Epigenetic State Research Articles

Evidence of innate training in bovine γδ T cells following subcutaneous BCG administration.

The Bacillus Calmette Guerin (BCG) vaccine has been shown to induce non-specific protection against diseases other than tuberculosis in vaccinated individuals, attributed to the induction of trained immunity. We have previously demonstrated that BCG administration induces innate immune training in mixed peripheral blood mononuclear cells and monocytes in calves. Gamma Delta (γδ) T cells are non-conventional T cells that exhibit innate and adaptive immune system features. They are in higher proportion in the peripheral blood of cattle than humans or rodents and play an essential role in bovine immune response to pathogens. In the current study, we determined if BCG administration induced innate immune training in bovine γδ T cells. A group of 16 pre-weaned Holstein calves (2-4 d age) were enrolled in the study and randomly assigned to vaccine and control groups (n=8/group). The vaccine group received two doses of 106 colony forming units (CFU) BCG Danish strain subcutaneously, separated by 2 weeks. The control group remained unvaccinated. Gamma delta T cells were purified from peripheral blood using magnetic cell sorting three weeks after receiving the 1st BCG dose. We observed functional changes in the γδ T cells from BCG-treated calves shown by increased IL-6 and TNF-α cytokine production in response to in vitro stimulation with Escherichia coli LPS and PAM3CSK4. ATAC-Seq analysis of 78,278 regions of open chromatin (peaks) revealed that γδ T cells from BCG-treated calves had an altered epigenetic status compared to cells from the control calves. Differentially accessible peaks (DAP) found near the promoters of innate immunity-related genes like Siglec14, Irf4, Ifna2, Lrrfip1, and Tnfrsf10d were 1 to 4-fold more accessible in cells from BCG-treated calves. MOTIF enrichment analysis of the sequences within DAPs, which explores transcription factor binding motifs (TFBM) upstream of regulatory elements, revealed TFBM for Eomes and IRF-5 were among the most enriched transcription factors. GO enrichment analysis of genes proximal to the DAPs showed enrichment of pathways such as regulation of IL-2 production, T-cell receptor signaling pathway, and other immune regulatory pathways. In conclusion, our study shows that subcutaneous BCG administration in pre-weaned calves can induce innate immune memory in the form of trained immunity in γδ T cells. This memory is associated with increased chromatin accessibility of innate immune response-related genes, thereby inducing a functional trained immune response evidenced by increased IL-6 and TNF-α cytokine production.

DNA methylation landscapes in DIPG reveal methylome variability that can be modified pharmacologically.

Diffuse intrinsic pontine glioma (DIPG) is a uniformly lethal brainstem tumor of childhood, driven by histone H3 K27M mutation and resultant epigenetic dysregulation. Epigenomic analyses of DIPG have shown global loss of repressive chromatin marks accompanied by DNA hypomethylation. However, studies providing a static view of the epigenome do not adequately capture the regulatory underpinnings of DIPG cellular heterogeneity and plasticity. To address this, we performed whole-genome bisulfite sequencing on a large panel of primary DIPG specimens and applied a novel framework for analysis of DNA methylation variability, permitting the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG has a markedly disordered epigenome with increasingly stochastic DNA methylation at genes regulating pluripotency and developmental identity, potentially enabling cells to sample diverse transcriptional programs and differentiation states. The DIPG epigenetic landscape was responsive to treatment with the hypomethylating agent decitabine, which produced genome-wide demethylation and reduced the stochasticity of DNA methylation at active enhancers and bivalent promoters. Decitabine treatment elicited changes in gene expression, including upregulation of immune signaling such as the interferon response, STING, and MHC class I expression, and sensitized cells to the effects of histone deacetylase inhibition. This study provides a resource for understanding the epigenetic instability that underlies DIPG heterogeneity. It suggests the application of epigenetic therapies to constrain the range of epigenetic states available to DIPG cells, as well as the use of decitabine in priming for immune-based therapies.

Bottom-up data integration in polymer models of chromatin organisation

Cellular functions crucially depend on the precise execution of complex biochemical reactions taking place on the chromatin fiber in the tightly packed environment of the cell nucleus. Despite the availability of large data sets probing this process from multiple angles, bottom-up frameworks which allow the incorporation of the sequence-specific nature of biochemistry in a unified model of 3D chromatin structure remain scarce. Here we propose SEMPER (Sequence Enhanced Magnetic PolymER), a novel stochastic polymer model which naturally incorporates observational data about sequence-driven biochemical processes, such as binding of transcription factor proteins, in a 3D model of chromatin structure. We introduce a novel approximate Bayesian algorithm to quantify a posteriori the relative importance of various factors, including the polymeric nature of DNA, in determining chromatin epigenetic state, thus providing a transparent way to generate biological hypotheses. While accurate prediction of contact frequencies (a problem already extensively studied in the literature) is not our main aim, as a byproduct of the inference procedure and without additional input from the genome 3D structure, our model can predict with reasonable accuracy some notable and nontrivial conformational features of chromatin folding within the nucleus. Our work highlights the importance of introducing physically realistic statistical models for predicting chromatin states from epigenetic data and opens the way to a new class of more systematic approaches to interpreting epigenomic data.

Oncogenes and cancer associated thrombosis: what can we learn from single cell genomics about risks and mechanisms?

Single cell analysis of cancer cell transcriptome may shed a completely new light on cancer-associated thrombosis (CAT). CAT causes morbid, and sometimes lethal complications in certain human cancers known to be associated with high risk of venous thromboembolism (VTE), pulmonary embolism (PE) or arterial thromboembolism (ATE), all of which worsen patients' prognosis. How active cancers drive these processes has long evaded scrutiny. While "unspecific" microenvironmental effects and consequences of patient care (e.g., chemotherapy) have been implicated in pathogenesis of CAT, it has also been suggested that oncogenic pathways driven by either genetic (mutations), or epigenetic (methylation) events may influence the coagulant phenotype of cancer cells and stroma, and thereby modulate the VTE/PE risk. Consequently, the spectrum of driver events and their downstream effector mechanisms may, to some extent, explain the heterogeneity of CAT manifestations between cancer types, molecular subtypes, and individual cases, with thrombosis-promoting, or -protective mutations. Understanding this molecular causation is important if rationally designed countermeasures were to be deployed to mitigate the clinical impact of CAT in individual cancer patients. In this regard, multi-omic analysis of human cancers, especially at a single cell level, has brought a new meaning to concepts of cellular heterogeneity, plasticity, and multicellular complexity of the tumour microenvironment, with profound and still relatively unexplored implications for the pathogenesis of CAT. Indeed, cancers may contain molecularly distinct cellular subpopulations, or dynamic epigenetic states associated with different profiles of coagulant activity. In this article we discuss some of the relevant lessons from the single cell "omics" and how they could unlock new potential mechanisms through which cancer driving oncogenic lesions may modulate CAT, with possible consequences for patient stratification, care, and outcomes.

Heritable changes of epialleles near genes in maize can be triggered in the absence of CHH methylation.

Trans-chromosomal interactions resulting in changes in DNA methylation during hybridization have been observed in several plant species. However, little is known about the causes or consequences of these interactions. Here, we compared DNA methylomes of F1 hybrids that are mutant for a small RNA biogenesis gene, Mop1 (Mediator of paramutation1), with that of their parents, wild-type siblings, and backcrossed progeny in maize (Zea mays). Our data show that hybridization triggers global changes in both trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM), most of which involved changes in CHH methylation. In more than 60% of these TCM differentially methylated regions (DMRs) in which small RNAs are available, no significant changes in the quantity of small RNAs were observed. Methylation at the CHH TCM DMRs was largely lost in the mop1 mutant, although the effects of this mutant varied depending on the location of these DMRs. Interestingly, an increase in CHH at TCM DMRs was associated with enhanced expression of a subset of highly expressed genes and suppressed expression of a small number of lowly expressed genes. Examination of the methylation levels in backcrossed plants demonstrates that both TCM and TCdM can be maintained in the subsequent generation, but that TCdM is more stable than TCM. Surprisingly, although increased CHH methylation in most TCM DMRs in F1 plants required Mop1, initiation of a new epigenetic state of these DMRs did not require a functional copy of this gene, suggesting that initiation of these changes is independent of RNA-directed DNA methylation.

Plasma growth hormone pulses induce male-biased pulsatile chromatin opening and epigenetic regulation in adult mouse liver.

Sex differences in plasma growth hormone (GH) profiles, pulsatile in males and persistent in females, regulate sex differences in hepatic STAT5 activation linked to sex differences in gene expression and liver disease susceptibility, but little is understood about the fundamental underlying, GH pattern-dependent regulatory mechanisms. Here, DNase-I hypersensitivity site (DHS) analysis of liver chromatin accessibility in a cohort of 18 individual male mice established that the endogenous male rhythm of plasma GH pulse-stimulated liver STAT5 activation induces dynamic, repeated cycles of chromatin opening and closing at several thousand liver DHS and comprises a novel mechanism conferring male bias to liver chromatin accessibility. Strikingly, a single physiological replacement dose of GH given to hypophysectomized male mice restored, within 30 min, liver STAT5 activity and chromatin accessibility at 83% of the dynamic, pituitary hormone-dependent male-biased DHS. Sex-dependent transcription factor binding patterns and chromatin state analysis identified key genomic and epigenetic features distinguishing this dynamic, STAT5-driven mechanism of male-biased chromatin opening from a second GH-dependent mechanism operative at static male-biased DHS, which are constitutively open in male liver. Dynamic but not static male-biased DHS adopt a bivalent-like epigenetic state in female liver, as do static female-biased DHS in male liver, albeit using distinct repressive histone marks in each sex, namely, H3K9me3 at male-biased DHS in female liver and H3K27me3 at female-biased DHS in male liver. Moreover, sex-biased H3K36me3 marks are uniquely enriched at static sex-biased DHS, which may serve to keep these sex-dependent hepatocyte enhancers free of H3K27me3 repressive marks and thus constitutively open. Pulsatile chromatin opening stimulated by endogenous, physiological hormone pulses is thus one of two distinct GH-determined mechanisms for establishing widespread sex differences in hepatic chromatin accessibility and epigenetic regulation, both closely linked to sex-biased gene transcription and the sexual dimorphism of liver function.

Plasma growth hormone pulses induce male-biased pulsatile chromatin opening and epigenetic regulation in adult mouse liver

Sex differences in plasma growth hormone (GH) profiles, pulsatile in males and persistent in females, regulate sex differences in hepatic STAT5 activation linked to sex differences in gene expression and liver disease susceptibility, but little is understood about the fundamental underlying, GH pattern-dependent regulatory mechanisms. Here, DNase-I hypersensitivity site (DHS) analysis of liver chromatin accessibility in a cohort of 18 individual male mice established that the endogenous male rhythm of plasma GH pulse-stimulated liver STAT5 activation induces dynamic, repeated cycles of chromatin opening and closing at several thousand liver DHS and comprises a novel mechanism conferring male bias to liver chromatin accessibility. Strikingly, a single physiological replacement dose of GH given to hypophysectomized male mice restored, within 30 min, liver STAT5 activity and chromatin accessibility at 83% of the dynamic, pituitary hormone-dependent male-biased DHS. Sex-dependent transcription factor binding patterns and chromatin state analysis identified key genomic and epigenetic features distinguishing this dynamic, STAT5-driven mechanism of male-biased chromatin opening from a second GH-dependent mechanism operative at static male-biased DHS, which are constitutively open in male liver. Dynamic but not static male-biased DHS adopt a bivalent-like epigenetic state in female liver, as do static female-biased DHS in male liver, albeit using distinct repressive histone marks in each sex, namely, H3K9me3 at male-biased DHS in female liver and H3K27me3 at female-biased DHS in male liver. Moreover, sex-biased H3K36me3 marks are uniquely enriched at static sex-biased DHS, which may serve to keep these sex-dependent hepatocyte enhancers free of H3K27me3 repressive marks and thus constitutively open. Pulsatile chromatin opening stimulated by endogenous, physiological hormone pulses is thus one of two distinct GH-determined mechanisms for establishing widespread sex differences in hepatic chromatin accessibility and epigenetic regulation, both closely linked to sex-biased gene transcription and the sexual dimorphism of liver function.

Epigenomic Profiling Advises Therapeutic Potential of Leukotriene Receptor Inhibitors for a Subset of Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype, with a poor survival rate compared to others subtypes. For a long time, chemotherapy was the only systemic treatment for TNBC, and the identification of actionable molecular targets might ultimately improve the prognosis for TNBC patients. We performed a genome-wide analysis of DNA methylation at CpG islands on a collection of one hundred ten breast carcinoma samples and six normal breast tissue samples using reduced representation bisulfite sequencing with the XmaI restriction enzyme (XmaI-RRBS) and identified a subset of TNBC samples with significant hypomethylation at the LTB4R/LTB4R2 genes' CpG islands, including CpG dinucleotides covered with cg12853742 and cg21886367 HumanMethylation 450K microarray probes. Abnormal DNA hypomethylation of this region in TNBC compared to normal samples was confirmed by bisulfite Sanger sequencing. Gene expression generally anticorrelates with promoter methylation, and thus, the promoter hypomethylation detected and confirmed in our study might be revealed as an indirect marker of high LTB4R/LTB4R2 expression using a simple methylation-sensitive PCR test. Analysis of RNA-seq expression and DNA methylation data from the TCGA dataset demonstrates that the expression of the LTB4R and LTB4R2 genes significantly negatively correlates with DNA methylation at both CpG sites cg12853742 (R = -0.4, p = 2.6 × 10-6; R = -0.21, p = 0.015) and cg21886367 (R = -0.45, p = 7.3 × 10-8; R = -0.24, p = 0.005), suggesting the upregulation of these genes in tumors with abnormal hypomethylation of their CpG island. Kaplan-Meier analysis using the TCGA-BRCA gene expression and clinical data revealed poorer overall survival for TNBC patients with an upregulated LTB4R. To this day, only the leukotriene inhibitor LY255283 has been tested on an MCF-7/DOX cell line, which is a luminal A breast cancer molecular subtype. Other studies compare the effects of Montelukast and Zafirlukast (inhibitors of the cysteinyl leukotriene receptor, which is different from LTB4R/LTB4R2) on the MDA-MB-231 (TNBC) cell line, with high methylation and low expression levels of LTB4R. In our study, we assess the therapeutic effects of various drugs (including leukotriene receptor inhibitors) with the DepMap gene effect and drug sensitivity data for TNBC cell lines with hypomethylated and upregulated LTB4R/LTB4R2 genes. LY255283, Minocycline, Silibinin, Piceatannol, Mitiglinide, 1-Azakenpaullone, Carbetocin, and Pim-1-inhibitor-2 can be considered as candidates for the additional treatment of TNBC patients with tumors demonstrating LTB4R/LTB4R2 hypomethylation/upregulation. Finally, our results suggest that the epigenetic status of leukotriene B4 receptors is a novel, potential, predictive, and prognostic biomarker for TNBC. These findings might improve individualized therapy for TNBC patients by introducing new therapeutic adjuncts as anticancer agents.

World Trade Center Exposure, DNA Methylation Changes, and Cancer: A Review of Current Evidence.

Introduction: Known carcinogens in the dust and fumes from the destruction of the World Trade Center (WTC) towers on 9 November 2001 included metals, asbestos, and organic pollutants, which have been shown to modify epigenetic status. Epigenome-wide association analyses (EWAS) using uniform (Illumina) methodology have identified novel epigenetic profiles of WTC exposure. Methods: We reviewed all published data, comparing differentially methylated gene profiles identified in the prior EWAS studies of WTC exposure. This included DNA methylation changes in blood-derived DNA from cases of cancer-free "Survivors" and those with breast cancer, as well as tissue-derived DNA from "Responders" with prostate cancer. Emerging molecular pathways related to the observed DNA methylation changes in WTC-exposed groups were explored and summarized. Results: WTC dust exposure appears to be associated with DNA methylation changes across the genome. Notably, WTC dust exposure appears to be associated with increased global DNA methylation; direct dysregulation of cancer genes and pathways, including inflammation and immune system dysregulation; and endocrine system disruption, as well as disruption of cholesterol homeostasis and lipid metabolism. Conclusion: WTC dust exposure appears to be associated with biologically meaningful DNA methylation changes, with implications for carcinogenesis and development of other chronic diseases.

Abstract A042: Targeting vulnerabilities arising from global DNA hypomethylation in cancer

Abstract Background: DNA methylation alterations are a universal feature of cancer. In addition to site specific gain of DNA methylation (hypermethylation), a global loss of methylation (hypomethylation) was noted in most cancer genomes. Whereas numerous studies have focused on the role of hypermethylation in disease progression, less is known about the biology of hypomethylation in cancer. Recently, we have identified tumors across all major cancer types that are characterized by severe loss of DNA methylation. Our preliminary data suggest that these DNA hypomethylated cancers (hereafter DHMCs) show distinct alterations influencing gene expression and tumor microenvironment composition. Objective: We hypothesize that since DNA methylation is critical for genome organization and gene expression. Severe global hypomethylation is likely to result in distinct epigenomic and genomic alterations. We therefore investigate common molecular changes associated with DNA hypomethylation to develop novel therapeutic approaches targeting unique vulnerabilities arising in DHMCs. Study design: To delineate the biology of DHMCs, we determined methylation changes with validated orthogonal methods in large representative cohorts of patient samples (The Cancer Genome Atlas (TCGA), University of Washington rapid autopsy cohorts), patient derived xenografts and cancer cell lines. We assessed the patterns of common molecular alterations by performing genome wide epigenome mapping experiments and by defining common driver gene events in DHMCs. We further determined tumor cell intrinsic therapeutic vulnerabilities in broad pharmacologic and CRISPR-Cas9 genomic screens. Results: We observed that DNA hypomethylation is a relatively common feature of solid tumors affecting overall 15-20% of all cancers, with highest rates observed in urothelial, melanoma, hepatocellular and lung and head and neck squamous cell carcinoma. In in vivo tumor models of DHMCs, we observe distinct shifts in epigenetic states upon DNA hypomethylation. Most notably, we found that DHMCs are characterized by unique intrinsic drug sensitivities to drug classes affecting core histone levels. Impact: This is the first study to show druggable vulnerabilities arising from DNA hypomethylation. Collectively, these studies define a novel epigenetic subtype of cancer, determine its clinical and molecular features, and pave the way for new targeted therapies. Citation Format: Pallabi Mustafi, Brian Hanratty, Ilsa Coleman, Radhika Patel, Adil Mohamed, Jay Sarthy, Gavin Ha, Michael Haffner. Targeting vulnerabilities arising from global DNA hypomethylation in cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A042.

Dietary supplementation of pomegranate extract alters the Caspase-3 and VEGF promoter epigenetic methylation status and protein levels in breast cancer patients undergoing chemotherapy

Dietary supplementation of pomegranate extract alters the Caspase-3 and VEGF promoter epigenetic methylation status and protein levels in breast cancer patients undergoing chemotherapy

Abstract B080: Non-genetic determinants driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer

Abstract The FDA approval of the KRAS G12C inhibitor (G12Ci) sotorasib and the advancement of similar drugs into the clinic marks a major milestone in treating KRAS G12C non-small cell lung cancer (NSCLC). However, not all patients respond (sotorasib – ORR = 37.1%, adagrasib - 43%, JDQ443 – 35%), motivating preclinical and clinical investigation into mechanisms of intrinsic and acquired resistance. For instance, clinical studies have reported on-target KRAS mutations and preclinical studies have demonstrated mitogen-activated protein kinase (MAPK) feedback reactivation including EGFR, SHP2, and WT RAS signaling. In response to targeted therapies, sub-populations of cells can enter quiescence or specific epigenetic-driven states that confer drug tolerance. However, epigenetic states defining drug-tolerant persister populations and contributing to adaptive resistance to KRAS G12Ci have not been reported. Using a high-complexity DNA-barcoding system and mathematical modeling, we observed that pre-existing resistant clones to KRAS G12Ci gain clonal fitness during treatment independent of growth rate. Using single-cell profiling and RNA-lineage tracing barcoded systems, we identified distinct subpopulations of cells defined by specific chromatin and transcriptional states, including activation of interferon-gamma genes (ISG) and increased chromatin accessibility for pioneer transcription factors, which may contribute to MAPK reactivation-driven resistance. These results suggest a potential role of pioneer transcription factor-activated interferon-gamma response in driving MAPK feedback reactivation in KRAS G12Ci- resistant tumor subpopulations. Citation Format: Chendi Li, Christopher J Graser, Qian Qin, Usman M Syed, Barbara Karakyriakou, Sarah E Clark, Anne Y Saiki, Paul E Hughes, Christopher Ott, Luca Pinello, Franziska Michor, Aaron N Hata. Non-genetic determinants driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B080.

Iguratimod Regulates CD4+ T-Cell Homeostasis and Function By Restoring PINK1/Parkin-Mediated Mitophagy in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP), an acquired autoimmune disease, is characterized by immune-mediated platelet destruction and suppressed platelet production, resulting in isolated thrombocytopenia. Subpopulation imbalance and dysfunction of CD4+ T cells are involved in the pathogenesis of ITP. However, the exact mechanism of CD4+ T-cell abnormalities is not fully understood. Mitophagy, a process that eliminates damaged and dysfunctional mitochondria, has conventionally been considered the primary mechanism responsible for mitochondrial quality control. Mitophagy deficiency has been implicated in a series of inflammatory and autoimmune diseases, such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). However, whether it plays a role in ITP immune abnormalities and its specific mechanism remain unclear. Iguratimod is a small molecule compound that is widely used as a novel antirheumatic drug in the treatment of rheumatoid arthritis. Iguratimod inhibits the production of several inflammatory cytokines, including IL-1, IL-6, IL-8, and tumor necrosis factor (TNF), by enhancing mitophagy. Our previous clinical study demonstrated the efficacy of iguratimod in the treatment of ITP. This study aimed to explore whether iguratimod regulates CD4+ T-cell homeostasis and function by enhancing mitophagy in ITP. Methods BM CD4+ T cells were isolated from ITP patients and healthy donors. The subgroup of CD4+ T cells was analyzed by flow cytometry. ELISA was used to evaluate cytokine and chemokine levels in bone marrow plasma. Mitochondrial mass, morphology, quality and mitophagy were evaluated by flow cytometry, confocal microscopy, electron microscopy and western blotting. RNA sequencing and metabolomics were performed. Epigenetic state alteration was assessed by a transposase-accessible chromatin assay with high throughput sequencing (ATAC-seq). Iguratimod-treated BM-derived ITP-CD4+ T cells and an ITP active mouse model were used to explore its regulation of mitophagy and effects on T-cell homeostasis and function. Results Th1/Th2 imbalance and Treg reduction were observed in the bone marrow of ITP patients, consistent with a previous report. Flow cytometry and confocal microscopy revealed an increase in mitochondrial content and mtROS in CD4+ T cells but a decrease in mitochondrial membrane potential. Furthermore, electron microscopy revealed structural damage to mitochondria in ITP-CD4+ T cells. These data confirmed an accumulation of damaged mitochondria in ITP-CD4+ T cells. The expression levels of LC3II/LC3I, PINK1 and parkin decreased in CD4+ T cells, and the colocalization of mitochondria and lysosomes decreased, which suggested compromised PINK1/parkin-mediated mitophagy in ITP-CD4+ T cells. RNA-seq revealed transcriptional changes associated with mitochondrial function, mitophagy and metabolic pathways in ITP-CD4+ T cells. Moreover, metabolomics showed alterations in TCA cycle metabolites. Since epigenetic programs are regulated by metabolites, ATAC-seq was performed to obtain the epigenetic state. We found that ITP-CD4+ T cells altered chromatin accessibility in regions with T cell differentiation, transcription factors and T-cell function. After in vitro treatment with iguratimod, the Th1/Th2 imbalance was corrected, and the number of Tregs was restored. The content, morphology and membrane potential of mitochondria in ITP-CD4+ T cells were restored after iguratimod intervention. The expression level of PINK1 and parkin and the colocalization of mitochondria and lysosomes were increased, suggesting an improvement in mitophagy. In vivo, iguratimod accelerated platelet recovery in an ITP active mouse model. After treatment, the Th1/Th2 imbalance and the Treg number were restored, and the mitophagy of CD4+ T cells was corrected in the bone marrow of the ITP mouse model. Conclusion PINK1/Parkin-mediated mitophagy deficiency contributed to mitochondrial dysfunction and altered the metabolism of ITP-CD4+ T cells, which affected their differentiation and function by regulating the epigenetic state of immune response genes. Iguratimod could restore CD4+ T-cell subpopulations and treat ITP by enhancing mitophagy.

Dissecting Lineage and Cell State-Specific Epigenetic Evolution in Relapsed Acute Myeloid Leukemia

Introduction: The prognosis for relapsed Acute Myeloid Leukemia (AML) following initial treatment and remission remains poor, largely due to a limited number of therapies with proven clinical efficacy. Epigenetic states and evolution are known to contribute to the initiation of AML, however, the role of epigenetic evolution in relapse remains understudied, and the effects of lineage-specific epigenetic changes at relapse are unknown. We hypothesized that epigenetic modifications confined to specific lineages or differentiation states may contribute to disease resistance and relapse. To test this, we generated single cell ATACseq data from four AML patients at initial diagnosis and at relapse post-induction chemotherapy. We utilized mapping of AML scATACseq to scATAC-derived healthy hematopoietic chromatin states, lineage trajectory analysis, and gene and transcription factor accessibility analysis to identify epigenetic changes occurring at discrete points in different AML differentiation trajectories. Methods: Malignant AML cells were purified from paired samples collected from four patients at diagnosis and relapse via FACS and were sequenced using the 10x chromium scATAC platform. ArchR, Monocle3, and custom R scripts were used for all analyses. AML cell scATAC data was mapped to a healthy hematopoietic scATAC reference using Latent Semantic Indexing (LSI) and projection onto a single cell ATAC-seq manifold derived from healthy hematopoietic cell types. Closest healthy cell types were identified for each AML cell, differentiation trajectories were generated using monocole3, and gene and transcription factor accessibility was quantified across the different AML differentiation states and lineages. Results: Our analysis showed varying patterns of lineage bias across AMLs. Some samples exhibited changes in differentiation state at relapse, becoming either less differentiated or less myeloid-like. Analysis of differences in TF activity between diagnosis and relapse within myeloid and lymphoid trajectories revealed lineage-specific and differentiation state-specific changes in TF activity. In patient SU360, overall differentiation states did not change, and AML cells at both diagnosis and relapse were primarily myeloid-like with a smaller lymphoid-like population. In this sample, GATA family TFs, which were active in progenitor-like cells at diagnosis, became significantly more active in terminally differentiated AML cells at relapse. No specific chromatin changes were observed in lymphoid-like AML cells at relapse. In patient SU484, where diagnosis cells were myeloid-differentiated and relapse cells were more progenitor-like, GATA and RUNX family TF activity, which was restricted to progenitor cells at diagnosis, showed broad activation in all cells at relapse, aligning with the overall de-differentiation observed in the AML. In SU142, a case with no significant change in overall chromatin or differentiation state at relapse, multiple SP family transcription factors showed significant activation specifically in progenitor-like AML cells at relapse. Conclusion: Our study of four distinct AMLs suggests the existence of lineage-specific epigenetic evolution in some relapsed AMLs. We found that epigenetic activation of specific transcription factors, such as GATA and RUNX, was restricted to certain steps of myeloid differentiation, suggesting that epigenetic changes contributing to resistance and relapse are relevant in specific AML lineages and differentiation states. Notably, we found that even in AMLs where overall epigenetic state remains stable, there is activation of specific TFs (e.g. SP1) in progenitor cells at relapse, consistent with literature demonstrating SP family TF relevance in Leukemic Stem Cell (LSC) drug resistance. These observations suggest that epigenetic modifications in less-differentiated AML cells may reflect changes occurring in LSCs, potentially contributing to resistance and relapse. Our study is limited due to a small sample size that precludes broad generalizations, but our findings provide a foundation for further characterization of AML relapse epigenetics. Ultimately, these findings suggest the existence of specific epigenetic changes at certain points in the AML differentiation hierarchy that contribute to relapse, warranting further exploration of cell state-specific epigenetic evolution.

ICF1-Syndrome-Associated DNMT3B Mutations Prevent De Novo Methylation at a Subset of Imprinted Loci during iPSC Reprogramming.

Parent-of-origin-dependent gene expression of a few hundred human genes is achieved by differential DNA methylation of both parental alleles. This imprinting is required for normal development, and defects in this process lead to human disease. Induced pluripotent stem cells (iPSCs) serve as a valuable tool for in vitro disease modeling. However, a wave of de novo DNA methylation during reprogramming of iPSCs affects DNA methylation, thus limiting their use. The DNA methyltransferase 3B (DNMT3B) gene is highly expressed in human iPSCs; however, whether the hypermethylation of imprinted loci depends on DNMT3B activity has been poorly investigated. To explore the role of DNMT3B in mediating de novo DNA methylation at imprinted DMRs, we utilized iPSCs generated from patients with immunodeficiency, centromeric instability, facial anomalies type I (ICF1) syndrome that harbor biallelic hypomorphic DNMT3B mutations. Using a whole-genome array-based approach, we observed a gain of methylation at several imprinted loci in control iPSCs but not in ICF1 iPSCs compared to their parental fibroblasts. Moreover, in corrected ICF1 iPSCs, which restore DNMT3B enzymatic activity, imprinted DMRs did not acquire control DNA methylation levels, in contrast to the majority of the hypomethylated CpGs in the genome that were rescued in the corrected iPSC clones. Overall, our study indicates that DNMT3B is responsible for de novo methylation of a subset of imprinted DMRs during iPSC reprogramming and suggests that imprinting is unstable during a specific time window of this process, after which the epigenetic state at these regions becomes resistant to perturbation.

Decelerated Epigenetic Aging in Long Livers

Epigenetic aging is a hot topic in the field of aging research. The present study estimated epigenetic age in long-lived individuals, who are currently actively being studied worldwide as an example of successful aging due to their longevity. We used Bekaert’s blood-based age prediction model to estimate the epigenetic age of 50 conditionally “healthy” and 45 frail long-livers over 90 years old. Frailty assessment in long-livers was conducted using the Frailty Index. The control group was composed of 32 healthy individuals aged 20–60 years. The DNA methylation status of 4 CpG sites (ASPA CpG1, PDE4C CpG1, ELOVL2 CpG6, and EDARADD CpG1) included in the epigenetic clock was assessed through pyrosequencing. According to the model calculations, the epigenetic age of long-livers was significantly lower than their chronological age (on average by 21 years) compared with data from the group of people aged 20 to 60 years. This suggests a slowing of epigenetic and potentially biological aging in long livers. At the same time, the obtained results showed no statistically significant differences in delta age (difference between the predicted and chronological age) between “healthy” long livers and long livers with frailty. We also failed to detect sex differences in epigenetic age either in the group of long livers or in the control group. It is possible that the predictive power of epigenetic clocks based on a small number of CpG sites is insufficient to detect such differences. Nevertheless, this study underscores the need for further research on the epigenetic status of centenarians to gain a deeper understanding of the factors contributing to delayed aging in this population.

Hematopoietic Stem/Progenitor Cell Intrinsic SAM Synthesis Is Required to Prevent p53 Pathway Activation By Maintaining DNA Stability

S-adenosylmethionine (SAM) is a principle methyl group donor to maintain epigenetic status. SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a (Methionine adenosyltransferase 2a). It has been reported that SAM reduction by methionine restriction or Mat2a inhibition suppresses the proliferation of leukemia cells to attract attentions as a potential therapeutic target for leukemia. To develop the therapy targeting methionine metabolism in leukemia, it is essential to elucidate the significance of methionine metabolism in non-leukemic hematopoiesis. However, still little is known about the importance of SAM synthesis in hematopoiesis, including hematopoietic stem/progenitor cells (HSPCs), in vivo. By using Mx1-Cre mediated Mat2a gene deletion in mice ( Mat2a fl/fl;Mx1-Cre mice), we investigated the roles of Mat2a in hematopoiesis in vivo. Reduction of Mat2a activity in Mat2a knockout (KO) mice in bone marrow (BM) was confirmed by ultra-high performance liquid chromatography tandem mass spectrometry analysis. Mat2a KO mice displayed severe pancytopenia in peripheral blood (PB). Mature cell fractions (Myeloid cells, B cells and T cells) and HSPCs (HSCs, MPP2s, MPP3s and MPP4s) in BM were also significantly decreased in Mat2a KO mice, suggesting that Mat2a is essential for the maintenance of hematopoiesis from HSPCs in vivo. To exclude the possible contributions of the effects induced by Mat2a KO in non-hematopoietic cells, we performed BM transplantation analyses in which hematopoietic cell specific KO or non-hematopoietic cell specific KO were achieved. These analyses clearly confirmed that HSPC intrinsic SAM synthesis is inevitable for the maintenance of hematopoiesis. To reveal the mechanistic insights of the regulatory roles for Mat2a in HSPCs, we performed RNA sequencing analysis ( Mat2a WT vs KO in HSPCs). GSEA (Gene Set Enrichment Analysis) using the Hallmark gene sets showed that p53 pathway was most highly enriched in Mat2a KO. In depth, genes downstream of p53 ( Bax, Apaf1, Bbc3, Casp3, Casp8, Fas, Cdkn1a etc.) were up-regulated by Mat2a KO, causing cell cycle arrest and apoptosis in HSPCs. On the other hand, gene expressions of the upstream of p53 ( Atr, Atm, Chek1, Chek2 etc.) were unchanged, suggesting that p53 activity was not increased at mRNA expression levels. In line with this, mRNA expression levels of Trp53 was unchanged, but total protein amount of p53 was increased in Mat2a KO HSPCs. Since DNA stability is known as a critical regulator of p53 pathway, we analyzed gH2AX levels as DNA damage marker and found that gH2AX levels were increased in Mat2a KO HSPCs. To be noted, expressions of genes involved in Fanc pathway ( Fanca, Fancc, Fancd2, Fance, Fancg, Fancl, Fanci etc), which is responsible for the DNA maintenance, were broadly down-regulated in Mat2a KO HSPCs at transcriptomic levels. Therefore, the Fanc pathway repression by SAM synthesis inhibition could cause DNA damage and p53 pathway dependent loss of maintenance in Mat2a KO HSPCs. To further understand the importance of SAM synthesis under stress hematopoiesis. We analyzed Mat2a expression changes in HSPCs by irradiation (5.5 Gy/mouse). Intriguingly, Mat2a expression was up-regulated by irradiation. We also found that dietary methionine restriction (MR) in mice prevents its hematopoietic recovery from irradiation stress. To be noted, long term MR (24 weeks) did not show any particular compared to control diet. Therefore, methionine metabolism possesses significant roles at least in the situation where acute hematopoietic recovery is required. Collectively, in this study, we found that cell intrinsic SAM synthesis is required for the maintenance (proliferation and survival) of HSPCs in vivo. SAM might be required for the prevention of DNA damage and p53 pathway activation by supporting Fanc pathway gene expressions. Methionine and/or SAM availability might possess critical roles for the regeneration of non-leukemic hematopoiesis. Further investigation about the significance of SAM synthesis in non-leukemic hematopoiesis is required for the proper establishment of SAM synthesis targeting therapy in cancers.

Ubtf- Tandem Duplications Are Recurrent Somatic Genetic Events in Adult AML and Associate with Additional Structural Variants and a Distinct Gene Expression Profile (Alliance)

Background The gene upstream binding transcription factor ( UBTF ) encodesa nucleolar protein that is a key regulator of the epigenetic status of ribosomal DNA and RNA transcription. Tandem duplications (TDs) of the UBTF gene have been initially identified in pediatric patients (pts) with acute myeloid leukemia (AML) and were recently also reported as relatively rare, but recurrent genetic events in adult AML based on targeted re-sequencing efforts (1.2%-3%). Their presence was associated with dysplastic bone marrow features, and co-occurrence of select genetic features, including FLT3-internal tandem duplications (ITDs) and WT1 mutations, and inferior outcome. However, associated biologic features and affected pathways have not yet been thoroughly investigated, and may provide further insights into leukemogenesis and identify potentially targetable vulnerabilities. Thus, we set out to assess the frequency of UBTF-TDs in a large cohort of adult AML pts (n=807) treated on frontline protocols of the Alliance for Clinical Trials in Oncology with complete RNA sequencing data, comprehensive targeted mutation profiling, centrally reviewed cytogenetic data and clinical information available, and to gain further insights into UBTF-TDs via comprehensive integrated genomic profiling. Methods UBTF-TD detection was performed via the RNAseq-based RNAmut and EnFusion algorithm. To gain insights into associated molecular features, all UBTF-TD + pts underwent paired tumor-normal whole-exome sequencing. To understand associated pathways and downstream effects, differential gene expression (DGE) and gene set enrichment analyses (GSEA) were performed. Results UBTF-TDs were detected in 6/807 pts (0.7%). In line with previous studies, these 6 UBTF-TD + pts were young, with a median age of 35y (range: 19-59y). Three pts had extramedullary disease, 2 with skin and 1 with gingival involvement. All but 1 of the 6 pts were cytogenetically normal. Clinically, pts did poorly after treatment with intensive chemotherapy induction, with a CR rate of only 33%. No pt was alive 2.5 y after diagnosis. In paired tumor/normal exome sequencing, UBTF-TD + pts had a median mutation burden of 21 non-synonymous variants in coding genes (range: 17-27), with a notable presence of larger insertions/deletions (indels), loss of heterozygosity (LOH) or TDs as co-occurring somatic genetic lesions (median: 4, range: 2-5). There was a 100% co-occurrence of UBTF-TDs with FLT3-ITDs. In addition, pts harbored larger indels in WT1 (n=2), BPTF, CEBPA, Corf53, KRTAP5-7, NFE2, PTPRN2, RNF144B, SOWAHC and ZCCHC14. Also, analysis of copy-number variations and LOH revealed LOH in 5 pts, in chromosomes 13 (n=3), 15 (n=1) and 18 (n=1), suggestive of a propensity to the acquisition of structural variants. In unsupervised clustering analysis of the RNAseq data, UBTF-TD + pts clustered with FLT3-ITD + pts but formed a relatively distinct subcluster in principal component and t-distributed stochastic neighbor embedding analyses. In supervised clustering comparing 6 UBTF-TD +/ FLT3-ITD + and 196 UBTF-TD -/ FLT3-ITD + pts, 2 UBTF-TD - pts clustered together with the UBTF-TD +/ FLT3-ITD + group, suggesting either presence of an undetected TD or genetic phenocopy. UBTF expression was higher in UBTF-TD pts. DGE analysis of UBTF-TD +/ FLT3-ITD + and UBTF-TD -/ FLT3-ITD + pts (Fig 1) showed distinct upregulation of the locus of the stem cell and chemoresistance marker ALDH1A3, including the associated long non-coding RNAs AC015712.7/AC015712.2 in UBTF-TD + pts, which was confirmed by a comparison of those pts to AML pts who had neither TD (n=605). GSEA indicated a downregulation of inflammation pathways and several metabolic pathways of UBTF-TD +/ FLT3-ITD + compared to FLT3-ITD + pts (Fig 2). Conclusions UBTF-TDs are rare, but recurrent genetic events in adult AML, associated with high chemotherapy resistance. Our results highlight a susceptibility of those pts to the acquisition of structural variants and suggest a distinct associated biology, with upregulation of ALDH1A3 as possible contributing resistance mechanism. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov Identifiers: NCT00048958 (CALGB 8461), NCT00899223 (CALGB 9665), NCT00900224 (CALGB 20202); https://acknowledgments.alliancefound.org

Compound Loss of Dnmt3a and Kmt2c in Myeloid Malignancies

Monosomy 7, or loss of 7q (-7/del(7q)), is one the most common chromosomal anomalies in myelodysplastic syndromes (MDS, ~10% of patients), a malignant disorder of the blood system with few treatment options. There are currently no targeted therapies available for -7/del(7q) MDS, and patients progress to a deadly acute myeloid leukemia (AML). The 7q segment is rich in genes that are implicated in MDS pathophysiology, including KMT2C encoding a histone H3 lysine 4 methyltransferase (depositing H3K4me1) that marks active gene enhancers during myeloid differentiation. Co-occurring DNA methyltransferase DNMT3A mutations are significantly overrepresented in -7/del(7q) MDS, especially in the absence of other chromosomal aberrations (23%). Individually, loss in DNMT3A activity in hematopoietic stem cells (HSCs) has shown bias towards self-renewal due to a failure to shut off stem-cell-related genes, whereas a loss of KMT2C leads to aberrant myelopoiesis. Generally, only one allele of KMT2C and DNMT3A is lost in MDS, suggesting that these haploinsufficient tumor suppressors may need to cooperate to drive disease. We hypothesized that disruption of Dnmt3a cooperated with loss of Kmt2c creating an aberrant epigenetic state that may accelerate development of MDS and progression to AML. To address this, we generated mice that lacked one copy of both Kmt2c and Dnmt3a in the bone marrow by conditional deletion with a hematopoietic-specific inducible Mx1-driven Cre recombinase. After inducing Cre-mediated recombination by poly(I:C) treatment creating Kmt2c(+/del):Dnmt3a(+/del) animals, the hematopoietic stem and progenitor compartment was subjected to comprehensive immunophenotypic and functional characterization. In serial competitive bone marrow repopulation assays, cells with combined heterozygous loss of both Dnmt3a and Kmt2c demonstrated increased competitive advantage in secondary transplants and retained ability to reconstitute tri-lineage hematopoiesis in tertiary transplants, unlike single gene deletion and wild-type controls. Consistently, immunophenotypic analysis of the bone marrow compartment 16 weeks after the third transplant demonstrated persistence of the primitive long-term hematopoietic stem cells (LT-HSCs) defined as Lineage -Sca1 +cKit + (LSK) CD150 +CD48 -, which were absent in other genotypes. Further, we found expansion of the LT-HSCs in aged 80-week old Kmt2c(+/del):Dnmt3a(+/del) animals that were also characterized by a gain in self-renewal as seen by their ability to serially replate in semisolid media (MethoCult GM3434). To test if combined loss of Kmt2c and Dnmt3a led to disease development, next we established a large cohort of animals non-competitively transplanted with Kmt2c(+/del):Dnmt3a(+/del), single gene deletion, and wild-type control bone marrow, and initiated a tumor watch by monthly monitoring the composition of their peripheral blood by CBCs and flow cytometry. Sixty weeks after transplantation, over a half of double knock-out animals developed myeloid disease characterized by neutrophilia and/or monocytosis accompanied by anemia, occasional thrombocytopenia, and splenomegaly most consistent with myelodysplasia. Immunophenotypically, we found expanded LSK populations (LT-HSCs specifically) in the bone marrow and evidence of extramedullary hematopoiesis in the spleen. At the same time, the majority of mice with single gene deletion and all wild-type controls remained healthy. Overall, our studies demonstrate that combined loss of Kmt2c and Dnmt3a results in the development of myelodysplastic disease with long latency through endowing the HSCs with enhanced self-renewal capacity. We are currently interrogating the epigenetic profiles and gene expression patterns resulting from combined disruption of Dnmt3a and Kmt2c aiming to identify gene targets and signaling networks for future therapeutic intervention. Better understanding of the molecular pathogenesis of the -7/del(7q) MDS with concurrent DNMT3A mutations is critical to develop more effective treatment approaches and to improve outcomes for MDS patients.

Epigenetic Scarring Leads to Irreversible NK Cell Dysfunction in Myeloid Malignancies

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) belong to the same spectrum of myeloid malignancies and have extremely poor outcomes in the relapsed/refractory setting. Despite advances in the understanding of the pathogenesis and molecular mechanisms of these disorders, and incremental improvements in treatment regimens, patients with MDS and AML often relapse and fail to achieve cure. These and other factors underscore the urgent need for new therapeutic alternatives that will improve the clinical outcomes of these patients. Immunotherapy using checkpoint molecule inhibitors and adoptive cell therapy using autologous immune effector cells have been mostly unsuccessful in patients with MDS and AML. This could be due to the immunosuppressive tumor microenvironment in the bone marrow niche or to intrinsic dysfunction in the immune effector cells of these patients. Natural killer (NK) cells are innate lymphocytes that play an important role in cancer immune surveillance and have distinct advantages over T cells as candidates for immunotherapy. Myeloid blasts are inherently susceptible to NK cell-mediated killing as they express many of the ligands recognized by NK cell activating receptors. However, malignant myeloid blasts are capable of adapting and developing defense mechanisms that allow them to evade NK cell-mediated cytotoxicity. Little is known about the mechanisms of NK cell immune evasion developed by myeloid blasts. Here, we show that NK cells from patients with myeloid malignancies display a global dysfunction with severely impaired secretory function and killing capacity. Single cell RNAseq and mass cytometry experiments revealed that these NK cells from MDS and AML patients display an exhausted phenotype at the transcriptomic and proteomic levels. We also show that these NK cells have an altered metabolism compared with age matched healthy control NK cells. We show that this dysfunction is mediated by a crosstalk between myeloid blasts and NK cells and cell-cell contact dependent release of transforming growth factor beta (TGF-β). This crosstalk leads to a profound epigenetic reprogramming of NK cells driven by transcription factors known to mediate exhaustion and immune suppression. Myeloid blast-induced NK dysfunction and epigenetic state of exhaustion can be prevented by pharmacologically inhibiting the TGF-β pathway or knockout of TGFBR2 in NK cells. However, our data reveal that once this dysfunction occurs it is irreversible owing to epigenetic scarring driven by the transcription factor BATF. In fact, we show that TGF-β induces the expression of BATF in NK cells, which in turn mediates a gene regulatory program leading to NK cell dysfunction by driving the expression of exhaustion and inhibitory genes (e.g. HAVCR2, ENTPD1, CTLA4, TGFBR2). Collectively, our findings reveal a novel mechanism of NK cell immune evasion manifested by stable epigenetic rewiring and inactivation of NK cells by myeloid blasts. Our data support the use of allogeneic sources for adoptive NK cell therapy in combination with strategies aiming at preventing immune suppression to treat myeloid malignancies rather than therapies aiming at reversing or rescuing the function of autologous NK cells.