Linking Aging to Cancer: The Role of Chromatin Biology.

Epigenetic changes have been established to be a hallmark of aging, which implies that aging science requires collaborating with the field of chromatin biology. DNA methylation patterns, changes in relative abundance of histone post-translational modifications (PTMs), and chromatin remodeling are the central players in modifying chromatin structure. Aging is commonly associated with an overall increase in chromatin instability, loss of homeostasis and decondensation. However, numerous publications have highlighted that the link between aging and chromatin changes is not nearly as linear as previously expected. This complex interplay of these epigenetics elements during the lifetime of an organism likely contribute to cellular senescence, genomic instability, and disease susceptibility. Yet, the causal links between these phenomena still need to be fully unraveled. In this perspective article, we discuss potential future directions of aging chromatin biology.

Evidence for negative selection in human primary fibroblasts to tolerate high somatic mutation loads upon treatment with multiple low doses of N-ethyl-N-nitrosourea.

High-throughput sequencing at the single-cell and single-molecule level has shown that mutation rate is much higher in somatic cells than in the germline, with thousands of mutations accumulating with age in most human tissues. While there is now ample evidence that some of these mutations can clonally amplify and lead to disease, most notably cancer, the total burden of mutations a cell can tolerate without functional decline remains unknown. Here we addressed this question by exposing human primary fibroblasts multiple times to low doses of N-ethyl-N-nitrosourea (ENU) and quantitatively analyzing somatic mutation burden using single-cell whole genome sequencing. The results indicate that individual cells can sustain ∼60,000 single-nucleotide variants (SNVs) with only a slight adverse effect on growth rate. We found evidence for selection against potentially deleterious variants in gene coding regions as well as depletion of mutations in sequences associated with genetic pathways expressed in these human fibroblasts, most notably those relevant for maintaining basic cellular function and growth. However, no evidence of negative selection was found for variants in non-coding regions. We conclude that actively proliferating fibroblasts can tolerate very high levels of somatic mutations without major adverse effects on growth rate via negative selection against damaging coding mutations. Since most tissues in adult organisms have very limited capacity to select against mutations based on a growth disadvantage, these results suggest that a causal effect of somatic mutations in aging and disease cannot be ruled out.

An iron rheostat controls hematopoietic stem cell fate.

Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron-particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells.

αKG-mediated carnitine synthesis promotes homologous recombination via histone acetylation.

Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. Here we identified that depletion of alpha-ketoglutarate (αKG) sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. αKG is required for the activity of αKG-dependent dioxygenases (αKGDDs), and prior work has shown that changes in αKGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 αKGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, αKG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation, while histone methylation was affected but dispensable. The increase in histone acetylation via αKG-dependent carnitine synthesis promoted HR-mediated DNA repair through site- and substrate-specific histone acetylation. These data demonstrate for the first time that HR-proficiency is mediated through αKG directly influencing histone acetylation via carnitine synthesis and provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

Functional Contribution and Clinical Implication of Cancer-Associated Fibroblasts in Glioblastoma.

PURPOSE: The abundance and biological contribution of cancer-associated fibroblasts (CAF) in glioblastoma (GBM) are poorly understood. Here, we aim to uncover its molecular signature, cellular roles, and potential tumorigenesis implications. EXPERIMENTAL DESIGN: We first applied single-cell RNA sequencing (RNA-seq) and bioinformatics analysis to identify and characterize stromal cells with CAF transcriptomic features in human GBM tumors. Then, we performed functional enrichment analysis and in vitro assays to investigate their interactions with malignant GBM cells. RESULTS: We found that CAF abundance was low but significantly correlated with tumor grade, poor clinical outcome, and activation of extracellular matrix remodeling using three large cohorts containing bulk RNA-seq data and clinical information. Proteomic analysis of a GBM-derived CAF line and its secretome revealed fibronectin (FN1) as a critical candidate factor mediating CAF functions. This was validated using in vitro cellular models, which demonstrated that CAF-conditioned media and recombinant FN1 could facilitate the migration and invasion of GBM cells. In addition, we showed that CAFs were more abundant in the mesenchymal-like state (or subtype) than in other states of GBMs. Interestingly, cell lines resembling the proneural state responded to the CAF signaling better for the migratory and invasive phenotypes. CONCLUSIONS: Overall, this study characterized the molecular features and functional impacts of CAFs in GBM, alluding to novel cell interactions mediated by CAFs in the GBM microenvironment.

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma.

Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 60 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Moreover, DLK1 is highly expressed in several adult cancer types, including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG), hepatoblastoma, and small cell lung cancer (SCLC), suggesting potential clinical benefit beyond neuroblastoma. Taken together, our study demonstrates the utility of comprehensive cancer surfaceome characterization and credentials DLK1 as an immunotherapeutic target. Highlights: Plasma membrane enriched proteomics defines surfaceome of neuroblastomaMulti-omic data integration prioritizes DLK1 as a candidate immunotherapeutic target in neuroblastoma and other cancersDLK1 expression is driven by a super-enhancer DLK1 silencing in neuroblastoma cells results in cellular differentiation ADCT-701, a DLK1-targeting antibody-drug conjugate, shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma preclinical models.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCC) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The first group shows decreased proliferation, genome instability, and increased sensitivity to genotoxic agents, such as PARP1/2 inhibitors. In contrast, the H3K36M HNSCC models with steady H3K27me3 levels do not exhibit these characteristics unless H3K27me3 levels are elevated, either by DNA hypomethylating agents or by inhibiting the H3K27me3 demethylases KDM6A/B. Mechanistically, we found that H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, we found that aberrant H3K27me3 levels induced by H3K36M expression is not a bona fide epigenetic mark in HNSCC since it requires continuous expression of H3K36M to be inherited. Moreover, increased sensitivity of H3K36M HNSCC models to PARP1/2 inhibitors solely depends on the increased H3K27me3 levels. Indeed, aberrantly high H3K27me3 levels decrease BRCA1 and FANCD2-dependent DNA repair, resulting in higher sensitivity to DNA breaks and replication stress. Finally, in support of our in vitro findings, a PARP1/2 inhibitor alone reduce tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a H3K36M HNSCC xenograft model with consistent H3K27me3 levels, a combination of PARP1/2 inhibitors and agents that upregulate H3K27me3 proves to be successful. In conclusion, our findings underscore a delicate balance between H3K36 and H3K27 methylation, essential for maintaining genome stability. This equilibrium presents promising therapeutic opportunities for patients with H3K36me-deficient tumors.

Urine Proteomics Link Complement Activation with Interstitial Fibrosis/Tubular Atrophy in Lupus Nephritis Patients.

BACKGROUND: Intrarenal complement activation has been implicated in the pathogenesis of tubulointerstitial fibrosis in lupus nephritis (LN) based on prior animal studies. The assembly of the membrane attack complex (MAC) by complement C5b to C9 on the cell membrane leads to cytotoxic pores and cell lysis, while CD59 inhibits MAC formation by preventing C9 from joining the complex. We hypothesize that complement activation and imbalance between complement activation and inhibition, as defined by increased production of individual complement components and uncontrolled MAC activation relative to CD59 inhibition, are associated with interstitial fibrosis and tubular atrophy (IFTA) in LN and correlate with the key mediators of kidney fibrosis- transforming growth factor receptors beta (TGFRß), platelet-derived growth factor beta (PDGFß) and platelet-derived growth factor receptor beta (PDGFRß). METHODS: We included urine samples from 46 adults and pediatric biopsy-proven lupus nephritis patients who underwent clinically indicated kidney biopsies between 2010 and 2019. We compared individual urinary complement components and the urinary C9-to-CD59 ratio between LN patients with moderate/severe IFTA and none/mild IFTA. IFTA was defined as none/mild (<25% of interstitium affected) versus moderate/severe (≥ 25% of interstitium affected). Proteomics analysis was performed using mass spectrometry (Orbitrap Fusion Lumos, Thermo Scientific) and processed by the Proteome Discoverer. Urinary complement proteins enriched in LN patients with moderate/severe IFTA were correlated with serum creatinine, TGFßR1, TGFßR2, PDGFß, and PDGFRß. RESULTS: Of the 46 LN patients included in the study, 41 (89.1%) were women, 20 (43.5%) self-identified as Hispanic or Latino, and 26 (56.5%) self-identified as Black or African American. Ten of the 46 (21.7%) LN patients had moderate/severe IFTA on kidney biopsy. LN patients with moderate/severe IFTA had an increased urinary C9-to-CD59 ratio [median 0.91 (0.83-1.05) vs 0.81 (0.76-0.91), p=0.01]. Urinary C3 and CFI levels in LN patients with moderate/severe IFTA were higher compared to those with none/mild IFTA [C3 median (IQR) 24.4(23.5-25.5) vs. 20.2 (18.5-22.2), p= 0.02], [CFI medium (IQR) 28.8 (21.8-30.6) vs. 20.4 (18.5-22.9), p=0.01]. Complement C9, CD59, C3 and CFI correlated with TGFßR1, PDGFß, and PDGFRß, while C9, CD59 and C3 correlated with TGFßR2. CONCLUSION: This study is one of the first to compare the urinary complement profile in LN patients with moderate/severe IFTA and none/mild IFTA in human tissues. This study identified C3, CFI, and C9-to-CD59 ratio as potential markers of tubulointerstitial fibrosis in LN.

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability.

Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here we discover a checkpoint-regulated cascade of chromatin signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fibre approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favoured by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.

Prophylactic effect of chronic immunosuppression in a mouse model of CSF-1 receptor-related leukoencephalopathy.

Mutations leading to colony-stimulating factor-1 receptor (CSF-1R) loss-of-function or haploinsufficiency cause CSF1R-related leukoencephalopathy (CRL), an adult-onset disease characterized by loss of myelin and neurodegeneration, for which there is no effective therapy. Symptom onset usually occurs in the fourth decade of life and the penetrance of disease in carriers is high. However, familial studies have identified a few carriers of pathogenic CSF1R mutations that remain asymptomatic even in their seventh decade of life, raising the possibility that the development and severity of disease might be influenced by environmental factors. Here we report new cases in which long-term glucocorticoid treatment is associated with asymptomatic status in elder carriers of pathogenic CSF-1R mutations. The main objective of the present study was to investigate the link between chronic immunosuppression initiated pre-symptomatically and resistance to the development of symptomatic CRL, in the Csf1r+/- mouse model. We show that chronic prednisone administration prevents the development of memory, motor coordination and social interaction deficits, as well as the demyelination, neurodegeneration and microgliosis associated with these deficits. These findings are in agreement with the preliminary clinical observations and support the concept that pre-symptomatic immunosuppression is protective in patients carrying pathogenic CSF1R variants associated with CRL. Proteomic analysis of microglia and oligodendrocytes indicates that prednisone suppresses processes involved in microglial activation and alleviates senescence and improves fitness of oligodendrocytes. This analysis also identifies new potential targets for therapeutic intervention.

Proteomics and phosphoproteomics profiling in glutamatergic neurons and microglia in an iPSC model of Jansen de Vries Syndrome.

Background: Jansen de Vries Syndrome (JdVS) is a rare neurodevelopmental disorder (NDD) caused by gain-of-function (GOF) truncating mutations in PPM1D exons 5 or 6. PPM1D is a serine/threonine phosphatase that plays an important role in the DNA damage response (DDR) by negatively regulating TP53 (P53). JdVS-associated mutations lead to the formation of a truncated PPM1D protein that retains catalytic activity and has a GOF effect because of reduced degradation. Somatic PPM1D exons 5 and 6 truncating mutations are well-established factors in a number of cancers, due to excessive dephosphorylation and reduced function of P53 and other substrates involved in DDR. Children with JdVS have a variety of neurodevelopmental, psychiatric, and physical problems. In addition, a small fraction has acute neuropsychiatric decompensation apparently triggered by infection or severe non-infectious environmental stress factors. Methods: To understand the molecular basis of JdVS, we developed an induced pluripotent stem cell (iPSC) model system. iPSCs heterozygous for the truncating variant (PPM1D+/tr), were made from a patient, and control lines engineered using CRISPR-Cas9 gene editing. Proteomics and phosphoprotemics analyses were carried out on iPSC-derived glutamatergic neurons and microglia from three control and three PPM1D+/tr iPSC lines. We also analyzed the effect of the TLR4 agonist, lipopolysaccharide, to understand how activation of the innate immune system in microglia could account for acute behavioral decompensation. Results: One of the major findings was the downregulation of POGZ in unstimulated microglia. Since loss-of-function variants in the POGZ gene are well-known causes of autism spectrum disorder, the decrease in PPM1D+/tr microglia suggests this plays a role in the neurodevelopmental aspects of JdVS. In addition, neurons, baseline, and LPS-stimulated microglia show marked alterations in the expression of several E3 ubiquitin ligases, most notably UBR4, and regulators of innate immunity, chromatin structure, ErbB signaling, and splicing. In addition, pathway analysis points to overlap with neurodegenerative disorders. Limitations: Owing to the cost and labor-intensive nature of iPSC research, the sample size was small. Conclusions: Our findings provide insight into the molecular basis of JdVS and can be extrapolated to understand neuropsychiatric decompensation that occurs in subgroups of patients with ASD and other NDDs.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

The role of histone H3 lysine demethylases in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults with an average survival of 15-18 months. Part of its malignancy derives from epigenetic regulation that occurs as the tumor develops and after therapeutic treatment. Specifically, enzymes involved in removing methylations from histone proteins on chromatin, i.e., lysine demethylases (KDMs), have a significant impact on GBM biology and reoccurrence. This knowledge has paved the way to considering KDMs as potential targets for GBM treatment. For example, increases in trimethylation of histone H3 on the lysine 9 residue (H3K9me3) via inhibition of KDM4C and KDM7A has been shown to lead to cell death in Glioblastoma initiating cells. KDM6 has been shown to drive Glioma resistance to receptor tyrosine kinase inhibitors and its inhibition decreases tumor resistance. In addition, increased expression of the histone methyltransferase MLL4 and UTX histone demethylase are associated with prolonged survival in a subset of GBM patients, potentially by regulating histone methylation on the promoter of the mgmt gene. Thus, the complexity of how histone modifiers contribute to glioblastoma pathology and disease progression is yet to be fully understood. To date, most of the current work on histone modifying enzymes in GBM are centered upon histone H3 demethylase enzymes. In this mini-review, we summarize the current knowledge on the role of histone H3 demethylase enzymes in Glioblastoma tumor biology and therapy resistance. The objective of this work is to highlight the current and future potential areas of research for GBM epigenetics therapy.

DPP4 inhibition impairs senohemostasis to improve plaque stability in atherosclerotic mice.

Senescent vascular smooth muscle cells (VSMCs) accumulate in the vasculature with age and tissue damage and secrete factors that promote atherosclerotic plaque vulnerability and disease. Here, we report increased levels and activity of dipeptidyl peptidase 4 (DPP4), a serine protease, in senescent VSMCs. Analysis of the conditioned media from senescent VSMCs revealed a unique senescence-associated secretory phenotype (SASP) signature comprising many complement and coagulation factors; silencing or inhibiting DPP4 reduced these factors and increased cell death. Serum samples from persons with high risk for cardiovascular disease contained high levels of DPP4-regulated complement and coagulation factors. Importantly, DPP4 inhibition reduced senescent cell burden and coagulation and improved plaque stability, while single-cell resolution of senescent VSMCs reflected the senomorphic and senolytic effects of DPP4 inhibition in murine atherosclerosis. We propose that DPP4-regulated factors could be exploited therapeutically to reduce senescent cell function, reverse senohemostasis, and improve vascular disease.

MAT Gain of Activity Mutation in Helicobacter pylori Is Associated with Resistance to MTAN Transition State Analogues.

Helicobacter pylori is found in the gut lining of more than half of the world's population, causes gastric ulcers, and contributes to stomach cancers. Menaquinone synthesis in H. pylori relies on the rare futalosine pathway, where H. pylori 5'-methylthioadenosine nucleosidase (MTAN) is proposed to play an essential role. Transition state analogues of MTAN, including BuT-DADMe-ImmA (BTDIA) and MeT-DADMe-ImmA (MTDIA), exhibit bacteriostatic action against numerous diverse clinical isolates of H. pylori with minimum inhibitory concentrations (MIC's) of <2 ng/mL. Three H. pylori BTDIA-resistant clones were selected under increasing BTDIA pressure. Whole genome sequencing showed no mutations in MTAN. Instead, resistant clones had mutations in metK, methionine adenosyltransferase (MAT), feoA, a regulator of the iron transport system, and flhF, a flagellar synthesis regulator. The mutation in metK causes expression of a MAT with increased catalytic activity, leading to elevated cellular S-adenosylmethionine. Metabolite analysis and the mutations associated with resistance suggest multiple inputs associated with BTDIA resistance. Human gut microbiome exposed to MTDIA revealed no growth inhibition under aerobic or anaerobic conditions. Transition state analogues of H. pylori MTAN have potential as agents for treating H. pylori infection without disruption of the human gut microbiome or inducing resistance in the MTAN target.

Proximity labeling reveals a new in vivo network of interactors for the histone demethylase KDM5.

BACKGROUND: KDM5 family proteins are multi-domain regulators of transcription that when dysregulated contribute to cancer and intellectual disability. KDM5 proteins can regulate transcription through their histone demethylase activity in addition to demethylase-independent gene regulatory functions that remain less characterized. To expand our understanding of the mechanisms that contribute to KDM5-mediated transcription regulation, we used TurboID proximity labeling to identify KDM5-interacting proteins. RESULTS: Using Drosophila melanogaster, we enriched for biotinylated proteins from KDM5-TurboID-expressing adult heads using a newly generated control for DNA-adjacent background in the form of dCas9:TurboID. Mass spectrometry analyses of biotinylated proteins identified both known and novel candidate KDM5 interactors, including members of the SWI/SNF and NURF chromatin remodeling complexes, the NSL complex, Mediator, and several insulator proteins. CONCLUSIONS: Combined, our data shed new light on potential demethylase-independent activities of KDM5. In the context of KDM5 dysregulation, these interactions may play key roles in the alteration of evolutionarily conserved transcriptional programs implicated in human disorders.

Post-Translational Modifications of Histone Variants in the Absence and Presence of a Methionine-Depleting Enzyme in Normal and Cancer Cells.

Methionine is an essential amino acid involved in the formation of polyamines and a precursor metabolite for DNA and protein methylation. The dependence of cancer cells on methionine has triggered extensive investigations aimed at its targeting for cancer therapy, including the exploitation as a therapeutic tool of methionine γ-lyase (MGL), a bacterial enzyme that degrades methionine, capable of inhibiting cancer cells growth due to methionine starvation. We have exploited the high-resolution power of mass spectrometry to compare the effects of reduced availability of the methyl donor SAM, induced by MGL treatment, on the post-translational modifications of the histone tails in normal Hs27 and cancer HT-29 cells. In the absence of MGL, our analysis detected a three-fold higher relative abundance of trimethylated K25 of H1.4 in HT-29 than Hs27 cells, and a complex pattern of methylated, unmethylated and acetylated peptides in H2 and H3.3. In the presence of MGL, in HT-29, the peptide H2A1_4_11 is predominantly unmodified with mono-methylated K5 increasing upon treatment, whereas in Hs27 cells, H2A1_4_11 is monomethylated at K5 and K9 with these marks decreasing upon treatment. The time dependence of the effects of MGL-mediated methionine depletion on PTMs of histone variants in HT-29 cancer cells was also monitored. Overall, our present data on histone variants H1, H2A, H2B as well as H3.3 integrated with our previous studies on histones H3 and H4, shed light on the epigenetic modifications associated with methionine starvation and associated cancer cell death.