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Ferroptosis is a type of cell death caused by radical-driven lipid peroxidation, leading to membrane damage and rupture. Here we show that enzymatically produced sulfane sulfur (S 0 ) species, specifically hydropersulfides, scavenge endogenously generated free radicals and, thereby, suppress lipid peroxidation and ferroptosis. By providing sulfur for S 0 biosynthesis, cysteine can support ferroptosis resistance independently of the canonical GPX4 pathway. Our results further suggest that hydropersulfides terminate radical chain reactions through the formation and self-recombination of perthiyl radicals. The autocatalytic regeneration of hydropersulfides may explain why low micromolar concentrations of persulfides suffice to produce potent cytoprotective effects on a background of millimolar concentrations of glutathione. We propose that increased S 0 biosynthesis is an adaptive cellular response to radical-driven lipid peroxidation, potentially representing a primordial radical protection system. Enzymatically generated sulfane sulfur species called hydropersulfides terminate free radical chain reactions to prevent oxidative membrane damage and ferroptosis induction.

Colorectal cancer (CRC) patients have been shown to possess an altered gut microbiome. Diet is a well-established modulator of the microbiome, and thus, dietary interventions might have a beneficial effect on CRC. An attenuating effect of the ketogenic diet (KD) on CRC cell growth has been previously observed, however the role of the gut microbiome in driving this effect remains unknown. Here, we describe a reduced colonic tumor burden upon KD consumption in a CRC mouse model with a humanized microbiome. Importantly, we demonstrate a causal relationship through microbiome transplantation into germ-free mice, whereby alterations in the gut microbiota were maintained in the absence of continued selective pressure from the KD. Specifically, we identify a shift toward bacterial species that produce stearic acid in ketogenic conditions, whereas consumers were depleted, resulting in elevated levels of free stearate in the gut lumen. This microbial product demonstrates tumor-suppressing properties by inducing apoptosis in cancer cells and decreasing colonic Th17 immune cell populations. Taken together, the beneficial effects of the KD are mediated through alterations in the gut microbiome, including, among others, increased stearic acid production, which in turn significantly reduces intestinal tumor growth. Attenuating effects of the ketogenic diet on colorectal cancer (CRC) cell growth has been previously described. Here, using a mouse model of CRC with a humanized microbiome, the authors identify a shift toward gut bacterial species that produce stearic acid in ketogenic conditions, resulting in elevated levels of free stearate in the gut lumen, which they then show exhibits tumor-suppressing properties.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR‐activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN ‐amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc − . The identification of LRP8 as a specific vulnerability of MYCN ‐amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet‐unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high‐risk neuroblastoma and potentially other MYCN ‐amplified entities. Synopsis The low‐density lipoprotein receptor (LRP8) was identified as a critical suppressor of ferroptosis in MYCN‐amplified neuroblastoma. Blocking selenium/selenocysteine uptake mechanisms via LRP8 offers a selective strategy to induce ferroptosis and disrupt GPX4 function. Ferroptosis, a cell death modality, is gaining interest as a therapeutic approach against challenging tumors. GPX4 is crucial for suppressing ferroptosis, but suitable in vivo inhibitors are lacking, limiting translation to cancer therapies. Genome‐wide and single‐cell CRISPR‐activation screens reveal LRP8 as a critical ferroptosis suppressor in MYCN‐amplified neuroblastoma. Blocking selenium/selenocysteine uptake via LRP8 disrupts GPX4 function and selectively induces ferroptotic cell death. LRP8 dependency emerges as the result of the low system Xc − activity suggesting that targeting LRP8 could be explore in other entities such as AML and lymphoma. Graphical Abstract The low‐density lipoprotein receptor (LRP8) was identified as a critical suppressor of ferroptosis in MYCN‐amplified neuroblastoma. Blocking selenium/selenocysteine uptake mechanisms via LRP8 offers a selective strategy to induce ferroptosis and disrupt GPX4 function.

The mono-ADP-ribosylhydrolase MacroD1 has been recently reported to localize to mitochondria exclusively. However, the extent and means by which MacroD1 regulates metabolic homeostasis remains unclear. Here we show that the absence of MacroD1 in mice decreased mitochondrial load and negatively impacted muscle function, reducing maximal exercise capacity. Knockdown of MacroD1 in C2C12 myoblast cells amplified the production of reactive oxygen species which ultimately resulted in increased mitochondrial fission. Proteomic and metabolomic profiling showed that loss of MacroD1 re-routed metabolite flux from glucose to the pentose-phosphate cycle instead of the tricarboxylic acid cycle to support the production of antioxidants, including glutathione and NADPH. This resulted in increased glucose uptake and dependency both in vitro and in vivo. Hence, our research establishes MacroD1 as a regulator of metabolic homeostasis, which ensures the coordination of cellular carbohydrate flux and optimal mitochondrial function. Here, the authors show that MacroD1 is important for mitochondrial integrity and function. Lack of MacroD1 resulted in impaired cellular respiration which was particularly detrimental for cells and organs with high energetic requirements, such as skeletal muscle.

Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr . Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3β axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism.

Zellweger spectrum disorders (ZSDs) are rare autosomal recessive conditions belonging to the larger group of peroxisome biogenesis disorders. The most prevalent form of ZSD is caused by mutations in the PEX1 gene, which encodes an AAA ATPase protein. Cells lacking functional PEX1 fail to import proteins crucial for the formation of competent peroxisomes, resulting in aberrant structures called ghost peroxisomes . Peroxisome dysfunction leads to the accumulation of compounds that are normally metabolized in this compartment, including very long-chain fatty acids (VLCFAs), pristanic and phytanic acids, as well as deficiency in compounds that are normally formed in this organelle, including docosahexaenoic acid (DHA) and plasmalogen precursors. Patients with a complete lack of PEX1 function develop severe symptoms and have a poor prognosis, with death in the first year of life. In the absence of effective treatments for ZSD, advancing our understanding of this complex multisystem disorder remains essential for uncovering new therapeutic opportunities. To this end, we generated and characterized a zebrafish model with Pex1 loss-of-function. Surprisingly, despite the early onset of disease-relevant features, about 10% of pex1 –/– zebrafish reached adulthood. However, this resilience was short-lived, as none of the mutant fish survived beyond one year. Histopathological analysis of the liver in adult pex1 –/– mutants revealed a profound peroxisomal import deficiency and severe vacuolation. Moreover, key metabolic hallmarks of ZSDs, including accumulation of VLCFAs and methyl-branched fatty acids phytanic and pristanic acid, were consistently detected in larval and adult pex1 –/– mutants. Transcriptomics analysis in pex1 –/– larvae revealed upregulation of ER-stress responses and pexophagy, as well as dysregulation of neurophysiological processes and visual perception. The latter findings were corroborated by abnormal locomotor behavior in the larvae and by disrupted outer nuclear and retinal layer architecture in adult mutant animals. The described zebrafish pex1 model provides a versatile in vivo platform to uncover novel disease-relevant pathways in ZSD and to investigate the physiological impact of VLCFAs and methyl-branched fatty acids. Its relative tolerance to Pex1 loss-of-function circumvents the early lethality observed in mouse models, enabling the study of ZSD pathophysiology beyond early developmental stages and offering a valuable tool for preclinical therapeutic exploration.

Recent advances in the genomics of glioblastoma (GBM) led to the introduction of molecular neuropathology but failed to translate into treatment improvement. This is largely attributed to the genetic and phenotypic heterogeneity of GBM, which are considered the major obstacle to GBM therapy. Here, we use advanced human GBM-like organoid (LEGO: L aboratory E ngineered G lioblastoma-like O rganoid) models and provide an unprecedented comprehensive characterization of LEGO models using single-cell transcriptome, DNA methylome, metabolome, lipidome, proteome, and phospho-proteome analysis. We discovered that genetic heterogeneity dictates functional heterogeneity across molecular layers and demonstrates that NF1 mutation drives mesenchymal signature. Most importantly, we found that glycerol lipid reprogramming is a hallmark of GBM, and several targets and drugs were discovered along this line. We also provide a genotype-based drug reference map using LEGO-based drug screen. This study provides new human GBM models and a research path toward effective GBM therapy.

Background Targeted therapy interventions using tyrosine kinase inhibitors (TKIs) provide encouraging treatment responses in patients with ALK -rearranged lung adenocarcinomas, yet resistance occurs almost inevitably. In addition to tumor cell-intrinsic resistance mechanisms, accumulating evidence suggests that cancer-associated fibroblasts (CAFs) within the tumor microenvironment contribute to therapy resistance. This study aimed to investigate CAF-driven molecular networks that shape the therapeutic susceptibility of ALK -driven lung adenocarcinoma cells. Methods Three-dimensional (3D) spheroid co-cultures comprising ALK -rearranged lung adenocarcinoma cells and CAFs were utilized to model the tumor microenvironment. Single-cell RNA sequencing was performed to uncover transcriptional differences between TKI-treated homotypic and heterotypic spheroids. Functional assays assessed the effects of CAF-conditioned medium and CAF-secreted factors on tumor cell survival, proliferation, lipid metabolism, and downstream AKT signaling. The therapeutic potential of targeting metabolic vulnerabilities was evaluated using pharmacological inhibition of lipid metabolism and by ferroptosis induction. Results CAFs significantly diminished the apoptotic response of lung tumor cells to ALK inhibitors while simultaneously enhancing their proliferative capacity. Single-cell RNA sequencing identified lipogenesis-associated genes as a key transcriptional difference between TKI-treated homotypic and heterotypic lung tumor spheroids. CAF-conditioned medium and the CAF-secreted factors HGF and NRG1 activated AKT signaling in 3D-cultured ALK-rearranged lung tumor cells, leading to increased de novo lipogenesis and suppression of lipid peroxidation. These metabolic adaptations were critical for promoting tumor cell survival and fostering therapy resistance. Notably, both dual inhibition of ALK and the lipid-regulatory factor SREBP-1, as well as co-treatment with ferroptosis inducers such as erastin or RSL3, effectively disrupted the CAF-driven metabolic-supportive niche and restored sensitivity of resistant lung tumor spheroids to ALK inhibition. Conclusions This study highlights a critical role for CAFs in mediating resistance to ALK-TKIs by reprogramming lipid metabolism in ALK-rearranged lung cancer cells. It suggests that targeting these metabolic vulnerabilities, particularly through inhibition of lipid metabolism or induction of ferroptosis, could provide a novel therapeutic approach to overcome resistance and improve patient outcomes.

Food supplementation with a fiber mix of guar gum and chickpea flour represents a promising approach to reduce the risk of type 2 diabetes mellitus (T2DM) by attenuating postprandial glycemia. To investigate the effects on postprandial metabolic fluxes of glucose-derived metabolites in response to this fiber mix, a randomized, cross-over study was designed. Twelve healthy, male subjects consumed three different flatbreads either supplemented with 2% guar gum or 4% guar gum and 15% chickpea flour or without supplementation (control). The flatbreads were enriched with ~2% of 13C-labeled wheat flour. Blood was collected at 16 intervals over a period of 360 min after bread intake and plasma samples were analyzed by GC-MS based metabolite profiling combined with stable isotope-assisted metabolomics. Although metabolite levels of the downstream metabolites of glucose, specifically lactate and alanine, were not altered in response to the fiber mix, supplementation of 4% guar gum was shown to significantly delay and reduce the exogenous formation of these metabolites. Metabolic modeling and computation of appearance rates revealed that the effects induced by the fiber mix were strongest for glucose and attenuated downstream of glucose. Further investigations to explore the potential of fiber mix supplementation to counteract the development of metabolic diseases are warranted.

SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.