Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation.

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.

DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells.

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

Newly developed oral bioavailable EHMT2 inhibitor as a potential epigenetic therapy for Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is the prototypic genomic disorder resulting from deficiency of paternally expressed genes in the human chromosome 15q11-q13 region. The unique molecular mechanism involving epigenetic modifications renders PWS as the most attractive candidate to explore a proof-of-concept of epigenetic therapy in humans. The premise is that epigenetic modulations could reactivate the repressed PWS candidate genes from the maternal chromosome and offer therapeutic benefit. Our prior study identifies an EHMT2/G9a inhibitor, UNC0642, that reactivates the expression of PWS genes via reduction of H3K9me2. However, low brain permeability and poor oral bioavailability of UNC0642 preclude its advancement into translational studies in humans. In this study, a newly developed inhibitor, MS152, modified from the structure of UNC0642, has better brain penetration and greater potency and selectivity against EHMT2/G9a. MS152 reactivated maternally silenced PWS genes in PWS patient fibroblasts and in brain and liver tissues of PWS mouse models. Importantly, the molecular efficacy of oral administration is comparable with the intraperitoneal route. MS152 treatment in newborns ameliorates the perinatal lethality and poor growth, maintaining reactivation in a PWS mouse model at postnatal 90 days. Our findings provide strong support for MS152 as a first-in-class inhibitor to advance the epigenetic therapy of PWS in humans.

A phase II study of guadecitabine combined with irinotecan vs regorafenib or TAS-102 in irinotecan-refractory metastatic colorectal cancer patients.

DNA methyltransferase inhibitors (DNMTi) have demonstrated benefit in reversing resistance to systemic therapies for several cancer types. In a phase II trial of guadecitabine and irinotecan compared to regorafenib or TAS-102 in pts with advanced mCRC refractory to irinotecan. Patients with mCRC refractory to irinotecan were randomized 2:1 to guadecitabine and irinotecan (Arm A) vs standard of care regorafenib or TAS-102 (Arm B) on a 28-day cycle. Between January 15, 2016 and October 24, 2018, 104 pts were randomized at four international sites, with 96 pts undergoing treatment, 62 in Arm A and 34 in Arm B. Median overall survival was 7.15 months for Arm A and 7.66 months for Arm B (HR 0.93, 95% CI: 0.58-1.47, P = .75). The Kaplan-Meier rates of progression free survival at 4 months were 32% in Arm A and 26% in Arm B. Common ≥Grade 3 treatment related adverse events in Arm A were neutropenia (42%), anemia (18%), diarrhea (11%), compared to Arm B pts with neutropenia (12%), anemia (12%). Guadecitabine and irinotecan had similar OS compared to standard of care TAS-102 or regorafenib, with evidence of target modulation. Clinical trial information: NCT01896856.

Identification of epigenetic modifiers essential for growth and survival of AML1/ETO-positive leukemia.

Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with dysregulation of epigenetic modifiers. The AML1/ETO (RUNX1/MTG8) fusion protein, caused by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes. We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive and negative cell lines. We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1 inhibitor decitabine in a patient with an AML1/ETO-positive AML. Bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 973 differentially expressed genes (DEGs). Three genes (PARP2, PRKCD, and SMARCA4) were both downregulated after AML1/ETO induction, and identified in shRNA screens. In conclusion, using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. DNMT1 and ATR were validated and are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy.

Targeting histone deacetylases in head and neck squamous cell carcinoma: molecular mechanisms and therapeutic targets.

The onerous health and economic burden associated with head and neck squamous cell carcinoma (HNSCC) is a global predicament. Despite the advent of novel surgical techniques and therapeutic protocols, there is an incessant need for efficacious diagnostic and therapeutic targets to monitor the invasion, metastasis and recurrence of HNSCC due to its substantial morbidity and mortality. The differential expression patterns of histone deacetylases (HDACs), a group of enzymes responsible for modifying histones and regulating gene expression, have been demonstrated in neoplastic tissues. However, there is limited knowledge regarding the role of HDACs in HNSCC. Consequently, this review aims to summarize the existing research findings and explore the potential association between HDACs and HNSCC, offering fresh perspectives on therapeutic approaches targeting HDACs that could potentially enhance the efficacy of HNSCC treatment. Additionally, the Cancer Genome Atlas (TCGA) dataset, CPTAC, HPA, OmicShare, GeneMANIA and STRING databases are utilized to provide supplementary evidence on the differential expression of HDACs, their prognostic significance and predicting functions in HNSCC patients.

Emerging DNA Methylome Targets in FLT3-ITD-Positive Acute Myeloid Leukemia: Combination Therapy with Clinically Approved FLT3 Inhibitors.

OPINION STATEMENT: The internal tandem duplication (ITD) mutation of the FMS-like receptor tyrosine kinase 3 (FLT3-ITD) is the most common mutation observed in approximately 30% of acute myeloid leukemia (AML) patients. It represents poor prognosis due to continuous activation of downstream growth-promoting signaling pathways such as STAT5 and PI3K/AKT. Hence, FLT3 is considered an attractive druggable target; selective small FLT3 inhibitors (FLT3Is), such as midostaurin and quizartinib, have been clinically approved. However, patients possess generally poor remission rates and acquired resistance when FLT3I used alone. Various factors in patients could cause these adverse effects including altered epigenetic regulation, causing mainly abnormal gene expression patterns. Epigenetic modifications are required for hematopoietic stem cell (HSC) self-renewal and differentiation; however, critical driver mutations have been identified in genes controlling DNA methylation (such as DNMT3A, TET2, IDH1/2). These regulators cause leukemia pathogenesis and affect disease diagnosis and prognosis when they co-occur with FLT3-ITD mutation. Therefore, understanding the role of different epigenetic alterations in FLT3-ITD AML pathogenesis and how they modulate FLT3I's activity is important to rationalize combinational treatment approaches including FLT3Is and modulators of methylation regulators or pathways. Data from ongoing pre-clinical and clinical studies will further precisely define the potential use of epigenetic therapy together with FLT3Is especially after characterized patients' mutational status in terms of FLT3 and DNA methlome regulators.

An Overview of Epigenetic Changes in the Parkinson's Disease Brain.

Parkinson's disease is a progressive neurodegenerative disorder, predominantly of the motor system. Although some genetic components and cellular mechanisms of Parkinson's have been identified, much is still unknown. In recent years, emerging evidence has indicated that non-DNA-sequence variation (in particular epigenetic mechanisms) is likely to play a crucial role in the development and progression of the disease. Here, we present an up-to-date overview of epigenetic processes including DNA methylation, DNA hydroxymethylation, histone modifications and non-coding RNAs implicated in the brain of those with Parkinson's disease. We will also discuss the limitations of current epigenetic research in Parkinson's disease, the advantages of simultaneously studying genetics and epigenetics, and putative novel epigenetic therapies.

Dietary molecules and experimental evidence of epigenetic influence in cancer chemoprevention: An insight.

The world-wide rate of incidence of cancer disease has been only modestly contested by the past and current preventive and interventional strategies. Hence, the global effort towards novel ideas to contain the disease still continues. Constituents of human diets have in recent years emerged as key regulators of carcinogenesis, with studies reporting their inhibitory potential against all the three stages vis-a-vis initiation, promotion and progression. Unlike drugs which usually act on single targets, these dietary factors have an advantage of multi-targeted effects and pleiotropic action mechanisms, which are effective against cancer that manifest as a micro-evolutionary and multi-factorial disease. Since most of the cellular targets have been identified and their consumption considered relatively safe, these diet-derived agents often appear as molecules of interest in repurposing strategies. Currently, many of these molecules are being investigated for their ability to influence the aberrant alterations in cell's epigenome for epigenetic therapy against cancer. Targeting the epigenetic regulators is a new paradigm in cancer chemoprevention which acts to reverse the warped-up epigenetic alterations in a cancer cell, thereby directing it towards a normal phenotype. In this review, we discuss the significance of dietary factors and natural products as chemopreventive agents. Further, we corroborate the experimental evidence from existing literature, reflecting the ability of a series of such molecules to act as epigenetic modifiers in cancer cells, by interfering with molecular events that map the epigenetic imprints such as DNA methylation, histone acetylation and non-coding RNA mediated gene regulation.

Epigenetic remodeling of the immune landscape in cancer: therapeutic hurdles and opportunities.

The tumor immune microenvironment represents a sophisticated ecosystem where various immune cell subtypes communicate with cancer cells and stromal cells. The dynamic cellular composition and functional characteristics of the immune landscape along the trajectory of cancer development greatly impact the therapeutic efficacy and clinical outcome in patients receiving systemic antitumor therapy. Mounting evidence has suggested that epigenetic mechanisms are the underpinning of many aspects of antitumor immunity and facilitate immune state transitions during differentiation, activation, inhibition, or dysfunction. Thus, targeting epigenetic modifiers to remodel the immune microenvironment holds great potential as an integral part of anticancer regimens. In this review, we summarize the epigenetic profiles and key epigenetic modifiers in individual immune cell types that define the functional coordinates of tumor permissive and non-permissive immune landscapes. We discuss the immunomodulatory roles of current and prospective epigenetic therapeutic agents, which may open new opportunities in enhancing cancer immunotherapy or overcoming existing therapeutic challenges in the management of cancer.

Structure-based drug design of potential inhibitors of FBXW8, the substrate recognition component of Cullin-RING ligase 7.

FBXW8 plays an irreplaceable role in the substrate recognition of ubiquitin-dependent proteolysis, which further regulates cell cycle progression and signal transduction. However, the abnormal expression of FBXW8 triggers malignancy, inflammation, and autophagy irregulation. FBXW8 is considered as an effective therapeutic target for Cullin-RING ligase 7 (CRL7)-related cancers. Still, the lack of selective inhibitors hinders further therapeutic development and limits the exploration of its biological mechanism. This study constructed an integrated protocol that combines pharmacophore modeling, structure-based virtual screening, and Molecular Dynamic Simulation. It was then used as a screening query to identify hit compounds targeted at the substrate recognition site of FBXW8 from a large-scale compound database including 120 million compounds. Then, ten lead compounds were retrieved by using molecular docking analysis and ADMET prediction. Finally, MD simulations were performed to validate the binding stability of selected drug candidates. The result indicated that three newly obtained compounds, namely ZINC96179876, ZINC72174069, and ZINC97730272, might be potent FBXW8 inhibitors against CRL7-related cancers such as endometrial cancer.

Downregulation of epithelial sodium channel (ENaC) activity in cystic fibrosis cells by epigenetic targeting.

The pathogenic mechanism of cystic fibrosis (CF) includes the functional interaction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with the epithelial sodium channel (ENaC). The reduction of ENaC activity may constitute a therapeutic option for CF. This hypothesis was evaluated using drugs that target the protease-dependent activation of the ENaC channel and the transcriptional activity of its coding genes. To this aim we used: camostat, a protease inhibitor; S-adenosyl methionine (SAM), showed to induce DNA hypermethylation; curcumin, known to produce chromatin condensation. SAM and camostat are drugs already clinically used in other pathologies, while curcumin is a common dietary compound. The experimental systems used were CF and non-CF immortalized human bronchial epithelial cell lines as well as human bronchial primary epithelial cells. ENaC activity and SCNN1A, SCNN1B and SCNN1G gene expression were analyzed, in addition to SCNN1B promoter methylation. In both immortalized and primary cells, the inhibition of extracellular peptidases and the epigenetic manipulations reduced ENaC activity. Notably, the reduction in primary cells was much more effective. The SCNN1B appeared to be the best target to reduce ENaC activity, in respect to SCNN1A and SCNN1G. Indeed, SAM treatment resulted to be effective in inducing hypermethylation of SCNN1B gene promoter and in lowering its expression. Importantly, CFTR expression was unaffected, or even upregulated, after treatments. These results open the possibility of CF patients' treatment by epigenetic targeting.

Non-Coding RNAs Regulating Mitochondrial Functions and the Oxidative Stress Response as Putative Targets against Age-Related Macular Degeneration (AMD).

Age-related macular degeneration (AMD) is an ever-increasing, insidious disease which reduces the quality of life of millions of elderly people around the world. AMD is characterised by damage to the retinal pigment epithelium (RPE) in the macula region of the retina. The origins of this multi-factorial disease are complex and still not fully understood. Oxidative stress and mitochondrial imbalance in the RPE are believed to be important factors in the development of AMD. In this review, the regulation of the mitochondrial function and antioxidant stress response by non-coding RNAs (ncRNAs), newly emerged epigenetic factors, is discussed. These molecules include microRNAs, long non-coding RNAs, and circular non-coding RNAs. They act mainly as mRNA suppressors, controllers of other ncRNAs, or by interacting with proteins. We include here examples of these RNA molecules which affect various mitochondrial processes and antioxidant signaling of the cell. As a future prospect, the possibility to manipulate these ncRNAs to strengthen mitochondrial and antioxidant response functions is discussed. Non-coding RNAs could be used as potential diagnostic markers for AMD, and in the future, also as therapeutic targets, either by suppressing or increasing their expression. In addition to AMD, it is possible that non-coding RNAs could be regulators in other oxidative stress-related degenerative diseases.

Tumor cell-intrinsic RIG-I signaling governs synergistic effects of immunogenic cancer therapies and checkpoint inhibitors in mice.

Immunogenic cancer therapies, including radiation and hypomethylating agents, such as 5-azacytidine, rely on tumor cell-intrinsic activation of the RNA receptor RIG-I for their synergism with immune checkpoint inhibitors. Possible RIG-I ligands are small nuclear RNA (snRNA) and endogenous retroviral elements (ERV) leaking from the nucleus during programmed cell death.

Epigenetic therapy in combination with a multi-epitope cancer vaccine targeting shared tumor antigens for high-risk myelodysplastic syndrome - a phase I clinical trial.

BACKGROUND: Standard care for patients with high-risk myelodysplastic syndrome (MDS) is hypomethylating agents such as azacitidine (AZA), which can induce expression of methylated tumor-associated antigens and therefore potentiate immunotherapeutic targeting. METHOD: In this phase 1 trial, we combined AZA with a therapeutic peptide vaccine targeting antigens encoded from NY-ESO-1, MAGE-A3, PRAME, and WT-1, which have previously been demonstrated to be upregulated by AZA treatment. RESULT: Five patients who had responded to AZA monotherapy were included in the study and treated with the vaccine. The combination therapy showed only few adverse events during the study period, whereof none classified as serious. However, no specific immune responses could be detected using intracellular cytokine staining or ELISpot assays. Minor changes in the phenotypic composition of immune cells and their expression of stimulatory and inhibitory markers were detected. All patients progressed to AML with a mean time to progression from inclusion (TTP) of 5.2 months (range 2.8 to 7.6). Mean survival was 18.1 months (range 10.9 to 30.6) from MDS diagnosis and 11.3 months (range 4.3 to 22.2) from inclusion. Sequencing of bone marrow showed clonal expansion of malignant cells, as well as appearance of novel mutations. CONCLUSION: The patients progressed to AML with an average time of only five months after initiating the combination therapy. This may be unrelated to the experimental treatment, but the trial was terminated early as there was no sign of clinical benefit or immunological response. Why the manuscript is especially interesting This study is the first to exploit the potential synergistic effects of combining a multi-peptide cancer vaccine with epigenetic therapy in MDS. Although our results are negative, they emphasize challenges to induce immune reactivity in patients with high-risk MDS.

Targeting lncRNA/Wnt axis by flavonoids: A promising therapeutic approach for colorectal cancer.

Despite the dramatic advances in our understanding of the etiology of colorectal cancer (CRC) in recent decades, effective therapeutic strategies are still urgently needed. Oncogenic mutations in the Wnt/ß-Catenin pathway are hallmarks of CRC. Moreover, long non-coding RNAs (lncRNAs) as molecular managers are involved in the initiation, progression, and metastasis of CRC. Therefore, it is important to further explore the interaction between lncRNAs and Wnt/ß-Catenin signaling pathway for targeted therapy of CRC. Natural phytochemicals have not toxicity and can target carcinogenesis-related pathways. Growing evidences suggest that flavonoids are inversely associated with CRC risk. These bioactive compounds could target carcinogenesis pathways of CRC and reduced the side effects of anti-cancer drugs. The review systematically summarized the progress of flavonoids targeting lncRNA/Wnt axis in the investigations of CRC, which will provide a promising therapeutic approach for CRC and develop nutrition-oriented preventive strategies for CRC based on epigenetic mechanisms. In the field, more epidemiological and clinical trials are required in the future to verify feasibility of targeting lncRNA/Wnt axis by flavonoids in the therapy and prevention of CRC.

Cell surface GRP78 signaling: An emerging role as a transcriptional modulator in cancer.

Cancer cells acquire dysregulated gene expression to establish specific transcriptional dependencies and their underlying mechanisms that are ultimately responsible for this addictions have not been fully elucidated. Glucose-regulated protein 78 (GRP78) is a stress-inducible, multifunctional, prosurvival, endoplasmic reticulum chaperone in the heat shock protein 70 family. Expression of cell surface GRP78 (CS-GRP78) is associated with increased malignant behavior and resistance to chemotherapy and radiotherapy by endowing various cancer cells with increased proliferative ability, altered metabolism, improved survival, and augmented invasive and metastatic potential. Emerging evidence has highlighted an unusual role of CS-GRP78 in regulating transcription factors (TFs) by mediating various signaling pathways involved in malignant transformation, metabolic reprogramming, and tumor progression. During the last decade, we targeted CS-GRP78 with C38 monoclonal antibody (C38 Mab) in numerous studies, which have highlighted the epigenetic interplay between CS-GRP78 and various TFs including c-MYC, Yes-associated protein/transcriptional coactivator with PDZ-binding motif, c-Fos, and histone acetylation to potentiate subsequent modulation of tumorigenesis, invasion, and metastasis. Here, we summarize the current state of knowledge about the role of CS-GRP78 in cancer development and progression, including epigenetic regulation and sheds light on CS-GRP78 as vulnerable target for cancer therapy. Overall, this review focuses on the mechanisms of TFs that are behind the transcriptional dysregulation in cancer and lays the groundwork for rational therapeutic use of C38 Mab based on CS-GRP78 biology.

Editing the Epigenome: Reshaping the Genomic Landscape.

The eukaryotic epigenome has an instrumental role in determining and maintaining cell identity and function. Epigenetic components such as DNA methylation, histone tail modifications, chromatin accessibility, and DNA architecture are tightly correlated with central cellular processes, while their dysregulation manifests in aberrant gene expression and disease. The ability to specifically edit the epigenome holds the promise of enhancing understanding of how epigenetic modifications function and enabling manipulation of cell phenotype for research or therapeutic purposes. Genome engineering technologies use highly specific DNA-targeting tools to precisely deposit epigenetic changes in a locus-specific manner, creating diverse epigenome editing platforms. This review summarizes these technologies and insights from recent studies, describes the complex relationship between epigenetic components and gene regulation, and highlights caveats and promises of the emerging field of epigenome editing, including applications for translational purposes, such as epigenetic therapy and regenerative medicine.

A novel epigenetic drug conjugating flavonoid and HDAC inhibitor confer suppression of acute myeloid leukemogenesis.

Epigenetic dysregulation has long been identified as a key driver of leukemogenesis in acute myeloid leukemia (AML). However, epigenetic drugs such as histone deacetylase inhibitors (HDACis) targeting epigenetic alterations in AML have obtained only limited clinical efficiency without clear mechanism. Fortunately, we screened out a novel epigenetic agent named Apigenin-Vorinostat-Conjugate (AVC), which provides us a possibility to handle the heterogeneous malignancy. Its inhibition on HDACs was presented by HDACs expression, enzyme activity, and histone acetylation level. Its efficacy against AML was detected by cell viability assay and tumor progression of AML mouse model. Apoptosis is the major way causing cell death. We found that AVC efficiently suppresses leukemogenesis while sparing the normal human cells. Kasumi-1 cells are at least 20-fold higher sensitive to AVC (IC50 = 0.024 µM) than vorinostat (IC50 = 0.513 µM) and Ara-C (IC50 = 0.4366 µM). Furthermore, it can efficiently regress the tumorigenesis in AML mouse model while keeping the pivotal organs safe, demonstrating a feasibility and favorable safety profile in treatment of AML. Collectively, these preclinical data suggest a promising potential utilizing flavonoid-HDACi-conjugate as a next-generation epigenetic drug for clinical therapy against AML.