Cell Death Discovery

BackgroundThe histone methyltransferase EZH2, enzymatic core of the trimeric polycomb repressive complex 2 (PRC2), has been shown to promote small cell lung cancer (SCLC) survival through epigenetic silencing of multiple targets including Class I MHC molecules (HLA-A/B) and DNA repair factors (SLFN11). Treatment of SCLC cells with EZH2 inhibitors in vitro can reactivate expression of these genes and result in therapeutic response to immune checkpoint inhibition (ICI) and chemotherapy. Here, we investigate the impact of EZH1/2 dual inhibition on 3D chromatin structure and its relationship to transcriptional regulation in neuroendocrine (NE) SCLC. ResultsEmploying Micro-C, a micrococcal nuclease-based 3D genome mapping technique, we show that EZH1/2 inhibition with Valemetostat induced significant changes at multiple genome organizational levels (compartment, topological associated domain, and chromatin loop) without incurring cell death in NE SCLC. Alterations in 3D genome permissive for transcriptional activation were correlated with increased chromatin accessibility (ATAC-sequencing) and expression of target genes (transcriptome profiling). Known transcription factor motif discovery revealed enrichment of non-NE motifs (e.g., REST) in regions with gained chromatin accessibility in Valemetostat-treated cells, consistent with results from gene set enrichment analysis demonstrating NE to non-neuroendocrine lineage shift. Notably, EZH1/2 inhibition reactivated Class I MHC expression by facilitating enhancer-promoter looping. ConclusionOur results demonstrate that repression of a subset of EZH2 targets including Class I MHC genes is affected through modulation of 3D genome structure to the level of chromatin looping and further support clinical investigation of EZH2 inhibition in boosting therapeutic efficacy of ICI in SCLC patients.

Following our laboratorys earlier observations on systemic damage inflicted by sev-Gal4 driven activated Ras (sev>RasV12) over-expression in Drosophila larval eye discs, we now show that sev>RasV12 expressing males suffer enhanced eye roughening and pupal death than female sibs because the former have significantly greater Ras levels in ommatidial cells than in female counterpart. In normally developing ommatidial cells, TBPH/TDP-43 was more abundant in cytoplasm in male than in female eye discs. The sev>RasV12 expression reduced nuclear TBPH in female eye discs but caused no apparent change in males. Caz/Fus, an interacting partner of TBPH, was significantly downregulated in sev>RasV12 eye discs, more so in males. Significant reduction in the microtubule binding protein Futsch in eye discs of sev>RasV12 larvae of either sexes but female-specific elevation of Fas2 appears to be due to the above normal developmental differences in TBPH and Caz in female and male ommatidial cells and because Sxl, the master regulator of sex-determination, is present only in females. In view of known auto-regulatory loop between Fas2 and Ras, we suggest that elevated levels of Fas2 cause levels of Ras to be much less elevated in sev>RasV12 female eye discs than in male sibs. This results in greater local and systemic damage in males. These findings have general and clinical relevance since perturbed Ras signaling is a major factor in several diseases, including cancer.

Doxorubicin (DOX) is an effective anti-cancer drug; however it can cause cardiotoxicity by inducing DNA double-strand breaks in cardiomyocytes. Cardiotoxicity can manifest immediately or years following treatment. Most human in vitro models of DOX-induced cardiotoxicity (DIC) focus on the acute effects of DOX treatment. To understand the long-term effects, we profiled the global gene expression response to DOX exposure over time. We treated iPSC-derived cardiomyocytes from six individuals with DOX for 24 hours and assayed responses after 0, 24 and 144 hours of recovery. DNA damage, determined by {gamma}H2AX expression, is induced following DOX treatment and is resolved by the final recovery timepoint. We identified both acute and chronic gene expression response signatures. The chronic signature, representing 501 genes, is enriched for p53 target genes and DNA damage response genes compared to acute response genes. P53 target genes are persistently activated, and DNA damage response genes are progressively downregulated over time. Our results suggest an altered cell state following repair of double-strand breaks that is distinct from pre-exposed cells. DOX response genes with persistent changes in expression can be applied to the design of toxicity biomarkers or therapeutic targets.

Extracellular vesicles (EVs) are increasingly recognized as critical mediators of intercellular communication, not least during cellular stress or therapy. While EV signalling is well-studied in various tissues, its role in the prepubertal testicular environment is not well understood. Chemotherapy, commonly used in paediatric oncology, poses a significant risk to spermatogonial stem cells (SSCs) and may affect long-term fertility in cancer survivors. The role of EVs in chemotherapy-induced testicular damage in these patients is unknown and may be important for developing new fertility preservation methods. Immortalised murine Sertoli (TM4) and spermatogonial (GC1-spg) cell lines were used to investigate cisplatin-induced changes in EV biogenesis, release, and function in an in vitro model of the prepubertal testicular microenvironment. Our findings indicate that cisplatin significantly increases EV secretion and internalisation by recipient cells. Notably, EVs from cisplatin-exposed Sertoli cells exhibit a novel pro-apoptotic phenotype when co-cultured with chemotherapy-naive Sertoli cells. Proteomic profiling of these EVs shows enrichment of apoptosis-regulatory proteins including caspases, activating Caspase-3/7 in recipient Sertoli cells. Conversely, germ cells exposed to Sertoli cell-derived EVs displayed reduced levels of apoptosis as well as a chemoprotective role to germ cells undergoing treatment with cisplatin. These findings indicate a dual role for Sertoli cell-derived EVs in mediating (1) apoptosis in Sertoli cells and (2) protection of germ cells following cisplatin exposure. The presence of pro-apoptotic molecules, especially caspases, in chemotherapy-induced Sertoli cell EVs provides mechanism for the induction of somatic cell apoptosis. Furthermore, their protective effects on germ cells demonstrate the complexity of EV-mediated signalling between testicular cell types. Manipulating EV biogenesis and cargo loading could be a promising approach to reduce chemotherapy-related gonadotoxicity and preserve fertility in childhood cancer patients.

Primordial germ cells (PGCs) are the population of cells that, in the human embryo, specify day 12 post-fertilization, and form the precursor cells for the future egg or sperm cells. Although in vitro differentiation of PGCs from human stem cells has been achieved, these primordial germ cell-like cells (hPGCLCs) fail to further mature. The reason for this is unclear. Previous studies in mice revealed that several specific metabolic changes occur during the maturation of these cells, which are essential for their developmental progress. However, very little is known about the metabolic profile of human primordial germ cells. In the severe scarcity of human PGCs, hPGCLCs serve as a research model to study PGC formation. To investigate this, we differentiated hPGCLCs using induced-pluripotent stem cells and performed a mass spectrometry analysis to establish their metabolome and proteome. These cells revealed distinct metabolic profile, with changes particularly at the proteome level. This included a shift between canonical and non-canonical citric acid cycle in hPGCLC, downregulation of late-stage glycolysis and reduction of nucleotide de novo synthesis. By providing an integrative map of these metabolic networks, we aim to provide insight on the influence of metabolism on human PGC development that could help improve methods for in vitro differentiation and maturation hPGCLCs.

Development of motoneurons from stem cells is characterized by a change from glycolytic to oxidative metabolism. Since this transition remains poorly understood, we examined it at five distinct differentiation stages from hiPSC to motoneuron. While a direct comparison of hiPSCs and mature motoneurons confirmed the expected glycolytic-to-oxidative shift, the intermediate stages showed that the conversion was not monotonic. After an initial drop of glycolysis at the hiPSC-to-neuroepithelial transition, late neuroepithelial cells showed intermittent peaks of the glycolytic marker lactate dehydrogenase A and the metabolic regulator TIGAR. Furthermore, the lactate-produced-to-glucose-consumed ratio remained elevated. A fully oxidative phenotype was only assumed upon progress from neural progenitors to motoneurons, portrayed by a definitive drop of the lactate-produced-to-glucose-consumed ratio, an increase of mitochondrial membrane charging, and shifts from lactate dehydrogenase A to B, from pyruvate dehydrogenase to anaplerotic pyruvate carboxylase, and from Mitofusin 1 to 2. Together, our data show that metabolic maturation in human motoneurons does not occur as a simple switch. Instead, it unfolds through distinct stages in a directional yet nonlinear manner.

The equilibrium between cell death and cell division is crucial for maintaining tissue homeostasis in a multicellular organism. Apoptosis plays an essential role in preserving homeostasis and hence occurs in a coordinated manner. However, inhibition of apoptosis is one of the hallmarks of cancer. Apoptosis Inhibitor 5 (Api5), an anti-apoptotic protein, is upregulated in various cancers, including ovarian, bladder, cervical, and lung cancers. Studies have demonstrated that altered expression of Api5 leads to the transformation of non-tumorigenic breast epithelial cells. However, the mechanism regulating this process is not well-elucidated. Our study demonstrates that overexpression of Api5 increased FGF2 (Fibroblast Growth Factor 2) levels both at protein and transcript levels. We studied the mechanistic details of changes in morphology, proliferation, and polarity observed upon FGF2/FGFR1 deregulation in Api5-overexpressing cells. Deciphering the signalling mechanism underlying Api5-FGF2-mediated breast tumorigenesis revealed that the PDK1/Akt and Ras/MAPK/ERK pathways regulated multiple transformation phenotypes. PDK1/Akt enhanced proliferation and altered morphology during initial stages, whereas Ras/MAPK/ERK regulated polarity disruption, proliferation, and reduced apoptosis during later stages of morphogenesis. In conclusion, this study provides insights into the signalling mechanism regulating the transformation phenotypes associated with Api5 overexpression in a non-tumorigenic breast epithelial cell line.

Astrocytes play a key role in neuronal homeostasis and in various neural disorders. The generation of astrocytes from neural progenitor cells (NPCs) and its functions are under a complex control of several signaling networks and transcription factors. In this study, we demonstrate that the transcription factor, GLIS similar 3 (GLIS3), which has been implicated in several neurodegenerative diseases, is highly expressed in astrocytes, and is required for the efficient differentiation of human NPCs into astrocytes. Loss of GLIS3 function greatly impairs astrocytes differentiation, resulting in reduced expression of astrocyte markers, whereas expression of exogenous GLIS3 restores the induction of astrocyte specific genes indicating a critical role for GLIS3 in astrocyte differentiation. Integrated transcriptomic and cistromic analyses revealed that GLIS3 directly regulates the transcription of several astrocyte-associated genes, including GFAP, SLC1A2, NFIA, and ATF3, in coordination with lineage-determining factors, such as STAT3, NFIA, and SOX9. We hypothesize that GLIS3 dysfunction disrupts this transcriptional network thereby contributing to astrocyte-associated neurological disorders. Identification of GLIS3 as a key regulator of astrocyte differentiation and gene expression will advance our understanding of its role in neurodegenerative diseases and may provide a new therapeutic target.

Ferroptosis is a form of regulated cell death that is characterized by iron-dependent lipid peroxidation. This process is regulated by specific metabolites, the lipid composition of the cells, redox-active iron, and antioxidant mechanisms. Although numerous regulators have been identified over the past decade, exploring other mechanisms, particularly from non-coding genomic regions, can build a thorough understanding of the multifaceted regulatory processes underlying ferroptosis. MicroRNAs (miRNAs) play a crucial role in gene regulation and cellular functions. Through a CRISPR KO screen, we identified miR-940 as a negative regulator of ferroptosis. Overexpression of miR-940 in several cell lines consistently suppressed ferroptosis induced by system xc- inhibition. Notably, multiple cancer patient cohorts with elevated miR-940 levels exhibit reduced survival. Integrated bioinformatic, transcriptomic, and proteomic analyses revealed that miR-940 decreases the expression of ACSL4, LPCAT3, DMT1, and NCOA4, and simultaneously increases levels of GPX4. Pharmacological inhibition of GPX4 attenuated the protective effect of miR-940, indicating that its primary anti-ferroptotic activity is mediated through GPX4. Overall, this gene rewiring is associated with reduced levels of redox-active iron and diminished lipid peroxidation, consistent with ferroptosis suppression. These findings suggest that miR-940 coordinates ferroptosis inhibition, which presents a novel regulatory layer for therapeutic exploration in susceptible cancers.

Circulating stem and progenitor cells (SPCs), including mesenchymal stromal cells (MSCs) and hematopoietic stem/progenitor cells (HSPCs), are mobilised after tissue injury but their temporal behaviour after hemorrhagic shock (HS) and relationship to cytokine milieus and outcome remain unclear. In a prospective observational cohort at JPN Apex Trauma Centre, AIIMS, New Delhi we studied 100 participants: 50 trauma patients with hemorrhagic shock and traumatic brain injury (HS index group), 25 trauma patients without HS, and 25 minor-injury controls. Peripheral blood was collected at admission (day 0) for all groups and additionally at days 3, 7 and 14 for the HS group. PBMCs were phenotyped by flow cytometry (HSPC markers: CD45, CD123, CD38, CD34; MSC markers: CD105, CD73, CD90) and serum SDF-1, VEGF-A, EGF, GRO- and GRO-{beta}, GM-CSF and G-CSF were measured by ELISA; group and time effects were evaluated with mixed-effects models and correlations by Spearman tests (two-tailed p<0.05). At admission, trauma patients without HS had significantly higher MSC and HSPC-like populations versus controls (p<0.0001). In the HS cohort SPC percentages rose modestly at day 0-3 then declined sharply by days 7-14 (time effect p<0.0001); non-survivors exhibited significantly higher early SPC and cytokine levels that persisted until death while survivors showed an early rise followed by decline (outcome and time interaction p<0.0001). All cytokines were up-regulated in trauma groups, peaked at day 0-3 in HS patients, and correlated positively with SPC counts (notably SDF-1, VEGF-A, G-CSF, Gro- and GM-CSF; Spearman p<0.05); higher early SPC and cytokine signatures associated with greater organ dysfunction (higher SOFA) and with timing of sepsis. These findings indicate that trauma provokes an early SPC and cytokine response that in HS is followed by later decline, and that persistent early elevation predicts worse outcomes, suggesting serial SPC and cytokine profiling may have prognostic value and identify an early therapeutic window for regenerative or immunomodulatory interventions.

Epidermal Growth factor (EGF) signaling is associated with (oncogenic) proliferation. Conversely, EGF-family ligands are able to trigger a differentiation program in cultured cells, an effect attributed to ligand affinity and EGFR phosphorylation. How EGF/EGFR driven proliferation-differentiation dynamics underlie tissue self-renewal has not been addressed. We show that culturing mouse small intestinal organoids (mSIOs) without EGF enhanced EGFR expression and base phosphorylation while maintaining a balanced development of proliferative crypts and differentiated villi. Addition of EGF or EREG triggers receptor endocytosis, reducing cell-surface and expression levels. While EGF promoted crypt proliferation, EREG promoted both proliferation and villus differentiation compared to untreated controls. Removal or re-introduction of EGF or EREG proved sufficient to induce development comparable to constant presence of ligands over 96h. Sub-saturating concentrations of EGF led to increased villus differentiation, resembling EREG treatments, suggesting that control over EGFR endocytic cycle ultimately regulates the balance of proliferation and differentiation in mSIOs SummaryExpression and signaling competency at the plasma membrane of EGFR drives crypt proliferation vs villus differentiation by medium ligand-composition, aiding mouse intestinal organoids self-renewal and regeneration.

BackgroundN4-acetylcytidine (ac4C) modification plays a critical role in cancer development. Exploring ac4C modification in laryngeal squamous cell carcinoma (LSCC) may help elucidate its pathogenesis. MethodsLSCC-related datasets were obtained from GEO. After preprocessing and annotating single-cell data, malignant cells were identified by CNV scoring and further divided into subpopulations. Malignant epithelial cells (MECs) were identified and subclustered based on ac4C-related gene activity. Prognostic genes were screened using Cox regression and machine-learning approaches, followed by validation in clinical samples using qPCR. The biological and immunological relevance of these genes was further explored through immune infiltration, immunotherapy response, and mutation analyses. ResultsThe 14,465 identified MECs were classified into five subgroups (MEC1-5), among which MEC3 showed the strongest association with the ac4C gene set. Machine-learning analysis of MEC3-derived genes yielded seven prognostic markers, including BARX1, FHL2, NXPH4, PKMYT1, TNFAIP8L1, CRLF1, and CENPP. qPCR confirmed their differential expression between tumor and adjacent normal tissues. These genes were significantly associated with alterations in the tumor immune microenvironment, with high-risk patients showing increased immune infiltration and immune activity. ConclusionSeven ac4C-related prognostic genes were identified that may contribute to LSCC progression by modulating the tumor immune microenvironment, providing potential therapeutic insights.

Post-acute sequelae of SARS-CoV-2 infection (PASC), commonly referred to as Long COVID, comprise a constellation of persistent, recurrent, or newly emerging symptoms that may endure for months or years following acute infection. Beyond respiratory impairment, PASC is characterized by a wide spectrum of extrapulmonary manifestations, among which neurological and neuropsychiatric symptoms are highly prevalent. Reported features include olfactory dysfunction with loss of smell and taste, fatigue, neuroinflammation, cognitive and memory impairment, depression, and anxiety, with some symptoms persisting up to one year post-infection. Despite increasing recognition of these complications, the molecular mechanisms underlying post-COVID neurological sequelae remain poorly defined. In this study, we employed a label-free quantitative (LFQ) proteomics approach to investigate protein alterations in olfactory neuroepithelium-derived stem cells (ONEs), a unique population of neural progenitors located in the olfactory mucosa at the interface between the respiratory system and both the peripheral and central nervous systems. Due to their anatomical exposure and susceptibility to SARS-CoV-2, ONEs represent a highly relevant translational model for exploring virus-associated neurobiological processes. ONEs derived from healthy donors were incubated with serum from either asymptomatic PCR-positive individuals (AS; n=4) or critically ill hospitalized patients (CR; n=6). Proteomic profiling revealed a distinct differential protein expression pattern in ONEs exposed to CR serum compared with AS serum. Altered pathways were associated with viral infection responses, respiratory and cardiovascular dysfunction, and notably, cerebrovascular and nervous system disorders. These findings highlight the vulnerability of ONEs to systemic factors associated with severe COVID-19 and provide molecular insight into mechanisms potentially contributing to persistent neurological sequelae in PASC. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/710460v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@12cfda5org.highwire.dtl.DTLVardef@c0636borg.highwire.dtl.DTLVardef@bf303eorg.highwire.dtl.DTLVardef@1f861e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intervertebral disc degeneration is characterized by inflammation, extracellular matrix breakdown, and neurovascular ingrowth, processes that contribute to discogenic, chronic back pain. The transient receptor potential canonical 6 (TRPC6) channel is a calcium-permeable ion channel implicated in inflammation and pain signaling in multiple tissues; however, its functional role in human disc cells remain unknown. Here, we investigated the expression, activation, and downstream consequences of TRPC6 activation using Hyp9, a pharmacological activator of TRPC6. TRPC6 transcripts were consistently detected across all donors examined (n = 17). Functional TRPC6 activation induced a rapid, dose-dependent calcium (Ca2+) influx across 0.5-100 {micro}M Hyp9. TRPC6 activation did not reduce metabolic activity or increase cytotoxicity at concentrations commonly used for in vitro TRPC6 activation. Mechanistically, TRPC6 activation induced mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-{kappa}B) pathways, as demonstrated by increased phosphorylation of p38 and extracellular signal-regulated kinase (ERK), degradation of the inhibitor of {kappa}B-alpha (I{kappa}B-), and increased nuclear translocation of the NF-{kappa}B p65 subunit. Downstream of these early signaling events, TRPC6 activation elicited a robust inflammatory and catabolic response with upregulation of IL-6, IL-8, COX-2, MMP-1, MMP-3, NGF, and VEGF, with corresponding increases in protein secretion. These findings identify TRPC6 as an important signaling node linking calcium influx to inflammatory, catabolic, and neuro- and angiogenesis-associated pathways in disc cells, highlighting TRPC6 as a potential therapeutic target in degenerative disc disease. HighlightsO_ST_ABSWhat are the main findings?C_ST_ABSO_LITRPC6 is endogenously expressed in human intervertebral disc cells, and its activation induces rapid calcium influx that initiates MAPK and NF-{kappa}B signaling pathways. C_LIO_LITRPC6 activation initiates a broad inflammatory and degenerative program, elevating the expression of IL-6, IL-8, COX-2, MMP-1, MMP-3, NGF, and VEGF. C_LI What are the implications of the main findings?O_LITRPC6 functions as a key upstream regulator linking calcium influx with inflammatory, matrix-degrading, and neuro-angiogenic processes central to disc degeneration and discogenic back pain. C_LIO_LIPharmacological targeting of TRPC6 may offer a novel therapeutic approach to suppress early inflammatory signaling, limit extracellular matrix breakdown, and reduce neurovascular ingrowth in degenerative disc disease. C_LI

Intervertebral disc degeneration, a leading cause of low back pain with incompletely elucidated molecular mechanisms, was studied via integrated in vivo/vitro approaches. This study first reveals that lactic acid accelerates intervertebral disc degeneration by inducing cartilage endplate stem cells senescence and DNA damage, thereby activating the P16/P21/P53-centered senescence pathway. In a rat tail vertebra puncture-induced intervertebral disc degeneration model, degenerated discs exhibited increased lactic acid levels, narrowed intervertebral spaces, and disrupted nucleus pulposus structure (P<0.05). In vitro, 0/2/6/10 mM lactic acid dose-dependently suppressed cartilage endplate stem cells viability (10 mM group: 15.7% of the control), elevated intracellular reactive oxygen species (ROS, 2.8-fold relative to the control), induced G0 cell cycle arrest (10 mM group: 85.63%), reduced EdU-positive cells (8.62%), and increased {beta}-galactosidase-positive cells (10 mM group: 33.06%) and {gamma}-H2AX foci (all P<0.01).Molecularly, lactic acid significantly upregulated P16 (2.1-fold), P21 (3.1-fold), P53 (2.4-fold), and {gamma}-H2AX (1.8-fold). In vivo intervertebral disc injection confirmed a positive correlation between lactic acid concentration and intervertebral disc degeneration severity. This study clarifies lactic acids role in intervertebral disc degeneration via the "oxidative stress-cell cycle arrest-cellular senescence" axis, advancing understanding of intervertebral disc degeneration pathogenesis and providing a basis for targeted therapies against lactic acid metabolism.

Photoreceptor integrity depends on the precise coordination of membrane trafficking and signal transmission. Despite their well-known roles in germline biology, the functions of PIWI family proteins in post-mitotic neuronal cells remain unclear. We investigated the role of HIWI2 in photoreceptor-derived 661W cells. Silencing of HIWI2 resulted in a significant decrease in the early endosomal marker, Rab5, and its effector EEA1, and reduced expression of the recycling endosome marker Rab11, indicating poor endosomal sorting and receptor recycling. In contrast, the marker for late endosomes, Rab7, was significantly upregulated, suggesting a shift toward degradative trafficking pathways, in line with increased receptor breakdown. These trafficking shifts led to the degradation of EphA2 and EphB2 receptors, as confirmed by a phospho-proteome receptor tyrosine kinase array and further supported by immunoblotting, and were accompanied by a compensatory increase in Akt phosphorylation. Furthermore, HIWI2 deficiency impaired cell motility in wound-healing assays. These results propose HIWI2 as a critical regulator of endosomal sorting and Eph receptor stability, providing a novel link between the PIWI pathway and photoreceptor integrity.

Epithelial to mesenchymal transition (EMT) is a process of trans-differentiation important for development, inflammation and cancer. Transforming Growth Factor-{beta} (TGF{beta}) is a physiologically relevant inducer of EMT. We had recently characterized the adenosine analogue, adenosine kinase inhibitor 5-Iodotubercidin (5-ITu), as a compound which preferentially sensitizes MK2-deficient cells to TNF-induced, RIPK1-dependent cell death. Here we investigated the effect of 5-ITu on TGF{beta}-induced EMT. 5-ITu suppressed TGF{beta}-induced morphological changes and migration in A549 (lung cancer) and PANC1 (pancreatic cancer) cell lines. Consistent with these effects, there was significant suppression of EMT markers as indicated by qPCR, immunoblotting and immunofluorescence and confocal microscopy. Mechanistic investigations revealed that 5-ITu-mediated EMT suppression was independent of adenosine kinase inhibition and RIPK1 activation. 5-ITu suppressed NF{kappa}B activity in cells undergoing EMT and IKK inhibition phenocopied the effect of 5-ITu on EMT. Kinase assays revealed IKK{beta} as a potential direct target of 5-ITu. We identified a TGF{beta}-associated, NF{kappa}B-dependent gene signature consisting of 4 genes, which are differentially regulated upon 5-ITu treatment. Interestingly, this 4 gene signature could predict survival in lung and pancreatic cancer. The identification of this role for the multitarget kinase inhibitor 5-ITu in NF{kappa}B activity-dependent EMT, in addition to RIPK1-dependent necroptosis has potential implications in anticancer strategies.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disease associated with repeated head injuries (RHI) commonly experienced by contact sport athletes, military personnel, and domestic abuse victims. Despite growing recognition of CTE, the molecular mechanisms underlying disease progression remain poorly understood. This study aims to identify proteomic alterations associated with CTE pathology and clinical features to elucidate key biological pathways involved in disease pathogenesis. SomaScan 7k high-throughput proteomics was performed on 204 dorsolateral prefrontal cortex samples from the Boston University CTE Center Brain Bank. We identified differentially expressed proteins associated with CTE, hyperphosphorylated tau (ptau) pathology, duration of contact sports play, dementia status, and Cognitive Difficulty Scale (CDS) scores. Gene set enrichment analysis revealed that proteasome subunit proteins and related pathways were strongly associated with CTE progression and correlated with years of contact sports play. Reduction in ribosomal proteins and pathways was closely associated with ptau burden. Additionally, multiple models demonstrated significant alterations in MAPK-related cell signaling pathways. These findings advance our understanding of CTE progression and identify mechanisms correlated with key pathological features of the disease. Validation of these results could inform the development of diagnostics and treatments for CTE.

BackgroundSmall-cell lung cancer (SCLC) represents 15% of lung cancers and with a 5-year survival rate under 7% remains one of the deadliest malignancies. Although initially responsive to chemotherapy, rapid recurrence and resistance are common. Epigenetic modifications, particularly DNA methylation, contribute to tumor progression and therapy resistance. Guadecitabine, a hypomethylating agent (HMA), has shown promising clinical activity when combined with carboplatin in preclinical models. We evaluated the combination of guadecitabine with carboplatin as a second-line treatment for extensive-stage SCLC (NCT03913455). Here we report methylome changes in peripheral blood mononuclear cell (PBMCs) collected at baseline and during treatment from patients on the trial. ResultsPMBC DNA was analyzed using Infinium HumanMethylationEPIC v1.0 bead chips. Data were processed and differentially methylated positions (DMPs) were identified and analyzed for pathway enrichment using bioinformatic approaches and immune deconvolution analyses were conducted to investigate the impact on immune cell composition. Direct comparison of PBMCs between cycle 2 day 5 (C2D5; post-treatment) vs cycle 1 day 1 (C1D1; pre-treatment) revealed a greater number of hypomethylated DMPs (380 DMPs in C2D5 vs C1D1 PBMCs; p < 0.05, |{beta}| > 20%). Moreover, when first compared with normal PBMCs from cancer-free controls, the number of hypomethylated DMPs was even greater in C2D5 than in C1D1 (1,771 vs 237 DMPs, respectively; p < 0.05, |{beta}| > 20%). Long interspersed nucleotide elements-1 (LINE-1) were also significantly hypomethylated in PBMCs after HMA treatment (C2D5), compared to C1D1. Pathway analysis of hypomethylated DMPs revealed significant alterations in key signaling pathways including NF-{kappa}B, Rho GTPase, pulmonary fibrosis, and p75 NTR in C1D1 vs C2D5. When normal PBMCs were compared to C1D1 PBMCs, changes in IL-3 signaling, Fc{gamma} receptor-mediated phagocytosis, and molecular mechanisms of cancer were observed. Deconvolution analysis revealed a significantly higher percentage of monocytes in C1D1 PBMCs vs normal PBMCs. However, after HMA treatment, percentages of monocytes and B cells decreased, while eosinophil percentage increased in C1D1 compared to C2D5 PBMCs. ConclusionIn the first study on the global impact of HMA treatment on PBMC methylomes in SCLC patients, DNA methylation changes associated with biological pathways related to PBMC function reveal shifts in distinct immune cell populations. SummaryMethylome changes in peripheral blood mononuclear cell (PBMCs) from small cell lung cancer (SCLC) patients treated with an epigenetic therapy revealed global hypomethylation and altered cancer signaling processes associated with tumor progression, immune response, therapy resistance and significant change in the proportion of immune cells. Integrating blood-based methylation biomarkers into clinical trials of epigenetic therapy and methylomic analysis of PBMCs provides direct monitoring of treatment effects in cancer patients, which may improve patient selection and enable real-time response assessment in patients receiving hypomethylating agents.

Ovarian cancer (OC) remains a lethal malignancy with limited therapeutic options, underscoring the need for the identification of novel agents. Natural products like clove (Syzygium aromaticum) have shown promising anti-cancer activity, but their mechanism in OC is poorly understood. This study investigates the anti-tumour effects and underlying mechanisms of a clove aqueous extract (CAE) on a panel of patient-derived OC cells. We found that CAE significantly inhibited cellular proliferation and induced cell death in a time-and dose-dependent manner. Mechanistically, CAE induced profound cellular stress, activating the transcription factor ATF-2. This was accompanied by a significantly increased lysosomal stress response, as evidenced by increased lysosomal mass/acidity, and a pathogenic hyperpolarisation of the mitochondrial membrane potential ({Delta}{Psi}m). The bioenergetic crisis induced as a consequence resulted in a sharp reduction in cellular oxygen consumption rate (OCR). Notably, the sensitivity to CAE-induced lysosomal and mitochondrial dysfunction varied across cell lines, revealing distinct phenotypic responses. Our results demonstrate that clove extract exerts its anti-tumour effects by orchestrating a multi-organellar stress response, positioning lysosomal disruption as a central event in its mechanism of action. This study provides a strong rationale for the further development of clove-based interventions for OC.