Autophagy-inducing nutritional interventions in experimental and clinical oncology.

Numerous pro-autophagic dietary interventions are being investigated for their potential cancer-preventive or therapeutic effects. This applies to different fasting regimens, methionine restriction and ketogenic diets. In addition, the supplementation of specific micronutrients such as nicotinamide (vitamin B3) or spermidine induces autophagy. In humans, leanness, plant-based diets (that may lead to partial methionine restriction) and high dietary uptake of spermidine are associated with a low incidence of cancers. Moreover, clinical trials have demonstrated the capacity of nicotinamide to prevent non-melanoma skin carcinogenesis. Multiple interventional trials are evaluating the capacity of autophagy-inducing regimens to improve the outcome of chemotherapy and immunotherapy. Here, we discuss the mechanistic underpinnings of autophagy induction by nutritional interventions, as well as the mechanisms through which autophagy induction in malignant or immune cells improves anticancer immunosurveillance.

Immunostimulatory effects of vitamin B5 improve anticancer immunotherapy.

Vitamin B5 (panthotenic acid), the precursor of coenzyme A (CoA), is contained in most food items and is produced by the intestinal microbiota. A recent study published in Cell Metabolism reports that vitamin B5 and CoA favor the differentiation of CD8+ cytotoxic T cells into interleukin-22 (IL-22)-producing Tc22 cells, likely through fueling mitochondrial metabolism. Importantly, in a small cohort of melanoma patients, the plasma levels of vitamin B5 positively correlate with responses to PD-1-targeted immunotherapy. Moreover, in mice, supplementation with vitamin B5 increases the efficacy of PD-L1-targeted cancer immunotherapy, and in vitro culture of T cells with CoA enhances their antitumor activity upon adoptive transfer into mice. These finding suggest that vitamin B5 is yet another B vitamin that stimulates anti-cancer immunosurveillance.

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.

Regulatory approval of photoimmunotherapy: photodynamic therapy that induces immunogenic cell death.

In September 2020, the Japanese government approved cetuximab saratolacan (previously known as RM-1929, commercial name: Akalux) for the treatment of unresectable locally advanced or recurrent head and neck cancer. Cetuximab saratolacan is a chemical conjugate of the photosensitizer IR700 with cetuximab, which targets EGFR. The treatment consists in the intravenous injection of cetuximab saratolacan, which binds to head and neck cancer cells expressing high levels of EGFR, followed by illumination of the tumor with red light (690 nm) for photodynamic therapy. This approach causes immunogenic cell death in malignant tissues, thus triggering a potent anticancer immune response.

Combination treatments with hydroxychloroquine and azithromycin are compatible with the therapeutic induction of anticancer immune responses.

Amid controversial reports that COVID-19 can be treated with a combination of the antimalarial drug hydroxychloroquine (HCQ) and the antibiotic azithromycin (AZI), a clinical trial (ONCOCOVID, NCT04341207) was launched at Gustave Roussy Cancer Campus to investigate the utility of this combination therapy in cancer patients. In this preclinical study, we investigated whether the combination of HCQ+AZI would be compatible with the therapeutic induction of anticancer immune responses. For this, we used doses of HCQ and AZI that affect whole-body physiology (as indicated by a partial blockade in cardiac and hepatic autophagic flux for HCQ and a reduction in body weight for AZI), showing that their combined administration did not interfere with tumor growth control induced by the immunogenic cell death inducer oxaliplatin. Moreover, the HCQ+AZI combination did not affect the capacity of a curative regimen (cisplatin + crizotinib + PD-1 blockade) to eradicate established orthotopic lung cancers in mice. In conclusion, it appears that HCQ+AZI does not interfere with the therapeutic induction of therapeutic anticancer immune responses.

On-target versus off-target effects of drugs inhibiting the replication of SARS-CoV-2.

The current epidemic of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for the development of inhibitors of viral replication. Here, we performed a bioinformatic analysis of published and purported SARS-CoV-2 antivirals including imatinib mesylate that we found to suppress SARS-CoV-2 replication on Vero E6 cells and that, according to the published literature on other coronaviruses is likely to act on-target, as a tyrosine kinase inhibitor. We identified a cluster of SARS-CoV-2 antivirals with characteristics of lysosomotropic agents, meaning that they are lipophilic weak bases capable of penetrating into cells. These agents include cepharentine, chloroquine, chlorpromazine, clemastine, cloperastine, emetine, hydroxychloroquine, haloperidol, ML240, PB28, ponatinib, siramesine, and zotatifin (eFT226) all of which are likely to inhibit SARS-CoV-2 replication by non-specific (off-target) effects, meaning that they probably do not act on their 'official' pharmacological targets, but rather interfere with viral replication through non-specific effects on acidophilic organelles including autophagosomes, endosomes, and lysosomes. Imatinib mesylate did not fall into this cluster. In conclusion, we propose a tentative classification of SARS-CoV-2 antivirals into specific (on-target) versus non-specific (off-target) agents based on their physicochemical characteristics.

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy.

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.

Crizotinib - a tyrosine kinase inhibitor that stimulates immunogenic cell death.

Crizotinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of non-small cell lung cancers (NSLCL) and lymphomas expressing activating translocations or mutations of oncogenic tyrosine kinases (in particular ALK and ROS1). We recently observed that high-dose (final concentration in vivo: ~10 µM) crizotinib can induce immunogenic cell death (ICD) in cancer cells lacking ALK/ROS1 activation through off-target effects that require the inhibition of several other tyrosine kinases. When combined with cisplatin (which alone does not induce ICD), crizotinib sensitizes NSCLC models to subsequent immunotherapy with PD-1 blockade, allowing to cure more than 90% of established orthotopic cancers. Of note, simultaneous treatment of mice with cisplatin, crizotinib and PD-1 blocking antibodies causes acute hepatotoxicity that can be avoided by a sequential regimen involving initial treatment with cisplatin plus crizotinib, followed by PD-1 blockade one week later. It will be important to translate these results obtained in mice into a clinical trial in NSCLC patients.

The flavonoid 4,4'-dimethoxychalcone promotes autophagy-dependent longevity across species.

Ageing constitutes the most important risk factor for all major chronic ailments, including malignant, cardiovascular and neurodegenerative diseases. However, behavioural and pharmacological interventions with feasible potential to promote health upon ageing remain rare. Here we report the identification of the flavonoid 4,4'-dimethoxychalcone (DMC) as a natural compound with anti-ageing properties. External DMC administration extends the lifespan of yeast, worms and flies, decelerates senescence of human cell cultures, and protects mice from prolonged myocardial ischaemia. Concomitantly, DMC induces autophagy, which is essential for its cytoprotective effects from yeast to mice. This pro-autophagic response induces a conserved systemic change in metabolism, operates independently of TORC1 signalling and depends on specific GATA transcription factors. Notably, we identify DMC in the plant Angelica keiskei koidzumi, to which longevity- and health-promoting effects are ascribed in Asian traditional medicine. In summary, we have identified and mechanistically characterised the conserved longevity-promoting effects of a natural anti-ageing drug.

Identification of pharmacological inhibitors of conventional protein secretion.

The retention using selective hooks (RUSH) system allows to withhold a fluorescent biosensor such as green fluorescent protein (GFP) fused to a streptavidin-binding peptide (SBP) by an excess of streptavidin molecules that are addressed to different subcellular localizations. Addition of biotin competitively disrupts this interaction, liberating the biosensor from its hook. We constructed a human cell line co-expressing soluble secretory-SBP-GFP (ss-SBP-GFP) and streptavidin within the endoplasmic reticulum (ER) lumen and then used this system to screen a compound library for inhibitors of the biotin-induced release of ss-SBP-GFP via the conventional Golgi-dependent protein secretion pathway into the culture supernatant. We identified and validated a series of molecularly unrelated drugs including antianginal, antidepressant, anthelmintic, antipsychotic, antiprotozoal and immunosuppressive agents that inhibit protein secretion. These compounds vary in their capacity to suppress protein synthesis and to compromise ER morphology and Golgi integrity, as well as in the degree of reversibility of such effects. In sum, we demonstrate the feasibility and utility of a novel RUSH-based phenotypic screening assay.

Identification of pharmacological agents that induce HMGB1 release.

The translocation of the protein high mobility group box 1 (HMGB1) from the nucleus to the cytoplasm and its secretion or passive release through the permeabilized plasma membrane, constitutes a major cellular danger signal. Extracellular HMGB1 can interact with pattern recognition receptors to stimulate pro-inflammatory and immunostimulatory pathways. Here, we developed a screening assay to identify pharmacological agents endowed with HMGB1 releasing properties. For this, we took advantage of the "retention using selective hooks" (RUSH) system in which a streptavidin-NLS3 fusion protein was used as a nuclear hook to sequestrate streptavidin-binding peptide (SBP) fused with HMGB1 and green fluorescent protein (GFP). When combined with biotin, which competitively disrupts the interaction between streptavidin-NLS3 and HMGB1-SBP-GFP, immunogenic cell death (ICD) inducers such as anthracyclines were able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. This system, was used in a high-content screening (HCS) campaign for the identification of HMGB1 releasing agents. Hits fell into three functional categories: known ICD inducers, microtubule inhibitors and epigenetic modifiers. These agents induced ICD through a panoply of distinct mechanisms. Their effective action was confirmed by multiple methods monitoring nuclear, cytoplasmic and extracellular HMGB1 pools, both in cultured human or murine cells, as well as in mouse plasma.

[Antitumoral immunization during cancer chemotherapy]. / Immunisation par les chimiothérapies anti- cancéreuses: le point en 2012.

Most anticancer agents are thought to act through direct induction of tumoral, stromal and endothelial cell death by apoptosis or necrosis. In a 2008 issue of Bulletin de l'Académie Nationale de Médecine, we described an alternative (or complementary) theory whereby the immune system participates in the antitumoral effects of some chemotherapy or radiotherapy regimens by promoting an immunogenic cell death pathway. In particular, we showed the critical importance of two pre-mortem stressors that determine the immunogenicity of dying tumor cells. The first, an ER stress response culminating in calreticuline exposure at the tumor cell surface, is mandatory for the uptake and efficient phagocytosis of apoptotic bodies by dendritic cells. In the second, autophagy leads to the release of ATP by dying tumor cells, resulting in the recruitment of inflammatory phagocytes and antigen-presenting cells, and also triggering the inflammasome that causes IL-1beta release and CD8+ T cell polarization. The tumor microenvironment changes following chemotherapy, favoring sequential accumulation of a series of innate and cognate effectors that act in a coordinated fashion to promote tumor eradication. These findings will help to identify immune predictors of the response to conventional anticancer treatments and to design innovative combinatorial immunochemotherapy regimens.

Cell death assays for drug discovery.

Cell death has an important role in many human diseases, and strategies aimed at modulating the associated pathways have been successfully applied to treat various disorders. Indeed, several clinically promising cytotoxic and cytoprotective agents with potential applications in cancer, ischaemic and neurodegenerative diseases have recently been identified by high-throughput screening (HTS), based on appropriate cell death assays. Given that different cell death modalities may be dysregulated in different diseases, it is becoming increasingly clear that such assays need to not only quantify the extent of cell death, but they must also be able to distinguish between the various pathways. Here, we systematically describe approaches to accurately quantify distinct cell death pathways, discuss their advantages and pitfalls, and focus on those techniques that are amenable to HTS.