Oncogenic Deregulation of EZH2 as an Opportunity for Targeted Therapy in Lung Cancer

As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically-engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways leading to epigenetic transformation independent from canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a novel and potent EZH2 inhibitor JQEZ5 that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer

Targeting Chromatin Complexes in Myeloid Malignancies and Beyond: From Basic Mechanisms to Clinical Innovation

The aberrant function of chromatin regulatory networks (epigenetics) is a hallmark of cancer promoting oncogenic gene expression. A growing body of evidence suggests that the disruption of specific chromatin-associated protein complexes has therapeutic potential in malignant conditions, particularly those that are driven by aberrant chromatin modifiers. Of note, a number of enzymatic inhibitors that block the catalytic function of histone modifying enzymes have been established and entered clinical trials. Unfortunately, many of these molecules do not have potent single-agent activity. One potential explanation for this phenomenon is the fact that those drugs do not profoundly disrupt the integrity of the aberrant network of multiprotein complexes on chromatin. Recent advances in drug development have led to the establishment of novel inhibitors of protein–protein interactions as well as targeted protein degraders that may provide inroads to longstanding effort to physically disrupt oncogenic multiprotein complexes on chromatin. In this review, we summarize some of the current concepts on the role epigenetic modifiers in malignant chromatin states with a specific focus on myeloid malignancies and recent advances in early-phase clinical trials

Molecular dynamics simulations and computational design of inhibitors of protein lysine methyltransferase EZH2

Enhancer of Zeste Homolog 2 (EZH2) je epigenetski enzim koji vrši selektivno metilovanje lizina 27 na histonu H3 (H3K27). Trimetilovani H3K27 je represivan epigenetski signal, cime EZH2 utice na smanjenje transkripcije ciljnih gena. Prekomerna aktivnost EZH2, izazvana povecanom ekspresijom ili mutacijama njegovog katalitickog domena, dovedena je u vezu sa vecim brojem maligniteta kod ljudi, a inhibicija ovog enzima smatra se perspektivnom strategijom u razvoju novih antitumorskih lekova. Prvi selektivni inhibitori EZH2 otkriveni su 2012. godine i njihova biološka karakterizacija potvrdila je terapijski potencijal inhibicije ovog epigenetskog regulatora. Iako postoji potreba za otkricem novih inhibitora, znacajnu prepreku u njihovom dizajnu do sada je predstavljao nedostatak trodimenzionalne strukture kompleksa EZH2 i okarakterisanih liganada. Cilj istraživanja u okviru ove doktorske disertacije bio je da se primenom racunarskih metoda uspostavi i validira model vezivnog mesta EZH2, i da se, zatim, uspostavljeni model upotrebi u dizajnu novih inhibitora kao potencijalnih antitumorskih lekova. U prvoj fazi istraživanja izvršeno je homologo modeliranje katalitickog domena EZH2 koji sadrži vezivno mesto za kofaktor, a za koje se kompetitivno vezuju svi poznati inhibitori. Pocetni model usavršen je primenom simulacija konvencionalne i ubrzane molekularne dinamike (MD), a zatim je daljim simulacijama istraživano kako 13 inhibitora piridonske strukture stupa u interakciju sa vezivnim mestom na enzimu. Korišcenjem prikupljenih trajektorija izvršena je procena slobodne energije vezivanja inhibitora zasnovana na molekularnoj mehanici uz generalizovani Bornov model rastvaraca (MM/GBSA). Izracunate energije upore ene su sa dostupnim eksperimentalnim podacima i utvr eno je dobro slaganje izme u vrednosti koje ukazuje na validnost uspostavljenog modela. Daljom analizom trajektorija i primenom racunarske mutageneze u alanin identifikovane su intermolekulske interakcije koje kljucno doprinose aktivnosti i selektivnosti proucavanih inhibitora. Na ovim osnovama, definisan je farmakoforni model inhibitora EZH2 a njegovom validacijom potvr ena je mogucnost uspešne identifikacije i inhibitora baziranih na drugim osnovnim strukturama. Efikasnost mnogih antitumorskih lekova ogranicava razvoj rezistencije tui morskih celija, pri cemu je opisan veci broj mehanizama koji dovode do pojave rezistencije...methylation of lysine 27 on histone H3. Trimethylated H3K27 constitutes a repressive epigenetic mark, making EZH2 capable of transcriptional silencing of target genes. Aberrant EZH2 activity, caused by either overexpression or point mutations in the catalytic domain, has been related to a number of human malignancies, making EZH2 inhibition a promising strategy in the development of novel anticancer treatments. First disclosure of selective EZH2 inhibitors in 2012 and their biological characterization has confirmed the therapeutic potential of inhibiting this epigenetic regulator. While there is an outstanding need for the discovery of novel EZH2 inhibitors, the lack of a three-dimensional structure of EZH2 complexed with one of its ligands has been a significant obstacle to this end. The aim of research conducted as part of this doctoral dissertation was to establish and validate a model of EZH2 binding site using computational methods, and to subsequently utilize the established model as a foundation in the design of novel potential EZH2 inhibitors. In the first stage of our research, homology modeling of the EZH2 catalytic domain was conducted, which contains the cofactor binding site targeted by all known inhibitors. The initial model was refined through conventional and accelerated molecular dynamics (MD) simulations. Next, structures of EZH2 complexed with 13 different pyridone inhibitors were simulated in order to study the key interactions participating in ligand binding. An assessment of inhibitors’ binding free energy was performed using the molecular mechanics/generalized Born surface area (MM/GBSA) method. The computed energies were correlated to available experimental data and good agreement was found supporting the validity of the established model. Further analysis of MD trajectories and computational alanine scanning facilitated the definition of key intermolecular interactions that contribute to the observed potency and selectivity of the studied inhibitors. On this basis, a pharmacophore model of EZH2 inhibitors was proposed and validated. It demonstrated good capabilities in identifying truly active molecules, including those not based on a pyridone scaffold, in presence of decoys. The efficacy of anticancer drugs can become limited with the onset of resiiv stance that can develop in cancer cells through various mechanisms..

Epigenetic-mediated apoptosis in aggressive B-cell lymphomas

Lymphomas are a complex group of cancers arising from mature lymphoid cells. Despite advances in the treatment and management of this disease, it remains a health challenge as response to treatment and survival outcomes differ in various lymphoma subtypes. The exact causes driving lymphomagenesis remain unknown, and although several risk factors have been identified, the complexity of this disorder indicates that different molecular mechanisms drive the disease. One primary event evident in this disease is the presence of epigenetic alterations. Epigenetic alterations could arise due to changes in the pattern of deoxyribonucleic acid (DNA) methylation or histone modifications. Enhancer of zeste homolog 2 (EZH2) modifies histone and mediates gene silencing by selectively catalyzing lysine 27 trimethylation on histone H3 (H3K27me3). Consequently, genes responsible for tumor suppression and differentiation are repressed, cells undergo uncontrolled proliferation, and this leads to tumor formation. EZH2 has been mentioned to be a driver oncogene that plays an important role in tumor initiation and progression, especially when overexpressed or when gain-of function mutations occur in this gene. These hotspot gain-of-function mutations (Tyr646, Ala682 and Ala692) that result in increased EZH2 activity, are associated with increased H3K27me3 in several cancer types including lymphomas. Therefore, EZH2 inhibition could be an alternative strategy for lymphoma therapy. We treated aggressive B-cell lymphoma cell lines with 3-Deazaneplanocin A (DZNep), an epigenetic drug that inhibits EZH2 indirectly and promotes apoptosis in various tumor entities including lymphomas. We aimed to investigate the apoptotic efficacy of DZNep in these lymphoma cell lines, to understand the mechanisms of resistance to DZNep and to determine predictive biomarkers that could be of importance for EZH2 inhibition with DZNep. Using a combination of molecular and cell biological techniques such as flow cytometry, Western blot, Sanger sequencing, fluorescence in situ hybridization (FISH) and real-time reverse transcriptase polymerase chain reaction (RT-PCR), we showed that DZNep possesses a strong apoptotic and anti-proliferative effect on B-cell lymphoma cell lines. This effect was independent of the lymphoma type, the presence of EZH2 gain-of-function mutations or the presence of known prognostic lymphoma biomarkers such as translocations of MYC, BCL2 and BCL6. To investigate the molecular mechanism behind resistance to DZNep, we generated a DZNep-resistant clone from a B-cell lymphoma cell line that was initially sensitive to DZNep. This clone was used as a model to elucidate this mechanism. Upon molecular characterization, comparison of this clone with the parent cell line of origin revealed differences in the karyotype, copy number alterations, proliferation rate and response to DZNep. Clonality studies of the rearranged immunoglobulin genes however, demonstrated that the resistant clone and the parent cell line were clonally identical. Whole exome sequencing performed on this clone in relation to its corresponding control revealed among others, a genomic amplification of the AHCY gene - a direct target of DZNep. Methods such as copy number variation assays, FISH, gene expression assays and immunohistochemistry were used to validate this massive AHCY gain at the DNA, chromosomal, transcriptional and translational levels respectively. Functional validation of AHCY showed that in a DZNep-sensitive osteosarcoma cell line, AHCY overexpression was associated with resistance to DZNep. Unfortunately, reproduction of AHCY function in lymphoma cell lines was not successful due to technical reasons. Further evaluation of the whole exome sequencing data from DZNep-resistant cell lines demonstrated in one of the cell lines, a mutation in the AHCY gene. This mutation was predicted from online databases to have a likely damaging effect on the protein. This study thus presents alterations in the AHCY gene as a potential mechanism of resistance to DZNep. It also reveals that molecular mechanisms of acquired resistance are quite distinct from those of intrinsic resistance to DZNep, which themselves, may further differ from one DZNep-resistant lymphoma entity to the next. AHCY could therefore, be a promising biomarker to evaluate prior to the initiation of therapy with DZNep

Peptide Technologies in the Development of Chemical Tools for Chromatin‐Associated Machinery

Discerning a mechanistic understanding of the cause‐and‐effect relationships between chromatin post‐translational modifications (PTMs) and DNA accessibility for replication, transcription, and repair is an elusive goal being pursued using molecular and cellular biology, biochemistry, and more recently chemical inhibition. Chemical intervention of the chromatin‐associated complexes that regulate PTM maintenance and chromatin structure faces numerous challenges due to the broad surface‐groove interactions between many of these proteins and histones; yet, the increasing interest in understanding chromatin‐modifying complexes suggests tractable lead compounds will be critical for elucidating the mechanisms of chromatin dysregulation in disease states and validating the druggability of these domains. Peptides and peptidomimetics afford several advantages to efficient inhibitor development including a rational starting point, modular assembly, and retention of secondary structure. Numerous peptide technologies have been employed in the chromatin field to characterize substrate interactions, evaluate ligand selectivity, and optimize potent peptidomimetic inhibitors. We describe the progress and advantages of these efforts, and provide a perspective on their implications for future chemical probe and drug discovery efforts. Drug Dev Res 78 : 300–312, 2017. © 2017 Wiley Periodicals, Inc

Histone modifiers are oxygen sensors.

Hypoxia signals directly to chromatin via histone demethylases to alter gene expression</jats:p

Discovery of Small Molecule Inhibitors for Histone Methyltransferases in Cancer

Cancer is the second leading cause of mortality in the United States. There are several therapeutic regimens employed to mitigate the mortality rate of cancer. This includes the use of chemotherapy, radiation, immunotherapy, and precision medicine/targeted therapy. Targeted therapy involves the use of drugs that target a specific pathway or biomolecule compromised in cancer for cancer treatment. Aberrant expression of epigenetic enzymes has been well documented for their contribution in driving tumorigenesis and other cancer hallmarks. Hence, there is an urgent need for novel drug discovery and development in epigenetics to help combat various cancer morbidities. Herein, we review the roles and consequences of dysregulated function of several epigenetic enzymes, with a focus on histone methyltransferases (HMTs). Additionally, we discussed the current efforts made in the development of small molecule inhibitors for a few representative HMTs implicated in different cancers. Furthermore, the common screening assays used in discovering potent small molecule inhibitors were also detailed in this chapter. Overall, this book chapter highlights the significance of targeting HMTs in different cancers and the clinical application potentials/limitations faced by the developed or emerging small molecule inhibitors of HMTs for the purpose of cancer therapy