PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition triggers antitumor immunity.

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.

Targeted Degradation of PARP14 Using a Heterobifunctional Small Molecule.

PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types, influencing pro-tumor macrophage polarization as well as suppressing the antitumor inflammation response by modulating IFN-γ and IL-4 signaling. PARP14 is a 203 kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates. PARP14 also contains three macrodomains and a WWE domain which are binding modules for mono-ADP-ribose and poly-ADP-ribose, respectively, in addition to two RNA recognition motifs. Catalytic inhibitors of PARP14 have been shown to reverse IL-4 driven pro-tumor gene expression in macrophages, however it is not clear what roles the non-enzymatic biomolecular recognition motifs play in PARP14-driven immunology and inflammation. To further understand this, we have discovered a heterobifunctional small molecule designed based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+ -binding site and recruits cereblon to ubiquitinate it and selectively target it for degradation.

A potent and selective PARP14 inhibitor decreases protumor macrophage gene expression and elicits inflammatory responses in tumor explants.

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.

In Vitro and Cellular Probes to Study PARP Enzyme Target Engagement.

Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD+) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD+ binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes. The in vitro assays use less enzyme than previously described activity assays, enabling discrimination of inhibitor potencies in the single-digit nanomolar range, and the cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.

Forced Self-Modification Assays as a Strategy to Screen MonoPARP Enzymes.

Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD+, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.

Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.

Characterization of Inhibitor Binding Through Multiple Inhibitor Analysis: A Novel Local Fitting Method.

Understanding inhibitor binding modes is a key aspect of drug development. Early in a drug discovery effort these considerations often impact hit finding strategies and hit prioritization. Multiple inhibitor experiments, where enzyme inhibition is measured in the presence of two simultaneously varied inhibitors, can provide valuable information about inhibitor binding. These experiments utilize the inhibitor concentration dependence of the observed combined inhibition to determine the relationship between two compounds. In this way, it can be determined whether two inhibitors bind exclusively, independently, synergistically, or antagonistically. Novel inhibitors can be tested against each other or reference compounds to assist hit classification and characterization of inhibitor binding. In this chapter, we discuss the utility and design of multiple inhibitor experiments and present a new local curve fitting method for analyzing these data utilizing IC50 replots. The IC50 replot method is analogous to that used for determining mechanisms of inhibition with respect to substrate, as originally proposed by Cheng and Prusoff (Cheng and Prusoff Biochem Pharmacol 22: 3099-3108, 1973). The IC50 replot generated by this method reveals distinct patterns that are diagnostic of the nature of the interaction between two inhibitors. Multiple inhibition of the histone methyltransferase EZH2 by EPZ-5687 and the reaction product S-adenosylhomocysteine is presented as an example of the method.

Novel Oxindole Sulfonamides and Sulfamides: EPZ031686, the First Orally Bioavailable Small Molecule SMYD3 Inhibitor.

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies.

Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP.

The protein methyltransferase (PMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor and its catalytic activity, has been reported to be regulated by a partner protein ATF7IP. Here, we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed and 1:1 stoichiometric complexes were purified for comparison against SETDB1 enzyme alone. We employed both radiometric flashplate-based and SAMDI mass spectrometry assays to follow methylation on histone H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated, or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex efficiently catalyze both monomethylation and dimethylation of H3K9 peptide substrates. The activity of the binary complex was 4-fold lower than SETDB1 alone. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between SETDB1 and the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in a distributive manner, and this too was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9. Our results indicate that ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting alternate roles, such as affecting cellular localization or mediating interaction with additional binding partners.

The Importance of Being Me: Magic Methyls, Methyltransferase Inhibitors, and the Discovery of Tazemetostat.

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.

Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.

A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies.

EPZ011989, A Potent, Orally-Available EZH2 Inhibitor with Robust in Vivo Activity.

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.

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.

A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening.

Demethylation of histones by lysine demethylases (KDMs) plays a critical role in controlling gene transcription. Aberrant demethylation may play a causal role in diseases such as cancer. Despite the biological significance of these enzymes, there are limited assay technologies for study of KDMs and few quality chemical probes available to interrogate their biology. In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work.

Synergistic Anti-Tumor Activity of EZH2 Inhibitors and Glucocorticoid Receptor Agonists in Models of Germinal Center Non-Hodgkin Lymphomas.

Patients with non-Hodgkin lymphoma (NHL) are treated today with a cocktail of drugs referred to as CHOP (Cyclophosphamide, Hydroxyldaunorubicin, Oncovin, and Prednisone). Subsets of patients with NHL of germinal center origin bear oncogenic mutations in the EZH2 histone methyltransferase. Clinical testing of the EZH2 inhibitor EPZ-6438 has recently begun in patients. We report here that combining EPZ-6438 with CHOP in preclinical cell culture and mouse models results in dramatic synergy for cell killing in EZH2 mutant germinal center NHL cells. Surprisingly, we observe that much of this synergy is due to Prednisolone - a glucocorticoid receptor agonist (GRag) component of CHOP. Dramatic synergy was observed when EPZ-6438 is combined with Prednisolone alone, and a similar effect was observed with Dexamethasone, another GRag. Remarkably, the anti-proliferative effect of the EPZ-6438+GRag combination extends beyond EZH2 mutant-bearing cells to more generally impact germinal center NHL. These preclinical data reveal an unanticipated biological intersection between GR-mediated gene regulation and EZH2-mediated chromatin remodeling. The data also suggest the possibility of a significant and practical benefit of combining EZH2 inhibitors and GRag that warrants further investigation in a clinical setting.

Reaction coupling between wild-type and disease-associated mutant EZH2.

EZH2 and EZH1 are protein methyltransferases (PMTs) responsible for histone H3, lysine 27 (H3K27) methylation. Trimethylation of H3K27 (H3K27me3) is a hallmark of many cancers, including non-Hodgkin lymphoma (NHL). Heterozygous EZH2 point mutations at Tyr641, Ala677, and Ala687 have been observed in NHL. The Tyr641 mutations enhance activity on H3K27me2 but have weak or no activity on unmethylated H3K27, whereas the Ala677 and Ala687 mutations use substrates of all methylation states effectively. It has been proposed that enzymatic coupling of the wild-type and mutant enzymes leads to the oncogenic H3K27me3 mark in mutant-bearing NHL. We show that coupling with the wild-type enzyme is needed to achieve H3K27me3 for several mutants, but that others are capable of achieving H3K27me3 on their own. All forms of PRC2 (wild-type and mutants) display kinetic signatures that are consistent with a distributive mechanism of catalysis.

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.

Convergent evolution of chromatin modification by structurally distinct enzymes: comparative enzymology of histone H3 Lys²7 methylation by human polycomb repressive complex 2 and vSET.

H3K27 (histone H3 Lys27) methylation is an important epigenetic modification that regulates gene transcription. In humans, EZH (enhancer of zeste homologue) 1 and EZH2 are the only enzymes capable of catalysing methylation of H3K27. There is great interest in understanding structure-function relationships for EZH2, as genetic alterations in this enzyme are thought to play a causal role in a number of human cancers. EZH2 is challenging to study because it is only active in the context of the multi-subunit PRC2 (polycomb repressive complex 2). vSET is a viral lysine methyltransferase that represents the smallest protein unit capable of catalysing H3K27 methylation. The crystal structure of this minimal catalytic protein has been solved and researchers have suggested that vSET might prove useful as an EZH2 surrogate for the development of active site-directed inhibitors. To test this proposition, we conducted comparative enzymatic analysis of human EZH2 and vSET and report that, although both enzymes share similar preferences for methylation of H3K27, they diverge in terms of their permissiveness for catalysing methylation of alternative histone lysine sites, their relative preferences for utilization of multimeric macromolecular substrates, their active site primary sequences and, most importantly, their sensitivity to inhibition by drug-like small molecules. The cumulative data led us to suggest that EZH2 and vSET have very distinct active site structures, despite the commonality of the reaction catalysed by the two enzymes. Hence, the EZH2 and vSET pair of enzymes represent an example of convergent evolution in which distinct structural solutions have developed to solve a common catalytic need.

Drugging the human methylome: an emerging modality for reversible control of aberrant gene transcription.

Protein and DNA methylation have emerged as critical mechanisms for the control of regulated gene transcription. In humans, the addition, recognition and removal of methyl groups are orchestrated by at least 344 proteins that we collectively refer to as the 'methylome'. The large size of the methylome likely reflects the importance of precise control over this small covalent modification. An increasing number of reports implicating the misregulation of methylation in disease make the proteins governing this modification attractive target for small molecule drug discovery. In light of the emerging opportunities for the development of therapeutics that modulate methylation-dependent pathways, this review examines the protein families that constitute the methylome, with emphasis on the methylation of arginine and lysine residues of proteins. Genetic aberrations that give rise to disease are highlighted, in addition to recent proof-of-concept successes in the development of small molecule modulators of methylome constituents.

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.