Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus.

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.

Identification of Small Molecules Affecting the Secretion of Therapeutic Antibodies with the Retention Using Selective Hook (RUSH) System.

Unlocking cell secretion capacity is of paramount interest for the pharmaceutical industry focused on biologics. Here, we leveraged retention using a selective hook (RUSH) system for the identification of human osteosarcoma U2OS cell secretion modulators, through automated, high-throughput screening of small compound libraries. We created a U2OS cell line which co-expresses a variant of streptavidin addressed to the lumen-facing membrane of the endoplasmic reticulum (ER) and a recombinant anti-PD-L1 antibody. The heavy chain of the antibody was modified at its C-terminus, to which a furin cleavage site, a green fluorescent protein (GFP), and a streptavidin binding peptide (SBP) were added. We show that the U2OS cell line stably expresses the streptavidin hook and the recombinant antibody bait, which is retained in the ER through the streptavidin-SBP interaction. We further document that the addition of biotin to the culture medium triggers the antibody release from the ER, its trafficking through the Golgi where the GFP-SBP moiety is clipped off, and eventually its release in the extra cellular space, with specific antigen-binding properties. The use of this clone in screening campaigns led to the identification of lycorine as a secretion enhancer, and nigericin and tyrphostin AG-879 as secretion inhibitors. Altogether, our data support the utility of this approach for the identification of agents that could be used to improve recombinant production yields and also for a better understanding of the regulatory mechanism at work in the conventional secretion pathway.

3,4-dimethoxychalcone induces autophagy and reduces neointimal hyperplasia and aortic lesions in mouse models of atherosclerosis.

Autophagy inducers can prevent cardiovascular aging and age-associated diseases including atherosclerosis. Therefore, we hypothesized that autophagy-inducing compounds that act on atherosclerosis-relevant cells might have a protective role in the development of atherosclerosis. Here we identified 3,4-dimethoxychalcone (3,4-DC) as an inducer of autophagy in several cell lines from endothelial, myocardial and myeloid/macrophagic origin, as demonstrated by the aggregation of the autophagosome marker GFP-LC3 in the cytoplasm of cells, as well as the downregulation of its nuclear pool indicative of autophagic flux. In this respect, 3,4-DC showed a broader autophagy-inducing activity than another chalcone (4,4- dimethoxychalcone), spermidine and triethylene tetramine. Thus, we characterized the potential antiatherogenic activity of 3,4-DC in two different mouse models, namely, (i) neointima formation with smooth muscle expansion of vein segments grafted to the carotid artery and (ii) genetically predisposed ApoE-/- mice fed an atherogenic diet. In the vein graft model, local application of 3,4-DC was able to maintain the lumen of vessels and to reduce neointima lesions. In the diet-induced model, intraperitoneal injections of 3,4-DC significantly reduced the number of atherosclerotic lesions in the aorta. In conclusion, 3,4-DC stands out as an autophagy inducer with potent antiatherogenic activity.

A microbiota-modulated checkpoint directs immunosuppressive intestinal T cells into cancers.

Antibiotics (ABX) compromise the efficacy of programmed cell death protein 1 (PD-1) blockade in cancer patients, but the mechanisms underlying their immunosuppressive effects remain unknown. By inducing the down-regulation of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the ileum, post-ABX gut recolonization by Enterocloster species drove the emigration of enterotropic α4ß7+CD4+ regulatory T 17 cells into the tumor. These deleterious ABX effects were mimicked by oral gavage of Enterocloster species, by genetic deficiency, or by antibody-mediated neutralization of MAdCAM-1 and its receptor, α4ß7 integrin. By contrast, fecal microbiota transplantation or interleukin-17A neutralization prevented ABX-induced immunosuppression. In independent lung, kidney, and bladder cancer patient cohorts, low serum levels of soluble MAdCAM-1 had a negative prognostic impact. Thus, the MAdCAM-1-α4ß7 axis constitutes an actionable gut immune checkpoint in cancer immunosurveillance.

Assessment of eIF2α phosphorylation during immunogenic cell death.

Immunogenic cell death (ICD) is a modality of cellular demise that when it is induced by certain anticancer treatments can ignite an adaptive anticancer immune response. ICD is characterized by the emission of a specific set of danger-associated molecular patterns (DAMPs) including calreticulin exposure at the plasma membrane, ATP liberation, HMGB1 exodus and type-I IFN release. The apical signaling triggering the appearance of these hallmarks involves the phosphorylation on serine 51 of the α-subunit of eukaryotic initiation factor 2 (EIF2), a key protein in the orchestration of endoplasmic reticulum (ER) stress responses. EIF2α can be phosphorylated by a family of four EIF2A kinases: EIF2AK1-4 (best known as heme regulated inhibitor, HRI, protein kinase R, PKR, protein kinase R-like endoplasmic reticulum kinase, PERK, and general control non-derepressible 2, GCN2), that each respond to a specific type of cellular stress. Here, we describe different techniques to investigate the biochemical pathways leading to eIF2α phosphorylation in the context of ICD.

Cancer cell-autonomous overactivation of PARP1 compromises immunosurveillance in non-small cell lung cancer.

BACKGROUND: High activity of poly(ADP-ribose) polymerase-1 (PARP1) in non-small cell lung cancer (NSCLC) cells leads to an increase in immunohistochemically detectable PAR, correlating with poor prognosis in patients with NSCLC, as well as reduced tumor infiltration by cytotoxic T lymphocytes (CTLs). Intrigued by this observation, we decided to determine whether PARP1 activity in NSCLC cells may cause an alteration of anticancer immunosurveillance. METHODS: Continuous culture of mouse NSCLC cells in the presence of cisplatin led to the generation of cisplatin-resistant PARhigh clones. As compared with their parental controls, such PARhigh cells formed tumors that were less infiltrated by CTLs when they were injected into immunocompetent mice, suggesting a causative link between high PARP1 activity and compromised immunosurveillance. To confirm this cause-and-effect relationship, we used CRISPR/Cas9 technology to knock out PARP1 in two PARhigh NSCLC mouse cell lines (Lewis lung cancer [LLC] and tissue culture number one [TC1]), showing that the removal of PARP1 indeed restored cisplatin-induced cell death responses. RESULTS: PARP1 knockout (PARP1KO) cells became largely resistant to the PARP inhibitor niraparib, meaning that they exhibited less cell death induction, reduced DNA damage response, attenuated metabolic shifts and no induction of PD-L1 and MHC class-I molecules that may affect their immunogenicity. PARhigh tumors implanted in mice responded to niraparib irrespective of the presence or absence of T lymphocytes, suggesting that cancer cell-autonomous effects of niraparib dominate over its possible immunomodulatory action. While PARhigh NSCLC mouse cell lines proliferated similarly in immunocompetent and T cell-deficient mice, PARP1KO cells were strongly affected by the presence of T cells. PARP1KO LLC tumors grew more quickly in immunodeficient than in immunocompetent mice, and PARP1KO TC1 cells could only form tumors in T cell-deficient mice, not in immunocompetent controls. Importantly, as compared with PARhigh controls, the PARP1KO LLC tumors exhibited signs of T cell activation in the immune infiltrate such as higher inducible costimulator (ICOS) expression and lower PD-1 expression on CTLs. CONCLUSIONS: These results prove at the genetic level that PARP1 activity within malignant cells modulates the tumor microenvironment.

BCG therapy downregulates HLA-I on malignant cells to subvert antitumor immune responses in bladder cancer.

Patients with high-risk, nonmuscle-invasive bladder cancer (NMIBC) frequently relapse after standard intravesical bacillus Calmette-Guérin (BCG) therapy and may have a dismal outcome. The mechanisms of resistance to such immunotherapy remain poorly understood. Here, using cancer cell lines, freshly resected human bladder tumors, and samples from cohorts of patients with bladder cancer before and after BCG therapy, we demonstrate 2 distinct patterns of immune subversion upon BCG relapse. In the first pattern, intracellular BCG infection of cancer cells induced a posttranscriptional downregulation of HLA-I membrane expression via inhibition of autophagy flux. Patients with HLA-I-deficient cancer cells following BCG therapy had a myeloid immunosuppressive tumor microenvironment (TME) with epithelial-mesenchymal transition (EMT) characteristics and dismal outcomes. Conversely, patients with HLA-I-proficient cancer cells after BCG therapy presented with CD8+ T cell tumor infiltrates, upregulation of inflammatory cytokines, and immune checkpoint-inhibitory molecules. The latter patients had a very favorable outcome. We surmise that HLA-I expression in bladder cancers at relapse following BCG does not result from immunoediting but rather from an immune subversion process directly induced by BCG on cancer cells, which predicts a dismal prognosis. HLA-I scoring of cancer cells by IHC staining can be easily implemented by pathologists in routine practice to stratify future treatment strategies for patients with urothelial cancer.

High-throughput label-free detection of DNA-to-RNA transcription inhibition using brightfield microscopy and deep neural networks.

Drug discovery is in constant evolution and major advances have led to the development of in vitro high-throughput technologies, facilitating the rapid assessment of cellular phenotypes. One such phenotype is immunogenic cell death, which occurs partly as a consequence of inhibited RNA synthesis. Automated cell-imaging offers the possibility of combining high-throughput with high-content data acquisition through the simultaneous computation of a multitude of cellular features. Usually, such features are extracted from fluorescence images, hence requiring labeling of the cells using dyes with possible cytotoxic and phototoxic side effects. Recently, deep learning approaches have allowed the analysis of images obtained by brightfield microscopy, a technique that was for long underexploited, with the great advantage of avoiding any major interference with cellular physiology or stimulatory compounds. Here, we describe a label-free image-based high-throughput workflow that accurately detects the inhibition of DNA-to-RNA transcription. This is achieved by combining two successive deep convolutional neural networks, allowing (1) to automatically detect cellular nuclei (thus enabling monitoring of cell death) and (2) to classify the extracted nuclear images in a binary fashion. This analytical pipeline is R-based and can be easily applied to any microscopic platform.

High throughput screening for autophagy.

Robotized high throughput screening allows for the assessment of autophagy in a large number of samples. Here, we describe a drug discovery platform for the phenotypic identification of novel autophagy inducers by means of automated cell biology workflows employing robotized cell culture, sample preparation and data acquisition. In this setting, fluorescent biosensor cells that express microtubule-associated proteins 1A/1B light chain 3B (best known as LC3) conjugated to green fluorescent protein (GFP), are utilized together with automated high content microscopy for the image-based assessment of autophagy. In sum, we detail a drug discovery screening workflow from high throughput sample preparation and processing to data acquisition and analysis.

Oleate-induced aggregation of LC3 at the trans-Golgi network is linked to a protein trafficking blockade.

Oleate, the most abundant endogenous and dietary cis-unsaturated fatty acid, has the atypical property to cause the redistribution of microtubule-associated proteins 1A/1B light chain 3B (referred to as LC3) to the trans-Golgi network (TGN), as shown here. A genome-wide screen identified multiple, mostly Golgi transport-related genes specifically involved in the oleate-induced relocation of LC3 to the Golgi apparatus. Follow-up analyses revealed that oleate also caused the retention of secreted proteins in the TGN, as determined in two assays in which the secretion of proteins was synchronized, (i) an assay involving a thermosensitive vesicular stomatitis virus G (VSVG) protein that is retained in the endoplasmic reticulum (ER) until the temperature is lowered, and (ii) an isothermic assay involving the reversible retention of the protein of interest in the ER lumen and that was used both in vitro and in vivo. A pharmacological screen searching for agents that induce LC3 aggregation at the Golgi apparatus led to the identification of "oleate mimetics" that share the capacity to block conventional protein secretion. In conclusion, oleate represents a class of molecules that act on the Golgi apparatus to cause the recruitment of LC3 and to stall protein secretion.

Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response.

The integrated stress response manifests with the phosphorylation of eukaryotic initiation factor 2α (eIF2α) on serine residue 51 and plays a major role in the adaptation of cells to endoplasmic reticulum stress in the initiation of autophagy and in the ignition of immune responses. Here, we report that lysosomotropic agents, including azithromycin, chloroquine, and hydroxychloroquine, can trigger eIF2α phosphorylation in vitro (in cultured human cells) and, as validated for hydroxychloroquine, in vivo (in mice). Cells bearing a non-phosphorylatable eIF2α mutant (S51A) failed to accumulate autophagic puncta in response to azithromycin, chloroquine, and hydroxychloroquine. Conversely, two inhibitors of eIF2α dephosphorylation, nelfinavir and salubrinal, enhanced the induction of such autophagic puncta. Altogether, these results point to the unexpected capacity of azithromycin, chloroquine, and hydroxychloroquine to elicit the integrated stress response.

Everolimus and plicamycin specifically target chemoresistant colorectal cancer cells of the CMS4 subtype.

Colorectal cancers (CRC) can be classified into four consensus molecular subtypes (CMS), among which CMS1 has the best prognosis, contrasting with CMS4 that has the worst outcome. CMS4 CRC is notoriously resistant against therapeutic interventions, as demonstrated by preclinical studies and retrospective clinical observations. Here, we report the finding that two clinically employed agents, everolimus (EVE) and plicamycin (PLI), efficiently target the prototypic CMS4 cell line MDST8. As compared to the prototypic CMS1 cell line LoVo, MDST8 cells treated with EVE or PLI demonstrated stronger cytostatic and cytotoxic effects, increased signs of apoptosis and autophagy, as well as a more pronounced inhibition of DNA-to-RNA transcription and RNA-to-protein translation. Moreover, nontoxic doses of EVE and PLI induced the shrinkage of MDST8 tumors in mice, yet had only minor tumor growth-reducing effects on LoVo tumors. Altogether, these results suggest that EVE and PLI should be evaluated for their clinical activity against CMS4 CRC.

IGF1 receptor inhibition amplifies the effects of cancer drugs by autophagy and immune-dependent mechanisms.

BACKGROUND: Pharmacological autophagy enhancement constitutes a preclinically validated strategy for preventing or treating most major age-associated diseases. Driven by this consideration, we performed a high-content/high-throughput screen on 65 000 distinct compounds on a robotized fluorescence microscopy platform to identify novel autophagy inducers. RESULTS: Here, we report the discovery of picropodophyllin (PPP) as a potent inducer of autophagic flux that acts on-target, as an inhibitor of the tyrosine kinase activity of the insulin-like growth factor-1 receptor (IGF1R). Thus, PPP lost its autophagy-stimulatory activity in cells engineered to lack IGF1R or to express a constitutively active AKT serine/threonine kinase 1 (AKT1) mutant. When administered to cancer-bearing mice, PPP improved the therapeutic efficacy of chemoimmunotherapy with a combination of immunogenic cytotoxicants and programmed cell death 1 (PDCD1, better known as PD-1) blockade. These PPP effects were lost when tumors were rendered PPP-insensitive or autophagy-incompetent. In combination with chemotherapy, PPP enhanced the infiltration of tumors by cytotoxic T lymphocytes, while reducing regulatory T cells. In human triple-negative breast cancer patients, the activating phosphorylation of IGF1R correlated with inhibited autophagy, an unfavorable local immune profile, and poor prognosis. CONCLUSION: Altogether, these results suggest that IGF1R may constitute a novel and druggable therapeutic target for the treatment of cancer in conjunction with chemoimmunotherapies.

Multifaceted modes of action of the anticancer probiotic Enterococcus hirae.

A deviated repertoire of the gut microbiome predicts resistance to cancer immunotherapy. Enterococcus hirae compensated cancer-associated dysbiosis in various tumor models. However, the mechanisms by which E. hirae restored the efficacy of cyclophosphamide administered with concomitant antibiotics remain ill defined. Here, we analyzed the multifaceted modes of action of this anticancer probiotic. Firstly, E. hirae elicited emigration of thymocytes and triggered systemic and intratumoral IFNγ-producing and CD137-expressing effector memory T cell responses. Secondly, E. hirae activated the autophagy machinery in enterocytes and mediated ATG4B-dependent anticancer effects, likely as a consequence of its ability to increase local delivery of polyamines. Thirdly, E. hirae shifted the host microbiome toward a Bifidobacteria-enriched ecosystem. In contrast to the live bacterium, its pasteurized cells or membrane vesicles were devoid of anticancer properties. These pleiotropic functions allow the design of optimal immunotherapies combining E. hirae with CD137 agonistic antibodies, spermidine, or Bifidobacterium animalis. We surmise that immunological, metabolic, epithelial, and microbial modes of action of the live E. hirae cooperate to circumvent primary resistance to therapy.

Phosphorylation of eukaryotic initiation factor-2α (eIF2α) in autophagy.

The integrated stress response is characterized by the phosphorylation of eukaryotic initiation factor-2α (eIF2α) on serine 51 by one out of four specific kinases (EIF2AK1 to 4). Here we provide three series of evidence suggesting that macroautophagy (to which we refer to as autophagy) induced by a variety of distinct pharmacological agents generally requires this phosphorylation event. First, the induction of autophagic puncta by various distinct compounds was accompanied by eIF2α phosphorylation on serine 51. Second, the modulation of autophagy by >30 chemically unrelated agents was partially inhibited in cells expressing a non-phosphorylable (S51A) mutant of eIF2α or lacking all four eIF2α kinases, although distinct kinases were involved in the response to different autophagy inducers. Third, inhibition of eIF2α phosphatases was sufficient to stimulate autophagy. In synthesis, it appears that eIF2α phosphorylation is a central event for the stimulation of autophagy.

Inhibition of transcription by dactinomycin reveals a new characteristic of immunogenic cell stress.

Chemotherapy still constitutes the standard of care for the treatment of most neoplastic diseases. Certain chemotherapeutics from the oncological armamentarium are able to trigger pre-mortem stress signals that lead to immunogenic cell death (ICD), thus inducing an antitumor immune response and mediating long-term tumor growth reduction. Here, we used an established model, built on artificial intelligence to identify, among a library of 50,000 compounds, anticancer agents that, based on their molecular descriptors, were predicted to induce ICD. This algorithm led us to the identification of dactinomycin (DACT, best known as actinomycin D), a highly potent cytotoxicant and ICD inducer that mediates immune-dependent anticancer effects in vivo. Since DACT is commonly used as an inhibitor of DNA to RNA transcription, we investigated whether other experimentally established or algorithm-selected, clinically employed ICD inducers would share this characteristic. As a common leitmotif, a panel of pharmacological ICD stimulators inhibited transcription and secondarily translation. These results establish the inhibition of RNA synthesis as an initial event for ICD induction.

ColocalizR: An open-source application for cell-based high-throughput colocalization analysis.

The microscopic assessment of the colocalization of fluorescent signals has been widely used in cell biology. Although imaging techniques have drastically improved over the past decades, the quantification of colocalization by measures such as the Pearson correlation coefficient or Manders overlap coefficient, has not changed. Here, we report the development of an R-based application that allows to (i) automatically segment cells and subcellular compartments, (ii) measure morphology and texture features, and (iii) calculate the degree of colocalization within each cell. Colocalization can thus be studied on a cell-by-cell basis, permitting to perform statistical analyses of cellular populations and subpopulations. ColocalizR has been designed to parallelize tasks, making it applicable to the analysis of large data sets. Its graphical user interface makes it suitable for researchers without specific knowledge in image analysis. Moreover, results can be exported into a wide range of formats rendering post-analysis adaptable to statistical requirements. This application and its source code are freely available at https://github.com/kroemerlab/ColocalizR.

A fluorescent biosensor-based platform for the discovery of immunogenic cancer cell death inducers.

Systemic anticancer immunity can be reinstated via the induction of immunogenic cell death (ICD) in malignant cells. Thus, certain classes of cytotoxic compounds, for example, anthracyclines, oxaliplatin and taxanes are endowed with the capacity to act on cancer cells to ignite premortem stress pathways that lead to the surface exposure of calreticulin (CALR) and the cellular release of adenosine triphosphate, annexin A1, high mobility group B1 and type-1 interferons. Altogether, these alterations constitute the hallmarks of ICD. Here we report the design of a discovery pipeline for the identification of novel ICD inducers by means of a phenotypic screening platform. The use of fluorescent biosensors as proxies for the manifestation of ICD hallmarks has enabled the exploration of large collections of chemical compounds by automatized screening routines. Imaging-based assessment and phenotypic selection led to the identification of potential ICD inducers that could be validated further invitro and invivo, confirming that bona fide ICD inducers possess the capacity to induce immunological long-term memory and to confer resistance against rechallenge with syngeneic tumors. Machine learning algorithms analyzing the physicochemical properties of ICD inducers can assist in the preselection of compounds with potential ICD-stimulatory properties, further accelerating the screening efforts designed to develop new immunotherapeutic agents.

Squaramide-based synthetic chloride transporters activate TFEB but block autophagic flux.

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.

Author Correction: Squaramide-based synthetic chloride transporters activate TFEB but block autophagic flux.

In the version of this article originally submitted, it was stated that the first three authors (Shaoyi_ Than, Yan Wang, Wei Xie) had contributed equally. However, in the published version this information was missing.