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Preclinical data have suggested that small molecule inhibitors for the histone methyltransferase EZH2 represent potential new treatment modalities for Non-Hodgkin lymphomas (NHL) expressing EZH2 change of function mutations. Our group has previously reported that selective inhibition of EZH2 results in specific killing of lymphoma cells bearing EZH2 mutations in vitro and in vivo, with minimal effects on non-mutant lymphoma cells [Knutson et al. Nature Chemical Biology 2012; Keilhack et al. Blood (ASH Annual Meeting Abstracts) 2012, 120, Abstract 3712]. Since epigenetic changes have been suggested to be involved in resistance of cancer cells to many anticancer agents, we studied EPZ-6438 (or E7438), our clinical stage EZH2 inhibitor, in combination with standard of care agents for NHL, second line therapies or targeted therapies that are being explored in this indication. With continuous exposure to EPZ-6438, cell-based assays of two different EZH2 mutant cell lines demonstrated combination benefits with all components of the CHOP chemotherapy regime, second line therapies but also with several targeted therapies (for instance other epigenetic drugs, PI3K pathway or other inhibitors). These effects were not observed in an EZH2 wild type lymphoma cell line of the activated B cell type. Strong combination benefit with CHOP was also observed in two different EZH2 mutant xenograft models. For instance, in the SUDHL6 Y646N xenograft model neither EPZ-6438 nor CHOP chemotherapy alone induced a significant antitumor effect, yet their combination produced durable tumor regressions even after cessation of dosing (figure 1). Importantly, this effect was preserved when doxorubicin was omitted from the CHOP chemotherapy regime in a third study with another EZH2 mutant xenograft model. Subsequently we showed that glucocorticoid receptor agonism may be a key mechanism of the combination benefit observed with CHOP, as the antiproliferative effect of EPZ-6438 was enhanced by either prednisolone or dexamethasone alone, in several EZH2 mutant lymphoma cell lines (in vitro). Taken together these data suggest that the single agent activity of EPZ-6438 in EZH2 mutant NHL may be further enhanced and expanded through rational combination strategies. Disclosures: Johnston: Epizyme: Employment, Equity Ownership, stock options Other. Knutson:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Warholic:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Klaus:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Wigle:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Iwanowicz:Epizyme, Inc.: Employment, Equity Ownership, stock options Other. Littlefield:Eisai Inc.: Employment. Porter Scott:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, 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, Patents & Royalties, stock options Other; Mersana: Membership on an entity’s Board of Directors or advisory committees. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Kuntz:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Keilhack:Epizyme, Inc.: Employment, Equity Ownership, Patents & Royalties, stock options Other. Raimondi:Epizyme, Inc: Employment, Equity Ownership, Patents & Royalties, stock options Other.

Abstract 3712 The coupled enzymatic activity of wild-type and mutant EZH2 results in hyper-trimethylation of histone H3 lysine 27 (H3K27), which drives lymphomagenesis in heterozygous patients bearing the EZH2 mutations. Our group has previously reported that selective inhibition of EZH2 in cell culture results in selective killing of lymphoma cells bearing EZH2 mutations, with minimal effect on non-mutant lymphoma cells, suggesting that EZH2 enzymatic activity is a required driver of proliferation in the mutant-bearing cells [Knutson et al. (2012) Nature Chemical Biology, in press]. Through iterative medicinal chemistry we have developed a selective inhibitor of EZH2 with good pharmacological properties, E7438. E7438 binds to the enzyme in a manner competitive with S-adenosyl methionine (SAM) and a Ki for wild-type EZH2 of 2.5 ± 0.5 nM. The compound potently inhibits all known mutants of EZH2 that have been identified in non-Hodgkin lymphoma (NHL) patient samples. E7438 displays about 35-fold less activity against the closely related enzyme EZH1, and is >4500-fold selective with respect to all other protein methyltransferases tested. Lymphoma cells treated with E7438 display concentration- and time-dependent loss of H3K27 methylation with no effect on the methylation status of any other histone sites. The loss of H3K27 methylation results in selective killing of EZH2 mutant-bearing lymphoma cell lines. E7438 displays good oral bioavailability and pharmacokinetic properties. Various EZH2 mutant-bearing human lymphoma tumors were subcutaneously implanted in nude, SCID or NSG mice. Oral administration of E7438 to tumor bearing mice resulted in significant anti-tumor activity. The responses ranged from dose-dependent tumor growth inhibition to complete and sustained regressions. For example, KARPAS422 tumors in nude mice showed complete tumor elimination after 28 days of dosing, with mice remaining tumor free for up to 90 days after treatment cessation. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Mice and rats tolerated E7438 administration well at doses representing high multiples of doses that show antitumor activity in mice. Activity against the EZH2 target in both species was demonstrated by dose-dependent diminution of H3K27me3 levels, assessed by ELISA, in samples of tumor, bone marrow, skin and peripheral blood mononuclear cells (PBMCs). Highly sensitive detection of existing H3K27me3 signal could also be observed with this ELISA assay in drug-naïve samples of human PBMCs. The ability to measure dose-dependent changes in H3K27me3 levels in skin and PBMCs portends the use of signal from these surrogate tissues as a non-invasive pharmacodynamics biomarker in human clinical trials. Disclosures: Keilhack: Epizyme Inc.: Employment, Equity Ownership. Yokoi:Eisai Co., Ltd.: Employment. Knutson:Epizyme Inc.: Employment, Equity Ownership. Wigle:Epizyme Inc.: Employment, Equity Ownership. Warholic:Epizyme Inc.: Employment, Equity Ownership. Kawano:Eisai Co., Ltd.: Employment. Minoshima:Eisai Co., Ltd.: Employment. Huang:Eisai Inc.: Employment. Kuznetsov:Eisai Inc.: Employment. Kumar:Eisai Inc.: Employment. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Allain:Epizyme Inc.: Employment, Equity Ownership. Raimondi:Epizyme Inc.: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Uenaka:Eisai Co., Ltd.: Employment. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Kuntz:Epizyme Inc.: Employment, Equity Ownership.

Juvenile Myelomonocytic Leukemia (JMML) is a clonal hematopoietic disorder of childhood that is principally characterized by proliferation of cells of the granulocytic and monocytic lineages. Previous studies showed that Ras or Nf1 mutations contribute to ~65% cases of JMML. More recently, mutations in the protein tyrosine phosphatase Shp2 (PTPN11) have been found in ~35% of sporadic JMML, and at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and childhood B-cell acute lymphoblastic leukemia (B-ALL). Interestingly, germline mutations of Shp2 cause the autosomal dominant genetic disorder Noonan Syndrome (NS) and NS patients may have an increased incidence of JMML. Nearly all disease-associated Shp2 mutations affect residues known to control catalytic activity. NS and JMML mutations can involve the same residues, but when they do, the latter are less conservative, suggesting that they may be more activated. We have compared the biochemical and biological effects of NS and JMML mutations. When produced as recombinant proteins in bacteria, nearly all NS mutations studied showed increased PTP activity. However, the JMML mutations E76K and D61Y have the highest activity, resembling the fully active mutant E76A that we defined earlier. Retroviral-mediated expression of E76K or D61Y, but not wild type (WT) Shp2 in murine bone marrow (BM) cells results in cytokine-independent myeloid colony outgrowth, as well as hypersensitivity to both IL-3 and GM-CSF. Notably, NS- associated mutants (e.g., D61G or N308D) also could transform BM cells to factor-independence, but yielded far fewer colonies. Transformation by the leukemia-associated E76K mutant required the phosphatase activity and intact FLVRE motifs in the Shp2 SH2 domains. Gab2, a major Shp2 SH2 domain binding protein, also was required for transformation. When retrovirally transduced BM cells were transplanted into lethally irradiated recipients, E76K and D61Y but not WT Shp2 evoked fatal myeloproliferative disease (MPD) in 30–40% of recipients characterized by splenomegaly, leukocytosis, neutrophilia and monocytosis with occasional anemia and thrombocytopenia. Another ~20% showed less severe MPD at early stage and ultimately succumbed to T-ALL at later times. Histological analysis of mice dying MPD revealed BM and spleen packed with myeloid cells and livers with perivascular myeloid infiltrates. Flow cytometry on BM and spleen confirmed the presence of MPD. Moreover, BM from E76K and D61Y transplanted recipients exhibited enhanced factor-independent colony formation. Furthermore, mast cells derived from E76K and D61Y transplanted mice exhibit increased proliferation and enhanced activation of Erk, Akt and Stat5 in response to IL-3. In contrast, mice with a knock-in mutation of NS (D61G) displayed only a mild MPD. These data support a model in which leukemia-associated mutants of Shp2 are strong hypermorphs that enhance signaling via βc cytokines, whereas NS mutants are less severe gain of function alleles. Our results provide the first evidence that Shp2 mutants have a causal role in leukemogenesis and therefore, Shp2 is the first bona fide PTP proto-oncogene.