The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection.

Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment.

In vivo reduction of RAD51-mediated homologous recombination triggers aging but impairs oncogenesis.

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant-negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti-tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging.

CDKN1A is a target for phagocytosis-mediated cellular immunotherapy in acute leukemia.

Targeting the reprogramming and phagocytic capacities of tumor-associated macrophages (TAMs) has emerged as a therapeutic opportunity for cancer treatment. Here, we demonstrate that tumor cell phagocytosis drives the pro-inflammatory activation of TAMs and identify a key role for the cyclin-dependent kinase inhibitor CDKN1A (p21). Through the transcriptional repression of Signal-Regularity Protein α (SIRPα), p21 promotes leukemia cell phagocytosis and, subsequently, the pro-inflammatory reprogramming of phagocytic macrophages that extends to surrounding macrophages through Interferon γ. In mouse models of human T-cell acute lymphoblastic leukemia (T-ALL), infusion of human monocytes (Mos) engineered to overexpress p21 (p21TD-Mos) leads to Mo differentiation into phagocytosis-proficient TAMs that, after leukemia cell engulfment, undergo pro-inflammatory activation and trigger the reprogramming of bystander TAMs, reducing the leukemic burden and substantially prolonging survival in mice. These results reveal p21 as a trigger of phagocytosis-guided pro-inflammatory TAM reprogramming and highlight the potential for p21TD-Mo-based cellular therapy as a cancer immunotherapy.

Succinate: A Serum Biomarker of SDHB-Mutated Paragangliomas and Pheochromocytomas.

CONTEXT: Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors that are frequently associated with succinate dehydrogenase (SDH) germline mutations. When mutated, SDH losses its function, thus leading to succinate accumulation. OBJECTIVE: In this study, we evaluated serum succinate levels as a new metabolic biomarker in SDHx-related carriers. METHODS: Retrospective monocentric study of 88 PPGL patients (43 sporadic, 35 SDHB, 10 SDHA/C/D), 17 tumor-free familial asymptomatic carriers (13 SDHB, 4 SDHC/D), and 60 healthy controls. Clinical, biological, and imaging data were reviewed. Serum succinate levels (n = 280) were quantified by an ultra-performance liquid chromatography coupled to a tandem mass spectrometry method and correlated to SDHx mutational status, disease extension, and other biological biomarkers. RESULTS: Serum succinate levels > 7 µM allowed identification of tumor-free asymptomatic SDHB-mutated cases compared to a healthy control group (100% specificity; 85% sensitivity). At PPGL diagnosis, SDHB-mutated patients had a significantly increased median succinate level (14 µM) compared to sporadic patients (8 µM) (P < 0.01). Metastatic disease extension was correlated to serum succinate levels (r = 0.81). In the SDHB group, patients displaying highest tumor burdens showed significant increased succinate levels compared to the sporadic group (P < 0.0001). CONCLUSIONS: In this pilot study, we showed that serum succinate level is an oncometabolic biomarker that should be useful to identify SDHB-related carriers. Succinate levels are also a marker of metabolic tumor burden in patients with a metastatic PPGL and a potential marker of treatment response and follow-up.

Oxazaphosphorines combined with immune checkpoint blockers: dose-dependent tuning between immune and cytotoxic effects.

BACKGROUND: Oxazaphosphorines (cyclophosphamide (CPA), ifosfamide (IFO)) are major alkylating agents of polychemotherapy protocols but limiting their toxicity and increasing their efficacy could be of major interest. Oxazaphosphorines are prodrugs that require an activation by cytochrome P450 (CYP). CPA is mainly metabolized (>80%) to phosphoramide mustard while only 10%-50% of IFO is transformed in the alkylating entity, isophosphoramide mustard and 50%-90% of IFO release chloroacetaldehyde, a nephrotoxic and neurotoxic metabolite. Geranyloxy-IFO (G-IFO) was reported as a preactivated IFO to circumvent the toxic pathway giving directly the isophosphoramide mustard without CYP metabolization. The similarity in structure of CPA and IFO and the similarity in metabolic balance of CPA and G-IFO have led us to explore immunomodulatory effect of these components in mice and to investigate the combination of these oxazaphosphorines with immune checkpoint blockers (ICB). METHODS: The investigation of the immunomodulatory properties of IFO and G-IFO compared with CPA has been conducted through immune cell phenotyping by flow cytometry and analysis of the cytokine profile of T cells after ex-vivo restimulation. T cell-mediated antitumor efficacy was confirmed in CD4+ and CD8+ T cell-depleted mice. A combination of oxazaphosphorines with an anti-programmed cell death 1 (PD-1) antibody has been studied in MCA205 tumor-bearing mice. RESULTS: Studies on a MCA205 mouse model have demonstrated a dose-dependent effect of IFO and G-IFO on T cell immunity. These components in particular favored Th1 polarization when used at low dose (150 and eq. 100 mg/kg, respectively). Antitumor activity at low dose was abolished in mice depleted in CD4+ and CD8+ T cells. G-IFO at low dose (eq. 100 mg/kg) in combination with anti-PD-1 antidody showed high synergistic antitumor efficacy compared with IFO. CONCLUSION: Oxazaphosphorines are characterized by a dual mechanism of antitumor action; low-dose schedules should be preferred in combination with ICB, and dose escalation was found to have better utility in polychemotherapy protocols where a conventional direct cytotoxic anticancer effect is needed. G-IFO, the novel oxazaphosphorine drug, has shown a better metabolic index compared with IFO as its metabolization gives mainly the alkylating mustard as CPA (and not IFO) and a best potential in combination with ICB.

SUGT1 controls susceptibility to HIV-1 infection by stabilizing microtubule plus-ends.

Understanding the viral-host cell interface during HIV-1 infection is a prerequisite for the development of innovative antiviral therapies. Here we show that the suppressor of G2 allele of skp1 (SUGT1) is a permissive factor for human immunodeficiency virus (HIV)-1 infection. Expression of SUGT1 increases in infected cells on human brain sections and in permissive host cells. We found that SUGT1 determines the permissiveness to infection of lymphocytes and macrophages by modulating the nuclear import of the viral genome. More importantly, SUGT1 stabilizes the microtubule plus-ends (+MTs) of host cells (through the modulation of microtubule acetylation and the formation of end-binding protein 1 (EB1) comets). This effect on microtubules favors HIV-1 retrograde trafficking and replication. SUGT1 depletion impairs the replication of HIV-1 patient primary isolates and mutant virus that is resistant to raltegravir antiretroviral agent. Altogether our results identify SUGT1 as a cellular factor involved in the post-entry steps of HIV-1 infection that may be targeted for new therapeutic approaches.

Emerging use of senolytics and senomorphics against aging and chronic diseases.

Senescence is a state of cell cycle arrest that plays an important role in embryogenesis, wound healing and protection against cancer. Senescent cells also accumulate during aging and contribute to the development of age-related disorders and chronic diseases, such as atherosclerosis, type 2 diabetes, osteoarthritis, idiopathic pulmonary fibrosis, and liver disease. Molecules that induce apoptosis of senescent cells, such as dasatinib, quercetin, and fisetin, produce health benefits and extend lifespan in animal models. We describe here the mechanism of action of senolytics and senomorphics, many of which are derived from plants and fungi. We also discuss the possibility of using such compounds to delay aging and treat chronic diseases in humans.

HIV-1 Envelope Overcomes NLRP3-Mediated Inhibition of F-Actin Polymerization for Viral Entry.

Purinergic receptors and nucleotide-binding domain leucine-rich repeat containing (NLR) proteins have been shown to control viral infection. Here, we show that the NLR family member NLRP3 and the purinergic receptor P2Y2 constitutively interact and regulate susceptibility to HIV-1 infection. We found that NLRP3 acts as an inhibitory factor of viral entry that represses F-actin remodeling. The binding of the HIV-1 envelope to its host cell receptors (CD4, CXCR4, and/or CCR5) overcomes this restriction by stimulating P2Y2. Once activated, P2Y2 enhances its interaction with NLRP3 and stimulates the recruitment of the E3 ubiquitin ligase CBL to NLRP3, ultimately leading to NLRP3 degradation. NLRP3 degradation is permissive for PYK2 phosphorylation (PYK2Y402∗) and subsequent F-actin polymerization, which is required for the entry of HIV-1 into host cells. Taken together, our results uncover a mechanism by which HIV-1 overcomes NLRP3 restriction that appears essential for the accomplishment of the early steps of HIV-1 entry.

CSRP3 mediates polyphenols-induced cardioprotection in hypertension.

Berries contain bioactive polyphenols, whose capacity to prevent cardiovascular diseases has been established recently in animal models as well in human clinical trials. However, cellular processes and molecular targets of berries polyphenols remain to be identified. The capacity of a polyphenol-enriched diet (i.e., blueberries, blackberries, raspberries, strawberry tree fruits and Portuguese crowberries berries mixture) to promote animal survival and protect cardiovascular function from salt-induced hypertension was evaluated in a chronic salt-sensitive Dahl rat model. The daily consumption of berries improved survival of Dahl/salt-sensitive rats submitted to high-salt diet and normalized their body weight, renal function and blood pressure. In addition, a prophylactic effect was observed at the level of cardiac hypertrophy and dysfunction, tissue cohesion and cardiomyocyte hypertrophy. Berries also protected the aorta from fibrosis and modulated the expression of aquaporin-1, a channel involved in endothelial water and nitric oxide permeability. Left ventricle proteomics analysis led to the identification of berries and salt metabolites targets, including cystein and glycin-rich protein 3 (CSRP3), a protein involved in myocyte cytoarchitecture. In neonatal rat ventricular cardiomyocytes, CSRP3 was validated as a target of a berries-derived polyphenol metabolite, 4-methylcatechol sulfate, at micromolar concentrations, mimicking physiological conditions of human plasma circulation. Accordingly, siRNA silencing of CSRP3 and 4-methylcatechol sulfate pretreatment reversed cardiomyocyte hypertrophy and CSRP3 overexpression induced by phenylephrine. Our systemic study clearly supports the modulation of CSRP3 by a polyphenol-rich berries diet as an efficient cardioprotective strategy in hypertension-induced heart failure.

AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine.

AGuIX® are sub-5 nm nanoparticles made of a polysiloxane matrix and gadolinium chelates. This nanoparticle has been recently accepted in clinical trials in association with radiotherapy. This review will summarize the principal preclinical results that have led to first in man administration. No evidence of toxicity has been observed during regulatory toxicity tests on two animal species (rodents and monkeys). Biodistributions on different animal models have shown passive uptake in tumours due to enhanced permeability and retention effect combined with renal elimination of the nanoparticles after intravenous administration. High radiosensitizing effect has been observed with different types of irradiations in vitro and in vivo on a large number of cancer types (brain, lung, melanoma, head and neck…). The review concludes with the second generation of AGuIX nanoparticles and the first preliminary results on human.

Anticancer chemotherapy and radiotherapy trigger both non-cell-autonomous and cell-autonomous death.

Even though cell death modalities elicited by anticancer chemotherapy and radiotherapy have been extensively studied, the ability of anticancer treatments to induce non-cell-autonomous death has never been investigated. By means of multispectral imaging flow-cytometry-based technology, we analyzed the lethal fate of cancer cells that were treated with conventional anticancer agents and co-cultured with untreated cells, observing that anticancer agents can simultaneously trigger cell-autonomous and non-cell-autonomous death in treated and untreated cells. After ionizing radiation, oxaliplatin, or cisplatin treatment, fractions of treated cancer cell populations were eliminated through cell-autonomous death mechanisms, while other fractions of the treated cancer cells engulfed and killed neighboring cells through non-cell-autonomous processes, including cellular cannibalism. Under conditions of treatment with paclitaxel, non-cell-autonomous and cell-autonomous death were both detected in the treated cell population, while untreated neighboring cells exhibited features of apoptotic demise. The transcriptional activity of p53 tumor-suppressor protein contributed to the execution of cell-autonomous death, yet failed to affect the non-cell-autonomous death by cannibalism for the majority of tested anticancer agents, indicating that the induction of non-cell-autonomous death can occur under conditions in which cell-autonomous death was impaired. Altogether, these results reveal that chemotherapy and radiotherapy can induce both non-cell-autonomous and cell-autonomous death of cancer cells, highlighting the heterogeneity of cell death responses to anticancer treatments and the unsuspected potential contribution of non-cell-autonomous death to the global effects of anticancer treatment.

Tumour spheres with inverted polarity drive the formation of peritoneal metastases in patients with hypermethylated colorectal carcinomas.

Metastases account for 90% of cancer-related deaths; thus, it is vital to understand the biology of tumour dissemination. Here, we collected and monitored >50 patient specimens ex vivo to investigate the cell biology of colorectal cancer (CRC) metastatic spread to the peritoneum. This reveals an unpredicted mode of dissemination. Large clusters of cancer epithelial cells displaying a robust outward apical pole, which we termed tumour spheres with inverted polarity (TSIPs), were observed throughout the process of dissemination. TSIPs form and propagate through the collective apical budding of hypermethylated CRCs downstream of canonical and non-canonical transforming growth factor-ß signalling. TSIPs maintain their apical-out topology and use actomyosin contractility to collectively invade three-dimensional extracellular matrices. TSIPs invade paired patient peritoneum explants, initiate metastases in mice xenograft models and correlate with adverse patient prognosis. Thus, despite their epithelial architecture and inverted topology TSIPs seem to drive the metastatic spread of hypermethylated CRCs.

Bimodal fluorescence/129Xe NMR probe for molecular imaging and biological inhibition of EGFR in Non-Small Cell Lung Cancer.

Although Non-Small Cell Lung Cancer (NSCLC) is one of the main causes of cancer death, very little improvement has been made in the last decades regarding diagnosis and outcomes. In this study, a bimodal fluorescence/129Xe NMR probe containing a xenon host, a fluorescent moiety and a therapeutic antibody has been designed to target the Epidermal Growth Factor Receptors (EGFR) overexpressed in cancer cells. This biosensor shows high selectivity for the EGFR, and a biological activity similar to that of the antibody. It is detected with high specificity and high sensitivity (sub-nanomolar range) through hyperpolarized 129Xe NMR. This promising system should find important applications for theranostic use.

Time dependent modulation of tumor radiosensitivity by a pan HDAC inhibitor: abexinostat.

Despite prominent role of radiotherapy in lung cancer management, there is an urgent need for strategies increasing therapeutic efficacy. Reversible epigenetic changes are promising targets for combination strategies using HDAC inhibitors (HDACi). Here we evaluated on two NSCLC cell lines, the antitumor effect of abexinostat, a novel pan HDACi combined with irradiation in vitro in normoxia and hypoxia, by clonogenic assays, demonstrating that abexinostat enhances radiosensitivity in a time dependent way with mean SER10 between 1.6 and 2.5 for A549 and H460. We found, by immunofluorescence staining, flow cytometry assays and western blotting, in abexinostat treated cells, increasing radio-induced caspase dependent apoptosis and persistent DNA double-strand breaks associated with decreased DNA damage signalling and repair. Interestingly, we demonstrated on nude mice xenografts that abexinostat potentiates tumor growth delay in combined modality treatments associating not only abexinostat and irradiation but also when adding cisplatin. Altogether, our data demonstrate in vitro and in vivo anti-tumor effect potentiation by abexinostat combined with irradiation in NSCLC. Moreover, our work suggests for the first time to our knowledge promising triple combination opportunities with HDACi, irradiation and cisplatin which deserves further investigations and could be of major interest in the treatment of NSCLC.

Macrophage biology plays a central role during ionizing radiation-elicited tumor response.

Radiation therapy is one of the major therapeutic modalities for most solid tumors. The anti-tumor effect of radiation therapy consists of the direct tumor cell killing, as well as the modulation of tumor microenvironment and the activation of immune response against tumors. Radiation therapy has been shown to promote immunogenic cells death, activate dendritic cells and enhance tumor antigen presentation and anti-tumor T cell activation. Radiation therapy also programs innate immune cells such as macrophages that leads to either radiosensitization or radioresistance, according to different tumors and different radiation regimen studied. The mechanisms underlying radiation-induced macrophage activation remain largely elusive. Various molecular players such as NF-κB, MAPKs, p53, reactive oxygen species, inflammasomes have been involved in these processes. The skewing to a pro-inflammatory phenotype thus results in the activation of anti-tumor immune response and enhanced radiotherapy effect. Therefore, a comprehensive understanding of the mechanism of radiation-induced macrophage activation and its role in tumor response to radiation therapy is crucial for the development of new therapeutic strategies to enhance radiation therapy efficacy.

NOX2-dependent ATM kinase activation dictates pro-inflammatory macrophage phenotype and improves effectiveness to radiation therapy.

Although tumor-associated macrophages have been extensively studied in the control of response to radiotherapy, the molecular mechanisms involved in the ionizing radiation-mediated activation of macrophages remain elusive. Here we show that ionizing radiation induces the expression of interferon regulatory factor 5 (IRF5) promoting thus macrophage activation toward a pro-inflammatory phenotype. We reveal that the activation of the ataxia telangiectasia mutated (ATM) kinase is required for ionizing radiation-elicited macrophage activation, but also for macrophage reprogramming after treatments with γ-interferon, lipopolysaccharide or chemotherapeutic agent (such as cisplatin), underscoring the fact that the kinase ATM plays a central role during macrophage phenotypic switching toward a pro-inflammatory phenotype through the regulation of mRNA level and post-translational modifications of IRF5. We further demonstrate that NADPH oxidase 2 (NOX2)-dependent ROS production is upstream to ATM activation and is essential during this process. We also report that the inhibition of any component of this signaling pathway (NOX2, ROS and ATM) impairs pro-inflammatory activation of macrophages and predicts a poor tumor response to preoperative radiotherapy in locally advanced rectal cancer. Altogether, our results identify a novel signaling pathway involved in macrophage activation that may enhance the effectiveness of radiotherapy through the reprogramming of tumor-infiltrating macrophages.

Entosis: The emerging face of non-cell-autonomous type IV programmed death.

The present review summarizes recent experimental evidences about the existence of the non-cell-autonomous death entosis in physiological and pathophysiological contexts, discusses some aspects of this form of cell death, including morphological, biochemical and signaling pathways that distinguish non-cell-autonomous demises from other death modalities and propose to define this new modality of death as type IV programmed cell death.

Modulating Both Tumor Cell Death and Innate Immunity Is Essential for Improving Radiation Therapy Effectiveness.

Radiation therapy is one of the cornerstones of cancer treatment. In tumor cells, exposure to ionizing radiation (IR) provokes DNA damages that trigger various forms of cell death such as apoptosis, necrosis, autophagic cell death, and mitotic catastrophe. IR can also induce cellular senescence that could serve as an additional antitumor barrier in a context-dependent manner. Moreover, accumulating evidence has demonstrated that IR interacts profoundly with tumor-infiltrating immune cells, which cooperatively drive treatment outcomes. Recent preclinical and clinical successes due to the combination of radiation therapy and immune checkpoint blockade have underscored the need for a better understanding of the interplay between radiation therapy and the immune system. In this review, we will present an overview of cell death modalities induced by IR, summarize the immunogenic properties of irradiated cancer cells, and discuss the biological consequences of IR on innate immune cell functions, with a particular attention on dendritic cells, macrophages, and NK cells. Finally, we will discuss their potential applications in cancer treatment.

Can immunostimulatory agents enhance the abscopal effect of radiotherapy?

Ionising radiation (IR) may harm cancer cells through a rare indirect out-of-field phenomenon described as the abscopal effect. Increasing evidence demonstrates that radiotherapy could be capable of generating tumour-specific immune responses. On the other hand, effects of IR also include inhibitory immune signals on the tumour microenvironment. Following these observations, and in the context of newly available immunostimulatory agents in metastatic cancers (anti-cytotoxic T lymphocyte-associated antigen 4 and programmed cell death protein-1 or -ligand 1 [PD1 or PDL-1]), there is a remarkable potential for synergistic combinations of IR with such agents that act through the reactivation of immune surveillance. Here, we present and discuss the pre-clinical and clinical rationale supporting the enhancement of the abscopal effect of IR on the blockade of immune checkpoints and discuss the evolving potential of immunoradiotherapy.