Roy M. Pollock

Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor.

Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.

A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells

EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkins lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.

MLL-Rearranged Leukemia Is Dependent on Aberrant H3K79 Methylation by DOT1L

The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3, and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSCs). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSCs. Inactivation of Dot1l led to downregulation of direct MLL-AF9 targets and an MLL translocation-associated gene expression signature, whereas global gene expression remained largely unaffected. Suppression of MLL translocation-associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.

Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency (kcat/K) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.

Potent inhibition of DOT1L as treatment of MLL-fusion leukemia

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.

Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2

Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.

Selective Inhibition of EZH2 by EPZ-6438 Leads to Potent Antitumor Activity in EZH2-Mutant Non-Hodgkin Lymphoma

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842–54. ©2014 AACR.

A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.

Conformational adaptation drives potent, selective and durable inhibition of the human protein methyltransferase DOT1L.

DOT1L is the human protein methyltransferase responsible for catalyzing the methylation of histone H3 on lysine 79 (H3K79). The ectopic activity of DOT1L, associated with the chromosomal translocation that is a universal hallmark of MLL‐rearranged leukemia, is a required driver of leukemogenesis in this malignancy. Here, we present studies on the structure–activity relationship of aminonucleoside‐based DOT1L inhibitors. Within this series, we find that improvements in target enzyme affinity and selectivity are driven entirely by diminution of the dissociation rate constant for the enzyme–inhibitor complex, leading to long residence times for the binary complex. The biochemical Ki and residence times measured for these inhibitors correlate well with their effects on intracellular H3K79 methylation and MLL‐rearranged leukemic cell killing. Crystallographic studies reveal a conformational adaptation mechanism associated with high‐affinity inhibitor binding and prolonged residence time; these studies also suggest that conformational adaptation likewise plays a critical role in natural ligand interactions with the enzyme, hence, facilitating enzyme turnover. These results provide critical insights into the role of conformational adaptation in the enzymatic mechanism of catalysis and in pharmacologic intervention for DOT1L and other members of this enzyme class.

Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l

The t(6;11)(q27;q23) is a recurrent chromosomal rearrangement that encodes the MLLAF6 fusion oncoprotein and is observed in patients with diverse hematologic malignancies. The presence of the t(6;11)(q27;q23) has been linked to poor overall survival in patients with AML. In this study, we demonstrate that MLL-AF6 requires continued activity of the histone-methyltransferase DOT1L to maintain expression of the MLL-AF6-driven oncogenic gene-expression program. Using gene-expression analysis and genome-wide chromatin immunoprecipitation studies followed by next generation sequencing, we found that MLL-fusion target genes display markedly high levels of histone 3 at lysine 79 (H3K79) dimethylation in murine MLL-AF6 leukemias as well as in ML2, a human myelomonocytic leukemia cell line bearing the t(6;11)(q27;q23) translocation. Targeted disruption of Dot1l using a conditional knockout mouse model inhibited leukemogenesis mediated by the MLL-AF6 fusion oncogene. Moreover, both murine MLL-AF6-transformed cells as well as the human MLL-AF6-positive ML2 leukemia cell line displayed specific sensitivity to EPZ0004777, a recently described, selective, small-molecule inhibitor of Dot1l. Dot1l inhibition resulted in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6-transformed cells. These results indicate that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation.