Epigenetic Proteins Research Articles

Epigenetic DNA Methylation and Protein Homocysteinylation: Key Players in Hypertensive Renovascular Damage.

Hypertension has been a threat to the health of people, the mechanism of which, however, remains poorly understood. It is clinically related to loss of nephron function, glomerular sclerosis, or necrosis, resulting in renal functional declines. The mechanisms underlying hypertension's development and progression to organ damage, including hypertensive renal damage, remain to be fully elucidated. As a developing approach, epigenetics has been postulated to elucidate the phenomena that otherwise cannot be explained by genetic studies. The main epigenetic hallmarks, such as DNA methylation, histone acetylation, deacetylation, noncoding RNAs, and protein N-homocysteinylation have been linked with hypertension. In addition to contributing to endothelial dysfunction and oxidative stress, biologically active gases, including NO, CO, and H2S, are crucial regulators contributing to vascular remodeling since their complex interplay conducts homeostatic functions in the renovascular system. Importantly, epigenetic modifications also directly contribute to the pathogenesis of kidney damage via protein N-homocysteinylation. Hence, epigenetic modulation to intervene in renovascular damage is a potential therapeutic approach to treat renal disease and dysfunction. This review illustrates some of the epigenetic hallmarks and their mediators, which have the ability to diminish the injury triggered by hypertension and renal disease. In the end, we provide potential therapeutic possibilities to treat renovascular diseases in hypertension.

The BET protein inhibitor apabetalone (RVX-208) prevents doxorubicin-induced endothelial senescence

Abstract Background Accumulation of senescent endothelial cells (ECs) plays a pivotal role in cardiometabolic disorders and lifespan reduction(1). Yet, only a few senolytics are known to date, partly due to the poor grasp of the molecular mechanisms that control the senescence survival programme. Bromo-and extraterminal domain (BET) proteins, known epigenetic reader proteins, recognize acetylated histone tails and regulate gene transcription. RVX-208, an FDA-approved BET inhibitor, has shown to modulate transcriptional programs involved in inflammation, cancer, and renal disease(2). Yet, its potential role in EC senescence and cardio-oncology remains elusive. Purpose Our study aims to investigate whether pharmacological modulation of BET proteins by RVX-208 can mitigate doxorubicin-induced endothelial senescence. Methods Human aortic endothelial cells (HAECs) were exposed to different concentrations of Doxo (50nM-500nM) for 48 hours. Cell morphology changes, cell cycle arrest, senescence associated secreted phenotype (SASP) release, upregulation of SA-b gal activity, DNA damage response (gH2AX) and expression of p21, p16 and p53 were used as molecular markers of cellular senescence. Conditioned media was collected from Doxo-treated ECs and processed for molecular analyses or used for treatment of healthy, non-senescent ECs. To test whether RVX-208 exerts any senolytic or senomorphic effect, HAECs were treated with Doxo (100 nM) in presence or absence of RVX-208 (5mM-20mM) for 48 hrs. RNA-seq and proteomic profiling were performed in vehicle and Doxo-treated ECs. Results Doxo-treated ECs showed a dose-dependent increase in senescence markers (p16, p21 and p53) and DNA-damage response (gH2AX). FACS analysis revealed an accumulation of cells in G2/M phase in Doxo-treated ECs (100 nM). Of interest, treatment with RVX-208 (15mM) prevented the upregulation of senescent markers (p16, p21 and p53) while rescuing alterations in cell morphology and reducing the number of SA-b gal positive cells. RVX-208 prevented Doxo-induced upregulation of genes involved in inflammation, oxidative stress and mitochondrial dysfunction as shown by RNA-seq and proteomic analysis. Conditioned media from Doxo-treated ECs showed an enrichment of SASP-associated markers (IL6, CCL2, IL8, TIMP2 and PDGF-b) as shown by dot-blot arrays. Exposure of healthy ECs to conditioned media from senescent ECs recapitulated aging features. Notably, RVX-208 blunted the release of SASP markers (IL6, CCL2 and IL8) from Doxo-treated ECs. Mechanistically, we found enhanced BET protein occupancy (BRD4) with concomitant enrichment of activating chromatin marks (H3K27Ac and H3K4me) on the promoter of senescence genes COL1A2, CDKN1C and SESN3. Conclusion Targeting BET proteins with RVX-208 may offer a promising therapeutic strategy to prevent endothelial aging in cancer patients undergoing cardiotoxic therapies.

Chem-CRISPR/dCas9FCPF: a platform for chemically induced epigenome editing.

Epigenetic aberration is one of the major driving factors in human cancer, often leading to acquired resistance to chemotherapies. Various small molecule epigenetic modulators have been reported. Nonetheless, outcomes from animal models and clinical trials have underscored the substantial setbacks attributed to pronounced on- and off-target toxicities. To address these challenges, CRISPR/dCas9 technology is emerging as a potent tool for precise modulation of epigenetic mechanism. However, this technology involves co-expressing exogenous epigenetic modulator proteins, which presents technical challenges in preparation and delivery with potential undesirable side effects. Recently, our research demonstrated that Cas9 tagged with the Phe-Cys-Pro-Phe (FCPF)-peptide motif can be specifically targeted by perfluorobiphenyl (PFB) derivatives. Here, we integrated the FCPF-tag into dCas9 and established a chemically inducible platform for epigenome editing, called Chem-CRISPR/dCas9FCPF. We designed a series of chemical inhibitor-PFB conjugates targeting various epigenetic modulator proteins. Focusing on JQ1, a panBET inhibitor, we demonstrate that c-MYC-sgRNA-guided JQ1-PFB specifically inhibits BRD4 in close proximity to the c-MYC promoter/enhancer, thereby effectively repressing the intricate transcription networks orchestrated by c-MYC as compared with JQ1 alone. In conclusion, our Chem-CRISPR/dCas9FCPF platform significantly increased target specificity of chemical epigenetic inhibitors, offering a viable alternative to conventional fusion protein systems for epigenome editing.

Abstract C072: Bmi1 expression is upregulated in response to injury/stress in the pancreas

Abstract Pancreatic homeostasis is regulated in part through epigenetic mechanisms that restrict or enable access of pancreatic lineage transcription factors to their target genes. These epigenetic protein cascades are also required for regeneration of the pancreas after injury. B-cell Moloney murine leukemia virus integration site 1 (Bmi1), a central protein in the Polycomb repressor complex 1 (PRC1), has been shown to be required for the regeneration of the pancreas after injury and its absence prevents pancreatic neoplasia in mice. Previous work also suggests that Bmi1 marks a subset of acinar cells contributing to pancreatic renewal and tissue maintenance. It remains unclear how Bmi1 expression levels are regulated in response to pancreatic stress and injury. Here we assess the baseline expression of Bmi1 in pancreatic tissue with a genetic reporter mouse model as well as changes in Bmi1 mRNA levels at different time points after the induction of pancreatitis in wild-type mice via RNA in situ hybridization (RNAscope). We also compare the ability of the murine pancreas to regenerate after acute pancreatitis in the presence and absence of Bmi1. Our data indicates that rather than a specific subpopulation, most, if not all, acinar cells in the pancreas express Bmi1 during their life and specific loss of Bmi1 in the pancreas impairs regeneration after acute pancreatitis. We also show an increase in Bmi1 mRNA levels within the pancreas shortly after the induction of acute pancreatitis which reduces as the pancreas resumes its normal histology. Altogether, our data indicate that Bmi1 expression is dynamic, responsive to injury and necessary for pancreatic homeostasis and regeneration. Citation Format: Nur Muhammad Renollet, Joyce K Thompson, Emily Wu, Osama Alkhalili, Nina Steele, Filip Bednar. Bmi1 expression is upregulated in response to injury/stress in the pancreas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C072.

The Effects of Maternal Nutrient Restriction during Mid to Late Gestation with Realimentation on Fetal Metabolic Profiles in the Liver, Skeletal Muscle, and Blood in Sheep.

Poor maternal nutrition during gestation negatively affects offspring growth and metabolism. To evaluate the impact of maternal nutrient restriction and realimentation on metabolism in the fetal liver, skeletal muscle, and circulation, on day 50 of gestation, ewes (n = 48) pregnant with singletons were fed 100% (CON) or 60% (RES) of requirements until day 90 of gestation, when a subset of ewes (n = 7/treatment) were euthanized, and fetal samples were collected. The remaining ewes were maintained on a current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to an alternative diet (CON-RES, RES-CON; n = 7/treatment). On day 130 of gestation, the remaining ewes were euthanized, and fetal samples were collected. Fetal liver, longissimus dorsi (LD), and blood metabolites were analyzed using LC-MS/MS, and pathway enrichment analysis was conducted using MetaboAnalyst. Then, 600, 518, and 524 metabolites were identified in the liver, LD, and blood, respectively, including 345 metabolites that were present in all three. Nutrient restriction was associated with changes in amino acid, carbohydrate, lipid, and transulfuration/methionine metabolic pathways, some of which were alleviated by realimentation. Fetal age also affected metabolite abundance. The differential abundance of metabolites involved in amino acid, methionine, betaine, and bile acid metabolism could impact fetal epigenetic regulation, protein synthesis, lipid metabolism, and signaling associated with glucose and lipid metabolism.

Betulin: a novel triterpenoid anti-cancerous agent targeting cervical cancer through epigenetic proteins.

Worldwide, cervical cancer (CCa) is a major killer of women. As the conventional drugs used to treat cervical cancer are expensive and expose severe side effects, there is a growing demand to search for novel modifications. Therefore, in the current investigation employing a bioinformatic approach, we explored triterpenoids for their anti-cancer efficacy by targeting cervical cancer epigenetic proteins, namely DNMT3A, HDAC4, and KMT2C. The study utilized molecular docking, ADMET assay, Molecular Dynamic simulation, and DFT calculation to unveil Betulin (BE) as the potential lead compound. Comparative analysis with that standard drug indicated that BE has a better glide score with the target protein KM2TC (- 9.893 kcal/mol), HDAC4 (- 9.720 kcal/mol), and DNMT3A (- 7.811 kcal/mol), which depicts that BE could be a potent inhibitor of these three epigenetic proteins and exhibits favorable pharmacokinetic, pharmacodynamics and toxicity properties. Molecular Dynamics simulation revealed noteworthy structural stability and compactness. DFT analysis revealed higher molecular activity of BE and showed the most increased kinetic stability (δE = 0.254647 eV). Further, we employed In vitro analysis through MTT assay and found that BE has IC50 of 15 µg/ml. In conclusion, BE can potentially treat CCa upon further investigations using in vivo models for better understanding.

SMCHD1 activates the expression of genes required for the expansion of human myoblasts.

SMCHD1 is an epigenetic regulatory protein known to modulate the targeted repression of large chromatin domains. Diminished SMCHD1 function in muscle fibers causes Facioscapulohumeral Muscular Dystrophy (FSHD2) through derepression of the D4Z4 chromatin domain, an event which permits the aberrant expression of the disease-causing gene DUX4. Given that SMCHD1 plays a broader role in establishing the cellular epigenome, we examined whether loss of SMCHD1 function might affect muscle homeostasis through additional mechanisms. Here we show that acute depletion of SMCHD1 results in a DUX4-independent defect in myoblast proliferation. Genomic and transcriptomic experiments determined that SMCHD1 associates with enhancers of genes controlling cell cycle to activate their expression. Amongst these cell cycle regulatory genes, we identified LAP2 as a key target of SMCHD1 required for the expansion of myoblasts, where the ectopic expression of LAP2 rescues the proliferation defect of SMCHD1-depleted cells. Thus, the epigenetic regulator SMCHD1 can play the role of a transcriptional co-activator for maintaining the expression of genes required for muscle progenitor expansion. This DUX4-independent role for SMCHD1 in myoblasts suggests that the pathology of FSHD2 may be a consequence of defective muscle regeneration in addition to the muscle wasting caused by spurious DUX4 expression.

A Review of the Bromodomain and Extraterminal Domain Epigenetic Reader Proteins: Function on Virus Infection and Cancer.

The BET (bromodomain and extraterminal domain) family of proteins, particularly BRD4 (bromodomain-containing protein 4), plays a crucial role in transcription regulation and epigenetic mechanisms, impacting key cellular processes such as proliferation, differentiation, and the DNA damage response. BRD4, the most studied member of this family, binds to acetylated lysines on both histones and non-histone proteins, thereby regulating gene expression and influencing diverse cellular functions such as the cell cycle, tumorigenesis, and immune responses to viral infections. Given BRD4's involvement in these fundamental processes, it is implicated in various diseases, including cancer and inflammation, making it a promising target for therapeutic development. This review comprehensively explores the roles of the BET family in gene transcription, DNA damage response, and viral infection, discussing the potential of targeted small-molecule compounds and highlighting BET proteins as promising candidates for anticancer therapy.

Molecular dynamics simulations of nucleosomes are coming of age

AbstractUnderstanding the function of eukaryotic genomes, including the human genome, is undoubtedly one of the major scientific challenges of the 21st century. The cornerstone of eukaryotic genome organization is nucleosomes—elementary building blocks of chromatin about 10 nm in size that wrap DNA around an octamer of histone proteins. Nucleosomes are integral players in all genomic processes, including transcription, DNA replication and repair. They mediate genome regulation at the epigenetic level, bridging the discrete nature of the genetic information encoded in DNA with the analog physical nature of the intermolecular interactions required to access that information. Due to their relatively large size and dynamic nature, nucleosomes are difficult objects for experimental characterization. Molecular dynamics (MD) simulations have emerged over the years as a useful tool to complement experimental studies. Particularly in recent years, advances in computing power, refinement of MD force fields and codes have opened up new frontiers in terms of simulation timescales and quality for nucleosomes and related systems. It has become possible to elucidate in atomistic detail their functional dynamics modes such as DNA unwrapping and sliding, to characterize the effects of epigenetic modifications, DNA and protein sequence variation on nucleosome structure and stability, to describe the mechanisms governing nucleosome interactions with chromatin‐associated proteins and the formation of supranucleosome structures. In this review, we systematically analyzed all‐atom MD simulation studies of nucleosomes and related structures published since 2018 and discussed their relevance in the context of older studies, experimental data, and related coarse‐grained and multiscale studies.This article is categorized under: Software > Molecular Modeling Molecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo Methods Structure and Mechanism > Computational Biochemistry and Biophysics

Alternative splicing of lncRNA LAIR fine-tunes the regulation of neighboring yield-related gene LRK1 expression in rice.

The diversity in alternative splicing of long noncoding RNAs (lncRNAs) poses a challenge for functional annotation of lncRNAs. Moreover, little is known on the effects of alternatively spliced lncRNAs on crop yield. In this study, we cloned nine isoforms resulting from the alternative splicing of the lncRNA LAIR in rice. The LAIR isoforms are generated via alternative 5'/3' splice sites and different combinations of specific introns. All LAIR isoforms activate the expression of the neighboring LRK1 gene and enhance yield-related rice traits. In addition, there are slight differences in the binding ability of LAIR isoforms to the epigenetic modification-related proteins OsMOF and OsWDR5, which affect the enrichment of H4K16ac and H3K4me3 at the LRK1 locus, and consequently fine-tune the regulation of LRK1 expression and yield-related traits. These differences in binding may be caused by polymorphic changes to the RNA secondary structure resulting from alternative splicing. It was also observed that the composition of LAIR isoforms was sensitive to abiotic stress. These findings suggest that the alternative splicing of LAIR leads to the formation of a functional transcript population that precisely regulates yield-related gene expression, which may be relevant for phenotypic polymorphism-based crop breeding under changing environmental conditions.

Physical modeling of nucleosome clustering in euchromatin resulting from interactions between epigenetic reader proteins

Euchromatin is an accessible phase of genetic material containing genes that encode proteins with increased expression levels. The structure of euchromatin in vitro has been described as a 30-nm fiber formed from ordered nucleosome arrays. However, recent advances in microscopy have revealed an in vivo euchromatin architecture that is much more disordered, characterized by variable-length linker DNA and sporadic nucleosome clusters. In this work, we develop a theoretical model to elucidate factors contributing to the disordered in vivo architecture of euchromatin. We begin by developing a 1D model of nucleosome positioning that captures the interactions between bound epigenetic reader proteins to predict the distribution of DNA linker lengths between adjacent nucleosomes. We then use the predicted linker lengths to construct 3D chromatin configurations consistent with the physical properties of DNA within the nucleosome array, and we evaluate the distribution of nucleosome cluster sizes in those configurations. Our model reproduces experimental cluster-size distributions, which are dramatically influenced by the local pattern of epigenetic marks and the concentration of reader proteins. Based on our model, we attribute the disordered arrangement of euchromatin to the heterogeneous binding of reader proteins and subsequent short-range interactions between bound reader proteins on adjacent nucleosomes. By replicating experimental results with our physics-based model, we propose a mechanism for euchromatin organization in the nucleus that impacts gene regulation and the maintenance of epigenetic marks.

Asymmetric allostery in estrogen receptor-α homodimers drives responses to the ensemble of estrogens in the hormonal milieu

The estrogen receptor-α (ER) is thought to function only as a homodimer but responds to a variety of environmental, metazoan, and therapeutic estrogens at subsaturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations-receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining the binding of the same ligand in crystal structures of ER in the agonist vs. antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist vs. antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from the ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric vs. dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing different modes for ligand-dependent regulation of ER activity.

The detection of a strong episignature for Chung–Jansen syndrome, partially overlapping with Börjeson–Forssman–Lehmann and White–Kernohan syndromes

Chung-Jansen syndrome is a neurodevelopmental disorder characterized by intellectual disability, behavioral problems, obesity and dysmorphic features. It is caused by pathogenic variants in the PHIP gene that encodes for the Pleckstrin homology domain-interacting protein, which is part of an epigenetic modifier protein complex. Therefore, we hypothesized that PHIP haploinsufficiency may impact genome-wide DNA methylation (DNAm). We assessed the DNAm profiles of affected individuals with pathogenic and likely pathogenic PHIP variants with Infinium Methylation EPIC arrays and report a specific and sensitive DNAm episignature biomarker for Chung–Jansen syndrome. In addition, we observed similarities between the methylation profile of Chung–Jansen syndrome and that of functionally related and clinically partially overlapping genetic disorders, White–Kernohan syndrome (caused by variants in DDB1 gene) and Börjeson–Forssman–Lehmann syndrome (caused by variants in PHF6 gene). Based on these observations we also proceeded to develop a common episignature biomarker for these disorders. These newly defined episignatures can be used as part of a multiclass episignature classifier for screening of affected individuals with rare disorders and interpretation of genetic variants of unknown clinical significance, and provide further insights into the common molecular pathophysiology of the clinically-related Chung–Jansen, Börjeson–Forssman–Lehmann and White–Kernohan syndromes.

Chronic kidney disease activates the HDAC6-inflammatory axis in the heart and contributes to myocardial remodeling in mice: inhibition of HDAC6 alleviates chronic kidney disease-induced myocardial remodeling.

Chronic kidney disease (CKD) adversely affects the heart. The underlying mechanism and the interplay between the kidney and the heart are still obscure. We examined the cardiac effect using the unilateral ureteral obstruction (UUO)-induced CKD pre-clinical model in mice. Echocardiography, histopathology of the heart, myocardial mRNA expression of ANP and BNP, the extent of fibrotic (TGF-β, α-SMA, and collagen I) and epigenetic (histone deacetylases, namely HDAC3, HDAC4, and HDAC6) proteins, and myocardial inflammatory response were assessed. Six weeks of post-UUO surgery, we observed a compromised left-ventricular wall thickness and signs of cardiac hypertrophy, accumulation of fibrosis associated, and inflammatory proteins in the heart. In addition, we observed a perturbation of epigenetic proteins, especially HDAC3, HDAC4, and HDAC6, in the heart. Pharmacological inhibition of HDAC6 using ricolinostat (RIC) lessened cardiac damage and improved left-ventricular wall thickness. The RIC treatment substantially restored the serum cardiac injury markers, namely creatine kinase-MB and lactate dehydrogenase (LDH) activities, ANP and BNP mRNA expression, and heart histological changes. The extent of myocardial fibrotic proteins, phospho-NF-κB (p65), and pro-inflammatory cytokines (TNF-α, IL-18, and IL-1β) were significantly decreased in the RIC treatment group. Further findings revealed the CKD-induced infiltration of CD3, CD8a, CD11c, and F4/80 positive inflammatory cells in the heart. Treatment with RIC substantially reduced the myocardial infiltration of these inflammatory cells. From these findings, we believe that CKD-induced myocardial HDAC6 perturbation has a deteriorative effect on the heart, and inhibition of HDAC6 can be a promising approach to alleviate CKD-induced myocardial remodeling.

PharmaCore: The Automatic Generation of 3D Structure-Based Pharmacophore Models from Protein/Ligand Complexes.

In this work, we present PharmaCore: a new, completely automatic workflow aimed at generating three-dimensional (3D) structure-based pharmacophore models toward any target of interest. The proposed approach relies on using cocrystallized ligands to create the input files for generating the pharmacophore hypotheses, integrating not only the three-dimensional structural information on the ligand but also data concerning the binding mode of these molecules put in the protein cavity. We developed a Python library that, starting from the specific UniProt ID of the protein under investigation as the only element that requires user intervention, subsequently collects and aligns the corresponding structures bearing a known ligand in a fully automated fashion, bringing them all into the same coordinate system. The protocol includes a final phase in which the aligned small molecules are used to produce the pharmacophore hypotheses directly onto the protein structure using a specific software, e.g., Phase (Schrödinger LLC). To validate the entire procedure and highlight the possible applications in the field of drug discovery and repositioning, we first generated pharmacophores for soluble epoxide hydrolase (sEH) and compared with already-published ones. Then, we reproduced the binding profile of a reported selective binder of ATAD2 bromodomain (AM879), testing it against a panel of 1741 pharmacophores related to 16 epigenetic proteins and automatically generated with PharmaCore, finally disclosing putative unprecedented off-targets. The computational predictions were successfully validated with AlphaScreen assays, highlighting the applicability of the proposed workflow in drug discovery and repositioning. Finally, the process was also validated on tankyrase 2 and SARS-CoV-2 MPro, confirming the robustness of PharmaCore.

LINC00152: Potential driver oncogene in pan-cancer.

Long noncoding RNAs (lncRNA) are a class of non-coding RNAs greater than 200 bp in length with limited peptide-coding function. The transcription of LINC00152 is derived from chromosome 2p11.2. Many studies prove that LINC00152 influences the progression of various tumors via promoting the tumor cells malignant phenotype, chemoresistance, and immune escape. LINC00152 is regulated by multiple transcription factors and DNA hypomethylation. In addition, LINC00152 participates in the regulation of complex molecular signaling networks through epigenetic regulation, protein interactions, and competitive endogenous RNA (ceRNA). Here, we provide a systematic review of the upstream regulatory factors of LINC00152 expression level in different types of tumors. In addition, we revisit the main functions and mechanisms of LINC00152 as driver oncogene and biomarker in pan-cancer. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Methods > RNA Analyses in Cells RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

DEEP-EP: Identification of epigenetic protein by ensemble residual convolutional neural network for drug discovery

Epigenetic proteins (EP) play a role in the progression of a wide range of diseases, including autoimmune disorders, neurological disorders, and cancer. Recognizing their different functions has prompted researchers to investigate them as potential therapeutic targets and pharmacological targets. This paper proposes a novel deep learning-based model that accurately predicts EP. This study introduces a novel deep learning-based model that accurately predicts EP. Our approach entails generating two distinct datasets for training and evaluating the model. We then use three distinct strategies to transform protein sequences to numerical representations: Dipeptide Deviation from Expected Mean (DDE), Dipeptide Composition (DPC), and Group Amino Acid (GAAC). Following that, we train and compare the performance of four advanced deep learning models algorithms: Ensemble Residual Convolutional Neural Network (ERCNN), Generative Adversarial Network (GAN), Convolutional Neural Network (CNN), and Gated Recurrent Unit (GRU). The DDE encoding combined with the ERCNN model demonstrates the best performance on both datasets. This study demonstrates deep learning's potential for precisely predicting EP, which can considerably accelerate research and streamline drug discovery efforts. This analytical method has the potential to find new therapeutic targets and advance our understanding of EP activities in disease.

Abstract 3314: Molecular profiling of covalent menin inhibitor, BMF-219, in KRAS-mutated solid tumors

Abstract Menin is an epigenetic protein that drives oncogenic function through transcriptional control directed by its various cofactors such as MLL1 (KMT2A) and MYC. BMF-219 is a covalent menin inhibitor currently under investigation in a dose-finding study evaluating safety and tolerability in adults with KRAS-driven Non-Small Cell Lung Cancer (NSCLC), Pancreatic Adenocarcinoma (PDAC), or Colorectal Cancer (CRC) (NCT NCT05631574). We previously showed that BMF-219 induces effective killing of solid tumor cells irrespective of their KRAS mutation subtype (AACR 2022, abstract 2665) and perturbs genomic binding of MYC and its binding partner MAX, in acute leukemia cells (ASH Abstract 2021). To probe into the underlying mechanism and the potential pathways engaged, BMF-219-induced effects were evaluated in CRC, PDAC and NSCLC cell lines. Cells treated with relevant concentrations of BMF-219 versus vehicle were assessed for menin and MYC protein levels, along with treatment effects on cell cycle and induction of apoptosis. In addition, bulk RNA-seq analysis was performed on the cell lines treated with BMF-219 in a time course study. BMF-219 induces a dose-dependent decrease in menin protein levels in the solid tumor cell lines tested following 24 hours treatment, with the least impact observed in a KRAS WT cell line. The effect of BMF-219 on c-MYC protein levels were variable and highly cell-type dependent, with the CRC metastatic tumor cell line SW620 exhibiting the highest reduction. Differential expression (DE) analysis of bulk RNA-seq data comparing BMF-219 treated versus vehicle treated cells, identified significant alterations in transcript abundance at 6- and 24-hours post treatment. The magnitude of change in the transcript abundance showed a dose-dependent response without significant alteration of a panel of housekeeping genes. GSEA analysis identified pathways associated with cell cycle, cell division and MYC-regulated were significantly differentially expressed across multiple cell lines. These data suggest similar molecular pathways are altered by BMF-219 across KRAS-mutated solid tumor cells. Additional insights into key pathways and modulators will be reported. Collectively, these data suggest that the functional pathway alterations induced by BMF-219 in solid tumor cells are likely pan-KRAS and are similar across different solid tumor indications. This provides further support for advancing the clinical investigation of BMF-219 in KRAS-driven solid tumors. Citation Format: Amin A. Momin, Tripta Rughwani, Nida Tanataweethum, Kasia Mordec, Raechel Lin, Megan Chin, Daniel Lu, Priyanka Somanath, Mini Balakrishnan, Thomas Butler. Molecular profiling of covalent menin inhibitor, BMF-219, in KRAS-mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3314.

Abstract 3226: PRMT1 is a critical dependency in clear cell renal cell carcinoma with roles in post- transcriptional regulation and DNA damage response

Abstract The majority of sporadic renal cell carcinoma (RCC) cases present as the clear cell subtype (ccRCC) and are associated with the biallelic inactivation of the von Hippel-Lindau (VHL) gene, leading to constitutive activation of the cellular hypoxia response and deleterious alterations to gene expression, metabolism, and growth. Despite the near ubiquity of VHL loss, evidence suggests that it alone is insufficient to drive tumor establishment. Additional loss of function (LOF) mutations in epigenetic regulatory proteins including PBRM1, SETD2 and BAP1 have been identified as tumor drivers. The frequency and penetrance of these mutations in epigenetic regulators implicates a vulnerability in ccRCC that may be exploited to develop new therapies. To explore this vulnerability, the Ailles lab completed a screen of the Structural Genomics Consortium’s (SGC) epiprobe library - a panel of inhibitors targeting an array of epigenetic regulators in patient derived ccRCC models. Among the several promising targets identified, a particularly favorable inhibition profile was noted for MS023, an inhibitor of the type I protein arginine methyltransferase family (PRMT1/3/4/6 and 8). PRMTs transfer methyl groups to nuclear histones and cytoplasmic targets, influencing gene expression, mRNA processing and viability. This growth inhibition was corroborated using an additional type I PRMT compound, GSK3368715. CRISPR-mediated deletion of type I PRMT targets indicated that PRMT1 is the main enzyme responsible for growth inhibition in our ccRCC models. Furthermore, genetic depletion of PRMT1 via shRNA phenocopied pharmacological inhibition and proliferative growth was restored in the presence of MS023 by overexpressing PRMT1. Finally, MS023 treatment of mice bearing ccRCC cell line and patient derived xenografts led to significant inhibition of tumor growth in vivo. Transcriptomic profiling of chemical treatment and PRMT1 genetic depletion demonstrated that PRMT1 inhibition triggers a significant down regulation of genes related to cell cycle progression and DNA damage. ccRCC cells treated with MS023 accumulated significant levels of DNA damage before undergoing cell death, with several DDR-related proteins being down-regulated. Proximity ligation and proteomic analysis (Bio-ID) revealed that PRMT1’s interactors skew heavily towards RNA metabolism regulators and widespread alterations in mRNA processing were noted with MS023 treatment. Evidence continues to mount that PRMT1 is implicated in the biology of cancer, but to our knowledge, no thorough investigations have been performed in the context of ccRCC. Our data suggests that inhibition of PRMT1 inhibits processing of mRNA transcripts, compromises DDR mechanisms, leading to an accumulation of double stranded breaks, cytostasis and cell death. Thus PRMT1 represents a viable therapeutic target in ccRCC. Citation Format: Joseph Walton, Karen Arevalo, Angel SN Ng, Jalna Meens, Christina Karamboulas, Panagiotis Prinos, Brian Raught, Eric Chen, Cheryl Arrowsmith, Laurie Ailles. PRMT1 is a critical dependency in clear cell renal cell carcinoma with roles in post- transcriptional regulation and DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3226.

Abstract 3239: Targeting the GAS41 YEATS domain in non-small cell lung cancer with novel small molecules

Abstract Background: Glioma amplified sequence 41 (GAS41) belongs to the Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain family of epigenetic reader proteins. This domain preferentially recognizes acetylated lysine residues on chromatin to facilitate gene expression by recruiting chromatin remodeling and transcription activating complexes. The YEATS4 gene encoding for GAS41 protein is frequently amplified in NSCLC patient samples relative to normal lung tissue, and GAS41 protein is overexpressed in NSCLC cells. In knockdown experiments using NSCLC xenograft mouse models, GAS41 has been shown to be essential for NSCLC tumor growth. In mutagenesis studies, the YEATS domain residues responsible for acetylation reader activity were found to be essential for NSCLC cellular proliferation and tumor progression. Thus, the YEATS domain of GAS41 represents an attractive target for the development of small-molecule inhibitors as potential therapeutics for NSCLC. Results: We developed the first-in-class small-molecule inhibitors for the GAS41 YEATS domain. Using an NMR-based fragment screen, we identified a hit with low millimolar binding affinity. Structure-based drug design followed by extensive medicinal chemistry optimization yielded the potent and selective inhibitor DLG-369 with sub-micromolar inhibition of the GAS41 YEATS domain interaction with acylated histones. Using the NanoBRET platform, we showed that DLG-369 selectively disrupts the association of GAS41 YEATS with chromatin in mammalian cells. Furthermore, DLG-369 inhibits proliferation of NSCLC cell lines without affecting normal lung fibroblasts. In transcriptomic experiments, DLG-369 treatment regulates expression of multiple genes in NSCLC cells, including upregulation of the critical tumor suppressor genes CDKN1A and CYR61 and downregulation of chemokine genes CXCL5 and CXCR4 associated with NSCLC progression. Conclusions: We developed DLG-369, a potent inhibitor of the GAS41 YEATS domain with activity in NSCLC cellular models. DLG-369 potently binds to the YEATS domain acetylation reader pocket and disrupts GAS41 recognition of acetylation in cells. DLG-369 potently reduces proliferation and drives differential expression of key genes associated with NSCLC. DLG-369 can serve as a chemical probe to further interrogate the biological role of the GAS41 YEATS domain in NSCLC and other cancers for potential therapeutic implications. Citation Format: Alyssa Winkler, Dymytrii Listunov, Brian Linhares, Joshua Ray, Sidney Weaver, Sergey Zolov, Venkat Keshamouni, Jolanta Grembecka, Tomaszc Cierpicki. Targeting the GAS41 YEATS domain in non-small cell lung cancer with novel small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3239.