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The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE263176 | GEO | 2024/04/04

REPOSITORIES: GEO

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CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress

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

The thioredoxin-1 system is essential for fueling DNA synthesis during T cell metabolic reprogramming and proliferation

Project description:The thioredoxin-1 (Trx1) system is a key player of the cellular redox balance and a sensor of energy and glucose metabolism. Here we report critical c-Myc-dependent activation of the Trx1 system during glucose-driven T cell expansion during development and immune responses but endogenous Trx1-repression during quiescence. Thioredoxin-reductase-1 (Txnrd1)-deletion in CD4-CD8- thymocytes prevented their expansion, while deletion in CD4+CD8+ thymocytes did not affect further maturation and peripheral homeostasis of T-cells. Moreover, B cell development was Txnrd1-independent. Txnrd1 was critical for expansion of activated T cells. Unbiased metabolomics revealed that Txnrd1 is necessary for donating reducing equivalent to ribonucleotide reductase (RNR) at the last step of nucleotide biosynthesis. Impaired availability of 2’-deoxyribonucleotides induced the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal role of the Trx1 system in metabolic reprograming of thymic and peripheral T cells and provide a rationale for targeting of Txnrd1 in T-cell leukemia.

2018-03-15 | GSE107090 | GEO

EZH2 deficient T-cell acute lymphoblastic leukemia is sensitized to CHK1 inhibition through enhanced replication stress

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

Phosphoproteomics analysis of CHK1 inhibitor treatment

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

EZH2 deficient T-cell acute lymphoblastic leukemia is sensitized to CHK1 inhibition through enhanced replication stress

Project description:Loss-of-function mutations of EZH2, the enzymatic component of PRC2, have been associated with poor outcome and chemotherapy resistance in T-cell acute lymphoblastic leukemia (T-ALL). Using isogenic T-ALL cells, with and without CRISPR/Cas9-induced EZH2-inactivating mutations, we performed a cell-based synthetic lethal drug screen. EZH2 deficient cells exhibited increased sensitivity to structurally diverse inhibitors of CHK1, an interaction that could be validated genetically. 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, as well as in a primary cell model of PRC2 mutant ALL. Mechanistically, EZH2 deficiency resulted in a gene expression signature of immature T-ALL cells, marked transcriptional upregulation of MYCN, increased replication stress, and enhanced dependency on CHK1 for cell survival. Lastly, we demonstrate this phenotype is mediated through de-repression of a distal PRC2-regulated MYCN enhancer. In conclusion, we highlight a novel and clinically exploitable pathway in high-risk EZH2 mutated T-ALL.

2020-05-31 | GSE147198 | GEO

Accumulation of auranofin in white adipose tissues lowers leptin levels and exerts anti-diabetic effects

Project description:Low-grade, sustained inflammation in white adipose tissue (WAT) characterizes obesity and frequently coincides with insulin resistance and type 2 diabetes (T2D). However, pharmacological targeting of WAT inflammation lacks durable therapeutic effects. Through a computational screen, we identified the FDA-approved rheumatoid arthritis drug auranofin is a putative small molecule for obesity treatment. We discovered that allometrically scaled safe auranofin doses homed to WAT and improved insulin sensitivity in obese wild-type mice. To assess the transcriptional changes underlying the beneficial effects of auranofin, we performed RNA-seq analysis on adipose and liver tissue after 4 weeks of treatment with vehicle or auranofin.

2022-10-06 | GSE202935 | GEO

Expression data of CHK1 shRNA knockdown and control shRNA in U2OS cells

Project description:Homologous recombination-mediated DNA repair deficiency (HRD) predisposes to cancer development, but also provides therapeutic opportunities. Here, we identified an HRD gene signature that robustly predicted HRD status. Unexpectedly, concurrent loss of PTEN in BRCA1-deficient cells might extensively rewire the HR repair network and confer resistance to PARP inhibitor, partially through over-expression of TTK. We used the HRD gene signature as a drug discovery tool and found several PARP-inhibitor-synergizing agents through the connectivity map. Thus gene expression profiling can be used to define the functional status of the HR repair network providing prognostic and therapeutic information. Various shRNAs that target genes involved in homologous recombination (HR) were transfected in MCF-10A non-transformed breast cells lines. Stable HR gene knockdown MCF-10A cells were seeded 200000 at 10 cm plate. Cells were harvested after 48 hours culturing and used for gene expression profiling. The shRNA that target CHK1 gene was transfected in human osteosarcoma U2OS cell line by lentiviral particles and selected stable CHK1 knockdown U2OS cells. Scrambled control shRNA-transfected U2OS cells were applying as control. Both stable CHK1 knockdown and control U2OS cells were seeded with 2 x 10^5 cells at 10 cm culture plate. Cells were cultured in McCOY 5A medium with 10% FBS and harvested after 48 hours culturing. mRNA was extracted from collected cells and performing gene expression profiling. Three biological replicates were applied.

2014-01-24 | E-GEOD-54267 | biostudies-arrayexpress

Calreticulin mutated hematopoietic cells are vulnerable to ATR-CHK1 pathway inhibition

Project description:Disease frameshift mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF). To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we aimed to search for small molecule drugs that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89,172 compounds using isogenic cell lines and identified several hits targeting the ATR-CHK1 pathway. The selective inhibitory effect of these candidate compounds was validated in a co-culture assay of CALR mutated and wild type cells. Of the tested hit compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing CALR wild type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to the drug treatment, suggesting that the vulnerability to ATR-CHK1 inhibition is specifically caused by the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. They also promoted S-phase cell cycle arrest and blocked the completion of DNA replication in CALR mutated cells. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by the oncogenic CALR mutation, suggesting an intrinsic vulnerability of these cells to CHK1 perturbation. This study indicates that ATR-CHK1 pathway is a potential therapeutic target in CALR mutated hematopoietic cells.

2021-09-07 | PXD026803 | Pride

Oxidized thioredoxin-1 restrains the NLRP1 inflammasome [mBMDM]

Project description:The danger signals that activate the NLRP1 inflammasome have yet to be firmly established. NLRP1 undergoes autoproteolysis to generate N-terminal (NT) and C-terminal (CT) fragment, which importantly, is a necessary step for its check-point regulation by the DPP9 ternary complex and the mechanistic activation of NLRP1 through functional degradation. Here, we report an added layer of regulatory complexity to NLRP1 activity, in the form of a repressive interaction that NLRP1 forms with the oxidized, but not reduced, form of thioredoxin-1 (TRX1). Loss of TRX1 destabilizes the NT fragment of NLRP1 and promotes enhanced inflammasome activation. The TRX1 interaction occurs through the NACHT-LRR of NLRP1 and requires nucleotide binding in its ATPase domain. In addition, we found that several patient-derived and ATPase-inactivating mutations in the NACHT-LRR region hyperactive the inflammasome by destabilize protein folding and are also shown to abrogate TRX1 binding. Thus, NLRP1 appears to detect intracellular reductive stress through a decrease in the fraction of intracellular oxidized TRX1, which enhances protein disorder, leading to inflammasome signaling. These findings link the cellular redox environment to NLRP1-mediated innate immunity.

2022-11-04 | GSE215035 | GEO

Oxidized thioredoxin-1 restrains the NLRP1 inflammasome [NTERT-1]

2022-11-04 | GSE215034 | GEO

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