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Abstract Background DNA hypermethylation and hotspot mutations were frequently observed in the upstream and core promoter of telomerase reverse transcriptase (TERT), respectively, and they were associated with increased TERT expression and adverse clinical outcomes in thyroid cancer. In TERT promoter mutant cancer cells, the hypomethylated TERT mutant allele was active and the hypermethylated TERT wild-type allele was silenced. However, whether and how the upstream promoter methylation regulates TERT expression in TERT mutation-negative cells were largely unknown. Methods DNA demethylating agents 5-azacytidine and decitabine and a genomic locus-specific demethylation system based on dCas9-TET1 were used to assess the effects of TERT upstream promoter methylation on TERT expression, cell growth and apoptosis of thyroid cancer cells. Regulatory proteins binding to TERT promoter were identified by CRISPR affinity purification in situ of regulatory elements (CAPTURE) combined with mass spectrometry. The enrichments of selected regulatory proteins and histone modifications were evaluated by chromatin immunoprecipitation. Results The level of DNA methylation at TERT upstream promoter and expression of TERT were significantly decreased after treatment with 5-azacytidine or decitabine in TERT promoter wild-type thyroid cancer cells. Genomic locus-specific demethylation of TERT upstream promoter induced TERT downregulation, along with cell apoptosis and growth inhibition. Consistently, demethylating agents sharply inhibited the growth of thyroid cancer cells harboring hypermethylated TERT but had little effect on cells with TERT hypomethylation. Moreover, we identified that the chromatin remodeling protein CHD4 binds to methylated TERT upstream promoter and promotes its transcription by suppressing the enrichment of H3K9me3 and H3K27me3 at TERT promoter. Conclusions This study uncovered the mechanism of promoter methylation mediated TERT activation in TERT promoter mutation-negative thyroid cancer cells and indicated TERT upstream promoter methylation as a therapeutic target for thyroid cancer.

BRAF or RAS mutation-induced aberrant activation of the mitogen-activated protein kinase (MAPK) pathway is frequently observed in human cancers. As the key downstream node of MAPK pathway, ERK1/2 is as an important therapeutic target. GDC-0994 (ravoxertinib), an orally bioavailable, highly selective small-molecule inhibitor of ERK1/2, showed acceptable safety and pharmacodynamic profile in a recent phase I clinical trial. In this study, we investigated dependence of the anti-tumor effect of ERK inhibitor GDC-0994 on genetic alterations in the MAPK pathway. The results showed that GDC-0994 sharply inhibited cell proliferation and colony formation and induced remarkable G1 phase cell-cycle arrest in cancer cells harboring BRAF mutation but had little effect on cell behaviors in most RAS mutant or wild-type cell lines. The expression of a large number of genes, particularly the genes in the cell cycle pathway, were significantly changed after GDC-0994 treatment in BRAF mutant cells, while no remarkable expression change of such genes was observed in wild-type cells. Moreover, GDC-0994 selectively inhibited tumor growth in a BRAF mutant xenograft mice model. Our findings demonstrate a BRAF mutation-dependent anti-tumor effect of GDC-0994 and provide a rational strategy for patient selection for ERK1/2 inhibitor treatment.

Abstract Background Hotspot mutations in the promoter of telomerase reverse transcriptase (TERT) gene are the most common genetic variants in hepatocellular carcinoma (HCC) and associated with poor prognosis of the disease. However, no drug was currently approved for treating TERT promoter mutation positive HCC patients. Here, we aim to explore the potential therapeutic strategy for targeting TERT promoter mutation in HCC. Methods The Liver Cancer Model Repository database was used for screening potential drugs to selectively suppress the growth of TERT promoter mutant HCC cells. RNA‐seq, CRISPR‐Cas9 technology and siRNA transfection were performed for mechanistic studies. Cell counting kit‐8 (CCK8) assay and the xenograft tumour models were used for cell growth detection in vitro and in vivo, respectively. Cell apoptosis and cell cycle arrest were analysed by Annexin V‐FITC staining and/or propidium iodide staining. Results PLK1 inhibitors were remarkably more sensitive to HCC cells harbouring TERT promoter mutation than wild‐type cells in vitro and in vivo, which were diminished after TERT promoter mutation was edited to the wild‐type nucleotide. Comparing the HCC cells with wild‐type promoter of TERT, PLK1 inhibitors specifically downregulated Smad3 to regulate TERT for inducing apoptosis and G2/M arrest in TERT mutant HCC cells. Moreover, knockout of Smad3 counteracted the effects of PLK1 inhibitors in TERT mutant HCC cells. Finally, a cooperative effect of PLK1 and Smad3 inhibition was observed in TERT mutant cells. Conclusions PLK1 inhibition selectively suppressed the growth of TERT mutant HCC cells through Smad3, thus contributed to discover a novel therapeutic strategy to treat HCC patients harbouring TERT promoter mutations. Key points TERT promoter mutation confers sensitivity to PLK1 inhibitors in HCC. The selective growth inhibition of TERT mutant HCC cells induced by PLK1 inhibitor was mediated by Smad3. Combined inhibition of PLK1 and Smad3 showed a cooperative anti‐tumor effect in TERT mutant HCC cells.

Shape-memory materials hold great potential to impart medical devices with functionalities useful during implantation, locomotion, drug delivery, and removal. However, their clinical translation is limited by a lack of non-invasive and precise methods to trigger and control the shape recovery, especially for devices implanted in deep tissues. In this study, the application of image-guided high-intensity focused ultrasound (HIFU) heating is tested. Magnetic resonance-guided HIFU triggered shape-recovery of a device made of polyurethane urea while monitoring its temperature by magnetic resonance thermometry. Deformation of the polyurethane urea in a live canine bladder (5 cm deep) is achieved with 8 seconds of ultrasound-guided HIFU with millimeter resolution energy focus. Tissue sections show no hyperthermic tissue injury. A conceptual application in ureteral stent shape-recovery reduces removal resistance. In conclusion, image-guided HIFU demonstrates deep energy penetration, safety and speed.

Developing an ambulatory assist lung (AAL) for patients who need continuous extracorporeal membrane oxygenation has been associated with several design objectives, including the design of compact components, optimization of gas transfer efficiency, and reduced thrombogenicity. In an effort to address thrombogenicity concerns with currently utilized component biomaterials, a low molecular weight water soluble siloxane‐functionalized zwitterionic sulfobetaine (SB‐Si) block copolymer was coated on a full‐scale AAL device set via a one pot aqueous circulation coating. All device parts including hollow fiber bundle, housing, tubing and cannular were successfully coated with increasing atomic compositions of the SB block copolymer and the coated surfaces showed a significant reduction of platelet deposition while gas exchange performance was sustained. However, water solubility of the SB‐Si was unstable, and the coating method, including oxygen plasma pretreatment on the surfaces were considered inconsistent with the objective of developing a simple aqueous coating. Addressing these weaknesses, SB block copolymers were synthesized bearing epoxy or epoxy‐silane groups with improved water solubility (SB‐EP & SB‐EP‐Si) and no requirement for surface pretreatment (SB‐EP‐Si). An SB‐EP‐Si triblock copolymer showed the most robust coating capacity and stability without prior pretreatment to represent a simple aqueous circulation coating on an assembled full‐scale AAL device. [ABSTRACT FROM AUTHOR]

This study investigates the neuroprotective potential of STAT3 inhibition in reducing oxidative stress-induced neuronal damage and apoptosis, a major factor contributing to the onset and progression of neurodegenerative diseases, including Alzheimer's disease (AD). Our findings demonstrate that STAT3 inhibitors significantly enhance cell survival and reduce apoptosis in SH-SY5Y cells exposed to hydrogen peroxide. These protective effects are mediated through the ERK/CREB signaling pathway rather than direct suppression of STAT3 phosphorylation. Further analysis revealed that the ERK pathway is a critical mediator of CREB activation following STAT3 inhibition. The protective effects of STAT3 inhibitors were significantly reduced in the presence of the ERK inhibitor PD98059, underscoring the importance of the ERK/CREB axis in neuroprotection. We observed that STAT3 inhibitors promote CREB phosphorylation, leading to the upregulation of immediate early genes such as c-Fos, c-Jun, Arc, Egr-1, NR4A1, and Homer1a, as well as BDNF. These genes are crucial for synaptic plasticity and long-term memory formation, suggesting that STAT3 inhibition may ameliorate cognitive impairments in neurodegenerative conditions. Our results highlight the potential of STAT3 inhibitors to counteract oxidative stress and enhance cognitive functions by modulating the ERK/CREB signaling pathway. These findings provide valuable insights into the molecular mechanisms of STAT3 inhibition and support its therapeutic potential for treating neurodegenerative diseases., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethical Approval: Not applicable. Consent to Participate: Not applicable. Consent for Publication: Not applicable., (© 2024. The Author(s).)

Various combination therapies have been investigated to overcome the limitations of using immune checkpoint inhibitors. However, determining the optimal combination therapy remains challenging. To overcome the therapeutical limitation, we conducted a translational research to elucidate the mechanisms by which AXL inhibition enhances the anti-tumor effects when combined with anti-PD-1 antibody therapy. Herein, we demonstrated improved antitumor effects through combination treatment with denfivontinib and pembrolizumab which resulted in enhanced differentiation into effector CD4+ and CD8+ memory T cells, accompanied by an increase in IFN-γ expression in the YHIM-2004 xenograft model derived from patients with NSCLC. Concurrently, a reduction in the number of immunosuppressive M2 macrophages and myeloid-derived suppressor cells was observed. Mechanistically, denfivontinib potentiated the NOD-like receptor pathway, thereby facilitating the NLRP3 inflammasome formation. This leads to macrophage activation via the NF-kB signaling pathway activation. We have confirmed that the positive interaction between macrophages and T cells arises from the enhanced antigen-presenting machinery of activated macrophages. Furthermore, the observed tumor effects in AXL knock-out mice confirmed that AXL inhibition by denfivontinib enhances the anti-tumor effects, thus opening new avenues for therapeutic interventions aimed at overcoming limitations in immunotherapy. To demonstrate the extent to which our findings reflect clinical results, we analyzed bulk-RNA sequencing data from 21 NSCLC patients undergoing anti-PD-1 immunotherapy. The NLRP3 inflammasome score influenced enhanced immune responses in patient data undergoing anti-PD-1 immunotherapy, suggesting a role for NLRP3 inflammasome in activating immune responses during treatment.

Purpose: We aimed to comprehensively analyze the immune cell and stromal components of tumor microenvironment at the single-cell level and identify tumor heterogeneity among the major top-derived oncogene mutations in non-small cell lung cancer (NSCLC) using single-cell RNA sequencing (scRNA-seq) data., Materials and Methods: The scRNA-seq dataset utilized in this study comprised 64369 primary tumor tissue cells from 21 NSCLC patients, focusing on mutations in EGFR , ALK , BRAF , KRAS , TP53 , and the wild-type., Results: Tumor immune microenvironment (TIM) analysis revealed differential immune responses across NSCLC mutation subtypes. TIM analysis revealed different immune responses across the mutation subtypes. Two mutation clusters emerged: KRAS , TP53 , and EGFR + TP53 mutations (MC1); and EGFR , BRAF , and ALK mutations (MC2). MC1 showed higher tertiary lymphoid structures signature scores and enriched populations of C2-T-IL7R, C3-T/NK-CXCL4, C9-T/NK-NKG, and C1-B-MS4A1 clusters than cluster 2. Conversely, MC2 cells exhibited higher expression levels of TNF , IL1B , and chemokines linked to alternative immune pathways. Remarkably, co-occurring EGFR and TP53 mutations were grouped as MC1. EGFR + TP53 mutations showed upregulation of peptide synthesis and higher synthetic processes, as well as differences in myeloid and T/NK cells compared to EGFR mutations. In T/NK cells, EGFR + TP53 mutations showed a higher expression of features related to cell activity and differentiation, whereas EGFR mutations showed the opposite., Conclusion: Our research indicates a close association between mutation types and tumor microenvironment in NSCLC, offering insights into personalized approaches for cancer diagnosis and treatment., Competing Interests: The authors have no potential conflicts of interest to disclose., (© Copyright: Yonsei University College of Medicine 2024.)

Purpose: By utilizing both protein and mRNA expression patterns, we can identify more detailed and diverse immune cells, providing insights into understanding the complex immune landscape in cancer ecosystems., Materials and Methods: This study was performed by obtaining publicly available Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) data of peripheral blood mononuclear cells (PBMCs) from the Gene Expression Omnibus database. A total of 94674 total cells were analyzed, of which 32412 were T cells. There were 228 protein features and 16262 mRNA features in the data. The Seurat package was used for quality control and preprocessing, principal component analysis was performed, and Uniform Manifold Approximation and Projection was used to visualize the clusters. Protein and mRNA levels in the CITE-seq were analyzed., Results: We observed that a subset of T cells in the clusters generated at the protein level divided better. By identifying mRNA markers that were highly correlated with the CD4 and CD8 proteins and cross-validating CD26 and CD99 markers using flow cytometry, we found that CD4 + and CD8 + T cells were better discriminated in PBMCs. Weighted Nearest Neighbor clustering results identified a previously unobserved T cell subset., Conclusion: In this study, we used CITE-seq data to confirm that protein expression patterns could be used to identify cells more precisely. These findings will improve our understanding of the heterogeneity of immune cells in the future and provide valuable insights into the complexity of the immune response in health and disease., Competing Interests: The authors have no potential conflicts of interest to disclose., (© Copyright: Yonsei University College of Medicine 2024.)