Preclinical Characterization of a Potent, Selective Inhibitor of the Protein Methyltransferase DOT1L for Use in the Treatment of MLL-Rearranged Leukemia.
Authors: Pollock Roy M , Daigle Scott R , Therkelsen Carly A , Basavapathruni Aravind , Jin Lei , Allain Christina J , Klaus Christine R , Raimondi Alejandra , Scott Margaret Porter , Chesworth Richard , Moyer Mikel P , Copeland Robert A , Richon Victoria M , Olhava Edward J 
Published: October 2019

Abstract
Abstract 2379
The enzymatic activity of the protein methyltransferase (PMT) DOT1L has been shown to be a driver of cell proliferation in MLL-rearranged leukemia. Our group has previously reported the design of potent and selective aminonucleoside inhibitors of DOT1L [Daigle et al. (2011) Cancer Cell 20: 53–65; Basavapathruni et al. (2012) Chem. Biol. Drug Design, in press]. Structure-guided design, together with robust biochemical and biological assays, was used to optimize the potency, selectivity and pharmacological features of the aminonucleosides, resulting in the compound EPZ-5676. EPZ-5676 is an S-adenosyl methionine (SAM) competitive inhibitor of DOT11L that displays a Ki value of 80 pM and a drug-target residence time of > 24 hours. The compound is highly selective for DOT1L, demonstrating > 37,000-fold selectivity against all other PMTs tested. Crystallographic studies reveal that the high affinity, durable inhibition of DOT1L by EPZ-5676 has its origin in a conformational adaptation of the protein that attends inhibitor binding, extending the compound binding pocket to include novel recognition elements beyond the SAM binding active site. Treatment of leukemia cells with EPZ-5676 results in concentration- and time-dependent reduction of H3K79 methylation without effect on the methylation status of other histone sites. The reduction of H3K79 methylation leads to inhibition of key MLL target genes and selective, apoptotic cell killing in MLL-rearranged leukemia cells, but has minimal impact on non-rearranged cells. EPZ-5676 is highly soluble in aqueous solution and can thus be formulated for intravenous administration. The effective pharmacokinetic half-life of EPZ-5676 in systemic circulation has been measured to be 0.25 and 1.5 h in rats and dogs, respectively. A nude rat subcutaneous xenograft model of MLL-rearranged leukemia has been established. Continuous intravenous infusion of EPZ-5676 for 21 days in this model leads to dose-dependent anti-tumor activity. At the highest dose, complete tumor regressions are achieved with no regrowth for up to 32 days after the cessation of treatment (Figure 1).
Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. . / No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented.

Disclosures:
Pollock: Epizyme: Employment, Equity Ownership. Daigle:Epizyme: Employment, Equity Ownership. Therkelsen:Epizyme: Employment, Equity Ownership. Basavapathruni:Epizyme: Employment, Equity Ownership. Jin:Epizyme: Employment, Equity Ownership. Allain:Epizyme: Employment, Equity Ownership. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Raimondi:Epizyme: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Olhava:Epizyme: Employment.