MAIT cells monitor intestinal dysbiosis and contribute to host protection during colitis.

Intestinal inflammation shifts microbiota composition and metabolism. How the host monitors and responds to such changes remains unclear. Here, we describe a protective mechanism by which mucosal-associated invariant T (MAIT) cells detect microbiota metabolites produced upon intestinal inflammation and promote tissue repair. At steady state, MAIT ligands derived from the riboflavin biosynthesis pathway were produced by aerotolerant bacteria residing in the colonic mucosa. Experimental colitis triggered luminal expansion of riboflavin-producing bacteria, leading to increased production of MAIT ligands. Modulation of intestinal oxygen levels suggested a role for oxygen in inducing MAIT ligand production. MAIT ligands produced in the colon rapidly crossed the intestinal barrier and activated MAIT cells, which expressed tissue-repair genes and produced barrier-promoting mediators during colitis. Mice lacking MAIT cells were more susceptible to colitis and colitis-driven colorectal cancer. Thus, MAIT cells are sensitive to a bacterial metabolic pathway indicative of intestinal inflammation.

Inherited C-terminal TREX1 variants disrupt homology-directed repair to cause senescence and DNA damage phenotypes in Drosophila, mice, and humans.

Age-related microangiopathy, also known as small vessel disease (SVD), causes damage to the brain, retina, liver, and kidney. Based on the DNA damage theory of aging, we reasoned that genomic instability may underlie an SVD caused by dominant C-terminal variants in TREX1, the most abundant 3'-5' DNA exonuclease in mammals. C-terminal TREX1 variants cause an adult-onset SVD known as retinal vasculopathy with cerebral leukoencephalopathy (RVCL or RVCL-S). In RVCL, an aberrant, C-terminally truncated TREX1 mislocalizes to the nucleus due to deletion of its ER-anchoring domain. Since RVCL pathology mimics that of radiation injury, we reasoned that nuclear TREX1 would cause DNA damage. Here, we show that RVCL-associated TREX1 variants trigger DNA damage in humans, mice, and Drosophila, and that cells expressing RVCL mutant TREX1 are more vulnerable to DNA damage induced by chemotherapy and cytokines that up-regulate TREX1, leading to depletion of TREX1-high cells in RVCL mice. RVCL-associated TREX1 mutants inhibit homology-directed repair (HDR), causing DNA deletions and vulnerablility to PARP inhibitors. In women with RVCL, we observe early-onset breast cancer, similar to patients with BRCA1/2 variants. Our results provide a mechanistic basis linking aberrant TREX1 activity to the DNA damage theory of aging, premature senescence, and microvascular disease.

Activation of lysosomal iron triggers ferroptosis in cancer.

Iron catalyses the oxidation of lipids in biological membranes and promotes a form of cell death referred to as ferroptosis1-3. Identifying where this chemistry takes place in the cell can inform the design of drugs capable of inducing or inhibiting ferroptosis in various disease-relevant settings. Whereas genetic approaches have revealed underlying mechanisms of lipid peroxide detoxification1,4,5, small molecules can provide unparalleled spatiotemporal control of the chemistry at work6. Here, we show that the ferroptosis inhibitor liproxstatin-1 (Lip-1) exerts a protective activity by inactivating iron in lysosomes. Based on this, we designed the bifunctional compound fentomycin that targets phospholipids at the plasma membrane and activates iron in lysosomes upon endocytosis, promoting oxidative degradation of phospholipids and ferroptosis. Fentomycin effectively kills primary sarcoma and pancreatic ductal adenocarcinoma cells. It acts as a lipolysis-targeting chimera (LIPTAC), preferentially targeting iron-rich CD44high cell-subpopulations7,8 associated with the metastatic disease and drug resistance9,10. Furthermore, we demonstrate that fentomycin also depletes CD44high cells in vivo and reduces intranodal tumour growth in an immunocompetent murine model of breast cancer metastasis. These data demonstrate that lysosomal iron triggers ferroptosis and that lysosomal iron redox chemistry can be exploited for therapeutic benefits.

PSL Chemical Biology Symposia: Recent Progress in Ferroptosis.

This symposium is the 5th PSL (Paris Sciences & Lettres) Chemical Biology meeting (2015, 2016, 2019, 2023, 2024) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition hosted around 150 participants and was focused on the burgeoning field of ferroptosis, its mechanism and implications in health and disease. While not initially planned, it was felt that the next large Ferroptosis venue (CSHA, China) would not happen before late 2024. A discussion involving Conrad, Birsoy, Ubellacker, Brabletz and Rodriguez next to lake Como in Italy sponsored by the DKFZ, prompted us to fill in this gap and to organize a Ferroptosis meeting in Paris beforehand.

Protocol to expand and CRISPR-Cas9 genomic edit murine MAIT cells for subsequent in vivo studies.

Generating knockout mice for target molecules in specific T cell populations, without subset-specific promoters, is time-consuming and costly. Here, we describe steps for enriching mucosal-associated invariant T cells from the thymus, expanding them in vitro and performing a CRISPR-Cas9 knockout. We then detail procedure for injecting the knockout cells into wounded Cd3ε-/- mice and characterizing them in the skin. For complete details on the use and execution of this protocol, please refer to du Halgouet et al. (2023).1.

PSL Chemical Biology Symposia Third Edition: A Branch of Science in its Explosive Phase.

This symposium is the third PSL (Paris Sciences & Lettres) Chemical Biology meeting (2016, 2019, 2023) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette (2013, 2014), under the directorship of Professor Max Malacria, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition was postponed twice for the reasons that we know. This has given us the opportunity to invite additional speakers of great standing. This year, Institut Curie hosted around 300 participants, including 220 on site and over 80 online. The pandemic has had, at least, the virtue of promoting online meetings, which we came to realize is not perfect but has its own merits. In particular, it enables those with restricted time and resources to take part in events and meetings, which can now accommodate unlimited participants. We apologize to all those who could not attend in person this time due to space limitation at Institut Curie.

Role of MR1-driven signals and amphiregulin on the recruitment and repair function of MAIT cells during skin wound healing.

Tissue repair processes maintain proper organ function following mechanical or infection-related damage. In addition to antibacterial properties, mucosal associated invariant T (MAIT) cells express a tissue repair transcriptomic program and promote skin wound healing when expanded. Herein, we use a human-like mouse model of full-thickness skin excision to assess the underlying mechanisms of MAIT cell tissue repair function. Single-cell RNA sequencing analysis suggested that skin MAIT cells already express a repair program at steady state. Following skin excision, MAIT cells promoted keratinocyte proliferation, thereby accelerating healing. Using skin grafts, parabiosis, and adoptive transfer experiments, we show that MAIT cells migrated into the wound in a T cell receptor (TCR)-independent but CXCR6 chemokine receptor-dependent manner. Amphiregulin secreted by MAIT cells following excision promoted wound healing. Expression of the repair function was probably independent of sustained TCR stimulation. Overall, our study provides mechanistic insights into MAIT cell wound healing function in the skin.

Iron-Sensitive Prodrugs That Trigger Active Ferroptosis in Drug-Tolerant Pancreatic Cancer Cells.

Persister cancer cells represent rare populations of cells resistant to therapy. Cancer cells can exploit epithelial-mesenchymal plasticity to adopt a drug-tolerant state that does not depend on genetic alterations. Small molecules that can interfere with cell plasticity or kill cells in a cell state-dependent manner are highly sought after. Salinomycin has been shown to kill cancer cells in the mesenchymal state by sequestering iron in lysosomes, taking advantage of the iron addiction of this cell state. Here, we report the chemo- and stereoselective synthesis of a series of structurally complex small molecule chimeras of salinomycin derivatives and the iron-reactive dihydroartemisinin. We show that these chimeras accumulate in lysosomes and can react with iron to release bioactive species, thereby inducing ferroptosis in drug-tolerant pancreatic cancer cells and biopsy-derived organoids of pancreatic ductal adenocarcinoma. This work paves the way toward the development of new cancer medicines acting through active ferroptosis.

Expeditive Synthesis of Potent C20-epi-Amino Derivatives of Salinomycin against Cancer Stem-Like Cells.

As a continuation of our studies toward the development of small molecules to selectively target cancer stem cells (CSCs), a library of 18 novel derivatives of salinomycin (Sal), a naturally occurring polyether ionophore, was synthesized with a good overall yield using a one-pot Mitsunobu-Staudinger procedure. Compared to the parent structure, the newly synthesized products contained the mono- or disubstituted C20-epi-amine groups. The biological activity of these compounds was evaluated against human mammary mesenchymal HMLER CD24low/CD44high cells, a well-established model of breast CSCs, and its isogenic epithelial cell line (HMLER CD24high/CD44low) lacking CSC properties. Importantly, the vast majority of Sal derivatives were characterized by low nanomolar activities, comparing favorably with previous data in the literature. Furthermore, some of these derivatives exhibited a higher selectivity for the mesenchymal state compared to the reference Sal and ironomycin, representing a promising new series of compounds with anti-CSC activity.

Rapid Access to Ironomycin Derivatives by Click Chemistry.

Salinomycin, a natural carboxylic polyether ionophore, shows a very interesting spectrum of biological activities, including selective toxicity toward cancer stem cells (CSCs). Recently, we have developed a C20-propargylamine derivative of salinomycin (ironomycin) that exhibits more potent activity in vivo and greater selectivity against breast CSCs compared to the parent natural product. Since ironomycin contains a terminal alkyne motif, it stands out as being an ideal candidate for further functionalization. Using copper-catalyzed azide-alkyne cycloaddition (CuAAC), we synthesized a series of 1,2,3-triazole analogs of ironomycin in good overall yields. The in vitro screening of these derivatives against a well-established model of breast CSCs (HMLER CD24low/CD44high) and its corresponding epithelial counterpart (HMLER CD24high/CD44low) revealed four new products characterized by higher potency and improved selectivity toward CSCs compared to the reference compound ironomycin. The present study highlights the therapeutic potential of a new class of semisynthetic salinomycin derivatives for targeting selectively the CSC niche and highlights ironomycin as a promising starting material for the development of new anticancer drug candidates.

CD8+T cell responsiveness to anti-PD-1 is epigenetically regulated by Suv39h1 in melanomas.

Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an "epigenetic checkpoint" for tumor immunity.

Small Molecule Regulators of Ferroptosis.

Ferroptosis is a dedicated mode of cell death involving iron, reactive oxygen species and lipid peroxidation. Involved in processes such as glutathione metabolism, lysosomal iron retention or interference with lipid metabolism, leading either to activation or inhibition of ferroptosis. Given the implications of ferroptosis in diseases such as cancer, aging, Alzheimer and infectious diseases, new molecular mechanisms underlying ferroptosis and small molecules regulators that target those mechanisms have prompted a great deal of interest. Here, we discuss the current scenario of small molecules modulating ferroptosis and critically assess what is known about their mechanisms of action.

Inclusion complex vs. conjugation of hydrophobic photosensitizers with ß-cyclodextrin: Improved disaggregation and photodynamic therapy efficacy against glioblastoma cells.

Self-aggregation of hydrophobic porphyrin-based photosensitizers (PSs) in aqueous biological environment decreases their bioavailability and in vivo therapeutic efficacy, which hampers their clinical use in photodynamic therapy (PDT). In the current study, we explore three new supramolecular systems based of hydrophobic PSs (i.e. 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP) or 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (P1COOH)) non-covalently or covalently attached to ß-CD. The two non-covalent solid inclusion complexes (ß-CD)2/mTHPP and [(ß-CD)/P1COOH]4 are prepared by a new co-precipitation@lyophilization combined method and the covalent conjugate ß-CD-P1 by click chemistry. The binding type effect and effectiveness on the disaggregation in aqueous medium and in vitro PDT efficacy against glioblastoma cancer cells of PSs are investigated for the three ß-CD/PS systems. The findings reveal a remarkable improvement of the disaggregation and in vitro PDT activity of these ß-CD/PS systems compared to the free PSs, except for [(ß-CD)/P1COOH]4 inclusion complex caused by J-type self-aggregation of the inclusion complex in tetrameric form. ß-CD-P1 conjugate shows the higher in vitro PDT efficacy compared to the other ß-CD/PS systems. Overall, the results indicate that the disaggregation in aqueous medium and in vitro PDT activity of hydrophobic PSs can be improved by their binding to ß-CD and the covalent binding is the best approach.

Salinomycin Derivatives Kill Breast Cancer Stem Cells by Lysosomal Iron Targeting.

Salinomycin (1) exhibits a large spectrum of biological activities including the capacity to selectively eradicate cancer stem cells (CSC), making it and its derivatives promising candidates for the development of drug leads against CSC. It has been previously shown that salinomycin and its C20-propargylamine derivative (Ironomycin (2)) accumulate in lysosomes and sequester iron in this organelle. Herein, a library of salinomycin derivatives is reported, including products of C20-amination, C1-esterification, C9-oxidation, and C28-dehydration. The biological activity of these compounds is evaluated against transformed human mammary epithelial HMLER CD24low /CD44high cells, a well-established model of breast CSC, and HMLER CD24high /CD44low cells deprived of CSC properties. Unlike other structural alterations, derivative 4, which displays a cyclopropylamine at position C20, showed a strikingly low IC50 value of 23 nm against HMLER CD24low /CD44high cells. This study provides highly selective molecules to target the CSC niche, a potential interesting advance for drug development to prevent cancer resistance.

A global approach for the development of photodynamic therapy of peritoneal metastases regardless of their origin.

Surgical management of peritoneal metastases raises the problem of the theoretical spread of the entire peritoneal surface. Intraperitoneal photodynamic therapy (IntraPDT) has been limited by the lack of specificity of photosensitizers (PS) and difficulties to bring light into the abdominal cavity. Recent data in ovarian cancer may give development opportunities for IntraPDT. Intraperitoneal PDT could be an option but the level of evidence of research in this topic must increase. Our opinion is that the most important is to have a realistic idea of what we can objectively expect from PDT and the feasibility of its daily application. At the time of personalized medicine, it is mandatory to select population eligible for a targeted PS administration and who could benefit from the process. The design of a specific PS for each subtype of cancers seems essential to avoid side effects on healthy tissue. On the contrary, our progress on lighting solutions can be beneficial for all patients with an indication of IntraPDT regardless of the origin of PM. A common lighting system developed for all cancers eligible for IntraPDT could be adapted with light source of specific wavelength to activate dedicated PS.

An Efficient Photodynamic Therapy Treatment for Human Pancreatic Adenocarcinoma.

To date, pancreatic adenocarcinoma (ADKP) is a devastating disease for which the incidence rate is close to the mortality rate. The survival rate has evolved only 2-5% in 45 years, highlighting the failure of current therapies. Otherwise, the use of photodynamic therapy (PDT), based on the use of an adapted photosensitizer (PS) has already proved its worth and has prompted a growing interest in the field of oncology. We have developed a new photosensitizer (PS-FOL/PS2), protected by a recently published patent (WO2019 016397-A1, 24 January 2019). This photosensitizer is associated with an addressing molecule (folic acid) targeting the folate receptor 1 (FOLR1) with a high affinity. Folate binds to FOLR1, in a specific way, expressed in 100% of ADKP or over-expressed in 30% of cases. The first objective of this study is to evaluate the effectiveness of this PS2-PDT in four ADKP cell lines: Capan-1, Capan-2, MiapaCa-2, and Panc-1. For this purpose, we first evaluated the gene and protein expression of FOLR1 on four ADKP cell lines. Subsequently, we evaluated PS2's efficacy in our cell lines and we assessed the impact of PDT on the secretome of cancer cells and its impact on the immune system. Finally, we evaluate the PDT efficacy on a humanized SCID mouse model of pancreatic cancer. In a very interesting way, we observed a significant increase in the proliferation of activated-human PBMC when cultured with conditioned media of ADKP cancer cells subjected to PDT. Furthermore, to evaluate in vivo the impact of this new PS, we analyzed the tumor growth in a humanized SCID mice model of pancreatic cancer. Four conditions were tested: Untreated, mice (nontreated), mice with PS (PS2), mice subjected to illumination (Light only), and mice subjected to illumination in the presence of PS (PDT). We noticed that the mice subjected to PDT presented a strong decrease in the growth of the tumor over time after illumination. Our investigations have not only suggested that PS2-PDT is an effective therapy in the treatment of PDAC but also that it activates the immune system and could be considered as a real adjuvant for anti-cancer vaccination. Thus, this new study provides new treatment options for patients in a therapeutic impasse and will provide a new arsenal in the fight against PDAC.

New Targeted Gold Nanorods for the Treatment of Glioblastoma by Photodynamic Therapy.

This study describes the employment of gold nanorods (AuNRs), known for their good reputation in hyperthermia-based cancer therapy, in a hybrid combination of photosensitizers (PS) and peptides (PP). We report here, the design and the synthesis of this nanosystem and its application as a vehicle for the selective drug delivery and the efficient photodynamic therapy (PDT). AuNRs were functionalized by polyethylene glycol, phototoxic pyropheophorbide-a (Pyro) PS, and a "KDKPPR" peptide moiety to target neuropilin-1 receptor (NRP-1). The physicochemical characteristics of AuNRs, the synthesized peptide and the intermediate PP-PS conjugates were investigated. The photophysical properties of the hybrid AuNRs revealed that upon conjugation, the AuNRs acquired the characteristic properties of Pyro concerning the extension of the absorption profile and the capability to fluoresce (Φf = 0.3) and emit singlet oxygen (ΦΔ = 0.4) when excited at 412 nm. Even after being conjugated onto the surface of the AuNRs, the molecular affinity of "KDKPPR" for NRP-1 was preserved. Under irradiation at 652 nm, in vitro assays were conducted on glioblastoma U87 cells incubated with different PS concentrations of free Pyro, intermediate PP-PS conjugate and hybrid AuNRs. The AuNRs showed no cytotoxicity in the absence of light even at high PS concentrations. However, they efficiently decreased the cell viability by 67% under light exposure. This nanosystem possesses good efficiency in PDT and an expected potential effect in a combined photodynamic/photothermal therapy guided by NIR fluorescence imaging of the tumors due to the presence of both the hyperthermic agent, AuNRs, and the fluorescent active phototoxic PS.

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology.

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment.

Use of Cyclodextrins in Anticancer Photodynamic Therapy Treatment.

Photodynamic therapy (PDT) is mainly used to destroy cancerous cells; it combines the action of three components: a photoactivatable molecule or photosensitizer (PS), the light of an appropriate wavelength, and naturally occurring molecular oxygen. After light excitation of the PS, the excited PS then reacts with molecular oxygen to produce reactive oxygen species (ROS), leading to cellular damage. One of the drawbacks of PSs is their lack of solubility in water and body tissue fluids, thereby causing low bioavailability, drug-delivery efficiency, therapeutic efficacy, and ROS production. To improve the water-solubility and/or drug delivery of PSs, using cyclodextrins (CDs) is an interesting strategy. This review describes the in vitro or/and in vivo use of natural and derived CDs to improve antitumoral PDT efficiency in aqueous media. To achieve these goals, three types of binding modes of PSs with CDs are developed: non-covalent CD⁻PS inclusion complexes, covalent CD⁻PS conjugates, and CD⁻PS nanoassemblies. This review is divided into three parts: (1) non-covalent CD-PS inclusion complexes, covalent CD⁻PS conjugates, and CD⁻PS nanoassemblies, (2) incorporating CD⁻PS systems into hybrid nanoparticles (NPs) using up-converting or other types of NPs, and (3) CDs with fullerenes as PSs.