Rationale: Tumor cell epigenetics, especially chromosome accessibility, has been reported to be closely related to the tumor immune landscape and immunotherapy. However, the exact mechanism remains unknown. Methods: Whole-exome sequencing was used to analyze 13 colorectal tumor samples treated with PD1 immunotherapy. The assays for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing were used to detect tumor cells' chromosome accessibility status and screen regulatory pathways. Results: Polybromo-1 (PBRM1) was one of the 12 genes with the highest frequency of somatic mutations associated with immunotherapy sensitivity. PBRM1/Pbrm1 deficiency in colorectal cancer promoted PD-1 immunotherapy sensitivity and chemotaxis of CD8+ T and NK cells in the microenvironment in vivo and in vitro. ATAC sequencing revealed that deletion of Pbrm1, a critical component of the SWI/SNF complex, increased chromosomal accessibility in tumor cells and triggered the release of cytokines, such as CCL5 and CXCL10, by activating the NF-κB signaling pathway. Application of ACBL1, a PROC inhibitor of PBRM1, in BALB/C mice or colorectal patient-derived tumor organoids (PDTOs) significantly promoted the sensitivity to PD1 antibody immunotherapy. Conclusions: Our study established that PBRM1/Pbrm1 deficiency was positively correlated with PD1 immunotherapeutic sensitivity in colorectal cancer. The underlying molecular mechanisms involved regulation of chromosome accessibility, activation of the NF-κB signaling pathway, and immune cell infiltration in the microenvironment. These findings identify potential molecular targets for enhancing immunotherapy for colorectal cancer.

Proteins play a central role in regulating biological functions, and various pathways regulate their synthesis and secretion. Endoplasmic reticulum-associated protein degradation (ERAD) is crucial for monitoring protein synthesis and processing unfolded or misfolded proteins in actively growing tumor cells. However, the role of the multiple ERAD complexes in liver cancer remains unclear.

The Gram-positive bacterium Listeria monocytogenes is a highly adaptable pathogen capable of causing severe foodborne infections, particularly in vulnerable populations. During infection, L. monocytogenes uses a variety of virulence factors to invade and multiply within host cells. The transcriptional regulator PrfA controls the expression of these virulence factors and is essential for the intracellular lifestyle of L. monocytogenes. Long-chain unsaturated free fatty acids (FFAs) have long been recognized for their antimicrobial activity and were recently shown to inhibit PrfA-dependent virulence gene expression in L. monocytogenes. To date, the antimicrobial and anti-virulent activities of FFAs have been primarily studied in laboratory strains. However, to fully evaluate their potential as anti-infective agents, it is essential to assess the effects of long-chain FFAs on clinically relevant isolates, including outbreak strains associated with high-fat food products. Here, we demonstrate that five different clinically relevant L. monocytogenes isolates are sensitive to the antimicrobial activity of long-chain unsaturated FFAs. Furthermore, at subinhibitory concentrations, these FFAs inhibit PrfA-regulated expression of virulence factors across all tested strains and reduce their invasive potential in non-phagocytic cells. These findings underscore the potential of long-chain unsaturated FFAs in developing new preventive strategies against L. monocytogenes strains associated with severe foodborne infections.

BACKGROUNDImmune checkpoint blockade (ICB) is an effective treatment in a subset of patients diagnosed with head and neck squamous cell carcinoma (HNSCC); however, the majority of patients are refractory.METHODSIn a nonrandomized, open-label Phase 1b clinical trial, participants with recurrent and/or metastatic (R/M) HNSCC were treated with low-dose 5-azacytidine (5-aza) daily for either 5 or 10 days in combination with durvalumab and tremelimumab after progression on ICB. The primary objective was to assess the biologically effective dose of 5-aza as determined by molecular changes in paired baseline and on-treatment tumor biopsies; the secondary objective was safety.RESULTSThirty-eight percent (3 of 8) of participants with evaluable paired tissue samples had a greater-than 2-fold increase from baseline in IFN-γ signature and CD274 (programmed cell death protein 1 ligand, PD-L1) expression within the tumor microenvironment (TME), which was associated with increased CD8+ T cell infiltration and decreased infiltration of CD4+ T regulatory cells. The mean neutrophil-to-lymphocyte ratio (NLR) decreased by greater than 50%, from 14.2 (SD 22.6) to 6.9 (SD 5.2). Median overall survival (OS) was 16.3 months (95% CI 1.9, NA), 2-year OS rate was 24.7% (95% CI: 4.5%, 53.2%), and 58% (7 of 12) of treated participants demonstrated prolonged OS of greater than 12 months.CONCLUSIONOur findings suggest that low-dose 5-aza can reprogram systemic host immune responses and the local TME to increase IFN-γ and PD-L1 expression. The increased expression of these established biomarkers correlated with prolonged OS upon ICB rechallenge.TRIAL REGISTRATIONClinicalTrials.gov NCT03019003.FUNDINGNIH/NCI P01 CA240239.

Despite advances in the development of direct KRAS inhibitors, KRAS-mutant cancers continue to exhibit resistance to the currently available therapies. Here, we identified REGγ as a mutant KRAS-associated factor that enhanced REGγ transcription through the KRAS intermediate NRF2, suggesting that the REGγ-proteasome is a potential target for pan-KRAS inhibitor development. We elucidated a mechanism involving the KRAS/NRF2/REGγ regulatory axis, which links activated KRAS to the ATP- and ubiquitin-independent proteasome. We subsequently developed RLY01, a REGγ-proteasome inhibitor that effectively suppressed tumor growth in KRAS-mutant cancer models and lung cancer organoids. Notably, the combination of RLY01 and the KRASG12C inhibitor AMG510 exhibited enhanced antitumor efficacy in KRASG12C cancer cells. Collectively, our data support the hypothesis that KRAS mutations enhance the capacity of the REGγ-proteasome by increasing REGγ expression, highlighting the potential of ubiquitin-independent proteasome inhibition as a therapeutic approach for pan-KRAS-mutant cancers.

In saline alkaline soils, microplastics inevitably form a combined stress with NaCl to limit crop growth, but the molecular mechanisms of their toxic effects remain vague and inadequate. We analyzed the molecular mechanisms underlying the response of maize seedlings to single or combined stresses of MPs and NaCl by means of combined metabolomic and transcriptomic analyses. MPs and NaCl single or combined stresses reduced plant fresh weight by 36.78 %, 50.65 % and 73.97 %, respectively. Analyses showed 2476 differentially expressed genes (DEGs) and 809 differential metabolites (DMs) for MPs, 2306 DEGs and 901 DMs for NaCl, and 2706 DEGs and 938 DMs for the combined stresses, compared to CK. Single or combined stresses mainly altered amino acid synthesis and phenylpropane biosynthetic metabolic pathways. Stress up-regulated glutamine synthetase (glnA), alanine transaminase (ALT), aspartate aminotransferase (ASP), ornithine carbamoyl transferase (argF), and glycine hydroxymethyl transferase (SHM) genes expression and promotes glutamine, 2-oxoglutarate, glutamate, fumarate, arginine, aspartate, L-isoleucine, L-valine, and serine synthesis. NaCl stimulated phenylpropanoid biosynthesis (tyrosine, 4-coumarate, and ferulate), whereas MPs decreased it. In addition, both individual or combined NaCl and MPs stress increased the expression of cinnamyl-alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) to promote sinapaldehyde synthesis. Our study provides a molecular perspective on the response of crops, such as maize, to individual or combined NaCl and MPs stress.

Alternative pre-mRNA splicing (AS) is a crucial regulatory layer of gene expression in eukaryotes. AS patterns can change in response to abiotic and biotic stress, allowing cellular functions to adapt to environmental conditions. Here, we examined the effects of cellular stress-inducing chemicals on AS-mediated gene regulation in Arabidopsis thaliana by investigating the alternatively spliced forms of SERINE-ARGININE PROTEIN30 (SRp30) and U1-70 K, encoding splicing factors, as well as ASCORBATE PEROXIDASE3 (APX3) and FOLYLPOLYGLUTAMATE SYNTHASE3 (FPGS3), encoding enzymes important for stress responses. Disrupting key cellular activities, including nitric oxide metabolism, ATPase activity, plastid function, and genome stability, affected AS patterns in Arabidopsis. Stress treatment altered the abundance of uridine-rich small nuclear RNAs (UsnRNAs), especially U1 snRNAs, which are essential non-coding RNA components of U1 small nuclear ribonucleoproteins (U1 snRNPs), suggesting that abnormalities in AS are partially mediated by changes in U1 snRNA levels. The shoot redifferentiation defectice2-1 (srd2-1) mutant defective for snRNA transcription was hypersensitive for stress treatment, since it showed changes in AS patterns at lower concentrations of stress inducers to compare with the wild type. Together, our data suggest that cellular stress can influence gene expression in plants by regulating AS, which is partially regulated by UsnRNA levels through the SRD2-mediated snRNA transcription.

Previous studies have demonstrated that silica can activate the NLRP3 inflammasome, and that macrophage phagocytosis is an essential step in this process. Although carbon is the primary component of coal dust particles, it also contains other impurities such as free silica, clay, sulfides, and carbonate minerals. Additional research is still necessary to discover if NLRP3 can be triggered due to the low silica content of coal dust particles. The purpose of this study is to investigate whether coal dust particles can induce the translation and transcription of NLRP3 inflammasome components in rat alveolar macrophages (NR8383) and whether Cytochalasin B may inhibit this process. According to the findings of our research, coal dust particles can upregulate the NLRP3 inflammasome components and IL-1β, a downstream inflammatory component. Furthermore, LPS and coal dust particles work in concert to raise the NLRP3 inflammasome components protein and transcript level in macrophages. Interestingly, the protein and transcript level of NLRP3 inflammasome components dramatically dropped when cells were concomitantly exposed to the actin polymerization inhibitor Cytochalasin B. This suggests that cellular uptake is required for coal dust particles to exert their pro-inflammatory effect.

Successful immune checkpoint inhibitor (ICI) therapy occurs in only a fraction of melanoma patients, and yet all patients are susceptible to potentially serious ICI-related side-effects. No current biomarkers robustly predict ICI treatment response in melanoma patients. In this study we sought to identify methylome and transcriptome markers which have the potential to predict immunotherapy response in melanoma patients ahead of treatment with anti-PD1 ICI monotherapy. Using Infinium MethylationEPIC microarrays, we analysed DNA methylation profiles of >850,000 CpG sites in pre-treatment melanoma tissues from patients administered anti-PD-1 monotherapy as first-line treatment. In addition, we analysed transcriptomes using RNA-seq. DNA methylation and gene expression data were then statistically compared to patient response to anti-PD1 therapy. We identified 2579 DNA hypomethylation and hypermethylation alterations correlating with melanoma response to anti-PD1 therapy. An integrative analysis of DNA methylomes and transcriptomes identified a subset of 35 loci, 13 of which were significantly differentially methylated in both initial discovery and external validation datasets. Functional enrichment analysis of hypomethylated sites (p-value <0.05) in non-responders was associated with "Formation of the cornified envelope", "Regulation of epithelial cell proliferation", and "Purine-containing compound metabolic process". We have identified novel integrated DNA methylation and gene expression markers, which correlate with anti-PD1 treatment response in melanoma patients. These findings suggest a relationship between tumour-associated genomic DNA methylation, gene expression patterns, and anti-PD1 ICI immunotherapy response in melanoma patients.

Micro/Nano-plastics (M/NPs) have emerged as a globally concerning pollutant. However, research on the phytotoxicity of M/NPs on plant secondary metabolism and the underlying molecular mechanisms is still limited. Pepper, a widely cultivated vegetable, is rich in flavonoids, which are a class of important secondary metabolites found throughout the plant kingdom with multiple biological functions. In this study, we conducted a detailed assessment of the physiological toxicity of Polyethylene Terephthalate microplastics (PET-MPs) on the growth of pepper seedlings. Results showed that PET-MPs significantly inhibited pepper growth, particularly root development. Moreover, PET-MPs exposure resulted in a burst of ROS, causing oxidative damage. KEGG pathways analysis illustrated that PET-MPs significantly altered the flavonoid biosynthesis and phenylpropanoid biosynthesis pathways at both the metabolome and transcriptome levels. Weighted gene correlation network analysis (WGCNA) identified ten structural genes and nine transcription factor genes that play pivotal roles in regulating flavonoid biosynthesis. In summary, this study elucidates the alterations in the flavonoid composition, along with the underlying gene regulatory network governing flavonoid metabolism under PET-MPs exposure in pepper. These findings enhance our comprehension of MPs pollution and provide valuable insights for the development of sustainable agro-ecosystems and food security in the future.

In cuttings, detached leaves or stems are exposed to many stresses during the root regeneration process. Here, we show that the detached Arabidopsis thaliana leaf can tolerate mild osmotic stress and still regenerate roots. Under stress conditions, wounding and stress upregulate the jasmonate (JA) signaling pathway transcription factor gene MYC2 and the abscisic acid (ABA) signaling pathway transcription factor gene ABA INSENSITIVE5 (ABI5). The MYC2-ABI5 complex upregulates the expression of β-GLUCOSIDASE18 (BGLU18), which releases ABA from ABA glucose ester, resulting in ABA accumulation in the detached leaf. Mutations in MYC2, ABI5, and BGLU18 lead to the loss of stress tolerance and defects in root regeneration under osmotic stress. The successive application of JA and ABA can enhance the root regeneration ability in Arabidopsis and poplar cuttings. Overall, the JA-mediated wound signaling pathway and the ABA-mediated stress signaling pathway collaboratively amplify ABA signals to protect root regeneration under stress conditions.

Auxin-induced xylem formation in angiosperms is negatively regulated by thermospermine, whose biosynthesis is also induced by auxin. In Arabidopsis thaliana, loss-of-function mutants of ACL5, which encodes thermospermine synthase, exhibit a dwarf phenotype accompanied by excessive xylem formation. Studies of suppressor mutants that recover from the acl5 dwarf phenotype suggest that thermospermine alleviates the inhibitory effect of an upstream open-reading frame (uORF) on the main ORF translation of SAC51 mRNA. Many suppressor mutations for acl5 have been mapped to the uORF conserved in the SAC51 family or to ribosomal protein genes, such as RPL10A, RPL4A, and RACK1A. In this study, we identified newly isolated acl5 suppressors, sac501, sac504, and sac506, which are additional alleles of RPL10A and the uORFs of SAC51 family members, SACL1 and SACL3, respectively. To investigate whether acl5-suppressor alleles of ribosomal genes broadly affect translation of uORF-containing mRNAs, we examined GUS activity in several 5'-GUS fusion constructs. Our results showed that these alleles enhanced GUS activity in SAC51 and SACL3 5'-fusion constructs but had no effect on other 5'-fusion constructs unrelated to thermospermine response. This suggests that these ribosomal proteins are specifically involved in the thermospermine-mediated regulation of mRNA translation.

Riboswitch-mediated control of gene expression without the interference of potentially immunogenic proteins is a promising approach for the development of tailor-made tools for biological research and the advancement of gene therapies. However, the current selection of applicable ligands for synthetic riboswitches is limited and strategies have mostly relied on de novo selection of aptamers. Here, we show that the bacterial xanthine I riboswitch aptamer recognizes oxypurinol, the active metabolite of the widely prescribed anti-gout drug allopurinol (Zyloprim®). We have characterized the aptamer/oxypurinol interaction and present a crystal structure of the oxypurinol-bound aptamer, revealing a binding mode similar to that of the cognate ligand xanthine. We then constructed artificial oxypurinol-responsive riboswitches that showed functionality in human cells. By optimizing splicing-based oxypurinol riboswitches using three different strategies, transgene expression could be induced by >100-fold. In summary, we have developed recombinant RNA switches enabling on-demand regulation of gene expression in response to an established and safe drug.

Global warming poses a significant threat to reproductive health of rabbits. Sustainable nutritional strategies are crucial for ensuring rabbit production and maintaining food security under these challenging conditions. This study sought to assess the protective benefits of dietary boswellia essential oil nano-emulsion (BEON) against oxidative stress, immune dysregulation, ferroptosis, and organ damage in female rabbits exposed to severe thermal stress. A total of 120 female rabbits were divided into four groups of 30 rabbits each. The rabbits were fed a basal diet supplemented with 0 (BEON0), 0.25 (BEON0.25), 0.5 (BEON0.5), and 1.0 (BEON1.0) mL of BEON per kilogram of diet. Results demonstrated that the BEON1.0 group exhibited significantly higher levels of IgG, superoxide dismutase (SOD), and glutathione peroxidase (GPx), while the BEON0.25 group showed elevated levels of IgM, catalase, and total antioxidant capacity (TAC) (P < 0.05). All BEON treatments significantly reduced malondialdehyde (MDA) levels (P < 0.01). Serum levels of progesterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were significantly elevated in the BEON0.5 and BEON1.0 groups compared to the control group (P < 0.01). A significant decrease in adipokine levels was observed in all BEON-supplemented groups compared to the control group (P < 0.05). All BEON groups demonstrated a modulation of ferroptosis pathways, characterized by decreased heat shock protein 70 (HSP70) expression and upregulated expression of glutathione peroxidase 4 (GPX4) and cystine transporter solute carrier 7A11 (SLC7A11) in ovarian tissues (P < 0.01). Furthermore, DNA methyltransferase 1 (DNMT1) expression increased in a dose-dependent manner with increasing BEON supplementation. Histological analysis revealed an improvement in the architecture of the liver, uterine horns, and ovarian tissues in rabbits fed BEON. Integrating BEON at doses of 0.5-1.0 mL/kg diet significantly improved reproductive performance in stressed female rabbits. PCA and correlation analyses demonstrated a positive correlation between BEON supplementation and immune function, reproductive hormone levels, and antioxidant status, while a negative correlation was observed with MDA and adipokine concentrations in rabbit serum. In conclusion, BEON supplementation demonstrates promise as a sustainable nutritional strategy for the rabbit industry, particularly in mitigating the challenges posed by global warming.

We aimed to restore MHC-I expression on the surface of solid tumors including breast cancer and melanoma cells to regain sensitivity to immunotherapy and suppress metastatic progression. We screened a natural compound library and identified macbecin II as a reagent that upregulates MHC-I expression and induces antigen-dependent cell death in pre-invasive and invasive breast cancer models. Furthermore, we employed active immunotherapy using engineered small extracellular vesicles from dendritic cells (DCs) as a tumor vaccine (IL2-ep13nsEV) in combination with macbecin II for personalized breast cancer treatment. We found that macbecin II induced MHC-I-dependent antigen presentation and that IL2-ep13nsEV synergized with macbecin II inducing cell death, reducing metastasis, and boosting immune cell infiltration. In addition, macbecin II potentiated the effects of anti-PD-1 immunotherapy in suppressing tumor growth and metastasis. Mechanistically, macbecin II upregulated MHC-I expression post-translationally by rescuing it from lysosomal degradation. Our findings revealed a strong efficacy of macbecin II in regulating MHC-I expression and following antigen-dependent cell death. Therefore, combining active immunotherapies and macbecin II represents an effective strategy to prevent growth and progression of solid tumors including breast cancer and melanoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality, characterized by a high rate of postoperative recurrence and poor long-term survival outcomes. Structural maintenance of chromosome 4 (SMC4) is frequently overexpressed in various types of cancer and plays a pivotal role in tumor cell growth, migration, and invasion. Bioinformatics analysis has revealed a significant correlation between the tumor-node metastasis (TNM) stage (P < 0.01) and SMC4 expression (P < 0.05), and SMC4 was associated with poor prognosis in HCC. Furthermore, SMC4 was identified as an independent prognostic factor for HCC. Ubiquitin-specific peptidase 39 (USP39) was found whether the regulation was observed to affect protein synthesis or stability through bioinformatics analysis and immunoprecipitation. The expression levels and cellular localization of SMC4 and USP39 in hepatoma cells were evaluated using quantitative real-time PCR (qPCR), western blotting, and immunohistochemistry (IHC), all of which indicated significantly elevated expression of USP39 and SMC4 in HCC. The roles of the SMC4/USP39 were further investigated through several assays, including the 3-(4,5-Dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and wound healing assay. The results demonstrated that USP39/SMC4 plays a crucial role in enhancing the viability and proliferation of HepG2 cells. Additionally, bioinformatics analysis identified ZNF207 and TIAL1 as potential target proteins of SMC4. Drug-resistant hepatoma cell lines were established, and both MTT and EdU assays were performed to assess cell viability and proliferation. The results demonstrated that HepG2/5-FU cells regained their sensitivity to 5-FU following the knockdown of SMC4. Additionally, the knockdown of either TIAL1 or ZNF207 also restored 5-FU sensitivity in HepG2/5-FU cells, effectively inhibiting cell viability and proliferation. Our study underscores the significant role of the USP39/SMC4 in HCC development and suggests that SMC4 may contribute to the regulation of drug resistance in hepatoma cell lines, potentially through interactions with TIAL1 and ZNF207.

Aging is a major risk factor for disease, and developing effective pharmaceutical interventions to improve healthspan and promote longevity has become a high priority for society. One of the molecular pathways related to longevity in various model organisms revolves around lowering AKT1 levels. This prompted our in silico drug screen for small molecules capable of mimicking the transcriptional effects of AKT1 knockdown. We found topoisomerase inhibitors as a top candidate longevity-drug class. Evaluating multiple compounds from this class in C. elegans revealed that the topoisomerase inhibitor amonafide has the greatest benefit on healthspan and lifespan. Intriguingly, the longevity effect of amonafide was not solely dependent on DAF-16/FOXO, the canonical pathway for lifespan extension via AKT1 inhibition. We performed RNA-seq on amonafide-treated worms and revealed a more youthful transcriptional signature, including the activation of diverse molecular and cellular defense pathways. We found the mitochondrial unfolded protein response (UPRmt) regulator afts-1 to be crucial for both improved healthspan and extended lifespan upon amonafide treatment. Moreover, healthspan was partially dependent on the immune response transcription factor zip-2 and the integrated stress response transcription factor atf-4. We further examined the potential of amonafide in age-related disease. Treating a C. elegans model for Parkinson's disease with amonafide improved mobility. In conclusion, we identified amonafide as a novel geroprotector, which activates mitochondrial-, pathogen-, and xenobiotic-associated defense responses that-though more studies are needed-may serve as a candidate for Parkinson's disease therapy.

Cisplatin is one of the principal chemotherapeutic agents used for esophageal cancer (EC) treatment; however, EC mortality remains high. It is therefore imperative to find new therapeutic targets and approaches to potentiate the chemotherapeutic efficacy of cisplatin. Angiotensin II receptor type 1 (AGTR1) is a potential therapeutic target in multiple cancer types. In the present study, RNA‑sequencing analysis of EC and normal esophageal tissues was performed and AGTR1 was identified as a differentially expressed gene that is markedly downregulated in recurrent and metastasized EC. AGTR1 upregulation in the esophageal squamous cell carcinoma cell lines, KYSE‑150 and EC109, promoted their chemosensitivity to cisplatin both in vitro and in vivo. Additionally, AGTR1 suppressed the metastasis‑relevant traits of EC cells, as evidenced by the reduced migration, invasion and wound healing of EC cells with higher AGTR1 expression levels. Moreover, AGTR1 overexpression in EC cells upregulated intracellular Ca2+ levels, reduced ATP levels and mitochondrial membrane potentials, which was accompanied by enhanced mitochondrial pathway apoptosis. Notably, either AGTR1 overexpression or treatments with the calcium channel blocker, fendiline, caused Ca2+ influx and promoted mitochondria‑dependent apoptosis in KYSE‑150 cells in vitro. These effects were augmented when both AGTR1 overexpression and fendiline stimulation were imposed in the absence or presence of cisplatin treatments. Furthermore, fendiline administration enhanced the chemosensitivity of cisplatin in an EC xenograft mouse model. Collectively, these findings offer an alternative treatment option and provide mechanistic insights into using fendiline to potentiate the chemotherapy efficacy of cisplatin in treating EC.

Rationale: TFEB activation is associated with prolonged survival in LUAD patients, suggesting potential benefits of TFEB agonists in LUAD treatment. In this study, we identify ginkgetin (GK), derived from Ginkgo folium, as a natural TFEB agonist, which has demonstrated promising anticancer effects in our previous research. TFEB activation has been shown to promote GPX4 degradation, inducing ferroptosis; however, the specific E3 ligases, deubiquitinating enzymes (DUBs), and types of polyubiquitination chains involved remain unclear. The unique mechanisms associated with natural compounds like GK may help elucidate the underlying biological processes. Here, we describe a novel biological event involved in the lysosomal degradation of GPX4 induced by TFEB activation through the utilization of GK. Methods: TFEB activation was induced with GK, and TFEB knockout cells were generated using CRISPR-Cas9. The activity of TFEB and its relationship with ferroptosis were assessed by immunoprecipitation, labile iron pool and lysosomal activity assays. The types of polyubiquitination chains, E3 ligases, and DUBs involved in GPX4 degradation were analyzed using LC-MS, immunoprecipitation, and immunofluorescence. These findings were further validated in an orthotopic xenograft SCID mouse model. Results: GK binds to and activates TFEB, leading to TFEB-mediated lysosomal activation and GPX4 degradation, which induces ferroptosis in LUAD cells. These effects were impaired in TFEB knockout cells. Mechanistically, K48-linked polyubiquitination of GPX4 was required for GK induced GPX4 lysosomal translocation. TFEB knockout reduced both K48-linked ubiquitination and lysosomal translocation of GPX4. Additionally, GK promotes the binding of TFEB and TRIM25. TRIM25 and USP5 were found to competitively bind to GPX4, with TFEB activation favoring TRIM25 binding to GPX4 and reducing the interaction of USP5 and GPX4. These findings were confirmed in a xenograft SCID mouse model using TFEB knockout LUAD cells. Conclusion: This study identifies, for the first time, GK as a promising TFEB agonist for LUAD treatment. TFEB activation promotes TRIM25-mediated K48-linked polyubiquitination and lysosomal degradation of GPX4, driving ferroptosis. This ferroptosis-driven mechanism offers a novel strategy to enhance ferroptosis-based anti-LUAD therapies.

Rationale: Mantle-cell lymphoma (MCL) remains an aggressive and incurable cancer. Accumulating evidence reveals that abnormal iron metabolism plays an important role in tumorigenesis and in cancer progression of many tumors. Based on these data, we searched to identify alterations of iron homeostasis in MCL that could be exploited to develop novel therapeutic strategies. Methods: Analysis of the iron metabolism gene expression profile of a cohort of patients with MCL enables the identification of patients with a poor outcome who might benefit from an iron homeostasis-targeted therapy. We analyzed the therapeutic interest of ironomycin, known to sequester iron in the lysosome and to induce ferroptosis. Results: In a panel of MCL cell lines, ironomycin inhibited MCL cell growth at nanomolar concentrations compared with conventional iron chelators. Ironomycin treatment resulted in ferroptosis induction and decreased cell proliferation rate, with a reduced percentage of cells in S-phase together with Ki67 and Cyclin D1 downregulation. Ironomycin treatment induced DNA damage response, accumulation of DNA double-strand breaks, and activated the Unfolded Protein Response (UPR). We validated the therapeutic interest of ironomycin in primary MCL cells of patients. Ironomycin demonstrated a significant higher toxicity in MCL cells compared to normal cells from the microenvironment. We tested the therapeutic interest of combining ironomycin with conventional treatments used in MCL. We identified a synergistic effect when ironomycin is combined with Ibrutinib, Bruton's tyrosine kinase (BTK) inhibitor, associated with a strong inhibition of B-Cell receptor (BCR) signaling. Conclusion: Altogether, these data underline that MCL patients my benefit from targeting iron homeostasis using ironomycin alone or in combination with conventional MCL treatments.