Epigenetic Inhibitors Research Articles

Oleic acid-related anti-inflammatory effects in force-stressed PdL fibroblasts are mediated by H3 lysine acetylation associated with altered IL10 expression

ABSTRACT The initiation of a spatially and temporally limited inflammation is essential for tissue and bone remodelling by the periodontal ligament (PdL) located between teeth and alveolar bone. Nutritional components may cause alterations in the inflammatory response of PdL fibroblasts to mechanical stress such as those occurring during orthodontic tooth movement (OTM). Recently, we reported an attenuated pro-inflammatory response of human PdL fibroblasts (HPdLFs) to compressive forces when stimulated with oleic acid (OA), a monounsaturated fatty acid particularly prominent in the Mediterranean diet. Fatty acids could serve as alternative source of acetyl-CoA, thereby affecting epigenetic histone marks, such as histone 3 lysine acetylation (H3Kac) in a lipid metabolism-dependent manner. In this study, we aimed to investigate the extent to which OA exerts its anti-inflammatory effect in compressed HPdLFs via changes in H3Kac. Six-hour compressed HPdLFs showed increased H3Kac when cultured with OA. Inhibition of histone deacetylases resulted in a comparable IL10-increase as observed in compressed OA-cultures. In contrast, inhibition of histone acetyltransferases, particularly p300/CBP, in compressed HPdLFs exposed to OA normalized the inflammatory response to control levels. OA-dependent increased association of H3Kac to IL10 promoter regions in compressed HPdLFs further strengthened the assumption that OA exhibits its anti-inflammatory properties via modulation of this epigenetic mark. In conclusion, our study strongly suggests that nutritional components can directly affect PdL cells via changes in their epigenetic code. Since epigenetic inhibitors are already widely used clinically, they may hold promise for novel approaches for personalized orthodontic treatment that incorporates nutritional and metabolism-related changes.

Immune Infiltration and N(6)-Methyladenosine ncRNA Isoform Detection in Acute Lung Injury.

Acute lung injury (ALI) is a severe form of sepsis that is associated with a high rate of morbidity and death in critically ill individuals. The emergence of ALI is the result of several factors at work. Case mortality rates might range from 40% to 70%. Researchers have discovered that epigenetic alterations are important in the pathophysiology of ALI and that using epigenetic inhibitors may help reduce symptoms. In embryonic development, circadian rhythm, the cell cycle, and cancer, methylation of m6A seems to be relevant all along the way. According to recent research, posttranscriptional methylation is a key player in the development of alveolar lymphoma. In this study, we clustered ALI based on m6A-related factors, analyzed different classes of immune cell enrichment and inflammatory cytokine expression, screened clustered differential genes for ALI to construct coexpression networks, screened key ALI genes potentially regulated by m6A modifications, and then typed the disease based on key genes to compare the consistency of different clustering results. Our findings have revealed a hitherto undiscovered prognostic sign and a therapeutic target for ALI therapy in m6A and immune invading cells, respectively.

MicroRNAs and their signaling pathway in mycosis fungoides.

Background:Oncogenic microRNAs, a kind of stable epigenetic inhibitors, often deregulated in Mycosis fungoides (MF) which affect the skin and tend to transform and spread.Results:Previous studies investigating the de-expression of microRNA in MF patients skin biopsies identified that they were not only regulated by signaling pathway, but also regulated other signaling pathway. Furthermore, studies have elucidated the molecular mechanisms of the STAT signaling pathway that can promote a great diversity of miRNA expression via cytokine binding receptors, activating Janus kinase-3 and STAT proteins. But some non-STAT signaling pathway with mircoRNA de-expression in MF was incomplete.Conclusion:Taken together, these studies demonstrate that microRNA may be used as the prognosis, progression and diagnose of MF, as they can not only control MF cell proliferation, but also induce MF cell apoptosis.

Acetyl-CoA metabolism drives epigenome change and contributes to carcinogenesis risk in fatty liver disease

BackgroundThe incidence of non-alcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma (HCC) is increasing worldwide, but the steps in precancerous hepatocytes which lead to HCC driver mutations are not well understood. Here we provide evidence that metabolically driven histone hyperacetylation in steatotic hepatocytes can increase DNA damage to initiate carcinogenesis.MethodsGlobal epigenetic state was assessed in liver samples from high-fat diet or high-fructose diet rodent models, as well as in cultured immortalized human hepatocytes (IHH cells). The mechanisms linking steatosis, histone acetylation and DNA damage were investigated by computational metabolic modelling as well as through manipulation of IHH cells with metabolic and epigenetic inhibitors. Chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) and transcriptome (RNA-seq) analyses were performed on IHH cells. Mutation locations and patterns were compared between the IHH cell model and genome sequence data from preneoplastic fatty liver samples from patients with alcohol-related liver disease and NAFLD.ResultsGenome-wide histone acetylation was increased in steatotic livers of rodents fed high-fructose or high-fat diet. In vitro, steatosis relaxed chromatin and increased DNA damage marker γH2AX, which was reversed by inhibiting acetyl-CoA production. Steatosis-associated acetylation and γH2AX were enriched at gene clusters in telomere-proximal regions which contained HCC tumour suppressors in hepatocytes and human fatty livers. Regions of metabolically driven epigenetic change also had increased levels of DNA mutation in non-cancerous tissue from NAFLD and alcohol-related liver disease patients. Finally, genome-scale network modelling indicated that redox balance could be a key contributor to this mechanism.ConclusionsAbnormal histone hyperacetylation facilitates DNA damage in steatotic hepatocytes and is a potential initiating event in hepatocellular carcinogenesis.

Abstract 2957: Inhibition of epigenetic regulator menin is a novel therapeutic approach for high-risk neuroblastoma

Abstract Controlling epigenetic factors such as histone methyltransferases and demethylases has recently emerged as an attractive therapeutic approach for different cancers. KMT2 family protein KMT2A or MLL1 is a H3K4 methyltransferase, which is known to play an essential role in cancer development. MLL1 forms a COMPASS complex to function as an epigenetic regulator and Menin is one of the specific and required binding partner for the activation and function of this epigenetic enzyme. Menin acts as a tumor suppressor protein, and inhibition of Menin-MLL1 interaction has been shown to destabilize the MLL1 epigenetic complex, inhibits H3K4 methyltransferase activity, and hinder the oncogenic potential of various cancers. High-risk neuroblastoma (NB) is the most common extracranial solid pediatric tumor and accounts for almost 15% of all pediatric cancer-related deaths. Recent reports indicate that aberrant activation of epigenetic regulators may drive NB progression and relapse. Therefore, in the present study, we used a specific small-molecule inhibitor, MI-503 to disrupt Menin-MLL1 interaction, and to further understand the role of Menin in pediatric NB. We used different NB cell lines including MYCN-non-amplified (SH-SY5Y, SK-N-AS, CHLA-255) and MYCN-amplified (NGP, LAN-5, IMR-32), and performed cytotoxicity and Colonogenic assays to determine the effect of Menin inhibition on NB proliferation. Results showed that MI-503 significantly inhibits NB proliferation and colony formation capacity in a dose-dependent manner in contrast to controls. Further, we performed apoptosis and cell cycle assays and observed that MI-503 significantly induces apoptosis as detected by the increase of Annexin-V positive early apoptotic cells, and blocks cell cycle progression at the S phase in all NB cell lines tested. Additionally, Western blot assays demonstrated an overall inhibition of H3K3me3 levels in NB cells in response to MI-503. To further determine the effects of Menin inhibition on NB tumor growth, we developed a NB 3D spheroid tumor model that recapitulate in vivo tumor growth for solid tumors. Treatment with MI-503 significantly inhibits spheroidal tumor growth by inducing tumor cell death in a dose-dependent manner. Overall, these data highlight the role of epigenetic regulation, MLL1 complex, and of Menin in NB growth and tumorigenicity. In our future efforts, we will further elucidate the effects of MI-503 and the role of Menin in NB by using in vivo tumor models. Epigenetic inhibitor such as MI-503 holds significant potential for further clinical development and as a novel therapeutic approach for NB. Citation Format: Rameswari Chilamakuri, Saurabh Agarwal. Inhibition of epigenetic regulator menin is a novel therapeutic approach for high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2957.

Abstract 721: Epigenetic intervention in the prevention of Lynch Syndrome Colorectal Cancer

Abstract Introduction: Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC) affecting >1 million Americans. LS is caused by germline mutations in one of four DNA mismatch repair (MMR) genes, generating several hundreds of mutations in microsatellite coding sequences. Although defects in the MMR machinery are the principal mechanism driving LS carcinogenesis, the role of the epigenome through histone and DNA methylation remains unexplored. Hypermethylation of MLH1 is detected in 15% of sporadic MMR-deficient CRC and also in LSCRC, indicating a pivotal role of DNA methylation in LSCRC. In this study, we investigated the landscape of DNA methylation and EZH2-mediated histone H3 lysine 27 methylation in LS tumor samples, and tested the preventive effects of GSK-503, on tumor formation using a novel LS mouse model. Methods: We profiled the somatic mutational, global DNA methylation landscape, and performed ChIP-seq in LS precancers and tumors. Patient-derived organoids (PDO) from colonic tissues treated with EZH2 and 5-AZA were used for gene expression analysis. LS mice (Villin-Cre;Msh2fl/fl;TgfβRII-KI) were treated for 9 weeks with EZH2 inhibitor GSK503 to assess the effect on polyp formation in vivo monitored through murine colonoscopy. Results: Our mutational profiling via whole-exome-seq and gene expression survey of 10 LSCRC and 33 pre-cancers showed accumulation of somatic mutations and aberrant expression patterns in key epigenetic regulators including EZH2 and DNMT3A, which suggests dysregulation of epigenetic control in LS. Global DNA methylation using RRBS in tumors of 3 LS patients showed that gene promoters were more hypermethylated when compared to matched normal samples. Immunohistochemistry data showed higher expression of EZH2 in tumors when compared to precancerous and normal colonic mucosa. ChIPseq analysis demonstrated high levels of H3K27me3 present in gene promoters of stem cell marker genes (WNT9A, LGR5, and ASCL2), and immune-related genes (CD4, CD8a, and HLA-E). Interestingly, 577 out of 2756 DNA hypermethylated genes from RRBS analysis displayed high levels of H3K27me3, thus indicating a central role of EZH2 in epigenetic programming of LS neoplasms. RNAseq data from PDOs treated with 5-AZA (DNMT inhibitor) and GSK503 (EZH2 inhibitor) showed that both epigenetic inhibitors led to the reactivation of silenced genes in colorectal carcinogenesis such as CDKN2A, RUNX3, IGFBP3, and immune genes, including CD40, CD4, SEMA3B, CXRC6/6, and ICAM1. Finally, a 9-week chemo-preventive treatment of a cohort of LS mice with GSK503 significantly decreased polyp formation when compared to control, and polyp reduction was associated with increased infiltration of cytotoxic CD8a+ Tcells and NK in the large intestine mucosa. Conclusions: These results demonstrate the chemopreventive potential of EZH2 inhibitors in prevention of LS-associated tumors through an immune modulation approach. Citation Format: Laura Reyes Uribe, Charles M Bowen, Kyera Evans, Hong Wu, Fahriye Duzagac, Krishna Sinha, Eduardo Vilar. Epigenetic intervention in the prevention of Lynch Syndrome Colorectal Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 721.

Abstract 3751: Altered prostatic HOX code is associated with decreased YAP1 expression during neuroendocrine prostate cancer development

Abstract Background: Neuroendocrine (NE) phenotype contributes to 25 to 30% androgen deprivation resistant prostate cancer cases. Until now, this aggressive phenotype is untreatable. Therefore, it is urgent to study the molecular mechanism of NE development to reveal therapeutic strategies. Methods: Data were downloaded from CCLE, cBioPortal, and GEO and analyzed by R. RT-qPCR, western-blot, Immunohistochemistry, and Immunofluorescence were used to assess gene expression. Results: Neuroendocrine prostate cancer (NEPCa) develops from prostate adenocarcinoma (AdPCa). We screened and identified the consensus differentially expressed genes across all public NEPCa transcriptome datasets. We found several HOX genes are included. HOXB13 is deceased but HOXB3, HOXD8 are increased in NEPCa. Based on the HOX code (total 39 HOX genes expression) theory, different cell lineages have distinct HOX codes and HOX codes can represent tissue identities, we established 42 HOX codes for 42 cells lineages including prostate through bioinformatics analysis of 1019 cancer cell lines transcriptome. We found NEPCa samples have changed prostatic HOX code. We also found YAP1, a previously characterized oncogene, is decreased in mRNA level in NEPCa than AdPCa. We confirmed the YAP1 protein is not detected in NEPCa human/mouse samples, PDX samples, and cell line samples (NCI-H660). We used YAP1 as a model. We hypothesized that the signaling involved in silencing YAP1 expression can also contribute to NE development. We first confirmed the loss of YAP1 expression in NEPCa cells is under epigenetic control by inducing YAP1 mRNA expression in primary mouse NEPCa cells through epigenetic inhibitors treatments (HDACs/DNA methyltransferase inhibitors). We characterized the YAP1 promoter through data mining chromatin ChIP-seq data and DNA methylation-seq data. We identified and cloned a 3kb DNA fragment across the YAP1 transcription start site as YAP1 regulatory element. We then built a YAP1-promoter-luciferase reporter using this fragment. We also generated a list of transcription factors that can bind the YAP1 regulatory element. We will then confirm the roles of these transcriptional factors in both regulating YAP1 and NE development. We also found YAP1 expression is co-expressed with many HOX genes in human samples. This suggested the potential regulatory function of HOX code to YAP1 expression. Conclusion: We proposed 2 critical events during NE development, changed prostatic HOX code and loss of YAP expression. We identified HOX code change as one of the potential regulatory mechanisms of YAP1. We then proposed to use YAP1 regulatory element as a model system to study the NE development mechanism. Funding: The LSUHS Office of Research SEED awards, NIH R01 CA226285, and Feist-Weiller Cancer Center pre-doc fellowship to SC. Acknowledgment: We acknowledge Feist-Weiller Cancer Center for travel support to SC. Citation Format: Siyuan Cheng, Shu Yang, Yingli Shi, Xiuping Yu. Altered prostatic HOX code is associated with decreased YAP1 expression during neuroendocrine prostate cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3751.

Small-Molecule-Mediated Split-Aptamer Assembly for Inducible CRISPR-dCas9 Transcription Activation.

Inducible CRISPR-dCas9 transcription system has become a powerful tool for transcription regulation and sensing. Here, we develop a new concept of small-molecule-mediated split-aptamer assembly for inducible CRISPR-dCas9 transcription activation, allowing quantitative detection and imaging of S-adenosyl methionine (SAM) in live cells. This inducible transcription system is designed by integrating one fragment of a split SAM aptamer to guide RNA (gRNA) and the other to MS2 arrays. SAM-mediated reassembly of the split fragments recruits an MCP-fused transcription activator to the gRNA-dCas9 complex, activating the expression of a near-infrared fluorescent protein for imaging. We demonstrate that this inducible transcription system achieves quantitative detection of SAM with high sensitivity in live cells. Our system shows that methionine adenosyltransferase 1A (MAT1A) and MAT2A can both catalyze SAM production in live cells and the SAM levels in cancer cells can be increased via upregulation of MAT1A mRNA by epigenetic inhibitors. This split-aptamer assembly strategy could afford a new approach for controlling the CRISPR-dCas9 system, enabling conditional transcription regulation in response to endogenous metabolites in live cells.

Histone Deacetylase 6 Inhibitor JS28 Prevents Pathological Gene Expression in Cardiac Myocytes.

BackgroundEpigenetic modulators have been proposed as promising new drug targets to treat adverse remodeling in heart failure. Here, we evaluated the potential of 4 epigenetic drugs, including the recently developed histone deacetylase 6 (HDAC6) inhibitor JS28, to prevent endothelin‐1 induced pathological gene expression in cardiac myocytes and analyzed the chromatin binding profile of the respective inhibitor targets.Methods and ResultsCardiac myocytes were differentiated and puromycin‐selected from mouse embryonic stem cells and treated with endothelin‐1 to induce pathological gene expression (938 differentially expressed genes, q<0.05). Dysregulation of gene expression was at least in part prevented by epigenetic inhibitors, including the pan‐BRD (bromodomain‐containing protein) inhibitor bromosporine (290/938 genes), the BET (bromodomain and extraterminal) inhibitor JQ1 (288/938), the broad‐spectrum HDAC inhibitor suberoylanilide hydroxamic acid (227/938), and the HDAC6 inhibitor JS28 (210/938). Although the 4 compounds were similarly effective toward pathological gene expression, JS28 demonstrated the least adverse effects on physiological gene expression. Genome‐wide chromatin binding profiles revealed that HDAC6 binding sites were preferentially associated with promoters of genes involved in RNA processing. In contrast, BRD4 binding was associated with genes involved in core cardiac myocyte functions, for example, myocyte contractility, and showed enrichment at enhancers and intronic regions. These distinct chromatin binding profiles of HDAC6 and BRD4 might explain the different effects of their inhibitors on pathological versus physiological gene expression.ConclusionsIn summary, we demonstrated, that the HDAC6 inhibitor JS28 effectively prevented the adverse effects of endothelin‐1 on gene expression with minor impact on physiological gene expression in cardiac myocytes. Selective HDAC6 inhibition by JS28 appears to be a promising strategy for future evaluation in vivo and potential translation into clinical application.

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D

Although chimeric antigen receptor T (CAR-T) cells have achieved remarkable successes in hematological malignancies, the efficacies of CAR-T cells against solid tumors remains unsatisfactory. Heterogeneous antigen expression is one of the obstacles on its effective elimination of solid cancer cells. DNAX-activating protein 10 (DAP10) interacts with natural killer group 2D (NKG2D), acting as an adaptor that targets various malignant cells for surveillance. Here, we designed a DAP10 chimeric receptor that utilized native NKG2D on T cells to target NKG2D ligand-expressing cancer cells. We then tandemly incorporated it with anti-glypican 3 (GPC3) single-chain variable fragment (scFv) to construct a dual-antigen-targeting system. T cells expressing DAP10 chimeric receptor (DAP10-T cells) displayed with an enhancement on both cytotoxicity and cytokine secretion against solid cancer cell lines, and its tandem connection with anti-GPC3 scFv (CAR GPC3-DAP10-T cells) exhibited a dual-antigen-targeting capacity on eliminating heterogeneous cancer cells in vitro and suppressing the growth of heterogeneous cancer in vivo. Thus, this novel dual-targeting system enabled a high efficacy on killing cancer cells and extended the recognition profile of CAR-T cells toward tumors, which providing a potential strategy on treatment of solid cancer clinically.

MODL-15. High-throughput combination drug screening identifies synergism between retinoic acid treatment and BCL-XL-inhibition in MYC(N)-driven medulloblastoma and neuroblastoma models

Abstract PURPOSE: Despite remarkable improvements in childhood cancer survival, certain high-risk entities including malignancies of the nervous system still carry dismal prognoses. Retinoic acid (RA) treatment has long been administered in high-risk neuroblastoma patients and preclinical data suggest a benefit in applying retinoids in selected brain tumor entities. However, limited single-agent efficacy and developing treatment resistance provide a rationale for investigating improved combination treatments. METHODS: We assessed the RA sensitivity of 16 cell lines and patient-derived cultures of pediatric nervous system tumors from the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) registry. Bulk RNA sequencing was performed for five sensitive models undergoing RA treatment. All models were screened for drug sensitivity to 76 clinically relevant drugs alone and combined with RA. Beneficial hit combinations were further investigated for synergy, as well as their effect on differentiation and apoptosis by high-content fluorescence microscopy and caspase activity assay. Results from in vivo experiments are pending. RESULTS: Group 3 medulloblastoma and high-risk neuroblastoma models were most sensitive to RA treatment leading to significant changes in gene expression of apoptotic regulation. Drug sensitivity screening revealed similar patterns of response to combination treatments in MYC-driven Group 3 medulloblastomas and MYCN-amplified neuroblastomas. Apoptosis regulating BCL-XL-inhibitors (A-1155463, navitoclax) were identified as top hits across RA sensitive models, whereas epigenetic BET family inhibitor I-BET151 and histone deacetylase inhibitor entinostat suggested entity-specific combination benefits in neuroblastoma and medulloblastoma models, respectively. In both entities, combining RA with navitoclax was synergistic and significantly increased caspase-3 activity. The combination led to a morphological shift from differentiation under RA treatment to cell death. CONCLUSION: Exploring combinations for RA treatment in pediatric nervous system tumors, we found that MYC(N)-driven medulloblastoma and neuroblastoma not only share responsiveness to RA treatment, but also demonstrate enhanced vulnerability in combination with BCL-XL-inhibition resulting in increased apoptosis.

Sensitization of Resistant Breast Cancer Cells with a Jumonji Family Histone Demethylase Inhibitor.

Simple SummaryUsing a cell culture model of resistant breast cancer cells with the phenotype that is often responsible for the early relapse of triple-negative breast cancer, namely, the persistence of these cells in reversible quiescence under a variety of challenges, we found that reprogramming the epigenome by treatment with JIB-04, a small-molecule inhibitor of Jumonji-family histone demethylases, sensitized resistant cells. We used this model of deep intrinsic resistance featuring many molecular mechanisms of achieving this phenotype to perform lengthy evaluations of less cytotoxic doses of JIB-04. We found that resistant cells derived from triple-negative inflammatory breast cancer cell lines were either much more sensitive to JIB-04 than the parental cell line or altered by the treatment such that they became sensitive to the chemotherapeutic drugs paclitaxel and doxorubicin. Notably, JIB-04 exposure increased PD-L1 expression in cancer cells, which means that JIB-04 may have clinical applications in improving the responses of triple-negative breast cancer to anti-PD-L1 therapy.In the present study, we evaluated JIB-04, a small-molecule epigenetic inhibitor initially discovered to inhibit cancer growth, to determine its ability to affect deep intrinsic resistance in a breast cancer model. The model was based on a function-based approach to the selection of cancer cells in a cell culture that can survive a variety of challenges in prolonged, but reversible, quiescence. These resistant cancer cells possessed a variety of mechanisms, including modifications of the epigenome and transcriptome, for generating a high degree of cellular heterogeneity. We found that long pretreatment with JIB-04 sensitized resistant triple-negative inflammatory breast cancer cells and their parental cell line SUM149 to the chemotherapeutic drugs doxorubicin and paclitaxel. Resistant cancer cells derived from another inflammatory breast cancer cell line, FC-IBC02, were considerably more sensitive to JIB-04 than the parental cell line. Investigating a mechanism of sensitization, we found that JIB-04 exposure increased the expression of PD-L1 in resistant cells, suggesting that JIB-04 may also sensitize resistant breast cancer cells to anti-PD-L1 immune therapy. Finally, these results support the usefulness of a cell culture-based experimental strategy for evaluating anticancer agents, such as JIB-04, that may halt cancer evolution and prevent the development of cancer resistance to currently used therapies.

Combined Inhibition of the Ataxia‐telangiectasia mutated and Rad3‐related Pathway and the G9a Methyltransferase Synergize to Reduce Pancreatic Cancer Cell Growth

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer‐related deaths in the United States with a devastating overall five‐year survival rate of only 10%. Consequently, there is an urgent need to identify innovative therapeutic strategies. PDAC exhibits chronic replication stress (RS), becoming reliant on a proficient Ataxia‐telangiectasia mutated and Rad3‐related (ATR) pathway to adapt to high RS levels in cells. This also presents an exploitable therapeutic vulnerability in tumors via strategies that enhance the endogenous RS to trigger replication catastrophe and cell death. Our laboratory is focused on utilizing epigenetic inhibitors in this context as novel therapeutic approaches. Recently, we have shown that combined inhibition of the major ATR effector, CHK1, and the G9a methyltransferase leads to replication catastrophe, providing an effective therapeutic approach for PDAC by priming replication failure through the epigenome rather than genomic damage. In the current study, we sought to investigate additional targets in the RS response pathway through direct ATR inhibition combined with G9a inhibition and perform mechanistic studies on DNA damage response checkpoints and cell cycle. We treated multiple PDAC cell lines with a combination of the ATR inhibitor, AZD6738, and either BRD4770 or UNC0642 to inhibit G9a at various concentrations to determine the effect on cell viability and cytotoxicity by live‐cell analysis. We find that the AZD6738‐BRD4770 and AZD6738‐UNC0642 combinations display synergistic effects to disrupt the RS response and inhibit PDAC growth. Overall, our work provides quantitative insights into a novel therapeutic opportunity for PDAC and expands the universality of a synergistic interaction resulting from simultaneous targeting of the ATR‐CHK1 RS pathway and the epigenomic regulator G9a.

Direct targeting of epigenetic regulator Menin inhibits neuroblastoma growth

Histone modifiers such as methyltransferases and demethylases have recently emerged as an attractive target to treat different cancers. Menin is a tumor suppressor protein and an essential oncogenic co‐factor of MLL1 COMPASS complex. Menin is a highly specific binding partner of the N‐terminus of MLL1 complex that have H3K4 methyltransferase activity. Menin‐MLL1 as an epigenetic regulator regulates the expression of target oncogenes, including HOXA, MEIS1, and AF9. Blocking menin‐MLL1 interaction destabilizes the COMPASS complex and hinders the oncogenic potential of cancer cells and cancer stem cells (CSCs). Neuroblastoma (NB) is an aggressive pediatric solid tumor develops during the early embryonic stage from extracranial sympathetic nervous system, and accounts for 10‐15% of all pediatric cancer‐related deaths. In the present study, we hypothesized that disrupting menin‐MLL1 interaction by using a specific small molecule inhibitor could disrupt COMPASS complex and overall inhibit NB growth, metastasis, and relapse. In the present study, we analyzed 1235 NB patients from different NB patient datasets and found that the upregulation of menin inversely correlate with overall survival of NB patients. We used six NB cell lines, including MYCN‐ amplified and ‐non‐amplified cell lines and performed cytotoxic assays. Results showed that inhibition of menin‐MLL1 interaction by MI‐503 significantly inhibits NB proliferation and colony formation capacity in contract to control treatments. Furthermore, MI‐503 significantly and in a dose‐dependent manner blocked NB cell cycle progression at S phase and enhanced apoptosis in treatment groups compared to controls. Additionally, we developed a 3D spheroidal tumor model for NB to mimic in vivo tumor conditions and tested the ability of MI‐503 in inhibiting tumor growth. Results showed significant inhibition of spheroidal tumor growth by enhancing tumor cell death in response to MI‐503 treatment. To further determine the effect of MI‐503 on inhibition of H3K4me3 levels, a key function of menin‐MLL1 COMPASS complex, we performed Western blot assays and observed a significant and dose dependent inhibition of H3K4me3 levels in response to MI‐503 treatment. Overall, our study highlights the role of menin‐MLL1 interaction in NB. Inhibition of menin‐MLL1 interaction by a specific small‐molecule inhibitor MI‐503 leads to the inhibition of epigenetic regulator functions and inhibits NB growth. Our future efforts will focus on further elucidating the role of menin in NB and to develop effective therapeutic strategies incorporating epigenetic inhibitors for NB patients.

Brain co-delivery of first-line chemotherapy drug and epigenetic bromodomain inhibitor for multidimensional enhanced synergistic glioblastoma therapy.

Glioblastoma (GBM) is a central nervous system tumor with poor prognosis due to the rapid development of resistance to mono chemotherapy and poor brain targeted delivery. Chemoimmunotherapy (CIT) combines chemotherapy drugs with activators of innate immunity that hold great promise for GBM synergistic therapy. Herein, we chose temozolomide, TMZ, and the epigenetic bromodomain inhibitor, OTX015, and further co-encapsulated them within our well-established erythrocyte membrane camouflaged nanoparticle to yield ApoE peptide decorated biomimetic nanomedicine (ABNM@TMZ/OTX). Our nanoplatform successfully addressed the limitations in brain-targeted drug co-delivery, and simultaneously achieved multidimensional enhanced GBM synergistic CIT. In mice bearing orthotopic GL261 GBM, treatment with ABNM@TMZ/OTX resulted in marked tumor inhibition and greatly extended survival time with little side effects. The pronounced GBM treatment efficacy can be ascribed to three key factors: (i) improved nanoparticle-mediated GBM targeting delivery of therapeutic agents by greatly enhanced blood circulation time and blood-brain barrier penetration; (ii) inhibited cellular DNA repair and enhanced TMZ sensitivity to tumor cells; (iii) enhanced anti-tumor immune responses by inducing immunogenic cell death and inhibiting PD-1/PD-L1 conjugation leading to enhanced expression of CD4+ and CD8+ T cells. The study validated a biomimetic nanomedicine to yield a potential new treatment for GBM.

Research Advance of Gene Mutation and Targeted Drug Therapy in Childhood Acute Myeloid Leukemia --Review

The clinical therapeutic regimen for acute myeloid leukemia (AML) is not significantly different between adults and children, which is mostly based on IA (idarubicin and cytosine arabinoside) induction chemotherapy. With the rapid development of sequencing technique, people's understandings towards the molecular and biological abnormalities of AML are increasing, diverse AML gene mutation-based targeted drugs have been rapidly developed and applied. In this review, several commonly gene mutations in AML (such as FLT3, NPM1 and C/EBPA) was described, and the therapeutic effects and differences of targeted drugs that used in clinical treatment or had been reported (like tyrosine kinase inhibitor, IDH1 mutation inhibitor and epigenetic modification inhibitor) in child and adult AML patients were summrized.

Temporal dynamics of HCMV gene expression in lytic and latent infections.

SummaryDuring productive human cytomegalovirus (HCMV) infection, viral genes are expressed in a coordinated cascade that conventionally relies on the dependencies of viral genes on protein synthesis and viral DNA replication. By contrast, the transcriptional landscape of HCMV latency is poorly understood. Here, we examine viral gene expression dynamics during the establishment of both productive and latent HCMV infections. We redefine HCMV gene expression kinetics during productive infection and reveal that viral gene regulation does not represent a simple sequential cascade; many viral genes are regulated by multiple independent modules. Using our improved gene expression classification combined with transcriptome-wide measurements of the effects of a wide array of epigenetic inhibitors on viral gene expression during latency, we show that a defining feature of latency is the unique repression of immediate-early (IE) genes. Altogether, we recharacterize HCMV gene expression kinetics and reveal governing principles of lytic and latent gene expression.

HSP90 Inhibition Overcomes Resistance to Molecular Targeted Therapy in BRAFV600E-mutant High-grade Glioma.

Molecular targeted therapy using BRAF and/or MEK inhibitors has been applied to BRAFV600E-mutant high-grade gliomas (HGG); however, the therapeutic effect is limited by the emergence of drug resistance. We established multiple paired BRAFV600E-mutant HGG patient-derived xenograft models based on tissues collected prior to and at relapse after molecular targeted therapy. Using these models, we dissected treatment-resistant mechanisms for molecular targeted therapy and explored therapeutic targets to overcome resistance in BRAFV600E HGG models in vitro and in vivo. We found that, despite causing no major genetic and epigenetic changes, BRAF and/or MEK inhibitor treatment deregulated multiple negative feedback mechanisms, which led to the reactivation of the MAPK pathway through c-Raf and AKT signaling. This altered oncogenic signaling primarily mediated resistance to molecular targeted therapy in BRAFV600E-mutant HGG. To overcome this resistance mechanism, we performed a high-throughput drug screening to identify therapeutic agents that potently induce additive cytotoxicity with BRAF and MEK inhibitors. We discovered that HSP90 inhibition combined with BRAF/MEK inhibition coordinately deactivated the MAPK and AKT/mTOR pathways, and subsequently induced apoptosis via dephosphorylation of GSK3β (Ser9) and inhibition of Bcl-2 family proteins. This mediated potent cytotoxicity in vitro and in vivo in refractory models with acquired resistance to molecular targeted therapy. The combination of an HSP90 inhibitor with BRAF or MEK inhibitors can overcome the limitations of the current therapeutic strategies for BRAFV600E-mutant HGG.

Biomimetic and responsive nanoparticles loading JQ1 for dual-targeting treatment of vascular restenosis via multiple actions

• The biomimetic and responsive DDS had optimized JQ1 release behavior and rapid homing to injured site. • The DDS inherited natural specificity for VSMCs from platelets and obtained dual-targeting function. • Pro-inflammatory cytokines secreted from macrophages were suppressed by the DDS. • The DDS can substantially inhibit VSMCs proliferation and intima hyperplasia with little effect on VECs. Postoperative restenosis remains the key problem of coronary arterial disease and seriously threatens human health. Currently, no effective strategies can thoroughly prevent restenosis owing to its complicated progression. We hypothesize the multi-effect drug of endothelium-protective epigenetic inhibitor (JQ1) may act well in anti-restenosis via exerting multiple functions and acting on different cells. Furthermore, homing to endothelium injured site and pH-triggered drug release can enhance targeted therapeutic efficacy by JQ1 preferential accumulation at injured site and in vascular smooth muscle cells (VSMCs). To verify our hypothesis and develop targeted drug delivery system (DDS) with controlled release ability for anti-restenosis, a pH-responsive nanoparticle was prepared to encapsulate JQ1 and further camouflaged by platelet membrane (PM). The resulting biomimetic and responsive DDS showed optimized drug release behavior and rapid homing to injured site. Moreover, the internalization of DDS in VSMCs was much higher than other groups without PM, which indicated that it inherited natural specificity for VSMCs from PM and obtained dual-targeting function to enhance therapeutic efficacy. The DDS presented significant anti-inflammatory effect via inhibiting NF-κB signaling pathway and secretion of pro-inflammatory cytokines from macrophages. Besides, VSMCs proliferation was suppressed by the DDS with little effect on vascular endothelial cell proliferation and in vivo re-endothelialization at suitable JQ1 concentration. More importantly, the DDS substantially inhibited intima hyperplasia without obvious pathological variation on main organs using a mouse model of carotid artery injury. In summary, we created a versatile DDS for the promotion of JQ1 targeting delivery and responsive release at injured site to mitigate restenosis. The DDS demonstrated outstanding performance via a synergistic and comprehensive effect of multiple factors for anti-restenosis, providing a promising method for clinical applications.

Epigenomic reprogramming via HRP2-MINA dictates response to proteasome inhibitors in multiple myeloma with t(4;14) translocation.

The chromosomal t(4;14) (p16;q32) translocation drives high expression of histone methyltransferase nuclear SET domain–containing 2 (NSD2) and plays vital roles in multiple myeloma (MM) evolution and progression. However, the mechanisms of NSD2-driven epigenomic alterations in chemoresistance to proteasome inhibitors (PIs) are not fully understood. Using a CRISPR/Cas9 sgRNA library in a bone marrow–bearing MM model, we found that hepatoma-derived growth factor 2 (HRP2) was a suppressor of chemoresistance to PIs and that its downregulation correlated with a poor response and worse outcomes in the clinic. We observed suppression of HRP2 in bortezomib-resistant MM cells, and knockdown of HRP2 induced a marked tolerance to PIs. Moreover, knockdown of HRP2 augmented H3K27me3 levels, consequentially intensifying transcriptome alterations promoting cell survival and restriction of ER stress. Mechanistically, HRP2 recognized H3K36me2 and recruited the histone demethylase MYC-induced nuclear antigen (MINA) to remove H3K27me3. Tazemetostat, a highly selective epigenetic inhibitor that reduces H3K27me3 levels, synergistically sensitized the anti-MM effects of bortezomib both in vitro and in vivo. Collectively, these results provide a better understanding of the origin of chemoresistance in patients with MM with the t(4;14) translocation and a rationale for managing patients with MM who have different genomic backgrounds.