Theranostics 2021; 11(17):8172-8184. doi:10.7150/thno.56737

Research Paper

Small-molecule inhibitor of AF9/ENL-DOT1L/AF4/AFF4 interactions suppresses malignant gene expression and tumor growth

Fangrui Wu1,2,*, Shenyou Nie1,*, Yuan Yao1,*, Tong Huo1,*, Xin Li1, Xiaowei Wu1, Jidong Zhao1, Yi-Lun Lin1, Yinjie Zhang1, Qianxing Mo3, Yongcheng Song1,2,✉

1. Department of Pharmacology and Chemical Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.
2. Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.
3. H. Lee Moffitt Cancer Center & Research Institute, 12902 USF Magnolia Drive, Tampa, FL 33612, USA.
* These authors contributed equally.

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Citation:
Wu F, Nie S, Yao Y, Huo T, Li X, Wu X, Zhao J, Lin YL, Zhang Y, Mo Q, Song Y. Small-molecule inhibitor of AF9/ENL-DOT1L/AF4/AFF4 interactions suppresses malignant gene expression and tumor growth. Theranostics 2021; 11(17):8172-8184. doi:10.7150/thno.56737. Available from https://www.thno.org/v11p8172.htm

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Abstract

Chromosome translocations involving mixed lineage leukemia (MLL) gene cause acute leukemia with a poor prognosis. MLL is frequently fused with transcription cofactors AF4 (~35%), AF9 (25%) or its paralog ENL (10%). The AHD domain of AF9/ENL binds to AF4, its paralog AFF4, or histone-H3 lysine-79 (H3K79) methyltransferase DOT1L. Formation of AF9/ENL/AF4/AFF4-containing super elongation complexes (SEC) and the catalytic activity of DOT1L are essential for MLL-rearranged leukemia. Protein-protein interactions (PPI) between AF9/ENL and DOT1L/AF4/AFF4 are therefore a potential drug target.

Methods: Compound screening followed by medicinal chemistry was used to find inhibitors of such PPIs, which were examined for their biological activities against MLL-rearranged leukemia and other cancer cells.

Results: Compound-1 was identified to be a novel small-molecule inhibitor of the AF9/ENL-DOT1L/AF4/AFF4 interaction with IC50s of 0.9-3.5 µM. Pharmacological inhibition of the PPIs significantly reduced SEC and DOT1L-mediated H3K79 methylation in the leukemia cells. Gene profiling shows compound-1 significantly suppressed the gene signatures related to onco-MLL, DOT1L, HoxA9 and Myc. It selectively inhibited proliferation of onco-MLL- or Myc-driven cancer cells and induced cell differentiation and apoptosis. Compound-1 exhibited strong antitumor activity in a mouse model of MLL-rearranged leukemia.

Conclusions: The AF9/ENL-DOT1L/AF4/AFF4 interactions are validated to be an anticancer target and compound-1 is a useful in vivo probe for biological studies as well as a pharmacological lead for further drug development.

Keywords: MLL-rearranged leukemia, Super elongation complexes, Protein-protein interaction, Small-molecule inhibitor, Cancer therapeutics