Critical role of Aquaporin-1 and telocytes in infantile hemangioma response to propranolol beta blockade.

Propranolol, a nonselective ß-adrenergic receptor (ADRB) antagonist, is the first-line therapy for severe infantile hemangiomas (IH). Since the incidental discovery of propranolol efficacy in IH, preclinical and clinical investigations have shown evidence of adjuvant propranolol response in some malignant tumors. However, the mechanism for propranolol antitumor effect is still largely unknown, owing to the absence of a tumor model responsive to propranolol at nontoxic concentrations. Immunodeficient mice engrafted with different human tumor cell lines were treated with anti-VEGF bevacizumab to create a model sensitive to propranolol. Proteomics analysis was used to reveal propranolol-mediated protein alteration correlating with tumor growth inhibition, and Aquaporin-1 (AQP1), a water channel modulated in tumor cell migration and invasion, was identified. IH tissues and cells were then functionally investigated. Our functional protein association networks analysis and knockdown of ADRB2 and AQP1 indicated that propranolol treatment and AQP1 down-regulation trigger the same pathway, suggesting that AQP1 is a major driver of beta-blocker antitumor response. Examining AQP1 in human hemangioma samples, we found it exclusively in a perivascular layer, so far unrecognized in IH, made of telocytes (TCs). Functional in vitro studies showed that AQP1-positive TCs play a critical role in IH response to propranolol and that modulation of AQP1 in IH-TC by propranolol or shAQP1 decreases capillary-like tube formation in a Matrigel-based angiogenesis assay. We conclude that IH sensitivity to propranolol may rely, at least in part, on a cross talk between lesional vascular cells and stromal TCs.

Xenopus: An in vivo model for studying skin response to ultraviolet B irradiation.

Ultraviolet B (UVB) in sunlight cause skin damage, ranging from wrinkles to photoaging and skin cancer. UVB can affect genomic DNA by creating cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidine (6-4) photoproducts (6-4PPs). These lesions are mainly repaired by the nucleotide excision repair (NER) system and by photolyase enzymes that are activated by blue light. Our main goal was to validate the use of Xenopus laevis as an in vivo model system for investigating the impact of UVB on skin physiology. The mRNA expression levels of xpc and six other genes of the NER system and CPD/6-4PP photolyases were found at all stages of embryonic development and in all adult tissues tested. When examining Xenopus embryos at different time points after UVB irradiation, we observed a gradual decrease in CPD levels and an increased number of apoptotic cells, together with an epidermal thickening and an increased dendricity of melanocytes. We observed a quick removal of CPDs when embryos are exposed to blue light versus in the dark, confirming the efficient activation of photolyases. A decrease in the number of apoptotic cells and an accelerated return to normal proliferation rate was noted in blue light-exposed embryos compared with their control counterparts. Overall, a gradual decrease in CPD levels, detection of apoptotic cells, thickening of epidermis, and increased dendricity of melanocytes, emulate human skin responses to UVB and support Xenopus as an appropriate and alternative model for such studies.

Is Infantile Hemangioma a Neuroendocrine Tumor?

Infantile hemangioma (IH) is the most common infantile tumor, affecting 5-10% of newborns. Propranolol, a nonselective ß-adrenergic receptor (ADRB) antagonist, is currently the first-line treatment for severe IH; however, both its mechanism of action and its main cellular target remain poorly understood. Since betablockers can antagonize the effect of natural ADRB agonists, we postulated that the catecholamine produced in situ in IH may have a role in the propranolol response. By quantifying catecholamines in the IH tissues, we found a higher amount of noradrenaline (NA) in untreated proliferative IHs than in involuted IHs or propranolol-treated IHs. We further found that the first three enzymes of the catecholamine biosynthesis pathway are expressed by IH cells and that their levels are reduced in propranolol-treated tumors. To study the role of NA in the pathophysiology of IH and its response to propranolol, we performed an in vitro angiogenesis assay in which IH-derived endothelial cells, pericytes and/or telocytes were incorporated. The results showed that the total tube formation is sensitive to propranolol only when exogenous NA is added in the three-cell model. We conclude that the IH's sensitivity to propranolol depends on crosstalk between the endothelial cells, pericytes and telocytes in the context of a high local amount of local NA.

Leukemia inhibitory factor signaling in Xenopus embryo: Insights from gain of function analysis and dominant negative mutant of the receptor.

Leukemia inhibitory factor (LIF) is a cytokine member of the interleukin 6 family (IL6) of cytokines. It signals through a heterodimer receptor complex that consists of the LIF receptor (or LIFR formerly known as gp190) and the Interleukin 6 signal transducer (or IL6ST formerly known as gp130). LIF signaling is mediated mainly by signal transducer and activator of transcription 3 (STAT3) and has a wide variety of biological activities with pleiotropic effects on many cell types and organs among which are stem cell renewal and implantation process in mammalian embryo. Despite the wealth of data on LIF in mammalian cells, there is a paucity of information on its functions in lower vertebrates. Here, we provide information on the status and the function of LIF signaling in Xenopus amphibian. The IL6 cytokine family is highly conserved in Xenopus genome both at ligands and receptors levels. All cytokines and receptors of the family, except oncostatin M (OSM) and IL27, can be identified in the genome including the orthologs of LIF, cardiotrophin 1 (CTF1), ciliary neurotrophic factor (CNTF), cardiotrophin like cytokine factor 1 (CLCF1), LIFR, IL6ST, IL6R, IL11RA and CNTFR. Lif mRNA is zygotically expressed after midblastula transition while lifr and il6st are maternally expressed. We have investigated the functions of LIF in Xenopus early development with a gain-of-function analysis combined to the use of a dominant negative form of the receptor. The overexpression of Xenopus lif in embryo activates STAT3 phosphorylation and induces a dramatic phenotype where embryos are ventralised and show a reduction of anterior structures with microcephaly. This results mainly from BMP signal stimulation and antagonism towards IGF signals. In addition, most embryos develop tumor-like cell masses according to both autonomous and non-autonomous processes. Through the use of a dominant negative form of the receptor, we demonstrate for the first time that a functional LIF signaling is required for normal vertebrate kidney development. Owing to its experimental advantages, the Xenopus embryo constitutes a useful model to identify the molecular actors that may account for the pleiotropic functions of LIF and their role in vertebrate development.

Energy metabolism in skin cancers: A therapeutic perspective.

Skin cancers are the most common cancers worldwide. The incidence of common skin cancers, including basal cell carcinomas (BCCs), squamous cell carcinomas (SCCs) and melanomas, continues to rise by 5 to 7% per year, mainly due to ultraviolet (UV) exposure and partly because of aging. This suggests an urgent necessity to improve the level of prevention and protection for skin cancers as well as developing new prognostic and diagnostic markers of skin cancers. Moreover, despite innovative therapies especially in the fields of melanoma and carcinomas, new therapeutic options are needed to bypass resistance to targeted therapies or treatment's side effects. Since reprogramming of cellular metabolism is now considered as a hallmark of cancer, some of the recent findings on the role of energy metabolism in skin cancer initiation and progression as well as its effect on the response to targeted therapies are discussed in this review. This article is part of a Special Issue entitled Mitochondria in cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

Delving into the Metabolism of Sézary Cells: A Brief Review.

Primary cutaneous lymphomas (PCLs) are a heterogeneous group of lymphoproliferative disorders caused by the accumulation of neoplastic T or B lymphocytes in the skin. Sézary syndrome (SS) is an aggressive and rare form of cutaneous T cell lymphoma (CTCL) characterized by an erythroderma and the presence of atypical cerebriform T cells named Sézary cells in skin and blood. Most of the available treatments for SS are not curative, which means there is an urgent need for the development of novel efficient therapies. Recently, targeting cancer metabolism has emerged as a promising strategy for cancer therapy. This is due to the accumulating evidence that metabolic reprogramming highly contributes to tumor progression. Genes play a pivotal role in regulating metabolic processes, and alterations in these genes can disrupt the delicate balance of metabolic pathways, potentially contributing to cancer development. In this review, we discuss the importance of targeting energy metabolism in tumors and the currently available data on the metabolism of Sézary cells, paving the way for potential new therapeutic approaches aiming to improve clinical outcomes for patients suffering from SS.

Xenopus as a model system for studying pigmentation and pigmentary disorders.

Human pigmentary disorders encompass a broad spectrum of phenotypic changes arising from disruptions in various stages of melanocyte formation, the melanogenesis process, or the transfer of pigment from melanocytes to keratinocytes. A large number of pigmentation genes associated with pigmentary disorders have been identified, many of them awaiting in vivo confirmation. A more comprehensive understanding of the molecular basis of pigmentary disorders requires a vertebrate animal model where changes in pigmentation are easily observable in vivo and can be combined to genomic modifications and gain/loss-of-function tools. Here we present the amphibian Xenopus with its unique features that fulfill these requirements. Changes in pigmentation are particularly easy to score in Xenopus embryos, allowing whole-organism based phenotypic screening. The development and behavior of Xenopus melanocytes closely mimic those observed in mammals. Interestingly, both Xenopus and mammalian skins exhibit comparable reactions to ultraviolet radiation. This review highlights how Xenopus constitutes an alternative and complementary model to the more commonly used mouse and zebrafish, contributing to the advancement of knowledge in melanocyte cell biology and related diseases.

Loss of epidermal hypoxia-inducible factor-1α accelerates epidermal aging and affects re-epithelialization in human and mouse.

In mouse and human skin, HIF-1α is constitutively expressed in the epidermis, mainly in the basal layer. HIF-1α has been shown to have crucial systemic functions: regulation of kidney erythropoietin production in mice with constitutive HIF-1α epidermal deletion, and hypervascularity following epidermal HIF-1α overexpression. However, its local role in keratinocyte physiology has not been clearly defined. To address the function of HIF-1α in the epidermis, we used the mouse model of HIF-1α knockout targeted to keratinocytes (K14-Cre/Hif1a(flox/flox)). These mice had a delayed skin phenotype characterized by skin atrophy and pruritic inflammation, partly mediated by basement membrane disturbances involving laminin-332 (Ln-332) and integrins. We also investigated the relevance of results of studies in mice to human skin using reconstructed epidermis and showed that HIF-1α knockdown in human keratinocytes impairs the formation of a viable reconstructed epidermis. A diminution of keratinocyte growth potential, following HIF-1α silencing, was associated with a decreased expression of Ln-322 and α6 integrin and ß1 integrin. Overall, these results indicate a role of HIF-1α in skin homeostasis especially during epidermal aging.

The E3 ubiquitin ligase FBXL6 controls the quality of newly synthesized mitochondrial ribosomal proteins.

In mammals, about 99% of mitochondrial proteins are synthesized in the cytosol as precursors that are subsequently imported into the organelle. The mitochondrial health and functions rely on an accurate quality control of these imported proteins. Here, we show that the E3 ubiquitin ligase F box/leucine-rich-repeat protein 6 (FBXL6) regulates the quality of cytosolically translated mitochondrial proteins. Indeed, we found that FBXL6 substrates are newly synthesized mitochondrial ribosomal proteins. This E3 binds to chaperones involved in the folding and trafficking of newly synthesized peptide and to ribosomal-associated quality control proteins. Deletion of these interacting partners is sufficient to hamper interactions between FBXL6 and its substrate. Furthermore, we show that cells lacking FBXL6 fail to degrade specifically mistranslated mitochondrial ribosomal proteins. Finally, showing the role of FBXL6-dependent mechanism, FBXL6-knockout (KO) cells display mitochondrial ribosomal protein aggregations, altered mitochondrial metabolism, and inhibited cell cycle in oxidative conditions.

Cancer cell survival following DNA damage-mediated premature senescence is regulated by mammalian target of rapamycin (mTOR)-dependent Inhibition of sirtuin 1.

DNA-damaging agents can induce premature senescence in cancer cells, which contributes to the static effects of cancer. However, senescent cancer cells may re-enter the cell cycle and lead to tumor relapse. Understanding the mechanisms that control the viability of senescent cells may be helpful in eliminating these cells before they can regrow. Treating human squamous cell carcinoma (SCC) cells with the anti-cancer compounds, resveratrol and doxorubicin, triggered p53-independent premature senescence by invoking oxidative stress-mediated DNA damage. This process involved the mTOR-dependent phosphorylation of SIRT1 at serine 47, resulting in the inhibition of the deacetylase activity of SIRT1. SIRT1 phosphorylation caused concomitant increases in p65/RelA NF-κB acetylation and the expression of an anti-apoptotic Bfl-1/A1. SIRT1 physically interacts with the mTOR-Raptor complex, and a single amino acid substitution in the TOS (TOR signaling) motif in the SIRT1 prevented Ser-47 phosphorylation and Bfl-1/A1 induction. The pharmacologic and genetic inhibition of mTOR, unphosphorylatable S47A, or F474A TOS mutants restored SIRT1 deacetylase activity, blocked Bfl-1/A1 induction, and sensitized prematurely senescent SCC cells for apoptosis. We further show that the treatment of UVB-induced SCCs with doxorubicin transiently stabilized tumor growth but was followed by tumor regrowth upon drug removal in p53(+/-)/SKH-1 mice. The subsequent treatment of stabilized SCCs with rapamycin decreased tumor size and induced caspase-3 activation. These results demonstrate that the inhibition of SIRT1 by mTOR fosters survival of DNA damage-induced prematurely senescent SCC cells via Bfl-1/A1 in the absence of functional p53.

XPC silencing in normal human keratinocytes triggers metabolic alterations through NOX-1 activation-mediated reactive oxygen species.

Cancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the inter-relationships between genomic instability, reactive oxygen species (ROS) generation, and metabolic alterations during neoplastic transformation. We showed that knockdown of XPC (XPC(KD)) in normal human keratinocytes results in metabolism remodeling through NADPH oxidase-1 (NOX-1) activation, which in turn leads to increased ROS levels. While enforcing antioxidant defenses by overexpressing catalase, CuZnSOD, or MnSOD could not block the metabolism remodeling, impaired NOX-1 activation abrogates both alteration in ROS levels and modifications of energy metabolism. As NOX-1 activation is observed in human squamous cell carcinomas (SCCs), the blockade of NOX-1 could be a target for the prevention and the treatment of skin cancers.

Hypoxia-inducible factor-1alpha regulates the expression of nucleotide excision repair proteins in keratinocytes.

The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1alpha (HIF-1alpha) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1alpha increased XPC mRNA expression due to competition between HIF-1alpha and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1alpha protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1alpha also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1alpha is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1alpha downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1alpha in the repair of UVB-induced DNA damage.

XPC multifaceted roles beyond DNA damage repair: p53-dependent and p53-independent functions of XPC in cell fate decisions.

Xeroderma pigmentosum group C protein (XPC) acts as a DNA damage recognition factor for bulky adducts and as an initiator of global genome nucleotide excision repair (GG-NER). Novel insights have shown that the role of XPC is not limited to NER, but is also implicated in DNA damage response (DDR), as well as in cell fate decisions upon stress. Moreover, XPC has a proteolytic role through its interaction with p53 and casp-2S. XPC is also able to determine cellular outcomes through its interaction with downstream proteins, such as p21, ARF, and p16. XPC interactions with effector proteins may drive cells to various fates such as apoptosis, senescence, or tumorigenesis. In this review, we explore XPC's involvement in different molecular pathways in the cell and suggest that XPC can be considered not only as a genomic caretaker and gatekeeper but also as a tumor suppressor and cellular-fate decision maker. These findings envisage that resistance to cell death, induced by DNA-damaging therapeutics, in highly prevalent P53-deficent tumors might be overcome through new therapeutic approaches that aim to activate XPC in these tumors. Moreover, this review encourages care providers to consider XPC status in cancer patients before chemotherapy in order to improve the chances of successful treatment and enhance patients' survival.

Rewiring Lipid Metabolism by Targeting PCSK9 and HMGCR to Treat Liver Cancer.

Alterations in lipid handling are an important hallmark in cancer. Our aim here is to target key metabolic enzymes to reshape the oncogenic lipid metabolism triggering irreversible cell breakdown. We targeted the key metabolic player proprotein convertase subtilisin/kexin type 9 (PCSK9) using a pharmacological inhibitor (R-IMPP) alone or in combination with 3-hydroxy 3-methylglutaryl-Coenzyme A reductase (HMGCR) inhibitor, simvastatin. We assessed the effect of these treatments using 3 hepatoma cell lines, Huh6, Huh7 and HepG2 and a tumor xenograft in chicken choriorallantoic membrane (CAM) model. PCSK9 deficiency led to dose-dependent inhibition of cell proliferation in all cell lines and a decrease in cell migration. Co-treatment with simvastatin presented synergetic anti-proliferative effects. At the metabolic level, mitochondrial respiration assays as well as the assessment of glucose and glutamine consumption showed higher metabolic adaptability and surge in the absence of PCSK9. Enhanced lipid uptake and biogenesis led to excessive accumulation of intracellular lipid droplets as revealed by electron microscopy and metabolic tracing. Using xenograft experiments in CAM model, we further demonstrated the effect of anti-PCSK9 treatment in reducing tumor aggressiveness. Targeting PCSK9 alone or in combination with statins deserves to be considered as a new therapeutic option in liver cancer clinical applications.

Vitiligo Skin T Cells Are Prone to Produce Type 1 and Type 2 Cytokines to Induce Melanocyte Dysfunction and Epidermal Inflammatory Response Through Jak Signaling.

Vitiligo is a T cell-mediated inflammatory skin disorder characterized by the loss of epidermal melanocytes. However, the contribution of melanocytes to the physiopathology of the disease in response to the T-cell microenvironment remains unclear. Here, using NanoString technology and multiplex ELISA, we show that active vitiligo perilesional skin is characterized by prominent type 1 and 2 associated immune responses. The vitiligo skin T-cell secretome downregulated melanocyte function and adhesion while increasing melanocyte mitochondrial metabolism and expression of inflammatory cytokines and chemokines by epidermal cells. The Jak1/2 inhibitor ruxolitinib strongly inhibited such effects on epidermal cells. Our data highlight that vitiligo is more complex than previously thought, with prominent combined activities of both T helper type 1- and T helper type 2-related cytokines inducing inflammatory responses of epidermal cells. Melanocytes do not appear only to be a target of T cells in vitiligo but could actively contribute to perpetuate inflammation. Jak inhibitors could prevent the impact of T cells on epidermal cells and pigmentation, highlighting their potential clinical benefit in vitiligo.

Lung Tumor Growth Promotion by Tobacco-Specific Nitrosamines Involves the ß2-Adrenergic Receptors-Dependent Stimulation of Mitochondrial REDOX Signaling.

Aims: Lung cancer is the leading cause of cancer death worldwide, and tobacco smoking is a recognized major risk factor for lung tumor development. We analyzed the effect of tobacco-specific nitrosamines (TSNAs) on human lung adenocarcinoma metabolic reprogramming, an emergent hallmark of carcinogenesis. Results: A series of in vitro and in vivo bioenergetic, proteomic, metabolomic, and tumor biology studies were performed to analyze changes in lung cancer cell metabolism and the consequences for hallmarks of cancer, including tumor growth, cancer cell invasion, and redox signaling. The findings revealed that nicotine-derived nitrosamine ketone (NNK) stimulates mitochondrial function and promotes lung tumor growth in vivo. These malignant properties were acquired from the induction of mitochondrial biogenesis induced by the upregulation and activation of the beta-2 adrenergic receptors (ß2-AR)-cholinergic receptor nicotinic alpha 7 subunit (CHRNAα7)-dependent nitrosamine canonical signaling pathway. The observed NNK metabolic effects were mediated by TFAM overexpression and revealed a key role for mitochondrial reactive oxygen species and Annexin A1 in tumor growth promotion. Conversely, ectopic expression of the mitochondrial antioxidant enzyme manganese superoxide dismutase rescued the reprogramming and malignant metabolic effects of exposure to NNK and overexpression of TFAM, underlining the link between NNK and mitochondrial redox signaling in lung cancer. Innovation: Our findings describe the metabolic changes caused by NNK in a mechanistic framework for understanding how cigarette smoking causes lung cancer. Conclusion: Mitochondria play a role in the promotion of lung cancer induced by tobacco-specific nitrosamines. Antioxid. Redox Signal. 36, 525-549.

Characterization of NADPH Oxidase Expression and Activity in Acute Myeloid Leukemia Cell Lines: A Correlation with the Differentiation Status.

In acute myeloid leukemia (AML), a low level of reactive oxygen species (ROS) is associated with leukemic stem cell (LSC) quiescence, whereas a high level promotes blast proliferation. ROS homeostasis relies on a tightly-regulated balance between the antioxidant and oxidant systems. Among the oxidants, NADPH oxidases (NOX) generate ROS as a physiological function. Although it has been reported in AML initiation and development, the contribution of NOX to the ROS production in AML remains to be clarified. The aim of this study was to investigate the NOX expression and function in AML, and to examine the role of NOX in blast proliferation and differentiation. First, we interrogated the NOX expression in primary cells from public datasets, and investigated their association with prognostic markers. Next, we explored the NOX expression and activity in AML cell lines, and studied the impact of NOX knockdown on cell proliferation and differentiation. We found that NOX2 is ubiquitously expressed in AML blasts, and particularly in cells from the myelomonocytic (M4) and monocytic (M5) stages; however, it is less expressed in LSCs and in relapsed AML. This is consistent with an increased expression throughout normal hematopoietic differentiation, and is reflected in AML cell lines. Nevertheless, no endogenous NOX activity could be detected in the absence of PMA stimulation. Furthermore, CYBB knockdown, although hampering induced NOX2 activity, did not affect the proliferation and differentiation of THP-1 and HL-60 cells. In summary, our data suggest that NOX2 is a marker of AML blast differentiation, while AML cell lines lack any NOX2 endogenous activity.

Targeting the mitochondrial trifunctional protein restrains tumor growth in oxidative lung carcinomas.

Metabolic reprogramming is a common hallmark of cancer, but a large variability in tumor bioenergetics exists between patients. Using high-resolution respirometry on fresh biopsies of human lung adenocarcinoma, we identified 2 subgroups reflected in the histologically normal, paired, cancer-adjacent tissue: high (OX+) mitochondrial respiration and low (OX-) mitochondrial respiration. The OX+ tumors poorly incorporated [18F]fluorodeoxy-glucose and showed increased expression of the mitochondrial trifunctional fatty acid oxidation enzyme (MTP; HADHA) compared with the paired adjacent tissue. Genetic inhibition of MTP altered OX+ tumor growth in vivo. Trimetazidine, an approved drug inhibitor of MTP used in cardiology, also reduced tumor growth and induced disruption of the physical interaction between the MTP and respiratory chain complex I, leading to a cellular redox and energy crisis. MTP expression in tumors was assessed using histology scoring methods and varied in negative correlation with [18F]fluorodeoxy-glucose incorporation. These findings provide proof-of-concept data for preclinical, precision, bioenergetic medicine in oxidative lung carcinomas.

Downregulation of Glutamine Synthetase, not glutaminolysis, is responsible for glutamine addiction in Notch1-driven acute lymphoblastic leukemia.

The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.

Trisk 95 as a novel skin mirror for normal and diabetic systemic glucose level.

Developing trustworthy, cost effective, minimally or non-invasive glucose sensing strategies is of great need for diabetic patients. In this study, we used an experimental type I diabetic mouse model to examine whether the skin would provide novel means for identifying biomarkers associated with blood glucose level. We first showed that skin glucose levels are rapidly influenced by blood glucose concentrations. We then conducted a proteomic screen of murine skin using an experimental in vivo model of type I diabetes and wild-type controls. Among the proteins that increased expression in response to high blood glucose, Trisk 95 expression was significantly induced independently of insulin signalling. A luciferase reporter assay demonstrated that the induction of Trisk 95 expression occurs at a transcriptional level and is associated with a marked elevation in the Fluo-4AM signal, suggesting a role for intracellular calcium changes in the signalling cascade. Strikingly, these changes lead concurrently to fragmentation of the mitochondria. Moreover, Trisk 95 knockout abolishes both the calcium flux and the mitochondrial phenotype changes indicating dependency of glucose flux in the skin on Trisk 95 function. The data demonstrate that the skin reacts robustly to systemic blood changes, and that Trisk 95 is a promising biomarker for a glucose monitoring assembly.