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CDK2-mediated TNFa Upregulation as a Mechanism of Cytotoxicity in Acute Leukemia

ABSTRACT: Although inhibitors of the kinases CHK1, ATR and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines, including TP53 null acute myeloid leukemia (AML) and BCR/ABL-positive acute lymphoid leukemia (ALL), in vitro and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, CHK1 downregulation or CHK1 inhibitor treatment triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNF production and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically CHK1 inhibitor resistant exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2  AP1  TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to antileukemic cell action of CHK1, ATR and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand-induced killing that can potentially be exploited for acute leukemia treatment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE131912 | GEO | 2020/12/28

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

Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition

Project description:Excessive genomic instability coupled with abnormalities in DNA repair pathways induce high levels of “replication stress” when cancer cells propagate. Rather than hampering cancer cell proliferation, novel treatment strategies are turning their attention toward targeting cell cycle checkpoint kinases (such as ATR, CHK1, WEE1 and others) along the DNA damage response and replicative stress response pathways, thereby allowing unrepaired DNA damage to be carried forward towards mitotic catastrophe and apoptosis. The selective inhibitor of ATR kinase elimusertib (BAY 1895344), has demonstrated preclinical and clinical monotherapy activity; however, reliable predictive biomarkers of treatment benefit are still lacking. In this study, using gene expression profiling of 24 cell lines from different cancer types and in a panel of ovarian cancer cell lines, we found that nuclear-specific enrichment of checkpoint kinase 1 (CHK1) correlated with increased sensitivity to elimusertib. Using an advanced multispectral imaging system in subsequent cell line-derived xenograft specimens, we showed a trend between nuclear phosphorylated-CHK1 (pCHK1) staining and increased sensitivity to the ATR inhibitor elimusertib, indicating the potential value of pCHK1 expression as a predictive biomarker of ATR inhibitor sensitivity.

2022-11-09 | GSE217579 | GEO

TRRAP-mediated regulation of Wee1

Project description:The G2 DNA damage checkpoint inhibits Cdc2 and mitotic entry through the dual regulation of Wee1 and Cdc25 by the Chk1 effector kinase. Up-regulation of Chk1 by mutation or overexpression bypasses the requirement for up-stream regulators or DNA damage to promote a G2 cell cycle arrest. We screened in fission yeast for mutations that rendered cells resistant to overexpressed Chk1. We identified a mutation in tra1, which encodes one of two homologs of TRRAP, an ATM/R-related pseudokinase that scaffolds several histone acetyltransferase (HAT) complexes. Inhibition of histone deacetylases reverts the resistance to overexpressed Chk1, suggesting this phenotype is due to a HAT activity, though expression of checkpoint and cell cycle genes is not greatly affected. Cells with mutant or deleted tra1 activate Chk1 normally and are checkpoint proficient. However, these cells are semi-wee even when overexpressing Chk1, and accumulate inactive Wee1 protein. The Cdr (changed division response) kinases Cdr1 and Cdr2 are negative regulators of Wee1, and while best characterized in the cellular response to limited nutrition, we show that they are required for the Tra1-dependent alterations to Wee1 function. This identifies Tra1 as another component controlling the timing of entry into mitosis via Cdc2 activation. Two and three independent microaaray experiments were done for wild type and the mutant (tra1-1) respectively.

2010-04-11 | E-GEOD-12674 | biostudies-arrayexpress

A network-based approach reveals a novel mechanism of resistance to TKI-therapy in AML

Project description:Internal tandem duplications (ITDs) in the FLT3 gene are frequently identified and confer a poor prognosis in patient affected by acute myeloid leukemia (AML). The insertion site of the ITDs in FLT3 significantly impacts the sensitivity to tyrosine kinase inhibitors (TKIs) therapy, affecting patient’s clinical outcome. To decipher the molecular mechanisms driving the different sensitivity to TKIs therapy of FLT3-ITD cells, we used high-sensitive mass spectrometry-based (phospho)proteomics and deep sequencing. Here, we developed a novel generally-applicable data analysis strategy, dubbed “Signaling Profiler”, that supports the integration of unbiased large-scale datasets with literature-derived signaling networks. The approach resulted in the generation of FLT3-ITDs specific predictive models and reveales a crucial and conserved role of the WEE1-CDK1 axis in TKIs resistance. Remarkably, pharmacological inhibition of the WEE1 kinase significantly synergizes and strengths the pro-apoptotic effect of TKIs therapy in cell lines and patient-derived primary blasts. In conclusion, this work proposes a new molecular mechanism of TKIs resistance in AML and suggests a combination therapy as option to improve therapeutic efficacy.

2023-03-10 | PXD033953 | Pride

Targeted sequencing DDR genes in cancer stem cells

Project description:Colorectal cancer stem cells (CSCs) show heterogeneous levels of constitutive replication stress leading distinct dependence on the replication stress response. The aim of the projects was to find genetic, epigenetic, phenotypic factors associated with the resistance of colorectal CSCs to inhibitors of the ATR/CHK1 pathway, which regulates of the replication stress response. Different pairs of colorectal CSCs sensitive and resistant to ATR or CHK1 inhibitors were analyzed

| EGAS00001004892 | EGA

Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment II

Project description:Decitabine (DAC) is used clinically for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells shows that mitotic regulation plays a pivotal role in DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations and antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, the overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation, while highlighting the potent activity of DAC to perturb mitosis through aberrant DNMT1-DNA covalent bonds.

2023-08-14 | GSE240570 | GEO

BRCA1 Represses DNA Replication Fork Firing and Prevents Mitotic Catastrophe through Antagonizing Estrogen Signaling during Pregnancy

Project description:The mammary gland at early stages of pregnancy undergoes fast cell proliferation, yet the mechanism to ensure its genome integrity is largely unknown. Here we show that pregnancy enhances expression of genes involved in numerous pathways, including most genes encoding replisomes. In mouse mammary glands, replisome genes are positively regulated by estrogen/ERa signaling but negatively regulated by BRCA1. Upon DNA damage, BRCA1 deficiency markedly enhances DNA replication initiation. BRCA1 deficiency also preferably impairs DNA replication checkpoints mediated by ATR and CHK1 but not by WEE1, which inhibits DNA replication initiation through CDC7-MCM2 pathway and enables BRCA1-deficient cells to avoid further genomic instability. Thus, BRCA1 and WEE1 inhibit DNA replication initiation in a parallel manner to ensure genome stability for mammary gland development during pregnancy.

2018-12-11 | GSE92342 | GEO

TRRAP-mediated regulation of Wee1

2010-04-11 | GSE12674 | GEO

Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment

Project description:Decitabine (DAC) is used clinically for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To elucidate its exact mechanism of action, we performed a genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells and revealed that mitotic regulation plays a pivotal role in DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations and antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, the overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to perturb mitosis through aberrant DNMT1-DNA covalent bonds. This clinically revised mode of action is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway.

2023-08-14 | GSE240439 | GEO

SETD2 loss and ATR inhibition synergistically promote cGAS signaling and immunotherapy response in renal cell carcinoma

Project description:Immune checkpoint blockade (ICB) demonstrates durable clinical benefit only in a minority of renal cell carcinoma (RCC) patients. Identifying molecular features that determine response and developing approaches to enhance the response remain an urgent clinical need. Here we found that, in multiple RCC cell lines, targeting the ATR-CHK1 axis with pharmacological inhibitors increased cytosolic DNA accumulation, activated the cGAS-IRF3-dependent cytosolic DNA sensing pathway, and resulted in the inflammatory cytokine expression. SETD2 mutated RCC cell lines or tumor samples were associated with preferential ATR-CHK1 activation over ATM-CHK2 activation. SETD2 knockdown promoted the cytosolic DNA sensing pathway and conferred greater sensitivity in response to ATR-CHK1 inhibition. In murine Renca tumors, Setd2 knockdown and ATR inhibitor VE822 synergistically promoted cytosolic DNA sensing pathway, immune cell infiltration, and immune checkpoint protein expression. Setd2 deficient Renca tumors demonstrated greater vulnerability to ICB monotherapy or in combination with VE822 than Setd2 proficient tumors. SETD2 mutations were associated with a higher response rate and prolonged overall survival in ICB-treated RCC patients, but not in non-ICB-treated RCC patients. This study provides a mechanism-based guidance to develop more personalized combination therapy regimens for RCC patients with SETD2 mutations.

2023-07-25 | GSE234732 | GEO

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