Acyl-coenzyme A (CoA)–binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is an extracellular feedback regulator of autophagy. Here, we report that injection of a monoclonal antibody neutralizing ACBP/DBI (α-DBI) protects the murine liver against ischemia/reperfusion damage, intoxication by acetaminophen and concanavalin A, and nonalcoholic steatohepatitis caused by methionine/choline-deficient diet as well as against liver fibrosis induced by bile duct ligation or carbon tetrachloride. α-DBI downregulated proinflammatory and profibrotic genes and upregulated antioxidant defenses and fatty acid oxidation in the liver. The hepatoprotective effects of α-DBI were mimicked by the induction of ACBP/DBI-specific autoantibodies, an inducible Acbp/Dbi knockout or a constitutive Gabrg2 F77I mutation that abolishes ACBP/DBI binding to the GABA A receptor. Liver-protective α-DBI effects were lost when autophagy was pharmacologically blocked or genetically inhibited by knockout of Atg4b . Of note, α-DBI also reduced myocardium infarction and lung fibrosis, supporting the contention that it mediates broad organ-protective effects against multiple insults.
e15091 Background: Local anesthetics (LA) are often used during surgery in order to control postoperative pain. Observational retrospective studies showed a significant and unexpected decrease of relapse and increased overall survival after LA injection during solid cancer removal. We hypothesized that LAs used as stand-alone treatment or combined with conventional anticancer therapy may induce cytotoxic effects on cancer cells and trigger antitumor responses against dead-cell antigens. Methods: Cell stress and cell death modalities were investigated in vitro by means of specific biosensors in human osteosarcoma (U2OS) cells after treatment with LAs such as bupivacaine, chloroprocaine, levobupivacaine, lidocaine, ropivacaine and prilocaine. Moreover tumor growth and overall survival were studied in solid tumor models (MCA 205 fibrosarcoma) established in immunocompetent C57Bl/6 mice treated under general anesthesia with LAs (lidocaine or ropivacaine) alone or combined with immune checkpoint blockade (anti-PD-1). Ethical Committee: CEEA IRCIV/IGR n°26, French Ministry of Research, Ref:16946/2018100309413893v2. Results: In vitro, the tested LAs triggered autophagy and induced all arms of the integrated stress response including the phosphorylation of eIF2alpha, the activation of ATF4, the splicing of XBP1 and the proteolytic cleavage of ATF6. Cell stress was followed by apoptotic cell death, and both were inhibited when eIF2alpha kinase 3 (EIF2AK3) was genetically removed by CRISPR/CAS9, while the removal of EIF2AK1, EIF2AK2 and EIF2AK4 had no effects on cellular demise. LAs also triggered mitochondrial dysfunction, mimicking the effect of mitochondrial uncouplers (such as rotenone and CCCP) characterized by respiratory chain and inhibition of a mitochondrial transmembrane enzyme (carnitine-acylcarnitine translocase). In vivo, LAs induced effective tumor growth reduction and improved survival of immunocompetent but not immunodeficient mice, suggesting the ignition of anticancer immune responses. These anticancer immune effects were significantly potentiated when LAs were combined with PD-1 immune checkpoint blockade. LAs and mitochondrial uncouplers failed to induce anticancer effects in MCA205 tumors that were unable to induce ER stress (due to EIF2AK3 knockout) or autophagy (due to AtG5 KO), suggesting that both premortem stress responses are indispensable for inducing anticancer immunity. Conclusions: LAs induce EIF2AK3-dependent cancer cell stress including autophagy and the integrated stress response, which is followed by mitochondrial toxicity and cell death both in vitro and in cancers established in mice. These effects trigger therapeutically relevant anticancer immune responses that can be further potentiated by means of PD-1 blockade. These preclinical observations suggest that the use of LAs during oncological surgery improves clinical outcome by inducing anticancer immunity.
Abstract The hypomethylating agent 5-azacytidine (AZA) is the first-line treatment for AML patients unfit for intensive chemotherapy. The effect of AZA results in part from T-cell cytotoxic responses against MHC-I-associated peptides (MAPs) deriving from hypermethylated genomic regions such as cancer-testis antigens (CTAs), or endogenous retroelements (EREs). However, evidence supporting higher ERE MAPs presentation after AZA treatment is lacking. Therefore, using proteogenomics, we examined the impact of AZA on the repertoire of MAPs and their source transcripts. AZA-treated AML upregulated both CTA and ERE transcripts, but only CTA MAPs were presented at greater levels. Upregulated ERE transcripts triggered innate immune responses against double-stranded RNAs but were degraded by autophagy, and not processed into MAPs. Autophagy resulted from the formation of protein aggregates caused by AZA-dependent inhibition of DNMT2. Autophagy inhibition had an additive effect with AZA on AML cell proliferation and survival, increased ERE levels, increased pro-inflammatory responses, and generated immunogenic tumor-specific ERE-derived MAPs. Finally, autophagy was associated with a lower abundance of CD8 + T-cell markers in AML patients expressing high levels of EREs. This work demonstrates that AZA-induced EREs are degraded by autophagy and shows that inhibiting autophagy can improve the immune recognition of AML blasts in treated patients.
Abstract Cystic fibrosis is a disease caused by defective function of a chloride channel coupled to a blockade of autophagic flux. It has been proposed to use synthetic chloride transporters as pharmacological agents to compensate insufficient chloride fluxes. Here, we report that such chloride anionophores block autophagic flux in spite of the fact that they activate the pro-autophagic transcription factor EB (TFEB) coupled to the inhibition of the autophagy-suppressive mTORC1 kinase activity. Two synthetic chloride transporters ( SQ1 and SQ2 ) caused a partially TFEB-dependent relocation of the autophagic marker LC3 to the Golgi apparatus. Inhibition of TFEB activation using a calcium chelator or calcineurin inhibitors reduced the formation of LC3 puncta in cells, yet did not affect the cytotoxic action of SQ1 and SQ2 that could be observed after prolonged incubation. In conclusion, the squaramide-based synthetic chloride transporters studied in this work (which can also dissipate pH gradients) are probably not appropriate for the treatment of cystic fibrosis yet might be used for other indications such as cancer.
// Allan Sauvat 1, 2, 3 , Yidan Wang 1, 2, 3, 4 , Florian Segura 1, 2, 3 , Sabrina Spaggiari 1, 2, 3 , Kevin Müller 1, 2, 3 , Heng Zhou 1, 2, 3, 4 , Lorenzo Galluzzi 1, 2, 5, 6 , Oliver Kepp 1, 2, 3 , Guido Kroemer 1, 2, 3, 6, 7 1 Equipe 11 labellisée par la Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France 2 INSERM, U1138, Paris, France 3 Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France 4 Faculté de Medecine, Université Paris-Sud, Le Kremlin-Bicêtre, France 5 Gustave Roussy Cancer Campus, Villejuif, France 6 Faculté de Medecine, Université Paris Descartes, Sorbonne Paris Cité, Paris, France 7 Pôle de Biologie, Hopitâl Européen George Pompidou, AP-HP, Paris, France Correspondence to: Oliver Kepp, e-mail: oliver.kepp@gustaveroussy.fr Guido Kroemer, e-mail: kroemer@orange.fr Keywords: apoptosis, necrosis, high-throughput screening, drug discovery Received: January 09, 2015 Accepted: January 31, 2015 Published: March 25, 2015 ABSTRACT Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4’,6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC 6 (3)) and caspase activation (based on YO-PRO ® -3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.
