Development of ChIPseq method using pathological specimens and identification of disease-specific super-enhancer

• Project/Area Number: 19K22542
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 49:Pathology, infection/immunology, and related fields
• Research Institution: Saitama Medical University
• Principal Investigator: Yamada Taketo 埼玉医科大学, 医学部, 教授 (60230463)
• Co-Investigator(Kenkyū-buntansha): 林 睦 慶應義塾大学, 医学部(信濃町), 助教 (60327575) ; 西田 浩子 慶應義塾大学, 医学部(信濃町), 助教 (80317130)
• Project Period (FY): 2019-06-28 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2020: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2019: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: スーパーエンハンサー / ホルマリン固定パラフィン包埋組織 / クロマチン免疫沈降塩基配列決定法 / モノクローナル抗体 / がん / FFPE / 病理検体 / ChIPseq法

Outline of Research at the Start

疾患特異的スーパーエンハンサー(SE)の同定や構造解析は、エピジェネティクス研究のみならず、SEを治療標的とする上でも重要である。本研究は、クロマチン免疫沈降塩基配列決定法(ChIPseq)で鍵となる変性架橋蛋白質に対するモノクローナル抗体の確立を通じてDNA蛋白質複合体の抽出やDNA断片化の至適化を進め、ホルマリン固定パラフィン（FFPE）検体での高感度ChIPseq法を開発し疾患特異的SEを同定するものである。本法の確立は、疾患について詳細な臨床情報や病理診断があり、また膨大な蓄積がある病理FFPE検体を用いるため、疾患特異的SEの同定やそれに基づくバイオマーカーの探索が可能となる。

Outline of Final Research Achievements

Super-enhancer (SE), which is composed of large clusters of enhancers densely loaded with mediator complex, transcription factors, and chromatin regulators, drive high expression of genes implicated in disease-specific manner. In this study, the novel method for SE identification in pathology archives as formaldehyde-fixed paraffin-embedded(FFPE) materials was developed by chromatin immunoprecipitation sequencing (ChIPseq) using new monoclonal antibodies against degenerated antigens such as H3K9, H3K27, BRD4 and MED1 with high affinity to antigens in FFPE samples. This improved ChIPseq protocol was used for the validation of disease-specific epigenetic biomarkers in FFPE human samples. As a result, some biomarkers for SE were detected in colon carcinoma cells using FFPE-ChiPseq.

Academic Significance and Societal Importance of the Research Achievements

疾患特異的SE解析はSEを治療標的とする上で重要である。SE解析は、生細胞で行われFFPE検体を用いた解析は、2010年にFanelli、2016年にCejasが新法を開発したが、その後の報告はなく汎用性の面で新法が必要と考えた。そこで変性SE構成分子に対する新規MoAbが必須と考え、変性架橋蛋白質に対するMoAbの確立を突破口として、DNA蛋白質複合体の抽出やDNA断片化の至適化を進めることでFFPE高感度ChIPseq法の開発を行った。本法は疾患の詳細な臨床情報や病理診断があり、膨大な蓄積がある病理FFPE検体を用いた疾患特異的SEの同定やそれに基づくバイオマーカーの探索に有用と考える。

Research Products

• [Journal Article] Serum soluble CD26/DPP4 titer variation is a potential prognostic biomarker in cancer therapy with a humanized anti-CD26 antibody. (2021)
  • Author(s): Kaneko Y, Hatano R, Hirota N, Isambert N, Trillet-Lenoir V, You B, Alexandre J, Zalcman G, Valleix F, Podoll T, Umezawa Y, Takao S, Iwata S, Hosono O, Taguchi T, Yamada T, Dang NH, Ohnuma K, Angevin E, Morimoto C.
  • Journal Title: Biomark Res Volume: 9 Issue: 1 Pages: 21-21
  • DOI: 10.1186/s40364-021-00273-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Arf1 and Arf6 Synergistically Maintain Survival of T Cells during Activation (2020)
  • Author(s): Sumiyoshi Mami、Kotani Yui、Ikuta Yuki、Suzue Kazutomo、Ozawa Madoka、Katakai Tomoya、Yamada Taketo、Abe Takaya、Bando Kana、Koyasu Shigeo、Kanaho Yasunori、Watanabe Toshio、Matsuda Satoshi
  • Journal Title: The Journal of Immunology Volume: 206 Issue: 2 Pages: 366-375
  • DOI: 10.4049/jimmunol.2000971
  • Peer Reviewed / Open Access
• [Journal Article] Single-cell atlas of colonic CD8+ T-cells in ulcerative colitis. (2020)
  • Author(s): Corridoni D, Antanaviciute A, Gupta T, Fawkner-Corbett D, Aulicino A, Jagielowicz M, Parikh K, Repapi E, Taylor S, Ishikawa D, Hatano R, Yamada T, Xin W, Slawinki H, Bowden R, Napolitani G, Brain O, Morimoto C, Koohy H, Simmons A.
  • Journal Title: Nat Med Volume: 26 Issue: 9 Pages: 1480-1490
  • DOI: 10.1038/s41591-020-1003-4
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Development of novel monoclonal antibodies with specific binding affinity for denatured human CD26 in formalin-fixed paraffin-embedded and decalcified specimens (2019)
  • Author(s): Hatano R, Yamada T, Madokoro H, Otsuka H, Komiya E, Itoh T, Narita Y, Iwata S, Yamazaki H, Matsuoka S, Dang NH, Morimoto C
  • Journal Title: PLoS ONE Volume: 14 Issue: 6 Pages: e0218330-e0218330
  • DOI: 10.1371/journal.pone.0218330
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Monoclonal Antibody Therapies in Multiple Myeloma: A Challenge to Develop Novel Targets (2019)
  • Author(s): Nishida H, Yamada T
  • Journal Title: Journal of Oncology Volume: 2019 Pages: 6084012-6084012
  • DOI: 10.1155/2019/6084012
  • Peer Reviewed
• [Journal Article] Novel antibody-drug conjugate with anti-CD26 humanized monoclonal antibody and transcription factor IIH (TFIIH) inhibitor, triptolide, inhibits tumor rrowth via impairing mRNA synthesis (2019)
  • Author(s): Hayashi M, Madokoro H, Yamada K, Nishida H, Morimoto C, Sakamoto M, Yanagawa H, Yamada T
  • Journal Title: Cancers Volume: 11 Issue: 8 Pages: 1138-1138
  • DOI: 10.3390/cancers11081138
  • Peer Reviewed / Open Access
• [Presentation] Anti-CD26 Humanized Monoclonal Antibody Conjugated to Triptolide Inhibits Tumor Cell Growth via Transportation into Nucleus and Impaired RNA Polymerase II (2019)
  • Author(s): Hayashi M,Madokoro H, Yamada T
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] Anti-CD26 humanized antibody-triptolide conjugate trasnports into nucleus and inhibits RNA polymerase II activity (2019)
  • Author(s): 山田 健人、坂元 亨宇、森本 幾夫、林 睦
  • Organizer: 第78回日本癌学会総会（
• [Presentation] Humanized anti-CD26 monoclonal antibody targets clonogenic side population cells in multiple myeloma (2019)
  • Author(s): Nishida H, Yamada T
  • Organizer: 第81回日本血液学会学術集会