Discovery of a Potent and Selective Tyrosine Kinase 2 Inhibitor: TAK-279.

TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30, a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.

Identification of Two Non-Peptidergic Small Molecule Inhibitors of CBX2 Binding to K27 Trimethylated Oligonucleosomes.

The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.

MEK inhibitors overcome resistance to BET inhibition across a number of solid and hematologic cancers.

BET inhibitors exhibit broad activity in cancer models, making predictive biomarkers challenging to define. Here we investigate the biomarkers of activity of the clinical BET inhibitor GSK525762 (I-BET; I-BET762) across cancer cell lines and demonstrate that KRAS mutations are novel resistance biomarkers. This finding led us to combine BET with RAS pathway inhibition using MEK inhibitors to overcome resistance, which resulted in synergistic effects on growth and survival in RAS pathway mutant models as well as a subset of cell lines lacking RAS pathway mutations. GSK525762 treatment up-regulated p-ERK1/2 levels in both RAS pathway wild-type and mutant cell lines, suggesting that MEK/ERK pathway activation may also be a mechanism of adaptive BET inhibitor resistance. Importantly, gene expression studies demonstrated that the BET/MEK combination uniquely sustains down-regulation of genes associated with mitosis, leading to prolonged growth arrest that is not observed with either single agent therapy. These studies highlight a potential to enhance the clinical benefit of BET and MEK inhibitors and provide a strong rationale for clinical evaluation of BET/MEK combination therapies in cancer.

Small-Molecule Targets in Immuno-Oncology.

Advances in understanding the role and molecular mechanisms underlying immune surveillance and control of (pre)malignancies is revolutionizing clinical practice in the treatment of cancer. Presently, multiple biologic drugs targeting the immune checkpoint proteins PD(L)1 or CTLA4 have been approved and/or are in advanced stages of clinical development for many cancers. In addition, combination therapy with these agents and other immunomodulators is being intensively explored with the aim of improving primary response rates or prolonging overall survival. The effectiveness of cancer immunotherapy with biologics is spurring research in alternate approaches including small-molecule-mediated targeting of intracellular pathways modulating the innate and adaptive immune response. This focus of this review is on some of the key intracellular pathways where the development of a small-molecule therapeutic is attractive, tractable, and potentially synergistic with extracellular biologic-mediated immune checkpoint blockade.

Role of Histone-Modifying Enzymes and Their Complexes in Regulation of Chromatin Biology.

In 1964, Alfrey and colleagues proposed that acetylation and methylation of histones may regulate RNA synthesis and described "the possibility that relatively minor modifications of histone structure, taking place on the intact protein molecule, offer a means of switching-on or off RNA synthesis at different loci along the chromosome" [Allfrey, V., Faulkner, R., and Mirsky, A. (1964) Proc. Natl. Acad. Sci. U.S.A. 51, 786]. Fifty years later, this prescient description provides a simple but conceptually accurate model for the biological role of histone post-translational modifications (PTMs). The basic unit of chromosomes is the nucleosome, with double-stranded DNA wrapped around a histone protein oligomer. The "tails" of histone proteins are post-translationally modified, which alters the physical properties of nucleosomes in a manner that impacts gene accessibility for transcription and replication. Enzymes that catalyze the addition and removal of histone PTMs, histone-modifying enzymes (HMEs), are present in large protein complexes, with DNA-binding proteins, ATP-dependent chromatin remodeling enzymes, and epigenetic reader proteins that bind to post-translationally modified histone residues [Arrowsmith, C. H., Bountra, C., Fish, P. V., Lee, K., and Schapira, M. (2012) Nat. Rev. Drug Discovery 11, 384-400]. The activity of HME complexes is coordinated with that of other chromatin-associated complexes that, together, regulate gene transcription, DNA repair, and DNA replication. In this context, the enzymes that catalyze addition and removal of histone PTMs are an essential component of the highly regulated mechanism for accessing compacted DNA. To fully understand the function of HMEs, the structure of nucleosomes, their natural substrate, will be described. Each major class of HMEs subsequently will be discussed with regard to its biochemistry, enzymatic mechanism, and biological function in the context of a prototypical HME complex.

A DNA Hypomethylation Signature Predicts Antitumor Activity of LSD1 Inhibitors in SCLC.

Epigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity.

A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation.

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival.

Control of NF-kB activity in human melanoma by bromodomain and extra-terminal protein inhibitor I-BET151.

The transcription factor NF-kappaB (NF-kB) is a key regulator of cytokine and chemokine production in melanoma and is responsible for symptoms such as anorexia, fatigue, and weight loss. In addition, NF-kB is believed to contribute to progression of the disease by upregulation of cell cycle and anti-apoptotic genes and to contribute to resistance against targeted therapies and immunotherapy. In this study, we have examined the ability of the bromodomain and extra-terminal (BET) protein inhibitor I-BET151 to inhibit NF-kB in melanoma cells. We show that I-BET151 is a potent, selective inhibitor of a number of NF-kB target genes involved in induction of inflammation and cell cycle regulation and downregulates production of cytokines such as IL-6 and IL-8. SiRNA studies indicate that BRD2 is the main BET protein involved in regulation of NF-kB and that I-BET151 caused transcriptional downregulation of the NF-kB subunit p105/p50. These results suggest that BET inhibitors may have an important role in treatment of melanoma where activation of NF-kB may have a key pathogenic role.

SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer.

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

Cancer epigenetics drug discovery and development: the challenge of hitting the mark.

Over the past several years, there has been rapidly expanding evidence of epigenetic dysregulation in cancer, in which histone and DNA modification play a critical role in tumor growth and survival. These findings have gained the attention of the drug discovery and development community, and offer the potential for a second generation of cancer epigenetic agents for patients following the approved "first generation" of DNA methylation (e.g., Dacogen, Vidaza) and broad-spectrum HDAC inhibitors (e.g., Vorinostat, Romidepsin). This Review provides an analysis of prospects for discovery and development of novel cancer agents that target epigenetic proteins. We will examine key examples of epigenetic dysregulation in tumors as well as challenges to epigenetic drug discovery with emerging biology and novel classes of drug targets. We will also highlight recent successes in cancer epigenetics drug discovery and consider important factors for clinical success in this burgeoning area.

Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762.

The bromodomain and extraterminal (BET) protein BRD2-4 inhibitors hold therapeutic promise in preclinical models of hematologic malignancies. However, translation of these data to molecules suitable for clinical development has yet to be accomplished. Herein we expand the mechanistic understanding of BET inhibitors in multiple myeloma by using the chemical probe molecule I-BET151. I-BET151 induces apoptosis and exerts strong antiproliferative effect in vitro and in vivo. This is associated with contrasting effects on oncogenic MYC and HEXIM1, an inhibitor of the transcriptional activator P-TEFb. I-BET151 causes transcriptional repression of MYC and MYC-dependent programs by abrogating recruitment to the chromatin of the P-TEFb component CDK9 in a BRD2-4-dependent manner. In contrast, transcriptional upregulation of HEXIM1 is BRD2-4 independent. Finally, preclinical studies show that I-BET762 has a favorable pharmacologic profile as an oral agent and that it inhibits myeloma cell proliferation, resulting in survival advantage in a systemic myeloma xenograft model. These data provide a strong rationale for extending the clinical testing of the novel antimyeloma agent I-BET762 and reveal insights into biologic pathways required for myeloma cell proliferation.

Long residence time inhibition of EZH2 in activated polycomb repressive complex 2.

EZH2/PRC2 catalyzes transcriptionally repressive methylation at lysine 27 of histone H3 and has been associated with numerous cancer types. Point mutations in EZH2 at Tyr641 and Ala677 identified in non-Hodgkin lymphomas alter substrate specificity and result in increased trimethylation at histone H3K27. Interestingly, EZH2/PRC2 is activated by binding H3K27me3 marks on histones, and this activation is proposed as a mechanism for self-propagation of gene silencing. Recent work has identified GSK126 as a potent, selective, SAM-competitive inhibitor of EZH2 capable of globally decreasing H3K27 trimethylation in cells. Here we show that activation of PRC2 by an H3 peptide trimethylated at K27 is primarily an effect on the rate-limiting step (kcat) with no effect on substrate binding (Km). Additionally, GSK126 is shown to have a significantly longer residence time of inhibition on the activated form of EZH2/PRC2 as compared to unactivated EZH2/PRC2. Overall inhibition constant (Ki*) values for GSK126 were determined to be as low as 93 pM and appear to be driven by slow dissociation of inhibitor from the activated enzyme. The data suggest that activation of EZH2 allows the enzyme to adopt a conformation that possesses greater affinity for GSK126. The long residence time of GSK126 may be beneficial in vivo and may result in durable target inhibition after drug systemic clearance.

Inhibition of BET bromodomain proteins as a therapeutic approach in prostate cancer.

BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. Small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.

BET inhibition silences expression of MYCN and BCL2 and induces cytotoxicity in neuroblastoma tumor models.

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.

EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations.

In eukaryotes, post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumorigenesis and correlates with poor prognosis in several tumour types. Additionally, somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 occur in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. The Y641 residue is the most frequently mutated residue, with up to 22% of germinal centre B-cell DLBCL and follicular lymphoma harbouring mutations at this site. These lymphomas have increased H3K27 tri-methylation (H3K27me3) owing to altered substrate preferences of the mutant enzymes. However, it is unknown whether specific, direct inhibition of EZH2 methyltransferase activity will be effective in treating EZH2 mutant lymphomas. Here we demonstrate that GSK126, a potent, highly selective, S-adenosyl-methionine-competitive, small-molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and markedly inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.

Development and validation of reagents and assays for EZH2 peptide and nucleosome high-throughput screens.

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [(3)H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation.

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27).

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.

ALDH1A1 is a novel EZH2 target gene in epithelial ovarian cancer identified by genome-wide approaches.

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy in the United States. EZH2 silences gene expression through trimethylating lysine 27 on histone H3 (H3K27Me3). EZH2 is often overexpressed in EOC and has been suggested as a target for EOC intervention. However, EZH2 target genes in EOC remain poorly understood. Here, we mapped the genomic loci occupied by EZH2/H3K27Me3 using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and globally profiled gene expression in EZH2-knockdown EOC cells. Cross-examination of gene expression and ChIP-seq revealed a list of 60 EZH2 direct target genes whose expression was upregulated more than 1.5-fold upon EZH2 knockdown. For three selected genes (ALDH1A1, SSTR1, and DACT3), we validated their upregulation upon EZH2 knockdown and confirmed the binding of EZH2/H3K27Me3 to their genomic loci. Furthermore, the presence of H3K27Me3 at the genomic loci of these EZH2 target genes was dependent upon EZH2. Interestingly, expression of ALDH1A1, a putative marker for EOC stem cells, was significantly downregulated in high-grade serous EOC (n = 53) compared with ovarian surface epithelial cells (n = 10, P < 0.001). Notably, expression of ALDH1A1 negatively correlated with expression of EZH2 (n = 63, Spearman r = -0.41, P < 0.001). Thus, we identified a list of 60 EZH2 target genes and established that ALDH1A1 is a novel EZH2 target gene in EOC cells. Our results suggest a role for EZH2 in regulating EOC stem cell equilibrium via regulation of ALDH1A1 expression.

Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2.

The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2.