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Cell Chemical Biology Oct 2023KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including...
KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.
Topics: Humans; Female; Breast Neoplasms; Histones; Histone Acetyltransferases; Signal Transduction; Cell Line, Tumor
PubMed: 37557181
DOI: 10.1016/j.chembiol.2023.07.005 -
Journal of Experimental & Clinical... Sep 2023FAT4 (FAT Atypical Cadherin 4) is a member of the cadherin-associated protein family, which has been shown to function as a tumor suppressor by inhibiting proliferation...
BACKGROUND
FAT4 (FAT Atypical Cadherin 4) is a member of the cadherin-associated protein family, which has been shown to function as a tumor suppressor by inhibiting proliferation and metastasis. The Wnt/β-catenin pathway activation is highly associated with PD-L1-associated tumor immune escape. Here, we report the mechanism by which FAT4 overexpression regulates anti-tumor immunity in cervical cancer by inhibiting PD-L1 N-glycosylation and cell membrane localization in a β-catenin-dependent manner.
METHODS
FAT4 expression was first detected in cervical cancer tissues and cell lines. Cell proliferation, clone formation, and immunofluorescence were used to determine the tumor suppressive impact of FAT4 overexpression in vitro, and the findings were confirmed in immunodeficient and immunocomplete mice xenografts. Through functional and mechanistic experiments in vivo and in vitro, we investigated how FAT4 overexpression affects the antitumor immunity via the β-catenin/STT3/PD-L1 axis.
RESULTS
FAT4 is downregulated in cervical cancer tissues and cell lines. We determined that FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1). FAT4 overexpression decreases programmed death-ligand 1 (PD-L1) mRNA expression at the transcriptional level, and causes aberrant glycosylation of PD-L1 via STT3A at the post-translational modifications (PTMs) level, leading to its endoplasmic reticulum (ER) accumulation and polyubiquitination-dependent degradation. We found that FAT4 overexpression promotes aberrant PD-L1 glycosylation and degradation in a β-catenin-dependent manner, thereby increasing cytotoxic T lymphocyte (CTL) activity in immunoreactive mouse models.
CONCLUSIONS
These findings address the basis of Wnt/β-catenin pathway activation in cervical cancer and provide combination immunotherapy options for targeting the FAT4/β-catenin/STT3/PD-L1 axis. Schematic cartoons showing the antitumor immunity mechanism of FAT4. (left) when Wnts bind to their receptors, which are made up of Frizzled proteins and LRP5/6, the cytoplasmic protein DVL is activated, inducing the aggregation of degradation complexes (AXIN, GSK3β, CK1, APC) to the receptor. Subsequently, stable β-catenin translocates into the nucleus and binds to TCF/LEF and TCF7L2 transcription factors, leading to target genes transcription. The catalytically active subunit of oligosaccharyltransferase, STT3A, enhances PD-L1 glycosylation, and N-glycosylated PD-L1 translocates to the cell membrane via the ER-to-Golgi pathway, resulting in immune evasion. (Right) FAT4 exerts antitumor immunity mainly through following mechanisms: (i) FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1); (ii) FAT4 inhibits PD-L1 and STT3A transcription in a β-catenin-dependent manner and induces aberrant PD-L1 glycosylation and ubiquitination-dependent degradation; (iii) Promotes activation of cytotoxic T lymphocytes (CTL) and infiltration into the tumor microenvironment.
Topics: Animals; Female; Humans; Mice; B7-H1 Antigen; beta Catenin; Cadherins; Glycogen Synthase Kinase 3 beta; Tumor Microenvironment; Tumor Suppressor Proteins; Uterine Cervical Neoplasms
PubMed: 37658376
DOI: 10.1186/s13046-023-02758-2 -
Cell May 2024Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine-learning-based approach to predict antimicrobial peptides (AMPs)...
Novel antibiotics are urgently needed to combat the antibiotic-resistance crisis. We present a machine-learning-based approach to predict antimicrobial peptides (AMPs) within the global microbiome and leverage a vast dataset of 63,410 metagenomes and 87,920 prokaryotic genomes from environmental and host-associated habitats to create the AMPSphere, a comprehensive catalog comprising 863,498 non-redundant peptides, few of which match existing databases. AMPSphere provides insights into the evolutionary origins of peptides, including by duplication or gene truncation of longer sequences, and we observed that AMP production varies by habitat. To validate our predictions, we synthesized and tested 100 AMPs against clinically relevant drug-resistant pathogens and human gut commensals both in vitro and in vivo. A total of 79 peptides were active, with 63 targeting pathogens. These active AMPs exhibited antibacterial activity by disrupting bacterial membranes. In conclusion, our approach identified nearly one million prokaryotic AMP sequences, an open-access resource for antibiotic discovery.
PubMed: 38843834
DOI: 10.1016/j.cell.2024.05.013 -
Frontiers in Aging 2023Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by a range of clinical symptoms, including poikiloderma, juvenile cataracts, short... (Review)
Review
Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by a range of clinical symptoms, including poikiloderma, juvenile cataracts, short stature, sparse hair, eyebrows/eyelashes, nail dysplasia, and skeletal abnormalities. While classically associated with mutations in the gene, which encodes a DNA helicase involved in DNA replication and repair, three additional genes have been recently identified in RTS: , encoding a subunit of the APC/C complex; which encodes a nuclease/helicase involved in DNA repair; and , encoding a poorly characterized protein implicated in excitatory synapse formation and splicing. Here, we review the clinical spectrum of RTS patients, analyze the genetic basis of the disease, and discuss molecular functions of the affected genes, drawing some novel genotype-phenotype correlations and proposing avenues for future studies into this enigmatic disorder.
PubMed: 38021400
DOI: 10.3389/fragi.2023.1296409 -
Frontiers in Neuroscience 2023Epilepsy is one of the most common neurologic disorders that is characterized by recurrent seizures, and depending on the type of seizure, it could lead to a severe... (Review)
Review
Epilepsy is one of the most common neurologic disorders that is characterized by recurrent seizures, and depending on the type of seizure, it could lead to a severe outcome. Epilepsy's mechanism of development is not fully understood yet, but some of the common features of the disease are blood-brain barrier disruption, microglia activation, and neuroinflammation. Those are also targets of activated protein C (APC). In fact, by downregulating thrombin, known as a pro-inflammatory, APC acts as an anti-inflammatory. APC is also an anti-apoptotic protein, instance by blocking p53-mediated apoptosis. APC's neuroprotective effect could prevent blood-brain barrier dysfunction by acting on endothelial cells. Furthermore, through the downregulation of proapoptotic, and proinflammatory genes, APC's neuroprotection could reduce the effect or prevent epilepsy pathogenesis. APC's activity acts on blood-brain barrier disruption, inflammation, and apoptosis and causes neurogenesis, all hallmarks that could potentially treat or prevent epilepsy. Here we review both Activated Protein C and epilepsy mechanism, function, and the possible association between them.
PubMed: 37901428
DOI: 10.3389/fnins.2023.1251017 -
Science Advances Nov 2023infection is a major risk factor for the development of gastric cancer. The bacteria reside in close proximity to gastric surface mucous as well as stem and progenitor...
infection is a major risk factor for the development of gastric cancer. The bacteria reside in close proximity to gastric surface mucous as well as stem and progenitor cells. Here, we take advantage of wild-type and genetically engineered murine gastric organoids and organoid-derived monolayers to study the cellular targets of -induced DNA damage and replication stress and to explore possible interactions with preexisting gastric cancer driver mutations. We find using alkaline comet assay, single-molecule DNA fiber assays, and immunofluorescence microscopy of DNA repair foci that induces transcription-dependent DNA damage in actively replicating, Leucine-rich-repeat containing G-Protein-Coupled Receptor 5 (Lgr5)-positive antral stem and progenitor cells and their Troy-positive corpus counterparts, but not in other gastric epithelial lineages. Infection-dependent DNA damage is aggravated by inactivation, but not by or loss, or overexpression. Our data suggest that induces DNA damage in stem and progenitor cells, especially in settings of hyperproliferation due to constitutively active Wnt signaling.
Topics: Animals; Humans; Mice; DNA Damage; Genes, Tumor Suppressor; Helicobacter Infections; Helicobacter pylori; Receptors, G-Protein-Coupled; Stem Cells; Stomach Neoplasms
PubMed: 37967175
DOI: 10.1126/sciadv.adh0322 -
Journal of Hepatology Jul 2024The effectiveness of immune checkpoint inhibitor (ICI) therapy for hepatocellular carcinoma (HCC) is limited by treatment resistance. However, the mechanisms underlying...
BACKGROUND & AIMS
The effectiveness of immune checkpoint inhibitor (ICI) therapy for hepatocellular carcinoma (HCC) is limited by treatment resistance. However, the mechanisms underlying immunotherapy resistance remain elusive. We aimed to identify the role of CT10 regulator of kinase-like (CRKL) in resistance to anti-PD-1 therapy in HCC.
METHODS
Gene expression in HCC specimens from 10 patients receiving anti-PD-1 therapy was identified by RNA-sequencing. A total of 404 HCC samples from tissue microarrays were analyzed by immunohistochemistry. Transgenic mice (Alb-Cre/Trp53) received hydrodynamic tail vein injections of a CRKL-overexpressing vector. Mass cytometry by time of flight was used to profile the proportion and status of different immune cell lineages in the mouse tumor tissues.
RESULTS
CRKL was identified as a candidate anti-PD-1-resistance gene using a pooled genetic screen. CRKL overexpression nullifies anti-PD-1 treatment efficacy by mobilizing tumor-associated neutrophils (TANs), which block the infiltration and function of CD8 T cells. PD-L1 TANs were found to be an essential subset of TANs that were regulated by CRKL expression and display an immunosuppressive phenotype. Mechanistically, CRKL inhibits APC (adenomatous polyposis coli)-mediated proteasomal degradation of β-catenin by competitively decreasing Axin1 binding, and thus promotes VEGFα and CXCL1 expression. Using human HCC samples, we verified the positive correlations of CRKL/β-catenin/VEGFα and CXCL1. Targeting CRKL using CRISPR-Cas9 gene editing (CRKL knockout) or its downstream regulators effectively restored the efficacy of anti-PD-1 therapy in an orthotopic mouse model and a patient-derived organotypic tumor spheroid model.
CONCLUSIONS
Activation of the CRKL/β-catenin/VEGFα and CXCL1 axis is a critical obstacle to successful anti-PD-1 therapy. Therefore, CRKL inhibitors combined with anti-PD-1 could be useful for the treatment of HCC.
IMPACT AND IMPLICATIONS
Here, we found that CRKL was overexpressed in anti-PD-1-resistant hepatocellular carcinoma (HCC) and that CRKL upregulation promotes anti-PD-1 resistance in HCC. We identified that upregulation of the CRKL/β-catenin/VEGFα and CXCL1 axis contributes to anti-PD-1 tolerance by promoting infiltration of tumor-associated neutrophils. These findings support the strategy of bevacizumab-based immune checkpoint inhibitor combination therapy, and CRKL inhibitors combined with anti-PD-1 therapy may be developed for the treatment of HCC.
Topics: Carcinoma, Hepatocellular; Liver Neoplasms; Animals; Humans; Mice; Drug Resistance, Neoplasm; Immune Checkpoint Inhibitors; Adaptor Proteins, Signal Transducing; Neutrophil Infiltration; Programmed Cell Death 1 Receptor; Mice, Transgenic; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Male; Chemokine CXCL1
PubMed: 38403027
DOI: 10.1016/j.jhep.2024.02.009 -
Current Protocols Nov 2023The MHC II-EGFP knock-in mouse model enables us to visualize and track MHC-II-expressing cells in vivo by expressing enhanced green fluorescent protein (EGFP) fused to...
The MHC II-EGFP knock-in mouse model enables us to visualize and track MHC-II-expressing cells in vivo by expressing enhanced green fluorescent protein (EGFP) fused to the MHC class II molecule under the MHC II beta chain promoter. Using this model, we can easily identify MHC-II-expressing cells, including dendritic cells, B cells, macrophages, and ILC3s, which play a key role as antigen-presenting cells (APCs) for CD4 T cells. In addition, we can also precisely identify and analyze APC-containing tissues and organs. Even after fixation, EGFP retains its fluorescence, so this model is suitable for immunofluorescence studies, facilitating an unbiased characterization of the histological context, especially with techniques such as light-sheet fluorescence microscopy. Furthermore, the MHC II-EGFP knock-in mouse model is valuable for studying the molecular mechanisms of MHC II gene regulation and expression by making it possible to correlate MHC II expression (MHC II-EGFP) with surface fraction through antibody detection, thereby shedding light on the intricate regulation of MHC II expression. Overall, this model is an essential asset for quantitative and systems immunological research, providing insights into immune cell dynamics and localization, with a tool for precise cell identification and with the ability to study MHC II gene regulation, thus furthering the understanding of immune responses and underlying mechanisms © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Characterization of antigen-specific MHC II loading compartment tubulation toward the immunological synapse Basic Protocol 2: Characterization of overall versus surface MHC II expression Basic Protocol 3: Identification and preparation of the lymphoid organs Basic Protocol 4: Quantification of APC content in lymphoid organs by fluorescence stereomicroscopy Basic Protocol 5: Quantification and measurement of intestinal lymphoid tissue by light-sheet fluorescence stereomicroscopy Basic Protocol 6: Visualization of corneal APCs Basic Protocol 7: Quantification of MHC II cells in maternal milk by flow cytometry Support Protocol 1: Cell surface staining and flow cytometry analysis of spleen mononuclear cells.
Topics: Animals; Mice; Green Fluorescent Proteins; Antigen-Presenting Cells; B-Lymphocytes; Cell Membrane; Disease Models, Animal
PubMed: 37934124
DOI: 10.1002/cpz1.925