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Introduction: Treatment options of lung cancer (LC) comprise radiotherapy, and/or combined treatment approaches, including chemotherapy, immunotherapy and targeted therapies based on the tumoral molecular profile. Following curative-intent first-line therapies, clinical surveillance involves serial CT imaging. However, such surveillance can detect only macroscopic disease recurrence and is frequently inconclusive. NGS has been utilized to help identify and monitor treatment plans. Nucleosomes, complexes of DNA and histones proteins, are released during cell death into blood circulation. Trimethylation of lysine 27 on histone H3 (H3K27Me3), catalyzed by enhancer of zeste homolog 2 (EZH2), is a crucial epigenetic process in tumorigenesis. We investigated if H3K27Me3-nucleosome concentration could be a biomarker for molecular residual disease (MRD). Patients and Methods: Plasmas were retrospectively collected from patients with advanced LC during treatment (CIRCAN’s cohort, n= 200) and from healthy donors (n=100). We carried out standard targeted NGS on paired plasmas. Samples were divided in two sub-groups based on genetical results: ctDNA negative (n=120) or positive (n=80) for presence of somatic alterations. Concentration of circulating H3K27Me3-nucleosome was measured using chemiluminescent Nu.Q® immunoassay (Belgian Volition SRL, Belgium). Results: Significantly elevated concentrations of H3K27Me3-nucleosomes were found in LC plasmas during the follow-up of patients compared to healthy donors (median 14.9 ng/ml vs 6.15 ng/ml, respectively, p Conclusions: High levels of Nu.Q® H3K27Me3 could allow physicians to detect MRD in LC patients following treatment with curative intent. This could be achieved by monitoring testing of patients at defined intervals of treatment and recovery, alongside imaging, to incorporate analyses of evolving molecular landscapes during treatment. In this setting, the H3K27Me3-nucleosome quantification, to complete the molecular exploration of cfDNA is highly encouraging, especially in advanced NSCLC, where re-tissue biopsies are impractical, expensive, and may cause undue harm. H3K27Me3-nucleosome quantification may also be useful in patient identification for specific treatments such as EZH2 inhibitor, but this requires investigation. Citation Format: Emmanuel Grolleau, Julie Candiracci, Arnaud Gauthier, Gaelle Lescuyer, David Barthelemy, Christine Haon, Florence Geiguer, Margaux Raffin, Nathalie Hardat, Julie Balandier, Rémi Rabeuf, Anne-Sophie Wozny, Guillaume Rommelaere, Claire Rodriguez-Lafrasse, Fabien Subtil, Sébastien Couraud, Marielle Herzog, Lea Payen-Gay. Circulating H3K27 nucleosomes to monitor lung cancer patients during treatment, a universal biomarker quantifying the molecular residual disease (MRD) in plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2163.

NETosis is an innate immune response occurring after infection or inflammation: activated neutrophils expel decondensed DNA in complex with histones into the extracellular environment in a controlled manner. It activates coagulation and fuels the risk of thrombosis. Human pregnancy is associated with a mild proinflammatory state characterized by circulatory neutrophil activation which is further increased in complicated pregnancies, placenta-mediated complications being associated with an increased thrombotic risk. This aberrant activation leads to an increased release of nucleosomes in the blood flow. The aim of our study was to initially quantify nucleosome-bound histones in normal pregnancy and in placenta-mediated complication counterpart. We analyzed the role of histones on extravillous trophoblast function. Circulating nucleosome-bound histones H3 (Nu.QH3.1, Nu.QH3PanCit, Nu.QH3K27me3) and H4 (Nu.QH4K16Ac) were increased in complicated pregnancies. In vitro using the extravillous cell line HTR-8/SVNeo, we observed that free recombinant H2B, H3, and H4 inhibited migration in wound healing assay, but only H3 also blocked invasion in Matrigel-coated Transwell experiments. H3 and H4 also induced apoptosis, whereas H2B did not. Finally, the negative effects of H3 on invasion and apoptosis could be restored with enoxaparin, a low-molecular-weight heparin (LMWH), but not with aspirin. Different circulating nucleosome-bound histones are increased in complicated pregnancy and this would affect migration, invasion, and induce apoptosis of extravillous trophoblasts. Histones might be part of the link between the risk of thrombosis and pregnancy complications, with an effect of LMWH on both.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Despite recent advances in therapy modalities, the overall survival of GBM patients remains poor. GBM diagnosis relies on neuroimaging techniques. However, confirmation via histopathological and molecular analysis is necessary. Given the intrinsic limitations of such techniques, liquid biopsy (mainly via blood samples) emerged as a non-invasive and easy-to-implement alternative that could aid in both the diagnosis and the follow-up of GBM patients. Cancer cells release tumoral content into the bloodstream, such as circulating tumor DNA, circulating microRNAs, circulating tumor cells, extracellular vesicles, or circulating nucleosomes: all these could serve as a marker of GBM. In this narrative review, we discuss the current knowledge, the advantages, and the disadvantages of each circulating biomarker so far proposed. ispartof: CANCERS vol:14 issue:14 ispartof: location:Switzerland status: published

e20078 Background: There are over 700,000 new cases of non-Hodgkin lymphoma (NHL), acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) diagnosed globally each year and approximately 415,000 deaths. The non-specific symptoms of lymphoma and leukemia often delay diagnosis. We investigated the circulating levels of intact nucleosomes containing the histone H3.1 isoform (Nu.Q-H3.1) in a variety of solid tumors, NHL, AML, ALL, and in healthy subjects. Methods: We measured levels of Nu.Q-H3.1 in plasma samples taken from 62 healthy volunteers (mean age = 45 yrs) and 329 patients diagnosed with cancer (mean age = 56 yrs), including 25 patients diagnosed with each of cancer of the bladder, bone, brain, oesophagus, cervix, skin, head & neck or melanoma, 21 patients diagnosed with uterine cancer as well as with NHL (n = 25), AML (n = 25) and ALL (n = 8). The cohort included samples taken from patients at diagnosis and at relapse. Whole blood samples were collected in EDTA plasma tubes, double-centrifuged at 1500 rcf for 15 minutes within 2 hrs of blood draw, after which plasma was transferred to a cryotube and frozen immediately until analysis. Plasma samples (20µl) were analyzed in duplicate for Nu.Q-H3.1 using an ELISA method developed and validated to CLSI guidelines. Results: We observed elevated levels of Nu.Q-H3.1 in the patients diagnosed with various cancers. Only 14 of 271 patients with a solid tumor had levels > 200ng/ml. In contrast the median Nu.Q-H3.1 levels observed for NHL, AML and ALL were 276, 284 and 585ng/ml respectively. The median nucleosome level in 62 healthy subjects was 40ng/ml; the highest level was 198ng/ml. The AUC for all patients diagnosed with NHL, AML or ALL (n = 54) vs healthy volunteers was 91% with a sensitivity of 74% at 95% specificity. The AUC for the subset of patients newly diagnosed with NHL, AML or ALL (n = 31) vs healthy volunteers was 92% with a sensitivity of 81% at 95% specificity. Conclusions: Elevated nucleosome levels have been reported for a number of diseases. Our early results indicate that levels of Nu.Q-H3.1 are particularly elevated in haematological malignancies and may be a useful diagnostic tool warranting further study.

A controlled regulation of mitochondrial mass through either the production (biogenesis) or the degradation (mitochondrial quality control) of the organelle represents a crucial step for proper mitochondrial and cell function. Key steps of mitochondrial biogenesis and quality control are overviewed, with an emphasis on the role of mitochondrial chaperones and proteases that keep mitochondria fully functional, provided the mitochondrial activity impairment is not excessive. In this case, the whole organelle is degraded by mitochondrial autophagy or "mitophagy". Beside the maintenance of adequate mitochondrial abundance and functions for cell homeostasis, mitochondrial biogenesis might be enhanced, through discussed signalling pathways, in response to various physiological stimuli, like contractile activity, exposure to low temperatures, caloric restriction and stem cells differentiation. In addition, mitochondrial dysfunction might also initiate a retrograde response, enabling cell adaptation through increased mitochondrial biogenesis. Although the examples cited above suggest that the control of mitochondrial mass, through biogenesis or quality control, is beneficial to the cell/organism demands, the data generated from diverse pathologies suggest that modulations in mitochondrial abundance/functions may participate to the pathogenesis. Increased mitochondrial abundance is generally described to characterize mitochondrial myopathies as well as most cancers, and is generally accompanied by qualitative modifications of mitochondria, thereby affecting programmed cells death susceptibility. On the contrary, in ageing, obesity and type 2 diabetes, mitochondrial biogenesis and functions are generally down-regulated. The development of insulin resistance, favoured by an impaired mitochondrial function, tends to reduce the abundance of the organelle, through a vicious circle. J. Cell. Physiol. © 2011 Wiley-Liss, Inc.

Mutations in the mitochondrial DNA can lead to the development of mitochondrial diseases such as Myoclonic Epilepsy with Ragged Red Fibers (MERRF) or Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS). We first show that human 143B-derived cybrid cells harboring either the A8344G (MERRF) or the A3243G (MELAS) mutation, are more prone to undergo apoptosis then their wild-type counterpart, when challenged with various apoptotic inducers such as staurosporine, etoposide and TRAIL. In addition, investigating the mechanisms underlying A8344G cybrid cells hypersensitivity to staurosporine-induced cell death, we found that staurosporine treatment activates caspases independently of cytochrome c release in both wild-type and mutated cells. Caspases are activated, at least partly, through the activation of calcium-dependent calpain proteases, a pathway that is more strongly activated in mutated cybrid cells than in wild-type cells exposed to staurosporine. These results suggest that calcium homeostasis perturbation induced by mitochondrial dysfunction could predispose cells to apoptosis, a process that could take part into the progressive cell degeneration observed in MERRF syndrome, and more generally in mitochondrial diseases.

Hepatocellular carcinoma is one of the most frequent tumor types worldwide and oxidative stress represents a major risk factor in pathogenesis of liver diseases leading to HCC. Nuclear factor erythroid 2-related factor (Nrf2) is a transcription factor activated by oxidative stress that governs the expression of many genes which constitute the antioxidant defenses of the cell. In addition, oxidative stress activates AMP-activated protein kinase (AMPK), which has emerged in recent years as a kinase that controls the redox-state of the cell. Since both AMPK and Nrf2 are involved in redox homeostasis, we investigated whether there was a crosstalk between the both signaling systems in hepatocarcinoma cells. Here, we demonstrated that AMPK activator AICAR, in contrary to the A769662 allosteric activator, induces Nrf2 activation and concomitantly modulates the basal redox state of the hepatocarcinoma cells. When the expression of Nrf2 is knocked down, AICAR failed to induce its effect on redox state. These data highlight a major role of Nrf2 signaling pathway in mediating the AICAR effect on basal oxidative state. Furthermore, we demonstrated that AICAR metabolization by the cell is required to induce Nrf2 activation while, the silencing of AMPK does not have any effect on Nrf2 activation. This suggests that AICAR-induced Nrf2 activation is independent of AMPK activity. In conclusion, we identified AICAR as a potent modulator of the redox state of human hepatocarcinoma cells, via the Nrf2 signaling pathway and in an AMPK-independent mechanism. © 2014 Elsevier Inc.

We show that mitochondrial DNA (mtDNA)-depleted 143B cells are hypersensitive to staurosporine-induced cell death as evidenced by a more pronounced DNA fragmentation, a stronger activation of caspase-3, an enhanced poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, and a more dramatic cytosolic release of cytochrome c. We also show that B-cell CLL/lymphoma-2 (Bcl-2), B-cell lymphoma extra large (Bcl-XL), and myeloid cell leukemia-1 (Mcl-1) are constitutively less abundant in mtDNA-depleted cells, that the inhibition of Bcl-2 and Bcl-XL can sensitize the parental cell line to staurosporine-induced apoptosis, and that overexpression of Bcl-2 or Bcl-XL can prevent the activation of caspase-3 in ρ0143B cells treated with staurosporine. Moreover, the inactivation of cathepsin B with CA074-Me significantly reduced cytochrome c release, caspase-3 activation, PARP-1 cleavage, and DNA fragmentation in mtDNA-depleted cells, whereas the pan-caspase inhibitor failed to completely prevent PARP-1 cleavage and DNA fragmentation in these cells, suggesting that caspase-independent mechanisms are responsible for cell death even if caspases are activated. Finally, we show that cathepsin B is released in the cytosol of ρ0 cells in response to staurosporine, suggesting that the absence of mitochondrial activity leads to a facilitated permeabilization of lysosomal membranes in response to staurosporine.

Tumor hypoxia is a common characteristic of most solid tumors and is correlated with poor prognosis for patients partly because hypoxia promotes resistance to cancer therapy. Hypoxia selects cancer cells that are resistant to apoptosis and allows the onset of mechanisms that promote cancer cells survival including autophagy. Previously, we showed that human hepatoma HepG2 cells were protected under hypoxia against the etoposide-induced apoptosis. In this study, respective putative contribution of autophagy and BNIP3 in the protection conferred by hypoxia against the etoposide-induced apoptosis was investigated. We report that autophagy is induced by etoposide, a process that is not affected by hypoxic conditions. Using Atg5 siRNA, we show that etoposide-induced autophagy promotes apoptotic cell death under normoxia but not under hypoxia. Then, we investigated whether the hypoxia-induced protein BNIP3 could explain the different effect of autophagy on cell death under hypoxia or normoxia. We show that the silencing of BNIP3 does not affect autophagy whatever the pO2 but participates in the protective effect of hypoxia against etoposide-induced apoptosis. Together, these results suggest that autophagy might be involved in etoposide-induced cell death only under normoxia and that BNIP3 is a major effector of the protective mechanism conferred by hypoxia to protect cancer cells against etoposide-induced apoptotic cell death. © 2010 Elsevier Inc.