Novel 14q32.2 paternal deletion encompassing the whole DLK1 gene associated with Temple syndrome.

BACKGROUND: Temple syndrome (TS14) is a rare imprinting disorder caused by maternal UPD14, imprinting defects or paternal microdeletions which lead to an increase in the maternal expressed genes and a silencing the paternally expressed genes in the 14q32 imprinted domain. Classical TS14 phenotypic features include pre- and postnatal short stature, small hands and feet, muscular hypotonia, motor delay, feeding difficulties, weight gain, premature puberty along and precocious puberty. METHODS: An exon array comparative genomic hybridization was performed on a patient affected by psychomotor and language delay, muscular hypotonia, relative macrocephaly, and small hand and feet at two years old. At 6 years of age, the proband presented with precocious thelarche. Genes dosage and methylation within the 14q32 region were analyzed by MS-MLPA. Bisulfite PCR and pyrosequencing were employed to quantification methylation at the four known imprinted differentially methylated regions (DMR) within the 14q32 domain: DLK1 DMR, IG-DMR, MEG3 DMR and MEG8 DMR. RESULTS: The patient had inherited a 69 Kb deletion, encompassing the entire DLK1 gene, on the paternal allele. Relative hypermethylation of the two maternally methylated intervals, DLK1 and MEG8 DMRs, was observed along with normal methylation level at IG-DMR and MEG3 DMR, resulting in a phenotype consistent with TS14. Additional family members with the deletion showed modest methylation changes at both the DLK1 and MEG8 DMRs consistent with parental transmission. CONCLUSION: We describe a girl with clinical presentation suggestive of Temple syndrome resulting from a small paternal 14q32 deletion that led to DLK1 whole-gene deletion, as well as hypermethylation of the maternally methylated DLK1-DMR.

Metformin and Glucose Concentration as Limiting Factors in Retinal Pigment Epithelial Cell Viability and Proliferation.

Metformin is a well-established drug for the treatment of type 2 diabetes; however, the mechanism of action has not been well described and many aspects of how it truly acts are still unknown. Moreover, regarding in vitro experiments, the glycaemic status when metformin is used is generally not considered, which, added to the suprapharmacological drug concentrations that are commonly employed in research, has resulted in gaps of its mechanism of action. The aim of this study was to determine how glucose and metformin concentrations influence cell culture. Considering that diabetic retinopathy is one of the most common complications of diabetes, a retinal pigment epithelial cell line was selected, and cell viability and proliferation rates were measured at different glucose and metformin concentrations. As expected, glucose concentration by itself positively influenced cell proliferation rates. When the metformin was considered, results were conditioned, as well, by metformin concentration. This conditioning resulted in cell death when high concentrations of metformin were used under physiological concentrations of glucose, while this did not happen when clinically relevant concentrations of metformin were used independently of glucose status. Our study shows the importance of in vitro cell growth conditions when drug effects such as metformin's are being analysed.

A personalized medicine approach identifies enasidenib as an efficient treatment for IDH2 mutant chondrosarcoma.

BACKGROUND: Sarcomas represent an extensive group of malignant diseases affecting mesodermal tissues. Among sarcomas, the clinical management of chondrosarcomas remains a complex challenge, as high-grade tumours do not respond to current therapies. Mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes are among the most common mutations detected in chondrosarcomas and may represent a therapeutic opportunity. The presence of mutated IDH (mIDH) enzymes results in the accumulation of the oncometabolite 2-HG leading to molecular alterations that contribute to drive tumour growth. METHODS: We developed a personalized medicine strategy based on the targeted NGS/Sanger sequencing of sarcoma samples (n = 6) and the use of matched patient-derived cell lines as a drug-testing platform. The anti-tumour potential of IDH mutations found in two chondrosarcoma cases was analysed in vitro, in vivo and molecularly (transcriptomic and DNA methylation analyses). FINDINGS: We treated several chondrosarcoma models with specific mIDH1/2 inhibitors. Among these treatments, only the mIDH2 inhibitor enasidenib was able to decrease 2-HG levels and efficiently reduce the viability of mIDH2 chondrosarcoma cells. Importantly, oral administration of enasidenib in xenografted mice resulted in a complete abrogation of tumour growth. Enasidenib induced a profound remodelling of the transcriptomic landscape not associated to changes in the 5 mC methylation levels and its anti-tumour effects were associated with the repression of proliferative pathways such as those controlled by E2F factors. INTERPRETATION: Overall, this work provides preclinical evidence for the use of enasidenib to treat mIDH2 chondrosarcomas. FUNDING: Supported by the Spanish Research Agency/FEDER (grants PID2022-142020OB-I00; PID2019-106666RB-I00), the ISC III/FEDER (PI20CIII/00020; DTS18CIII/00005; CB16/12/00390; CB06/07/1009; CB19/07/00057); the GEIS group (GEIS-62); and the PCTI (Asturias)/FEDER (IDI/2021/000027).

The thyroid hormone enhances mouse embryonic fibroblasts reprogramming to pluripotent stem cells: role of the nuclear receptor corepressor 1.

Introduction: Pluripotent stem cells can be generated from somatic cells by the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc. Methods: Mouse embryonic fibroblasts (MEFs) were transduced with the Yamanaka factors and generation of induced pluripotent stem cells (iPSCs) was assessed by formation of alkaline phosphatase positive colonies, pluripotency gene expression and embryod bodies formation. Results: The thyroid hormone triiodothyronine (T3) enhances MEFs reprogramming. T3-induced iPSCs resemble embryonic stem cells in terms of the expression profile and DNA methylation pattern of pluripotency genes, and of their potential for embryod body formation and differentiation into the three major germ layers. T3 induces reprogramming even though it increases expression of the cyclin kinase inhibitors p21 and p27, which are known to oppose acquisition of pluripotency. The actions of T3 on reprogramming are mainly mediated by the thyroid hormone receptor beta and T3 can enhance iPSC generation in the absence of c-Myc. The hormone cannot replace Oct4 on reprogramming, but in the presence of T3 is possible to obtain iPSCs, although with low efficiency, without exogenous Klf4. Furthermore, depletion of the corepressor NCoR (or Nuclear Receptor Corepressor 1) reduces MEFs reprogramming in the absence of the hormone and strongly decreases iPSC generation by T3 and also by 9cis-retinoic acid, a well-known inducer of reprogramming. NCoR depletion also markedly antagonizes induction of pluripotency gene expression by both ligands. Conclusions: Inclusion of T3 on reprogramming strategies has a potential use in enhancing the generation of functional iPSCs for studies of cell plasticity, disease and regenerative medicine.

A complex interplay between H2A.Z and HP1 isoforms regulates pericentric heterochromatin.

Pericentric heterochromatin (PCH) plays an essential role in the maintenance of genome integrity and alterations in PCH have been linked to cancer and aging. HP1 α, ß, and γ, are hallmarks of constitutive heterochromatin that are thought to promote PCH structure through binding to heterochromatin-specific histone modifications and interaction with a wide range of factors. Among the less understood components of PCH is the histone H2A variant H2A.Z, whose role in the organization and maintenance of PCH is poorly defined. Here we show that there is a complex interplay between H2A.Z and HP1 isoforms in PCH. While the loss of HP1α results in the accumulation of H2A.Z.1 in PCH, which is associated with a significant decrease in its mobile fraction, H2A.Z.1 binds preferentially to HP1ß in these regions. Of note, H2A.Z.1 downregulation results in increased heterochromatinization and instability of PCH, reflected by accumulation of the major epigenetic hallmarks of heterochromatin in these regions and increased frequency of chromosome aberrations related to centromeric/pericentromeric defects. Our studies support a role for H2A.Z in genome stability and unveil a key role of H2A.Z in the regulation of heterochromatin-specific epigenetic modifications through a complex interplay with the HP1 isoforms.

A human genome editing-based MLL::AF4 ALL model recapitulates key cellular and molecular leukemogenic features.

Cellular ontogeny and MLL breakpoint site influence the capacity of MLL-edited CD34+ hematopoietic cells to initiate and recapitulate infant patients' features in pro-B-cell acute lymphoblastic leukemia (B-ALL). We provide key insights into the leukemogenic determinants of MLL-AF4+ infant B-ALL.

CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

BACKGROUND: Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated. RESULTS: In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence. CONCLUSIONS: These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.

DPPA3-HIF1α axis controls colorectal cancer chemoresistance by imposing a slow cell-cycle phenotype.

Tumor relapse is linked to rapid chemoresistance and represents a bottleneck for cancer therapy success. Engagement of a reduced proliferation state is a non-mutational mechanism exploited by cancer cells to bypass therapy-induced cell death. Through combining functional pulse-chase experiments in engineered cells and transcriptomic analyses, we identify DPPA3 as a master regulator of slow-cycling and chemoresistant phenotype in colorectal cancer (CRC). We find a vicious DPPA3-HIF1α feedback loop that downregulates FOXM1 expression via DNA methylation, thereby delaying cell-cycle progression. Moreover, downregulation of HIF1α partially restores a chemosensitive proliferative phenotype in DPPA3-overexpressing cancer cells. In cohorts of CRC patient samples, DPPA3 overexpression acts as a predictive biomarker of chemotherapeutic resistance that subsequently requires reduction in its expression to allow metastatic outgrowth. Our work demonstrates that slow-cycling cancer cells exploit a DPPA3/HIF1α axis to support tumor persistence under therapeutic stress and provides insights on the molecular regulation of disease progression.

Multi-omic integration of DNA methylation and gene expression data reveals molecular vulnerabilities in glioblastoma.

Glioblastoma (GBM) is one of the most aggressive types of cancer and exhibits profound genetic and epigenetic heterogeneity, making the development of an effective treatment a major challenge. The recent incorporation of molecular features into the diagnosis of patients with GBM has led to an improved categorization into various tumour subtypes with different prognoses and disease management. In this work, we have exploited the benefits of genome-wide multi-omic approaches to identify potential molecular vulnerabilities existing in patients with GBM. Integration of gene expression and DNA methylation data from both bulk GBM and patient-derived GBM stem cell lines has revealed the presence of major sources of GBM variability, pinpointing subtype-specific tumour vulnerabilities amenable to pharmacological interventions. In this sense, inhibition of the AP-1, SMAD3 and RUNX1/RUNX2 pathways, in combination or not with the chemotherapeutic agent temozolomide, led to the subtype-specific impairment of tumour growth, particularly in the context of the aggressive, mesenchymal-like subtype. These results emphasize the involvement of these molecular pathways in the development of GBM and have potential implications for the development of personalized therapeutic approaches.

Circulating miRNA expression in long-standing type 1 diabetes mellitus.

Type 1 diabetes is a chronic autoimmune disease which results in inefficient regulation of glucose homeostasis and can lead to different vascular comorbidities through life. In this study we aimed to analyse the circulating miRNA expression profile of patients with type 1 diabetes, and with no other associated pathology. For this, fasting plasma was obtained from 85 subjects. Next generation sequencing analysis was firstly performed to identify miRNAs that were differentially expressed between groups (20 patients vs. 10 controls). hsa-miR-1-3p, hsa-miR-200b-3p, hsa-miR-9-5p, and hsa-miR-1200 expression was also measured by Taqman RT-PCR to validate the observed changes (34 patients vs. 21 controls). Finally, through a bioinformatic approach, the main pathways affected by the target genes of these miRNAs were studied. Among the studied miRNAs, hsa-miR-1-3p expression was found significantly increased in patients with type 1 diabetes compared to controls, and positively correlated with glycated haemoglobin levels. Additionally, by using a bioinformatic approach, we could observe that changes in hsa-miR-1-3p directly affect genes involved in vascular development and cardiovascular pathologies. Our results suggest that, circulating hsa-miR-1-3p in plasma, together with glycaemic control, could be used as prognostic biomarkers in type 1 diabetes, helping to prevent the development of vascular complications in these patients.

Nicotinamide N-methyltransferase (NNMT) regulates the glucocorticoid signaling pathway during the early phase of adipogenesis.

Obesity is associated with adipose tissue dysfunction through the differentiation and expansion of pre-adipocytes to adipocytes (hyperplasia) and/or increases in size of pre-existing adipocytes (hypertrophy). A cascade of transcriptional events coordinates the differentiation of pre-adipocytes into fully differentiated adipocytes; the process of adipogenesis. Although nicotinamide N-methyltransferase (NNMT) has been associated with obesity, how NNMT is regulated during adipogenesis, and the underlying regulatory mechanisms, remain undefined. In present study we used genetic and pharmacological approaches to elucidate the molecular signals driving NNMT activation and its role during adipogenesis. Firstly, we demonstrated that during the early phase of adipocyte differentiation NNMT is transactivated by CCAAT/Enhancer Binding Protein beta (CEBPB) in response to glucocorticoid (GC) induction. We found that Nnmt knockout, using CRISPR/Cas9 approach, impaired terminal adipogenesis by influencing the timing of cellular commitment and cell cycle exit during mitotic clonal expansion, as demonstrated by cell cycle analysis and RNA sequencing experiments. Biochemical and computational methods showed that a novel small molecule, called CC-410, stably binds to and highly specifically inhibits NNMT. CC-410 was, therefore, used to modulate protein activity during pre-adipocyte differentiation stages, demonstrating that, in line with the genetic approach, chemical inhibition of NNMT at the early stages of adipogenesis impairs terminal differentiation by deregulating the GC network. These congruent results conclusively demonstrate that NNMT is a key component of the GC-CEBP axis during the early stages of adipogenesis and could be a potential therapeutic target for both early-onset obesity and glucocorticoid-induced obesity.

Maternal obesity and gestational diabetes reprogram the methylome of offspring beyond birth by inducing epigenetic signatures in metabolic and developmental pathways.

BACKGROUND: Obesity is a negative chronic metabolic health condition that represents an additional risk for the development of multiple pathologies. Epidemiological studies have shown how maternal obesity or gestational diabetes mellitus during pregnancy constitute serious risk factors in relation to the appearance of cardiometabolic diseases in the offspring. Furthermore, epigenetic remodelling may help explain the molecular mechanisms that underlie these epidemiological findings. Thus, in this study we explored the DNA methylation landscape of children born to mothers with obesity and gestational diabetes during their first year of life. METHODS: We used Illumina Infinium MethylationEPIC BeadChip arrays to profile more than 770,000 genome-wide CpG sites in blood samples from a paediatric longitudinal cohort consisting of 26 children born to mothers who suffered from obesity or obesity with gestational diabetes mellitus during pregnancy and 13 healthy controls (measurements taken at 0, 6 and 12 month; total N = 90). We carried out cross-sectional and longitudinal analyses to derive DNA methylation alterations associated with developmental and pathology-related epigenomics. RESULTS: We identified abundant DNA methylation changes during child development from birth to 6 months and, to a lesser extent, up to 12 months of age. Using cross-sectional analyses, we discovered DNA methylation biomarkers maintained across the first year of life that could discriminate children born to mothers who suffered from obesity or obesity with gestational diabetes. Importantly, enrichment analyses suggested that these alterations constitute epigenetic signatures that affect genes and pathways involved in the metabolism of fatty acids, postnatal developmental processes and mitochondrial bioenergetics, such as CPT1B, SLC38A4, SLC35F3 and FN3K. Finally, we observed evidence of an interaction between developmental DNA methylation changes and maternal metabolic condition alterations. CONCLUSIONS: Our observations highlight the first six months of development as being the most crucial for epigenetic remodelling. Furthermore, our results support the existence of systemic intrauterine foetal programming linked to obesity and gestational diabetes that affects the childhood methylome beyond birth, which involves alterations related to metabolic pathways, and which may interact with ordinary postnatal development programmes.

The TINCR ubiquitin-like microprotein is a tumor suppressor in squamous cell carcinoma.

The TINCR (Terminal differentiation-Induced Non-Coding RNA) gene is selectively expressed in epithelium tissues and is involved in the control of human epidermal differentiation and wound healing. Despite its initial report as a long non-coding RNA, the TINCR locus codes for a highly conserved ubiquitin-like microprotein associated with keratinocyte differentiation. Here we report the identification of TINCR as a tumor suppressor in squamous cell carcinoma (SCC). TINCR is upregulated by UV-induced DNA damage in a TP53-dependent manner in human keratinocytes. Decreased TINCR protein expression is prevalently found in skin and head and neck squamous cell tumors and TINCR expression suppresses the growth of SCC cells in vitro and in vivo. Consistently, Tincr knockout mice show accelerated tumor development following UVB skin carcinogenesis and increased penetrance of invasive SCCs. Finally, genetic analyses identify loss-of-function mutations and deletions encompassing the TINCR gene in SCC clinical samples supporting a tumor suppressor role in human cancer. Altogether, these results demonstrate a role for TINCR as protein coding tumor suppressor gene recurrently lost in squamous cell carcinomas.

The outcome of boosting mitochondrial activity in alcohol-associated liver disease is organ-dependent.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) accounts for 70% of liver-related deaths in Europe, with no effective approved therapies. Although mitochondrial dysfunction is one of the earliest manifestations of alcohol-induced injury, restoring mitochondrial activity remains a problematic strategy due to oxidative stress. Here, we identify methylation-controlled J protein (MCJ) as a mediator for ALD progression and hypothesize that targeting MCJ may help in recovering mitochondrial fitness without collateral oxidative damage. APPROACH AND RESULTS: C57BL/6 mice [wild-type (Wt)] Mcj knockout and Mcj liver-specific silencing (MCJ-LSS) underwent the NIAAA dietary protocol (Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 days, plus a single binge ethanol feeding at day 11). To evaluate the impact of a restored mitochondrial activity in ALD, the liver, gut, and pancreas were characterized, focusing on lipid metabolism, glucose homeostasis, intestinal permeability, and microbiota composition. MCJ, a protein acting as an endogenous negative regulator of mitochondrial respiration, is downregulated in the early stages of ALD and increases with the severity of the disease. Whole-body deficiency of MCJ is detrimental during ALD because it exacerbates the systemic effects of alcohol abuse through altered intestinal permeability, increased endotoxemia, and dysregulation of pancreatic function, which overall worsens liver injury. On the other hand, liver-specific Mcj silencing prevents main ALD hallmarks, that is, mitochondrial dysfunction, steatosis, inflammation, and oxidative stress, as it restores the NAD + /NADH ratio and SIRT1 function, hence preventing de novo lipogenesis and improving lipid oxidation. CONCLUSIONS: Improving mitochondrial respiration by liver-specific Mcj silencing might become a novel therapeutic approach for treating ALD.

PI3Kß-regulated ß-catenin mediates EZH2 removal from promoters controlling primed human ESC stemness and primitive streak gene expression.

The mechanism governing the transition of human embryonic stem cells (hESCs) toward differentiated cells is only partially understood. To explore this transition, the activity and expression of the ubiquitous phosphatidylinositol 3-kinase (PI3Kα and PI3Kß) were modulated in primed hESCs. The study reports a pathway that dismantles the restraint imposed by the EZH2 polycomb repressor on an essential stemness gene, NODAL, and on transcription factors required to trigger primitive streak formation. The primitive streak is the site where gastrulation begins to give rise to the three embryonic cell layers from which all human tissues derive. The pathway involves a PI3Kß non-catalytic action that controls nuclear/active RAC1 levels, activation of JNK (Jun N-terminal kinase) and nuclear ß-catenin accumulation. ß-Catenin deposition at promoters triggers release of the EZH2 repressor, permitting stemness maintenance (through control of NODAL) and correct differentiation by allowing primitive streak master gene expression. PI3Kß epigenetic control of EZH2/ß-catenin might be modulated to direct stem cell differentiation.

Classification of follicular-patterned thyroid lesions using a minimal set of epigenetic biomarkers.

Objective: The minimally invasive fine-needle aspiration cytology (FNAC) is the current gold standard for the diagnosis of thyroid nodule malignancy. However, the correct discrimination of follicular neoplasia often requires more invasive diagnostic techniques. The lack of suitable immunohistochemical markers to distinguish between follicular thyroid carcinoma and other types of follicular-derived lesions complicates diagnosis, and despite most of these tumours being surgically resected, only a small number will test positive for malignancy. As such, the development of new orthogonal diagnostic approaches may improve the accuracy of diagnosing thyroid nodules. Design: This study includes a retrospective, multi-centre training cohort including 54 fresh-frozen follicular-patterned thyroid samples and two independent, multi-centre validation cohorts of 103 snap-frozen biopsies and 33 FNAC samples, respectively. Methods: We performed a genome-wide genetic and epigenetic profiling of 54 fresh-frozen follicular-patterned thyroid samples using exome sequencing and the Illumina Human DNA Methylation EPIC platform. An extensive validation was performed using the bisulfite pyrosequencing technique. Results: Using a random forest approach, we developed a three-CpG marker-based diagnostic model that was subsequently validated using bisulfite pyrosequencing experiments. According to the validation cohort, this cost-effective method discriminates between benign and malignant nodules with a sensitivity and specificity of 97 and 88%, respectively (positive predictive value (PPV): 0.85, negative predictive value (NPV): 0.98). Conclusions: Our classification system based on a minimal set of epigenetic biomarkers can complement the potential of the diagnostic techniques currently available and would prioritize a considerable number of surgical interventions that are often performed due to uncertain cytology. Significance statement: In recent years, there has been a significant increase in the number of people diagnosed with thyroid nodules. The current challenge is their etiological diagnosis to discount malignancy without resorting to thyroidectomy. The method proposed here, based on DNA pyrosequencing assays, has high sensitivity (0.97) and specificity (0.88) for the identification of malignant thyroid nodules. This simple and cost-effective approach can complement expert pathologist evaluation to prioritize the classification of difficult-to-diagnose follicular-patterned thyroid lesions and track tumor evolution, including real-time monitoring of treatment efficacy, thereby stimulating adherence to health promotion programs.

Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells.

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages.

Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming.

The expression of the pluripotency factors OCT4, SOX2, KLF4, and MYC (OSKM) can convert somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Notably, partial and reversible reprogramming does not change cell identity but can reverse markers of aging in cells, improve the capacity of aged mice to repair tissue injuries, and extend longevity in progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome, and metabolome in naturally aged mice subject to a single period of transient OSKM expression. We found that this is sufficient to reverse DNA methylation changes that occur upon aging in the pancreas, liver, spleen, and blood. Similarly, we observed reversion of transcriptional changes, especially regarding biological processes known to change during aging. Finally, some serum metabolites and biomarkers altered with aging were also restored to young levels upon transient reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic, and metabolomic changes toward a younger configuration in multiple tissues and in the serum.

Epigenetic Profiling and Response to CD19 Chimeric Antigen Receptor T-Cell Therapy in B-Cell Malignancies.

BACKGROUND: Chimeric antigen receptor (CAR) T cells directed against CD19 (CART19) are effective in B-cell malignancies, but little is known about the molecular factors predicting clinical outcome of CART19 therapy. The increasingly recognized relevance of epigenetic changes in cancer immunology prompted us to determine the impact of the DNA methylation profiles of CART19 cells on the clinical course. METHODS: We recruited 114 patients with B-cell malignancies, comprising 77 patients with acute lymphoblastic leukemia and 37 patients with non-Hodgkin lymphoma who were treated with CART19 cells. Using a comprehensive DNA methylation microarray, we determined the epigenomic changes that occur in the patient T cells upon transduction of the CAR vector. The effects of the identified DNA methylation sites on clinical response, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, event-free survival, and overall survival were assessed. All statistical tests were 2-sided. RESULTS: We identified 984 genomic sites with differential DNA methylation between CAR-untransduced and CAR-transduced T cells before infusion into the patient. Eighteen of these distinct epigenetic loci were associated with complete response (CR), adjusting by multiple testing. Using the sites linked to CR, an epigenetic signature, referred to hereafter as the EPICART signature, was established in the initial discovery cohort (n = 79), which was associated with CR (Fisher exact test, P < .001) and enhanced event-free survival (hazard ratio [HR] = 0.36; 95% confidence interval [CI] = 0.19 to 0.70; P = .002; log-rank P = .003) and overall survival (HR = 0.45; 95% CI = 0.20 to 0.99; P = .047; log-rank P = .04;). Most important, the EPICART profile maintained its clinical course predictive value in the validation cohort (n = 35), where it was associated with CR (Fisher exact test, P < .001) and enhanced overall survival (HR = 0.31; 95% CI = 0.11 to 0.84; P = .02; log-rank P = .02). CONCLUSIONS: We show that the DNA methylation landscape of patient CART19 cells influences the efficacy of the cellular immunotherapy treatment in patients with B-cell malignancy.