Oncogenesis

Cancer metabolism rewiring is one of the hallmarks of cancer, enabling cancer cell survival in a nutrient deprived microenvironment. Key to this is nutrient scavenging where cancer cells rely on extracellular proteins, including extracellular matrix (ECM) components, to sustain their proliferation. ECM uptake is mediated by 2{beta}1 integrin, however it is not clear how this process is controlled by nutrient availability. Here we demonstrated that amino acid starvation promoted ECM internalisation, by inducing the expression of 2 integrin. Mechanistically, starvation-driven RAS/MAPK pathway activation in cells harbouring oncogenic RAS mutations and mTOR inhibition increased 2 integrin, while the GCN2-depedent integrated stress response was not required. Functionally, elevated 2 integrin levels promoted cell adhesion and migration in nutrient starved cells. Finally, 2 integrin was found upregulated in pancreatic tumours and correlated with poor prognosis in pancreatic adenocarcinoma patients. Together, these data indicate that the nutrient- starved pancreatic cancer microenvironment synergises with KRAS mutation to drive pancreatic cancer aggressiveness.

Well- and dedifferentiated liposarcomas (WDLPS and DDLPS) are characterized by extensive copy- number alterations rather than recurrent gene-inactivating mutations, obscuring the molecular mechanisms that drive disease progression and, critically, the transition from well-differentiated to the more aggressive dedifferentiated tumor states. Despite marked differences in clinical behavior and prognosis, the regulatory events underlying adipocytic lineage destabilization in DDLPS remain poorly understood. Here, we establish an in vivo model of retroperitoneal liposarcoma in Xenopus tropicalis through early embryonic mosaic perturbation of p53 and Rb pathway components. Combined disruption reproducibly induced retroperitoneal WDLPS development, demonstrating that pathway-level deregulation of the MDM2-p53 and CDK4-Rb axes is sufficient to initiate liposarcoma development in vivo. Strikingly, additional perturbation of transcriptional co-activator ep300 in this context resulted in increased tumor dedifferentiation, yielding lesions composed of spatially coexisting well- and dedifferentiated adipocytic states. In contrast, direct targeted disruption of downstream adipogenic regulators recurrently lost in human DDLPS, including cebpa, g0s2, and dgat2, failed to induce dedifferentiation in the same genetic context in vivo. These findings indicate that dedifferentiation cannot be explained by loss of downstream adipocytic effectors alone but instead reflects destabilization of higher-order regulatory programs governing adipocytic identity. Together, these results establish an in vivo model that closely reflects the clinical situation on a pathway level and provides initial mechanistic insight into how adipocytic differentiation may become destabilized during disease progression. This framework offers a foundation for future studies leveraging higher-order and multi-omic approaches to dissect the molecular processes underlying the WDLPS-to-DDLPS transition.

Colorectal cancer across sub-Saharan Africa presents a growing global health burden, with increasing cases and mortality linked to late diagnosis, limited healthcare access and lack of effective treatments. African patients typically present with aggressive disease marked by distinct genomic signatures, indicating the need for targeted treatment approaches. We integrated genetic modelling, phenotypic scoring, imaging and biochemical analysis to explore how mutations found in individual Nigerian colorectal cancer patients influence drug responsiveness. We used the data from Cancer Genome Atlas to identify mutation profiles specific to Nigerian patients. We then generated ten stable Drosophila melanogaster personalised patient avatar lines designed to model patient genomic profiles. This study focused on three lines; each line included oncogenic RAS plus targeting patient-specific variants. These models exhibited various phenotypes including altered larval size, gut size and reduced survival. Two of the three avatar lines showed improved survival, reduced hindgut proliferation zone expansion and restored redox balance after treatment with regorafenib and trametinib. Mirroring clinical patient responses, we found that response to therapy is dependent on the specific genetic profile of the tumour. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/714433v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@110518aorg.highwire.dtl.DTLVardef@5965a0org.highwire.dtl.DTLVardef@11f16a3org.highwire.dtl.DTLVardef@744a1_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIAfrican colorectal cancer showed distinct mutation patterns that contribute to tumour heterogeneity. C_LIO_LIPatient-derived Drosophila avatars were engineered using tumour-specific genetic mutations with key features of human colorectal cancer. C_LIO_LITreatment with targeted therapies showed responses patterned by tumour genotype. C_LIO_LIResponse patterns indicated the need for personalised for colorectal cancer therapies among diverse populations. C_LI

Chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 has transformed outcomes for children with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL), yet the influence of molecular subtype on outcomes remains unclear. We evaluated the impact of cytogenetic and molecular signatures on complete response (CR), overall survival (OS), and leukemia-free survival (LFS) after CD19 CAR-T therapy in eighty-six pediatric patients with R/R B-ALL treated with tisagenlecleucel. CR was assessed 30 days after infusion. Cytogenetic data were available for 84 patients and molecular profiling for 62. Survival analyses included 72 patients who received CD19 CAR-T as the sole cellular therapy. Seventy-seven patients achieved CR (89.5%). Pre-infusion bone marrow blasts of [&ge;]20% were associated with lower CR rates (53.8% vs 95.9%, p<0.0001) and significantly reduced OS and LFS (both p<0.0001). Among molecular markers, RAS mutations correlated with inferior OS (p=0.0222) and LFS (0.0402). In multivariate analysis, bone marrow blasts >20% and RAS mutations independently predicted inferior OS. Post CAR-T, CD19 negative relapses showed almost twice higher prevalence of RAS mutations (66% vs 37.5%). These findings highlight RAS mutations as a key molecular predictor of outcome after CD19 CAR-T therapy and suggest emergence of unique risk stratification for patients receiving CD19-targeting therapy.

Macrophages/osteoclasts are highly fusogenic cells that interact closely with bone-metastatic breast cancer cells. These cancer cells adapt to bone microenvironments by undergoing osteomimicry, acquiring bone-like phenotypes. Exploration using human breast cancer-bone metastases dataset revealed that a small population of epithelial breast cancer cells express osteoclast-like and osteomimicry genes at the single-cell level. Cell fusion and cell-in-cell (CIC) processes are two uncommon yet prognostically significant mechanisms in cancer. We showed that co-culture between murine breast cancer cells and osteoclasts yielded a unique osteoclast phenotype through dynamic cell-in-cell (CIC) interactions and fusion-like behaviours between pre-osteoclasts/mature osteoclasts and breast tumor cells, resulting in osteoclast-tumor hybrid-like cells. These tumor cell interactions characterized by membrane retention and nuclear adjacency to host nuclei were consistently observed throughout osteoclast differentiation. Single-cell sequencing analysis and interpretative assays on hybrid-like cells revealed altered extracellular matrix (ECM) modification processes, immunoregulatory, and cancer-associated pathways compared to unfused osteoclasts. Tumor cells co-cultured with osteoclasts expressed hematopoietic and osteoclast-lineage factors more strongly than tumor cells cultured alone with their effects amplified under direct cell-cell contact. The presence of these hybrid-like cells was validated in human breast cancer-bone metastases. We propose that disseminated bone-tropic breast cancer cells were stimulated by osteoclasts to undergo a non-canonical, dynamic osteoclast differentiation and CIC formation to form hybrid-like cells that may facilitate bone metastatic lesions.

Atypical teratoid rhabdoid tumor (ATRT) is a malignant brain tumor of children that has an overall survival of less than 40 percent even with aggressive therapy. We identified upregulation of the mitogen activated protein (MAP) kinase pathway in ATRT. The novel, brain-penetrant MEK inhibitor mirdametinib inhibited the growth of ATRT cell lines in culture at nanomolar concentrations. Mirdametinib suppressed proliferation as measured by BrdU incorporation and induced apoptosis as measured by cPARP and Annexin V staining. Monotherapy with mirdametinib extended the life of mice bearing orthotopic xenografts. Combination therapy with the brain-penetrant cyclin dependent kinase 4/6 inhibitor abemaciclib further suppressed growth and BrdU incorporation in ATRT cell lines representing all molecular subgroups. Mirdametinib and abemaciclib combined to extend survival of mice bearing orthotopic ATRT xenografts. In conclusion, mirdametinib has single agent activity against ATRT and combines with abemaciclib to decrease proliferation and extend survival in orthotopic xenograft models of ATRT.

Osteosarcoma is a malignant bone tumor with a high risk of metastatic relapse and poor outcomes due to primary and acquired chemoresistance. This highlights the medical need to develop effective targeted approaches to overcome chemoresistance. Recent studies have revealed the roles of metabolic reprogramming and mitochondria-nucleus crosstalk in osteosarcoma progression, indicating the potential of these cellular processes as therapeutic targets. The complex formed by mitochondrial apoptosis-inducing factor (AIF) and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) orchestrates the import and oxidative folding of cysteine-rich, nuclear-encoded proteins, thereby regulating key mitochondrial functions and metabolism. Here, we identified mitoxantrone as an inhibitor of the AIF/CHCHD4 mitochondrial import machinery and revealed a new mitoxantrone-induced metabolic vulnerability in some osteosarcoma cell line models, characterized by intracellular glutamine accumulation and an increase in nucleotide synthesis. As a result, synergy was found between mitoxantrone and the glutaminase inhibitor telaglenastat in both in vitro and in vivo osteosarcoma models. Collectively, our findings position the AIF/CHCHD4 complex as a druggable therapeutic target and provide a combination strategy for mitoxantrone/telaglenastat treatment to overcome metabolic adaptations and chemoresistance in osteosarcoma. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/716303v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1229e58org.highwire.dtl.DTLVardef@1c9af45org.highwire.dtl.DTLVardef@120d2borg.highwire.dtl.DTLVardef@11e8216_HPS_FORMAT_FIGEXP M_FIG C_FIG

The central nervous system (CNS) is a common site of metastatic spread for both non-small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases remain limited. Tyrosine kinase inhibitors have shown promising results in increasing the overall response in brain metastases, owing to their brain penetrance and increased effectiveness; however, their use is limited to the small group of tumors carrying specific oncogenic drivers. Among these, inhibitors with activity against neurotrophic tyrosine receptor kinases (NTRKs) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the brain metastatic niche and can, in a paracrine manner, activate NTRK function in cancer cells. Here we show that NTRKs are expressed in primary tumors, brain metastases, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous BDNF and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro, and effectively prevented brain metastatic colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for brain metastases of WT-NTRK+ lung cancers, and therefore, NTRK inhibitors can be used to target non-fusion NTRK function to prevent or decrease brain metastases. SIGNIFICANCEThese studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

Keratin-positive giant cell-rich tumor (KPGCT) is a newly described bone and soft tissue tumor. The tumor is characterized by scattered keratin-positive cells and the presence of HMGA2::NCOR2 fusions. It is not known if the HMGA2::NCOR2 fusion is located in the keratin-positive cells, and little is known about how KPGCT develops. KPGCT shares some histologic features with tenosynovial giant cell tumor (TGCT), a soft tissue tumor with CSF1 rearrangements. Single-nuclei RNA sequencing (snRNA-seq) and Xenium spatial transcriptomics were used to elucidate the mechanisms driving KPGCT and compare KPGCT to TGCT. We show that the neoplastic cells in KPGCT constitute only a minority of cells in the tumor, and that they co-express keratin, HMGA2 and CSF1. The neoplastic cells in KPGCT express no synovial markers, confirming KPGCT as a distinct entity, separate from TGCT. The bulk of the tumor consists of CSF1R-expressing macrophages and osteoclast-like giant cells, suggesting an important role for CSF1-CSF1R signaling. In addition, we find that the cells with the HMGA2 translocation show activation of the hippo signaling pathway, which is known to regulate CSF1 expression. We show that the CSF1-CSF1R axis, possibly regulated through the hippo signaling pathway, plays an important role in KPGCT. This axis likely stimulates the migration and proliferation of macrophages, which form the majority of cells in the tumor, as well as their differentiation into osteoclasts-like giant cells. These results provide a rationale for the use of CSF1R inhibitors, which have already shown efficacy in TGCT, as a therapy for KPGCT. SignificanceKeratin-positive giant cell-rich tumor (KPGCT) is a rare, newly described soft tissue and bone tumor. By examining this tumour on a single-cell level, we confirm the identity of the neoplastic cells on a molecular level, showing these form a minority of cells in the tumor. We show that activation of the hippo pathway in the neoplastic cells is a likely driver of tumorigenesis. Additionally, we show the neoplastic cells produce large amounts of CSF1, attracting the macrophages that form the majority of cells in the tumor. This finding gives supporting evidence for anecdotal reports of response to CSF1 inhibitor therapy. Finally, we identify key differences between KPGCT and tenosynovial giant cell tumor, a tumor that shares histological features with KPGCT.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and lacks effective therapies. The stimulator of interferon genes (STING) has been shown to both suppress and promote migration in various cancer types, but its role in TNBC remains unclear. To investigate this, we established STING-overexpressing murine TNBC cell lines and assessed their migratory and proliferative behavior. STING overexpression significantly suppressed cell migration without affecting cell proliferation. Furthermore, STING overexpression upregulated expression levels of Itgb1 and Itga6 significantly, but not Icam1, Cxcl3, Itgb2, Lama5, and Rhoa. These findings highlight the potential anti-migratory role of STING beyond immunomodulatory functions.

B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in hepatocellular carcinoma (HCC) and represents a promising therapeutic target. However, its roles in tumor cell adhesion, metastatic behavior and immune evasion--particularly in interactions with natural killer (NK) cells--remain incompletely understood. In the present study, B7-H3 was depleted using small interfering RNA and CRISPR/Cas9 in epithelial (Huh7 and HepG2) and mesenchymal (SNU449) HCC cell lines. Tumor cell survival, apoptosis, adhesion, migration and invasion were evaluated using functional assays. Expression of adhesion molecules and immune checkpoint proteins was assessed by flow cytometry and western blotting. Oncogenic signaling pathways were analyzed by examining phosphorylation of Akt, ERK, FAK and STAT3. NK cell-mediated cytotoxicity was assessed using primary human NK cells. B7-H3 depletion reduced clonogenic survival and increased apoptosis in mesenchymal HCC cells under anchorage-independent conditions. Loss of B7-H3 impaired cell adhesion, migration and invasion, accompanied by downregulation of PTGFRN, E-cadherin, integrin 3 and integrin V, and reduced cell-to-cell aggregation under anchorage-independent conditions. B7-H3 depletion also decreased surface expression of PD-L1, PD-L2 and CD47. Notably, B7-H3-deficient cells exhibited enhanced susceptibility to primary NK cell-mediated cytotoxicity. Mechanistically, B7-H3 promoted tumorigenic signaling through Akt/S6, MVP/ERK and FAK/Src pathways in epithelial cells, and through FAK/Src and JAK2/STAT3 pathways in mesenchymal cells. Together, these findings reveal previously unrecognized roles for B7-H3 in coordinating adhesion and NK immune evasion in HCC, and support its therapeutic targeting for next-generation immunotherapies.

La-related protein 1 (LARP1) is an RNA-binding protein that post-transcriptionally regulates mRNA with potential oncogenic role in multiple cancers; however, its function in endometrial cancer remains unknown. An analysis of the TCGA endometrial cancer cohort showed that overexpression of LARP1 is associated with shorter overall survival (OS) and progression-free interval (PFI) as indicated by Kaplan-Meier analysis. Functional in vitro studies revealed that LARP1 knockdown by two different siRNAs markedly suppressed cell viability and triggered apoptosis, as confirmed by increased protein levels of cleaved PARP1 and cleaved caspase-3. Mechanistically, LARP1 knockdown remarkably reduced E2F1 protein levels as confirmed by immunofluorescence and Western blotting. Clinically, co-overexpression of LARP1 and E2F1 further decreased OS and PFI, suggesting a co-operative oncogenic axis. Importantly, LARP1 knockdown enhanced the sensitivity of ISHI and HEC-1A endometrial cancer cell lines to carboplatin treatment. These findings suggest that LARP1 promotes endometrial cancer survival and resistance to chemotherapy, at least in part, through the regulation of E2F1 and suppression of apoptosis. Targeting LARP1 could represent a promising therapeutic strategy to suppress tumor growth and enhance sensitivity to platinum-based chemotherapy.

Cellular senescence exerts powerful non-cell autonomous effects through the senescencelzlassociated secretory phenotype (SASP). This SASP comprises soluble factors and extracellular vesicles (EVs). Although soluble SASP components can induce senescence in neigbouring cells, the specific contribution of EVs to paracrine senescence is poorly defined. Here, we show that EVs released by senescent tumor cells are necessary and sufficient to propagate senescence. Conditioned media from bleomycinlzlinduced senescent A549 cells triggered a permanent growth arrest with morphological changes and upregulation of senescence markers in recipient tumor cells. Pharmacological inhibition of EV biogenesis using GW4869 or genetic downregulation of the EV secretion mediator RAB27A markedly attenuates paracrine senescence without affecting soluble SASP factor secretion or the senescent state of producer cells. Proteomic characterization reveals that senescent EVs exhibit a distinct molecular signature enriched for extracellular components and processes related to wound healing and hemostasis. Importantly, purified senescent EVs, devoid of soluble SASP factors, fully recapitulated paracrine senescence induction. These findings identify senescent EVs as key autonomous SASP effectors and highlight vesicular pathways as potential therapeutic targets in cancer and therapylzlinduced senescence.

Prostate cancer progression is characterized by dysregulated lipid metabolism, with fatty acid synthase (FASN), the rate-limiting step in de novo lipogenesis (DNL), resulting in significant accumulation of saturated lipids. Here, we investigate whether pharmacologic FASN inhibition creates a metabolic state that increases reliance on exogenous polyunsaturated fatty acids (PUFAs). Inhibition of FASN profoundly alters membrane phospholipid composition, driving compensatory incorporation of PUFAs into membrane phospholipids, thus increasing susceptibility to lipid peroxidation and oxidative damage. Combined FASN inhibition and PUFA exposure increased reactive oxygen species, induced mitochondrial hyperpolarization, and enhanced lipid peroxidation in both hormone-sensitive and castration-resistant prostate cancer models. Marked inhibition of human and murine prostate cancer organoids is achieved ex vivo. In genetically engineered, DNL-reliant Hi-Myc mice, a diet enriched in PUFAs significantly inhibited invasive carcinoma compared to a saturated fat-enriched diet. Environmental PUFAs modulate and enhance the therapeutic efficacy of FASN-targeted strategies. These findings set the stage for pharmacologic and dietary intervention in prostate cancer patients.

Temozolomide (TMZ) resistance remains a major obstacle in glioblastoma (GBM) therapy, yet the metabolic adaptations underlying this phenotype are incompletely understood. Here, we performed integrative lipidomic, ultrastructural, and pathway analyses to define lipid metabolic reprogramming associated with TMZ resistance and failure of statin-mediated sensitization. Targeted LC-MS lipidomics quantified 322 lipid species across 25 lipid classes in TMZ-sensitive and TMZ-resistant U251 cells under basal conditions and following TMZ, simvastatin, or combination treatment. Multivariate analyses (PCA, PLS-DA, and volcano plots) revealed a robust and treatment-resilient lipidomic signature in resistant cells characterized by enrichment of lysophospholipids, sphingolipids, and cholesteryl esters, alongside depletion of glycerolipid and phospholipid pools. Complementary univariate analysis confirmed these changes at the species level, demonstrating consistent elevation of lysophosphatidylcholine/ethanolamine, glycosphingolipid subclasses, and cholesteryl esters, together with reductions in phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and diacylglycerol intermediates across multiple treatment conditions. In contrast, sensitive cells displayed dynamic lipid remodeling, including phosphatidylinositol and phosphatidylethanolamine enrichment associated with autophagic membrane expansion. KEGG pathway analysis linked the resistant phenotype to Rap1, PI3K-Akt, and phospholipase D signaling networks regulating vesicle trafficking and membrane homeostasis. Transmission electron microscopy confirmed a vesicle-rich intracellular architecture consistent with persistent autophagy flux blockade in resistant cells. Collectively, these findings define a stable lipid metabolic program characterized by lysophospholipid expansion and cholesteryl ester accumulation that supports membrane integrity and therapeutic resistance. Targeting lipid buffering and cholesterol storage pathways may represent a promising strategy to overcome chemoresistance in glioblastoma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/712341v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@178acd7org.highwire.dtl.DTLVardef@19b6a79org.highwire.dtl.DTLVardef@6b3904org.highwire.dtl.DTLVardef@16c3d01_HPS_FORMAT_FIGEXP M_FIG C_FIG Lipidomic and autophagy differences between non-resistant (NR) and temozolomide-resistant (R) glioblastoma cells. NR cells show dynamic lipid remodeling and treatment-dependent autophagy responses, whereas R cells maintain blocked autophagy flux and persistent enrichment of LPC, SM, and cholesteryl esters across treatments.

3-mercaptopyruvate sulfurtransferase (3-MST) is a mammalian enzyme that contributes to hydrogen sulfide and reactive sulfur species generation. Here we show that 3-MST is markedly upregulated in colorectal cancer stem cells (CSCs) and functions as a critical metabolic support mechanism for this therapy-resistant tumor cell population. CSCs exhibit low proliferation rate, high membrane rigidity and a metabolically restrained phenotype characterized by low oxidative phosphorylation rate, combined with a reduced rate of glycolysis. Genetic or pharmacological inhibition of 3-MST further suppresses cellular bioenergetics in CSCs, and this bioenergetic collapse impairs CSC proliferation, spheroid formation, migration and promotes cell death and attenuates tumor growth. Integrated transcriptomic, proteomic, metabolomic, and lipidomic analyses reveal extensive metabolic remodeling of the CSCs following 3-MST inhibition, including disruption of the glycolysis-TCA axis and marked remodeling of membrane lipid composition, including enrichment of ceramides and sphingolipids and increased incorporation of polyunsaturated phospholipids, resulting in increased membrane fluidity. 3-MST inhibition induced an activation of integrated stress pathways, proteotoxic stress responses and inflammatory signaling, linking the metabolic failure of CSCs to the induction of mixed-mode cell death. These findings identify 3-MST as a metabolic vulnerability in colorectal CSCs. Targeting this enzyme may be a translatable strategy to eliminate therapy-resistant tumor stem cell populations.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer characterized by a high abundance of cancer-associated fibroblasts (CAFs), which influence therapy response, tumor biology and tumor aggressiveness. CAFs are a heterogeneous cell type and previous single-cell RNA sequencing (scRNAseq) of PDAC tumors identified three main CAF subtypes: myofibroblastic, inflammatory and antigen-presenting CAFs (myCAF, iCAF, apCAF, respectively). However, scRNAseq on large patient cohorts is often not feasible due to costs and technical constraints. Therefore, bulk RNAseq deconvolution can be used to identify cell types within the heterogeneous tumor microenvironment. Here, Statescope deconvolution was used to identify different cell types of the tumor microenvironment within an early onset PDAC cohort, comprising 74 patients aged under 60. Three CAF populations were identified (iCAFs, myCAFs and desmoplastic CAFs), and their correlations with tumor microenvironment components, mutational signatures and survival were examined. iCAFs were associated with classical-like tumor cells, whereas myCAFs and desmoplastic CAFs correlated with basal-like tumor cells. Desmoplastic CAFs are associated with inflammatory granulocytes/neutrophils, while negatively associating with monocyte-derived macrophages and immature/transitional B cells. No associations were observed between mutational signatures and the abundance of CAF and epithelial tumor subtypes. Interestingly, a high abundance of CAFs, and specifically increased iCAF abundance, was associated with improved survival. This iCAF-mediated survival effect was predominantly apparent in female patients. All in all, deconvolution of bulk RNA sequencing data, followed by its integration with clinical and biological parameters, reveals the heterogeneity and prognostic implications of CAF subpopulations in the tumor microenvironment of early onset PDAC patients.

Obesity-driven metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are shaped by depot-specific adipose tissue dysfunction, including maladaptive expansion and visceral adipose tissue (VAT) fibrosis. Pirfenidone, an anti-fibrotic agent, improves experimental liver disease. However, its actions on adipose depots and adipose-liver crosstalk remain unclear. Here, we identify pirfenidone as a modulator of mechanistic target of rapamycin complex 1 (mTORC1)-dependent adipose tissue remodeling with divergent outputs in subcutaneous and visceral fat. In diet-induced obese MASH mice, pirfenidone decreased subcutaneous adipose tissue (SAT), inhibiting mTORC1-driven lipogenesis and enhancing oxidative lipid metabolism. Pirfenidone attenuated VAT fibrosis by suppressing an mTORC1-mothers against decapentaplegic homolog 3 (SMAD3)-yes-associated protein (YAP) axis and extracellular matrix gene programs. Pirfenidone also lowered hepatic triglycerides, improved steatosis and fibrosis, reduced hepatic mTORC1 activity. Conditioned medium from fibrotic adipocytes induced lipogenic, inflammatory, and pro-fibrotic programs in AML12, which effects that were blunted by pirfenidone. These data reveal adipose tissue-centered actions of pirfenidone that link mTORC1 remodeling to improved obesity-associated liver disease.

BackgroundDrug responses in pancreatic ductal adenocarcinoma (PDAC) vary sharply across in vitro culture formats, but most 2D-3D comparisons conflate microenvironmental cues with time-dependent cellular adaptation. As a result, conventional assays frequently overestimate drug efficacy and poorly reflect clinical pharmacology. Main findingsWe profiled MiaPaCa-2, PANC-1, and CFPAC-1 grown in an extracellular-matrix (ECM) hydrogel for 1-12 days, defining extended 3D cultures ([&ge;]10 days) as mature tumoroids, and quantified 72 h drug responses to a multi-class oncology panel using growth-rate (GR) metrics to normalize for proliferation across formats and durations. Prolonged 3D pre-culture induced broad tolerance, with typical 10-100x reductions in sensitivity to standards of care (5-fluorouracil, SN38, oxaliplatin, gemcitabine, paclitaxel), following a reproducible susceptibility hierarchy (MiaPaCa-2 > PANC-1 > CFPAC-1) after GR correction. In mature tumoroids, GR values closely approximated clinically observed plasma exposures (e.g., within <4x for 5-FU and <0.5x for gemcitabine), whereas 2D and short-term organoid assays markedly underestimated resistance, often by >100x, thereby overstating drug activity. Notably, CFPAC-1 exhibited increased sensitivity to SN38 and trametinib under mature-organoid conditions, demonstrating that microenvironmental conditioning can invert responses for selected mechanisms. Transcriptomic profiling revealed coordinated up-regulation of multiple ABC transporters with extended 3D residence, tracking resistance phenotypes across lines and implicating transporter-linked tolerance programs. SignificanceTogether, these data identify time-in-3D and the emergence of mature tumoroids as dominant, previously under-controlled determinants of PDAC pharmacology that both induce tolerance and unmask context-dependent vulnerabilities. We propose incorporating both short-term and mature-tumoroid screening arms into preclinical workflows, reporting pre-culture duration alongside GR-normalized effect sizes, and leveraging transporter-informed biomarkers to guide regimen prioritization and sequencing. This framework enhances physiological relevance, reproducibility, and translational fidelity in PDAC drug discovery.

The RUNX1 transcription factor is a critical regulator of hematopoiesis and frequently mutated in myeloid malignancies. In the myeloproliferative neoplasm, chronic myeloid leukemia (CML), secondary somatic RUNX1 mutations and RUNX1::MECOM/EVI1, are associated with tyrosine kinase inhibitor (TKI) resistance and progression to the blast-phase (BP-CML). Research has predominantly focussed on transcriptional dysregulation mediated by RUNX1 mutations in myeloid malignancies, whilst post-transcriptional dysregulation remains comparatively unexplored. To address this, we used orthogonal organic phase separation (OOPS), to characterise the RNA-binding proteome of RUNX1 deficient BP-CML cells. RUNX1 depleted BP-CML cells exhibited significant alterations to RBP abundance involved in stress response pathways and translation/ribosome-biogenesis (RiBi). Furthermore, RUNX1 depletion or expression of RUNX1::EVI1 in BP-CML cells induced expression and RNA binding activity of SPATS2L, a component of stress granules (SG); membraneless cytoplasmic condensates protecting mRNAs from degradation, promoting survival under stress. Whilst RUNX1 depletion increased SG-assembly, SPATS2L depletion reduced SG-assembly in BP-CML cells and inhibited the growth and survival of multiple BP-CML cell lines. The translation inhibitor homoharringtonine (HHT), used historically in TKI-resistant CML, ablated SG-assembly in BP-CML cells with RUNX1 depletion, and, primary BP-CML cells with LOF/hypomorphic RUNX1 mutations (characterised by defective DNA-binding/CBF{beta}-interaction) were preferentially sensitised to HHT. Finally, suppressing SPATS2L expression induced by RUNX1 depletion, increased the HHT-sensitivity of RUNX1 depleted BP-CML cells, suggesting SPATS2L contributes to therapeutic resistance in CML with RUNX1 mutations. This study suggests that SPATS2L and SG induction could be critical to RUNX1-mutant leukemias, and, provides preliminary evidence for a mutationally-targeted approach in CML with RUNX1 aberrations.