Analysis of dose-dependent RAS mediated survival signalings

• Project/Area Number: 18K06953
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 49010:Pathological biochemistry-related
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: Kurata Morito 東京医科歯科大学, 大学院医歯学総合研究科, 講師 (40451926)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2020: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2019: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2018: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: がん遺伝子 / CRISPR screening / RAS / NRAS / 癌遺伝子 / CRISPR library / CRISPR activation / 分子標的薬 / スクリーニング / CRISPR

Outline of Final Research Achievements

Mutations in RAS family are observed in 6-96% of cancer. However, the role of the magnitude of RAS activation in proliferation and cell cycle progression is not fully understood. To define the role of oncogenic NRAS dose in malignant behavior, we used a genetically engineered THP-1 cell line where the expression of NRAS-G12V is under the control of a doxycycline-responsive element. We established modified cell lines, named B11. To identify whole downstream of NRAS signals, CRISPR libraries were introduced into B11 and removing doxycycline to collect clones that can proliferate without NRAS signaling. As a result, 21 from the activation library. NRAS activation with Dox can induce the mRNA expression for DOHH and TAF6 in B11. DOHH and TAF6 are newly identified as a downstream of NRAS signaling

Academic Significance and Societal Importance of the Research Achievements

RASシグナルの下流として考えられているRAF-MEK-ERK-pathwayを阻害することで、一部の癌細胞では治療効果が期待できる。しかしながら、すべてのRAS変異癌細胞を制御する包括的な治療法の確立には至っていない。網羅的な解析を行えるCRISPR library によるスクリーニングを使用することで、NRASのシグナル経路に関わる新たな遺伝子を同定した。今後、更に詳細な検討が必要とされるものの、RAS経路の全貌を明らかにできる手法の確立に成功したと考えられる。

Research Products

• [Int'l Joint Research] University of Minnesota/Masonic cancer center/Department of Genetics, Cell Biology(米国)
• [Int'l Joint Research] David Largaespasa/University of Minnesota/Masonic cancer center(米国)
• [Int'l Joint Research] Zohar Sachs/University of Minnesota/Masonic cancer center(米国)
• [Journal Article] CRISPR screening identifies M1AP as a new MYC regulator with a promoter-reporter system (2020)
  • Author(s): Yamamoto Akiko、Kurata Morito、Onishi Iichiroh、Sugita Keisuke、Matsumura Miwa、Ishibashi Sachiko、Ikeda Masumi、Yamamoto Kouhei、Kitagawa Masanobu
  • Journal Title: PeerJ Volume: 8 Pages: e9046-e9046
  • DOI: 10.7717/peerj.9046
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The expression of MYC is strongly dependent on the circular PVT1 expression in pure Gleason pattern 4 of prostatic cancer (2020)
  • Author(s): Umemori Miyaka、Kurata Morito、Yamamoto Akiko、Yamamoto Kouhei、Ishibashi Sachiko、Ikeda Masumi、Tashiro Kojiro、Kimura Takahiro、Sato Shun、Takahashi Hiroyuki、Kitagawa Masanobu
  • Journal Title: Medical Molecular Morphology Volume: Jan 13 Online ahead of print Issue: 3 Pages: 156-167
  • DOI: 10.1007/s00795-020-00243-9
  • Peer Reviewed
• [Journal Article] CRISPR/Cas9 screeningとその応用 (特集 新しい解析技術の展開) The applications of CRISPR/Cas9 screening (2019)
  • Author(s): 大西威一郎 倉田盛人
  • Journal Title: 臨床免疫・アレルギー科 Clinical immunology & allergology Volume: 72(3) Pages: 287-292
• [Journal Article] Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays (2018)
  • Author(s): Kurata Morito、Wolf Natalie K.、Lahr Walker S.、Weg Madison T.、Kluesner Mitchell G.、Lee Samantha、Hui Kai、Shiraiwa Masano、Webber Beau R.、Moriarity Branden S.
  • Journal Title: PLOS ONE Volume: 13 Issue: 9 Pages: e0198714-e0198714
  • DOI: 10.1371/journal.pone.0198714
  • Peer Reviewed / Open Access
• [Presentation] Dose-dependentNRASG12V-mediated signaling controls cell cycle progression and leukemogenic signaling in a CRISPR/Cas9-modified human AML cell line (2020)
  • Author(s): Kurata Morito、Antony Marie L.、Noble Klara E.、Rathe Susan K.、Hirakouchi Haruka、Yamamoto Kouhei、Kitagawa Masanobu、Sachs Zohar、Largaespada David A.
  • Organizer: AACR Annual Meeting 2020
  • Int'l Joint Research
• [Presentation] NRAS発現量依存的な細胞老化シグナルの解析 (2019)
  • Author(s): 倉田盛人 山本浩平 Rathe Susan David Largaespada 北川昌伸
  • Organizer: 第108回日本病理学会総会 東京
• [Presentation] Discovery of cancer genes and pathways operative in PI3K activated mammary cancer (2019)
  • Author(s): Morito Kurata, Kouhei Yamamoto, Masanobu kitagawa, Jingmin Shu, Emily A. Pope, Wenlin Yuan, Wendy A. Hudson, Aaron L. Sarver, Nuri A. Temiz, Mark Sokolowski, Zora Modrusan, Eric Stawski, Stephen Durinck, Sekar Seshigiri, David A. Largaespada
  • Organizer: 第78回日本癌学会総会 大阪
• [Presentation] NRAS発現調整可能細胞株を用いたシグナリングの解析 (2018)
  • Author(s): 倉田盛人, 山本浩平, Noble-Orcutt Klara, 山本阿紀子, Sachs Zohar, David Largaespada, 北川昌伸
  • Organizer: 第107回日本病理学会総会
• [Presentation] NRAS発現調整可能システムとシグナルの解析 (2018)
  • Author(s): 倉田盛人, 山本浩平, 北川昌伸, Noble-Orcutt Klara、Susan K. Rathe, Alexandra Hilles heim, Zain Qarni, Zohar Sachs, David La rgaespada
  • Organizer: 第77回日本癌学会学術総会