ABSTRACT
PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Interferon Type I/metabolism , Neoplasms/drug therapy , Nucleoside Transport Proteins/genetics , Nucleoside Transport Proteins/metabolism , Small Molecule Libraries/administration & dosage , Adaptive Immunity/drug effects , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , HEK293 Cells , HeLa Cells , Humans , Mice , Neoplasms/genetics , Neoplasms/metabolism , Signal Transduction/drug effects , Small Molecule Libraries/pharmacology , Tumor Escape/drug effects , Xenograft Model Antitumor AssaysABSTRACT
PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 µM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Inflammation/drug therapy , Interleukin-4/antagonists & inhibitors , Kidney Neoplasms/drug therapy , Macrophages/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Dose-Response Relationship, Drug , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , HEK293 Cells , Humans , Inflammation/genetics , Inflammation/metabolism , Interleukin-4/genetics , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Structure , Poly(ADP-ribose) Polymerases/genetics , RAW 264.7 Cells , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Posttranslational methylation of histones plays a critical role in gene regulation. Misregulation of histone methylation can lead to oncogenic transformation. Enhancer of Zeste homologue 2 (EZH2) methylates histone 3 at lysine 27 (H3K27) and abnormal methylation of this site is found in many cancers. Tazemetostat, an EHZ2 inhibitor in clinical development, has shown activity in both preclinical models of cancer as well as in patients with lymphoma or INI1-deficient solid tumors. Herein we report the structure-activity relationships from identification of an initial hit in a high-throughput screen through selection of tazemetostat for clinical development. The importance of several methyl groups to the potency of the inhibitors is highlighted as well as the importance of balancing pharmacokinetic properties with potency.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Histones/metabolism , Methylation/drug effects , Polycomb Repressive Complex 2/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Animals , Drug Discovery , Enhancer of Zeste Homolog 2 Protein , Enzyme Inhibitors/pharmacokinetics , Humans , Mice , Polycomb Repressive Complex 2/metabolism , Protein Processing, Post-Translational/drug effects , Small Molecule Libraries/pharmacokinetics , Structure-Activity RelationshipABSTRACT
The study by Kelly and colleagues, published in the September 1, 2003, issue of Clinical Cancer Research, established the safety and biologic activity of the first-in-class histone deacetylase inhibitor, vorinostat, which was administered intravenously. Subsequent studies led to the development of oral vorinostat and the regulatory approval of vorinostat for cutaneous T-cell lymphomas, which opened the door for the next generation of inhibitors.
Subject(s)
Hematologic Neoplasms/drug therapy , Histone Deacetylase Inhibitors , Hydroxamic Acids/administration & dosage , Female , Humans , MaleABSTRACT
AIMS: Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines. RESULTS: We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors. INNOVATION: We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA. CONCLUSION: In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis).
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Drug Resistance, Neoplasm/drug effects , Hydroxamic Acids/pharmacology , Antineoplastic Agents/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Buthionine Sulfoximine/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/metabolism , Hydroxamic Acids/toxicity , Oxidation-Reduction/drug effects , Primary Cell Culture , VorinostatABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Lymphoma, Non-Hodgkin/drug therapy , Polycomb Repressive Complex 2/antagonists & inhibitors , Polycomb Repressive Complex 2/genetics , Pyridones/pharmacology , Animals , Apoptosis/drug effects , Biphenyl Compounds , Catalytic Domain/genetics , Cell Cycle/drug effects , Cell Line, Tumor , Female , Humans , Lymphoma, Non-Hodgkin/pathology , Male , Mice , Mice, SCID , Molecular Sequence Data , Morpholines , Point Mutation , Rats , Rats, Sprague-Dawley , Xenograft Model Antitumor AssaysABSTRACT
(2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (EPZ-5676) is a novel DOT1L histone methyltransferase inhibitor currently in clinical development for the treatment of MLL-rearranged leukemias. This report describes the preclinical pharmacokinetics and metabolism of EPZ-5676, an aminonucleoside analog with exquisite target potency and selectivity that has shown robust and durable tumor growth inhibition in preclinical models. The in vivo pharmacokinetics in mouse, rat and dog were characterized following i.v. and p.o. administration; EPZ-5676 had moderate to high clearance, low oral bioavailability with a steady-state volume of distribution 2-3 fold higher than total body water. EPZ-5676 showed biexponential kinetics following i.v. administration, giving rise to a terminal elimination half-life (t1/2 ) of 1.1, 3.7 and 13.6 h in mouse, rat and dog, respectively. The corresponding in vitro ADME parameters were also studied and utilized for in vitro-in vivo extrapolation purposes. There was good agreement between the microsomal clearance and the in vivo clearance implicating hepatic oxidative metabolism as the predominant elimination route in preclinical species. Furthermore, low renal clearance was observed in mouse, approximating to fu -corrected glomerular filtration rate (GFR) and thus passive glomerular filtration. The metabolic pathways across species were studied in liver microsomes in which EPZ-5676 was metabolized to three monohydroxylated metabolites (M1, M3 and M5), one N-dealkylated product (M4) as well as an N-oxide (M6).
Subject(s)
Antineoplastic Agents/pharmacokinetics , Benzimidazoles/pharmacokinetics , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Methyltransferases/antagonists & inhibitors , Animals , Antineoplastic Agents/blood , Benzimidazoles/blood , Blood Proteins/metabolism , Dogs , Hepatocytes/metabolism , Madin Darby Canine Kidney Cells , Male , Mice , Microsomes, Liver/metabolism , Permeability , Rats, Sprague-DawleyABSTRACT
Over the past decade, the number of new therapies developed for the treatment of rare diseases continues to increase. The most rapid growth has been in the development of new drugs for oncology indications. One focus in drug discovery for oncology indications is the development of targeted therapies for select patient subgroups characterized by genetic alterations. The identification of these patient subgroups has increased in the past decade and has resulted in a corresponding increase in the development of new drugs for genetically defined patient subgroups. As an example of the development of new therapeutics for rare indications, I describe here the drug discovery efforts leading to the development of DOT1L inhibitors for the treatment of MLL-rearranged leukemia.
Subject(s)
Antineoplastic Agents/therapeutic use , Drug Discovery/methods , Enzyme Inhibitors/therapeutic use , Leukemia/drug therapy , Methyltransferases/antagonists & inhibitors , Animals , Drug Discovery/history , Drug Discovery/trends , Gene Rearrangement , Histone-Lysine N-Methyltransferase , History, 21st Century , Humans , Leukemia/genetics , Leukemia/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolismABSTRACT
Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited.
Subject(s)
Antineoplastic Agents/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Acetylation/drug effects , Animals , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Enzyme Activation/drug effects , Fusion Proteins, bcr-abl/metabolism , HSP90 Heat-Shock Proteins/metabolism , Histone Deacetylase 1/antagonists & inhibitors , Histone Deacetylase 2/antagonists & inhibitors , Histone Deacetylase 6 , Histone Deacetylase Inhibitors/administration & dosage , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/administration & dosage , Hydroxamic Acids/pharmacology , Lymphoma/drug therapy , Lymphoma/metabolism , Lymphoma/mortality , Lymphoma/pathology , Mice , Vorinostat , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Benzimidazoles/pharmacology , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Leukemia/genetics , Leukemia/therapy , Methyltransferases/antagonists & inhibitors , Myeloid-Lymphoid Leukemia Protein/genetics , Animals , Cell Line, Tumor , Cell Proliferation , DNA Methylation , Dose-Response Relationship, Drug , Female , Histone Methyltransferases , Histones/metabolism , Humans , Neoplasm Transplantation , Protein Conformation , Rats , Rats, NudeABSTRACT
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.
Subject(s)
Apoptosis , Neoplasms/therapy , Polycomb Repressive Complex 2/antagonists & inhibitors , Rhabdoid Tumor/enzymology , Rhabdoid Tumor/genetics , Animals , Antineoplastic Agents/pharmacology , Biphenyl Compounds/pharmacology , Cell Line, Tumor , Cell Proliferation , Drug Design , Enhancer of Zeste Homolog 2 Protein , Epigenesis, Genetic , Gene Expression Profiling , HEK293 Cells , Histones/metabolism , Humans , Mice , Neoplasm Transplantation , Neoplasms/drug therapy , Neoplasms/pathology , Pyridines/pharmacologyABSTRACT
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.
Subject(s)
Cell Transformation, Neoplastic/genetics , Histone-Lysine N-Methyltransferase/genetics , Kinesins/genetics , Methyltransferases/physiology , Myeloid-Lymphoid Leukemia Protein/genetics , Myosins/genetics , Oncogene Proteins, Fusion/genetics , Adenosine/analogs & derivatives , Adenosine/pharmacology , Animals , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/metabolism , Cells, Cultured , Enzyme Inhibitors/pharmacology , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/physiology , Lysine/metabolism , Methyltransferases/antagonists & inhibitors , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Phenylurea Compounds/pharmacologyABSTRACT
EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's 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.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Histones/metabolism , Indazoles/pharmacology , Lymphoma/drug therapy , Lymphoma/pathology , Polycomb Repressive Complex 2/antagonists & inhibitors , Pyridones/pharmacology , Antineoplastic Agents/chemistry , Cell Cycle/drug effects , Cell Death/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Enhancer of Zeste Homolog 2 Protein , Enzyme Inhibitors/chemistry , Histones/chemistry , Humans , Indazoles/chemistry , Lymphoma/enzymology , Lymphoma/genetics , Lysine/metabolism , Methylation/drug effects , Molecular Structure , Point Mutation , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Pyridones/chemistry , Structure-Activity RelationshipABSTRACT
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 K(i) 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.
Subject(s)
Enzyme Inhibitors/chemistry , Methyltransferases/antagonists & inhibitors , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/metabolism , Histone-Lysine N-Methyltransferase , Histones/metabolism , Humans , Kinetics , Methylation , Methyltransferases/metabolism , Molecular Docking Simulation , Nucleosides/chemistry , Nucleosides/metabolism , Protein Binding , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
Heterozygous point mutations at Y641 and A677 in the EZH2 SET domain are prevalent in about 10-24% of Non-Hodgkin lymphomas (NHL). Previous studies indicate that these are gain-of-function mutations leading to the hypertrimethylation of H3K27. These EZH2 mutations may drive the proliferation of lymphoma and make EZH2 a molecular target for patients harboring these mutations. Here, another EZH2 SET domain point mutation, A687V, occurring in about 1-2% of lymphoma patients, is also shown to be a gain-of-function mutation that greatly enhances its ability to perform dimethylation relative to wild-type EZH2 and is equally proficient at catalyzing trimethylation. We propose that A687V EZH2 also leads to hypertrimethylation of H3K27 and may thus be a driver mutation in NHL.
Subject(s)
Lymphoma, Non-Hodgkin/enzymology , Lymphoma, Non-Hodgkin/genetics , Mutant Proteins/genetics , Neoplasm Proteins/genetics , Point Mutation , Polycomb Repressive Complex 2/genetics , Enhancer of Zeste Homolog 2 Protein , Heterozygote , Histones/chemistry , Histones/metabolism , Humans , Kinetics , Methylation , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Polycomb Repressive Complex 2/chemistry , Polycomb Repressive Complex 2/metabolism , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Mutations at tyrosine 641 (Y641F, Y641N, Y641S and Y641H) in the SET domain of EZH2 have been identified in patients with certain subtypes of non-Hodgkin lymphoma (NHL). These mutations were shown to change the substrate specificity of EZH2 for various methylation states of lysine 27 on histone H3 (H3K27). An additional mutation at EZH2 Y641 to cysteine (Y641C) was also found in one patient with NHL and in SKM-1 cells derived from a patient with myelodisplastic syndrome (MDS). The Y641C mutation has been reported to dramatically reduce enzymatic activity. Here, we demonstrate that while the Y641C mutation ablates enzymatic activity against unmethylated and monomethylated H3K27, it is superior to wild-type in catalyzing the formation of trimethylated H3K27 from the dimethylated precursor.
Subject(s)
DNA-Binding Proteins/metabolism , Histones/metabolism , Lysine/metabolism , Mutation , Substrate Specificity/genetics , Transcription Factors/metabolism , Animals , Base Sequence , Cell Line , Cysteine/chemistry , Cysteine/metabolism , DNA-Binding Proteins/genetics , Enhancer of Zeste Homolog 2 Protein , Histones/genetics , Humans , Lysine/chemistry , Methylation , Molecular Sequence Data , Polycomb Repressive Complex 2 , Sequence Analysis, DNA , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Enzyme Inhibitors/pharmacology , Leukemia, Biphenotypic, Acute/pathology , Methyltransferases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Animals , Cell Death/drug effects , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Gene Expression Profiling , Gene Expression Regulation, Leukemic/drug effects , Gene Rearrangement/drug effects , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histones/metabolism , Humans , Leukemia, Biphenotypic, Acute/genetics , Lysine/metabolism , Methylation/drug effects , Methyltransferases/metabolism , Mice , Myeloid-Lymphoid Leukemia Protein/metabolism , Oncogene Proteins, Fusion/metabolism , Small Molecule Libraries/administration & dosage , Small Molecule Libraries/chemistry , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Gene Rearrangement/genetics , Histones/metabolism , Lysine/metabolism , Methyltransferases/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Animals , Apoptosis , Cell Cycle , Cell Differentiation , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Genetic Loci/genetics , Hematopoiesis , Histone-Lysine N-Methyltransferase , Homeodomain Proteins/metabolism , Humans , Methylation , Mice , Myeloid Ecotropic Viral Integration Site 1 Protein , Myeloid Progenitor Cells/metabolism , Myeloid Progenitor Cells/pathology , Neoplasm Proteins/metabolism , Oncogene Proteins, Fusion/metabolism , Protein Processing, Post-TranslationalABSTRACT
A survey of the human genome was performed to understand the constituency of protein methyltransferases (both protein arginine and lysine methyltransferases) and the relatedness of their catalytic domains. We identified 51 protein lysine methyltransferase proteins based on similarity to the canonical Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain. Disruptor of telomeric silencing-1-like, a known protein lysine methyltransferase, did not fit within the protein lysine methyltransferase family, but did group with the protein arginine methyltransferases, along with 44 other proteins, including the METTL and NOP2/Sun domain family proteins. We show that a representative METTL, METTL11A, demonstrates catalytic activity as a histone methyltransferase. We also solved the co-crystal structures of disruptor of telomeric silencing-1-like with S-adenosylmethionine and S-adenosylhomocysteine bound in its active site. The conformation of both ligands is virtually identical to that found in known protein arginine methyltransferases, METTL and NOP2/Sun domain family proteins and is distinct from that seen in the Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain protein lysine methyltransferases. We have developed biochemical assays for 11 members of the protein methyltransferase target class and have profiled the affinity of three ligands for these enzymes: the common methyl-donating substrate S-adenosylmethionine; the common reaction product S-adenosylhomocysteine; and the natural product sinefungin. The affinity of each of these ligands is mapped onto the family trees of the protein lysine methyltransferases and protein arginine methyltransferases to reveal patterns of ligand recognition by these enzymes.
Subject(s)
Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Models, Molecular , Binding Sites , Crystallography, X-Ray , Humans , Inhibitory Concentration 50 , Ligands , Molecular Structure , PhylogenyABSTRACT
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 (k(cat)/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.