Epigenetic Function Research Articles

CHD4 Loss Alters Chromatin Accessibility Giving Rise to Sensitivity to DNA Damage Repair Compounds in Acute Myeloid Leukaemia

Background: Acute Myeloid Leukaemia (AML) is a heterogeneous group of blood and bone marrow disorders arising from myeloid precursors. Responses to current therapeutic options remain sub-optimal for many individuals, highlighting the need for novel therapeutic options; alongside stratifying patients by (epi)genetic alterations. Chromatin remodelling complexes (e.g. nucleosome remodelling and deacetylation complex (NuRD)), that control chromatin organization and gene expression, have emerged as key players in cancer. Cancer genome studies highlighted that modifications in NuRD occur in > 20% of cancers. NuRD regulates chromatin accessibility; CHD4, a core subunit, has a multitude of functions including epigenetic regulation, DNA Damage Repair (DDR), replication and cell cycle progression. Aims: To explore the molecular and epigenetic functions of CHD4 in AML using CRISPR-Cas9 isogenic knockout models (CHD4-/-), particularly the role of CHD4 on AML development and progression and evaluate if there is targetable therapeutic potential. Methods: TGCA datasets were assessed to identify impact of CHD4 expression on AML patient prognosis. Isogenic CHD4-/- models were generated using CRISPR-Cas9 in two AML cell lines (OCI AML 2 and OCI AML 3). Knockout models underwent functional phenotyping studies before inducing DNA damage at clinically relevant doses. Epigenetic mechanisms of CHD4 were assessed using multi-omic approaches including ATAC sequencing, RNA sequencing and proteomic analysis. A DNA Damage/Repair Compound Library (Selleckchem) was screened on CHD4-/- and CHD4 WT models using CellTox™ Green to identify DNA damage compounds that showed significant sensitivity in CHD-/- models and validation was assessed using CellTiter® Glo viability assay and co-culture assays. Combination indices (CI) were calculated using CompuSyn software. Results: Analysis of TCGA AML transcriptomic data (N=173), found that AML patients with intermediate risk cytogenetics and low CHD4 expression had a poorer 5-year survival compared to similar risk group patients with higher CHD4 levels (P=0.032). This indicates that lower CHD4 levels may result in a link between altered cellular transcription and patient survival causing a more aggressive disease phenotype in these patients. Phenotypical analysis of CHD4-/- models showed increased 53BP1 (P= 0.026) and PARP indicating DDR defect characterised by unrepaired dsDNA breaks. Defective DDR was shown by reduced proliferation with cell cycle analysis showing inter-S phase defect with accumulation in G1 and reduction in cells entering S and G2/M phases in the CHD4-/- models. With this profile being further exacerbated under cell exposure to clinically relevant irradiation induced stress. Chromatin accessibility was assessed via ATAC sequencing and showed enhanced open chromatin at areas impacting immune regulation within CHD4-/- cells. RNA and proteomic analysis both showed upregulation of genes involved in immune activation (P=6.96e-8) and cellular senescence (P=5.6e-5) pathways suggesting an altered immune profile in CHD4-/- cells with a potential to exploit this therapeutically. To identify potential therapeutic vulnerabilities in CHD4-/- cells we performed high throughput screening using a 160 compound DNA damaging library which showed an increased sensitivity of CHD4-/- cells to compounds targeting DNA/RNA synthesis pathways, including Mitomycin C, this indicated that loss of CHD4 results in hyper-increase in damage. Further investigation with Mitomycin C found that when in combination with Cytarabine yielded enhanced synergistic effects in CHD4-/- cells (CI 0.001-0.37) when compared to CHD4 WT (CI 0.002-3.1). There was also significantly less viable cells (P=0.016) 24-hours post combination treatment in CHD4-/- compared to WT (33% vs 56%), indicating that CHD4 loss is furthering the damage caused by improper cell cycle leading to cell death. Conclusion A cohort of AML patients with intermediate risk cytogenetics along with lower CHD4 levels have poorer 5-year survival. Lack of CHD4 is shown to cause epigenetic restructuring, gene abnormalities and defective DDR. We have identified Mitomycin C as a therapeutic potential in combination with Cytarabine in CHD4-/- models. Further investigation of this in combination and the mechanism of action could lead to improved outcome for this sub-group of patients.

PIM Kinases Regulate Super-Enhancer-Dependent Gene Expression in Diffuse Large B-Cell Lymphoma

The PIM kinase family consists of 3 proto-oncogenic proteins: PIM1, PIM2 and PIM3, expressed in numerous cancers, including diffuse large B-cell lymphoma (DLBCL). PIM kinases regulate crucial processes, such as proliferation, apoptosis, metabolism, and migration, therefore their inhibition is of great interest as a potential therapeutic strategy. However, the detailed mechanisms of PIM's oncogenic effects and the consequences of their inhibition in DLBCL remain insufficiently understood. Our earlier studies (Szydłowski et al., Blood 2017, Szydłowski et al., Cancer Res. 2021) highlighted immunomodulatory functions of PIM kinases in lymphoid malignancies, but also indicated that their inhibition has a profound impact on gene expression. Although earlier studies indicated that PIMs regulate certain transcription factors and/or regulate gene expression via epigenetic mechanisms through histone H3S10 phosphorylation, the detailed genomic functions of PIM kinases in modulating transcription in DLBCL remains unclear. In order to investigate the mechanisms and consequences of PIM kinase inhibition on gene expression in DLBCL cells, we first performed RNA sequencing-based transcriptome profiling in DLBCL cells treated with the pan-PIM and FLT3 inhibitor MEN1703, which is currently in clinical development. MEN1703 markedly downregulated the expression of transcripts controlled by super-enhancers (SE), i.e. large regulatory regions responsible for high levels of transcript expression of the key essential genes for the cell, including oncogenes. The analysis also suggested new biological processes controlled by PIMs, including the reduction of non-coding RNAs generated from the SE regions. To understand the mechanisms linking PIM inhibition with decreased SE-driven gene expression, we assessed PIM-dependent H3S10 phosphorylation in lymphoma cells. Since H3S10 phosphorylation facilitates MYST family KAT8 (MOF) acetyltransferase recruitment to certain promoters/enhancers, we evaluated the histone H3 and H4 acetylations in DLBCL cells following PIM inhibition. MEN1703 markedly reduced the H3K9Ac, H3K14Ac and H4 pan-acetylation, which was associated with reduced BRD4 binding to the SE regions and reduced RNAPII serine 5 (S5) phosphorylation, but not S3 or S7 phosphorylations. Together, these observations suggest the participation of PIM in the elongation phase of SE-dependent gene transcription via H3S10-dependent epigenetic mechanism. The effects of PIM inhibition on histone modifications and proliferation were confirmed by genetic silencing or CRISPR-Cas9 deletion of individual PIM kinases. To better characterize the function of H3S10 phosphorylation in DLBCL, non-phosphorylatable H3S10A mutants were introduced to DLBCL cells. Compared to wild type counterparts, cells with the mutated H3S10 exhibited decreased proliferation, indicating a significant biological role of this modification in DLBCL. The effect was potentiated in cells exposed to metabolic stress (starvation) conditions. Together, these studies confirm the epigenetic role of PIM kinases, especially in the regulation of SE-dependent gene transcription and suggest that inhibition of PIM activity leads to disturbances of SE integrity and DLBCL transcription. The results document new, epigenetic function of PIM kinases and present a novel cytotoxicity mechanism of their inhibition in DLBCL. They also indicate that PIM inhibition with MEN1703 can be a therapeutic option in DLBCL. Study supported aby Foundation For Polish Science TEAM Grant # POIR.04.04.00-00-5C84/17 and Polish National Science Centre Grant # 2018/29/B/NZ5/01471.

Epigenetic modifications in the accumulation and function of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are key players under various pathologic conditions, such as cancer. Epigenetic modifications such as DNA methylation, RNA-mediated processes, and histone modification can alter gene transcription, and thus regulating pathological process. Studies have shown that epigenetic modification contributes to the accumulation and function of MDSCs. This review summarizes the crosstalk between the epigenetic alterations and MDSCs functions, and briefly introduces how the accumulation and function of MDSCs caused by epigenetic modification impact on the disease development, which represents as a promising therapeutic strategy for the related disorders.

A quantitative structural analysis of AR-42 derivatives as HDAC1 inhibitors for the identification of promising structural contributors

ABSTRACT Alteration and abnormal epigenetic mechanisms can lead to the aberration of normal biological functions and the occurrence of several diseases. The histone deacetylase (HDAC) family of enzymes is one of the prime regulators of epigenetic functions modifying the histone proteins, and thus, regulating epigenetics directly. HDAC1 is one of those HDACs which have important contributions to cellular epigenetics. The abnormality of HDAC is correlated to the occurrence, progression, and poor prognosis in several disease conditions namely neurodegenerative disorders, cancer cell proliferation, metastasis, chemotherapy resistance, and survival in various cancers. Therefore, the progress of potent and effective HDAC1 inhibitors is one of the prime approaches to combat such diseases. In this study, both regression and classification-based molecular modelling studies were conducted on some AR-42 derivatives as HDAC1 inhibitors to elucidate the crucial structural aspects that are responsible for regulating their biological responses. This study revealed that the molecular polarizability, van der Waals volume, the presence of aromatic rings as well as the higher number of hydrogen bond acceptors might affect prominently their inhibitory activity and might be responsible for proper fitting and interactions at the HDAC1 active site to pertain effective inhibition.

Synthetic antibody-derived immunopeptide provides neuroprotection in glaucoma through molecular interaction with retinal protein histone H3.1.

Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs) as well as their axons leading to irreversible loss of sight. Medical management of the intraocular pressure (IOP) still represents the gold standard in glaucoma therapy, which only manages a single risk factor and does not directly address the neurodegenerative component of this eye disease. Recently, our group showed that antibody-derived immunopeptides (encoding complementarity-determining regions, CDRs) provide attractive glaucoma medication candidates and directly interfere its pathogenic mechanisms by different modes of action. In accordance with these findings, the present study showed the synthetic complementary-determining region 2 (CDR2) peptide (INSDGSSTSYADSVK) significantly increased RGC viability in vitro in a concentration-dependent manner (p < 0.05 using a CDR2 concentration of 50 μg/mL). Employing state-of the-art immunoprecipitation experiments, we confirmed that synthetic CDR2 exhibited a high affinity toward the retinal target protein histone H3.1 (HIST1H3A) (p < 0.001 and log2-fold change > 3). Furthermore, molecular dynamics (MD) simulations along with virtual docking analyses predicted potential CDR2-specific binding regions of HIST1H3A, which might represent essential post-translational modification (PTM) sites for epigenetic regulations. Quantitative mass spectrometry (MS) analysis of retinas demonstrated 39 proteins significantly affected by CDR2 treatment (p < 0.05). An up-regulation of proteins involved in the energy production (e.g., ATP5F1B and MT-CO2) as well as the regulatory ubiquitin proteasome system (e.g., PSMC5) was induced by the synthetic CDR2 peptide. On the other hand, CDR2 reduced metabolic key enzymes (e.g., DDAH1 and MAOB) as well as ER stress-related proteins (e.g., SEC22B and VCP) and these data were partially confirmed by microarray technology. Our outcome measurements indicate that specific protein-peptide interactions influence the regulatory epigenetic function of HIST1H3A promoting the neuroprotective mechanism on RGCs in vitro. In addition to IOP management, such synthetic peptides as CDR2 might serve as a synergistic immunotherapy for glaucoma in the future.

Effects of exosomes on tumor immunomodulation and their potential clinical applications (Review).

Exosomes are small extracellular vesicles containing proteins, nucleic acids and lipids, which can transmit information between cells, and can interfere with the epigenetic characteristics and functions of recipient cells. Tumor‑derived exosomes play a crucial role as communicators in the tumor microenvironment, and are involved in the occurrence and development of various tumors. The present review article summarizes the biogenesis of exosomes and their communication with local and remote cells, focusing on the function of tumor‑derived exosomes in the tumor microenvironment, including the promotion of angiogenesis, the induction of epithelial mesenchymal transformation, and the activation and inhibition of immune cells, as well as the effects of exosomes on the tumor microenvironment during microbial infections. Additionally, the effects of exosomes on tumor immunotherapy and the potential applications of exosomes as biomarkers, delivery vehicles and cancer vaccines in cancer diagnostics and therapeutics are discussed.

Vitamin C epigenetically controls osteogenesis and bone mineralization

Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2, in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C’s epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions.

Geographic encoding of transcripts enabled high-accuracy and isoform-aware deep learning of RNA methylation.

As the most pervasive epigenetic mark present on mRNA and lncRNA, N6-methyladenosine (m6A) RNA methylation regulates all stages of RNA life in various biological processes and disease mechanisms. Computational methods for deciphering RNA modification have achieved great success in recent years; nevertheless, their potential remains underexploited. One reason for this is that existing models usually consider only the sequence of transcripts, ignoring the various regions (or geography) of transcripts such as 3′UTR and intron, where the epigenetic mark forms and functions. Here, we developed three simple yet powerful encoding schemes for transcripts to capture the submolecular geographic information of RNA, which is largely independent from sequences. We show that m6A prediction models based on geographic information alone can achieve comparable performances to classic sequence-based methods. Importantly, geographic information substantially enhances the accuracy of sequence-based models, enables isoform- and tissue-specific prediction of m6A sites, and improves m6A signal detection from direct RNA sequencing data. The geographic encoding schemes we developed have exhibited strong interpretability, and are applicable to not only m6A but also N1-methyladenosine (m1A), and can serve as a general and effective complement to the widely used sequence encoding schemes in deep learning applications concerning RNA transcripts.

Trypanosoma brucei histones are heavily modified with combinatorial post-translational modifications and mark Pol II transcription start regions with hyperacetylated H2A.

Trypanosomes diverged from the main eukaryotic lineage about 600 million years ago, and display some unusual genomic and epigenetic properties that provide valuable insight into the early processes employed by eukaryotic ancestors to regulate chromatin-mediated functions. We analysed Trypanosoma brucei core histones by high mass accuracy middle-down mass spectrometry to map core histone post-translational modifications (PTMs) and elucidate cis-histone combinatorial PTMs (cPTMs). T. brucei histones are heavily modified and display intricate cPTMs patterns, with numerous hypermodified cPTMs that could contribute to the formation of non-repressive euchromatic states. The Trypanosoma brucei H2A C-terminal tail is hyperacetylated, containing up to five acetylated lysine residues. MNase-ChIP-seq revealed a striking enrichment of hyperacetylated H2A at Pol II transcription start regions, and showed that H2A histones that are hyperacetylated in different combinations localised to different genomic regions, suggesting distinct epigenetic functions. Our genomics and proteomics data provide insight into the complex epigenetic mechanisms used by this parasite to regulate a genome that lacks the transcriptional control mechanisms found in later-branched eukaryotes. The findings further demonstrate the complexity of epigenetic mechanisms that were probably shared with the last eukaryotic common ancestor.

The epigenetic role of proteasome subunit RPT6 during memory formation in female rats.

Reports of sex differences in the neurobiology of memory formation are becoming more prevalent. Despite this, much remains unknown about the role of sex in this process. We previously reported the first evidence of a novel epigenetic role for proteasome subunit RPT6 during memory formation in the hippocampus of male rodents whereby it associated with monoubiquitinated histone H2B (H2Bubi). Here, we used molecular, biochemical, and behavioral approaches to investigate whether RPT6 has a similar epigenetic role during memory formation in female rats. Following contextual fear conditioning, we found that RPT6 levels and DNA binding at regions coding for c-fos, the previously identified target of RPT6 in males, were unchanged in the hippocampus of females and that loss of RPT6 did not alter learning-induced increases in c-fos However, RPT6 was in complex with H2Bubi in the female hippocampus and this association increased with fear conditioning, suggesting that it could still retain an epigenetic function. Consistent with this, hippocampal siRNA-mediated knockdown of the RPT6-coding gene, Psmc5, impaired memory in females. These results suggest that while RPT6 does associate with epigenetic H2Bubi during memory formation in both males and females, it has sex-specific gene targets during the memory consolidation process.

Abstract P3098: Histone Modifying Genes In Heart Development And Disease

Sequencing of Congenital Heart Disease (CHD) patients identified variants in many histone modifying genes, suggesting that histone modification plays important roles in heart development and disease. However, how these histone modifying genes perform their epigenetic regulation function during heart development remain elusive, and whether these novel genetic variants identified from CHD patients are causal remain unclear. We found that most histone modifying genes are highly conserved from Drosophila to humans, and many are expressed in the developing heart in both systems. Using a high-throughput functional validation system that we generated for providing the “gene-level” disease association for candidate CHD genes, we screened lots of histone-modifying genes and identified many that are required for heart development in Drosophila. Among them, genes involved in histone H3K4 mono- and dimethylation are particularly important for heart development and appear to regulate evolutionarily conserved targets during heart development from Drosophila to mammals. We also generated Drosophila models for CHD patient-derived variants in histone-modifying genes to study how these variants affect the function of the human genes in the context of the fly heart development, to provide the “variant-level” evidence for their causal association with CHD.

Identification of Metabolic Syndrome-Related miRNA-mRNA Regulatory Networks and Key Genes Based on Bioinformatics Analysis.

Metabolic syndrome, which affects approximately one-quarter of the world's population, is a combination of multiple traits and is associated with high all-cause mortality, increased cancer risk, and other hazards. It has been shown that the epigenetic functions of miRNAs are closely related to metabolic syndrome, but epigenetic studies have not yet fully elucidated the regulatory network and key genes associated with metabolic syndrome. To perform data analysis and screening of potential differentially expressed target miRNAs, mRNAs and genes based on a bioinformatics approach using a metabolic syndrome mRNA and miRNA gene microarray, leading to further analysis and identification of metabolic syndrome-related miRNA-mRNA regulatory networks and key genes. The miRNA gene set (GSE98896) and mRNA gene set (GSE98895) of peripheral blood samples from patients with metabolic syndrome from the GEO database were screened, and set|logFC|> 1 and adjusted P < 0.05 were used to identify the differentially expressed miRNAs and mRNAs. Differentially expressed miRNA transcription factors were predicted using FunRich software and subjected to GO and KEGG enrichment analysis. Next, biological process enrichment analysis of differentially expressed mRNAs was performed with Metascape. Differentially expressed miRNAs and mRNAs were identified and visualized as miRNA-mRNA regulatory networks based on the complementary pairing principle. Data analysis of genome-wide metabolic syndrome-related mRNAs was performed using the gene set enrichment analysis (GSEA) database. Finally, further WGCNA of the set of genes most closely associated with metabolic syndrome was performed to validate the findings. A total of 217 differentially expressed mRNAs and 158 differentially expressed miRNAs were identified by screening the metabolic syndrome miRNA and mRNA gene sets, and these molecules mainly included transcription factors, such as SP1, SP4, and EGR1, that function in the IL-17 signalling pathway; cytokine-cytokine receptor interaction; proteoglycan syndecan-mediated signalling events; and the glypican pathway, which is involved in the inflammatory response and glucose and lipid metabolism. miR-34C-5P, which was identified by constructing a miRNA-mRNA regulatory network, could regulate DPYSL4 expression to influence insulin β-cells, the inflammatory response and glucose oxidative catabolism. Based on GSEA, metabolic syndrome is known to be closely related to oxidative phosphorylation, DNA repair, neuronal damage, and glycolysis. Finally, RStudio and DAVID were used to perform WGCNA of the gene sets most closely associated with metabolic syndrome, and the results further validated the conclusions. Metabolic syndrome is a common metabolic disease worldwide, and its mechanism of action is closely related to the inflammatory response, glycolipid metabolism, and impaired mitochondrial function. miR-34C-5P can regulate DPYSL4 expression and can be a potential research target. In addition, UQCRQ and NDUFA8 are core genes of oxidative phosphorylation and have also been identified as potential targets for the future treatment of metabolic syndrome.

Breastfeeding and Allergy Effect Modified by Genetic, Environmental, Dietary, and Immunological Factors.

Breastfeeding (BF) is the most natural mode of nutrition. Its beneficial effect has been revealed in terms of both the neonatal period and those of lifelong effects. However, as for protection against allergy, there is not enough data. In the current narrative review, the literature within the last five years from clinical trials and population-based studies on breastfeeding and allergy from different aspects was explored. The aim of this review was to explain how different factors could contribute to the overall effect of BF. Special consideration was given to accompanying exposure to cow milk, supplement use, the introduction of solid foods, microbiota changes, and the epigenetic function of BF. Those factors seem to be modifying the impact of BF. We also identified studies regarding BF in atopic mothers, with SCFA as a main player explaining differences according to this status. Conclusion: Based on the population-based studies, breastfeeding could be protective against some allergic phenotypes, but the results differ within different study groups. According to the new research in that matter, the effect of BF could be modified by different genetic (HMO composition), environmental (cesarean section, allergen exposure), dietary (SCFA, introduction of solid food), and immunologic factors (IgG, IgE), thus partially explaining the variance.

Epigenetic genes and epilepsy - emerging mechanisms and clinical applications.

An increasing number of epilepsies are being attributed to variants in genes with epigenetic functions. The products of these genes include factors that regulate the structure and function of chromatin and the placing, reading and removal of epigenetic marks, as well as other epigenetic processes. In this Review, we provide an overview of the various epigenetic processes, structuring our discussion around five function-based categories: DNA methylation, histone modifications, histone-DNA crosstalk, non-coding RNAs and chromatin remodelling. We provide background information on each category, describing the general mechanism by which each process leads to altered gene expression. We also highlight key clinical and mechanistic aspects, providing examples of genes that strongly associate with epilepsy within each class. We consider the practical applications of these findings, including tissue-based and biofluid-based diagnostics and precision medicine-based treatments. We conclude that variants in epigenetic genes are increasingly found to be causally involved in the epilepsies, with implications for disease mechanisms, treatments and diagnostics.

ZMYND8 is a master regulator of 27-hydroxycholesterol that promotes tumorigenicity of breast cancer stem cells.

27-Hydroxycholesterol (27-HC) is the most abundant oxysterol that increases the risk of breast cancer progression. However, little is known about epigenetic regulation of 27-HC metabolism and its role in breast tumor initiation. Using genetic mouse mammary tumor and human breast cancer models, we showed here that the histone reader ZMYND8 was selectively expressed in breast cancer stem cells (BCSCs) and promoted epithelial-mesenchymal transition (EMT), BCSC maintenance and self-renewal, and oncogenic transformation through its epigenetic functions, leading to breast tumor initiation. Mechanistically, ZMYND8 was a master transcriptional regulator of 27-HC metabolism. It increased cholesterol biosynthesis and oxidation but blocked cholesterol efflux and 27-HC catabolism, leading to accumulation of 27-HC in BCSCs. Consequently, 27-HC promoted EMT, oncogenic transformation, and tumor initiation through activation of liver X receptor. These findings reveal that ZMYND8 is an epigenetic booster that drives breast tumor initiation through metabolic reprogramming.

O-149 Personalized probiotics and food supplementation according to microbiome miRNA test improves the conception rate in multiple IVF failure patients

Abstract Study question Can the microbiota play a role in the development of infertility by affecting the epigenetic, immunologic, and/or biochemical functions of the host? Summary answer The customization of diet and a nutraceutical and probiotic supplementation according microbiome miRNA signature could increase the pregnancy and live birth rates. What is known already The normal gut microbiota has an impact on the normal function of the male and female reproductive tract by regulating the immunological changes associated with conception. Several miRNAs -a class of small noncoding RNA molecules that control gene expression at the post-transcriptional level- have been described to be associated with dysbiosis and an immune imbalance. We previously demonstrated that women with unexplained infertility could have a gut and vaginal microbiome imbalance with increased intestinal permeability linked to an overexpression of miR-21 and miR-155. Study design, size, duration Observational study including 287 multiple IVF failure infertile patients. They had an average age of 40y and they performed 4 IVF-ET within 10 years on average. Swab samples were collected from the vagina and rectum. Participants/materials, setting, methods Microbial composition by NGS and miRNA expression by real time PCR of vaginal and rectal samples were measured. Immunometabolic markers from blood (TPO, vitamin D and B12, insulin, LDL) were analyzed. Patients followed a customized food supplementation, according to the tested biomarkers, for 90 days. Main results and the role of chance We found that 80% of women showed increased miRNA levels associated with gut and vaginal microbiome imbalances. Deficient values of Vitamin D and/or B were found in 73.2% of tested patients, whereas 56.1% showed high LDL and or insulin values and 19.9% showed positive anti thyroid TPO antibodies. Based upon the elevated levels of miRNAs and immunometabolic markers (insulin, vitamin D, autoantibodies, LDL cholesterol and secretory IgA), patients completed a 60-90 day therapy prior to their next IVF-ET attempt with no clinical side effects. BMI measurements before and after the therapy were not significantly different. After normalization of the blood biomarkers, each patient underwent a next IVF -ET attempt. The total pregnancy rate resulted in 75% (215/287) of positive chemical pregnancy (hCG) and 73% (157/215) of clinically confirmed pregnancy (ultrasound). The live birth rate per intended egg retrieval (all embryos transfers) was 60% (129/215); 27 pregnancies resulted in first trimester losses and one fetal death after a severe preeclampsia. Limitations, reasons for caution A future study should be performed testing miRNA expression before and after the food supplementation to achieve a direct correlation between the microRNA test and the supplementation in a placebo control study in order to evaluate the efficacy of the personalized supplementation on improving pregnancy and live birth rates. Wider implications of the findings Immune system and microbiome modulation could be adress by customizing different strains of probiotics and nutraceuticals selected for their immunomodulatory capacity. An anti-inflammatory dose-specific effect to maintain a delicate immune balance and favor endometrial mucous vascularization and the subsequent placentation, should be personalized to achieve a healthy pregnancy. Trial registration number CEI 1590

Abstract 2966: The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia

Abstract MLL1 (KMT2A) translocations are found in ~10% of acute leukemia and give rise to an aggressive form of leukemia in infant, pediatric and adult patients. MLL1 fusion driven acute leukemia is characterized by deregulated activity of the Super Elongation Complex (SEC) and the H3K79 methyltransferase DOT1L, which alter the epigenetic landscape and transcription of pro-leukemic MLL1 fusion targets like HoxA9 and Meis1. The Eleven-Nineteen-Leukemia gene (ENL or MLLT1) is a common MLL1 fusion partner and a SEC component. The ENL protein contains a highly conserved N-terminal epigenetic reader YEATS domain that recognizes acetylated H3K9/K18/K27 (H3Kac hereinafter). Wild type ENL was recently found to be essential for leukemic cell growth, which is dependent on its YEATS domain interaction with H3Kac. While this finding highlighted the YEATS domain importance in wild type ENL function in leukemic cells, the inclusion and importance of the YEATS domain in MLL-ENL fusion protein remain to be elucidated. Here, we investigated the clinical relevance and importance of the ENL YEATS domain in MLL-ENL leukemias. We analyzed &amp;gt;300 t(11;19) MLL-ENL leukemia patients for the breakpoint location within the ENL gene and found that the YEATS domain is retained in the resultant MLL-ENL fusion protein in 84.1% of t(11;19) leukemia patients. We tested the importance of the YEATS domain in MLL-ENL mouse models and found that the YEATS domain and downstream sequence is required for MLL-ENL leukemogenesis in vivo. YEATS deletion decreased expression of pro-leukemic targets such as Meis1, an important factor for leukemic stem cells (LSC). To interrogate the contribution of the YEATS epigenetic reader function in MLL-ENL leukemogenesis, we introduced YEATS point mutations rendering the domain defective in interacting with H3Kac and found that this significantly increased leukemia latency in vivo. Further investigation revealed that YEATS point mutations disrupting H3Kac binding significantly decreased MLL-ENL LSC frequency while not affecting homing to the bone marrow. We attribute this LSC frequency change to altered Meis1 expression. Additionally, disruption of the YEATS epigenetic reader function in MLL-ENL leukemia cells does not induce differentiation, apoptosis nor cell cycle arrest. Therapeutically, we predicted the YEATS domain in MLL-ENL would sensitize MLL-ENL leukemia to YEATS domain inhibitors. Indeed, MLL-ENL leukemia cells are more sensitive to the ENL/AF9 YEATS domain inhibitor, SGC-iMLLT, compared to acute leukemia cells driven by other fusions. Together, our results demonstrate that YEATS-H3Kac binding plays an important role in MLL-ENL fusion mediated leukemogenesis. Our data establishes a strong rationale for future exploration of small molecules aimed at disrupting the YEATS-H3Kac interaction as a targeted therapeutics for treating t(11;19) leukemia patients. Citation Format: Hsiangyu Hu, Nirmalya Saha, Ejaz Ahmad, Yuting Yang, Lili Chen, Lauren Lachowski, Blaine Teahan, Sierrah Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, Andrew G. Muntean. The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia [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 2966.

Abstract 419: The development of BRPF1 degraders as a potential treatment for acute myeloid leukemia

Abstract Acetylation of histones and additional nuclear proteins is a key mechanism in the regulation of gene expression. Aberrant acetylation has been linked to a wide range of diseases including cancer, inflammation, and neurodevelopmental disorders. Histone acetylation is introduced by histone acetyltransferases complexes (HATs), where substrate specificity is dramatically enhanced by scaffolding proteins that activate and target them to specific chromatin sites. A protein of interest with both epigenetic acetyl reader and scaffolding function is the protein Bromodomain and PHD finger-containing protein 1 (BRPF1). The Protein contains domains of two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) Tudor domain. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as the catalytic MYST-family acetyltransferase subunit and the stable complex with Moz-Tif2 is known to lead to the development of human acute myeloid leukemia (AML). Given this, we chose to pursue the development of BRPF1 degraders to probe cancer disease biology especially in AML, where new therapies are required to overcome several unmet needs such as less-toxic treatments and relapsed/refractory disease paradigms. Here we present the design and synthesis of a range of BRPF1 degraders in highly desirable physicochemical space utilizing in-silico modeling. The prepared degraders which utilize multiple E3 ligases were then screened against cell lines harboring Mixed-lineage leukemia (MLL) translocations specifically the THP-1 cell line. In addition to this, we investigated the ability of the compounds to effectively degrade the target and suitability of our degraders for potential in-vivo exposure through a panel of routine ADMET assays. Citation Format: Daniel Joseph Glynn, Rosie Crampton, Thomas Pesnot, Andrew Scott, Anne-Chloe Nassoy, Ralph Kirk, Daniele Narducci, Gary Nelson, Lynette Ongiri, Habiba Begum, Vincent Rao, Matilda Bingham, Rhoanne McPherson, Darryl Turner. The development of BRPF1 degraders as a potential treatment for acute myeloid leukemia [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 419.

The Role of Myeloid Cells in GBM Immunosuppression.

Gliomas are intrinsic brain tumors that originate from glial cells. Glioblastoma (GBM) is the most aggressive glioma type and resistant to immunotherapy, mainly due to its unique immune environment. Dimensional data analysis reveals that the intra-tumoral heterogeneity of immune cell populations in the glioma microenvironment is largely made up of cells of myeloid lineage. Conventional therapies of combined surgery, chemotherapy and radiotherapy have achieved limited improvements in the prognosis of glioma patients, as myeloid cells are prominent mediators of immune and therapeutic responses—like immunotherapy resistance—in glioma. Myeloid cells are frequently seen in the tumor microenvironment (TME), and they are polarized to promote tumorigenesis and immunosuppression. Reprogramming myeloid cells has emerged as revolutionary, new types of immunotherapies for glioma treatment. Here we detail the current advances in classifying epigenetic, metabolic, and phenotypic characteristics and functions of different populations of myeloid cells in glioma TME, including myeloid-derived suppressor cells (MDSCs), glioma-associated microglia/macrophages (GAMs), glioma-associated neutrophils (GANs), and glioma-associated dendritic cells (GADCs), as well as the mechanisms underlying promotion of tumorigenesis. The final goal of this review will be to provide new insights into novel therapeutic approaches for specific targeting of myeloid cells to improve the efficacy of current treatments in glioma patients.

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.