Introduction

Chromosome translocations involving mixed lineage leukemia 1 (MLL1, also acknowledged as MLL/KMT2A) gene positioned at chromosome 11q23 is responsible for approximately 75% of acute leukemia in infants and 5–10% in children and adults. It is clinically described as either acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML). MLL1-rearranged (MLL1-r) ALL displays a poor prognosis with 5-year survival rates of <40%; similarly, MLL1-r AML also has low survival rates in younger as well as older patients (5-year of ~50%).

MLL is commonly fused with transcription cofactors AF4, AF9, and ENL. AF9 and ENL possess a YEATS and AHD domain, allowing them to bind with other partner proteins to execute the cell proliferation process in MLL. The AHD domain of AF9/ENL binds to AF4/AFF4/histone-H3 lysine-79 (H3K79) methyltransferase DOT1L. The formation of AF9/ENL/AF4/AFF4-comprising super elongation complexes (SEC) and the catalytic activity of DOT1L are crucial for MLL-rearranged leukemia. Protein-protein interactions (PPI) between AF9 AHD and DOT1L/AF4/AFF4 appear to be potential drug targets for MLL-r leukemia.

Methods

Compound screening followed by a medicinal chemistry strategy was applied to discover inhibitors of such PPIs that were examined for their biological activities against MLL-rearranged leukemia and other cancer cells. This strategy bestowed numerous potent inhibitors of AF9/ENL-DOT1L.

Results

Compound-1 was found to be an effective small-molecule inhibitor of the AF9/ENL-DOT1L/ AF4/AFF4 interaction with IC50s of 0.9-3.5 µM. Inhibition of the PPIs significantly reduced SEC and DOT1L-mediated H3K79 methylation in the leukemia cells. Gene profiling displays that this compound significantly suppressed the gene signatures related to onco-MLL, DOT1L, HoxA9, and Myc. Additionally, this compound inhibited the proliferation of onco-MLL- or Myc-driven cancer cells. Further, we modified this lead molecule to develop more effective PPI inhibitors using a medicinal chemistry approach. Following this approach, several more potent PPI inhibitors of AF9 AHD and DOT1L/AF4/AFF4 have been developed with effective cytotoxicity action against numerous leukemia cell lines.  

Conclusion

The AF9/ENL-DOT1L/AF4/AFF4 interactions are proven to be an effective anticancer target, and compound-1 is a valuable in vivo probe for biological studies. Further lead modification work bestowed numerous potent PPI inhibitors with promising cancer action against several leukemia cell lines.