Oncogenesis

BackgroundIn RAS-mutant tumors, ERK phosphorylates the mitochondrial fission GTPase DRP1 to promote mitochondrial fission. DRP1 activity is tumor-promoting in pancreatic and other RAS-driven cancers, but its role in therapeutic resistance is unknown. MethodsWe developed a panel of patient-derived pancreatic cancer cell lines resistant to the MEK inhibitor trametinib. We used immunofluorescence imaging, in vitro growth assays and orthotopic xenografts to determine the role of DRP1 in trametinib resistance. ResultsWe find that trametinib-resistant cells exhibit increased expression and phosphorylation of DRP1 compared to sensitive counterparts. Quantitative analysis of mitochondrial structure reveals that mitochondria in resistant cells are morphologically distinct and relatively smaller than sensitive cells treated with trametinib. Genetic and pharmacological inhibition of both c-Myc and CDK6 are sufficient to block DRP1 phosphorylation in resistant cells, suggesting that activation of a c-Myc-CDK6 signaling axis drives reactivation of mitochondrial fission in the absence of MAPK signaling. Importantly, deletion of DRP1 leads to either growth inhibition or re-sensitization to trametinib in resistant lines. ConclusionThese findings suggest DRP1 contributes to drug resistance, and that inhibition of mitochondrial fission might be a promising therapeutic strategy to combat resistance to MAPK and RAS inhibitors.

PurposeGlasdegib is a Sonic Hedgehog (SHH) pathway inhibitor used for treating newly diagnosed acute myeloid leukemia in elders or patients unfit for intensive chemotherapy. This study sought to demonstrate growth inhibition and increased apoptosis of B-cell acute lymphoblastic leukemia (B-ALL) in vitro under glasdegib, alone and combined with inotuzumab, using a novel co-culture system and validated chemosensitivity testing model to determine whether glasdegib with and without inotuzumab may represent a promising treatment strategy in B-ALL. MethodsSeven blood and marrow samples from B-ALL patients were co-cultured with HS-5 stromal cells in a co-culturing system designed to mimic the tumor microenvironment to maintain B-ALL cell viability for chemosensitivity testing under glasdegib and inotuzumab. ResultsCo-culturing improved B-ALL viability from four to nine days. Dosage-dependent responses to glasdegib were consistent among B-ALL samples on day four based on culture viability, and varied based on expressions of SSH genes GLI1, GLI3, SMO, and PTCH1. Combination with inotuzumab had varied effects on treatment response. ConclusionCo-culturing B-ALL cells with HS-5 stromal cells improves B-ALL growth and viability. Glasdegib with and without inotuzumab treatments impact the viability of co-cultured B-ALL cells by day four. SHH gene expressions suggest different B-ALL patients may be sensitive or resistant to glasdegib and inotuzumab.

Breast cancer is the most common malignancy in women, with triple-negative breast cancer (TNBC) representing the most aggressive subtype and carrying a poor metastatic prognosis. Metastasis requires tumor cells to cross the endothelial barrier, a process facilitated by tumor-derived extracellular vesicles (EVs), which can disrupt vascular integrity. Fluid shear stress (FSS), generated by blood flow, shapes endothelial physiology and may influence EV uptake, yet the mechanisms underlying TNBC-derived small EV (sEV) internalization remain unclear. Here, we investigated TNBC sEV-endothelial interactions using combined in silico and in vitro approaches. Human umbilical vein endothelial cells (HUVECs) were cultured under static or FSS conditions (20 dyn/cm{superscript 2}), followed by proteomic profiling and protein-protein interaction analyses with sEV proteomes. Uptake assays employed pharmacological inhibition (Dynasore, M{beta}CD, Pitstop2), Caveolin-1 (CAV-1) and Clathrin Heavy Chain (CLHC), siRNA-mediated knockdown, and junctional interaction analyses via confocal microscopy and co-immunoprecipitation. FSS downregulated proliferation- and angiogenesis-associated proteins while upregulating adhesion and cytoskeletal regulators assessed by proteomics. Network analysis identified clathrin- and caveolin-mediated endocytosis (CME and CavME), integrins, and early endosomes as central mediators of sEV uptake. Functionally, uptake was reduced by Pitstop2, M{beta}CD, and CAV-1/CLHC knockdown under static conditions, but silencing paradoxically enhanced uptake under FSS, suggesting compensatory flow-dependent pathways. Notably, under FSS, sEVs accumulated at endothelial junctions, colocalizing with VE-CAD and associating with CLDN5, indicating a potential disruption mechanism of adherens and tight junctions and consequent endothelial permeability. These findings identify CME and CavME as key uptake routes while underscoring FSS as a critical determinant of endothelial-tumor EV interactions. By revealing junctional targeting of sEVs, this work provides new mechanistic insight into vascular remodeling during metastasis and highlights EV pathways as potential therapeutic targets in TNBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/721946v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f91c5org.highwire.dtl.DTLVardef@2b4dc8org.highwire.dtl.DTLVardef@ff94f1org.highwire.dtl.DTLVardef@18b714b_HPS_FORMAT_FIGEXP M_FIG C_FIG Uptake and localization of sEVs on HUVEC under (a) static and (b) fluid shear-stress conditions. sEVs: Small Extracellular Vesicles. CME: Clathrin-mediated Endocytosis. CavME: Caveolin-mediated Endocytosis. CLDN5: Claudin-5. VE-CAD: Vascular Endothelial Cadherin. FSS: Fluid shear-stress.

Withdrawal StatementThe authors have withdrawn this manuscript to address issues related to data-use permission and authorship review. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm with very heterogeneous clinical and biological behavior. Among molecular variables, TP53 alterations are well-established adverse prognostic markers; however, MYC activation, which has been linked to disease progression, has not been completely defined in terms of clinical and biological impact, particularly in relation to TP53 status. Here, we investigated the effects of MYC overexpression according to TP53 status using clinical and transcriptomic data from CLL patients and novel cellular models. CLL patients with TP53WT and MYC overexpression exhibited significantly shorter time to first treatment and overall survival, indicating an aggressive disease course comparable to that of patients with TP53 alterations. Consistently, MYC overexpression in in vitro TP53WTmodels was associated with increased proliferation, enrichment of AKT/mTOR signaling and upregulation of genes involved in leukemogenesis and tumor progression such as FOXO6. Moreover, MYC overexpression was associated with increased sensitivity to venetoclax in TP53WT cells. By contrast, the concurrence of MYC overexpression and TP53 dysfunction conferred resistance to conventional CLL therapies such as BCL2 or BTK inhibitors. Of note, we identified a glycolysis inhibitor, in monotherapy or combined with BKT inhibitors, as a potential therapeutic strategy for CLL patients harboring MYC overexpression and TP53 alterations.

The PRESERVE trial (NCT04972097) is a prospective, single-arm pivotal IDE study evaluating focal irreversible electroporation (IRE) using the NanoKnife System for intermediate-risk prostate cancer. Men with Gleason Grade Group 2-3 disease underwent focal IRE and were followed for durability of oncologic control and safety. At 24 months, 68 patients completed follow-up with no new treatment failures identified. PSA levels were below baseline in 97% of patients, and one clinically triggered biopsy was negative for cancer. No new device- or procedure-related adverse events occurred beyond 12 months. These findings demonstrate durable efficacy and sustained safety of focal IRE.

Suppressor of cytokine signaling 1 (SOCS1) negative regulates inflammatory cytokine production and attenuates oncogenic growth factor signaling pathways. Reduced SOCS1 protein expression in human prostate cancer correlates with greater disease severity. To define the physiological functions of SOCS1 functions in the prostate, we conditionally ablated Socs1 in prostate epithelial cells of C57BL/6 mice. These Socs1{Delta}PE mice exhibited normal prostate development, maturation and lobular architecture. However, adult Socs1{Delta}PEmice developed progressive epithelial hyperplasia and inflammatory cell infiltration that were temporally and spatially distinct. SOCS1-deficient prostate showed increased epithelial cell proliferation and elevated oxidative stress markers, and prostate organoids recapitulated this hyperplasia phenotype. Diet-induced obesity exacerbated both hyperplasia and inflammation in SOCS1-deficient prostate. Upon transurethral infection with uropathogenic Escherichia coli UPEC1677 expressing the genotoxin colibactin, Socs1{Delta}PE mice developed invasive prostate cancer with complete loss of lobular architecture, whereas control mice developed hyperplasia and pre-neoplastic lesions. In vitro, SOCS1-deficient prostate organoid-derived epithelial cells exhibited increased DNA damage following exposure to UPEC1677. Deletion of the colibactin biosynthetic gene clbP in UPEC1677 abolished its ability to induce DNA damage in SOCS1-deficient cells and to drive prostate cancer in vivo. Proteomic analysis of prostate organoids revealed dysregulation of basal and luminal epithelial lineage markers and signaling pathway proteins that could promote neoplasia in SOCS1-deficient cells. Collectively, these findings establish an essential, epithelial cell-intrinsic role for SOCS1 in maintaining prostate tissue homeostasis by restraining proliferation, regulating lineage plasticity, limiting inflammation and oxidative stress, and conferring protection against genotoxic injury and neoplastic transformation.

Background: Modern therapy for childhood and adolescent leukemia requires accurate risk classification of genomic subtype. Although short-read next-generation sequencing (NGS)- based approaches provide comprehensive clinical diagnostics in limited, highly resourced settings, they remain expensive, slow, and inaccessible to most children worldwide. Transformative approaches are needed to improve diagnostic classification for leukemia globally. Methods: We simultaneously continued to develop an analytical pipeline NASVar (Nanopore variant calling for adaptive sampling), and conducted a multicenter, type-two hybrid clinical validation study of an Oxford Nanopore Technologies (ONT) adaptive-sampling whole-genome sequencing (asWGS) assay across hospitals with varying diagnostic resources. In preparation for implementation, a global panel developed a leukemia-based standardized gene set and consensus laboratory-developed test (LDT) validation guidelines. Measures of assay effectiveness compared to both conventional and orthogonal NGS methods, where available, were simultaneously collected with data to measure the implementation outcomes of feasibility, fidelity, appropriateness, and cost. Results: All four centers successfully completed the LDT validation, with minimal adaptations required for regulatory compliance. A total of 457 specimens were sequenced (331 B-ALL, 83 AML, 43 T-ALL). For the 210 B-ALL cases with locally resolved genomic subtypes defined by DNA alterations, asWGS was 100% concordant (210/210). Cases locally defined as B-other were resolved via asWGS with disease-defining DNA alterations in 47% (49/105) of cases. An additional 41% (43/105) of locally defined B-other cases were classified by incorporation of DNA methylation, and all 16 B-ALL patient-derived xenograft controls were correct, for a total of 96% (318/331) of all B-ALL cases in the cohort resolved with single assay asWGS. For AML, 97% (56/58) of cases with locally resolved genomic subtypes were identified by automated asWGS analysis, while an additional two cases were identified after targeted manual review. At Indus Hospital in Pakistan, the B-ALL and AML diagnostic genomic subtype yield increased from 28% with local standard of care diagnostic testing, to 84% with asWGS. The cost of reagents and consumables in the United States, assuming pooled three-plexing, was $343/sample. Based on the combined hybrid validation results, all centers are independently preparing for clinical return of results. Conclusions: ONT asWGS was successfully validated as a clinical assay in four diverse hospital settings. As a single, multi-omic platform that delivers value across the continuum of high-resource to resource-limited contexts, the approach offers a disruptive solution to address the global equity gap in cancer diagnostics.

Multiple myeloma remains a fatal, incurable disease. Most therapies are targeted to the cancer cell or T cell engagement. Little is known about the supporting myeloma microenvironment and its contribution to tumor fitness. Here, we expand upon the observation of human mast cells in the NSG-hIL6 myeloma patient derived xenograft mouse model to show mast cells decrease time to engraftment, promote increased myeloma engraftment and cause myeloma bone disease. We identify 10 mast cell secreted factors that together improve the survival of patient myeloma cells in vitro. Our results highlight the versatility of the NSG-hIL6 model to study microenvironmental interactions between human bone marrow cells and myeloma and confirm prior suggestions that clinical signs of disease, such as osteolytic lesions, may at least partially be related to non-malignant bone marrow microenvironmental cells, such as mast cells.

BackgroundMesothelioma (Me) is an aggressive cancer with limited response to conventional therapies. The tumors harsh microenvironment contributes to immune escape and therapy resistance and the effects of ICIs on Me are still unclear. Adenosine, an immunosuppressive molecule produced from AMP by the enzyme CD73, accumulates in hypoxic tumor areas. Elevated CD73 and adenosine receptor A2B (A2Br) levels on Me cells are linked to worse patient outcomes, indicating their important role in disease progression and potential as targets for treatment. AimThis study characterizes the Me-ME (micro environment) and evaluates the efficacy of TT-4 (A2B inibitor) and AB680 (CD73 inibitor), alone or with aPD-1, using 3D models in vitro and in vivo. MethodsCD73 and A2B receptor levels were quantified in tumor and normal samples using qRT-PCR and IHC. Cells lines were treated with CoCl2 to mimic hypoxia, then CD73, A2Br and related markers were analyzed. MSTO-211H and REN cells were silenced for CD73, grown as spheroids and adenosine release was measured. Co-culture spheroids of MSTO-211H and Jurkat cells were treated with AMP and CD73 inhibitor, then analyzed for viability and immune markers. An orthotopic Me model was established by injecting AB1-B/c-LUC cells and monitored by in vivo imaging. Proteomic analysis of spheroids was conducted to identify proteins and pathways involved. ResultsHypoxia boosts CD73 and A2Br expression in Me cells, leading to adenosine production via CD73. In 3D co-cultures, AB680 lowered Me cell viability and enhanced activation of Jurkat T cells. In mice, combining aPD-1 therapy with A2Br or CD73 inhibitors strongly reduced tumor growth. Proteomics identified 93 proteins influenced by adenosine signaling through A2B. ConclusionTargeting the adenosine pathway alongside PD-1 blockade offers a promising new immunotherapy strategy for Me.

The scaffold protein FRS2 is central to FGFR signaling, linking receptor activation to MAPK/ERK and PI3K/AKT pathways. Elevated FRS2 expression correlates with aggressive tumor phenotypes and poor prognosis across multiple cancers, including the pediatric cerebellar tumor medulloblastoma (MB). Here, we characterized FRS2s subcellular localization and interactome in MB cells, employing live-cell imaging, phosphoproteomics, immunoprecipitation, and APEX2-based proximity labeling. We found that increased FRS2 expression is associated with increased motile and invasive behavior in MB tumor cells. We furthermore identified novel candidate FRS2-associated proteins involved in actin cytoskeleton remodeling, cell junction assembly, and translation initiation, which indicate a growth factor-dependent reorganization of the FRS2 signalosome. Our data furthermore indicate a regulatory role of FRS2 in directing subcellular distribution of the cell junction and cell motility regulator TJP1. Our findings highlight the relevance of FRS2 as a mediator of cell motility and invasiveness and provide candidate proteins associated with FRS2 that are involved in cellular processes governing migration and invasion. This study thus provides a framework for exploring the FRS2 interactome as a possible target to attenuate FGFR-driven oncogenic processes with next-generation therapeutic strategies.

Platinum resistance remains a major barrier in Ovarian cancer (OC) treatment[1]. While hyperactivation of DNA damage response (DDR) is a hallmark of chemoresistance[2], the underlying epigenetic mechanisms driving this adaptation remain poorly understood. Here, we identify a novel post-transcriptional regulatory axis involving miR-221-5p that governs two critical DDR effectors: RAD18, which mediates DNA damage tolerance through trans-lesion synthesis (TLS)[3][4], and RAD51, the central recombinase for homologous recombination (HR)[5][6]. Although the miR-221/222 cluster is traditionally categorized as oncogenic[7][8], we demonstrate that the miR-221-5p arm functions as a potent tumor suppressor in OC. Bioinformatic and luciferase reporter assays confirmed that miR-221-5p directly targets the 3'UTRs of both RAD18 and RAD51. In OC clinical specimens and cell lines, miR-221-5p downregulation inversely correlates with RAD18/RAD51 expression. Functionally, miR-221-5p restoration suppressed platinum-induced PCNA mono-ubiquitination and HR, inducing a "functional BRCAness" that sensitized both established and patient-derived primary OC cells to carboplatin and PARP inhibition. Furthermore, in vivo disseminated xenograft models demonstrated that stable miR-221-5p expression significantly reduced tumor burden. Collectively, our results delineate a novel regulatory mechanism where loss of miR-221-5p drives chemoresistance by derepressing the RAD18/RAD51 axis, identifying this axis as a promising therapeutic target.

Chromophobe renal cell carcinoma (ChRCC) accounts for 5% of all renal cancer cases. Despite its generally indolent behavior and low mutational burden, there is no targeted therapy for metastatic ChRCC. Profilin-1 (Pfn1), a cytoskeletal regulator of actin and tubulin dynamics, has emerged as a potential oncogenic driver in several cancers including RCC, but its role in ChRCC, remains undefined. We observed elevated Pfn1 expression in stage IV ChRCC patients, implicating Pfn1 in advanced disease progression. To investigate this, we manipulated Pfn1 expressions in two ChRCC cell lines UOK276 and RCJ41M. Pfn1 knockdown (KD) significantly reduced proliferation, invasion, and colony formation, whereas Pfn1 overexpression (OE) in UOK276 enhanced ChRCC aggressive phenotypes. Pharmacological inhibition of Pfn1 significantly suppressed proliferation and clonogenic growth in both cell lines. Additionally, Pfn1 KD increased intracellular ROS accumulation, while overexpressed reduced ROS levels, linking cytoskeletal regulation to oxidative stress control. Together, these findings position Pfn1 as a critical mediator of ChRCC progression, linking cytoskeletal remodeling to aggressive tumor behavior. This work highlights Pfn1 as a potential therapeutic target and establishes a framework for cytoskeletal-focused strategies in advanced ChRCC.

The RNA polymerase-associated factor 1 complex (PAF1C) is an evolutionarily conserved transcription elongation complex that regulates RNA polymerase II-mediated transcription and chromatin modification. LEO1, a core subunit of PAF1C, has been implicated in developmental gene regulation, WNT signaling, and leukemogenesis; however, its role in solid tumor progression remains poorly understood. In this study, we found that although LEO1 expression is generally elevated in colorectal cancer (CRC), its expression is reduced in stage IV tumors and is associated with poor clinical outcomes. To investigate its function, we established LEO1 -deficient HCT116 cell line and performed transcriptomic analyses. Loss of LEO1 suppressed epithelial differentiation and developmental gene programs while inducing cell cycle delay. Despite these changes, LEO1-deficient cells exhibited aggressive phenotypes, including enlarged nuclei and increased expression of migration-associated genes, which were further enhanced under glucose deprivation. Motif analysis identified FOXM1 as a key regulator of these migration-related genes. Mechanistically, LEO1 deficiency promoted accelerated transcriptional activation of GRP78, a central regulator of endoplasmic reticulum (ER) stress adaptation. GRP78 was required for survival under ER stress conditions, and its inhibition suppressed both migration and migration-associated gene expression. In addition, transcriptomic analyses revealed upregulation of cholesterol metabolism-related genes in LEO1-deficient cells. Consistently, treatment with the HMG-CoA reductase inhibitor atorvastatin selectively impaired their survival, indicating cholesterol metabolic dependency. Collectively, these findings demonstrate that LEO1 loss promotes ER stress-adapted migration and cholesterol metabolic dependency in CRC, suggesting that these pathways may represent therapeutic vulnerabilities in metastatic LEO1-low CRC.

FLT3 inhibitors have improved outcomes in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), but responses are not durable. Notably, FLT3 inhibitors clear blasts from the blood, but not the bone marrow, a hypoxic niche. We investigated effects of hypoxia and the key nutrient glutamine on FLT3 inhibitor response. FLT3-ITD AML cell lines and patient blasts were cultured with FLT3 inhibitors under normoxia (21%) or hypoxia (<1% O2) with or without glutamine or the glutaminase inhibitor telaglenastat (CB-839). Cytotoxicity was measured in WST-1 assays and drug combination effects by Chou-Talalay analysis. Protein expression was measured by immunoblotting, turnover and proteasomal degradation by cycloheximide chase with and without MG-132, and mRNA expression by RT-qPCR. Effect of the ubiquitin ligase c-CBL was tested by siRNA knockdown. FLT3 inhibitor ICs were 3-5-fold higher in hypoxia than normoxia, associated with FLT3-ITD and p-STAT5 downregulation and accelerated FLT3-ITD proteasomal degradation (half-life, 1.0 vs. 2.5 hours). c-CBL expression increased in hypoxia, and c-CBL knockdown restored FLT3-ITD expression and FLT3 inhibitor sensitivity. Glutamine deprivation or telaglenastat treatment abrogated c-CBL upregulation in hypoxia and preserved FLT3-ITD and p-STAT5 expression and FLT3 inhibitor sensitivity. Telaglenastat synergized with FLT3 inhibitors in hypoxia, supporting clinical testing.

Background and AimsThe tumor microenvironment in colorectal cancer (CRC) is richly innervated, yet the contribution of the enteric nervous system (ENS) to CRC biology remains poorly defined. ENS neurons express proenkephalin (PENK), which can be processed by proprotein convertase 1/3 (PCSK1) to generate Methionine-enkephalin (M-ENK), a bioactive peptide with growth-regulatory potential. We hypothesized that an ENS-derived PCSK1-M-ENK axis restrains CRC proliferation through opioid growth factor receptor (OGFr) signaling and is modulated by stress-associated glucocorticoid receptor (GR) signaling and GLP1 receptor (GLP1R) activity. MethodsPublicly available human CRC single-cell RNA-sequencing datasets were analyzed for OGFr expression. PCSK1 and M-ENK expression in murine ENS and tumor-associated tissue was assessed by immunofluorescence. Functional studies were performed using murine CRC organoids, and primary murine ENS neurons in mono- and co-culture. CRC proliferation was quantified by EdU incorporation following treatment with recombinant M-ENK, recombinant PCSK1, OGFr synthetic ligand naloxone, or PCSK1 inhibitors. Effects of dexamethasone and liraglutide on PCSK1 expression in ENS-containing murine tissue were evaluated. ResultsOGFr was enriched in CRC cells and positively associated with KRAS gene expression. A subset of adult murine colonic myenteric neurons expressed PCSK1 and M-ENK. M-ENK dose-dependently suppressed proliferation of CRC organoid cells. ENS neurons also suppressed CRC proliferation in a PCSK1-dependent manner. Dexamethasone reduced, whereas liraglutide increased, PCSK1 expression. ConclusionsThese findings define a previously unrecognized ENS-derived neuro-oncologic pathway that is associated with reduced CRC cell proliferation and identify the GR/GLP1R-PCSK1-M-ENK axis as a potentially actionable therapeutic node. SummaryThis study identifies a neuronal PCSK1 - M-ENK pathway in the ENS that directly suppresses colorectal cancer growth through local OGFr activation, revealing a previously unrecognized neuropeptidergic mechanism of tumor control within the intestinal microenvironment.

Background & AimsHepatocellular carcinoma (HCC) frequently exhibits resistance to immune checkpoint inhibitors (ICIs), particularly in {beta} -catenin-driven tumors characterized by immune exclusion. While the Unfolded Protein Response (UPR) and the Integrated Stress Responses (ISR) enable tumor adaptation to metabolic stress their role in shaping tumor immunogenicity remains incompletely understood. We investigated whether ATF4, a central effector of the integrated stress response, couples metabolic reprogramming to suppression of anti-tumor immunity in HCC. MethodsWe combined transcriptomic analyses across three independent human HCC cohorts with mechanistic studies using an immunotherapy-resistant MYC/{beta}-catenin-driven murine HCC model. We integrated CRISPR/Cas9-mediated deletion of Atf4 with RNA-sequencing and targeted metabolomics. The impact of tumor-derived metabolites on macrophage differentiation and polarization was evaluated using primary bone marrow-derived cells. Therapeutic responses were evaluated in orthotopic and subcutaneous models treated with anti-PD-1 and anti-VEGFA. ResultsATF4 and XBP1 transcriptional signatures are selectively enriched in human HCC and associate with poor prognosis, vascular invasion, and an immunosuppressive myeloid-enriched tumor microenvironment. Genetic ablation of Atf4 markedly suppressed tumor growth in immunocompetent but not immunodeficient hosts, establishing a requirement for immune-mediated tumor control. Mechanistically, Atf4 loss downregulated Aldh18a1 and disrupted proline biosynthesis, resulting in extracellular proline depletion. This proline-deficient environment abrogated monocyte-to-macrophage differentiation and decreased M2 polarization, thereby reshaping the tumor microenvironment toward enhanced T cell infiltration and activation. Functionally, Atf4-deficient tumors exhibited restored sensitivity to anti-PD-1 monotherapy and showed pronounced responses to combined anti-PD-1/anti-VEGFA treatment in aggressive orthotopic models. ConclusionATF4 programs a proline-dependent metabolic axis that sustains macrophage-mediated immunosuppression and immune evasion in {beta}-catenin-driven HCC. Disruption of this pathway converts immune-excluded tumors into T cell-inflamed states and restores responsiveness to immunotherapy. By governing proline homeostasis and macrophage-mediated immunosuppression, ATF4 is a key metabolic checkpoint for immune evasion, linking stress adaptation to immune escape and a candidate therapeutic target in HCC. Impact and implicationsWe identify ATF4 as a crucial metabolic-immune orchestrator that sustains myeloid-driven immune evasion in {beta}-catenin-dependent HCC through proline-dependent circuitry. Disrupting the ATF4-proline axis converts immune-desert tumors into T cell-inflamed lesions by blocking macrophage differentiation, thereby sensitizing tumors to immune checkpoint therapy. This work positions ATF4 as a tractable therapeutic target to overcome immunotherapy resistance in HCC. Graphical abstract Highlights- ATF4 orchestrates an immunosuppressive tumor microenvironment in HCC by coupling metabolic stress adaptation to immune evasion. - Ablation of ATF4 disrupts proline biosynthesis, leading to a marked depletion of extracellular proline. - Cancer cell-derived proline availability contributes to macrophage differentiation and M2 polarization; its loss restores T cell-mediated anti-tumor surveillance and sensitizes beta-catenin-driven HCC to immune checkpoint blockade.

Cerebellum tissue invasion and dissemination are major drivers of recurrence and metastatic spread in medulloblastoma (MB), yet no invasion-inhibitory therapy is currently available. The serine/threonine kinase MAP4K4 is highly expressed in MB and promotes invasive behavior downstream of growth factor signaling, while its physiological postnatal downregulation suggests that disrupted developmental control may contribute to tumor pathogenesis. Here, we investigate pharmacological targeting of MAP4K4 using famlasertib, a CNS-penetrant and neuroprotective MAP4K inhibitor, as a strategy to suppress invasion in MB. Using 3D invasion assays, quantitative live-cell microscopy, and phospho-proteomics, we demonstrate that famlasertib markedly reduces invasive behavior and single-cell motility of MB cells. Zebrafish larval and tissue models further confirm anti-invasive efficacy without detectable developmental toxicity at effective concentrations. Mechanistically, MAP4K inhibition alters kinase signaling linked to cytoskeletal remodeling, leading to suppressed F-actin dynamics, increased cell clustering, and enhanced cortical accumulation of tight junction protein 1 (TJP1). Collectively, our findings confirm MAP4Ks as a therapeutic targetable regulators of MB invasion and establish famlasertib as a candidate migrastatic agent that restrains tumor cell dissemination while preserving developmental integrity.

Glioblastomas are incurable and lethal brain cancers. Immunotherapies offer new and promising treatment options for glioblastoma patients, but the highly immunosuppressive nature of these cancers presents a challenging clinical obstacle. Glioblastoma immune evasion is driven by cell-cell interactions in the tumor microenvironment and recent studies have identified astrocytes as important contributors to immune silencing [1, 2]. Cell plasticity is a key feature of reactive astrocytes that drives heterogeneous, pro- or anti-inflammatory states [3], but the molecular regulators of astrocyte-immune interactions remain incompletely understood. Here, we investigate whether cell plasticity of glioblastoma-associated astrocytes promotes or opposes tumor progression and show that loss of astrocyte plasticity results in T-cell recruitment and immune activation. We evaluate how astrocytic cell plasticity contributes to immune functions in the glioblastoma microenvironment using single cell sequencing from preclinical models, in vivo genetic perturbations and in vitro mouse and human experimental systems. We show that astrocytes surrounding glioblastoma express the stem cell-associated transcription factor, ZEB1, and that conditional-inducible astrocytic deletion of Zeb1 remarkably reduces glioblastoma growth and extends survival. Increased recruitment and activation of T cells in astrocytic Zeb1-deficient mouse models is linked to increased expression of the immunoattractant cytokine CXCL14, and viral delivery of CXCL14 in experimental glioblastoma models increases survival. Our data support that CXCL14 is a candidate therapeutic target for reprogramming the tumor microenvironment that can restrict and reduce glioblastoma growth and progression.

BackgroundObesity significantly increases the risk of prognosis and clinical outcomes in pancreatic ductal adenocarcinoma (PDAC). While research on the interactions between obesity and the tumor microenvironment (TME) is mostly confined to a few interactions at a time, leaving a gap in the comprehensive understanding of obesity-driven PDAC. We set out to develop a cell-type-resolved model of obesity-driven PDAC using bulk transcriptomic data to investigate TME changes. MethodsWe conducted an integrated transcriptomic analysis of PDAC patients from the CPTAC-3 cohort (n=140) stratified by BMI. A custom immune and stromal functional gene signature database covering 65 cell types was constructed, followed by LLM-assisted review, overlap control, and validation. BayesPrism deconvolution using matched single-cell references was used to derive expression profiles for each cell type. Stabl, a machine-learning algorithm, was used to identify BMI-associated signatures. Bayesian hierarchical modeling, using both continuous and categorical BMI change, was applied to estimate effect sizes and assess the statistical credibility of the signature changes using the 95% Highest Density Interval (HDI) excluding zero. Virtual multiplex immunofluorescence was generated from whole-slide H&E images using gigaTIME to assess the spatial manifestation of BMI-associated TME changes in tissue ResultsBulk pathway analysis showed that ECM homeostasis and primary immunodeficiency pathways deteriorated with increasing BMI. However, Bayesian modeling revealed cell-type-specific, non-linear dynamics. Stromal populations in overweight (OW) individuals were altered, with changes in ECM synthesis and inflammatory signaling that stabilized rather than intensified during obesity. Immune compartments also showed diverse trajectories: CD4+ T cells remained functional in OW but collapsed in obesity; CD8+ T cells progressed linearly from activation to chronic exhaustion. NK cells exhibited non-monotonic behavior, and monocyte and B cell lineages became impaired prior to clinical obesity. Cell-cell interaction analysis showed a shift from a T cell and dendritic cell-centric adaptive interactome in normal weight patients to a neutrophil-dominated inflammatory network in OW. Spatial analysis showed stromal-trapped CD8+ T cells were compressed closer to the tumor boundary with rising BMI. ConclusionsOverweight status represents a critical tipping point in tumor microenvironmental reprogramming, challenging linear models of obesity-associated immune modulation and suggesting that early metabolic interventions may prevent PDAC functional deterioration. Model is available at https://obese-pdac-model.streamlit.app/ O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/721695v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@b1c8cdorg.highwire.dtl.DTLVardef@1f61b7forg.highwire.dtl.DTLVardef@876c60org.highwire.dtl.DTLVardef@dc32b2_HPS_FORMAT_FIGEXP M_FIG C_FIG