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.

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.

Ether lipids influence cancer cell fate by modulating iron uptake.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with a high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, leading directly to significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

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.

Cooperative pro-tumorigenic adaptation to oncogenic RAS through epithelial-to-mesenchymal plasticity.

In breast cancers, aberrant activation of the RAS/MAPK pathway is strongly associated with mesenchymal features and stemness traits, suggesting an interplay between this mitogenic signaling pathway and epithelial-to-mesenchymal plasticity (EMP). By using inducible models of human mammary epithelial cells, we demonstrate herein that the oncogenic activation of RAS promotes ZEB1-dependent EMP, which is necessary for malignant transformation. Notably, EMP is triggered by the secretion of pro-inflammatory cytokines from neighboring RAS-activated senescent cells, with a prominent role for IL-6 and IL-1α. Our data contrast with the common view of cellular senescence as a tumor-suppressive mechanism and EMP as a process promoting late stages of tumor progression in response to signals from the tumor microenvironment. We highlighted here a pro-tumorigenic cooperation of RAS-activated mammary epithelial cells, which leverages on oncogene-induced senescence and EMP to trigger cellular reprogramming and malignant transformation.

A druggable copper-signalling pathway that drives inflammation.

Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

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.

Effects of iron modulation on mesenchymal stem cell-induced drug resistance in estrogen receptor-positive breast cancer.

Patients with estrogen receptor-positive (ER+) breast cancer, the most common subtype, remain at risk for lethal metastatic disease years after diagnosis. Recurrence arises partly because tumor cells in bone marrow become resistant to estrogen-targeted therapy. Here, we utilized a co-culture model of bone marrow mesenchymal stem cells (MSCs) and ER+ breast cancer cells to recapitulate interactions of cancer cells in bone marrow niches. ER+ breast cancer cells in direct contact with MSCs acquire cancer stem-like (CSC) phenotypes with increased resistance to standard antiestrogenic drugs. We confirmed that co-culture with MSCs increased labile iron in breast cancer cells, a phenotype associated with CSCs and disease progression. Clinically approved iron chelators and in-house lysosomal iron-targeting compounds restored sensitivity to antiestrogenic therapy. These findings establish iron modulation as a mechanism to reverse MSC-induced drug resistance and suggest iron modulation in combination with estrogen-targeted therapy as a promising, translatable strategy to treat ER+ breast cancer.

Small Molecule Inhibitors of Interferon-Induced JAK-STAT Signalling.

Interferons (IFN) are cytokines which, upon binding to cell surface receptors, trigger a series of downstream biochemical events including Janus Kinase (JAK) activation, phosphorylation of Signal Transducer and Activator of Transcription protein (STAT), translocation of pSTAT to the nucleus and transcriptional activation. Dysregulated IFN signalling has been linked to cancer progression and auto-immune diseases. Here, we report the serendipitous discovery of a small molecule that blocks IFNγ activation of JAK-STAT signalling. Further lead optimisation gave rise to a potent and more selective analogue that exerts its activity by a mechanism consistent with direct IFNγ targeting in vitro, which reduces bleeding in model of haemorrhagic colitis in vivo. This first-in-class small molecule also inhibits type I and III IFN-induced STAT phosphorylation in vitro. Our work provides the basis for the development of pan-IFN inhibitory drugs.

BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells.

Replication stress (RS) has a pivotal role in tumor initiation, progression, or therapeutic resistance. In this study, we depicted the mechanism of breast cancer stem cells' (bCSCs) response to RS and its clinical implication. We demonstrated that bCSCs present a limited level of RS compared with non-bCSCs in patient samples. We described for the first time that the spatial nuclear location of BMI1 protein triggers RS response in breast cancers. Hence, in bCSCs, BMI1 is rapidly located to stalled replication forks to recruit RAD51 and activate homologous-recombination machinery, whereas in non-bCSCs BMI1 is trapped on demethylated 1q12 megasatellites precluding effective RS response. We further demonstrated that BMI1/RAD51 axis activation is necessary to prevent cisplatin-induced DNA damage and that treatment of patient-derived xenografts with a RAD51 inhibitor sensitizes tumor-initiating cells to cisplatin. The comprehensive view of replicative-stress response in bCSC has profound implications for understanding and improving therapeutic resistance.

Targeting Cellular Iron Homeostasis with Ironomycin in Diffuse Large B-cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy. Although more than half of patients with DLBCL achieve long-term remission, the majority of remaining patients succumb to the disease. As abnormal iron homeostasis is implicated in carcinogenesis and the progression of many tumors, we searched for alterations in iron metabolism in DLBCL that could be exploited to develop novel therapeutic strategies. Analysis of the iron metabolism gene expression profile of large cohorts of patients with DLBCL established the iron score (IS), a gene expression-based risk score enabling identification of patients with DLBCL with a poor outcome who might benefit from a suitable targeted therapy. In a panel of 16 DLBCL cell lines, ironomycin, a promising lysosomal iron-targeting small molecule, inhibited DLBCL cell proliferation at nanomolar concentrations compared with typical iron chelators. Ironomycin also induced significant cell growth inhibition, ferroptosis, and autophagy. Ironomycin treatment resulted in accumulation of DNA double-strand breaks, delayed progression of replication forks, and increased RPA2 phosphorylation, a marker of replication stress. Ironomycin significantly reduced the median number of viable primary DLBCL cells of patients without major toxicity for nontumor cells from the microenvironment and presented low toxicity in hematopoietic progenitors compared with conventional treatments. Significant synergistic effects were also observed by combining ironomycin with doxorubicin, BH3 mimetics, BTK inhibitors, or Syk inhibitors. Altogether, these data demonstrate that a subgroup of high-risk patients with DLBCL can be identified with the IS that can potentially benefit from targeting iron homeostasis. SIGNIFICANCE: Iron homeostasis represents a potential therapeutic target for high-risk patients with DLBCL that can be targeted with ironomycin to induce cell death and to sensitize tumor cells to conventional treatments.

Persister cancer cells: Iron addiction and vulnerability to ferroptosis.

Ferroptosis is a unique type of non-apoptotic cell death resulting from the unrestrained occurrence of peroxidized phospholipids, which are subject to iron-mediated production of lethal oxygen radicals. This cell death modality has been detected across many organisms, including in mammals, where it can be used as a defense mechanism against pathogens or even harnessed by T cells to sensitize tumor cells toward effective killing. Conversely, ferroptosis is considered one of the main cell death mechanisms promoting degenerative diseases. Emerging evidence suggests that ferroptosis represents a vulnerability in certain cancers. Here, we critically review recent advances linking ferroptosis vulnerabilities of dedifferentiating and persister cancer cells to the dependency of these cells on iron, a potential Achilles heel for small-molecule intervention. We provide a perspective on the mechanisms reliant on iron that contribute to the persister cancer cell state and how this dependency may be exploited for therapeutic benefits.

Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death.

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. SIGNIFICANCE: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.

Transcription/Replication Conflicts in Tumorigenesis and Their Potential Role as Novel Therapeutic Targets in Multiple Myeloma.

Plasma cells (PCs) have an essential role in humoral immune response by secretion of antibodies, and represent the final stage of B lymphocytes differentiation. During this differentiation, the pre-plasmablastic stage is characterized by highly proliferative cells that start to secrete immunoglobulins (Igs). Thus, replication and transcription must be tightly regulated in these cells to avoid transcription/replication conflicts (TRCs), which could increase replication stress and lead to genomic instability. In this review, we analyzed expression of genes involved in TRCs resolution during B to PC differentiation and identified 41 genes significantly overexpressed in the pre-plasmablastic stage. This illustrates the importance of mechanisms required for adequate processing of TRCs during PCs differentiation. Furthermore, we identified that several of these factors were also found overexpressed in purified PCs from patients with multiple myeloma (MM) compared to normal PCs. Malignant PCs produce high levels of Igs concomitantly with cell cycle deregulation. Therefore, increasing the TRCs occurring in MM cells could represent a potent therapeutic strategy for MM patients. Here, we describe the potential roles of TRCs resolution factors in myelomagenesis and discuss the therapeutic interest of targeting the TRCs resolution machinery in MM.