Cell Death & Disease

Iron is essential for tumor proliferation and metabolic adaptation but becomes cytotoxic when unbuffered, creating a potential metabolic vulnerability. Ferritin, a conserved iron-storage complex, limits labile iron and establishes the upper threshold of iron tolerance in cancer cells. Here, we report the first ferritin heavy chain (FTH) knockout in a brain tumor model system. Although FTH loss was tolerated under basal conditions through adaptive remodeling of iron metabolism, it exposed profound vulnerabilities under iron stress. FTH deficiency lowered the threshold for iron toxicity, sensitizing medulloblastoma (MB) cells to both canonical ferroptosis and a mechanistically distinct iron-dependent cell death pathway. Oxidative iron stress impaired tumor growth and prolonged survival in orthotopic xenografts, whereas vitamin C-induced iron reduction triggered a selective, iron-dependent, but non-ferroptotic elimination of MB-like cells in tumor organoids. Notably, sensitivity to iron toxicity correlated strongly with cellular phenotype, with mesenchymal-like cells displaying greater susceptibility than epithelial-like counterparts. Collectively, these findings identify ferritin as a central regulator of iron tolerance in MB and establish iron toxicity, not via iron deprivation, as a therapeutically exploitable vulnerability. More broadly, this work provides a mechanistic framework for targeting iron metabolism through modulation of ferritin-dependent iron buffering and iron redox homeostasis in cancers. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/739635v1_ufig1.gif" ALT="Figure 1000"> View larger version (53K): org.highwire.dtl.DTLVardef@e8ba94org.highwire.dtl.DTLVardef@1317618org.highwire.dtl.DTLVardef@4060d7org.highwire.dtl.DTLVardef@95fac6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neuroblastoma (NB) is one of the most common solid malignancies in children, and high-risk patients have poor prognosis, underscoring the need for novel therapeutic agents. The small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various protein functions, and the first-in-class SUMOylation inhibitor TAK-981 (subasumstat) is currently in clinical trials in adult cancer patients. However, the role of SUMOylation in NB pathogenesis remains poorly characterized. We show that SUMOylation-catalyzing enzymes, SAE1, SAE2, and UBE2I, are highly expressed in malignant neuroendocrine cells, and their expression correlate with poor survival in NB patients and associate with advanced stage, MYCN amplifications, and downregulation of late differentiation markers. The peripheral nervous system lineage and NB cell lines are highly sensitive to the CRISPR knockout of SAE1 and SAE2, suggesting SUMOylation as promising therapeutic target in NB. Accordingly, TAK-981-mediated inhibition of SUMOylation reduces growth of NB cells in both in vitro and in vivo models through induction of apoptosis and perturbation of differentiation-associated pathways. Sensitivity to SUMOylation inhibition is greatest in NB cells with low expression of favorable late neuroblast markers and is independent of MYCN amplification status. Moreover, TAK-981 is effective in combination with differentiation-inducing all-trans retinoic acid and the DNA methyltransferase inhibitor decitabine. Mechanistically, combination of TAK-981 and retinoic acid potently downregulates retinoic acid receptor alpha (RAR) expression, whereas inhibition of SUMOylation combined with decitabine induces a strong accumulation of DNA damage. Taken together, these findings establish SUMO pathway components as potential prognostic markers and SUMOylation as a therapeutic vulnerability in high-risk NB.

Fibroblast growth factor 2 (FGF2) is frequently induced during ischemic retinal injury and has traditionally been considered a pro-angiogenic factor based largely on studies using exogenous FGF2 administration. However, its endogenous cellular origin and physiological role remain incompletely understood. Here, we used single-cell transcriptomic analysis combined with spatial validation and rod photoreceptor-specific genetic approaches to define the endogenous role of FGF2 during oxygen-induced retinopathy (OIR). We identify rod photoreceptors as a major cellular source of ischemia-induced FGF2. Notably, Fgf2 expression remained elevated during the regression of pathological neovascularization, revealing a temporal dissociation between neuronal stress responses and vascular remodeling. Single-cell analysis further showed that Fgf2 induction occurred within a coordinated photoreceptor stress-response program involving endothelin 2 (Edn2) and B-cell lymphoma 3 (Bcl3). This transcriptional signature was independently reproduced in the N-methyl-N-nitrosourea (MNU)-induced photoreceptor degeneration model. Rod-specific deletion of Fgf2 markedly increased photoreceptor apoptosis, indicating that endogenous FGF2 contributes to photoreceptor survival under ischemic stress. In contrast, neither genetic depletion nor overexpression of FGF2 altered pathological neovascularization or vaso-obliteration. Bidirectional manipulation of FGF2 further modulated the expression of representative stress-associated genes Edn2 and Bcl3, supporting FGF2 involvement in this injury-response program. Finally, receptor expression analysis revealed relatively limited endothelial expression of Fgfr1-Fgfr4 compared with VEGF receptors, suggesting a cellular basis for the distinct effects of endogenous FGF2 and VEGF signaling. Together, these findings identify endogenous retinal FGF2 as a photoreceptor-derived survival factor that is induced during stress but is insufficient to drive pathological angiogenesis. These results support a model in which neuronal adaptation and vascular remodeling represent partially distinct responses during ischemic retinal injury.

Background Vitiligo is an acquired depigmenting disorder characterised by melanocyte loss. While immune responses against melanocytes are implicated in its pathogenesis, melanocyte detachment, melanocyte senescence and aberrant epidermal differentiation are increasingly characterised as additional pathological features. Granzyme B (GzmB), classically recognised as an intracellular effector of perforin-dependent cytotoxicity, is increased in vitiligo lesions; however, its extracellular contribution to vitiligo pathology remains unclear. Objectives To determine whether extracellular GzmB contributes to depigmentation and epidermal pathological features relevant to vitiligo, and to define its cellular source and underlying mechanisms. Methods Human vitiligo skin samples, murine depigmentation models and cultured human epidermal cells were analysed. The functional effects of extracellular GzmB were examined by subcutaneous administration of recombinant GzmB in mice. A rhododendrol (RD)-induced leukoderma model was used to evaluate the disease relevance of GzmB and the therapeutic effect of topical GzmB inhibition with VTI-1002. Results GzmB-positive cells were markedly increased in active vitiligo lesions and were predominantly associated with tryptase-positive cells, with limited co-distribution with CD8 or perforin. Vitiligo lesions also showed melanocyte detachment, increased p16INK4A-positive melanocytes and aberrant keratinocyte differentiation. These pathological features were recapitulated in vivo by extracellular GzmB administration, accompanied by focal depigmentation. In cultured melanocytes, GzmB exerted no cytotoxic effects but reduced attachment strength and induced a senescence-associated phenotype characterised by decreased extracellular matrix- and adhesion-related gene expression, increased p16INK4A level, elevated senescence-associated {beta}-galactosidase activity and activation of transforming growth factor-{beta}/SMAD signalling. In keratinocytes, extracellular GzmB promoted aberrant differentiation associated with activation of p53 signalling. Topical inhibition of GzmB in RD-induced leukoderma attenuated depigmentation progression, melanocyte detachment, melanocyte senescence and abnormal epidermal differentiation. Conclusions Extracellular GzmB promotes depigmentation associated with inducing melanocyte detachment, melanocyte senescence and aberrant keratinocyte differentiation. These findings identify extracellular GzmB as a previously underrecognised pathogenic mediator and potential therapeutic target in vitiligo-associated depigmentation.

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.

Neuroblastoma (NB) is a pediatric malignancy that predominantly affects young children and can be associated with neurological complications even in the absence of direct central nervous system (CNS) invasion. The CNS is protected by specialized endothelial barriers whose integrity depends on tightly regulated intercellular junctions and extracellular matrix homeostasis. Here, we investigated whether extracellular vesicles (EVs) released by NB cells contribute to CNS endothelial barrier dysfunction and examined the underlying molecular mechanisms.

Adverse cardiac remodeling following myocardial infarction (MI) are driven by processes including autophagy and apoptosis. While microtubule-associated protein 1S (MAP1S) is known to regulate autophagy, its role in the cardiac pathological conditions remains unclear. This study aimed to elucidate the role of MAP1S and its underlying mechanisms in pathological cardiac remodeling. Following MI, MAP1S knockout mice exhibited increased mortality, impaired cardiac function, and elevated apoptosis. Similarly, MAP1S silencing in cultured cardiomyocytes augmented apoptosis under oxidative stress. Mechanistic investigation revealed potential link to the Hippo pathway. MAP1S knockdown in cardiomyocytes increased Mammalian Ste-20 like 1/2 (MST1/2) activation and reduced Yes-associated protein (YAP) activity, potentially explaining apoptosis regulation. Conversely, MAP1S overexpression reduced apoptosis and positively modulated autophagy. Importantly, in vivo modRNA-mediated MAP1S overexpression protected against apoptosis and adverse remodeling. This study reveals that MAP1S protects the heart from excessive apoptosis and adverse remodeling following MI, likely by modulating the Hippo signaling pathway.

Mitochondrial plasticity, characterized by the dynamic balance between glycolysis and oxidative phos-phorylation in response to genetic and microenvironmental changes, is a hallmark of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations had shown that the lysosomal sphingolipid-metabolizing enzyme {beta}-galactosylceramidase (GALC) rewires the lipid profile of mouse melanoma cells, exerting pro-oncogenic functions, gene silencing leading to a decreased oncogenic activity in murine and human melanoma cells. Here, we have focused on the mitochondrial sphingolipid composition and energetic metabolism in GALC knockout (KO) A2058 human melanoma cells. Targeted analysis of the mitochondrial sphingolipid profile, transcriptomic data, and mitochondrial structural and functional studies indicate that GALC loss drives a sphingolipid-mediated reprogramming of mitochondrial metabolism in absence of major structural alterations, characterized by bioenergetic insufficiency possibly due to ceramide- and sphingomyelin-driven impairment of respiratory chain function. Overall, these data indicate that GALC KO leads to a sphin-golipid-driven mitochondrial metabolic suppression and may provide novel information for the development of efficacious approaches in mitochondrial targeting melanoma therapies.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by high metastatic dissemination, therapy resistance, and poor clinical outcome. Inhibitor of differentiation 1 or ID1, is frequently overexpressed in PDAC and is associated with tumour progression and adverse clinical outcome. However, the mechanisms governing its post-transcriptional regulation remain insufficiently characterized. Here, we identify tumour-suppressive miR-615-5p as a regulator of ID1 expression in PDAC. Integrative in-silico target prediction prioritized miR-615-5p based on seed complementarity and thermodynamic stability with the ID1 3' -UTR. Expression analysis of available PDAC clinical datasets revealed reduced miR-615-5p expression associated with increased ID1 expression. Direct association was validated using luciferase reporter assays, where miR-615-5p suppressed 3' -UTR reporter activity of ID1 in a sequence dependent manner, while mutation of the predicted binding site attenuated this effect. Further biotinylated-RIP and AGO2-RIP assays demonstrated the co-enrichment of ID1 transcripts and miR-615-5p with AGO2 associated RISC complexes, while AntimiR mediated inhibition of miR-615-5p perturbs association between miR/ID1 to AGO2, supporting interaction specificity. Functionally, modulation of miR-615-5p altered ID1 expression and impacted PDAC cell migration in vitro. Mechanistic analyses further indicated that the miR-615-5p/ID1 axis influences autophagic flux where miR-615-5p mediated inhibition of autophagy suppresses ID1 dependent cellular migration. Collectively, these findings define a previously uncharacterized miRNA-dependent regulation of ID1 expression and link this axis to autophagy-associated migratory responses in PDAC cells. The study expands the post-transcriptional regulatory landscape of ID1 and provides a possible mechanism where suppression of miR-615-5p leads to ID1 overexpression and subsequent poor clinical outcome in PDAC cells.

The cGAS-STING pathway is an innate immune system that responds to cytoplasmic double-stranded DNA, but recent studies have highlighted its noncanonical role in facilitating lysosomal stress responses. Specifically, STING activation has been shown to trigger V-ATPase-ATG16L1-induced LC3 lipidation (VAIL) and to activate LRRK2, a Parkinson disease-associated kinase, but the detailed molecular mechanism and the physiological roles of the activation have remained unclear. Here, we found that STING activation in microglia induces the translocation of LRRK2 to the pericentrosomal area, where lysosomes and recycling endosomes (REs) concentrate. This process was VAIL-dependent, and LRRK2 was found to be localized to the organelles. The translocation of LRRK2 required intact microtubule structures and was mediated by LRRK2 kinase activity, its substrate Rab35 and the motor adaptor proteins including JIP4. All these proteins were concentrated at the pericentrosomal area upon STING stimulation. The hyperactivating G2019S mutation of LRRK2 promoted the pericentrosomal translocation of these proteins. Overall, we propose a novel STING-induced noncanonical pathway that involves LRRK2 to facilitate pericentrosomal organelle positioning.

BRCA2-mutated epithelial ovarian cancer (EOC) is deficient in homologous recombination (HR) repair and hypersensitive to PARP inhibitors. However, BRCA2-mutated EOC frequently develops PARP inhibitor resistance and the underlying mechanisms involving apoptosis evasion remain poorly understood. In this study, our bioinformatic analysis of clinical transcriptomic datasets revealed that increased expression of KLF4, a zinc finger transcription factor, was strongly associated with high-grade serous EOC subtype and reduced overall survival of patients. Using isogenic EOC cells, we demonstrated that BRCA2 mutation led to pronounced KLF4 up-regulation by PARP inhibition in an ATM-dependent manner. Silencing of KLF4 and its target gene NR4A1 enhanced olaparib-induced apoptosis. Inhibition of anti-apoptotic effectors using the BH3-mimetic navitoclax, but not the SMAC-mimetic birinapant, selectively sensitized BRCA2-mutated EOC cells to olaparib. Furthermore, KLF4 silencing abrogated olaparib-induced BCL-w and BCL-xL, while olaparib-induced cIAP2 was attenuated only by NR4A1 silencing in BRCA2-mutated EOC cells. In vivo, combined treatment of navitoclax and olaparib synergized to impede the progression of BRCA2-mutated EOC xenografts and prolong mouse survival time. Collectively, our investigations discovered KLF4 as a regulatory hub of DNA damage response and apoptosis evasion in BRCA2-mutated EOC. These findings support targeting KLF4-driven anti-apoptotic pathways as a rational strategy to overcome PARP inhibitor resistance.

Leptin receptor (LEPR) signaling has been implicated in multiple malignancies; however, its role in gastric tumors remains poorly defined. We previously demonstrated that mice with gastrointestinal epithelial cell-specific deletion of suppressor of cytokine signaling 3 (SOCS3 cKO), a negative feedback regulator of LEPR signaling, develop gastric tumors due to aberrant leptin production and LEPR activation. Here, we demonstrate that concurrent deletion of both Socs3 and Lepr (double knockout; DKO) under the same promoter substantially suppresses gastric tumorigenesis and markedly prolonged survival. Whereas SOCS3 cKO mice exhibited early stromal activation, increased TGF-{beta}1 production, accumulation of cancer-associated fibroblasts (CAFs) and collagen deposition, these tumor-promoting alterations were substantially attenuated in DKO mice. Additionally, DKO mice showed reduced inflammatory cytokine and chemokine signaling, decreased the accumulation of Gr-1+CD11b+ myeloid-derived suppressor cells, and reduced LEPR and TGF-{beta} signaling. Analysis of The Cancer Genome Atlas stomach adenocarcinoma cohort revealed high LEPR expression in the chromosomal instability and genomically stable subtypes, correlating with poor prognosis. Moreover, LEPR expression was mutually exclusive with CLDN18 and ERBB2, two major therapeutic biomarkers, and positively correlated with a CAF-related transcriptional signature. Our findings identify LEPR signaling in epithelial cells as a key driver of gastric tumorigenesis through promotion of stromal activation and tumor microenvironment development. They further highlight LEPR as a promising therapeutic target for patients with gastric cancer who are unlikely to benefit from current ERBB2/HER2- or CLDN18-directed therapies.

Colorectal cancer (CRC) exhibits substantial intratumoral heterogeneity, with differences in oncogenic driver mutations and apoptotic competence contributing to therapeutic resistance. Here, we investigated whether combined celecoxib and NDGA treatment (C+N) induces redox-mediated cell death via the p62/KEAP1 axis, offering a therapeutic approach that can overcome genetic diversity in CRC. We used two genetically distinct CRC cell lines, HCT116 and HT29, harbouring KRAS and BRAF mutations respectively, with differing p53 status, as models of tumor heterogeneity. C+N exhibited profound cytotoxicity in both cell lines, independent of COX-2/5-LOX expression and apoptotic competence. Additionally, C+N induced marked oxidative stress with mitochondrial ROS, lipid peroxidation, and labile ferrous iron accumulation, visualized as distinct JC-1 green mitochondrial puncta and FerroOrange red iron puncta in both cell lines. The antioxidant NAC reversed C+N-induced death, but inhibitors of apoptosis, autophagy, necroptosis, or ferroptosis did not, indicating a mechanism distinct from these pathways. Mechanistically, the combination induced p62 Ser349 phosphorylation, promoting KEAP1 degradation and NRF2 stabilization independently of ULK1-mediated autophagy; however, NRF2 stabilization was uncoupled from HO-1 induction. Our work reveals that C+N induces a ULK1/autophagy-independent, redox-mediated cell death mechanism that circumvents apoptotic competence, highlighting redox disruption as a genotype-independent therapeutic strategy to overcome CRC heterogeneity.

Intestinal epithelial barrier is a key feature of the healthy gut, and disruption of this barrier is a driver of mucosal inflammation. Integrity of the intestinal epithelial barrier is controlled by tight junctions (TJs) and adherens junctions (AJs) associated with the underlying actin cytoskeleton. Posttranslational modifications of actins affect the cytoskeletal assembly and dynamics, however whether such modifications regulate epithelial barriers remains unknown. SETD3 methyltransferase methylates actins at the His73 residue, thereby enhancing filamentous (F)-actin polymerization and turnover. This study examines the role of SETD3 in regulating structure and function of normal intestinal epithelial barrier, as well as its involvement in barrier disruption during mucosal inflammation. siRNA mediated knockdown of SETD3 in DLD-1 and HCA-7 human intestinal epithelial cells (IEC) resulted in several functional and morphological cell abnormalities. These include increased permeability of the IEC barrier, diminished assembly of the perijunctional F-actin belt, increased cell size, and delayed TJ reassembly during calcium switch. Consistently, SETD3 knockout (KO) mice displayed increased intestinal permeability and decreased labeling intensity of apical {beta}-actin as compared to wildtype controls. Furthermore, SETD3 KO mice showed increased cell stemness and proliferation in colonic epithelium without defects in AJ/TJ assembly and secretory cell differentiation. In addition, SETD3 KO mice developed more severe disease symptoms during dextran sodium sulfate (DSS)-induced colitis, which were accompanied by accelerated mucosal erosion and increased macrophage, T cell and neutrophil infiltration into colonic tissue. Overall, our data suggests that SETD3 acts as a novel positive regulator of the intestinal epithelial barrier and plays a protective role during intestinal mucosal damage and inflammation.

Psoriasis is a chronic skin disease characterized by red, scaly plaques mainly driven by dysregulated T cells that induce keratinocytes (KCs) proliferation and inflammation. Despite significant advancements in disease management through biologics and topical therapies, these treatments offer only temporary relief and fail to prevent disease recurrence. Considering the current therapeutic needs, we described herein a novel small molecule TEM1657, derived from 3,5-dicaffeoylquinic acid with anti-inflammatory potency in imiquimod-induced psoriasis mouse model as well as in Th17-stimulated KCs. Mechanistically, its action is mediated through modulation of mitochondrial dynamics, specifically via OPA1, an inner mitochondrial membrane protein that alters mitochondrial calcium influx, and further impacts cellular bioenergetics. Moreover, TEM1657 rapidly suppresses transcription of NFKBIZ and various other psoriasis-associated genes. These observations highlighted a pathway that represents an innovative target in psoriasis and other NFKBIZ-related disorders.

The association of cytoplasmic localization of VDR in TNBC tumors and disease aggressivity prompted investigation of its regulation by XPO1. In TNBC cell lines, the XPO1 inhibitors selinexor and eltanexor increased nuclear VDR localization. Their effects were particularly strong when combined with vitamin D treatment. Similarly, XPO1 knockdown increased nuclear VDR levels, both alone and in combination with vitamin D. These results identify XPO1 as an important regulator of VDR subcellular distribution. XPO1 inhibition also enhanced vitamin D-dependent VDR transcriptional activity. Indeed, selinexor, eltanexor, and XPO1 silencing increased vitamin D-dependent CYP24A1 reporter activity and the expression of genes such as CYP24A1 or CDH1. RNA sequencing showed that XPO1 modulation markedly reshaped the vitamin D transcriptional response. The affected genes were mainly involved in metabolism, cell-cycle regulation, protein localization, and cellular organization. Functional analyses demonstrated that cytoplasmic VDR promoted TNBC cell proliferation. Conversely, nuclear VDR was required for the antiproliferative effects of vitamin D. Accordingly, XPO1 inhibition or depletion potentiated vitamin D-mediated growth suppression. This finding suggested functional cooperation between XPO1-dependent export and VDR activity. Finally, high VDR expression was associated with poor disease-free or overall survival only in tumors with high XPO1 expression. Overall, XPO1 negatively regulates VDR nuclear localization and vitamin D responsiveness in TNBC. Targeting XPO1 may therefore enhance the therapeutic potential of vitamin D-based strategies in TNBC.

Neuroblastoma is a neural crest-derived pediatric malignancy characterized by marked cellular heterogeneity and variable differentiation status. Undifferentiated tumors are associated with aggressive clinical behavior, treatment resistance and poor outcome, highlighting the need to identify molecular mechanisms that sustain tumor cell plasticity and prevent differentiation. Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase involved in cell-cycle progression, DNA-damage responses and transcriptional regulation, and has previously been associated with neuroblastoma progression. However, its role in the control of neuroblastoma differentiation remains unclear. Here, we investigated the relationship between VRK1 expression, tumor differentiation and stem-like properties in human neuroblastoma. Analysis of patient tumor datasets and tissue microarrays showed that VRK1 expression is enriched in undifferentiated neuroblastoma and stage 4 tumors, and inversely correlates with established differentiation markers, including DDC, NCAM1 and S100B. This association was maintained in MYCN-non-amplified tumors, indicating that the relationship between VRK1 and differentiation is not dependent on MYCN status. Single-cell transcriptomic analyses further demonstrated elevated VRK1 expression in developmentally immature neural crest progenitor and Schwann cell precursor-like populations. Induction of neuronal or mesenchymal differentiation consistently reduced VRK1 expression in neuroblastoma cell lines and patient-derived cells. Conversely, VRK1 silencing promoted differentiation-marker expression, reduced nestin and Ki67 expression, and produced sustained differentiation-associated changes in xenograft tumors. VRK1 was also enriched in tumorsphere cultures that select for undifferentiated stem-like neuroblastoma cells. VRK1 depletion impaired tumorsphere growth, reduced intratumoral proliferation and altered the balance between undifferentiated cells and differentiated progeny, supporting a role for VRK1 in self-renewal and maintenance of progenitor-like tumor cells. Mechanistically, VRK1 expression positively correlated with the core stemness transcription factor SOX2 in neuroblastoma tumor cells. VRK1 knockdown reduced nuclear SOX2 abundance, whereas VRK1 overexpression increased SOX2 protein levels. In addition, analysis of the VRK1 locus identified an active chromatin configuration and potential SOX2-binding sites, consistent with a regulatory relationship between these factors. Together, these findings identify VRK1 as a regulator of the undifferentiated, proliferative and stem-like state in neuroblastoma. The VRK1-SOX2 axis may contribute to stabilizing tumor-cell immaturity and represents a potential target for differentiation based therapeutic strategies in high-risk neuroblastoma.

Variants at the CD2AP locus, which encodes a branched-actin regulator, are associated with late-onset Alzheimer's disease (AD), and CD2AP-deficient mice show increased blood-brain barrier (BBB) permeability. CD2AP is enriched in endothelial cells, but its function there is poorly understood. In endothelium, branched actin networks maintain cell-cell junctions and are also required for immune cells to cross through individual endothelial cells (transcellular transendothelial migration, TEM). We previously showed that the Arp2/3 activators N-WASP and WAVE2 contribute to these processes, but the capping protein (CP)-interacting proteins involved are unknown. Here we tested whether CD2AP, a CP-interacting protein recruited to cell-cell contacts, regulates both junction integrity and TEM. In primary human microvascular endothelial cells, shRNA knockdown of CD2AP increased TNF-induced monolayer permeability, measured by transendothelial electrical resistance, and altered VE-cadherin junction morphology. Unexpectedly, on soft hydrogels the same knockdown reduced TEM of NK-92 MI cells, lowering the proportion of transcellular events. Thus, loss of CD2AP weakens the endothelial barrier yet impairs a distinct, actin-dependent route of immune-cell passage. These findings suggest that CD2AP risk variants may affect the BBB through more than permeability, and they identify endothelial immune-cell trafficking as a testable link between CD2AP and AD.

The modulation of iron metabolism is increasingly explored as a therapeutic strategy in cancer, particularly through the induction of ferroptosis. Given the central role of ferritin as a major intracellular iron buffer, targeting ferritin represents an attractive strategy to disrupt iron homeostasis; however, the lack of pharmacological approaches capable of selectively targeting ferritin currently limits the therapeutic exploitation of this vulnerability. MMRi62, initially developed to target the MDM2-MDM4 axis, has been proposed to induce ferroptosis via ferritin degradation. Here, we revisited this hypothesis in the context of medulloblastoma (MB). Contrary to this proposed mechanism, we found that MMRi62 does not trigger ferroptosis but instead induces robust apoptotic cell death. This effect is observed in both p53-mutant DAOY and p53 wild-type HD-MB03 cells, as well as in c-Myc/OTX2-driven medulloblastoma-like tumours genetically induced in brain organoids. Although ferritin degradation occurs upon treatment, genetic dissection demonstrates that neither ferritin itself nor ferritinophagy are required for MMRi62-induced cytotoxicity. Together, these findings rule out ferritin-dependent mechanisms as primary drivers of cytotoxicity. Instead, we uncovered that MMi62 induces a profound rewiring of iron metabolism consistent with a canonical iron starvation response, accompanied by a marked reduction in intracellular iron levels. Consistently, the UV-visible spectroscopic data were in agreement with the proposed iron-chelating properties of MMRi62. Taken together, our findings identify iron depletion-driven apoptosis, rather than ferroptosis, independently of ferritin degradation, as the primary mechanism of MMRi62 cytotoxicity in MB, refining its mode of action and highlighting iron homeostasis as a therapeutic vulnerability.

MLLT1 (also named ENL) is a chromatin reader protein whose encoding gene was originally identified as a chromosomal translocation partner with MLL(KMT2A) in acute leukemia. However, its role in normal hematopoiesis has not been investigated. This study uncovers a critical role of Mllt1 in normal B cell lymphopoiesis. We found Mllt1 to be essential for early B lymphocyte development using a conditional Mllt1 knockout mouse model that we developed. A significant decrease of bone marrow B-lineage progenitors, splenic transitional B cells and peripheral blood B cells were observed in Mllt1del mice compared to control Mllt1fl/fl mice. Similarly, Mllt1 deletion in in vitro cultured B-enriched progenitor cells from Mllt1fl/fl; Rosa26CreERT2/+ mice resulted in reduced B cells, demonstrating the cell-intrinsic role of Mllt1 in this process. Direct MLLT1 target genes including Il7r and critical B-lineage transcription factors, Ebf1 and Pax5, were decreased following Mllt1 deletion. Gene set enrichment, gene ontology, and functional analyses of Mllt1-deficient cells showed significant alterations related to B cell development, critical relevant signaling pathways, DNA replication, and mitochondrial function. In vitro complementation with MLLT1 rescued the B cell phenotype observed with endogenous Mllt1 deletion; however, specific MLLT1 YEATS domain mutants lacking chromatin reader and RNA-binding functions were unable to rescue the phenotype. Taken together, our research demonstrates a previously unappreciated role for MLLT1 as critical for maintenance of B cell lymphopoiesis.