Chemical genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis via SYK activation
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA608435 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11165226.fastq.gz | Fastqsanger.gz | |||
| SRR11165227.fastq.gz | Fastqsanger.gz | |||
| SRR11165228.fastq.gz | Fastqsanger.gz | |||
| SRR11165229.fastq.gz | Fastqsanger.gz | |||
| SRR11165230.fastq.gz | Fastqsanger.gz |
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Project description:Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases.
| S-EPMC7316778 | biostudies-literature
Project description:Many neurodegenerative diseases are thought to be caused by impaired containment and/or disposal of neurotoxic material such as amyloid beta (Ab) and myelin debris. Indeed, recent human genome-wide association studies (GWAS) and animal model studies have begun to reveal critical roles for the brain’s professional phagocytes, microglia, as well as various innate immune receptors expressed by microglia in the control of neurotoxic material and subsequent neurodegenerative disease pathogenesis. Yet, the critical intracellular molecules that orchestrate the neuroprotective functions of microglia in degenerative disorders remain poorly understood. In our studies, we have identified the innate immune signaling molecule spleen tyrosine kinase (SYK) as a key regulator of microglial phagocytosis in neurodegenerative disease. We find that targeted deletion of SYK in microglia leads to exacerbated Ab deposition, aggravated neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer’s disease (AD). Furthermore, disruption of SYK signaling in this AD model was also shown to impede the development of disease-associated microglia (DAMs), alter AKT/GSK3b-signaling in microglia, and to cause severe deficits in the ability of microglia to phagocytose Ab. Importantly, these critical neuroprotective functions of SYK in microglia were not only restricted to Ab-driven models of neurodegeneration, as we found that SYK is also a critical regulator of microglial phagocytosis and DAM phenotype acquisition in demyelinating disease. Collectively, these results help to break new ground in our understanding of the key innate immune signaling molecules that instruct beneficial microglial functions in response to neurotoxic material. Moreover, these findings suggest that targeting SYK may offer a therapeutic strategy to treat a spectrum of neurodegenerative disorders.
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Project description:Many neurodegenerative diseases are thought to be caused by impaired containment and/or disposal of neurotoxic material such as amyloid beta (Ab) and myelin debris. Indeed, recent human genome-wide association studies (GWAS) and animal model studies have begun to reveal critical roles for the brain’s professional phagocytes, microglia, as well as various innate immune receptors expressed by microglia in the control of neurotoxic material and subsequent neurodegenerative disease pathogenesis. Yet, the critical intracellular molecules that orchestrate the neuroprotective functions of microglia in degenerative disorders remain poorly understood. In our studies, we have identified the innate immune signaling molecule spleen tyrosine kinase (SYK) as a key regulator of microglial phagocytosis in neurodegenerative disease. We find that targeted deletion of SYK in microglia leads to exacerbated Ab deposition, aggravated neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer’s disease (AD). Furthermore, disruption of SYK signaling in this AD model was also shown to impede the development of disease-associated microglia (DAMs), alter AKT/GSK3b-signaling in microglia, and to cause severe deficits in the ability of microglia to phagocytose Ab. Importantly, these critical neuroprotective functions of SYK in microglia were not only restricted to Ab-driven models of neurodegeneration, as we found that SYK is also a critical regulator of microglial phagocytosis and DAM phenotype acquisition in demyelinating disease. Collectively, these results help to break new ground in our understanding of the key innate immune signaling molecules that instruct beneficial microglial functions in response to neurotoxic material. Moreover, these findings suggest that targeting SYK may offer a therapeutic strategy to treat a spectrum of neurodegenerative disorders.
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Project description:Publication abstract: An incomplete view of the (epi)genetic events that drive melanoma initiation and progression has been a major barrier to rational development of effective therapeutics and prognostic diagnostics for melanoma patients. Recent approaches that integrate human melanoma genomic and transcriptomic data provide unprecedented opportunities to discover oncogenic melanoma drivers. One limitation, however, is that human melanoma genome exhibits a radically altered cytogenetic profile. There is therefore the need for biologically-meaningful approaches to identify and validate lesions that drive melanomagenesis. We combined comparative oncogenomic approaches with mouse modeling to identify new cancer genes/pathways that drive melanoma progression. Spontaneously acquired genetic alterations such as copy-number alterations and specific mutations in mouse tumors of defined genetic origin were identified and used to prioritize relevant lesions from the complex human melanoma genomes. This integrated effort confirmed the importance of several genes and pathways previously implicated in melanoma and identified new putative melanoma tumor suppressor genes. Genetic ablation of one such gene, c-Fes, cooperated with BRafv600E to accelerate melanomagenesis in mice. This comparative oncogenomic approach has therefore helped discover a series of novel melanoma tumor suppressor genes, including c-FES, with prognostic and therapeutic relevance in human melanoma.
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Project description:Background: Among all gynecologic malignancies, epithelial ovarian cancer has the highest case-to-fatality ratio. Most patients are diagnosed at advanced stages and recurrence is common and accounts for disease-related mortality. Spleen tyrosine kinase (SYK) is an emerging cancer-associated kinase upregulated in recurrent tumors and is amenable for inhibition using small compounds that have been studied in clinical trials for autoimmune diseases. Our previous proteomic analysis identified several novel SYK substrates including EGFR and ERBB2. Here, we investigated the cross-talk between these pathways in ovarian carcinomas. Methods: Immunohistochemistry and immunoblotting were utilized to assess SYK and EGFR phosphorylation in ovarian serous carcinomas. Association with survival was determined by Kaplan-Meier analysis and the log-rank test. To study its role in EGFR signaling, SYK activity was modulated using a small molecule inhibitor, a syngeneic knockout, and an active kinase inducible system. We applied RNA-seq to investigate the SYK-regulated EGF-induced transcriptome. Results: Intense immunoreactivity of active pSYK(Y525/526) correlated with poor overall survival in two independent ovarian cancer cohorts. SYK directly phosphorylated EGFR and ERBB2, while knockout of SYK reduces their phosphorylation. Phosphorylation levels of SYK(Y525/526) positively correlated with EGFR(Y1187) and STAT3(Y705). Active SYK reduced sensitivity to the EGFR/ERBB2 inhibitor, lapatinib, and SYK non-phosphorylatable EGFR mutant was more sensitive to paclitaxel. SYK modulated the EGF-induced transcriptome, supporting its involvement in EGFR/ERBB2-regulated transcriptional activity. Conclusions: Our findings suggest an upstream role of SYK in regulating the EGFR/ERBB2 signaling, and provide a biological rationale for targeting SYK in ovarian cancer therapy.
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Project description:Genetic studies have highlighted microglia as pivotal in orchestrating Alzheimer’s disease (AD). Microglia that adhere to Aβ plaques acquire a transcriptional signature, “diseaseassociated microglia” (DAM), which largely emanates from the TREM2-DAP12 receptor complex that transmits intracellular signals through the protein tyrosine kinase SYK. The human TREM2R47H variant associated with high AD risk fails to activate microglia via SYK. We found that SYK-deficient microglia cannot encase Aβ plaques, accelerating brain pathology and behavioral deficits. SYK deficiency impaired the PI3K-AKT-GSK3β-mTOR pathway, incapacitating anabolic support required for attaining the DAM profile. However, SYK-deficient microglia proliferated and advanced to an Apoe-expressing prodromal stage of DAM; this pathway relied on the adaptor DAP10, which also binds TREM2. Thus, microglial responses to Aβ involve non-redundant SYK- and DAP10-pathways. Systemic administration of an antibody against CLEC7A, a receptor that directly activates SYK, rescued microglia activation in mice expressing the TREM2R47H allele, unveiling new options for AD immunotherapy.
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