Histone Methyltransferase Inhibitor Research Articles

The HIF2α-dependent Upregulation of SETDB1 Facilitates Hypoxia-induced Functional and Phenotypical Changes of Pulmonary Microvascular Endothelial Cells.

Background: Emerging studies have reported the vital role of histone modification in the dysfunction of pulmonary vascular endothelial cells, which acts as the key reason to drive the hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension (PH). This study aims to investigate the role of a histone 3 lysine 9 (H3K9) methyltransferase, SET domain bifurcated 1 (SETDB1), in hypoxia-induced functional and phenotypical changes of pulmonary vascular endothelial cells. Methods: Primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were used as cell model. Specific knockdown and overexpression strategies were used to systematically determine the molecular regulation and function of SETDB1 in PMVECs. Results: SETDB1 is highly expressed and significantly upregulated in the pulmonary vascular endothelium of lung tissue isolated from SU5416/hypoxia-induced PH (SuHx-PH) rats, and also in pulmonary arterial endothelial cells (PAECs) from idiopathic pulmonary arterial hypertension (IPAH) patients, comparing to their respective controls. In primarily cultured rat PMVECs, treatment of hypoxia or CoCl2 induces significant upregulation of HIF2α, SETDB1 and H3K9me3. Specific knockdown and overexpression strategies indicate the hypoxia- or CoCl2-induced upregulation of SETDB1 is mediated through a HIF2α-dependent mechanism. Knockdown of SETDB1 significantly inhibits the hypoxia- or CoCl2-induced apoptosis, senescence and endothelial to mesenchymal transition (EndoMT) in rat PMVECs. Moreover, treatment of the specific inhibitor of histone methyltransferase, Chaetocin, effectively attenuates the disease pathogenesis of SuHx-PH in rat. Conclusions: Our results suggest that the HIF2α-dependent upregulation of SETDB1 facilitates hypoxia-induced functional and phenotypical changes of PMVECs, potentially contributing to the hypoxia-induced pulmonary vascular remodeling and PH.

Combined inhibition of histone methyltransferases EZH2 and DOT1L is an effective therapy for neuroblastoma.

The child cancer, neuroblastoma (NB), is characterised by a low incidence of mutations and strong oncogenic embryonal driver signals. Many new targeted epigenetic modifier drugs have failed in human trials as monotherapy. We performed a high-throughput, combination chromatin-modifier drug screen against NB cells. We screened 13 drug candidates in 78 unique combinations. We found that the combination of two histone methyltransferase (HMT) inhibitors: GSK343, targeting EZH2, and SGC0946, targeting DOT1L, demonstrated the strongest synergy across 8 NB cell lines, with low normal fibroblast toxicity. High mRNA expression of both EZH2 and DOT1L in NB tumour samples correlated with the poorest patient survival. Combination HMT inhibitor treatment caused activation of ATF4-mediated endoplasmic reticulum (ER) stress responses. In addition, glutathione and several amino acids were depleted by HMT inhibitor combination on mass spectrometry analysis. The combination of SGC0946 and GSK343 reduced tumour growth in comparison to single agents. Our results support further investigation of HMT inhibitor combinations as a therapeutic approach in NB.

Histone Methylation-Mediated Reproductive Toxicity to Consumer Product Chemicals in Caenorhabditis elegans: An Epigenetic Adverse Outcome Pathway (AOP).

The significance of histone methylation in epigenetic inheritance underscores its relevance to disease and the chronic effects of environmental chemicals. However, limited evidence of the causal relationships between chemically induced epigenetic changes and organismal-level effects hinders the application of epigenetic markers in ecotoxicological assessments. This study explored the contribution of repressive histone marks to reproductive toxicity induced by chemicals in consumer products in Caenorhabditis elegans, applying the adverse outcome pathway (AOP) framework. Triclosan (TCS) and tetrabromobisphenol A (TBBPA) exposures caused reproductive toxicity and altered histone methyltransferase (HMT) and histone demethylase (HDM) activities, increasing the level of trimethylation of H3K9 and H3K27. Notably, treatment with an H3K27-specific HMT inhibitor alleviated reproductive defects and the transcriptional response of genes related to vitellogenin, xenobiotic metabolism, and oxidative stress. Comparison of points of departure (PODs) based on calculated benchmark concentrations (BMCs) revealed the sensitivity of histone-modifying enzyme activities to these chemicals. Our findings suggest that the 'disturbance of HMT and HDM' can serve as the molecular initiating event (MIE) leading to reproductive toxicity in the epigenetic AOP for TCS and TBBPA. The study extended the biological applicability of these enzymes by identifying model species with analogous protein sequences and functions. This combined approach enhances the essentiality, empirical support, and taxonomic domain of applicability (tDOA), which are crucial considerations for ecotoxicological AOPs. Given the widespread use and environmental distribution of chemicals in consumer products, this study proposes histone-modifying enzyme activity as an effective screening tool for reproductive toxicants and emphasizes the integration of epigenetic mechanisms into a prospective ERA.

3-Deazaneplanocin A (DZNep): A Drug That Deserves a Second Look.

The emerging data compiled during the past five years on 3-deazaneplanocin (DZNep) provide compelling evidence to reevaluate this drug as a better alternative over the specific catalytic inhibitors of histone methyl transferases (HTMs). The indirect mechanism of DZNep via inhibition of AdoHcy-ase, once considered a liability due to possible side effects, has now shown to be rather beneficial as additional pathways targeted by DZNep are important contributors to its superior anticancer properties. Furthermore, DZNep has demonstrated the ability to induce proteasomal degradation of its target and reduce toxicity in combination with well-established antitumor therapies in animal models. In addition, DZNep has shown important effects in suppressing fibrosis and inflammation in liver, kidney, peritoneum, and airways. Finally, inhibition of mRNA m6A methylation by DZNep suppresses the synthesis of the viral genome in SARS-Cov-2 infection and promises to have important therapeutic value when combined with its potent antiviral efficacy and anti-inflammatory effects.

Epigenetic silencing of ZIC4 unveils a potential tumor suppressor role in pediatric choroid plexus carcinoma

Zic family member ZIC4 is a transcription factor that has been shown to be silenced in several cancers. However, understanding the regulation and function of ZIC4 in pediatric choroid plexus tumors (CPTs) remained limited. This study employed data mining and bioinformatics analysis to investigate the DNA methylation status of ZIC4 in CPTs and its correlation with patient survival. Our results unveiled ZIC4 methylation as a segregating factor, dividing CPT cohorts into two clusters, with hyper-methylation linked to adverse prognosis. Hyper-methylation of ZIC4 was confirmed in a choroid plexus carcinoma-derived cell line (CCHE-45) by bisulfite sequencing. Furthermore, our study demonstrated that demethylating agent and a histone methyltransferase inhibitor could reverse ZIC4 silencing. RNA sequencing and proteomic analysis showed that ZIC4 over-expression influenced genes and proteins involved in immune response, antigen processing and presentation, endoplasmic reticulum stress, and metabolism. Functionally, re-expressing ZIC4 negatively impacted cell proliferation and migration. Ultimately, these findings underscore ZIC4 hyper-methylation as a prognostic marker in CPTs and shed light on potential mechanisms underlying its tumor suppressor role in CPC. This insight paves the way for novel therapeutic targets in treating aggressive CPTs.

A Live-Cell Epigenome Manipulation by Photo-Stimuli-Responsive Histone Methyltransferase Inhibitor.

Post-translational modifications on the histone H3 tail regulate chromatin structure, impact epigenetics, and hence the gene expressions. Current chemical modulation tools, such as unnatural amino acid incorporation, protein splicing, and sortase-based editing, have allowed for the modification of histones with various PTMs in cellular contexts, but are not applicable for editing native chromatin. The use of small organic molecules to manipulate histone-modifying enzymes alters endogenous histone PTMs but lacks precise temporal and spatial control. To date, there has been no achievement in modulating histone methylation in living cells with spatiotemporal resolution. In this study, a new method is presented for temporally manipulating histone dimethylation H3K9me2 using a photo-responsive inhibitor that specifically targets the methyltransferase G9a on demand. The photo-caged molecule is stable under physiological conditions and cellular environments, but rapidly activated upon exposure to light, releasing the bioactive component that can immediately inhibit the catalytic ability of the G9a in vitro. Besides, this masked compound could also efficiently reactivate the inhibition of methyltransferase activity in living cells, subsequently suppress H3K9me2, a mark that regulates various chromatin functions. Therefore, the chemical system will be a valuable tool for manipulating the epigenome for therapeutic purposes and furthering the understanding of epigenetic mechanisms.

Epigenetic Modulations in Breast Cancer: An Emerging Paradigm in Therapeutic Implications.

Breast cancer, a heterogeneous and intricate disease, ranks among the leading causes of mortality in women. Restricted therapeutic choices, drug resistance, recurrence, and metastasis are the predominant conditions that lead to mortality. Accumulating evidence has shown breast cancer initiation and progression happen through a multifaceted and intricate process that involves numerous genetic and epigenetic alterations. The modulation of gene expression through epigenetic modifications, encompassing DNA methylation, histone alterations, and non-coding RNA regulation, has emerged as a fascinating field that represents a new avenue for breast cancer therapy. This review emphasizes various aberrant epigenetic regulations implicated in the onset and advancement of breast cancer. The critical epigenetic modifications closely associated with estrogen signaling, epithelial-to-mesenchymal transition (EMT), cancer stemness, and drug resistance have been discussed extensively. Moreover, it highlights current epi-drugs, including DNA modifying agents, histone acetyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors, and histone demethyltransferase inhibitors used for breast cancer treatment. Nonetheless, we described current investigations pertaining to combination therapy employing epi-drugs and future challenges.

Inhibition of histone methyltransferase promotes cognition and mitochondrial function in vascular dementia model

Vascular dementia (VD) is one of the most common forms of dementia worldwide, characterized by problems with reasoning, planning, judgment, and memory. This study investigated the effect of a histone methyltransferase inhibitor on cognition and mitochondrial function in a rat model of VD, as well as its impact on H2O2-induced neurotoxicity in hippocampal neuronal cultures. In the in vivo experiments, VD was induced by bilateral occlusion of the common carotid artery (CCA) for one month. The histone methyltransferase inhibitor, BIX01294, was administered intracerebroventricularly for one month (22.5 µg.kg−1 three times/week). On day 30, behavioral tests, including the novel object recognition test and elevated plus maze test, were conducted. Mitochondrial enzyme activities, including aconitase, α-ketoglutarate dehydrogenase (α-KG), complex I, and complex IV, were evaluated in the hippocampus of rats following CCA ligation. In the in vitro experiments, the effect of BIX01294 (50–600 μM) on H2O2 (400 µM)-induced cytotoxicity in hippocampal neuronal cells was assessed using the MTT assay. Flow cytometry was performed to evaluate apoptosis. Our findings revealed that BIX01294 effectively improved memory function, Krebs cycle enzyme activity, and mitochondrial function in the rat model of VD. Moreover, in vitro results showed that BIX01294 at a concentration of 100 µM significantly reversed the cytotoxicity and apoptosis induced by H2O2 in neuronal cells. These findings suggest that BIX01294 may have the potential to improve VD complications by reducing oxidative stress and inhibiting histone methylation.

SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

Ewing’s sarcoma (ES) represents a rare yet exceedingly aggressive neoplasm that poses a significant health risk to the pediatric and adolescent population. The clinical outcomes for individuals with relapsed or refractory ES are notably adverse, primarily attributed to the constrained therapeutic alternatives available. Despite significant advancements in the field, molecular pathology-driven therapeutic strategies have yet to achieve a definitive reduction in the mortality rates associated with ES. Consequently, there exists an imperative need to discover innovative therapeutic targets to effectively combat ES. To reveal the mechanism of the SETD8 (also known as lysine methyltransferase 5A) inhibitor UNC0379, cell death manners were analyzed with different inhibitors. The contributions of SETD8 to the processes of apoptosis and ferroptosis in ES cells were evaluated employing the histone methyltransferase inhibitor UNC0379 in conjunction with RNA interference techniques. The molecular regulatory mechanisms of SETD8 in ES were examined through the application of RNA sequencing (RNA-seq) and mass spectrometry-based proteomic analysis. Moreover, nude mouse xenograft models were established to explore the role of SETD8 in ES in vivo. SETD8, a sole nucleosome-specific methyltransferase that catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1), was found to be upregulated in ES, and its overexpression was associated with dismal outcomes of patients. SETD8 knockdown dramatically induced the apoptosis and ferroptosis of ES cells in vitro and suppressed tumorigenesis in vivo. Mechanistic investigations revealed that SETD8 facilitated the nuclear translocation of YBX1 through post-transcriptional regulatory mechanisms, which subsequently culminated in the transcriptional upregulation of RAC3. In summary, SETD8 inhibits the apoptosis and ferroptosis of ES cells through the YBX1/RAC3 axis, which provides new insights into the mechanism of tumorigenesis of ES. SETD8 may be a potential target for clinical intervention in ES patients.

Involvement of dysregulated hippocampal histone H3K9 methylation at the promoter of the BDNF gene in impaired memory extinction

RationaleSince the precise mechanisms of posttraumatic stress disorder (PTSD) remain unknown, effective treatment interventions have not yet been established. Impaired extinction of fear memory (EFM) is one of the core symptoms of PTSD and is associated with stress-induced epigenetic change in gene expression.ObjectivesIn this study, we examined whether the involvement of histone H3 lysine 9 dimethylation (H3K9me2) in EFM is mediated through brain-derived neurotrophic factor (BDNF) expression in the hippocampus, and whether BIX01294, a selective G9a and GLP histone methyltransferase inhibitor, could be treatment for impaired EFM in an animal model of PTSD.MethodsThe single prolonged stress (SPS) paradigm was used to model PTSD. We measured BDNF mRNA levels by RT-PCR, and H3K9me2 levels in the BDNF gene promoters by chromatin immunoprecipitation-qPCR. After undergoing contextual fear conditioning and hippocampal injection of BIX01294, male rats were subjected to extinction training and extinction testing and their freezing times and BDNF mRNA levels were measured.ResultsCompared to sham rats, SPS rats showed decreased BDNF mRNA levels 2 h after extinction training, no significant changes in levels of global H3K9me2 prior to extinction training, and increased levels of H3K9me2 in BDNF gene promoter IV, but not in BDNF gene promoter I. Administration of BIX01294 ameliorated the decrease in BDNF mRNA levels 2 h after extinction training and subsequently alleviated impaired EFM in extinction tests in SPS rats.ConclusionWe conclude that reduced hippocampal levels of BDNF mRNA due to increase in H3K9me2 levels may play a role in PTSD-associated EFM impairment, and BIX01294 could be a PTSD treatment option.

T Cells Spatially Regulate B Cell Receptor Signaling in Lymphomas through H3K9me3 Modifications.

Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is a subtype associated with poor survival outcomes. Despite identifying therapeutic targets through molecular characterization, targeted therapies have limited success. New strategies using immune-competent tissue models are needed to understand how DLBCL cells evade treatment. Here, synthetic hydrogel-based lymphoma organoids are used to demonstrate how signals in the lymphoid tumor microenvironment (Ly-TME) can alter B cell receptor (BCR) signaling and specific histone modifications, tri-methylation of histone 3 at lysine 9 (H3K9me3), dampening the effects of BCR pathway inhibition. Using imaging modalities, T cells increase DNA methyltransferase 3A expression and cytoskeleton formation in proximal ABC-DLBCL cells, regulated by H3K9me3. Expansion microscopy on lymphoma organoids reveals T cells increase the size and quantity of segregated H3K9me3 clusters in ABC-DLBCL cells. Findings suggest the re-organization of higher-order chromatin structures that may contribute to evasion or resistance to therapy via the emergence of novel transcriptional states. Treating ABC-DLBCL cells with a G9α histone methyltransferase inhibitor reverses T cell-mediated modulation of H3K9me3 and overcomes T cell-mediated attenuation of treatment response to BCR pathway inhibition. This study emphasizes the Ly-TME's role in altering DLBCL fate and suggests targeting aberrant signaling and microenvironmental cross-talk that can benefit high-risk patients.

Advances in Targeted Therapy for Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a rare cancer with high malignancy and aggressiveness on the pleural, caused by the following risk factors including asbestos inhalation, genetic factors, and genetic mutation. The present chemotherapy, antiangiogenic therapy, and immunotherapy methods are ineffective and the survival time of patients is very short. There is an urgent need to find potential therapeutic targets for MPM. At present, it has been found the following types of targets: gene mutation targets such as BRCA associated protein 1 (BAP1) and cyclin-dependent kinase 2A (CDKN2A); epigenetic targets such as lysine (K)-specific demethylase 4A (KDM4A) and lysine-specific demethylase 1 (LSD1), and signal protein targets such as glucose-regulated protein 78 (GRP78) and signal transducer and activator of transcription 3 (STAT3). So far, available clinical trials include phase II clinical trials of histone methyltransferase inhibitor Tazemetostat, poly (ADP-ribose) polymerase (PARP) inhibitor Rucaparib and cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor Abemaciclib, as well as phase I clinical trials of mesothelin-targeting chimeric antigen receptor T-cell immunotherapy (CAR-T) cell injection in the thoracic cavity and TEA domain family member (TEAD) inhibitor VT3989 and IK-930, and the results of these trials have showed certain clinical efficacy. .

The pharmacoepigenetic paradigm in cancer treatment.

Epigenetic modifications, characterized by changes in gene expression without altering the DNA sequence, play a crucial role in the development and progression of cancer by significantly influencing gene activity and cellular function. This insight has led to the development of a novel class of therapeutic agents, known as epigenetic drugs. These drugs, including histone deacetylase inhibitors, histone acetyltransferase inhibitors, histone methyltransferase inhibitors, and DNA methyltransferase inhibitors, aim to modulate gene expression to curb cancer growth by uniquely altering the epigenetic landscape of cancer cells. Ongoing research and clinical trials are rigorously evaluating the efficacy of these drugs, particularly their ability to improve therapeutic outcomes when used in combination with other treatments. Such combination therapies may more effectively target cancer and potentially overcome the challenge of drug resistance, a significant hurdle in cancer therapy. Additionally, the importance of nutrition, inflammation control, and circadian rhythm regulation in modulating drug responses has been increasingly recognized, highlighting their role as critical modifiers of the epigenetic landscape and thereby influencing the effectiveness of pharmacological interventions and patient outcomes. Epigenetic drugs represent a paradigm shift in cancer treatment, offering targeted therapies that promise a more precise approach to treating a wide spectrum of tumors, potentially with fewer side effects compared to traditional chemotherapy. This progress marks a step towards more personalized and precise interventions, leveraging the unique epigenetic profiles of individual tumors to optimize treatment strategies.

Retroelements in targeted antitumor therapy (review)

Background: Pathological expression of retroelements is determined in most malignant tumors, which reflects the pathogenesis of tumors, since activated retrotransposons cause genomic instability due to a cascade of mutual regulatory influence events between transposons, tumor suppressors and oncogenes. These relationships are reflected in the expression profiles of certain long non-coding RNAs and miRNAs in specific tumor tissues, since retroelements are key sources of non-coding RNAs. Since retroelements serve as drivers for the initiation and progression of carcinogenesis, it is promising to use drugs aimed at inhibiting their expression. The aim of the study: To determine the available methods of influencing retroelements in antitumor therapy, to evaluate the ways of their further improvement for the effective treatment of malignant neoplasms. Materials and methods: The databases Scopus, WoS, PubMed were used to analyze the role of retroelements in the development of tumors, methods of influencing them in the treatment of neoplasms. Results: Reverse transcriptase inhibitors have proven to be effective. Silencing of retrotransposons by acting on histone modifiers can lead to a weakening of the antitumor immune response; therefore, this approach requires RNA guides that direct the formation of heterochromatin in the area where strictly specific retroelements are located. A promising method is viral mimicry aimed at activating the interferon and T-cell response to artificially activated endogenous retroviruses. For this purpose, inhibitors of histone methyltransferases, DNA methyltransferases, and histone deacetylases are used. Their joint use is most effective, especially with PD-1 checkpoint blockers. The arrangement of retroelements in gene introns has become the basis for the development of spliceosomal targeted therapy, which also initiates an antitumor immune response to double-stranded RNA transcripts. We suggest the possibility of using antisense oligonucleotides targeting retroelements in anticancer therapy. Such approach is used to treat age-related macular degeneration, Fukuyama muscular dystrophy, amyotrophic lateral sclerosis. Antisense oligonucleotides targeting microRNAs, proto-oncogenes, and oncosuppressors are used in anticancer therapy and are closely related to retroelements, which could become promising targets for antisense oligonucleotides. Conclusion: In antitumor therapy, it is possible to use two diametrically opposed strategies for influencing retroelements: inhibition and activation. The most promising way is a combination of these methods with specific silencing of retrotransposons using guides (complementary RNAs) and stimulation of the expression of elements not involved in carcinogenesis, but activating the immune response.

Abstract 4378: Identification of novel TXNIP-dependent antitumor pathways in breast cancer

Abstract Thioredoxin-interacting protein (TXNIP) plays a critical role in glucose metabolism and redox signaling. TXNIP has recently emerged as a potent tumor suppressor in various cancer models. In previous studies, we found TXNIP was activated by UNC0642, an inhibitor of histone methyltransferase, in MDA-MB-231 breast cancer cells. TXNIP activation was coupled with significant suppression of cell proliferation and tumor growth. In contrast, TXNIP knockdown increased MDA-MB-231 cell proliferation and in tumor growth and metastasis in vivo. TXNIP-reconstitution in TXNIP-deficient HCC1954 cells reduced cell proliferation and migration coupled with increased reactive oxygen species production. TXNIP-reconstitution also significantly decreased mitochondrial respiration, mitochondrial membrane potential and glycolysis in HCC1954 cells. To understand the mechanisms underlying TXNIP’s antitumor activity, we performed co-immunoprecipitation and proteomic analyses to uncover TXNIP-interacting proteins. We identified calpastatin (CAST) as a novel TXNIP-interacting protein in breast cancer cells. CAST is an endogenous inhibitor of calpains, a family of calcium-activated cysteine proteases. Intriguingly, CAST association with TXNIP was readily detectable in TXNIP-low, but not in TXNIP-high breast cancer cells. The disassociation of CAST from TXNIP in TXNIP-high cells might allow CAST to inhibit calpain activity to augment TXNIP’s antitumor function. Through RNAseq analysis, we identified a positive correlation between TXNIP expression and IL-24 activation in various breast cancer cells, which were also confirmed by western blotting. IL-24 has emerged as an important cancer therapy target because of its role in promoting cancer-specific apoptosis. Our data also demonstrated that TXNIP upregulated the level of IL-24 and downregulated STAT-3. Moreover, TXNIP exerts multifaceted effects on BC development through the IL24-STAT3 signaling axis, deepening our understanding of its complex role in the disease. Taken together, these studies identified novel TNXIP-dependent pathways and their potential new mechanisms in breast cancer pathogenesis with significant clinical implications. Citation Format: Jasvinder Singh, Bindeshwar Sah, Robert Clarke, Liang Liu. Identification of novel TXNIP-dependent antitumor pathways in breast cancer [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 4378.

Abstract 444: Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells

Abstract Introduction. A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increases flux through various metabolic reactions including those involved in serine, one-carbon, glycine (SOG) and methionine. We hypothesized that the metabolism of lipids in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing S-adenosylmethionine (SAM) leading to histone methylation increases and changes in gene expression. Methods. MCF-10A cells exposed to octanoic acid (OA) were utilized for U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± the PHGDH inhibitor CBR-5884. ROS-induced redox changes were monitored live in cells transduced with ORP1-roGFP2 vectors. CUT&RUN was performed for H3K4me3. Expression of OA-induced genes was measured by rt-qPCR upon exposure to OA ± CBR-5884 or OA ± the histone methyltransferase (HMT) inhibitor Piribedil. Alkaline comet assay was done to detect DNA breaks. Single-cell RNA-seq (scRNAseq) was performed on tissue-derived breast microstructures exposed to ± OA. Results. Upon OA treatment, the Cancer index increased and 1C-THF was redirected to the methionine cycle increasing flux to methylation. OA significantly increased the production of SAM, GSH and 2-HG after 15 min. Blocking the first enzyme in the serine pathway, PHGDH, prevented these increases. After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p < 0.0001), peaking at 15 min. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01) including neural, EMT and ER- BC related genes. Motif analysis revealed an overrepresentation of binding sites for serine pathway transcriptional activators ATF3/4 (p < 0.05). Blocking PHGDH or HMT prevented OA-induced gene expression changes. Alkaline comet assay showed that OA significantly increased comet tails. scRNAseq revealed OA increased the numbers of cells within all cell clusters expressing ATF3 and PHGDH. Also observed was an increase in the percentage of basal BSL1, luminal progenitor LP3 and hormone sensing HS1 cells. Conclusions. Metabolism of OA results in a metabolic shift toward the SOG and methionine increasing the production of SAM, GSH and 2-HG which have implications for oncogenesis. The increased SAM fosters epigenetic phenotypic plasticity via altered histone methylation. The increased proportion of specific cell types likely reflects the survival of cells able to mitigate oxidative stress. Increased 2-HG may produce metabolic BRCAness by inhibiting 2-oxoglutarate-dependent dioxygenases. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Elizaveta V. Benevolenskaya, Maxim V. Frolov, Jason Locasale, Marcelo G. Bonini, Navdeep S. Chandel, Seema Khan, Susan Clare. Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells [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 444.

Role and potential therapeutic value of histone methyltransferases in drug resistance mechanisms in lung cancer.

Lung cancer, ranking second globally in both incidence and high mortality among common malignant tumors, presents a significant challenge with frequent occurrences of drug resistance despite the continuous emergence of novel therapeutic agents. This exacerbates disease progression, tumor recurrence, and ultimately leads to poor prognosis. Beyond acquired resistance due to genetic mutations, mounting evidence suggests a critical role of epigenetic mechanisms in this process. Numerous studies have indicated abnormal expression of Histone Methyltransferases (HMTs) in lung cancer, with the abnormal activation of certain HMTs closely linked to drug resistance. HMTs mediate drug tolerance in lung cancer through pathways involving alterations in cellular metabolism, upregulation of cancer stem cell-related genes, promotion of epithelial-mesenchymal transition, and enhanced migratory capabilities. The use of HMT inhibitors also opens new avenues for lung cancer treatment, and targeting HMTs may contribute to reversing drug resistance. This comprehensive review delves into the pivotal roles and molecular mechanisms of HMTs in drug resistance in lung cancer, offering a fresh perspective on therapeutic strategies. By thoroughly examining treatment approaches, it provides new insights into understanding drug resistance in lung cancer, supporting personalized treatment, fostering drug development, and propelling lung cancer therapy into novel territories.

Epigenetic small-molecule screen for inhibition and reversal of acinar ductal metaplasia in mouse pancreatic organoids.

Background: Acinar ductal metaplasia (ADM) is among the earliest initiating events in pancreatic ductal adenocarcinoma (PDAC) development. Methods: We developed a novel morphology-based screen using organoids from wildtype and p48Cre/+ (Cre) mice to discover epigenetic modulators that inhibit or reverse pancreatic ADM more effectively than the broad-spectrum HDAC inhibitor trichostatin A (TSA). Results: Of the 144 compounds screened, nine hits and two additional natural product HDAC inhibitors were validated by dose-response analysis. The class I HDAC inhibitors apicidin and FK228, and the histone methyltransferase inhibitor chaetocin demonstrated pronounced ADM inhibition and reversal without inducing significant cytotoxicity at 1µM. Thioester prodrug class I HDAC inhibitor largazole attenuated ADM while its disulfide homodimer was effective in both ADM inhibition and reversal. Prioritized compounds were validated for ADM reversal in p48Cre/+; LSL-KrasG12D/+ (KC) mouse organoids using both morphological and molecular endpoints. Molecular index analysis of ADM reversal in KC mouse organoids demonstrated improved activity compared to TSA. Improved prodrug stability translated into a stronger phenotypic and molecular response. RNA-sequencing indicated that angiotensinogen was the top inhibited pathway during ADM reversal. Conclusion: Our findings demonstrate a unique epigenetic mechanism and suggest that the phenotypic screen developed here may be applied to discover potential treatments for PDAC.

Abstract B009: Dual PARP and EHMT1/2 inhibition induces an interferon response and anti-tumor immunity in ovarian cancer

Abstract Ovarian cancer has a high propensity to develop therapy resistance. Epigenetic therapies can sensitize ovarian cancer cells to first-line treatments such as platinum-based chemotherapy and Poly (ADP-ribose) polymerase inhibitors (PARPi). Particularly, the reduction of histone 3 lysine 9 dimethylation (H3K9me2) via a euchromatin histone methyltransferase inhibitor (EHMTi) has been shown to sensitize PARPi-resistant cells to PARPi therapy. Additionally, cell growth defects were observed in PARPi-resistant cells treated with dual PARPi/EHMTi treatment compared to DMSO or single agent treatments. To determine the mechanism by which dual therapy sensitizes cells and reduces cell viability, bulk RNA-sequencing was performed on several PARPi-resistant cell lines treated with DMSO, PARPi, EHMTi, or dual PARPi/EHMTi. Transcriptomic analyses revealed that dual therapy increased interferon (IFN) signaling-related genes compared to DMSO or single agent treatments. Additionally, expression of transposable element (TE) transcripts, which can form double-stranded RNA (dsRNA) and induce an IFN response, was also increased in dual treated cells compared to controls. To determine if the reduction in cell viability is dependent on TEs and IFN signaling (i.e., viral mimicry), CRISPRi technology was used to silence intracellular sensors of dsRNA, RIGI and MDA5. Cell growth assays of these silenced cells showed that the reduction in cell growth observed with dual treatment was partially dependent on MDA5 expression. Based on the observed increase in IFN signaling and T cell recruiting cytokines, we performed an animal study using an immunogenic, PARPi-resistant model of ovarian cancer to determine if this dual therapy has any anti-tumor effects. Indeed, we observed that single and dual EHMTi therapy significantly reduced tumor burden and that this reduction was partially dependent on CD8 T cells. Lastly, to determine if dual therapy also induces a viral mimicry response in the human tumor microenvironment, we created ex vivo cultures of tumors resected from patients with ovarian cancer. We then treated the ex vivo tissues with DMSO, PARPi, EHMTi, and dual PARPi/EHMTi, and performed bulk RNA-sequencing and multispectral immunohistochemistry. From these analyses, we found that dual therapy upregulated TE transcripts and multiple immune-related pathways, and increased T cell activity in the tumor microenvironment. Altogether, our data suggest that dual therapy activates viral mimicry and promotes anti-tumor immunity in both PARPi-resistant and -sensitive ovarian cancer. Citation Format: Lily Nguyen, Zachary L. Watson, Elizabeth R. Woodruff, Kimberly R. Jordan, Edward Chuong, Benjamin G. Bitler. Dual PARP and EHMT1/2 inhibition induces an interferon response and anti-tumor immunity in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B009.

Future of epigenetic immunotherapy in kidney cancer

In clinical practice, immune checkpoint inhibition based on the use of antibodies against PD-1 (programmed death 1), PD-L1 (programmed death-ligand 1) and CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is actively used for treatment of kidney cancer. However, objective response to monotherapy with these drugs is observed only in 9–24 % of patients, and combinations with other anticancer drugs in most cases cause severe adverse reactions. At the same time, there is an increased risk of toxic liver damage, immune-dependent pneumonitis, and rash. Therefore, it is necessary to search for new methods of immunotherapy, the most promising of which is the method of viral mimicry based on epigenetic stimulation of retroelement expression. Double-stranded retroelement transcripts activate antiviral interferon response that induces apoptosis of tumor cells. To achieve this, inhibitors of DNA methyltransferase, deacetylase and histone methyltransferase are used which have been successfully applied to treat various malignant neoplasms. In the experiment, DNA methyltransferase inhibitor 5-aza-2-deoxytidine (decitabine) effectively inhibited clear cell renal cell carcinoma cells proliferation which indicates their potential in treatment of kidney cancer. However, similarly to other neoplasms, activation of retroelements in renal cell carcinoma serves as initiator of the tumor process as it leads to increased expression of oncogenes, inactivation of tumor suppressors, and genomic instability. Therefore, the method of viral mimicry requires a differentiated approach with inhibition of retroelements involved in carcinogenesis and simultaneous stimulation of expression of retrotransposons that are not involved in the mechanisms of tumor development and have immunogenic properties. For this, microRNAs derived from transposons can be used as guides for DNA methyltransferases. An analysis of scientific literature revealed 41 such microRNAs of which decreased expression in kidney cancer was established for miR-95, -887, -652, -585, -511, -502, -495, -493, -487b, -335; increased for miR-1249, -1266, -151a, -211, -2114, -2355, -28, -3144, -340, -342, -374a, -374b, -3934, -421, -545, -576, -582, -584, -616, -769; and specific expression in different tumor subtypes for miR-708, -577, -450b, -326, -3200, -31, -224, -192, -1271. Since activation of retroelements can lead to insertions into new genome loci with formation of new mutations involved in carcinogenesis, a promising direction in integrated immunotherapy of kidney cancer is the use of reverse transcriptase inhibitors.