Molecular Oncology

ObjectivesHomologous recombination repair (HRR) deficiency is associated with improved immunotherapy responses in non-small cell lung cancer (NSCLC) patients. The HRR genes BRCA1/2 are key regulators of DNA repair, yet their impact on the tumor microenvironment (TME) in lung adenocarcinoma (LUAD) remains unclear. MethodsUsing single-cell sequencing and multi-omics data, we characterized BRCA1/2 mutation-associated transcriptional programs, immune cell composition, and functional alterations in T cells, investigating the molecular and immune architecture of BRCA-mutant LUAD patients. ResultsBRCA1/2 mutations were associated with increased genomic instability and poor prognosis in LUAD patients, but predicted better clinical outcomes following immune checkpoint blockade (ICB) treatment. BRCA1 mutations correlated with an upregulated type I IFN/IFN-{gamma} signature and CD8+ T cell activation. BRCA2 mutations were associated with alveolar/stress/inflammatory responses and enhanced MHC-II antigen presentation, linked to CD4+ T cell differentiation. Both alterations coincided with reduced CD28 co-stimulation and CTL activity, hinting of immune evasion. We identified two tissue-resident memory T cell (Trm) subsets as predictors of clinical outcomes and ICB response. BRCA1 mutations were associated with CD8+ Trm expansion, whereas BRCA2 mutations linked to tumor CD4+ Trm expansion and peripheral T/NK cell cytotoxicity. Furthermore, a cancer-promoting program activated by BRCA1 mutation was vulnerable to histone deacetylase inhibitors, which inhibited LUAD tumor growth. ConclusionsThis study provides a preliminary characterization of the BRCA-mutant TME in LUAD patients, revealing distinct transcriptional and immune patterns that highlight differences in BRCA1/2-associated molecular architecture and offer a framework for improving therapy efficacy in LUAD.

BackgroundThis study investigates the role of the pioneer transcription factor FOXA1 as a master gene in sustaining epithelial cell polarization in early-stage lung adenocarcinoma. The partial loss of FOXA1 is explored to determine if it will affect plasticity and progression of lung adenocarcinoma. The study also addresses the transcriptional circuitry that links polarity defects to lysosome homeostasis. MethodsA multiomics approach was used to define the status of the chromatin in epithelial and mesenchymal states of A549 adenocarcinoma cells obtained with a newly synthetized TGF-{beta} receptor inhibitor or TGF-{beta} respectively. The study leveraged ATAC-seq, RNA sequencing, Cut&Tag sequencing of FOXA1 and histone marks profiling. The functional impact of FOXA1 was examined by partial silencing in vitro and by heterozygous FOXA1 deletion in a KrasG12D mouse model. Three-dimensional organoid culture, high-resolution electron microscopy, spatial transcriptomics and multiplex immunohistochemistry assessed carcinoma cell polarity, proliferation, the tumor microenvironment and organelle content. Group differences were evaluated with two-tailed t tests or one-way analysis of variance. ResultsFOXA1 binding and expression were highest in cells harboring an epithelial phenotype. In mouse KrasG12D LUAD tumors FOXA1 marked polarized, CDH1-positive cells; heterozygous loss diminished CDH1, disrupted apical-basal architecture, lowered organoid-forming efficiency and remodeled the immune microenvironment. Spatial transcriptomics and ultrastructural analyses showed that FOXA1-deficient carcinoma cells accumulated lysosomes, down-regulated vesicle fusion genes of the SNARE family and activated the lysosomal CLEAR gene network. FOXA1 occupied enhancers of lysosome-associated genes and competed with the transcription factor TFE3, thereby suppressing transcription of cathepsin B and cathepsin C and restricting lysosome biogenesis. ConclusionsFOXA1 is a central regulator that preserves epithelial cell polarity and limits lysosome formation in lung adenocarcinoma. Targeting the FOXA1-TFE3-lysosome axis may affect tumor plasticity and provide new therapeutic opportunities.

BackgroundMethylthioadenosine phosphorylase (MTAP) loss co-occurs with actionable genomic alterations in non-small cell lung cancer (NSCLC) and creates vulnerability to protein arginine methyltransferase 5 (PRMT5) inhibition. Estimates of prevalence of MTAP loss rely on next-generation sequencing which can underestimate copy losses. Moreover, the activity of PRMT5 inhibitors in oncogene-driven NSCLC is not well established. MethodsWe assessed MTAP expression by immunohistochemistry (IHC) in 243 NSCLCs (n=132 early stage, n=111 metastatic), including 33 specimens with paired lymph nodes. Antiproliferative activity of PRMT5 inhibitor monotherapy and in combination with tyrosine kinase inhibitors (TKIs) was evaluated in MTAP-deleted NSCLC cell lines harboring EGFR or KRAS mutations or ALK rearrangements. ResultsAmong 243 NSCLC specimens from 240 patients (72% with driver alterations, 90% adenocarcinoma), MTAP loss was identified in 43 (18%) specimens from 40 (17%) patients, including 18 (14%) early-stage and 22 (20%) metastatic tumors. MTAP loss occurred in 24% of stage 4 driver-positive NSCLCs versus 14% of driver-negative tumors (p=0.314). Twenty (61%) lung-nodal pairs demonstrated concordance; eight cases only exhibited decreased MTAP expression in nodes. Variable sensitivity to PRMT5 inhibitors was observed in 22 MTAP-deleted NSCLC cell lines (9 EGFR-mutant, 7 KRAS-mutant, 6 ALK-rearranged), with responses seen in TKI-sensitive and TKI-resistant lines. SDMA (symmetric dimethylarginine) expression did not predict PRMT5 inhibitor sensitivity. TKI + PRMT5 inhibitor combos had greater activity than monotherapy. ConclusionsMTAP loss occurs in 1-in-5 oncogene-driven metastatic NSCLCs. PRMT5 inhibitor activity is independent of TKI exposure, driver alteration, and SDMA expression and enhanced by addition of TKI. These findings support clinical evaluation of PRMT5 inhibitor + TKI combinations for advanced NSCLC.

BackgroundDespite extensive characterization of key oncogenic drivers, pancreatic ductal adenocarcinoma (PDAC) continues to exhibit profound molecular heterogeneity and inconsistent responses to standard therapies, including gemcitabine. The role of pathway-level alterations, particularly in the context of age at onset and therapeutic exposure, remains insufficiently defined. MethodsIn this study, we leveraged a conversational artificial intelligence framework (AI-HOPE-TP53 and AI-HOPE-PI3K) to enable precision oncology, driven interrogation of clinical and genomic data from 184 PDAC tumors, stratified by age at diagnosis and gemcitabine exposure. Using AI-enabled cohort construction and pathway-centric analyses, we evaluated alterations in TP53 and PI3K signaling networks, with findings validated through conventional statistical methods. ResultsTP53 pathway analysis revealed a significantly higher frequency of TP53 mutations in early-onset compared to late-onset PDAC among gemcitabine-treated patients (86.7% vs. 57.1%, p = 0.04), with a similar trend observed between treated and untreated early-onset cases (86.7% vs. 40%, p = 0.07). Notably, in late-onset PDAC patients not treated with gemcitabine, absence of TP53 pathway alterations was associated with improved overall survival (p = 0.011). Complementary analyses of the PI3K pathway demonstrated a higher prevalence of pathway alterations in late-onset gemcitabine-treated tumors compared to untreated counterparts (13.2% vs. 2.7%, p = 0.02). Importantly, among late-onset patients not receiving gemcitabine, those without PI3K pathway alterations exhibited significantly improved overall survival (p < 0.0001). ConclusionTogether, these findings identify distinct TP53 and PI3K pathway dependencies that are modulated by both age-of-onset and treatment exposure in PDAC. This work highlights the utility of conversational artificial intelligence in enabling rapid, integrative, and hypothesis-generating analyses within a precision oncology framework, supporting the identification of clinically relevant molecular stratification strategies for this aggressive disease.

Head and neck cancer (HNC) is highly prevalent in South-Asia, driven by additional region-specific exposures such as chewing tobacco and betel nut. Despite therapeutic advances, five-year survival rate remains around 50-60%, underscoring urgent need to identify novel therapeutic targets and improve disease-free survival. This study was designed to identify both somatic and germline drivers contributing to HNC pathogenesis. Through whole-exome sequencing of 103 patients, we detected mutations in known HNC drivers (TP53, CDKN2A, NOTCH1) as well as novel hotspots in several genes, including TRIM48, MAP3K19, and CDC20. A recurrent hotspot mutation (p.A187T) in POLQ gene was identified in patients with high tumor mutation burden and was absent in both TCGA and ICGC cohorts. Among known hotspots, the MYC p.T73A mutation was highly prevalent, occurring in over 50% of patients. As MYC is considered an "undruggable" target, alternative strategies targeting upstream regulators such as BRD4 with specific inhibitors may hold promise for South-Asian HNSCC patients harboring the p.T73A mutation. Copy-number variation analysis further revealed EGFR amplification and TP73 deletion in the majority of patients, highlighting additional layers of genomic dysregulation. Comparative genomic analyses showed no recurrent mutations in epigenetic regulators (ARID2, EP300, KMT2B/MLL2, KMT2D/MLL4, NSD1, and TET1). We report p.S456L germline variant in SDHA consistently among South-Asian cohorts. Patients with p.S456L mutation were younger than those without it, reflecting typical epidemiological signature of a genetic variant that increases susceptibility. Systematic molecular characterization of recurrent mutations is required to elucidate mechanism of action of these variants and to find actionable therapeutic targets.

Constitutively active KRAS mutations are highly prevalent in lung cancers, but the direct role of its downstream phosphatidylinositol 3-kinase (PI3K) pathway in tumor progression remains unclear. A previous study established the requirement for PIK3CA, the alpha catalytic isoform, in lung tumor development in mouse models with an intact Trp53 tumor suppressor. In this study, we further investigated the requirement for PIK3CA for tumor growth both in vitro and in vivo. We first generated a "KPA" cell line by genetically deleting Pik3ca from a murine lung adenocarcinoma "KP" cell line harboring oncogenic KrasG12D and lacking Trp53. We found that Pik3ca is not required for cell survival and growth in vitro, even under anchorage-independent conditions but reduced the growth rate by 20%. We next orthotopically implanted KP and KPA cells into syngeneic mice and found that PIK3CA is absolutely required for tumor progression, even in the absence of Trp53. Implantation of KP cells, or a "KPS" cell line lacking the Stk11 gene, led to rapid tumor growth and death of all host animals. In contrast, mice implanted with KPA cells all survived with no detectable lung tumors. The gene expression profiles from cultured cell lines suggest KPA cells may be vulnerable to oxidative stress. Indeed, we found KPA cells were more sensitive to hydrogen peroxide and diethyl maleate-induced oxidative stress as compared to KP and KPS cells. Together, these results demonstrate that PIK3CA is not required for lung cancer cell growth induced by mutant KRAS in vitro but is critically needed for in vivo progression and growth.

BackgroundTreatment optimization in HPV-associated oropharyngeal cancer (OPSCC) remains challenging, as recent de-escalation trials have shown limited success. Current patient selection strategies based on smoking history and TNM classification are insufficient, highlighting the need for robust, standardized prognostic biomarkers. We report the first validation of the Immunoscore (IS) for prognostic stratification in HPV-associated OPSCC. Patients and methodsWe analyzed 191 HPV-associated (p16+ and HPV DNA/RNA+) OPSCC patients from an international multicenter cohort (2015-2024), comprising a French monocentric retrospective training cohort (N = 48) and three validation cohorts: French monocentric retrospective (N = 48), French multicenter prospective (N = 50), and US multicenter retrospective (N = 45). IS is a standardized digital pathology assay quantifying CD3lJ and CD8lJ densities in tumor cores and invasive margins, with cut-offs defined in the training cohort and validated across cohorts. Associations with disease-free survival (DFS), time to recurrence (TTR) and overall survival (OS) were assessed, alongside 3RNA-seq and sequential immunofluorescence profiling of immune composition. ResultsMedian age 65; 80% male; 74% smokers; 66% T1-2; 82% N0-1 (AJCC8th). IS-High patients demonstrated superior 3-year DFS in the training and validation cohorts 1-3 (all log-rank P < 0.05). Multivariable analysis identified IS-Low as the strongest independent risk factor for DFS (HR 9.03; 95% CI: 4.02-20.31; P < 0.001). The model combining IS with clinical factors showed higher predictive accuracy for DFS (C-index 0.82) than clinical variables alone (0.7; P < 0.0001). Similar findings were observed for TTR and OS. IS-High tumors showed markedly higher enrichment of lymphoid and myeloid immune cell populations, contrasting with immune-poor signatures in IS-Low tumors. ConclusionsIS is a robust biomarker that outperforms standard clinical variables in both prognostic and predictive accuracy. The enriched cytotoxic immune infiltrate in IS-High tumors explains favorable outcomes and supports their suitability for treatment de-escalation. Prospective validation is warranted.

Tunneling nanotubes (TNTs) are actin-based cytoplasmic connections that can mediate intercellular transfer of various cellular cargo and have been implicated in cancer progression and chemoresistance. However, the signalling mechanisms driving their formation remain poorly understood. Given the frequent dysregulation of EGFR and c-Met signalling in non-small cell lung cancer (NSCLC), and prior evidence of TNTs in lung adenocarcinoma patient samples, we investigated the role of EGFR and c-Met receptor signalling crosstalk in TNT induction in A549 lung adenocarcinoma cells. Stimulation with EGF, HGF, or in combination induced a concentration dependent increase in the formation of TNTs. TNTs exhibited typical characteristics, including F-actin expression, non-adherence to the substratum and facilitated intercellular trafficking of lysosomes, mitochondria, and lipid vesicles. EGFR was identified as a novel component of TNTs, but had little co-localisation with the c-Met receptor. Co-stimulation with HGF and EGF did not produce consistent additive or synergistic effects on TNT formation, suggesting shared downstream signalling. Furthermore, although EGFR and c-Met inhibition blocked EGF- and HGF-induced TNTs respectively, inhibition of both receptors was required to suppress TNTs following dual HGF/EGF treatment. Interestingly, blocking the EGF receptor alongside c-Met resulted in a more potent inhibition of HGF-induced TNTs, indicating crosstalk. Furthermore, inhibition of downstream MEK and PI3K pathways reduced HGF- or EGF- induced TNT formation, but dual inhibition was required to completely block TNT formation in HGF+EGF co-stimulated cells. These findings reveal a novel convergence of EGFR and c-Met and their downstream MAPK/PI3K pathways in TNT regulation, which can have important clinical implications in combinatorial receptor and cell signalling pathway targeting in NSCLC.

BackgroundColorectal cancer (CRC) is a leading cause of cancer mortality. While early detection improves outcomes, current non-invasive tests often lack sensitivity for early-stage CRC and advanced precancerous lesions (APL). Stool-based host messenger RNA (mRNA) biomarkers offer a promising approach, though the most clinically useful candidates remain undefined. MethodsWe screened for mRNA biomarkers by first using bioinformatic analysis of tissue RNA-seq datasets to identify candidate genes with strong and ubiquitous differential expression in CRC versus normal tissues. The top 135 computationally predicted biomarkers were evaluated using "gold standard" RT-PCR on clinical stool samples across two independent cohorts. ResultsSeveral biomarkers, including PPBP, MYC, MMP7, and TGFBI, exhibited strong predictive power. Integrating top-performing markers through machine learning yielded an AUC of 0.98 for CRC and 0.76 for APL detection. The optimized panel demonstrated 98% sensitivity for CRC and 50% for APL, with a specificity of 90%. ConclusionsThis study derives a high-performance mRNA-based stool test for non-invasive CRC screening. Our findings demonstrate that a multi-marker panel achieves exceptional sensitivity and good specificity, providing a viable tool for clinical diagnostics.

PurposePancreatic ductal adenocarcinoma (PDAC) is characterized by a nutrient-deprived and hypoxic tumor microenvironment (TME) that imposes severe metabolic stress on cancer cells. Under these conditions, tumor cells frequently activate the integrated stress response (ISR) to adapt to TME and develop resistance to therapies. However, how TME components support tumor adaptation to mitochondrial metabolic stress remains incompletely understood. Here, we aimed to identify key metabolite involved in ISR adaptation under oxidative phosphorylation (OXPHOS) inhibition and to elucidate the metabolic symbiosis between cancer-associated fibroblasts (CAFs) and PDAC cells. MethodsWe integrated transcriptomic and metabolomic analyses with functional assays. ISR activation was evaluated by assessing the phosphorylation of eIF2 (p-eIF2) following treatment with carboxyamidotriazole orotate (CTO), an Complex I inhibitor. Metabolomic profiling was used to identify metabolites involved in ISR activation alleviation. Mouse models were used to assess therapeutic responses following depletion of the identified metabolite under CTO treatment. Genetic perturbation of Slc38a4 was performed to assess its functional role in tumor cell adaptation to metabolic stress. ResultsWe identified asparagine (ASN) as a critical metabolite supplied by CAFs to PDAC cells under OXPHOS inhibition. A minimum level of ASN is required for PDAC cells to execute ISR downstream adaptation. ASN depletion significantly enhanced the anti-tumor efficacy of OXPHOS inhibition both in vitro and in vivo. SLC38A4 emerged as a potential mediator of this interaction. SLC38A4 expression was associated with c-Myc, and its loss increased the sensitivity of PDAC cells to CTO-induced metabolic stress. ConclusionOur findings reveal a CAF-tumor metabolic crosstalk in which stromal-derived ASN supports PDAC cell adaptation to mitochondrial metabolic stress. Adaptive outcome of ISR signaling depends on the availability of key metabolic substrates such as ASN. When extracellular ASN supply is limited, the ATF4-dependent adaptive program collapses, converting ISR from a pro-survival response into a therapeutic vulnerability. SLC38A4 may function as a key mediator of this metabolic coupling and represents a potential target for enhancing the efficacy of OXPHOS inhibition in PDAC.

Inactivating mutations in BRCA1-associated protein 1 (BAP1) are observed in approximately 45% of primary and [~]85% of metastatic uveal melanoma (UM) cases and are strongly correlated with aggressive phenotypes and poor prognosis. However, the mechanistic contribution of BAP1 to tumor aggressiveness remains elusive. This study investigates the role of BAP1 loss in senescence and explores the potential therapeutic implications of targeting senescence pathway. Analysis of The Cancer Genome Atlas UM cohort revealed that BAP1-mutant tumors exhibited increased senescence pathway activity score, and elevated expression of multiple cytokines, chemokines, growth factors and matrix-remodeling enzymes related to senescence-associated secretory phase. Functional assays revealed that BAP1 loss promotes senescence hallmarks including upregulated p16, p21, and phospho-ATM proteins, increased {beta}-gal positive cells, accumulated {gamma}H2AX foci, depleted lamin B1, and reduced PARP1 cleavage and Ki67 levels. These effects were further exacerbated following radiation exposure. Importantly, BAP1 knockdown, alone or in combination with ionizing radiation, sensitized UM cells to senolytic agents, dasatinib and quercetin. In conclusion, our findings identify BAP1 loss as a driver of senescence and suggest that BAP1-mutant tumors may benefit from senolytics treatment.

It is now recognized that mitochondria play a crucial role in tumorigenesis, however, it has become clear that tumor metabolism varies significantly between cancer types. The failure of recent clinical trials aimed at directly targeting tumor respiration through oxidative phosphorylation inhibitors underscores the critical need for further studies providing an in-depth evaluation of mitochondrial bioenergetics. Accordingly, we comprehensively assessed the bulk tumor and mitochondrial metabolic phenotype in murine HER2-driven mammary cancer tumors and benign mammary tissue. Transcriptomic and proteomic profiling revealed a broad downregulation of mitochondrial genes/proteins in tumors, including OXPHOS subunits comprising Complexes I-IV. Despite reductions in tumor mitochondrial proteins, mitochondrial respiration was several-fold higher compared to benign mammary tissue, which persisted regardless of normalization method (wet weight, total protein content and when corrected for mitochondrial content). This upregulated respiratory capacity could not be explained by OXPHOS uncoupling, suggesting HER2 signaling regulates intrinsic mitochondrial bioenergetics. In further support, lapatinib, an EGFR/HER2 tyrosine kinase inhibitor, attenuated mitochondrial respiration in NF639 murine mammary tumor epithelial cells. Together, this data highlights that the typical correlation between mitochondrial content and respiratory capacity may not apply to all tumor types and implicates HER2-linked activation of mitochondrial respiration supporting tumorigenesis in this model.

BackgroundImmune checkpoint inhibitors (ICIs) targeting the programmed death (ligand)-1 (PD-1/PD-L1) axis, like pembrolizumab, have significantly improved survival in non-small cell lung cancer (NSCLC). However, less than 50% of patients respond. Identifying early-response biomarkers is crucial to personalize therapy, thereby preventing ineffective, expensive and potentially harmful treatment. MethodsWe applied a novel ex vivo immunopharmacological bioassay to assess pembrolizumab-dependent T cell signalling in baseline peripheral blood mononuclear cells (PBMCs) from 64 NSCLC patients. PBMCs were stimulated with anti-CD3/CD28 with or without pembrolizumab, and phosphorylation states of PD-1-dependent T cell receptor (TCR) signalling pathways were measured by spectral flow cytometry. A composite signalling score was calculated representing the net pembrolizumab-induced phosphorylation response and patients were classified as low, optimal and high modulation responders based on this signalling score. Associations with progression-free survival and overall survival (OS) were evaluated using univariate Cox regression. ResultsPatients with optimal baseline pembrolizumab-induced signalling scores exhibited significantly higher signalling score outcomes than those with low modulation (p < 0.0001) and lower than patients with excessive modulation (p < 0.01) and had significantly longer OS (HR = 2.83, p = 0.013; and HR = 12.05, p = 0.003, respectively). Notably, conventional pharmacodynamic parameters, including half-maximal effective concentration (EC50) for PD-1 receptor occupancy and maximum IL-2 production (Emax), were not associated with clinical outcomes, underscoring the unique predictive value of the phosphorylation-based signalling score. In vivo, pembrolizumab-induced T cell activation changes and TCR signalling inhibition post-treatment correlated with shorter survival (HRs = 1.33-1.95), consistent with our ex vivo findings. ConclusionsWe demonstrate that a pretreatment signalling score derived from ex vivo pembrolizumab-modulated T cell phosphorylation identifies clinical response in NSCLC. This functional bioassay offers a novel approach to identify patients most likely to benefit from ICI therapy, potentially enabling personalised treatment decisions before therapy initiation. Graphical abstract textOur findings reveal that pretreatment, pembrolizumab-dependent modulation of T cell phosphorylation identifies clinical response in NSCLC. Furthermore, we introduce an overall signalling score, reflecting the net phosphorylation profile, which could serve as a potential predictive biomarker to distinguish responders from non-responders, thereby supporting biomarker-driven therapeutic strategies.

Background: Gastric cancer surveillance in CDH1 pathogenic variant carriers is challenging, as predictors of localized (stage T1a) and advanced (stage >T1a) signet ring cell carcinoma (SRCC) are not well defined. We established the Group of investigAtors STriving toward Research In CDH1 (GASTRIC) consortium to identify clinicopathological factors associated with localized and advanced SRCC. Methods: A retrospective observational study (1998-2025) of CDH1 carriers across twelve academic centers was performed. Clinical, endoscopic, and pathological data were compared between carriers with and without SRCC on endoscopy, and between those with advanced versus localized or no cancer on gastrectomy specimens. Results: Overall, 390 CDH1 carriers from 235 families were included. Presence of SRCCs on endoscopy was significantly associated with thickened folds, nodularity, masses, and intestinal metaplasia, while gastritis was negatively associated. Of 196 carriers (52.4%) undergoing gastrectomy, 11 (5.6%) had advanced cancers, 10(90.9%) of which showed endoscopic abnormalities. Identification of SRCC on baseline endoscopy was the most sensitive feature for advanced disease (0.81) but had moderate specificity (0.74), whereas masses and thickened folds were highly specific (0.99 and 0.96, respectively) but less sensitive. Negative predictive values were high (0.94-1.0), while positive predictive values were modest (0.13-0.66). On multivariate analysis, masses and SRCC foci on baseline endoscopy were independent predictors of advanced disease. Conclusion: Among CDH1 carriers, absence of endoscopic findings was reassuring, whereas significance of detected endoscopic and pathological abnormalities was less certain. Advanced cancer occurred in a small number of carriers, with endoscopic abnormalities in nearly all cases. Endoscopic surveillance might be an alternative to surgery in carriers without worrisome mucosal findings.

BackgroundChemo-resistance remains a major clinical challenge in Oral Squamous Cell Carcinoma (OSCC), attributed to the intrinsically resistant cells. Although tumour-derived extracellular vesicles (EVs) have been implicated in cell-cell communication, their role in propagating chemo-resistance remains poorly defined. This study aims to identify salivary EV-associated miRNAs capable of predicting chemoresistance and to delineate the role of miR-1307-5p in modulating CSC-driven therapeutic refractoriness. MethodsSalivary EV-derived expression profile of miR-1307-5p was assessed by qPCR in chemo resistant OSCC patients and further validated in TCGA small RNA sequencing datasets. Expression was validated by qPCR and correlated with clinicopathological outcomes. Functional assays including cell-cycle analysis, apoptosis, migration/invasion, 3D spheroids, angiogenesis, and CAM assays were performed in miR-1307-5p inhibited CD44 CSC subpopulation compared to its vehicular control. Transcriptomic profiling cross-referencing with TCGA was conducted to identify potential novel targets of miR-1307-5p. Chemo-sensitisation was assessed by treating the knockdown chemo resistant cells with low dose cisplatin and validating it using in-vitro functional assays and orthotopic xenograft model. ResultsmiR-1307-5p was significantly elevated in salivary EVs of chemo resistant OSCC patients and correlated with poor overall survival (p = 0.03). The miRNA was markedly enriched in endogenously resistant CD44 CSCs. Silencing of miR-1307-5p induced G2/M arrest, triggered apoptosis, impaired invasion, and reduced angiogenesis both in-vitro and in ex-vivo assays. Transcriptomic profiling, TCGA validation, and integrative pathway analysis identified key oncogenic hubs which converge on PI3K-AKT, MAPK/ERK, and YAP signalling pathways governing EMT. Inhibition of miR-1307-5p restored cisplatin sensitivity in resistant CSCs, with low-dose cisplatin producing substantial tumour suppression in-vitro and in-vivo. Reduced CD44 expression in xenograft models confirmed CSC reprogramming. EVs from anti-miR-treated cells confer chemo sensitisation upon uptake by resistant CSCs. Xenograft models substantiated that EVs can initiate tumour formation and that EV-mediated delivery of anti-miR-1307-5p drives significant tumour regression. ConclusionThis study identifies salivary EV-derived miR-1307-5p as a clinically relevant biomarker of chemoresistance in OSCC and reveals its mechanistic role in sustaining CSC-driven therapeutic failure. Targeting miR-1307-5p offers a promising avenue for restoring cisplatin sensitivity and developing exosome-based therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/709730v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@19f88e0org.highwire.dtl.DTLVardef@d36b95org.highwire.dtl.DTLVardef@3c2579org.highwire.dtl.DTLVardef@c04ef5_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

BackgroundLung cancer, including Non-Small Cell Lung Carcinoma (NSCLC), is the leading cause of cancer mortality worldwide. Platinum salts are the gold standard chemotherapy for NSCLC but many patients develop resistance leading to disease progression. Identifying new therapeutic strategies to counteract resistance is crucial. Pharmacological compounds targeting core components of the spliceosome machinery have emerged as promising anti-cancer agents. However, their mechanisms of action remain to be elucidated in NSCLC. MethodsVarious NSCLC cell lines were used in 2D and 3D cultures or clonogenic assays. NSCLC Patient-Derived Xenografts were also used. SF3B1 was silenced by siRNA. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. Western-blot, immunofluorescence, SIRF analysis and DNA repair assays were done to assess globally the DNA damage response. RNA-Seq, RT-qPCR and RT-PCR studies were performed to identify gene and splicing events impacted by SF3B1 inhibition. Publicly available transcriptomic and proteomic data were analyzed. ResultsSF3B1 is a core component of the spliceosome machinery. We show that NSCLC cells with acquired resistance to platinum salts are vulnerable to pladienolide B, a SF3B1 inhibitor, or SF3B1 knock-down. Importantly, pladienolide B also slows down tumor growth of NSCLC Patient-Derived Xenografts (PDXs) poorly responsive to platinum salts. Mechanistically, we show that pladienolide B leads to genomic instability and apoptosis, that correlate with early transcription-dependent replication stress and DNA-PKcs activation, followed by the shutdown of ATR/DNA-PKcs-dependent signaling. In addition, pladienolide B profoundly regulates the expression and/or splicing, particularly exon skipping, of numerous genes involved in DNA repair, leading to decreased repair capacities of DNA double strand breaks. Although exon skipping events are mostly transient, skipping of exon 8 of MLH3, a gene involved in mismatch DNA repair, persisted along time. Finally, we show that pladienolide B counteracts resistance to platinum salts in NSCLC cells as well as PDXs, which correlates with enhanced MLH3 exon 8 skipping and decrease of ATR, DNA-PKCs and MLH3 protein levels. ConclusionsAs a whole, our data highlight the targeting of SF3B1 as a potential therapeutic strategy, alone or in combination, in NSCLC patients who escape platinum salts-based chemotherapy.

BackgroundHead and neck squamous cell carcinoma (HNSCC) comprises a heterogeneous group of epithelial malignancies associated with poor survival ({approx}50%), limited therapeutic options, and a lack of predictive biomarkers. Concurrent chemoradiotherapy (CRT) remains the standard treatment for advanced disease; however, many patients fail to respond, develop resistance, or eventually relapse. The development of three-dimensional organoid technology has enabled the generation of patient-derived organoids (PDOs), offering a promising platform for personalized therapeutic testing. MethodsWe established a biobank of HNSCC PDOs from fresh laryngeal and pharyngeal tumor samples, including human papillomavirus-positive (HPV+) cases. Organoid formation and expansion rates were analyzed in relation to clinical parameters. Selected representative PDOs were histologically and molecularly characterized. Additionally, several models were exposed to cisplatin and radiation to evaluate treatment response, and a subset was assessed for tumorigenicity in subcutaneous mouse models. ResultsFifty-seven PDO models were successfully established, long-term expanded, and cryopreserved. Prior chemotherapy and/or radiotherapy was identified as an independent negative predictor of organoid outgrowth and expansion capacity compared with treatment-naive samples. Histological features, including differentiation grade and immunohistochemical markers, were largely preserved and strongly correlated with the original tumors. PDOs displayed heterogeneous responses to cisplatin and radiotherapy, with HPV-positive models showing greater sensitivity, consistent with clinical observations. Global transcriptomic profiling revealed molecular subtypes concordant with established HNSCC classifications and suggested an additional subtype characterized by low MYC and mTORC1 transcriptional activity. ConclusionHNSCC PDOs faithfully recapitulate tumor histology and molecular diversity, providing a robust platform to investigate tumor biology and therapeutic response.

PurposeTreatment options of advanced oral squamous cell carcinomas (OSCC) are limited, and cisplatin toxicity and drug resistance are major clinical issues. Src is a central kinase that integrates multiple oncogenic pathways and a promising therapeutic target. However, Src inhibitors have shown suboptimal efficacy as monotherapies and their sensitivity in OSCC remains elusive. Experimental DesignWe examined the activation of major oncogenic signaling pathways and the antitumor effects of six Src inhibitors (dasatinib, ponatinib, vandetanib, saracatinib, PP2, bosutinib) in seven human OSCC cell lines (HSC-2, HSC-3, HSC-4, SAS, HO-1-u-1, CAL27, SCC-25). BALB/cAJcl nu/nu mice bearing CAL27 xenografts received dasatinib (30 mg/kg, intraperitoneally, daily), bosutinib (50 mg/kg, intraperitoneally, daily), cisplatin (2 mg/kg or 4 mg/kg, intraperitoneally, weekly), or combinations. Tumor volume, bioluminescence imaging, and body weight were monitored for 17 or 21 days, followed by histopathological assessment. ResultsThe activation of the key pathways, including Src and MAPK, considerably differed among the cell lines and was linked to heterogeneous sensitivity to Src inhibitors. Effective growth suppression required Src dephosphorylation and downstream MAPK pathway inhibition, which vary depending on the cell line. Additionally, combination treatment with a Src inhibitor and cisplatin showed additive antitumor effects, allowing the reduction of cisplatin doses by half without efficacy loss. Notably, dasatinib alone and in combination with cisplatin decreased tumor burden with characteristic internal tumor death in vivo. ConclusionsThese findings elucidate Src signaling dependency on OSCC and the potential of Src inhibition to decrease cisplatin toxicity, paving way for Src targeted therapeutic strategies.

Epidemiological studies suggest that vegetarian diets are associated with lower cancer incidence and mortality, an effect attributed in part to phytochemicals such as polyphenols and carotenoids. Although numerous in vitro experiments and investigations using immunodeficient rodent models report tumor-suppressive activities of phytochemicals, their impact on tumor progression in immunocompetent hosts remains insufficiently understood. Here, we examined the influence of a defined plant phytochemical mixture (PPM) on the growth of colon cancer liver metastases, both in vitro and in immunocompetent mice. Consistent with the prevailing literature, treatment of the murine colon cancer cell line MC38 with the PPM significantly reduced cell proliferation and survival in vitro. Strikingly, however, administration of the PPM to mice bearing MC38-derived hepatic metastases markedly accelerated tumor growth. Immunohistochemical analyses revealed a significantly increased accumulation of immune cells--specifically CD45 leukocytes and F4/80 macrophages--at the periphery of the metastatic lesions in PPM-treated animals. To assess the functional relevance of this inflammatory response, the PPM was combined with the anti-inflammatory drug prednisolone. This intervention resulted in significantly reduced metastatic burden, supporting the notion that the PPM exacerbates tumor progression through enhanced peritumoral inflammation. These findings highlight the importance of validating observations from cell culture and immunodeficient models in fully immunocompetent systems. They further emphasize that the immunomodulatory effects of plant phytochemicals warrant careful and comprehensive investigation.