424, a potent JAK inhibitor, than KSL cells. p53 restoration could ameliorate Jak2 V617F-transduced p53-null erythroleukemia. Taken together, our results show that p53 loss is sufficient for inducing leukemic transformation in Jak2 V617F-positive MPN.

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A TLR3-Specific Adjuvant Relieves Innate Resistance to PD-L1 Blockade without Cytokine Toxicity in Tumor Vaccine Immunotherapy

Takeda, Y., Kataoka, K., Yamagishi, J., Ogawa, S., Seya, T. and Matsumoto, M.

Cell Rep 19 ( 9 ) 1874 - 1887 2017.05

ISSN 2211-1247

Cancer patients having anti-programmed cell death-1 (PD-1)/PD ligand 1 (L1)-unresponsive tumors may benefit from advanced immunotherapy. Double-stranded RNA triggers dendritic cell (DC) maturation to cross-prime antigen-specific cytotoxic T lymphocytes (CTLs) via Toll-like receptor 3 (TLR3). The TLR3-specific RNA agonist, ARNAX, can induce anti-tumor CTLs without systemic cytokine/interferon (IFN) production. Here, we have developed a safe vaccine adjuvant for cancer that effectively implements anti-PD-L1 therapy. Co-administration of ARNAX with a tumor-associated antigen facilitated tumor regression in mouse models, and in combination with anti-PD-L1 antibody, activated tumor-specific CTLs in lymphoid tissues, enhanced CTL infiltration, and overcame anti-PD-1 resistance without cytokinemia. The TLR3-TICAM-1-interferon regulatory factor (IRF)3-IFN-beta axis in DCs exclusively participated in CD8+ T cell cross-priming. ARNAX therapy established Th1 immunity in the tumor microenvironment, upregulating genes involved in DC/T cell/natural killer (NK) cell recruitment and functionality. Human ex vivo studies disclosed that ARNAX+antigen induced antigen-specific CTL priming and proliferation in peripheral blood mononuclear cells (PBMCs), supporting the feasibility of ARNAX for potentiating anti-PD-1/PD-L1 therapy in human vaccine immunotherapy.

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Constitutional abnormalities of IDH1 combined with secondary mutations predispose a patient with Maffucci syndrome to acute lymphoblastic leukemia

Hirabayashi, S., Seki, M., Hasegawa, D., Kato, M., Hyakuna, N., Shuo, T., Kimura, S., Yoshida, K., Kataoka, K., Fujii, Y., Shiraishi, Y., Chiba, K., Tanaka, H., Kiyokawa, N., Miyano, S., Ogawa, S., Takita, J. and Manabe, A.

Pediatr Blood Cancer 2017.05

ISSN 1545-5017

Maffucci syndrome is a nonhereditary disorder caused by somatic mosaic isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) mutations and is characterized by multiple enchondromas along with hemangiomas. Malignant transformation of enchondromas to chondrosarcomas and secondary neoplasms, such as brain tumors or acute myeloid leukemia, are serious complications. A 15-year-old female with Maffucci syndrome developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). A somatic mutation in IDH1 was detected in hemangioma and leukemic cells. KRAS mutation and deletion of IKZF1 were detected in leukemic cells. Patients with Maffucci syndrome may, therefore, be at risk of BCP-ALL associated with secondary genetic events that affect lymphocyte differentiation.

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Genetic abnormalities in myelodysplasia and secondary acute myeloid leukemia: impact on outcome of stem cell transplantation

Yoshizato, T., Nannya, Y., Atsuta, Y., Shiozawa, Y., Iijima-Yamashita, Y., Yoshida, K., Shiraishi, Y., Suzuki, H., Nagata, Y., Sato, Y., Kakiuchi, N., Matsuo, K., Onizuka, M., Kataoka, K., Chiba, K., Tanaka, H., Ueno, H., Nakagawa, M. M., Przychodzen, B., Haferlach, C., Kern, W., Aoki, K., Itonaga, H., Kanda, Y., Sekeres, M. A., Maciejewski, J. P., Haferlach, T., Miyazaki, Y., Horibe, K., Sanada, M., Miyano, S., Makishima, H. and Ogawa, S.

Blood 129 ( 17 ) 2347 - 2358 2017.04

ISSN 1528-0020

Genetic alterations, including mutations and copy-number alterations, are central to the pathogenesis of myelodysplastic syndromes and related diseases (myelodysplasia), but their roles in allogeneic stem cell transplantation have not fully been studied in a large cohort of patients. We enrolled 797 patients who had been diagnosed with myelodysplasia at initial presentation and received transplantation via the Japan Marrow Donor Program. Targeted-capture sequencing was performed to identify mutations in 69 genes, together with copy-number alterations, whose effects on transplantation outcomes were investigated. We identified 1776 mutations and 927 abnormal copy segments among 617 patients (77.4%). In multivariate modeling using Cox proportional-hazards regression, genetic factors explained 30% of the total hazards for overall survival; clinical characteristics accounted for 70% of risk. TP53 and RAS-pathway mutations, together with complex karyotype (CK) as detected by conventional cytogenetics and/or sequencing-based analysis, negatively affected posttransplant survival independently of clinical factors. Regardless of disease subtype, TP53-mutated patients with CK were characterized by unique genetic features and associated with an extremely poor survival with frequent early relapse, whereas outcomes were substantially better in TP53-mutated patients without CK. By contrast, the effects of RAS-pathway mutations depended on disease subtype and were confined to myelodysplastic/myeloproliferative neoplasms (MDS/MPNs). Our results suggest that TP53 and RAS-pathway mutations predicted a dismal prognosis, when associated with CK and MDS/MPNs, respectively. However, for patients with mutated TP53 or CK alone, long-term survival could be obtained with transplantation. Clinical sequencing provides vital information for accurate prognostication in transplantation.

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Blastic transformation of juvenile myelomonocytic leukemia caused by the copy number gain of oncogenic KRAS

Osumi, T., Kato, M., Ouchi-Uchiyama, M., Tomizawa, D., Kataoka, K., Fujii, Y., Seki, M., Takita, J., Ogawa, S., Uchiyama, T., Ohki, K. and Kiyokawa, N.

Pediatr Blood Cancer 2017.02

ISSN 1545-5017

Previous studies have reported several cases of juvenile myelomonocytic leukemia (JMML) developing blastic transformation during an indolent clinical course, but the underlying mechanism of transformation is still not well understood. In this report, we describe a case of JMML with blastic transformation possibly caused by additional copy number gains of the KRAS mutant allele. We have discovered that the copy number gain of the mutant allele is an additional possible cause of blastic transformation in JMML.

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Regeneration of CD8alphabeta T Cells from T-cell-Derived iPSC Imparts Potent Tumor Antigen-Specific Cytotoxicity

Maeda, T., Nagano, S., Ichise, H., Kataoka, K., Yamada, D., Ogawa, S., Koseki, H., Kitawaki, T., Kadowaki, N., Takaori-Kondo, A., Masuda, K. and Kawamoto, H.

Cancer Res 76 ( 23 ) 6839 - 6850 2016.12

ISSN 1538-7445

Although adoptive transfer of cytotoxic T lymphocytes (CTL) offer a promising cancer therapeutic direction, the generation of antigen-specific CTL from patients has faced difficulty in efficient expansion in ex vivo culture. To resolve this issue, several groups have proposed that induced pluripotent stem cell technology be applied for the expansion of antigen-specific CTL, which retain expression of the same T-cell receptor as original CTL. However, in these previous studies, the regenerated CTL are mostly of the CD8alphaalpha+ innate type and have less antigen-specific cytotoxic activity than primary CTL. Here we report that, by stimulating purified iPSC-derived CD4/CD8 double-positive cells with anti-CD3 antibody, T cells expressing CD8alphabeta were generated and exhibited improved antigen-specific cytotoxicity compared with CD8alphaalpha+ CTL. Failure of CD8alphabeta T-cell production using the previous method was found to be due to killing of double-positive cells by the double-negative cells in the mixed cultures. We found that WT1 tumor antigen-specific CTL regenerated by this method prolonged the survival of mice bearing WT1-expressing leukemic cells. Implementation of our methods may offer a useful clinical tool. Cancer Res; 76(23); 6839-50. (c)2016 AACR.

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Variegated RHOA mutations in human cancers

Kataoka, K. and Ogawa, S.

Exp Hematol 44 ( 12 ) 1123 - 1129 2016.12

ISSN 1873-2399

RHOA is one of the most extensively investigated members of the Rho GTPase family of proteins and has long been implicated in malignant transformation as well as tumor invasion and metastasis. Recently, revolutionized sequencing platforms have revealed frequent RHOA mutations in a wide variety of human cancers, including angio-immunoblastic T-cell lymphoma, adult T-cell leukemia/lymphoma, germinal center B-cell lymphoma, diffuse-type gastric cancer, and other solid tumors. With their discrete positional distributions and types of amino acid substitution depending on cancer type, different RHOA mutations seem to have unique functional and biological properties. However, their impact on the development of different types of tumors remains controversial. Here, we present an overview of the current understanding of RHOA mutations in human cancers and highlight the unanticipated complexity of their genetic and biological properties.

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Somatic PHF6 mutations in 1760 cases with various myeloid neoplasms

Mori, T., Nagata, Y., Makishima, H., Sanada, M., Shiozawa, Y., Kon, A., Yoshizato, T., Sato-Otsubo, A., Kataoka, K., Shiraishi, Y., Chiba, K., Tanaka, H., Ishiyama, K., Miyawaki, S., Mori, H., Nakamaki, T., Kihara, R., Kiyoi, H., Koeffler, H. P., Shih, L. Y., Miyano, S., Naoe, T., Haferlach, C., Kern, W., Haferlach, T., Ogawa, S. and Yoshida, K.

Leukemia 30 ( 11 ) 2270 - 2273 2016.11

ISSN 1476-5551

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Cytoprotective autophagy maintains leukemia-initiating cells in murine myeloid leukemia

Sumitomo, Y., Koya, J., Nakazaki, K., Kataoka, K., Tsuruta-Kishino, T., Morita, K., Sato, T. and Kurokawa, M.

Blood 128 ( 12 ) 1614 - 24 2016.09

ISSN 1528-0020

Despite advances in the treatment of acute myeloid leukemia (AML), relapse and drug resistance frequently occur. Therefore, detailed mechanisms of refractoriness, including leukemia-initiating cell (LIC) biology, should be elucidated to treat AML. The self-degradative property of cytosolic macromolecules is central to autophagy and can contribute to homeostasis and stress response. Recent reports suggest the importance of autophagy in hematopoietic stem cells and various tumors. Thus, this study investigated the functional role of autophagy in AML maintenance and drug resistance using tamoxifen-inducible conditional knockout mice of Atg5 or Atg7, which are essential genes for autophagy, combined with an mixed lineage leukemia-eleven nineteen leukemia-induced murine AML model. Inactivation of autophagy by deletion of Atg5 or Atg7 prolonged survival in leukemic mice and reduced functional LICs. Atg7-deficient LICs displayed enhanced mitochondrial activity and reactive oxygen species production together with increased cell death. In addition, Atg7 deletion markedly decreased peripheral blood leukemia cells, concurrent with increased apoptosis, suggesting a higher dependency on autophagy compared with bone marrow leukemia cells. Finally, cytarabine (AraC) treatment activated autophagy in LICs, and Atg7 deletion potentiated the therapeutic effects of AraC, which included decreased LICs and prolonged survival, suggesting that autophagy contributes to AraC resistance. Our results highlight the intratumoral heterogeneity related to autophagy in AML and the unique role of autophagy in leukemia development and drug resistance.

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Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers

Kataoka, K., Shiraishi, Y., Takeda, Y., Sakata, S., Matsumoto, M., Nagano, S., Maeda, T., Nagata, Y., Kitanaka, A., Mizuno, S., Tanaka, H., Chiba, K., Ito, S., Watatani, Y., Kakiuchi, N., Suzuki, H., Yoshizato, T., Yoshida, K., Sanada, M., Itonaga, H., Imaizumi, Y., Totoki, Y., Munakata, W., Nakamura, H., Hama, N., Shide, K., Kubuki, Y., Hidaka, T., Kameda, T., Masuda, K., Minato, N., Kashiwase, K., Izutsu, K., Takaori-Kondo, A., Miyazaki, Y., Takahashi, S., Shibata, T., Kawamoto, H., Akatsuka, Y., Shimoda, K., Takeuchi, K., Seya, T., Miyano, S. and Ogawa, S.

Nature 534 ( 7607 ) 402 - 6 2016.06

ISSN 1476-4687

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3' region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3'-untranslated region (UTR). Disruption of the Pd-l1 3'-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3'-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

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Paroxysmal nocturnal hemoglobinuria induced by the occurrence of BCR-ABL in a PIGA mutant hematopoietic progenitor cell

Tominaga, R., Katagiri, T., Kataoka, K., Kataoka, K., Wee, R. K., Maeda, A., Gomyo, H., Mizuno, I., Murayama, T., Ogawa, S. and Nakao, S.

Leukemia 30 ( 5 ) 1208 - 10 2016.05

ISSN 1476-5551

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Mutational Landscape and Antiproliferative Functions of ELF Transcription Factors in Human Cancer

Ando, M., Kawazu, M., Ueno, T., Koinuma, D., Ando, K., Koya, J., Kataoka, K., Yasuda, T., Yamaguchi, H., Fukumura, K., Yamato, A., Soda, M., Sai, E., Yamashita, Y., Asakage, T., Miyazaki, Y., Kurokawa, M., Miyazono, K., Nimer, S. D., Yamasoba, T. and Mano, H.

Cancer Res 76 ( 7 ) 1814 - 24 2016.04

ISSN 1538-7445

ELF4 (also known as MEF) is a member of the ETS family of transcription factors. An oncogenic role for ELF4 has been demonstrated in hematopoietic malignancies, but its function in epithelial tumors remains unclear. Here, we show that ELF4 can function as a tumor suppressor and is somatically inactivated in a wide range of human tumors. We identified a missense mutation affecting the transactivation potential of ELF4 in oral squamous cell carcinoma cells. Restoration of the transactivation activity through introduction of wild-type ELF4 significantly inhibited cell proliferation in vitro and tumor xenograft growth. Furthermore, we found that ELF1 and ELF2, closely related transcription factors to ELF4, also exerted antiproliferative effects in multiple cancer cell lines. Mutations in ELF1 and ELF2, as in ELF4, were widespread across human cancers, but were almost all mutually exclusive. Moreover, chromatin immunoprecipitation coupled with high-throughput sequencing revealed ELF4-binding sites in genomic regions adjacent to genes related to cell-cycle regulation and apoptosis. Finally, we provide mechanistic evidence that the antiproliferative effects of ELF4 were mediated through the induction of HRK, an activator of apoptosis, and DLX3, an inhibitor of cell growth. Collectively, our findings reveal a novel subtype of human cancer characterized by inactivating mutations in the ELF subfamily of proteins, and warrant further investigation of the specific settings where ELF restoration may be therapeutically beneficial. Cancer Res; 76(7); 1814-24. (c)2016 AACR.

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DNMT3A R882 mutants interact with polycomb proteins to block haematopoietic stem and leukaemic cell differentiation

Koya, J., Kataoka, K., Sato, T., Bando, M., Kato, Y., Tsuruta-Kishino, T., Kobayashi, H., Narukawa, K., Miyoshi, H., Shirahige, K. and Kurokawa, M.

Nat Commun 7 10924 2016.03

ISSN 2041-1723

Despite the clinical impact of DNMT3A mutation on acute myeloid leukaemia, the molecular mechanisms regarding how this mutation causes leukaemogenesis in vivo are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant haematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are downregulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells, representing a DNA methylation-independent role of mutated DNMT3A. DNMT3A R882H also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, the DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), causing transcriptional silencing, revealing a DNA methylation-independent role of DNMT3A mutation. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. From our data, it is shown that DNMT3A mutants can block the differentiation of HSCs and leukaemic cells via PRC1. This interaction could be targetable in DNMT3A-mutated leukaemias.

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Variegated RHOA mutations in adult T-cell leukemia/lymphoma

Nagata, Y., Kontani, K., Enami, T., Kataoka, K., Ishii, R., Totoki, Y., Kataoka, T. R., Hirata, M., Aoki, K., Nakano, K., Kitanaka, A., Sakata-Yanagimoto, M., Egami, S., Shiraishi, Y., Chiba, K., Tanaka, H., Shiozawa, Y., Yoshizato, T., Suzuki, H., Kon, A., Yoshida, K., Sato, Y., Sato-Otsubo, A., Sanada, M., Munakata, W., Nakamura, H., Hama, N., Miyano, S., Nureki, O., Shibata, T., Haga, H., Shimoda, K., Katada, T., Chiba, S., Watanabe, T. and Ogawa, S.

Blood 127 ( 5 ) 596 - 604 2016.02

ISSN 1528-0020

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers.

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Genetic biomarkers for PD-1/PD-L1 blockade therapy

Kataoka, Keisuke and Ogawa, Seishi

Oncoscience 3 ( 11-12 ) 311 2016

BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and KLF4

Morita, K., Masamoto, Y., Kataoka, K., Koya, J., Kagoya, Y., Yashiroda, H., Sato, T., Murata, S. and Kurokawa, M.

Leukemia 29 ( 11 ) 2248 - 56 2015.11

ISSN 1476-5551

Although high brain and acute leukemia, cytoplasmic (BAALC) expression is a well-characterized poor prognostic factor in acute myeloid leukemia (AML), neither the exact mechanisms by which BAALC drives leukemogenesis and drug resistance nor therapeutic approaches against BAALC-high AML have been properly elucidated. In this study, we found that BAALC induced cell-cycle progression of leukemia cells by sustaining extracellular signal-regulated kinase (ERK) activity through an interaction with a scaffold protein MEK kinase-1 (MEKK1), which inhibits the interaction between ERK and MAP kinase phosphatase 3 (MKP3/DUSP6). BAALC conferred chemoresistance in AML cells by upregulating ATP-binding cassette proteins in an ERK-dependent manner, which can be therapeutically targeted by MEK inhibitor. We also demonstrated that BAALC blocks ERK-mediated monocytic differentiation of AML cells by trapping Kruppel-like factor 4 (KLF4) in the cytoplasm and inhibiting its function in the nucleus. Consequently, MEK inhibition therapy synergizes with KLF4 induction and is highly effective against BAALC-high AML cells both in vitro and in vivo. Our data provide a molecular basis for the role of BAALC in regulating proliferation and differentiation of AML cells and highlight the unique dual function of BAALC as an attractive therapeutic target against BAALC-high AML.

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Integrated molecular analysis of adult T cell leukemia/lymphoma

Kataoka, K., Nagata, Y., Kitanaka, A., Shiraishi, Y., Shimamura, T., Yasunaga, J., Totoki, Y., Chiba, K., Sato-Otsubo, A., Nagae, G., Ishii, R., Muto, S., Kotani, S., Watatani, Y., Takeda, J., Sanada, M., Tanaka, H., Suzuki, H., Sato, Y., Shiozawa, Y., Yoshizato, T., Yoshida, K., Makishima, H., Iwanaga, M., Ma, G., Nosaka, K., Hishizawa, M., Itonaga, H., Imaizumi, Y., Munakata, W., Ogasawara, H., Sato, T., Sasai, K., Muramoto, K., Penova, M., Kawaguchi, T., Nakamura, H., Hama, N., Shide, K., Kubuki, Y., Hidaka, T., Kameda, T., Nakamaki, T., Ishiyama, K., Miyawaki, S., Yoon, S. S., Tobinai, K., Miyazaki, Y., Takaori-Kondo, A., Matsuda, F., Takeuchi, K., Nureki, O., Aburatani, H., Watanabe, T., Shibata, T., Matsuoka, M., Miyano, S., Shimoda, K. and Ogawa, S.

Nat Genet 47 ( 11 ) 1304 - 15 2015.11

ISSN 1546-1718

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-kappaB 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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Targeted gene correction of RUNX1 in induced pluripotent stem cells derived from familial platelet disorder with propensity to myeloid malignancy restores normal megakaryopoiesis

Iizuka, H., Kagoya, Y., Kataoka, K., Yoshimi, A., Miyauchi, M., Taoka, K., Kumano, K., Yamamoto, T., Hotta, A., Arai, S. and Kurokawa, M.

Exp Hematol 43 ( 10 ) 849 - 57 2015.10

ISSN 1873-2399

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease.

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Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia

Yoshizato, T., Dumitriu, B., Hosokawa, K., Makishima, H., Yoshida, K., Townsley, D., Sato-Otsubo, A., Sato, Y., Liu, D., Suzuki, H., Wu, C. O., Shiraishi, Y., Clemente, M. J., Kataoka, K., Shiozawa, Y., Okuno, Y., Chiba, K., Tanaka, H., Nagata, Y., Katagiri, T., Kon, A., Sanada, M., Scheinberg, P., Miyano, S., Maciejewski, J. P., Nakao, S., Young, N. S. and Ogawa, S.

N Engl J Med 373 ( 1 ) 35 - 47 2015.07

ISSN 1533-4406

BACKGROUND: In patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immunosuppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the diagnosis of aplastic anemia. METHODS: We performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients. RESULTS: Somatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as acquired mutations. The prevalence of the mutations increased with age, and mutations had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- and BCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients. CONCLUSIONS: Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.).

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Mutational landscape and clonal architecture in grade II and III gliomas

Suzuki, H., Aoki, K., Chiba, K., Sato, Y., Shiozawa, Y., Shiraishi, Y., Shimamura, T., Niida, A., Motomura, K., Ohka, F., Yamamoto, T., Tanahashi, K., Ranjit, M., Wakabayashi, T., Yoshizato, T., Kataoka, K., Yoshida, K., Nagata, Y., Sato-Otsubo, A., Tanaka, H., Sanada, M., Kondo, Y., Nakamura, H., Mizoguchi, M., Abe, T., Muragaki, Y., Watanabe, R., Ito, I., Miyano, S., Natsume, A. and Ogawa, S.

Nat Genet 47 ( 5 ) 458 - 68 2015.05

ISSN 1546-1718

Grade II and III gliomas are generally slowly progressing brain cancers, many of which eventually transform into more aggressive tumors. Despite recent findings of frequent mutations in IDH1 and other genes, knowledge about their pathogenesis is still incomplete. Here, combining two large sets of high-throughput sequencing data, we delineate the entire picture of genetic alterations and affected pathways in these glioma types, with sensitive detection of driver genes. Grade II and III gliomas comprise three distinct subtypes characterized by discrete sets of mutations and distinct clinical behaviors. Mutations showed significant positive and negative correlations and a chronological hierarchy, as inferred from different allelic burdens among coexisting mutations, suggesting that there is functional interplay between the mutations that drive clonal selection. Extensive serial and multi-regional sampling analyses further supported this finding and also identified a high degree of temporal and spatial heterogeneity generated during tumor expansion and relapse, which is likely shaped by the complex but ordered processes of multiple clonal selection and evolutionary events.

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Evi1 defines leukemia-initiating capacity and tyrosine kinase inhibitor resistance in chronic myeloid leukemia

Sato, T., Goyama, S., Kataoka, K., Nasu, R., Tsuruta-Kishino, T., Kagoya, Y., Nukina, A., Kumagai, K., Kubota, N., Nakagawa, M., Arai, S., Yoshimi, A., Honda, H., Kadowaki, T. and Kurokawa, M.

Oncogene 33 ( 42 ) 5028 - 38 2014.10

ISSN 1476-5594

Relapse of chronic myeloid leukemia (CML) is triggered by stem cells with a reconstituting capacity similar to that of hematopoietic stem cells (HSCs) and CML stem cells are a source of resistance in drug therapy with tyrosine kinase inhibitors (TKIs). Ecotropic viral integration site 1 (EVI1), a key transcription factor in HSC regulation, is known to predict poor outcomes in myeloid malignancies, however, incapability of prospective isolation of EVI1-high leukemic cells precludes the functional evaluation of intraindividual EVI1-high cells. Introduction of CML into Evi1-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) knock-in mice, a versatile HSC-reporter strain, enables us to separate Evi1-high CML cells from the individual. Evi1-IRES-GFP allele models of CML in chronic phase (CML-CP), by retroviral overexpression of BCR-ABL and by crossing BCR-ABL transgenic mice, revealed that Evi1 is predominantly enriched in the stem cell fraction and associated with an enhanced proliferative as well as a leukemia-initiating capacity and that Evi1-high CML-CP cells exhibit resistance to TKIs. Overexpressing BCR-ABL and NUP98-HOXA9 in Evi1-IRES-GFP knock-in mice to model CML in blast crisis (CML-BC), in which Evi1-high cells turned to be a major population as opposed to a minor population in CML-CP models, showed that Evi1-high CML-BC cells have a greater potential to recapitulate the disease and appear resistant to TKIs. Furthermore, given that Evi1 heterozygosity ameliorates CML-CP and CML-BC development and that the combination of Evi1 and BCR-ABL causes acute myeloid leukemia resembling CML-BC, Evi1 could regulate CML development as a potent driver. In addition, in human CML-CP cases, we show that EVI1 is highly expressed in stem cell-enriched CD34+CD38-CD90+ fraction at single-cell level. This is the first report to clarify directly that Evi1-high leukemic cells themselves possess the superior potential to Evi1-low cells in oncogenic self-renewal, which highlights the role of Evi1 as a valuable and a functional marker of CML stem cells.

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Generation of induced pluripotent stem cells derived from primary and secondary myelofibrosis patient samples

Hosoi, M., Kumano, K., Taoka, K., Arai, S., Kataoka, K., Ueda, K., Kamikubo, Y., Takayama, N., Otsu, M., Eto, K., Nakauchi, H. and Kurokawa, M.

Exp Hematol 42 ( 9 ) 816 - 25 2014.09

ISSN 1873-2399

Induced pluripotent stem cells (iPS) derived from disease cells are expected to provide a new experimental material, especially for diseases from which samples are difficult to obtain. In this study, we generated iPS from samples from patients with primary and secondary myelofibrosis. The primary myelofibrosis cells had chromosome 13q deletions, and the secondary myelofibrosis (SMF) cells had JAK2V617F mutations. The myelofibrosis patient cell-derived iPS (MF-iPS) were confirmed as possessing these parental disease-specific genomic markers. The capacity to form three germ layers was confirmed by teratoma assay. By co-culture with specific feeder cells and cytokines, MF-iPS can re-differentiate into blood progenitor cells and finally into megakaryocytes. We found that mRNA levels of interleukin-8, one of the candidate cytokines related to the pathogenesis of myelofibrosis, was elevated predominantly in megakaryocytes derived from MF-iPS. Because megakaryocytes from myelofibrosis clones are considered to produce critical mediators to proliferate fibroblasts in the bone marrow and iPS can provide differentiated cells abundantly, the disease-specific iPS we established should be a good research tool for this intractable disease.

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Recurrent somatic mutations underlie corticotropin-independent Cushing's syndrome

Sato, Y., Maekawa, S., Ishii, R., Sanada, M., Morikawa, T., Shiraishi, Y., Yoshida, K., Nagata, Y., Sato-Otsubo, A., Yoshizato, T., Suzuki, H., Shiozawa, Y., Kataoka, K., Kon, A., Aoki, K., Chiba, K., Tanaka, H., Kume, H., Miyano, S., Fukayama, M., Nureki, O., Homma, Y. and Ogawa, S.

Science 344 ( 6186 ) 917 - 20 2014.05

ISSN 1095-9203

Cushing's syndrome is caused by excess cortisol production from the adrenocortical gland. In corticotropin-independent Cushing's syndrome, the excess cortisol production is primarily attributed to an adrenocortical adenoma, in which the underlying molecular pathogenesis has been poorly understood. We report a hotspot mutation (L206R) in PRKACA, which encodes the catalytic subunit of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), in more than 50% of cases with adrenocortical adenomas associated with corticotropin-independent Cushing's syndrome. The L206R PRKACA mutant abolished its binding to the regulatory subunit of PKA (PRKAR1A) that inhibits catalytic activity of PRKACA, leading to constitutive, cAMP-independent PKA activation. These results highlight the major role of cAMP-independent activation of cAMP/PKA signaling by somatic mutations in corticotropin-independent Cushing's syndrome, providing insights into the diagnosis and therapeutics of this syndrome.

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The IL-2/CD25 axis maintains distinct subsets of chronic myeloid leukemia-initiating cells

Kobayashi, C. I., Takubo, K., Kobayashi, H., Nakamura-Ishizu, A., Honda, H., Kataoka, K., Kumano, K., Akiyama, H., Sudo, T., Kurokawa, M. and Suda, T.

Blood 123 ( 16 ) 2540 - 9 2014.04

ISSN 1528-0020

Just as normal stem cells require niche cells for survival, leukemia-initiating cells (LICs) may also require niche cells for their maintenance. Chronic myeloid leukemia (CML) is caused by the activity of BCR-ABL, a constitutively active tyrosine kinase. CML therapy with tyrosine kinase inhibitors is highly effective; however, due to the persistence of residual LICs, it is not curative. Several factors are known to support CML LICs, but purification of LICs and a thorough understanding of their niche signals have not yet been achieved. Using a CML-like mouse model of myeloproliferative disease, we demonstrate that CML LICs can be divided into CD25(+)FcepsilonRIalpha(-) Lineage marker (Lin)(-) Sca-1(+)c-Kit(+) (F(-)LSK) cells and CD25(-)F(-)LSK cells. The CD25(+)F(-)LSK cells had multilineage differentiation capacity, with a preference toward cytokine-producing mast cell commitment. Although cells interconverted between CD25(-)F(-)LSK and CD25(+)F(-)LSK status, the CD25(+)F(-)LSK cells exhibited higher LIC capacity. Our findings suggest that interleukin-2 derived from the microenvironment and CD25 expressed on CML LICs constitute a novel signaling axis. The high levels of CD25 expression in the CD34(+)CD38(-) fraction of human CML cells indicate that CD25(+) LICs constitute an "LIC-derived niche" that could be preferentially targeted in therapy for CML.

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Positive feedback between NF-kappaB and TNF-alpha promotes leukemia-initiating cell capacity

Kagoya, Y., Yoshimi, A., Kataoka, K., Nakagawa, M., Kumano, K., Arai, S., Kobayashi, H., Saito, T., Iwakura, Y. and Kurokawa, M.

J Clin Invest 124 ( 2 ) 528 - 42 2014.02

ISSN 1558-8238

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that originates from leukemia-initiating cells (LICs). The identification of common mechanisms underlying LIC development will be important in establishing broadly effective therapeutics for AML. Constitutive NF-kappaB pathway activation has been reported in different types of AML; however, the mechanism of NF-kappaB activation and its importance in leukemia progression are poorly understood. Here, we analyzed myeloid leukemia mouse models to assess NF-kappaB activity in AML LICs. We found that LICs, but not normal hematopoietic stem cells or non-LIC fractions within leukemia cells, exhibited constitutive NF-kappaB activity. This activity was maintained through autocrine TNF-alpha secretion, which formed an NF-kappaB/TNF-alpha positive feedback loop. LICs had increased levels of active proteasome machinery, which promoted the degradation of IkappaBalpha and further supported NF-kappaB activity. Pharmacological inhibition of the proteasome complex markedly suppressed leukemia progression in vivo. Conversely, enhanced activation of NF-kappaB signaling expanded LIC frequency within leukemia cell populations. We also demonstrated a strong correlation between NF-kappaB activity and TNF-alpha secretion in human AML samples. Our findings indicate that NF-kappaB/TNF-alpha signaling in LICs contributes to leukemia progression and provide a widely applicable approach for targeting LICs.

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Identification of long-term repopulating hematopoietic stem cells by Evi1

Kataoka, Keisuke, Sato, Tomohiko and Kurokawa, Mineo

Inflammation and Regeneration 33 ( 4 ) 175 - 180 2013

ISSN 1880-9693

Ecotropic viral integration site 1, stem cell self-renewal and leukemogenesis

Kataoka, K. and Kurokawa, M.

Cancer Sci 103 ( 8 ) 1371 - 7 2012.08

ISSN 1349-7006

It has become evident that acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by a subset of self-renewing leukemia stem cells. Recent gene expression profile analysis of human leukemia stem cells and hematopoietic stem cell (HSC) populations identified a key transcriptional program shared by leukemia stem cells and HSC, which is associated with adverse outcomes in AML patients. One molecule that has been established as a pivotal regulator in fine-tuning of stem cell properties as well as a potent oncogenic determinant is ecotropic viral integration site 1 (EVI1). EVI1 is a transcription factor that has stem cell-specific expression pattern and is essential for the regulation of HSC self-renewal. This gene is notorious for its involvement in AML, as its activation confers extremely poor prognosis in patients with AML. Molecular analysis has identified a variety of gene products that are involved in HSC regulation as downstream targets or interacting proteins of EVI1. Thus, exploration of the molecular pathogenesis underlying EVI1-related leukemogenesis provides insight into how shared stemness transcriptional programs contribute to leukemia progression and therapeutic resistance in AML. Here, we review the current knowledge regarding the role of EVI1 in HSC self-renewal and leukemogenesis and highlight the relationship between stem cell self-renewal properties and adverse outcome in myeloid malignancies.

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AML1/RUNX1 functions as a cytoplasmic attenuator of NF-kappaB signaling in the repression of myeloid tumors

Nakagawa, M., Shimabe, M., Watanabe-Okochi, N., Arai, S., Yoshimi, A., Shinohara, A., Nishimoto, N., Kataoka, K., Sato, T., Kumano, K., Nannya, Y., Ichikawa, M., Imai, Y. and Kurokawa, M.

Blood 118 ( 25 ) 6626 - 37 2011.12

ISSN 1528-0020

Functional deregulation of transcription factors has been found in many types of tumors. Transcription factor AML1/RUNX1 is one of the most frequent targets of chromosomal abnormalities in human leukemia and altered function of AML1 is closely associated with malignant transformation of hematopoietic cells. However, the molecular basis and therapeutic targets of AML1-related leukemia are still elusive. Here, we explored immediate target pathways of AML1 by in vitro synchronous inactivation in hematopoietic cells. We found that AML1 inhibits NF-kappaB signaling through interaction with IkappaB kinase complex in the cytoplasm. Remarkably, AML1 mutants found in myeloid tumors lack the ability to inhibit NF-kappaB signaling, and human cases with AML1-related leukemia exhibits distinctly activated NF-kappaB signaling. Furthermore, inhibition of NF-kappaB signaling in leukemic cells with mutated AML1 efficiently blocks their growth and development of leukemia. These findings reveal a novel role for AML1 as a cytoplasmic attenuator of NF-kappaB signaling and indicate that NF-kappaB signaling is one of the promising therapeutic targets of hematologic malignancies with AML1 abnormality.

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Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity

Kataoka, K., Sato, T., Yoshimi, A., Goyama, S., Tsuruta, T., Kobayashi, H., Shimabe, M., Arai, S., Nakagawa, M., Imai, Y., Kumano, K., Kumagai, K., Kubota, N., Kadowaki, T. and Kurokawa, M.

J Exp Med 208 ( 12 ) 2403 - 16 2011.11

ISSN 1540-9538

Ecotropic viral integration site 1 (Evi1), a transcription factor of the SET/PR domain protein family, is essential for the maintenance of hematopoietic stem cells (HSCs) in mice and is overexpressed in several myeloid malignancies. Here, we generate reporter mice in which an internal ribosome entry site (IRES)-GFP cassette is knocked-in to the Evi1 locus. Using these mice, we find that Evi1 is predominantly expressed in long-term HSCs (LT-HSCs) in adult bone marrow, and in the hematopoietic stem/progenitor fraction in the aorta-gonad-mesonephros, placenta, and fetal liver of embryos. In both fetal and adult hematopoietic systems, Evi1 expression marks cells with long-term multilineage repopulating activity. When combined with conventional HSC surface markers, sorting according to Evi1 expression markedly enhances purification of cells with HSC activity. Evi1 heterozygosity leads to marked impairment of the self-renewal capacity of LT-HSCs, whereas overexpression of Evi1 suppresses differentiation and boosts self-renewal activity. Reintroduction of Evi1, but not Mds1-Evi1, rescues the HSC defects caused by Evi1 heterozygosity. Thus, in addition to documenting a specific relationship between Evi1 expression and HSC self-renewal activity, these findings highlight the utility of Evi1-IRES-GFP reporter mice for the identification and sorting of functional HSCs.

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The negative impact of female donor/male recipient combination in allogeneic hematopoietic stem cell transplantation depends on disease risk

Nannya, Y., Kataoka, K., Hangaishi, A., Imai, Y., Takahashi, T. and Kurokawa, M.

Transpl Int 24 ( 5 ) 469 - 76 2011.05

ISSN 1432-2277

Optimal donor selection is one of the key factors to enhance the success rate of allogeneic hematopoietic stem cell transplantation (HSCT). The effect of sex mismatch, especially the effect of Y chromosome mismatch in graft-versus-host disease (GVHD) direction (female donors to male recipients: denoted as FtoM mismatch) on overall survival (OS) has been controversial and not examined out of the patient population in Western countries. We retrospectively analyzed 225 cases of allogeneic HSCT and showed that FtoM mismatch confers a highly significant impact on OS (P < 0.005) in Japanese population. We demonstrated that this effect depends on the disease risk; for standard risk cases, this effect was significantly associated with poor outcome (for OS, P = 0.021), while for high risk cases, it had no effect on the results (for OS, P = 0.26). We further showed that FtoM mismatch was associated with nonrelapse mortality (P = 0.019) and most of them were GVHD-related in standard risk cases. In conclusion, FtoM mismatch has a significant impact on transplant outcome, especially in standard risk cases.

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Pretransplant predictors and posttransplant sequels of acute kidney injury after allogeneic stem cell transplantation

Kagoya, Y., Kataoka, K., Nannya, Y. and Kurokawa, M.

Biol Blood Marrow Transplant 17 ( 3 ) 394 - 400 2011.03

ISSN 1523-6536

Acute kidney injury (AKI) is a common complication after allogeneic stem cell transplantation (SCT). Although various risk factors for AKI have been reported, the influence of pretransplant comorbidity on the incidence of AKI has not been well investigated. We performed a retrospective analysis of 207 consecutive patients undergoing myeloablative or nonmyeloablative SCT between 2001 and 2009, using the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) as a representative of pretransplant comorbidities. According to Risk, Injury, Failure, Loss, and End-stage kidney disease (RIFLE) criteria, 158 patients (76.3%) developed AKI, and 92 patients (44.4%) developed severe AKI (RIFLE class I or class F) within 100 days after SCT. The cumulative incidence of severe AKI within 100 days in patients with an HCT-CI score 0, 1-2, and >/=3 was 21.3%, 48.8%, and 73.9%, respectively. In multivariate analysis, the HCT-CI was independently associated with severe AKI (HCT-CI 1-2: adjusted hazard ratio [HR] 2.42, P < .01; HCT-CI >/=3: adjusted HR 4.69, P < .01). In a landmark analysis, patients with severe AKI had a lower 3-year overall survival (OS) (39.3% versus 61.4%, P < .01), and a higher 3-year nonrelapse mortality (NRM) (40.8% versus 5.6%, P < .01) than those without AKI. Multivariate analysis showed that severe AKI was a significant risk factor for worse OS (HR: 2.10, P = .01) and NRM (HR: 6.15, P < .01). Thus, it is important to assess the HCT-CI to predict the incidence of AKI, which is a strong indicator of worse prognosis after SCT.

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Outcome and treatment of late-onset noninfectious pulmonary complications after allogeneic haematopoietic SCT

Ueda, K., Watadani, T., Maeda, E., Ota, S., Kataoka, K., Seo, S., Kumano, K., Hangaishi, A., Takahashi, T., Imai, Y., Ohtomo, K., Fukayama, M., Nannya, Y. and Kurokawa, M.

Bone Marrow Transplant 45 ( 12 ) 1719 - 27 2010.12

ISSN 1476-5365

Late-onset noninfectious pulmonary complications (LONIPCs) are life threatening for allogeneic hematopoietic SCT (allo-HSCT) recipients. However, the impact of LONIPCs on survival has not been properly evaluated and little is known about treatment efficacy. We retrospectively investigated 290 allo-HSCT recipients in our institute and reviewed the clinical aspects of 44 patients who had been diagnosed with LONIPCs. LONIPCs were significantly associated with higher rates of chronic GVHD (P<0001) and nonrelapse mortality (P=0.013), and lower rates of relapse (P=0.009). As a result of these effects, OS was significantly worse in those with LONIPCs (P=0.003). This result differs from a previous report. We then assessed short-term treatment response and final outcome. These results were defined by radiological findings, subjective symptoms, oxygen requirement and survival. Use of inhaled and systemic steroids did not affect either short-term response or final outcomes. However, administration of systemic corticosteroids earlier than at 21 days (median interval of time from onset of symptoms to systemic corticosteroids administration) was associated with a better outcome (P=0.054 for short-term response, and 0.016 for final outcome). Our study indicates that LONIPCs reduce OS, and early intervention with systemic corticosteroids may be effective.

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Plasma brain natriuretic peptide is associated with hepatic veno-occlusive disease and early mortality after allogeneic hematopoietic stem cell transplantation

Kataoka, K., Nannya, Y., Iwata, H., Seo, S., Kumano, K., Takahashi, T., Nagai, R. and Kurokawa, M.

Bone Marrow Transplant 45 ( 11 ) 1631 - 7 2010.11

ISSN 1476-5365

Fluid retention is characteristic of veno-occlusive disease (VOD). We hypothesized that plasma brain natriuretic peptide (BNP), a neurohormone secreted in response to volume expansion, may be associated with VOD after hematopoietic stem cell transplantation (HSCT). BNP was measured before and weekly after HSCT in 46 recipients. Sixteen patients developed VOD. BNP concentrations were similar before and on day 0 in patients with and without VOD, but were significantly higher on day 7 and later in those with VOD. Patients with VOD had significantly higher peak BNP concentrations before engraftment than those without VOD (median, 634.4 versus 80.9 pg ml(-)(1); P=0.01). Multivariate analysis showed that VOD was independently associated with BNP elevation (odds ratio, 50.1; 95% CI: 5.2-478.4; P<0.01). Landmark analysis at day 7 showed that patients with peak BNP concentration of >/= 180 pg ml(-)(1) had significantly worse 100-day survival than patients with peak BNP <180 pg ml(-)(1) (54 versus 91%; P<0.01). In multivariate analysis, BNP elevation before day 7 significantly predicted 100-day survival (hazard ratio 5.3; 95% CI: 1.1-24.3; P=0.03). These findings suggest that plasma BNP may serve as a diagnostic and prognostic marker of VOD.

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Post-transplant lymphoproliferative disorder after adult-to-adult living donor liver transplant: case series and review of literature

Kataoka, K., Seo, S., Sugawara, Y., Ota, S., Imai, Y., Takahashi, T., Fukayama, M., Kokudo, N. and Kurokawa, M.

Leuk Lymphoma 51 ( 8 ) 1494 - 501 2010.08

ISSN 1029-2403

Post-transplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplant. Although living donor liver transplant (LDLT) has been increasingly performed, PTLD after LDLT has not been well investigated. We aimed to determine the clinical characteristics of PTLD after LDLT. We investigated 323 consecutive patients undergoing adult-to-adult LDLT and identified three patients who developed biopsy-proven PTLD. All of them were seropositive for Epstein-Barr virus (EBV) and had hepatitis C virus-related cirrhosis at transplant. All three patients developed late-onset and monomorphic PTLD, including one diffuse large B-cell lymphoma and two Burkitt lymphomas with c-myc rearrangement. Two of them were EBV negative. The initial therapy included chemotherapy, rituximab, and immunosuppression withdrawal. One patient died of sepsis during treatment and two patients achieved complete responses. We showed a relatively low incidence and distinct clinicopathological features of PTLD after adult-to-adult LDLT, which might reflect the unique nature of LDLT.

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Positron emission tomography in the diagnosis and therapeutic monitoring of post-transplant lymphoproliferative disorder after cord blood transplantation

Kataoka, K., Seo, S., Ota, S., Takahashi, T. and Kurokawa, M.

Bone Marrow Transplant 45 ( 3 ) 610 - 2 2010.03

ISSN 1476-5365

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Differential prognostic impact of pretransplant comorbidity on transplant outcomes by disease status and time from transplant: a single Japanese transplant centre study

Kataoka, K., Nannya, Y., Ueda, K., Kumano, K., Takahashi, T. and Kurokawa, M.

Bone Marrow Transplant 45 ( 3 ) 513 - 20 2010.03

ISSN 1476-5365

This retrospective study examined the differences in the prognostic impact of the haematopoietic cell transplantation-specific comorbidity index (HCT-CI) on transplant outcomes by disease status and time from transplant in allogeneic haematopoietic stem cell transplantation (HSCT) recipients at a Japanese transplant centre. Of 187 patients, nonrelapse mortality (NRM) at 3 years was 9.6, 21.2 and 27.8% in the low-risk (score 0), intermediate-risk (score 1-2) and high-risk (score > or =3) HCT-CI groups, respectively (P=0.03). The corresponding overall survival (OS) at 3 years was 70.1, 60.5 and 38.9%, respectively (P<0.01). In multivariate analyses, high-risk HCT-CI significantly predicted higher NRM (relative risk, (RR) 2.44 (95% confidence interval, (CI) 1.02-5.85); P=0.04) and worse OS (RR 2.02 (95% CI 1.15-3.54); P=0.01). In the subgroup analysis according to disease status, the HCT-CI was associated with OS (P<0.01) and NRM (P=0.07) in patients with low-risk diseases, but not in those with high-risk diseases. Within patients who survived without relapse >1 year after HSCT, the HCT-CI did not predict OS (P=0.59) or NRM (P=0.31). These findings can be useful to determine the role of pretransplant comorbidity in allogeneic HSCT.

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Fulminant myocarditis after allogeneic bone marrow transplantation: successful cytomegalovirus therapy and mechanical circulatory support for bridge to recovery

Kataoka, K., Takahashi, T., Iwata, H., Hangaishi, A., Kumano, K. and Kurokawa, M.

Biol Blood Marrow Transplant 16 ( 1 ) 129 - 30 2010.01

ISSN 1523-6536

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Interstitial pneumonia associated with progression of myelodysplastic syndrome

Kataoka, K., Ichikawa, M., Hangaishi, A., Takahashi, T., Imai, Y. and Kurokawa, M.

Int J Hematol 89 ( 5 ) 718 - 9 2009.06

ISSN 1865-3774

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Influence of pretransplantation serum ferritin on nonrelapse mortality after myeloablative and nonmyeloablative allogeneic hematopoietic stem cell transplantation

Kataoka, K., Nannya, Y., Hangaishi, A., Imai, Y., Chiba, S., Takahashi, T. and Kurokawa, M.

Biol Blood Marrow Transplant 15 ( 2 ) 195 - 204 2009.02

ISSN 1523-6536

Iron overload might be an important contributor to nonrelapse mortality (NRM) in hematopoietic stem cell transplantation (HSCT). We studied 264 patients undergoing allogeneic HSCT for hematologic malignancies between 1996 and 2006, using pretransplantation serum ferritin as a surrogate marker of iron overload. At 5 years, patients in the high ferritin group (>or= 599 ng/mL) had a lower overall survival (OS; 33.0% versus 63.5%; P< .001) and a higher NRM (34.9% versus 13.7%; P< .001) than those in the low ferritin group (<599 ng/mL). Multivariate analyses showed that high pretransplantation serum ferritin was a significant risk factor for worse survival (relative risk [RR]=1.68; P= .05) and increased NRM (RR=2.47; P= .01). There was no significant difference in the cumulative incidence of relapse, and acute and chronic graft-versus-host disease (aGVHD, cGVHD) between the 2 groups. Patients in the high ferritin group were more likely to die of infection (P< .010) and organ failure (P< .019). Similar results were observed after dividing the patients according to the intensity of conditioning regimens. These findings emphasize the prognostic impact of pretransplantation serum ferritin in HSCT recipients.

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Successful engraftment following HLA-mismatched cord blood transplantation for patients with anti-HLA Abs

Kataoka, K., Yamamoto, G., Nannya, Y., Yoshimi, A., Okada, S., Asai, T., Kumano, K., Hangaishi, A., Takahashi, T., Chiba, S. and Kurokawa, M.

Bone Marrow Transplant 42 ( 2 ) 129 - 30 2008.07

ISSN 0268-3369

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Recurrence of primary hyperparathyroidism following spontaneous remission with intracapsular hemorrhage of a parathyroid adenoma

Kataoka, K., Taguchi, M., Takeshita, A., Miyakawa, M. and Takeuchi, Y.

J Bone Miner Metab 26 ( 3 ) 295 - 7 2008

ISSN 0914-8779

A few patients with primary hyperparathyroidism (PHPT) have a chance of spontaneous remission by either infarction of or hemorrhage into or around the parathyroid adenoma. In most cases, biochemical derangements associated with PHPT are permanently improved after spontaneous remission. Here we report a case with a recurrence of PHPT 4 months after spontaneous remission with acute intracapsular hemorrhage of parathyroid adenoma. In the literature, only two cases have been reported to have experienced a recurrence of clinical features of PHPT after infarction but not hemorrhage of parathyroid adenomas. Thus, the spontaneous remission of biological derangements in PHPT upon hemorrhage or infarction of parathyroid adenoma could be temporary. One should carefully observe such patients thereafter.

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