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3930 DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs or DNA Hypomethylating Agents in MLL-Rearranged Leukemia Cells

Program: Oral and Poster Abstracts
Session: 615. Acute Myeloid Leukemia: Therapy, excluding Transplantation: Poster III
Monday, December 9, 2013, 6:00 PM-8:00 PM
Hall E (Ernest N. Morial Convention Center)

Christine R Klaus*, Scott R. Daigle, PhD*, Dorothy Iwanowics*, L. Danielle Johnston*, Carly A Therkelsen*, Jesse J Smith, PhD*, Mikel P Moyer, PhD*, Robert A. Copeland, PhD, Edward J Olhava, PhD*, Margaret Porter Scott, PhD*, Roy M. Pollock, PhD* and Alejandra Raimondi*

Epizyme, Inc., Cambridge, MA

EPZ-5676 is a small molecule inhibitor of the histone methyltransferase DOT1L that is currently under clinical investigation as a potential therapy for acute leukemias bearing MLL-rearrangements. Gene knockout and small molecule inhibitor studies have demonstrated that DOT1L is required for MLL-fusion protein–mediated leukemogenesis in model systems. In preclinical studies EPZ-5676 promoted cell killing of acute leukemia lines bearing MLL translocations in vitro while sparing those without MLL gene translocations and also caused sustained tumor regressions in a rat xenograft model of MLL-rearranged leukemia [Daigle et al. Blood 2013]. To support potential future clinical scenarios, we evaluated the activity of EPZ-5676 in combination with current standard of care agents for acute leukemias as well as other chromatin modifying drugs in cell proliferation assays with three human acute leukemia cell lines; Molm-13 (MLL-AF9 expressing acute myeloid leukemia (AML)), MV4-11 (MLL-AF4 expressing acute biphenotypic leukemia cell line) and SKM-1 (non-MLL-rearranged AML). We established a high density combination platform suitable for testing the anti-proliferative activity of a complete titration matrix of two agents with multiple replicate points to enable generation of statistically meaningful results.  This platform was used to evaluate the anti-proliferative effects of EPZ-5676 combinations tested in a co-treatment model in which the second agent was added along with EPZ-5676 at the beginning of the assay, or in a pre-treatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. The drug combination analysis was performed using the Chou-Talalay method [Chou TC Pharmacological Reviews 2006].  Graphs representing values of combination index (CI) versus Fractional effect (Fa) known as Fa-CI plots were generated and synergy was evaluated.  Drug synergy was statistically defined by CI values less than 1, antagonism by CI >1 and additive effect by CI equal to 1.

We found that EPZ-5676 acts synergistically with the AML standard of care agents cytarabine or daunorubicin in Molm-13 and MV4-11 MLL-rearranged cell lines. However, in the non-rearranged SKM-1 cell line EPZ-5676 had no effect alone and did not act synergistically with cytarabine or daunorubicin.

Moreover, a persistent combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the standard of care agents (Figure 1), suggesting that EPZ-5676 sets up a durable altered chromatin state that enhances the effect of chemotherapeutic agents in MLL-rearranged cells. We are currently exploring the mechanism of action of this synergy in more detail.

Our evaluation of EPZ-5676 in conjunction with other chromatin modifying drugs also revealed a consistent combination benefit including synergy with DNA hypomethylating agents.

In summary, our results indicate that EPZ-5676 is highly efficacious as a single agent and is synergistic with other anticancer agents including AML standard of care drugs and DNA hypomethylating agents in MLL-rearranged cells.

Figure 1. Fa-CI plots show that EPZ-5676 and cytarabine act synergistically to induce an antiproliferative effect in the Molm-13 cell line in a pre-treatment model. (A) Ten-day continuous dosing of EPZ-5676 with addition of cytarabine at day 7 showed a range of fractional effects with CI values <1 denoting synergy. (B) EPZ-5676 was removed at day 7 prior to the addition of cytarabine showing durable combination benefit.

Disclosures: Klaus: Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Daigle: Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Iwanowics: Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Johnston: Epizyme, Inc: Employment, Equity Ownership, Stock Options Other. Therkelsen: Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Smith: Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Moyer: Epizyme, Inc.: Employment, Equity Ownership, Stock Options Other. Copeland: Epizyme Inc. : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Mersana: Membership on an entity’s Board of Directors or advisory committees. Olhava: Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Porter Scott: Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Pollock: Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Raimondi: Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, Stock Options Other.

*signifies non-member of ASH