創薬標的分子のスクリーニングのために近位ビオチン標識法（BioID）を用いてEZH2欠損細胞におけるEZH1の会合分子を499個同定したが、さらにCRISPRスクリーニング［発表論文１］を行い、EZH2欠損との合成致死性を示す分子を58個同定した。両アッセイで共通にヒットした分子を選び、アドバイジーである学生（薬学科３年生 田中祐貴君）と共に共免疫沈降―ウェスタンブロッティングや共培養の細胞増殖アッセイにより結果を再現した。欠損変異体も作製し、共免疫沈降―ウェスタンブロッティングで結合領域を同定した。また、リコンビナントタンパク質を作製し、in vitroアッセイによりEZH1-PRC2と直接結合することを示した。ChIP-seqによりこの分子のKOでクロマチン191箇所のEZH1リクルート量が減少することを明らかにした［招待公演１；学会発表１］。
【発表論文】
1. Kaito S, Aoyama K, Oshima M, Tsuchiya A, Miyota M, Yamashita M, Koide S, Nakajima-Takagi Y, Kozuka-Hata H, Oyama M, Yabushita T, Ueno M, Hirao A, Tohyama K, Yamaguchi K, Furukawa Y, Kosako H, Yoshimi A, Goyama S, Nannya Y, Ogawa S, Agger K, Helin K, Koseki H, Nishiyama A, Nakanishi M, and Iwama A, Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization, Nat. Commun., 2024 Aug 28;15(1):7359
【招待公演】
1. 青山和正 (口頭) ポリコーム抑制複合体の制御機構と創薬標的分⼦の探索 千葉大学薬学部 分⼦⼼⾎管薬理学セミナー (千葉, 2025.1 )
【学会発表】
1. 田中祐貴（薬学科３年生）、青山和正、菊池敬、田口想、東孝輔、中澤洋介、多胡めぐみ 「残存H3K27me3」を標的としたEZH2機能喪失型MDSの治療法の探索 [Exploration of therapeutic strategies targeting “retained H3K27me3” for EZH2-deficient MDS] 第145回日本薬学会年会（福岡, 2025.3）
For the screening of drug target molecules, previously we performed proximity biotin labeling (BioID) and identified 499 interacting molecules of EZH1 in EZH2-deficient cells. Subsequently, we conducted CRISPR screening [Published Paper 1] and identified 58 molecules that exhibited synthetic lethality with EZH2 deficiency.Among the molecules commonly identified in both assays, we selected key candidates and, together with my advisee, Yuki Tanaka (a third-year pharmacy student), validated the results using co-immunoprecipitation–Western blotting and co-culture cell proliferation assays.We also generated deletion mutants and identified binding regions through co-immunoprecipitation–Western blotting.Furthermore, we produced recombinant proteins and demonstrated direct binding to EZH1-PRC2 through in vitro assays.Using ChIP-seq, we revealed that knockout (KO) of this molecule led to a reduction in EZH1 recruitment at 191 chromatin loci [Invited Lecture 1; Conference Presentation 1].
[Published Paper]
1.Kaito S, Aoyama K, Oshima M, Tsuchiya A, Miyota M, Yamashita M, Koide S, Nakajima-Takagi Y, Kozuka-Hata H, Oyama M, Yabushita T, Ueno M, Hirao A, Tohyama K, Yamaguchi K, Furukawa Y, Kosako H, Yoshimi A, Goyama S, Nannya Y, Ogawa S, Agger K, Helin K, Koseki H, Nishiyama A, Nakanishi M, and Iwama A, Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization, Nat. Commun., 2024 Aug 28;15(1):7359.
[Invited Lecture]
1.Kazumasa Aoyama (Oral Presentation), Regulatory Mechanisms of Polycomb Repressive Complex and Exploration of Drug Discovery Target Molecules, Chiba University Faculty of Pharmaceutical Sciences, Molecular Cardiovascular Pharmacology Seminar (Chiba, 2025.1).
[Conference Presentation]
1.Yuuki Tanaka (Third-year Pharmacy Student), Kazumasa Aoyama, Takashi Kikuchi, So Taguchi, Kosuke Higashi, Yosuke Nakazawa, Megumi Tago, Exploration of Therapeutic Strategies Targeting “Retained H3K27me3” for EZH2-Deficient MDS, The 145th Annual Meeting of the Pharmaceutical Society of Japan (Fukuoka, 2025.3).
|
