Cardiac fibrosis is a hallmark of cardiac aging and a major contributor to development of heart failure. However, therapeutic strategies that specifically target cardiac fibrosis remain limited. In this study, we demonstrate that small-molecule compound ML216 exerts protective effects against aging-associated or β-adrenoceptor agonist isoproterenol-induced cardiac fibrosis in vitro or in vivo. Mechanistically, ML216 inhibits transforming growth factor-β1 (TGF-β1) signaling by reducing TGF-β1 protein levels, thereby attenuating Mothers against decapentaplegic homolog (SMAD) phosphorylation and downstream induction of connective tissue growth factor (CTGF). This leads to a marked suppression of fibrotic genes Col1a1, Cnn2, and Acta2, ultimately resulting in reduced fibrosis. Additionally, the inhibition of the TGF-β1 pathway alleviates cardiomyocytes apoptosis, which may further limit inflammatory responses and contributes to the overall attenuation of cardiac fibrosis. Collectively, these findings demonstrate that ML216 mitigates cardiac fibrosis through the inhibition of TGF-β1 pathway-mediated fibrotic signaling and apoptosis, highlighting its potential as a therapeutic candidate for the treatment of cardiac fibrosis.

The molecular mechanism underlying male reproductive toxicity associated with Perfluorooctanoic acid (PFOA), a persistent environmental endocrine disruptor (EDC), has not yet been fully elucidated. Six-week-old male C57BL/6 mice were treated with PFOA by oral gavage at 0, 1.25, 5, 10, and 20 mg/kg/day for 35 days to explore its toxic effects on the male reproductive system and the underlying mechanisms. Analyses of semen quality, testicular histopathology, and blood-testis barrier (BTB) integrity revealed that PFOA caused dose-dependent structural and functional damage to the BTB, leading to markedly reduced semen quality. Based on transcriptomic sequencing and differential gene enrichment analysis, the glycolytic pathway was identified as a key regulatory target for PFOA-induced damage to the reproductive system. Further validation revealed that PFOA exposure inhibited glycolysis-related enzymes (Hexokinase 1 (HK1), Glucose Transporter 1 (GLUT1), and Lactate Dehydrogenase A (LDHA)), reduced lactate production and ATP synthesis, lowered Pan-Kla and H3K18la levels, and diminished H3K18la enrichment at the Hk1, Glut1, and Ldha promoters, whereas exogenous sodium lactate reversed these changes. This study is the first to identify the "glycolysis-lactate-H3K18la" chain as a key regulator in PFOA-induced BTB damage and spermatogenesis impairment, offering a new theoretical foundation for understanding EDC-induced male reproductive toxicity.

The metabolic by-product of glycolysis, lactate, and lactylation-mediated protein modification are involved in various biological processes, including host antiviral responses. The spring viremia of carp virus (SVCV), a rhabdovirus that devastates aquaculture, relies on its multifunctional phosphoprotein (P) for replication and evasion of the host immune system. In this study, we found that SVCV infection increased lactate levels in zebrafish, with lactate modulating SVCV proliferation through lactylation of the viral P protein. ldha-null zebrafish exhibited reduced lactate levels and increased susceptibility to SVCV. Conversely, sodium lactate supplementation suppressed viral replication and improved the survival rate of infected zebrafish. Furthermore, we identified lactylation sites in the SVCV P protein. These findings revealed that lactate-mediated lactylation is a host defense mechanism against SVCV and could provide novel strategies for antiviral intervention in aquaculture.IMPORTANCELactylation is an emerging post-translational modification, yet its presence in viruses has rarely been investigated. This study identified the lactylation modification sites in the spring viremia of carp virus (SVCV) P protein and demonstrated that adding sodium lactate modifies the P protein and reduces SVCV replication. These results suggest that sodium lactate has antiviral properties and plays an important role in combating SVCV infection, while also revealing a novel modification of the SVCV viral protein within the host.

Grass carp (Ctenopharyngodon idella), which is farmed extensively throughout the world, has a key function in the aquaculture industry. But its haemorrhagic disease resulting from grass carp reovirus (GCRV) causes significant economic losses. Consequently, elucidating the GCRV tolerance mechanisms and identifying candidate genes for GCRV resistance are of great necessity. Here, we cloned the coding sequences (CDS) of grass carp tripartite motif containing 25 (gc-trim25) and sequence analysis revealed that gc-trim25 is conserved across species. GCRV infection induced gc-trim25 mRNA expression within most grass carp tissues and CIK cells. Additionally, gctrim25 enhanced interferon (IFN) activation that was induced by poly I:C-HMW and poly I:C-LMW. Furthermore, gc-trim25 enhanced antiviral gene expression and suppressed viral replication in an enzyme activitydependent manner upon infection with GCRV. Mechanistically, gc-trim25 interacts with retinoic acidinducible gene-i (rig-i) and melanoma-differentiation-associated gene-5 (mda5) for enhancing K63-linked gcrig-i and gc-mda5 ubiquitination, thus promoting the antiviral immunity. Furthermore, trim25-deficient zebrafish are more susceptible to GCRV infection. These findings help better understanding the effect of grass carp trim25 on antiviral innate immunity.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a key component of the innate immune response and initiates stimulator of interferon genes (STING)-dependent signaling in an ancient stress response. Hypoxia-inducible transcription factors 1α and 2α control the hypoxia response, another ancient stress response. This response regulates genes involved in adapting to hypoxia. We discovered that hypoxia-induced phosphatidylinositol 4,5-biphosphate (PIP2) depletion results in the relocation of cGAS from the plasma membrane, which suppresses hypoxia signaling. Mechanistically, cGAS enhances HIFα binding to HSC70/LAMP2A, thus leading to chaperone-mediated autophagy (CMA) of HIFα. Prolonged hypoxia enhances cGAS oligomerization, which reduces the ability of cGAS to promote HIFα degradation. Disrupting cGAS in mice and zebrafish resulted in the upregulation of hypoxia-inducible genes, a change that enhanced tolerance to hypoxia. These results suggest that cGAS attenuates hypoxia signaling by promoting CMA degradation of HIFα.

Hepatic lipotoxicity in type 2 diabetes promotes oxidative stress and ferroptosis, driving progressive liver injury, for which effective targeted therapies remain lacking. Here, we identify tacrolimus (TAC), a clinically established immunosuppressant, as an unexpected suppressor of hepatic ferroptosis in db/db diabetic mice. TAC administration markedly alleviated liver injury, fibrosis, and inflammation, accompanied by reduced oxidative stress and ferroptosis signatures. Transcriptomic profiling revealed enrichment of glutathione metabolism pathways in livers of TAC-treated db/db diabetic mice. Mechanistically, TAC inhibited ferroptosis in primary hepatocytes by activating the NRF2 pathway, increasing NRF2 protein abundance and its nuclear translocation in an SIRT7 deacetylase activity-dependent manner. Together, these findings uncover a previously unrecognized role of TAC in repressing ferroptosis through the SIRT7-NRF2 axis, highlighting ferroptosis modulation by TAC as a potential therapeutic strategy for diabetic liver diseases.

TRIM25 is an E3 ubiquitin ligase involved in various cellular processes due to its enzymatic activity. In particular, it plays a role in antiviral innate immunity. Here, we demonstrate that TRIM25 modulates hypoxia signaling. TRIM25 interacts with HIF-1α and HIF-2α, stabilizing them. TRIM25 catalyzes K11-linked polyubiquitination of HIF-1α at K719 and K721 and of HIF-2α at K709. This results in the stabilization of the proteins and enhanced hypoxia signaling. Moreover, TRIM25-mediated augmentation of hypoxia signaling depends on HIF-1α. Trim25-deficient mice are more sensitive to hypoxia, and zebrafish lacking trim25 show a similar phenotype. These data reveal TRIM25’s role in regulating hypoxia signaling and provide insight into a new mechanism that modulates the stabilization and activity of HIF-1α and HIF-2α.

Abstract Primary resistance to targeted therapies, immunotherapies, and gene therapies in NSCLC continues to be a significant challenge. TUSC2 tumor suppressor gene therapy has shown promising anti-tumor efficacy by overcoming resistance to targeted therapy and enhancing checkpoint blockade immunotherapy, including in a mutant KRAS/LKB1-driven immunotherapy-resistant NSCLC model. TUSC2 protein expression is downregulated or absent in over 80% of NSCLC and 100% of SCLC cases., TUSC2 mediates cancer cell death through several mechanisms: inhibiting MAPK and mTOR signaling pathways, arresting cell growth, inducing programmed cell death, and activating immune responses. We established models primarily resistant to TUSC2 gene therapy to find biomarkers indicative of TUSC2 gene therapy resistance in NSCLC patient-derived xenografts (PDXs), PDX-derived organoids (PDXOs), and cell lines. A panel of 10 NSCLC cell lines screened for TUSC2 sensitivity showed resistance in 50% of the cell lines, as assessed by annexin V staining and colony formation assays. We evaluated TUSC2 sensitivity in 12 NSCLC PDXOs using ATP-based viability assays in 3D culture following TUSC2 or empty vector transfection. While some PDXOs were highly responsive to TUSC2 within 72 hours post-transfection, 50% of PDXOs exhibited primary resistance. We developed TC314AR (Acquired Resistance) PDX tumors and xenograft models (A549, H1299, H23AR) in NSG mice and treated them with TUSC2 gene therapy. 20-30% of tumors in every model showed resistance, with no significant reduction in size compared to the control tumors after treatment. Protein expression profiling using reverse-phase protein array (RPPA) analysis of 500 proteins showed distinct expression signatures, with several candidate biomarkers significantly altered in resistant cell lines and PDXOs. RPPA analysis of residual tumors from both the xenograft and PDX models revealed significant but model-specific alterations in protein expression between responders and non-responders. Comparative analyses across the three models showed low expression of TROP2 and high expression of PTEN as potential biomarkers of primary resistance. Overexpression of TROP2 in H1299 and H460 cells increased TUSC2-induced apoptosis. These findings suggest that TROP2 and PTEN may serve as biomarkers to predict TUSC2 response and guide therapeutic strategies in NSCLC. Citation Format: Ismail M. Meraz, Renduo Song, Shuhong Wu, Yi Xu, Meng Feng, Lihui Gao, Chenghui Ren, Qi Wang, Jun Li, Mourad Majidi, Jing Wang, Mark Berger, Jack A. Roth. TROP2 and PTEN are biomarkers of primary resistance to TUSC2 gene therapy in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 391.

BACKGROUND:Tumour-draining lymph nodes (TDLNs) serve as the closest immunological hubs to the primary tumour site in colorectal cancer (CRC). Owing to their unique and irreplaceable anatomical advantage and dynamic immune cell repertoire, TDLNs represent an intensively studied immunological niche and are a potential therapeutic target. OBJECTIVE:This study aimed to map the immune microenvironment of CRC TDLNs to identify targetable immunomodulatory axes. DESIGN:Single-cell RNA sequencing (scRNA-seq) was performed on 23 quadruplet-matched samples, including primary tumours, adjacent normal tissues, tumour-free lymph nodes (TFLN) and tumour-invaded lymph nodes (TILN) from seven CRC patients. Mechanistically significant findings were further validated through in vitro functional assays, CRISPR knockout in primary regulatory T cells (Tregs), in vivo murine footpad-popliteal lymph node metastasis models with lipid nanoparticle-encapsulated siSPP1 (LNP-siSPP1) and/or anti-CD44 mAb, and multiomics analysis of independent CRC cohorts. RESULTS:scRNA-seq analysis delineated TILN-specific immunological landscapes dominated by SPP1+ macrophage expansion and active Treg differentiation niches, establishing TILNs as maturation hubs for Tregs versus TFLNs. Mechanistically, SPP1+ macrophages drove Treg differentiation into immunosuppressive CD137+ subsets via the SPP1-CD44 axis, which required NF-κB1 to directly bind the TNFRSF9 promoter. In vivo, LNP-siSPP1 plus anti-CD44 mAb synergistically suppressed lymph node metastasis, reduced CD137+ Tregs and enhanced CD8+ T cell function. Findings were consistently observed across all experimental models and patient-derived datasets. CONCLUSIONS:SPP1+ macrophages established an immunosuppressive niche in CRC TDLNs by promoting CD137+ Treg maturation via the SPP1-CD44-NF-κB1 axis. Targeting this axis with LNP-siSPP1 and anti-CD44 mAb might overcome Treg-mediated immunosuppression in CRC.

TROP2 antibody-drug conjugate sacituzumab govitecan (SG) is approved for treatment of advanced breast cancer. However, intrinsic and acquired resistance occur, leading to a need for novel therapies. We hypothesized that antagonists of inhibitor of apoptosis protein (IAP) enhance the activity of SG and its payload. In this preclinical study, we employed breast cancer models, including patient-derived xenografts (PDXs) and cell lines, to conduct combination therapies with SG and IAP inhibitors. In PDXs, birinapant enhanced the antitumor activity of several chemotherapeutics, especially irinotecan, a metabolic precursor of payload SN-38. We subsequently demonstrated that combinations of SG with birinapant or tolinapant had greater antitumor activity and significantly prolonged event-free survival compared with SG alone. In vitro, IAP antagonists significantly synergized with SG on inhibition of cell viability and colony formation, and on apoptosis induction. These findings suggest that SG combination with IAP inhibitors may represent an effective therapeutic strategy for breast cancer.

Megalobrama amblycephala is a species of high economic importance in Chinese freshwater aquaculture. However, its low hypoxia tolerance imposes growth restrictions and diminished survival rates, posing a major constraint to the sustainable development of the industry. Prolyl hydroxylase domain protein 3 (PHD3), a canonical suppressor of hypoxia signaling, inhibits the hypoxia-inducible factor (HIF) pathway via direct hydroxylation of HIF-1α or HIF-2α proteins. Nevertheless, its functional role and mechanistic underpinnings in hypoxia within M. amblycephala remain incompletely characterized. Under hypoxic stress, we found phd3 expression was significantly upregulated in oxygen-sensitive tissues (notably brain and gill) and embryos of M. amblycephala. Evolutionary and structural analyses demonstrated high conservation of the phd3 gene of M. amblycephala (Ma-phd3) across vertebrate lineages. Genetic knockout of phd3 in M. amblycephala markedly enhanced the expression of hypoxia-inducible genes under hypoxic conditions. Mechanistic investigations revealed that Ma-phd3 suppresses the transcriptional activity of HIF-1α and HIF-2α while concurrently mediating ubiquitin-proteasomal degradation of HIF-1α and HIF-2α proteins through its intrinsic hydroxylase activity. This study not only advances our understanding of the critical regulatory function exerted by phd3 in modulating the hypoxia signaling pathway but also nominates it as a promising candidate gene for the genetic improvement of hypoxia-tolerant M. amblycephala strains.

Differential gene expression analysis for covariates controlled.

Breast cancer remains the most common cancer type and second leading cause of cancer related deaths among women in the U.S. Despite progress in detection, surveillance, and targeted therapies, aggressive subtypes like triple-negative breast cancer (TNBC), along with challenges such as drug resistance and disease recurrence, remain significant clinical hurdles. To address these unmet needs in TNBC treatment, our group has developed a series of oridonin-derived analogs (oridonalogs), including CYD0618. These analogs exhibit enhanced drug-like properties and significantly improved potency against various cancers in both in vitro and in vivo models. This study aims to characterize the mechanism of action of CYD0618 against TNBC cells. Our findings demonstrate that CYD0618 effectively reduces the proliferation of TNBC cells in vitro with IC50s at the nanomolar level. Additionally, CYD0618 exhibits high potency against TNBC in vivo, significantly suppressing tumor growth and prolonging survival in a xenograft mouse model. Preliminary results reveal that CYD0618 inhibits activation of STAT3, a key transcription factor involved in proliferation, survival, and transformation of epithelial cells. Given that STAT3 is constitutively active in ∼70% of human cancers, there has been a tremendous effort to develop STAT3 targeting therapies; however, none have achieved FDA approval. In addition to STAT3, our study shows that CYD0618 also suppresses other STAT family transcription factors. Compared to selective STAT3 degradation by PROTAC treatment, poly-STAT targeting by CYD0618 exhibits superior activity against TNBC cells. These findings identify CYD0618 as a novel poly-STAT inhibitor with promising activity against TNBC. Gabrielle Vontz,Jun Li,Zhipin Liang,Jia Zhou,Qiang Shen. Targeting STAT pathways with oridonin derivatives to suppress triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 600.

Integrated gene set enrichment analysis of subgroups by body mass index BMI. This figure compares gene expression between overweight/obese (OW/OB) and normal (NL) BMI patients with metastatic melanoma across subgroups by sex, cohort, and tissue site. Supplementary Figure 2 presents a dotplot of genes differentially up- or downregulated in OW/OB patients versus NL BMI patients by subgroups. Red indicates upregulation in OW/OB verse NL.

BACKGROUND:Prostate cancer (PCa) presents a significant risk to the health of men, and its metastatic spread greatly affects patient survival rates and quality of life. This research investigated the role of DNA methyltransferase 1 (DNMT1) in the progression of PCa. METHODS:By performing bioinformatics analysis and in vivo and in vitro experiments, we investigated the expression levels of DNMT1 and LAMA2 in PCa. Additionally, we evaluated how they influence the proliferation and tumor microenvironment (TME) of PCa cells. RESULTS:DNMT1 was upregulated in PCa, whereas LAMA2 was downregulated. DNMT1 inhibited the expression of LAMA2 by promoting methylation of the LAMA2 promoter, thereby activating the PI3K/AKT signaling pathway, promoting the proliferation of PCa cells, and inducing M2 polarization of macrophages in the TME. Furthermore, DNMT1 promoted the release of the cytokines CCL5, VEGF, MMP9, and PTX3 by PC-3 cells and affected the TME. CONCLUSION:This research highlights the crucial function of DNMT1 in the progression of PCa, providing new strategies for the treatment of PCa, particularly therapeutic strategies that focus on DNA methylation and tumor-associated macrophages.

RNASeq data for the MDA cohort (n=61) with associated clinical info.

Compound soil drought and heat extremes are expected to occur more frequently with global warming, causing wide-ranging socio-ecological repercussions. Vegetation modulates air temperature and soil moisture through biophysical processes, thereby influencing the occurrence of such extremes. Global vegetation cover is broadly expected to increase under climate change, but it remains unclear whether vegetation greening will alleviate or aggravate future increases in compound soil drought-heat events. Here, using a suite of state-of-the-art model simulations, we show that the projected vegetation greening will increase the frequency of global compound soil drought-heat events, equivalent to 12-21% of the total increment at the end of 21st century. This increase is predominantly driven by reduced albedo and enhanced transpiration associated with increased leaf area. Although greening-induced transpiration enhancement has counteracting cooling and drying effects, the excessive water loss in the early growing season can lead to later soil moisture deficits, amplifying compound soil drought-heat extremes during the subsequent warm season. These changes are most pronounced in northern high latitudes and are dominated by the warming effect of CO2. Our study highlights the necessity of integrating vegetation biophysical effects into mitigation and adaptation strategies for addressing compound climate risks.

Immune cell analysis by BMI and sex. A. Immunohistochemistry (IHC) analysis of the MDA cohort stratified by BMI and sex. Line represents median +/- interquartile range; each dot represents a single tumor. B. IHC analysis of the Gide cohort stratified by BMI and sex. Line represents median +/- interquartile range; each dot represents a single tumor.

Immune cell analysis by BMI. A. Immunohistochemistry (IHC) analysis for CD8-, CD45RO-, FOXP3-, CD68-, GZMB-, PD-1-, LAG-3-, and CD3-positive cells in overweight/obese (OW/OB) verse normal (NL) tumors as defined by body mass index from the MD Anderson Cancer Center (MDA) cohort. Line represents median +/- interquartile range; each dot represents a single tumor. B. IHC analysis for PD-L1-, CD45RO-, FOXP3-, EOMES-, GZMB-, PD-1-, TBET-, and TBET:FOXP3-positive cells in OW/OB verse NL tumors as defined by body mass index from the Gide cohort. Line represents median +/- interquartile range; each dot represents a single tumor.

Direct metabolite measurements from a subset of The Cancer Genome Atlas (TCGA) cohort. Comparison of additional tricarboxylic acid cycle metabolites measured by liquid chromatography/mass spectrometry between metastatic melanoma tumors from overweight/obese (OW/OB) patients by body mass index (BMI) verse normal (NL) BMI from The Cancer Genome Atlas (TCGA). Lines represent mean +/- SEM; each dot represents a single tumor.