CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA664943 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12692088_1.fastq.gz | Fastqsanger.gz | |||
| SRR12692088_2.fastq.gz | Fastqsanger.gz | |||
| SRR12692089_1.fastq.gz | Fastqsanger.gz | |||
| SRR12692089_2.fastq.gz | Fastqsanger.gz | |||
| SRR12692090_1.fastq.gz | Fastqsanger.gz |
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Project description:While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A confers cellular resistance to CHK1 inhibitors and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase which dephosphorylates the WEE1 protein and rescues WEE1 from Ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1 inhibitors. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1 inhibitor plus a WEE1 inhibitor can overcome CHK1 inhibitor resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1 inhibitor sensitivity or resistance.
2021-01-01 | GSE158338 | GEO
Project description:SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.
2018-01-18 | GSE101516 | GEO
Project description:SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.
2018-01-18 | GSE101512 | GEO
Project description:SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.
2018-01-18 | GSE101515 | GEO
Project description:Inhibitors of checkpoint kinase 1 (CHK1),a central component of DNA damage and cell cycle checkpoint response, represent a promising new cancer therapy, but the global cellular functionsthey regulate through phosphorylationare poorly understood. To elucidate the CHK1-regulated phosphorylation network, we performed a global quantitative phosphoproteomics analysis, which revealed 142 phosphositeswhose phosphorylation levels were significantly different following treatment with the CHK1 inhibitor SCH 900776.
2020-04-23 | PXD015125 | Pride
Project description:While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.
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Project description:Post-translational modifications (PTM) of chromatin control the genomic environment for transcription, DNA replication and repair in response to cell stimuli1–3. Replication stress in budding and fission yeasts leads to abundant acetylation of histone H3 on lysine-56 (H3K56ac)4–6, but only trace levels of H3K56ac are detected in human cells7,8, implying that other histone modifications promote a repair-permissive environment. In budding yeast, genetic interactions between histone H3 lysine-56 and serine-57 substitutions suggest a possible role for serine-57 (H3S57) in responses to replication poisons9. In this study, we identify a phosphorylated form of H3S57 (H3S57ph) using phosphoproteomics in replicating Xenopus egg extracts, and show that it is a highly conserved histone modification which promotes responses to DNA replication stress in human cells. A kinome screen and functional experiments identified Checkpoint kinase 1 (CHK1) as the H3S57ph kinase; CHK1 inhibition eliminates H3S57ph and arrests cells in S-phase. Induction of replication stress increases H3S57ph, while disrupting H3S57ph reduces stalling of replication forks upon replication stress, inducing DNA damage. We identified two distinct mechanisms of action. First, H3S57ph interacts with specific DNA repair proteins, notably Rad50. Second, atomistic molecular dynamics simulations of the nucleosome core particle and in vitro assays indicate that H3S57ph interacts with the unacetylated side-chain of K56, thus loosening DNA-histone contacts. Our results suggest that H3S57ph is an effector of CHK1 that assists in processing stalled replication forks by increasing nucleosome mobility and promoting interactions with repair machinery, thereby limiting DNA damage upon replication stress.
2022-02-10 | PXD011328 | Pride
Project description:BACKGROUND: The deSUMOylase SENP2 exerts athero-protective effects by inhibiting endothelial cell (EC) activation through attenuating ERK5 and p53 SUMOylation. Publicly available datasets show that SENP2 S344 is phosphorylated by Checkpoint Kinase 1 (CHK1), but the functional role remains unknown. METHODS: Mouse SENP2 S343A (human S344A) phosphodeficient knock in (KI) mutant was generated by CRISPR/Cas9, and vascular-specific function was assessed via bone marrow transplantation (BMT). ECs from KI and wild type (WT) mice were exposed to smooth (laminar flow; l-flow) or grooved (disturbed flow; d-flow) cone-and-plate devices and characterized by RNA sequencing (RNA-seq). RESULTS: L-flow increased CHK1 S280 and SENP2 S344 phosphorylation, which inhibited ERK5 and p53 SUMOylation and atherogenesis in vivo. BMT-generated vascular specific SENP2 S344A KI showed more atherogenesis but thinner fibrous cap formation specifically in the aortic arch area (d-flow) compared to that of WT mice. Ionizing radiation (IR) decreased CHK1 expression and SENP2 S344 phosphorylation, which might account for differences between systemic and BMT-generated vascular specific SENP2 S344A KI models. RNA-seq data analysis showed that SENP2 S344 phosphorylation in ECs in response to l-flow inhibited EC activation and fibrotic changes without interfering EC lineage phenotype. Lastly, l-flow-induced expression of genes was regulated by SENP2 S344 phosphorylation through ERK5 activation and inhibited EC apoptosis. CONCLUSIONS: We uncovered a novel mechanism by which l-flow inhibits EC activation, including proliferation, migration, inflammation, and fibrotic changes, via upregulating CHK1-mediated SENP2 S344 phosphorylation to attenuate atherogenesis. We also uncovered a unique expression pattern of fibrotic changes without affecting EC lineage, which is distinct from endothelial-to-mesenchymal transition and therefore should be considered a unique type of EC activation for its potential role in vulnerable plaque formation.
2023-09-20 | GSE222511 | GEO
Project description:Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i’s) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.
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Project description:Loss-of-function mutations of EZH2, the enzymatic component of PRC2, are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) and have been associated with chemotherapy resistance and poor outcome. Using isogenic T-ALL cells, with and without CRISPR-induced EZH2-inactivating mutations, we performed a cell-based synthetic lethal drug screen. EZH2 deficient cells exhibited increased sensitivity to structurally diverse CHK1 inhibitors, an interaction that could be validated genetically. Notably, EZH2 deficient cells acquired a gene expression signature of immature T-ALL cells, with significant enrichment of MYC target genes. Mechanistically, EZH2 deficiency was associated with marked transcriptional upregulation of MYCN, increased replication stress, and enhanced dependency on CHK1 for cell survival. Furthermore, small molecule inhibition of CHK1 had efficacy in delaying tumor progression in isogenic EZH2 deficient, but not EZH2 wild-type T-ALL cells in vivo, suggesting there is an exploitable therapeutic window for CHK1 inhibitors in high risk EZH2 mutated T-ALL.
2020-08-28 | GSE127261 | GEO