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

Theranostics. 2021 Jul 13;11(17):8172-8184. doi: 10.7150/thno.56737. eCollection 2021.


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

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacology*
  • Gene Expression / drug effects
  • Histone-Lysine N-Methyltransferase / chemistry
  • Histone-Lysine N-Methyltransferase / drug effects
  • Histone-Lysine N-Methyltransferase / genetics
  • Leukemia, Myeloid, Acute* / drug therapy
  • Leukemia, Myeloid, Acute* / genetics
  • Mice
  • Oncogene Proteins, Fusion* / chemistry
  • Oncogene Proteins, Fusion* / drug effects
  • Oncogene Proteins, Fusion* / genetics
  • Oncogenes / drug effects
  • Protein Interaction Domains and Motifs
  • Transcriptional Elongation Factors / chemistry
  • Transcriptional Elongation Factors / drug effects
  • Transcriptional Elongation Factors / genetics


  • AFF4 protein, human
  • Antineoplastic Agents
  • Oncogene Proteins, Fusion
  • Transcriptional Elongation Factors
  • DOT1L protein, human
  • Dot1l protein, mouse
  • Histone-Lysine N-Methyltransferase