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Denosumab Enhances Antitumor Immunity by Suppressing SPP1 and Boosting Cytotoxic T Cells.
Denosumab, a RANK ligand inhibitor, is primarily used to prevent osteoclastogenesis in the treatment of conditions such as osteoporosis, bone metastasis, and giant cell tumor of bone (GCTB). RANK ligand also plays an important role in immunity by activating NF-κB and its target genes, including the osteopontin-coding gene SPP1 (also known as OPN), which is linked to CXCL9:SPP1 macrophage polarization and prognosis. In this study, we explored an additional role of denosumab in enhancing antitumor immunity in patients. Single-cell RNA sequencing was performed on nine human GCTB samples, including six untreated and three treated only with denosumab, to exclude confounding treatment factors linked with bone metastasis samples. We further analyzed paired samples collected before and after denosumab treatment from a cohort of nine patients with GCTB and conducted a pan-cancer analysis of 34 distinct types of cancers. Our single-cell analysis of GCTB resulted in a comprehensive cell atlas revealing an antitumor role of denosumab in inhibiting SPP1 expression and augmenting active cytotoxic T-cell abundance. Furthermore, we validated this immunomodulatory role of denosumab using the paired GCTB samples. Finally, the pan-cancer analysis supported a negative correlation between SPP1 and CD8A levels, with the CD8A:SPP1 ratio correlating with overall survival in 14 cancer types, which was superior to either CD8A or SPP1 alone. Our research provides clinical evidence that denosumab improves antitumor immunity by decreasing SPP1 expression and enhancing cytotoxic T-cell activity, serving as a milestone in the development of innovative use of denosumab and offering potential benefits to patients with elevated levels of SPP1.
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Human Body Single-Cell Atlas of 3D Genome Organization and DNA Methylation.
Higher-order chromatin structure and DNA methylation are critical for gene regulation, but how these vary across the human body remains unclear. We performed multi-omic profiling of 3D genome structure and DNA methylation for 86,689 single nuclei across 16 human tissues, identifying 35 major and 206 cell subtypes. We revealed extensive changes in CG and non-CG methylation across almost all cell types and characterized 3D chromatin structure at an unprecedented cellular resolution. Intriguingly, extensive discrepancies exist between cell types delineated by DNA methylation and genome structure, indicating that the role of distinct epigenomic features in maintaining cell identity may vary by lineage. This study expands our understanding of the diversity of DNA methylation and chromatin structure and offers an extensive reference for exploring gene regulation in human health and disease.
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Renal compartment-specific genetic variation analyses identify new pathways in chronic kidney disease.
Chronic kidney disease (CKD), a condition in which the kidneys are unable to clear waste products, affects 700 million people globally. Genome-wide association studies (GWASs) have identified sequence variants for CKD; however, the biological basis of these GWAS results remains poorly understood. To address this issue, we created an expression quantitative trait loci (eQTL) atlas for the glomerular and tubular compartments of the human kidney. Through integrating the CKD GWAS with eQTL, single-cell RNA sequencing and regulatory region maps, we identified novel genes for CKD. Putative causal genes were enriched for proximal tubule expression and endolysosomal function, where DAB2, an adaptor protein in the TGF-β pathway, formed a central node. Functional experiments confirmed that reducing Dab2 expression in renal tubules protected mice from CKD. In conclusion, compartment-specific eQTL analysis is an important avenue for the identification of novel genes and cellular pathways involved in CKD development and thus potential new opportunities for its treatment.
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Tie2 activity in cancer associated myofibroblasts serves as novel target against reprogramming of cancer cells to embryonic-like cell state and associated poor prognosis in oral carcinoma patients.
BACKGROUND: Myofibroblastic cancer-associated fibroblasts (CAF) in tumor stroma serves as an independent poor prognostic indicator, supporting higher stemness in oral cancer; however, the underlying biology is not fully comprehended. Here, we have explored the crucial role of Tunica Interna Endothelial Cell Kinase (Tie2/TEK) signaling in transition and maintenance of myofibroblastic phenotype of CAFs, and as possible link with the poor prognosis of head and neck squamous cell carcinoma (HNSCC) patients. METHODS: Bulk and single cell RNA-sequencing (scRNAseq) methods and in-depth bioinformatic analysis were applied for CAF and cancer cells co-culture for studying molecular relationships. In vitro 3D-spheroid-forming ability, expression of stemness markers, in vivo tumor formation ability in zebrafish embryo and syngeneic mouse allografts formation was conducted to test stemness, upon targeting CAF-specific Tie2 activity by gene silencing or with small molecule inhibitor. Immunohistochemistry analysis was performed to locate the distribution of Tie2 and αSMA in primary tumors of oral carcinoma. Prognosis in HNSCC patient cohort from The Cancer Genome Atlas (TCGA) study was analysed based on single sample gene set enrichment score (ssGSEA) and Kaplan-Meier analysis. RESULTS: Autocrine or exogenous TGFβ-induction in CAF led to the recruitment of histone deacetylase 2 (HDAC2) on the promoter of Tie2-antagonist, Angiopoietin-2 (ANGPT2), resulting in its downregulation, leading to phosphorylation of Tie2 (Y992) and subsequent activation of SRC (Y418). This led to SRC/ROCK mediated αSMA-positive stress-fiber formation with gain of myofibroblast phenotype. The CAF-specific Tie2-signaling was responsible for producing embryonic-like cell state in co-cultured cancer cells; with enhanced tumor initiating ability. Tie2 activity in CAF exerted the dynamic gene expression reprogramming, with the upregulation of 'cell migration' and downregulation of 'protein biosynthesis' related gene-regulatory-network modules in malignant cells. The AUCell scores calculated for gene signatures derived from these modules showed significant concordance in independently reported scRNAseq studies of HNSCC tumors and significant association with poor prognosis in HNSCC patient cohort. CONCLUSIONS: CAF-specific Tie2 activity may serve as direct stromal-target against cancer cell plasticity leading to poor prognosis of oral cancer patients. Overall, our work has provided wider applicability of Tie2-specific functions in tumor biology, along with its known role in endothelial cell-specific function.
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Nicotinamide-N-methyltransferase is a promising metabolic drug target for primary and metastatic clear cell renal cell carcinoma.
BACKGROUND: The metabolic enzyme nicotinamide-N-methyltransferase (NNMT) is highly expressed in various cancer entities, suggesting tumour-promoting functions. We systematically investigated NNMT expression and its metabolic interactions in clear cell renal cell carcinoma (ccRCC), a prominent RCC subtype with metabolic alterations, to elucidate its role as a drug target. METHODS: NNMT expression was assessed in primary ccRCC (n = 134), non-tumour tissue and ccRCC-derived metastases (n = 145) by microarray analysis and/or immunohistochemistry. Findings were validated in The Cancer Genome Atlas (kidney renal clear cell carcinoma [KIRC], n = 452) and by single-cell analysis. Expression was correlated with clinicopathological data and survival. Metabolic alterations in NNMT-depleted cells were assessed by nontargeted/targeted metabolomics and extracellular flux analysis. The NNMT inhibitor (NNMTi) alone and in combination with the inhibitor 2-deoxy-D-glucose for glycolysis and BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl-sulfide) for glutamine metabolism was investigated in RCC cell lines (786-O, A498) and in two 2D ccRCC-derived primary cultures and three 3D ccRCC air-liquid interface models. RESULTS: NNMT protein was overexpressed in primary ccRCC (p = 1.32 × 10-16 ) and ccRCC-derived metastases (p = 3.92 × 10-20 ), irrespective of metastatic location, versus non-tumour tissue. Single-cell data showed predominant NNMT expression in ccRCC and not in the tumour microenvironment. High NNMT expression in primary ccRCC correlated with worse survival in independent cohorts (primary RCC-hazard ratio [HR] = 4.3, 95% confidence interval [CI]: 1.5-12.4; KIRC-HR = 3.3, 95% CI: 2.0-5.4). NNMT depletion leads to intracellular glutamine accumulation, with negative effects on mitochondrial function and cell survival, while not affecting glycolysis or glutathione metabolism. At the gene level, NNMT-depleted cells upregulate glycolysis, oxidative phosphorylation and apoptosis pathways. NNMTi alone or in combination with 2-deoxy-D-glucose and BPTES resulted in inhibition of cell viability in ccRCC cell lines and primary tumour and metastasis-derived models. In two out of three patient-derived ccRCC air-liquid interface models, NNMTi treatment induced cytotoxicity. CONCLUSIONS: Since efficient glutamine utilisation, which is essential for ccRCC tumours, depends on NNMT, small-molecule NNMT inhibitors provide a novel therapeutic strategy for ccRCC and act as sensitizers for combination therapies.
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Prion protein regulates invasiveness in glioblastoma stem cells.
BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor driven by glioblastoma stem cells (GSCs), which represent an appealing target for therapeutic interventions. The cellular prion protein (PrPC), a scaffold protein involved in diverse cellular processes, interacts with various membrane and extracellular matrix molecules, influencing tumor biology. Herein, we investigate the impact of PrPC expression on GBM. METHODS: To address this goal, we employed CRISPR-Cas9 technology to generate PrPC knockout (KO) glioblastoma cell lines, enabling detailed loss-of-function studies. Bulk RNA sequencing followed by differentially expressed gene and pathway enrichment analyses between U87 or U251 PrPC-wild-type (WT) cells and PrPC-knockout (KO) cells were used to identify pathways regulated by PrPC. Immunofluorescence assays were used to evaluate cellular morphology and protein distribution. For assessment of protein levels, Western blot and flow cytometry assays were employed. Transwell and growth curve assays were used to determine the impact of loss-of-PrPC in GBM invasiveness and proliferation, respectively. Single-cell RNA sequencing analysis of data from patient tumors from The Cancer Genome Atlas (TCGA) and the Broad Institute of Single-Cell Data Portal were used to evaluate the correspondence between our in vitro results and patient samples. RESULTS: Transcriptome analysis of PrPC-KO GBM cell lines revealed altered expression of genes associated with crucial tumor progression pathways, including migration, proliferation, and stemness. These findings were corroborated by assays that revealed impaired invasion, migration, proliferation, and self-renewal in PrPC-KO GBM cells, highlighting its critical role in sustaining tumor growth. Notably, loss-of-PrPC disrupted the expression and localization of key stemness markers, particularly CD44. Additionally, the modulation of PrPC levels through CD44 overexpression further emphasizes their regulatory role in these processes. CONCLUSIONS: These findings establish PrPC as a modulator of essential molecules on the cell surface of GSCs, highlighting its potential as a therapeutic target for GBM.
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Multi-omics analysis identifies CCNB1 as a cell cycle factor driving glioblastoma progression and its inhibition by resveratrol.
Glioblastoma (GBM) is a fast-growing primary brain tumor with high mortality and recurrence rates. Dysregulation of the cell cycle is a hallmark of GBM, and cyclin B1 (CCNB1) is a key regulator of the cell cycle. However, the role of CCNB1 in GBM remains unclear. In this study, we found that CCNB1 mRNA and protein expression levels were significantly higher in GBM tissues than normal tissues. High CCNB1 mRNA expression was associated with poorer prognosis in GBM patients. Single-cell and spatial transcriptomics data revealed that CCNB1+ cells represent a proliferative subcluster in GBM, annotated as proliferative cells, and characterized by the upregulation of cell cycle-related pathways. CCNB1 inhibition decreased the proliferation of GBM cells and impaired cell cycle progression from S phase to G2/M. Additionally, resveratrol could inhibit the expression of CCNB1 and its interacting gene polo-like kinase 1 (PLK1). Importantly, through in vitro and in vivo experiments, we found that resveratrol suppressed GBM cell growth with low toxicity. CCNB1 silencing combined with resveratrol treatment further inhibited the proliferation of GBM cells. Collectively, these data suggest that CCNB1 is highly expressed in GBM and may promote GBM progression. Inhibition of CCNB1 may represent a potential therapeutic strategy for GBM.
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Integrative multiomics dissection of LSM7 as a prognostic biomarker and therapeutic target in hepatocellular carcinoma.
ObjectiveHepatocellular carcinoma (HCC) has a poor prognosis, necessitating novel biomarkers. The role of LSM7 (Like-Sm protein 7), an RNA-binding protein, in HCC remains unclear.MethodsWe employed a multiomics strategy using transcriptomic data from TCGA, GTEx, GEO, and ICGC to evaluate LSM7's expression and prognostic value. A protein-protein interaction (PPI) network was constructed via STRING, followed by Gene Ontology/KEGG/Gene Set Enrichment Analysis (GSEA) functional enrichment analysis. The relationship between LSM7 and the immune infiltration landscape was assessed using single-sample GSEA (ssGSEA) and the TISCH single-cell database. Drug sensitivity was analyzed using Genomics of Drug Sensitivity in Cancer, and structure-based virtual screening was performed on the ZINC library to identify potential LSM7 inhibitors. Key findings were validated in clinical samples and MHCC97H cells and Huh-7 cells using Western blot, IHC, and MTT assays.ResultsLSM7 was significantly overexpressed in HCC, correlating with adverse clinicopathological features (higher histological grade, elevated AFP levels, vascular invasion) and shorter overall survival, identifying it as an independent risk factor. Functionally, LSM7 was implicated in processes such as hormone regulation. Immune analysis revealed that high LSM7 expression was associated with altered immune cell abundance, upregulation of immune checkpoints, and a higher Tumor Immune Dysfunction and Exclusion score, suggesting a role in immune evasion. The high-LSM7 group showed increased sensitivity to drugs like sorafenib. Virtual screening identified Velpatasvir as a top LSM7-targeting candidate, with in vitro validation confirming that it inhibits MHCC97H cells and Huh-7 cells proliferation and downregulates LSM7 protein in a concentration- and time-dependent manner.ConclusionsBy integrating multiomics strategy and experimental validation, this study suggests that LSM7 may play a role in HCC progression and influence the tumor immune microenvironment. LSM7 may serve as a potential biomarker and therapeutic target, and Velpatasvir appears to be a candidate agent worthy of further investigation.
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Integrated Multi-Omics and Spatial Transcriptomics Reveal GUK1 as a Prognostic Biomarker Regulated by the TP53-HSF1 Axis in Breast Cancer.
Background: Guanylate kinase 1 (GUK1) is crucial for nucleotide metabolism, yet its impact on breast cancer (BC) progression remains poorly defined. The objective of the present study is to investigate GUK1 as a prognostic biomarker and therapeutic target. Methods: We employed a multi-omics approach integrating The Cancer Genome Atlas (TCGA) data, machine learning algorithm, High-Definition spatial transcriptomics (Visium HD), single-cell profiling, molecular docking and experimental validation including in vitro knockdown models and Surface Plasmon Resonance (SPR). Results: LASSO regression identified GUK1 as a key metabolic driver. High expression correlated significantly with poor survival and was most pronounced in Human Epidermal Growth Factor Receptor 2 (HER2)-positive and triple-negative subtypes. Spatial transcriptomics revealed GUK1 strongly colocalizes with expanding cancer cell nests, intensifying with disease stage. Single-cell analysis linked GUK1 overexpression to an immunosuppressive microenvironment enriched in exhausted T-cells. Clinically and molecularly, TP53 mutations are highly associated with HSF1 promoter hypomethylation and subsequent HSF1-mediated GUK1 upregulation. We experimentally confirmed this axis, showing that HSF1 or GUK1 knockdown significantly impaired cell migration and suppressed mTOR signaling. Furthermore, while high GUK1 levels predicted resistance to CDK4/6 inhibitors, they enhanced sensitivity to the PI3K/mTOR inhibitor Apitolisib. This therapeutic vulnerability was validated by SPR, which confirmed high-affinity binding between GUK1 and Apitolisib, and by cell viability assays where GUK1 depletion induced drug resistance. Conclusion: GUK1 serves as a robust prognostic biomarker regulated by the TP53-HSF1 axis. Its distinct spatial patterns, immune-suppressive associations, and experimentally validated role in modulating PI3K/mTOR inhibitor sensitivity position GUK1 as a promising target for precision oncology in invasive BC.
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Spatiotemporal dynamics of the cardioimmune niche during lesion repair.
The heart is one of the least regenerative organs in humans, and ischemic heart disease is the leading cause of death worldwide. Understanding the cellular and molecular processes that occur during cardiac wound healing is an essential prerequisite to reducing health burden and improving cardiac function after myocardial tissue damage. Here, by integrating single-cell RNA sequencing with high-resolution spatial transcriptomics, we reconstruct the spatiotemporal dynamics of the fibrotic niches after cardiac injury in adult mice. We reveal a complex multicellular network that regulates cardiac repair, including fibroblast proliferation silencing by Trem2high macrophages to prevent excessive fibrosis. We further discovered a rare population of progenitor-like cardiomyocytes after lesion, promoted by myeloid and lymphoid niche signals. Culturing non-regenerative mouse cardiomyocytes or human heart tissue with these niche factors reactivated progenitor gene expression and cell cycle activity. In summary, this spatiotemporal atlas provides valuable insights into the heterocellular interactions that control cardiac repair.