Korean Journal of Clinical Laboratory Science (대한임상검사과학회지)

Korean Society for Clinical Laboratory Science (대한임상검사과학회)
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Synergistic Effects of Combined PROTAC-based EZH2 Degrader and METTL3 Inhibitor in Burkitt's Lymphoma

버킷림프종에서 EZH2 분해제와 METTL3 억제제 병용의 상승 항암 효과

  • Minseo YU (Department of Biomedical Laboratory Science, Inje University) ;
  • Ra Eun KIM (Department of Biomedical Laboratory Science, Inje University) ;
  • Yurim JEONG (Department of Biomedical Laboratory Science, Inje University) ;
  • Hyewon JANG (Department of Biomedical Laboratory Science, Inje University) ;
  • Se Been KIM (Department of Biomedical Laboratory Science, Inje University) ;
  • Jung-Yeon LIM (Department of Biomedical Laboratory Science, Inje University)
  • 유민서 (인제대학교 임상병리학과) ;
  • 김라은 (인제대학교 임상병리학과) ;
  • 정유림 (인제대학교 임상병리학과) ;
  • 장혜원 (인제대학교 임상병리학과) ;
  • 김세빈 (인제대학교 임상병리학과) ;
  • 임정연 (인제대학교 임상병리학과)
  • Received : 2024.07.26
  • Accepted : 2024.09.02
  • Published : 2024.09.30
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Abstract

EZH2 is a methyltransferase that is a critical target for lymphoma treatment. However, it is not yet widely used in clinical settings. PROteolysis TArgeting Chimeras (PROTACs) represent a novel therapeutic strategy aimed at eliminating proteins that have been a challenging target using conventional small molecules. In our previous research, we compared the small molecules-based EZH2 inhibitor used in clinical settings with a PROTAC-based EZH2 degrader. We found that the PROTAC-based degrader was significantly more effective. Building on this, we further investigated the effects of combining the PROTAC-based EZH2 degrader (dEZH2) with a METTL3 inhibitor, both of which have demonstrated effectiveness in inhibiting cell proliferation and inducing apoptosis in Burkitt's lymphoma. Using the CCK-8 assay, we found that both drugs, alone and in combination, significantly inhibited Daudi and Ramos cell growth in a dose-dependent manner. The combined treatment markedly suppressed cell proliferation and induced apoptosis, as confirmed by Annexin V/PI staining. Our results revealed G2/M phase arrest with a significant decrease in the G0/G1 phase by flow cytometry. Our study also showed increased levels of cleaved PARP, cleaved caspase-3, tumor protein p53 (TP53), and PUMA using the western blot technique, indicating enhanced p53-dependent apoptosis. Our findings suggest that the combination therapy of dEZH2 and iMETTL3 could be a promising approach in the treatment of Burkitt's lymphoma.

PROteolysis TArgeting Chimeras (PROTACs)은 기존의 저분자(small moleule)로는 타겟팅이 어려웠던 단백질을 제거하기 위한 새로운 치료 전략을 제공한다. EZH2는 림프종 치료의 중요한 타겟인 메틸트랜스퍼라제(methyltransferase)이지만, 임상에서 널리 사용되지 않는다. 이전 연구에서 우리는 임상에서 사용되는 EZH2 억제제와 PROTAC 기반의 EZH2 분해제를 비교했으며, PROTAC 기반 분해제가 훨씬 더 효과적임을 확인했다. 이를 바탕으로, 우리는 PROTAC 기반의 EZH2 분해제와 기존 림프종 치료제인 METTL3 억제제를 병용하여 버킷림프종 세포의 증식과 세포 사멸에 미치는 영향을 조사했다. CCK-8 분석을 통해 두 약물이 단독 및 병용 처리 시 농도 의존적으로 Daudi 및 Ramos 세포 성장을 유의미하게 억제하는 것을 확인했다. 병용 치료는 세포 증식을 현저히 억제하고, annexin V/PI 염색을 통해 세포 사멸을 유도하는 것으로 확인되었다. 유세포 분석에서는 G0/G1 단계의 감소와 함께 G2/M 단계에서의 세포 주기 정지를 보였다. 웨스턴 블럿(western blot) 분석에서는 cleaved PARP, cleaved caspase-3, TP53 및 PUMA의 증가된 수준을 나타내어 p53 의존적인 세포 사멸이 강화됨을 증명했다. 우리는 이 연구 결과가 이 병용 요법이 버킷림프종 치료에 유망한 접근법임을 제안한다.

Keywords

Acknowledgement

This work was supported by grant from Inje University, 2023 (No. 20230017).

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