The troglitazone derivative EP13 disrupts energy metabolism through respiratory chain complex I inhibition in breast cancer cells and potentiates the antiproliferative effect of glycolysis inhibitors.

BACKGROUND: The metabolism of cancer cells generally differs from that of normal cells. Indeed, most cancer cells have a high rate of glycolysis, even at normal oxygen concentrations. These metabolic properties can potentially be exploited for therapeutic intervention. In this context, we have developed troglitazone derivatives to treat hormone-sensitive and triple-negative breast cancers, which currently lack therapeutic targets, have an aggressive phenotype, and often have a worse prognosis than other subtypes. Here, we studied the metabolic impact of the EP13 compound, a desulfured derivative of Δ2-troglitazone that we synthetized and is more potent than its parent compounds. METHODS: EP13 was tested on two triple-negative breast cancer cell lines, MDA-MB-231 and Hs578T, and on the luminal cell line MCF-7. The oxygen consumption rate (OCR) of the treated cell lines, Hs578T mammospheres and isolated mitochondria was measured using the XFe24 Seahorse analyser. ROS production was quantified using the MitoSOX fluorescent probe. Glycolytic activity was evaluated through measurement of the extracellular acidification rate (ECAR), glucose consumption and lactate production in extracellular medium. The synergistic effect of EP13 with glycolysis inhibitors (oxamate and 2-deoxyglucose) on cell cytotoxicity was established using the Chou-Talalay method. RESULTS: After exposure to EP13, we observed a decrease in the mitochondrial oxygen consumption rate in MCF7, MDA-MB-231 and Hs578T cells. EP13 also modified the maximal OCR of Hs578T spheroids. EP13 reduced the OCR through inhibition of respiratory chain complex I. After 24 h, ATP levels in EP13-treated cells were not altered compared with those in untreated cells, suggesting compensation by glycolysis activity, as shown by the increase in ECAR, the glucose consumption and lactate production. Finally, we performed co-treatments with EP13 and glycolysis inhibitors (oxamate and 2-DG) and observed that EP13 potentiated their cytotoxic effects. CONCLUSION: This study demonstrates that EP13 inhibits OXPHOS in breast cancer cells and potentiates the effect of glycolysis inhibitors.

Deciphering genetic and nongenetic factors underlying tumour dormancy: insights from multiomics analysis of two syngeneic MRD models of melanoma and leukemia.

BACKGROUND: Tumour dormancy, a resistance mechanism employed by cancer cells, is a significant challenge in cancer treatment, contributing to minimal residual disease (MRD) and potential relapse. Despite its clinical importance, the mechanisms underlying tumour dormancy and MRD remain unclear. In this study, we employed two syngeneic murine models of myeloid leukemia and melanoma to investigate the genetic, epigenetic, transcriptomic and protein signatures associated with tumour dormancy. We used a multiomics approach to elucidate the molecular mechanisms driving MRD and identify potential therapeutic targets. RESULTS: We conducted an in-depth omics analysis encompassing whole-exome sequencing (WES), copy number variation (CNV) analysis, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome and proteome investigations. WES analysis revealed a modest overlap of gene mutations between melanoma and leukemia dormancy models, with a significant number of mutated genes found exclusively in dormant cells. These exclusive genetic signatures suggest selective pressure during MRD, potentially conferring resistance to the microenvironment or therapies. CNV, histone marks and transcriptomic gene expression signatures combined with Gene Ontology (GO) enrichment analysis highlighted the potential functional roles of the mutated genes, providing insights into the pathways associated with MRD. In addition, we compared "murine MRD genes" profiles to the corresponding human disease through public datasets and highlighted common features according to disease progression. Proteomic analysis combined with multi-omics genetic investigations, revealed a dysregulated proteins signature in dormant cells with minimal genetic mechanism involvement. Pathway enrichment analysis revealed the metabolic, differentiation and cytoskeletal remodeling processes involved in MRD. Finally, we identified 11 common proteins differentially expressed in dormant cells from both pathologies. CONCLUSIONS: Our study underscores the complexity of tumour dormancy, implicating both genetic and nongenetic factors. By comparing genomic, transcriptomic, proteomic, and epigenomic datasets, our study provides a comprehensive understanding of the molecular landscape of minimal residual disease. These results provide a robust foundation for forthcoming investigations and offer potential avenues for the advancement of targeted MRD therapies in leukemia and melanoma patients, emphasizing the importance of considering both genetic and nongenetic factors in treatment strategies.

p65/RelA NF-κB fragments generated by RIPK3 activity regulate tumorigenicity, cell metabolism, and stemness characteristics.

Receptor-interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces caspase-mediated p65/RelA cleavage, resulting in N-terminal 1-361 and C-terminal 362-549 fragments. We show here that a noncleavable p65/RelA D361E mutant expressed in DA1-3b leukemia cells decreases mouse survival times and that coexpression of p65/RelA fragments increases the tumorigenicity of B16F1 melanoma cells. This aggressiveness in vivo did not correlate with NF-κB activity measured in vitro. The fragments and p65/RelA D361E mutant induced different expression profiles in DA1-3b and B16F1 cells. Stemness markers were affected: p65/RelA D361E increased ALDH activity in DA1-3b cells, and fragment expression increased melanoma sphere formation in B16/F1 cells. p65/RelA fragments and the D361E noncleavable mutant decreased oxidative or glycolytic cell metabolism, with differences observed between models. Thus, p65/RelA cleavage initiated by kinase-independent RIPK3 activity in cancer cells is not neutral and induces pleiotropic effects in vitro and in vivo that may vary across tumor types.

Mitochondrial spare respiratory capacity: Mechanisms, regulation, and significance in non-transformed and cancer cells.

Mitochondrial metabolism must constantly adapt to stress conditions in order to maintain bioenergetic levels related to cellular functions. This absence of proper adaptation can be seen in a wide array of conditions, including cancer. Metabolic adaptation calls on mitochondrial function and draws on the mitochondrial reserve to meet increasing needs. Among mitochondrial respiratory parameters, the spare respiratory capacity (SRC) represents a particularly robust functional parameter to evaluate mitochondrial reserve. We provide an overview of potential SRC mechanisms and regulation with a focus on its particular significance in cancer cells.

A New Strategy to Preserve and Assess Oxygen Consumption in Murine Tissues.

Mitochondrial dysfunctions are implicated in several pathologies, such as metabolic, cardiovascular, respiratory, and neurological diseases, as well as in cancer and aging. These metabolic alterations are usually assessed in human or murine samples by mitochondrial respiratory chain enzymatic assays, by measuring the oxygen consumption of intact mitochondria isolated from tissues, or from cells obtained after physical or enzymatic disruption of the tissues. However, these methodologies do not maintain tissue multicellular organization and cell-cell interactions, known to influence mitochondrial metabolism. Here, we develop an optimal model to measure mitochondrial oxygen consumption in heart and lung tissue samples using the XF24 Extracellular Flux Analyzer (Seahorse) and discuss the advantages and limitations of this technological approach. Our results demonstrate that tissue organization, as well as mitochondrial ultrastructure and respiratory function, are preserved in heart and lung tissues freshly processed or after overnight conservation at 4 °C. Using this method, we confirmed the repeatedly reported obesity-associated mitochondrial dysfunction in the heart and extended it to the lungs. We set up and validated a new strategy to optimally assess mitochondrial function in murine tissues. As such, this method is of great potential interest for monitoring mitochondrial function in cohort samples.

First-line Screening of OXPHOS Deficiencies Using Microscale Oxygraphy in Human Skin Fibroblasts: A Preliminary Study.

The diagnosis of mitochondrial diseases is a real challenge because of the vast clinical and genetic heterogeneity. Classically, the clinical examination and genetic analysis must be completed by several biochemical assays to confirm the diagnosis of mitochondrial disease. Here, we tested the validity of microscale XF technology in measuring oxygen consumption in human skin fibroblasts isolated from 5 pediatric patients with heterogeneous mitochondrial disorders. We first set up the protocol conditions to allow the determination of respiratory parameters including respiration associated with ATP production, proton leak, maximal respiration, and spare respiratory capacity with reproducibility and repeatability. Maximum respiration and spare capacity were the only parameters decreased in patients irrespective of the type of OXPHOS deficiency. These results were confirmed by high-resolution oxygraphy, the reference method to measure cellular respiration. Given the fact that microscale XF technology allows fast, automated and standardized measurements, we propose to use microscale oxygraphy among the first-line methods to screen OXPHOS deficiencies.

Melanoma metabolism contributes to the cellular responses to MAPK/ERK pathway inhibitors.

BACKGROUND: Besides its influence on survival, growth, proliferation, invasion and metastasis, cancer cell metabolism also greatly influences the cellular responses to molecular-targeted therapies. SCOPE OF THE REVIEW: To review the recent advances in elucidating the metabolic effects of BRAF and MEK inhibitors (clinical inhibitors of the MAPK/ERK pathway) in melanoma and discuss the underlying mechanisms involved in the way metabolism can influence melanoma cell death and resistance to BRAF and MEK inhibitors. We also underlined the therapeutic perspectives in terms of innovative drug combinations. MAJOR CONCLUSION: BRAF and MEK inhibitors inhibit aerobic glycolysis and induce high levels of metabolic stress leading to effective cell death by apoptosis in BRAF-mutated cancer cells. An increase in mitochondrial metabolism is required to survive to MAPK/ERK pathway inhibitors and the sub-population of cells that survives to these inhibitors are characterized by mitochondrial OXPHOS phenotype. Consequently, mitochondrial inhibition could be combined with oncogenic "drivers" inhibitors of the MAPK/ERK pathway for improving the efficacy of molecular-targeted therapy. GENERAL SIGNIFICANCE: Metabolism is a key component of the melanoma response to BRAF and/or MEK inhibitors. Mitochondrial targeting may offer novel therapeutic approaches to overwhelm the mitochondrial addiction that limits the efficacy of BRAF and/or MEK inhibitors. These therapeutic approaches might be quickly applicable to the clinical situation.

GILZ overexpression attenuates endoplasmic reticulum stress-mediated cell death via the activation of mitochondrial oxidative phosphorylation.

The Glucocorticoïd-induced leucine zipper (GILZ) protein has profound anti-inflammatory activities in haematopoietic cells. GILZ regulates numerous signal transduction pathways involved in proliferation and survival of normal and neoplastic cells. Here, we have demonstrated the potential of GILZ in alleviating apoptosis induced by ER stress inducers. Whereas the glucocorticoid, dexamethasone, protects from tunicamycin-induced cell death, silencing endogeneous GILZ in dexamethasone-treated cancer cells alter the capacity of glucocorticoids to protect from tunicamycin-mediated apoptosis. Under ER stress conditions, overexpression of GILZ significantly reduced activation of mitochondrial pathway of apoptosis by maintaining Bcl-xl level. GILZ protein affects the UPR signaling shifting the balance towards pro-survival signals as judged by down-regulation of CHOP, ATF4, XBP1s mRNA and increase in GRP78 protein level. Interestingly, GILZ sustains high mitochondrial OXPHOS during ER stress and cytoprotection mediated by GILZ is abolished in cells depleted of mitochondrial DNA, which are OXPHOS-deficient. These findings reveal a new role of GILZ, which acts as a cytoprotector against ER stress through a pathway involving mitochondrial OXPHOS.

Cigarette smoke alters the ability of human dendritic cells to promote anti-Streptococcus pneumoniae Th17 response.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation and impaired immune response to pathogens leading to bacteria-induced exacerbation of the disease. A defect in Th17 cytokines in response to Streptococcus pneumoniae, a bacteria associated with COPD exacerbations, has been recently reported. Dendritic cells (DC) are professional antigen presenting cells that drive T-cells differentiation and activation. In this study, we hypothesized that exposure to cigarette smoke, the main risk factor of COPD, might altered the pro-Th17 response to S. pneumoniae in COPD patients and human DC. METHODS: Pro-Th1 and -Th17 cytokine production by peripheral blood mononuclear cells (PBMC) from COPD patients was analyzed and compared to those from smokers and non-smokers healthy subjects. The effect of cigarette smoke extract (CSE) was analyzed on human monocyte-derived DC (MDDC) from controls exposed or not to S. pneumoniae. Bacteria endocytosis, maturation of MDDC and secretion of cytokines were assessed by flow cytometry and ELISA, respectively. Implication of the oxidative stress was analyzed by addition of antioxidants and mitochondria inhibitors. In parallel, MDDC were cocultured with autologous T-cells to analyze the consequence on Th1 and Th17 cytokine production. RESULTS: PBMC from COPD patients exhibited defective production of IL-1ß, IL-6, IL-12 and IL-23 to S. pneumoniae compared to healthy subjects and smokers. CSE significantly reduced S. pneumoniae-induced MDDC maturation, secretion of pro-Th1 and -Th17 cytokines and activation of Th1 and Th17 T-cell responses. CSE exposure was also associated with sustained CXCL8 secretion, bacteria endocytosis and mitochondrial oxidative stress. Antioxidants did not reverse these effects. Inhibitors of mitochondrial electron transport chain partly reproduced inhibition of S. pneumoniae-induced MDDC maturation but had no effect on cytokine secretion and T cell activation. CONCLUSIONS: We observed a defective pro-Th1 and -Th17 response to bacteria in COPD patients. CSE exposure was associated with an inhibition of DC capacity to activate antigen specific T-cell response, an effect that seems to be not only related to oxidative stress. These results suggest that new therapeutics boosting this response in DC may be helpful to improve treatment of COPD exacerbations.

Another facet to the anticancer response to lamellarin D: induction of cellular senescence through inhibition of topoisomerase I and intracellular Ros production.

Lamellarin D (LamD) is a marine alkaloid with broad spectrum antitumor activities. Multiple intracellular targets of LamD, which affect cancer cell growth and induce apoptosis, have been identified. These include nuclear topoisomerase I, relevant kinases (such as cyclin-dependent kinase 2) and the mitochondrial electron transport chain. While we have previously demonstrated that LamD at micromolar range deploys strong cytotoxicity by inducing mitochondrial apoptosis, mechanisms of its cytostatic effect have not yet been characterized. Here, we demonstrated that induction of cellular senescence (depicted by cell cycle arrest in G2 associated with ß-galactosidase activity) is a common response to subtoxic concentrations of LamD. Cellular senescence is observed in a large panel of cancer cells following in vitro or in vivo exposure to LamD. The onset of cellular senescence is dependent on the presence of intact topoisomerase I since topoisomerase I-mutated cells are resistant to senescence induced by LamD. LamD-induced senescence occurs without important loss of telomere integrity. Instead, incubation with LamD results in the production of intracellular reactive oxygen species (ROS), which are critical for senescence as demonstrated by the inhibitory effect of antioxidants. In addition, cancer cells lacking mitochondrial DNA also exhibit cellular senescence upon LamD exposure indicating that LamD can trigger senescence, unlike apoptosis, in the absence of functional mitochondria. Overall, our results identify senescence-associated growth arrest as a powerful effect of LamD and add compelling evidence for the pharmacological interest of lamellarins as potential anticancer agents.

Implication of Rac1 GTPase in molecular and cellular mitochondrial functions.

Rac1 is a member of the Rho GTPase family which plays major roles in cell mobility, polarity and migration, as a fundamental regulator of actin cytoskeleton. Signal transduction by Rac1 occurs through interaction with multiple effector proteins, and its activity is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The small protein is mainly anchored to the inner side of the plasma membrane but it can be found in endocellular compartments, notably endosomes and cell nuclei. The protein localizes also into mitochondria where it contributes to the regulation of mitochondrial dynamics, including both mitobiogenesis and mitophagy, in addition to signaling processes via different protein partners, such as the proapoptotic protein Bcl-2 and chaperone sigma-1 receptor (σ-1R). The mitochondrial form of Rac1 (mtRac1) has been understudied thus far, but it is as essential as the nuclear or plasma membrane forms, via its implication in regulation of oxidative stress and DNA damages. Rac1 is subject to diverse post-translational modifications, notably to a geranylgeranylation which contributes importantly to its mitochondrial import and its anchorage to mitochondrial membranes. In addition, Rac1 contributes to the mitochondrial translocation of other proteins, such as p53. The mitochondrial localization and functions of Rac1 are discussed here, notably in the context of human diseases such as cancers. Inhibitors of Rac1 have been identified (NSC-23766, EHT-1864) and some are being developed for the treatment of cancer (MBQ-167) or central nervous system diseases (JK-50561). Their effects on mtRac1 warrant further investigations. An overview of mtRac1 is provided here.

MYC dependency in GLS1 and NAMPT is a therapeutic vulnerability in multiple myeloma.

Multiple myeloma (MM) is an incurable hematological malignancy in which MYC alterations contribute to the malignant phenotype. Nevertheless, MYC lacks therapeutic druggability. Here, we leveraged large-scale loss-of-function screens and conducted a small molecule screen to identify genes and pathways with enhanced essentiality correlated with MYC expression. We reported a specific gene dependency in glutaminase (GLS1), essential for the viability and proliferation of MYC overexpressing cells. Conversely, the analysis of isogenic models, as well as cell lines dataset (CCLE) and patient datasets, revealed GLS1 as a non-oncogenic dependency in MYC-driven cells. We functionally delineated the differential modulation of glutamine to maintain mitochondrial function and cellular biosynthesis in MYC overexpressing cells. Furthermore, we observed that pharmaceutical inhibition of NAMPT selectively affects MYC upregulated cells. We demonstrate the effectiveness of combining GLS1 and NAMPT inhibitors, suggesting that targeting glutaminolysis and NAD synthesis may be a promising strategy to target MYC-driven MM.

NOD2 in monocytes negatively regulates macrophage development through TNFalpha.

Objective: It is believed that intestinal recruitment of monocytes from Crohn's Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes. Design: The monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs). Results: We observed a decrease in the frequency of mo-DCs in the colon of Nod2-deficient mice, despite a similar abundance of monocytes. This decrease was independent of the changes in the gut microbiota and dysbiosis caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted in a Nod2-deficient mixed bone marrow (BM) chimera. The use of pharmacological inhibitors revealed that activation of NOD2 during monocyte-derived cell development, dominantly inhibits mTOR-mediated macrophage differentiation in a TNFα-dependent manner. These observations were supported by the identification of a TNFα-dependent response to muramyl dipeptide (MDP) that is specifically lost when CD14-expressing blood cells bear a frameshift mutation in NOD2. Conclusion: NOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD.

Mitochondrial adaptation decreases drug sensitivity of persistent triple negative breast cancer cells surviving combinatory and sequential chemotherapy.

Triple negative breast cancer (TNBC) is an aggressive malignancy for which chemotherapy remains the standard treatment. However, between 3 and 5 years after chemotherapy, about half patients will relapse and it is essential to identify vulnerabilities of cancer cells surviving neoadujuvant therapy. In this study, we established persistent TNBC cell models after treating MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide, and then with paclitaxel, for a total of 18 weeks. The resulting chemo-persistent cell lines were more proliferative, both in vitro and in xenografted mice. Interestingly, MDA-MB-231 persistent cells became less sensitive to chemotherapeutic drugs, whereas SUM159-PT persistent cells kept similar sensitivity compared to control cells. The reduced sensitivity to chemotherapy in MDA-MB-231 persistent cells was found to be associated with an increased oxidative phosphorylation (OXPHOS) and modified levels of tricarboxylic acid cycle (TCA) intermediates. Integration of data from proteomics and metabolomics demonstrated TCA cycle among the most upregulated pathways in MDA-MB-231 persistent cells. The absence of glucose and pyruvate impeded OXPHOS in persistent cells, while the absence of glutamine did not. In contrast, OXPHOS was not modified in control cells independently of TCA substrates, indicating that MDA-MB-231 persistent cells evolved towards a more pyruvate dependent profile. Finally, the inhibition of pyruvate entry into mitochondria with UK-5099 reduced OXPHOS and re-sensitized persistent cells to therapeutic agents. Together, these findings suggest that targeting mitochondrial pyruvate metabolism may help to overcome mitochondrial adaptation of chemo-persistent TNBC.

Extracellular vesicles derived from nasopharyngeal carcinoma induce the emergence of mature regulatory dendritic cells using a galectin-9 dependent mechanism.

Nasopharyngeal carcinoma-derived small extracellular vesicles (NPCSEVs) have an immunosuppressive impact on the tumour microenvironment. In this study, we investigated their influence on the generation of tolerogenic dendritic cells and the potential involvement of the galectin-9 (Gal9) they carry in this process. We analysed the phenotype and immunosuppressive properties of NPCSEVs and explored the ability of DCs exposed to NPCSEVs (NPCSEV-DCs) to regulate T cell proliferation. To assess their impact at the pathophysiological level, we performed real-time fluorescent chemoattraction assays. Finally, we analysed phenotype and immunosuppressive functions of NPCSEV-DCs using a proprietary anti-Gal9 neutralising antibody to assess the role of Gal9 in this effect. We described that NPCSEV-DCs were able to inhibit T cell proliferation despite their mature phenotype. These mature regulatory DCs (mregDCs) have a specific oxidative metabolism and secrete high levels of IL-4. Chemoattraction assays revealed that NPCSEVs could preferentially recruit NPCSEV-DCs. Finally, and very interestingly, the reduction of the immunosuppressive function of NPCSEV-DCs using an anti-Gal9 antibody clearly suggested an important role for vesicular Gal9 in the induction of mregDCs. These results revealed for the first time that NPCSEVs promote the emergence of mregDCs using a galectin-9 dependent mechanism and open new perspectives for antitumour immunotherapy targeting NPCSEVs.

Regulation by survivin of cancer cell death induced by F14512, a polyamine-containing inhibitor of DNA topoisomerase II.

F14512, an epipodophyllotoxin derivative equipped with a spermine moiety, is selectively taken up by the polyamine transport system over-active in tumor cells. F14512 was identified as a selective anticancer agent with a broad spectrum of antitumor activities and is currently undergoing phase I clinical trial in onco-hematology. However, the mechanism by which F14512 exerts its selective effects on neoplastic cells remains poorly understood. In this study, using mainly P388 leukemia cells, we showed that activation of the DNA damage response by F14512 did not induce immediate apoptosis but resulted in an early growth arrest. F14512-induced G2 arrest was accompanied by the appearance of a senescence-like phenotype (characterized by an increased ß-galactosidase staining) with up-regulation of the cyclin-dependent kinase inhibitor p16, and cyclin D1. The early senescence-based cell cycle block was characterized by a marked increase of the level of the IAP protein survivin, but not cIAP2, in P388 cells as well as in three other leukemia and melanoma cell types. The Thr(34)-phosphorylated form of survivin was observed within 4 h after F14512 exposure. Inhibition of survivin by siRNA resulted in a switch from senescence-like growth arrest to apoptosis. Compared with the parental drug etoposide, F14512-induced DNA damage signaling pathway resulted in greater senescence like-growth arrest and delayed apoptosis. Collectively, our data show that senescence arrest and subsequent apoptosis are powerful mechanisms mediating the chemotherapeutic effects of F14512 and identify survivin as the molecular determinant responsible for a qualitative shift in cell fate from senescence to apoptosis upon treatment with F14512.

Comparison of the in vivo genotoxicity of electronic and conventional cigarettes aerosols after subacute, subchronic and chronic exposures.

Tobacco smoking is classified as a human carcinogen. A wide variety of new products, in particular electronic cigarettes (e-cigs), have recently appeared on the market as an alternative to smoking. Although the in vitro toxicity of e-cigs is relatively well known, there is currently a lack of data on their long-term health effects. In this context, the aim of our study was to compare, on a mouse model and using a nose-only exposure system, the in vivo genotoxic and mutagenic potential of e-cig aerosols tested at two power settings (18 W and 30 W) and conventional cigarette (3R4F) smoke. The standard comet assay, micronucleus test and Pig-a gene mutation assay were performed after subacute (4 days), subchronic (3 months) and chronic (6 months) exposure. The generation of oxidative stress was also assessed by measuring the 8-hydroxy-2'-deoxyguanosine and by using the hOGG1-modified comet assay. Our results show that only the high-power e-cig and the 3R4F cigarette induced oxidative DNA damage in the lung and the liver of exposed mice. In return, no significant increase in chromosomal aberrations or gene mutations were noted whatever the type of product. This study demonstrates that e-cigs, at high-power setting, should be considered, contrary to popular belief, as hazardous products in terms of genotoxicity in mouse model.

Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I.

SMIP004-7 is a small molecule inhibitor of mitochondrial respiration with selective in vivo anti-cancer activity through an as-yet unknown molecular target. We demonstrate here that SMIP004-7 targets drug-resistant cancer cells with stem-like features by inhibiting mitochondrial respiration complex I (NADH:ubiquinone oxidoreductase, complex I [CI]). Instead of affecting the quinone-binding site targeted by most CI inhibitors, SMIP004-7 and its cytochrome P450-dependent activated metabolite(s) have an uncompetitive mechanism of inhibition involving a distinct N-terminal region of catalytic subunit NDUFS2 that leads to rapid disassembly of CI. SMIP004-7 and an improved chemical analog selectively engage NDUFS2 in vivo to inhibit the growth of triple-negative breast cancer transplants, a response mediated at least in part by boosting CD4+ and CD8+ T cell-mediated immune surveillance. Thus, SMIP004-7 defines an emerging class of ubiquinone uncompetitive CI inhibitors for cell autonomous and microenvironmental metabolic targeting of mitochondrial respiration in cancer.

Antimetabolic cooperativity with the clinically approved l-asparaginase and tyrosine kinase inhibitors to eradicate CML stem cells.

OBJECTIVE: Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS: Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS: Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION: Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.

TRPC3 shapes the ER-mitochondria Ca2+ transfer characterizing tumour-promoting senescence.

Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca2+ load via negative regulation of IP3 receptor-mediated Ca2+ release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.