Long-term intermittent hypoxia in mice induces inflammatory pathways implicated in sleep apnea and steatohepatitis in humans.

Obstructive sleep apnea (OSA) induces intermittent hypoxia (IH), an independent risk factor for non-alcoholic fatty liver disease (NAFLD). While the molecular links between IH and NAFLD progression are unclear, immune cell-driven inflammation plays a crucial role in NAFLD pathogenesis. Using lean mice exposed to long-term IH and a cohort of lean OSA patients (n = 71), we conducted comprehensive hepatic transcriptomics, lipidomics, and targeted serum proteomics. Significantly, we demonstrated that long-term IH alone can induce NASH molecular signatures found in human steatohepatitis transcriptomic data. Biomarkers (PPARs, NRFs, arachidonic acid, IL16, IL20, IFNB, TNF-α) associated with early hepatic and systemic inflammation were identified. This molecular link between IH, sleep apnea, and steatohepatitis merits further exploration in clinical trials, advocating for integrating sleep apnea diagnosis in liver disease phenotyping. Our unique signatures offer potential diagnostic and treatment response markers, highlighting therapeutic targets in the comorbidity of NAFLD and OSA.

Nucleoside diphosphate kinases 1 and 2 regulate a protective liver response to a high-fat diet.

The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.

Aberrant activation of five embryonic stem cell-specific genes robustly predicts a high risk of relapse in breast cancers.

BACKGROUND: In breast cancer, as in all cancers, genetic and epigenetic deregulations can result in out-of-context expressions of a set of normally silent tissue-specific genes. The activation of some of these genes in various cancers empowers tumours cells with new properties and drives enhanced proliferation and metastatic activity, leading to a poor survival prognosis. RESULTS: In this work, we undertook an unprecedented systematic and unbiased analysis of out-of-context activations of a specific set of tissue-specific genes from testis, placenta and embryonic stem cells, not expressed in normal breast tissue as a source of novel prognostic biomarkers. To this end, we combined a strict machine learning framework of transcriptomic data analysis, and successfully created a new robust tool, validated in several independent datasets, which is able to identify patients with a high risk of relapse. This unbiased approach allowed us to identify a panel of five biomarkers, DNMT3B, EXO1, MCM10, CENPF and CENPE, that are robustly and significantly associated with disease-free survival prognosis in breast cancer. Based on these findings, we created a new Gene Expression Classifier (GEC) that stratifies patients. Additionally, thanks to the identified GEC, we were able to paint the specific molecular portraits of the particularly aggressive tumours, which show characteristics of male germ cells, with a particular metabolic gene signature, associated with an enrichment in pro-metastatic and pro-proliferation gene expression. CONCLUSIONS: The GEC classifier is able to reliably identify patients with a high risk of relapse at early stages of the disease. We especially recommend to use the GEC tool for patients with the luminal-A molecular subtype of breast cancer, generally considered of a favourable disease-free survival prognosis, to detect the fraction of patients undergoing a high risk of relapse.

Chromatin-associated YTHDC1 coordinates heat-induced reprogramming of gene expression.

Heat stress (HS) induces a cellular response leading to profound changes in gene expression. Here, we show that human YTHDC1, a reader of N6-methyladenosine (m6A) RNA modification, mostly associates to the chromatin fraction and that HS induces a redistribution of YTHDC1 across the genome, including to heat-induced heat shock protein (HSP) genes. YTHDC1 binding to m6A-modified HSP transcripts co-transcriptionally promotes expression of HSPs. In parallel, hundreds of the genes enriched in YTHDC1 during HS have their transcripts undergoing YTHDC1- and m6A-dependent intron retention. Later, YTHDC1 concentrates within nuclear stress bodies (nSBs) where it binds to m6A-modified SATIII non-coding RNAs, produced in an HSF1-dependent manner upon HS. These findings reveal that YTHDC1 plays a central role in a chromatin-associated m6A-based reprogramming of gene expression during HS. Furthermore, they support the model where the subsequent and temporary sequestration of YTHDC1 within nSBs calibrates the timing of this YTHDC1-dependent gene expression reprogramming.

Luspatercept (RAP-536) modulates oxidative stress without affecting mutation burden in myelodysplastic syndromes.

In low-risk myelodysplastic syndrome (LR-MDS), erythropoietin (EPO) is widely used for the treatment of chronic anemia. However, initial response to EPO has time-limited effects. Luspatercept reduces red blood cell transfusion dependence in LR-MDS patients. Here, we investigated the molecular action of luspatercept (RAP-536) in an in vitro model of erythroid differentiation of MDS, and also in a in vivo PDX murine model with primary samples of MDS patients carrying or not SF3B1 mutation. In our in vitro model, RAP-536 promotes erythroid proliferation by increasing the number of cycling cells without any impact on apoptosis rates. RAP-536 promoted late erythroid precursor maturation while decreasing intracellular reactive oxygen species level. RNA sequencing of erythroid progenitors obtained under RAP-536 treatment showed an enrichment of genes implicated in positive regulation of response to oxidative stress and erythroid differentiation. In our PDX model, RAP-536 induces a higher hemoglobin level. RAP-536 did not modify variant allele frequencies in vitro and did not have any effect against leukemic burden in our PDX model. These results suggest that RAP-536 promotes in vivo and in vitro erythroid cell differentiation by decreasing ROS level without any remarkable impact on iron homeostasis and on mutated allele burden.

SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation-Phosphorylation Cross-talk.

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. SIGNIFICANCE: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007.

Chronic Intermittent Hypoxia Increases Cell Proliferation in Hepatocellular Carcinoma.

Obstructive sleep apnea (OSA) syndrome is characterized by chronic intermittent hypoxia and is associated with an increased risk of all-cause mortality, including cancer mortality. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, characterized by increasing incidence and high mortality. However, the link between HCC and OSA-related chronic intermittent hypoxia remains unclear. Herein, we used a diethylnitrosamine (DEN)-induced HCC model to investigate whether OSA-related chronic intermittent hypoxia has an impact on HCC progression. To elucidate the associated mechanisms, we first evaluated the hypoxia status in the DEN-induced HCC model. Next, to simulate OSA-related intermittent hypoxia, we exposed cirrhotic rats with HCC to intermittent hypoxia during six weeks. We performed histopathological, immunohistochemical, RT-qPCR, and RNA-seq analysis. Chronic DEN injections strongly promoted cell proliferation, fibrosis, disorganized vasculature, and hypoxia in liver tissue, which mimics the usual events observed during human HCC development. Intermittent hypoxia further increased cell proliferation in DEN-induced HCC, which may contribute to an increased risk of HCC progression. In conclusion, our observations suggest that chronic intermittent hypoxia may be a factor worsening the prognosis of HCC.

Ectopic expression of a combination of 5 genes detects high risk forms of T-cell acute lymphoblastic leukemia.

BACKGROUND: T cell acute lymphoblastic leukemia (T-ALL) defines a group of hematological malignancies with heterogeneous aggressiveness and highly variable outcome, making therapeutic decisions a challenging task. We tried to discover new predictive model for T-ALL before treatment by using a specific pipeline designed to discover aberrantly active gene. RESULTS: The expression of 18 genes was significantly associated with shorter survival, including ACTRT2, GOT1L1, SPATA45, TOPAZ1 and ZPBP (5-GEC), which were used as a basis to design a prognostic classifier for T-ALL patients. The molecular characterization of the 5-GEC positive T-ALL unveiled specific characteristics inherent to the most aggressive T leukemic cells, including a drastic shut-down of genes located on the mitochondrial genome and an upregulation of histone genes, the latter characterizing high risk forms in adult patients. These cases fail to respond to the induction treatment, since 5-GEC either predicted positive minimal residual disease (MRD) or a short-term relapse in MRD negative patients. CONCLUSION: Overall, our investigations led to the discovery of a homogenous group of leukemic cells with profound alterations of their biology. It also resulted in an accurate predictive tool that could significantly improve the management of T-ALL patients.

Direct visualization of pre-protamine 2 detects protamine assembly failures and predicts ICSI success.

Histone-to-protamine transition is an essential step in the generation of fully functional spermatozoa in various mammalian species. In human and mouse, one of the two protamine-encoding genes produces a precursor pre-protamine 2 (pre-PRM2) protein, which is then processed and assembled. Here, we design an original approach based on the generation of pre-PRM2-specific antibodies to visualize the unprocessed pre-PRM2 by microscopy, flow cytometry and immunoblotting. Using mouse models with characterized failures in histone-to-protamine replacement, we show that pre-PRM2 retention is tightly linked to impaired nucleosome disassembly. Additionally, in elongating/condensing spermatids, we observe that pre-PRM2 and transition protein are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in the spermatozoa from 49 men of a series of infertile couples undergoing ICSI, which shed new light on the debated relation between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei embryo formation following ICSI, we evaluated the fertilization ability of the sperm in these 49 patients. Our results suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Hence, anti-pre-PRM2 antibodies are valuable tools that could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programmes to better understand the obscure process of histone-to-protamine transition.

AKR1B10, One of the Triggers of Cytokine Storm in SARS-CoV2 Severe Acute Respiratory Syndrome.

Preventing the cytokine storm observed in COVID-19 is a crucial goal for reducing the occurrence of severe acute respiratory failure and improving outcomes. Here, we identify Aldo-Keto Reductase 1B10 (AKR1B10) as a key enzyme involved in the expression of pro-inflammatory cytokines. The analysis of transcriptomic data from lung samples of patients who died from COVID-19 demonstrates an increased expression of the gene encoding AKR1B10. Measurements of the AKR1B10 protein in sera from hospitalised COVID-19 patients suggests a significant link between AKR1B10 levels and the severity of the disease. In macrophages and lung cells, the over-expression of AKR1B10 induces the expression of the pro-inflammatory cytokines Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß) and Tumor Necrosis Factor a (TNFα), supporting the biological plausibility of an AKR1B10 involvement in the COVID-19-related cytokine storm. When macrophages were stressed by lipopolysaccharides (LPS) exposure and treated by Zopolrestat, an AKR1B10 inhibitor, the LPS-induced production of IL-6, IL-1ß, and TNFα is significantly reduced, reinforcing the hypothesis that the pro-inflammatory expression of cytokines is AKR1B10-dependant. Finally, we also show that AKR1B10 can be secreted and transferred via extracellular vesicles between different cell types, suggesting that this protein may also contribute to the multi-organ systemic impact of COVID-19. These experiments highlight a relationship between AKR1B10 production and severe forms of COVID-19. Our data indicate that AKR1B10 participates in the activation of cytokines production and suggest that modulation of AKR1B10 activity might be an actionable pharmacological target in COVID-19 management.

Chidamide inhibits the NOTCH1-MYC signaling axis in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most dangerous hematological malignancies, with high tumor heterogeneity and poor prognosis. More than 60% of T-ALL patients carry NOTCH1 gene mutations, leading to abnormal expression of downstream target genes and aberrant activation of various signaling pathways. We found that chidamide, an HDAC inhibitor, exerts an antitumor effect on T-ALL cell lines and primary cells including an anti-NOTCH1 activity. In particular, chidamide inhibits the NOTCH1-MYC signaling axis by down-regulating the level of the intracellular form of NOTCH1 (NICD1) as well as MYC, partly through their ubiquitination and degradation by the proteasome pathway. We also report here the preliminary results of our clinical trial supporting that a treatment by chidamide reduces minimal residual disease (MRD) in patients and is well tolerated. Our results highlight the effectiveness and safety of chidamide in the treatment of T-ALL patients, including those with NOTCH1 mutations and open the way to a new therapeutic strategy for these patients.

DEN-Induced Rat Model Reproduces Key Features of Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The majority of HCC cases are associated with liver fibrosis or cirrhosis developing from chronic liver injuries. The immune system of the liver contributes to the severity of tissue damage, the establishment of fibrosis and the disease's progression towards HCC. Herein, we provide a detailed characterization of the DEN-induced HCC rat model during fibrosis progression and HCC development with a special focus on the liver's inflammatory microenvironment. Fischer 344 male rats were treated weekly for 14 weeks with intra-peritoneal injections of 50 mg/kg DEN. The rats were sacrificed before starting DEN-injections at 0 weeks, after 8 weeks, 14 weeks and 20 weeks after the start of DEN-injections. We performed histopathological, immunohistochemical, RT-qPCR, RNA-seq and flow cytometry analysis. Data were compared between tumor and non-tumor samples from the DEN-treated versus untreated rats, as well as versus human HCCs. Chronic DEN injections lead to liver damage, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and a modulated immune microenvironment that mimics the usual events observed during human HCC development. The RNA-seq results showed that DEN-induced liver tumors in the rat model shared remarkable molecular characteristics with human HCC, especially with HCC associated with high proliferation. In conclusion, our study provides detailed insight into hepatocarcinogenesis in a commonly used model of HCC, facilitating the future use of this model for preclinical testing.

ATAD2 controls chromatin-bound HIRA turnover.

Taking advantage of the evolutionary conserved nature of ATAD2, we report here a series of parallel functional studies in human, mouse, and Schizosaccharomyces pombe to investigate ATAD2's conserved functions. In S. pombe, the deletion of ATAD2 ortholog, abo1, leads to a dramatic decrease in cell growth, with the appearance of suppressor clones recovering normal growth. The identification of the corresponding suppressor mutations revealed a strong genetic interaction between Abo1 and the histone chaperone HIRA. In human cancer cell lines and in mouse embryonic stem cells, we observed that the KO of ATAD2 leads to an accumulation of HIRA. A ChIP-seq mapping of nucleosome-bound HIRA and FACT in Atad2 KO mouse ES cells demonstrated that both chaperones are trapped on nucleosomes at the transcription start sites of active genes, resulting in the abnormal presence of a chaperone-bound nucleosome on the TSS-associated nucleosome-free regions. Overall, these data highlight an important layer of regulation of chromatin dynamics ensuring the turnover of histone-bound chaperones.

CCM2-deficient endothelial cells undergo a ROCK-dependent reprogramming into senescence-associated secretory phenotype.

Cerebral cavernous malformation (CCM) is a cerebrovascular disease in which stacks of dilated haemorrhagic capillaries form focally in the brain. Whether and how defective mechanotransduction, cellular mosaicism and inflammation interplay to sustain the progression of CCM disease is unknown. Here, we reveal that CCM1- and CCM2-silenced endothelial cells expanded in vitro enter into senescence-associated secretory phenotype (SASP) that they use to invade the extracellular matrix and attract surrounding wild-type endothelial and immune cells. Further, we demonstrate that this SASP is driven by the cytoskeletal, molecular and transcriptomic disorders provoked by ROCK dysfunctions. By this, we propose that CCM2 and ROCK could be parts of a scaffold controlling senescence, bringing new insights into the emerging field of the control of ageing by cellular mechanics. These in vitro findings reconcile the known dysregulated traits of CCM2-deficient endothelial cells into a unique endothelial fate. Based on these in vitro results, we propose that a SASP could link the increased ROCK-dependent cell contractility in CCM2-deficient endothelial cells with microenvironment remodelling and long-range chemo-attraction of endothelial and immune cells.

The combined detection of Amphiregulin, Cyclin A1 and DDX20/Gemin3 expression predicts aggressive forms of oral squamous cell carcinoma.

BACKGROUND: Large-scale genetic and epigenetic deregulations enable cancer cells to ectopically activate tissue-specific expression programmes. A specifically designed strategy was applied to oral squamous cell carcinomas (OSCC) in order to detect ectopic gene activations and develop a prognostic stratification test. METHODS: A dedicated original prognosis biomarker discovery approach was implemented using genome-wide transcriptomic data of OSCC, including training and validation cohorts. Abnormal expressions of silent genes were systematically detected, correlated with survival probabilities and evaluated as predictive biomarkers. The resulting stratification test was confirmed in an independent cohort using immunohistochemistry. RESULTS: A specific gene expression signature, including a combination of three genes, AREG, CCNA1 and DDX20, was found associated with high-risk OSCC in univariate and multivariate analyses. It was translated into an immunohistochemistry-based test, which successfully stratified patients of our own independent cohort. DISCUSSION: The exploration of the whole gene expression profile characterising aggressive OSCC tumours highlights their enhanced proliferative and poorly differentiated intrinsic nature. Experimental targeting of CCNA1 in OSCC cells is associated with a shift of transcriptomic signature towards the less aggressive form of OSCC, suggesting that CCNA1 could be a good target for therapeutic approaches.

Metabolically controlled histone H4K5 acylation/acetylation ratio drives BRD4 genomic distribution.

In addition to acetylation, histones are modified by a series of competing longer-chain acylations. Most of these acylation marks are enriched and co-exist with acetylation on active gene regulatory elements. Their seemingly redundant functions hinder our understanding of histone acylations' specific roles. Here, by using an acute lymphoblastic leukemia (ALL) cell model and blasts from individuals with B-precusor ALL (B-ALL), we demonstrate a role of mitochondrial activity in controlling the histone acylation/acetylation ratio, especially at histone H4 lysine 5 (H4K5). An increase in the ratio of non-acetyl acylations (crotonylation or butyrylation) over acetylation on H4K5 weakens bromodomain containing protein 4 (BRD4) bromodomain-dependent chromatin interaction and enhances BRD4 nuclear mobility and availability for binding transcription start site regions of active genes. Our data suggest that the metabolism-driven control of the histone acetylation/longer-chain acylation(s) ratio could be a common mechanism regulating the bromodomain factors' functional genomic distribution.

Immediate and durable effects of maternal tobacco consumption alter placental DNA methylation in enhancer and imprinted gene-containing regions.

BACKGROUND: Although exposure to cigarette smoking during pregnancy has been associated with alterations of DNA methylation in the cord blood or placental cells, whether such exposure before pregnancy could induce epigenetic alterations in the placenta of former smokers has never been investigated. METHODS: Our approach combined the analysis of placenta epigenomic (ENCODE) data with newly generated DNA methylation data obtained from 568 pregnant women, the largest cohort to date, either actively smoking during their pregnancy or formerly exposed to tobacco smoking. RESULTS: This strategy resulted in several major findings. First, among the 203 differentially methylated regions (DMRs) identified by the epigenome-wide association study, 152 showed "reversible" alterations of DNA methylation, only present in the placenta of current smokers, whereas 26 were also found altered in former smokers, whose placenta had not been exposed directly to cigarette smoking. Although the absolute methylation changes were smaller than those observed in other contexts, such as in some congenital diseases, the observed alterations were consistent within each DMR. This observation was further supported by a demethylation of LINE-1 sequences in the placentas of both current (beta-coefficient (ß) (95% confidence interval (CI)), - 0.004 (- 0.008; 0.001)) and former smokers (ß (95% CI), - 0.006 (- 0.011; - 0.001)) compared to nonsmokers. Second, the 203 DMRs were enriched in epigenetic marks corresponding to enhancer regions, including monomethylation of lysine 4 and acetylation of lysine 27 of histone H3 (respectively H3K4me1 and H3K27ac). Third, smoking-associated DMRs were also found near and/or overlapping 10 imprinted genes containing regions (corresponding to 16 genes), notably including the NNAT, SGCE/PEG10, and H19/MIR675 loci. CONCLUSIONS: Our results pointing towards genomic regions containing the imprinted genes as well as enhancers as preferential targets suggest mechanisms by which tobacco could directly impact the fetus and future child. The persistence of significant DNA methylation changes in the placenta of former smokers supports the hypothesis of an "epigenetic memory" of exposure to cigarette smoking before pregnancy. This observation not only is conceptually revolutionary, but these results also bring crucial information in terms of public health concerning potential long-term detrimental effects of smoking in women.

PenDA, a rank-based method for personalized differential analysis: Application to lung cancer.

The hopes of precision medicine rely on our capacity to measure various high-throughput genomic information of a patient and to integrate them for personalized diagnosis and adapted treatment. Reaching these ambitious objectives will require the development of efficient tools for the detection of molecular defects at the individual level. Here, we propose a novel method, PenDA, to perform Personalized Differential Analysis at the scale of a single sample. PenDA is based on the local ordering of gene expressions within individual cases and infers the deregulation status of genes in a sample of interest compared to a reference dataset. Based on realistic simulations of RNA-seq data of tumors, we showed that PenDA outcompetes existing approaches with very high specificity and sensitivity and is robust to normalization effects. Applying the method to lung cancer cohorts, we observed that deregulated genes in tumors exhibit a cancer-type-specific commitment towards up- or down-regulation. Based on the individual information of deregulation given by PenDA, we were able to define two new molecular histologies for lung adenocarcinoma cancers strongly correlated to survival. In particular, we identified 37 biomarkers whose up-regulation lead to bad prognosis and that we validated on two independent cohorts. PenDA provides a robust, generic tool to extract personalized deregulation patterns that can then be used for the discovery of therapeutic targets and for personalized diagnosis. An open-access, user-friendly R package is available at https://github.com/bcm-uga/penda.