Structure and Activation Mechanism of the CHK2 DNA Damage Checkpoint Kinase

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Molecular cell (Impact Factor: 14.02). 09/2009; 35(6):818-29. DOI: 10.1016/j.molcel.2009.09.007
Source: PubMed

ABSTRACT The CHK2 protein kinase is an important transducer of DNA damage checkpoint signals, and its mutation contributes to hereditary and sporadic cancer. CHK2 activation is triggered by the phosphorylation of Thr68 by the DNA damage-activated ATM kinase. This leads to transient CHK2 dimerization, in part through intermolecular phosphoThr68-FHA domain interactions. Dimerization promotes kinase activation through activation-loop autophosphorylation, but the mechanism of this process has not been clear. The dimeric crystal structure of CHK2, described here, in conjunction with biochemical and mutational data reveals that productive CHK2 dimerization additionally involves intermolecular FHA-kinase domain and FHA-FHA interactions. Ile157, mutated in the Li-Fraumeni cancer-predisposition syndrome, plays a central role in the FHA-kinase domain interface, explaining the lack of dimerization and autophosphorylation of this mutant. In the dimer, the kinase active sites face each other in close proximity, indicating that dimerization may also serve to optimally position the kinase active sites for efficient activation loop transphosphorylation.

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    • "Phosphorylation of Chk1 leads to checkpoint activation and cell cycle arrest at the G1/S, intra S or G2/M phases. Despite their similarity in name, Chk1 and Chk2 differ substantially in the structure of their kinase pocket [18, 19] and in their cellular function with Chk1 suggested to be the major component responsible for responses to DNA damage. Inhibiting Chk1 following genotoxic stress (such as that induced by cytotoxic chemotherapy) results in checkpoint abrogation, inhibition of DNA repair and induction of cell death in cells with a defective p53 response [20, 21]. "
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    ABSTRACT: Background Chk1 inhibitors are currently in clinical trials as putative potentiators of cytotoxic chemotherapy drugs. Chk1 inhibitors may exhibit single agent anti-tumor activity in cancers with underlying DNA repair, DNA damage response or DNA replication defects. Methods Here we describe the cellular effects of the pharmacological inhibition of the checkpoint kinase Chk1 by the novel inhibitor V158411 in triple-negative breast cancer and ovarian cancer. Cytotoxicity, the effect on DNA damage response and cell cycle along with the ability to potentiate gemcitabine and cisplatin cytotoxicity in cultured cells was investigated. Western blotting of proteins involved in DNA repair, checkpoint activation, cell cycle and apoptosis was used to identify potential predictive biomarkers of Chk1 inhibitor sensitivity. Results The Chk1 inhibitors V158411, PF-477736 and AZD7762 potently inhibited the proliferation of triple-negative breast cancer cells as well as ovarian cancer cells, and these cell lines were sensitive compared to ER positive breast and other solid cancer cells lines. Inhibition of Chk1 in these sensitive cell lines induced DNA damage and caspase-3/7 dependent apoptosis. Western blot profiling identified pChk1 (S296) as a predictive biomarker of Chk1 inhibitor sensitivity in ovarian and triple-negative breast cancer and pH2AX (S139) in luminal breast cancer. Conclusions This finding suggests that Chk1 inhibitors either as single agents or in combination chemotherapy represents a viable therapeutic option for the treatment of triple-negative breast cancer. pChk1 (S296) tumor expression levels could serve as a useful biomarker to stratify patients who might benefit from Chk1 inhibitor therapy.
    BMC Cancer 08/2014; 14(1):570. DOI:10.1186/1471-2407-14-570 · 3.36 Impact Factor
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    • "The DDR can be activated by a range of endogenous and external insults including therapies currently used for the treatment of cancer such as ionizing radiation and cytotoxic chemotherapeutic agents such as gemcitabine, irinotecan and cisplatin [2,3]. Despite their similarity in name, Chk1 and Chk2 differ substantially in the structure of their kinase pocket [4,5] and in their cellular function with Chk1 suggested to be the major component responsible for responses to DNA damage [3,6,7]. Inhibiting Chk1 following genotoxic stress (such as that induced by cytotoxic chemotherapy) results in checkpoint abrogation, inhibition of DNA repair and induction of cell death in cells with a defective p53 response [8,9]. "
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    ABSTRACT: Background Chk1 forms a core component of the DNA damage response and small molecule inhibitors are currently being investigated in the clinic as cytotoxic chemotherapy potentiators. Recent evidence suggests that Chk1 inhibitors may demonstrate significant single agent activity in tumors with specific DNA repair defects, a constitutively activated DNA damage response or oncogene induced replicative stress. Methods Growth inhibition induced by the small molecule Chk1 inhibitor V158411 was assessed in a panel of human leukemia and lymphoma cell lines and compared to cancer cell lines derived from solid tumors. The effects on cell cycle and DNA damage response markers were further evaluated. Results Leukemia and lymphoma cell lines were identified as particularly sensitive to the Chk1 inhibitor V158411 (mean GI50 0.17 μM) compared to colon (2.8 μM) or lung (6.9 μM) cancer cell lines. Chk1 inhibition by V158411 in the leukemia and lymphoma cell lines induced DNA fragmentation and cell death that was both caspase dependent and independent, and prevented cells undergoing mitosis. An analysis of in vitro pharmacodynamic markers identified a dose dependent decrease in Chk1 and cyclin B1 protein levels and Cdc2 Thr15 phosphorylation along with a concomitant increase in H2AX phosphorylation at Ser139 following V158411 treatment. Conclusions These data support the further evaluation of Chk1 inhibitors in hematopoietic cancers as single agents as well as in combination with standard of care cytotoxic drugs.
    Molecular Cancer 06/2014; 13(1):147. DOI:10.1186/1476-4598-13-147 · 4.26 Impact Factor
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    • "Initially, Lee et al. [58] found that kinase activity of the p.I157T allele was comparable to the wild type. More recent studies have reported that the allele is at least partially defective in dimerization and autophosphorylation, binding and phosphorylating Cdc25, and binding BRCA1 [59-62]. In populations in which p.I157T and c.1100delC are both present at appreciable frequencies and have been subject to independent risk estimates, p.I157T does appear to confer increased risk of breast cancer, but the OR or penetrance associated with the missense substitution appears to be more modest than that associated with the frame shift c.1100delC [63]. "
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    ABSTRACT: Both protein-truncating variants and some missense substitutions in CHEK2 confer increased risk of breast cancer. However, no large-scale study has used full open reading frame mutation screening to assess the contribution of rare missense substitutions in CHEK2 to breast cancer risk. This absence has been due in part to a lack of validated statistical methods for summarizing risk attributable to large numbers of individually rare missense substitutions. Previously, we adapted an in silico assessment of missense substitutions used for analysis of unclassified missense substitutions in BRCA1 and BRCA2 to the problem of assessing candidate genes using rare missense substitution data observed in case-control mutation-screening studies. The method involves stratifying rare missense substitutions observed in cases and/or controls into a series of grades ordered a priori from least to most likely to be evolutionarily deleterious, followed by a logistic regression test for trends to compare the frequency distributions of the graded missense substitutions in cases versus controls. Here we used this approach to analyze CHEK2 mutation-screening data from a population-based series of 1,303 female breast cancer patients and 1,109 unaffected female controls. We found evidence of risk associated with rare, evolutionarily unlikely CHEK2 missense substitutions. Additional findings were that (1) the risk estimate for the most severe grade of CHEK2 missense substitutions (denoted C65) is approximately equivalent to that of CHEK2 protein-truncating variants; (2) the population attributable fraction and the familial relative risk explained by the pool of rare missense substitutions were similar to those explained by the pool of protein-truncating variants; and (3) post hoc power calculations implied that scaling up case-control mutation screening to examine entire biochemical pathways would require roughly 2,000 cases and controls to achieve acceptable statistical power. This study shows that CHEK2 harbors many rare sequence variants that confer increased risk of breast cancer and that a substantial proportion of these are missense substitutions. The study validates our analytic approach to rare missense substitutions and provides a method to combine data from protein-truncating variants and rare missense substitutions into a one degree of freedom per gene test.
    Breast cancer research: BCR 01/2011; 13(1):R6. DOI:10.1186/bcr2810 · 5.49 Impact Factor
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