Selective Killing of Mixed Lineage Leukemia Cells by a Potent Small-Molecule DOT1L Inhibitor

Abstract 780

Rearrangements of the mixed lineage leukemia (MLL) gene on chromosome 11q23 are found in over 70 % of infant leukemias, and approximately 10% of adult acute myeloid leukemias (AML). Patients with MLL-rearranged leukemias have aggressive disease with a poor prognosis. Recent studies suggest that DOT1L, a histone methyltransferase that methylates lysine 79 of histone H3 (H3K79), plays a fundamental role in the development and maintenance of this genetically defined subset of leukemia. Rearrangements of the MLL gene result in the expression of MLL-fusion proteins that gain the ability to recruit DOT1L to chromatin. This leads to aberrantly high levels of H3K79 methylation and gene expression at specific genomic loci, including HOXA9 and MEIS1 that are thought to promote leukemogenesis. These findings, together with studies demonstrating a key role for DOT1L in propagating the transforming activity of MLL-fusion proteins in model systems, support the development of inhibitors of this enzyme as targeted therapeutics for patients bearing MLL-rearranged leukemias. To this end, we have used mechanism-guided design to identify EPZ01, the first small molecule DOT1L inhibitor. This compound is a potent and specific inhibitor of DOT1L methyltransferase activity with a Ki of ~ 400 pM in biochemical assays. EPZ01 acts as a competitive inhibitor with the co-factor S-adenosyl-methionine (SAM), and demonstrates greater than 500-fold selectivity for DOT1L over other lysine and arginine histone methyltransferases. Incubation of MLL-rearranged leukemic cell lines with EPZ01 leads to a dramatic decrease in cellular H3K79 methylation but does not affect the methylation of other histone residues, including H3K4, H3K27, H3K36 and H3K9. Analysis of the effects of EPZ01 on the proliferation of a panel of acute lymphoid leukemia (ALL) or AML-derived human MLL-rearranged cell lines including SEMK2, MV4-11, RS4;11, MOLM-13 and THP-1, and non-rearranged leukemia cell lines including HL-60, Jurkat and U937, reveals anti-proliferative activity that is remarkably selective for cell lines bearing the MLL-rearrangement. EC50 values for inhibition of proliferation by EPZ01 are in the nanomolar to low micromolar range for all MLL-rearranged lines tested. In contrast, EPZ01 shows little or no effect on the proliferation of cells lacking an MLL-rearrangement despite an equal decrease in cellular H3K79 methylation. A more detailed analysis of the cellular effects of EPZ01 in MLL-rearranged cell lines reveals that treatment with the inhibitor causes a decrease in mRNA expression of known MLL-fusion target genes including HOXA9 and MEIS1, cell cycle arrest in G0/G1, an increase in expression of differentiation markers in MLL-rearranged AML cells and death by apoptosis. We are currently evaluating the effects of EPZ01 and related compounds in in vivo models of MLL-rearranged leukemia where preliminary results indicate that we are able to achieve inhibition of DOT1L activity. EPZ01 therefore represents the first example of a histone methyltransferase inhibitor that selectively kills tumor cells bearing a defined genetic lesion. These data provide compelling validation for the development of DOT1L inhibitors as targeted therapeutics for MLL-rearranged leukemias and we are currently working towards this goal.