Structural instability in an autophosphorylating kinase switch

We analyse a simple kinase model that exhibits bistability when there is no protein turnover, and show analytically that the property of being bistable is not necessarily conserved when degradation and synthesis of the kinase are taken into account

Functional analyses of ATM, ATR and Fanconi anemia proteins in lung carcinoma

Background: ATM and ATR are kinases implicated in a myriad of DNA-damage responses. ATM kinase inhibition radiosensitizes cells and selectively kills cells with Fanconi anemia (FA) gene mutations. ATR kinase inhibition sensitizes cells to agents that induce replication stress and selectively kills cells with ATM and TP53 mutations. ATM mutations and FANCF promoter-methylation are reported in lung carcinomas. Methods: We undertook functional analyses of ATM, ATR, Chk1 and FA proteins in lung cancer cell lines. We included Calu6 that is reported to be FANCL-deficient. In addition, the cancer genome atlas (TCGA) database was interrogated for alterations in: 1) ATM, MRE11A, RAD50 and NBN; 2) ATR, ATRIP and TOPBP1; and 3) 15 FA genes. Results: No defects in ATM, ATR or Chk1 kinase activation, or FANCD2 monoubiquitination were identified in the lung cancer cell lines examined, including Calu6, and major alterations in these pathways were not identified in the TCGA database. Cell lines were radiosensitized by ATM kinase inhibitor KU60019, but no cell killing by ATM kinase inhibitor alone was observed. While no synergy between gemcitabine or carboplatin and ATR kinase inhibitor ETP-46464 was observed, synergy between gemcitabine and Chk1 kinase inhibitor UCN-01 was observed in 54T, 201T and H460, and synergy between carboplatin and Chk1 kinase inhibitor was identified in 201T and 239T. No interactions between ATM, ATR and FA activation were observed by either ATM or ATR kinase inhibition in the lung cancer cell lines. Conclusions: Analyses of ATM serine 1981 and Chk1 serine 345 phosphorylation, and FANCD2 monoubiquitination revealed that ATM and ATR kinase activation and FA pathway signaling are intact in the lung cancer cell lines examined. As such, these posttranslational modifications may have utility as biomarkers for the integrity of DNA damage signaling pathways in lung cancer. Different sensitization profiles between gemcitabine and carboplatin and ATR kinase inhibitor ETP-46464 and Chk1 kinase inhibitor UCN-01 were observed and this should be considered in the rationale for Phase I clinical trial design with ATR kinase inhibitors

Tumor suppressor p53 binds with high affinity to CTG-CAG trinucleotide repeats and induces topological alterations in mismatched duplexes

DNA binding is central to the ability of p53 to function as a tumor suppressor. In line with the remarkable functional versatility of p53, which can act on DNA as a transcription, repair, recombination, replication, and chromatin accessibility factor, the modes of p53 interaction with DNA are also versatile. One feature common to all modes of p53-DNA interaction is the extraordinary sensitivity of p53 to the topology of its target DNA. Whereas the strong impact of DNA topology has been demonstrated for p53 binding to sequence-specific sites or to DNA lesions, the possibility that DNA structure-dependent recognition may underlie p53 interaction with other types of DNA has not been addressed until now. We demonstrate for the first time that conformationally flexible CTG·CAG trinucleotide repeats comprise a novel class of p53-binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in vivo. Our major finding is that p53 binds to CTG·CAG tracts by different modes depending on the conformation of DNA. Although p53 binds preferentially to hairpins formed by either CTG or CAG strands, it can also bind to linear forms of CTG·CAG tracts such as canonic B DNA or mismatched duplex. Intriguingly, by binding to a mismatched duplex p53 can induce further topological alterations in DNA, indicating that p53 may act as a DNA topology-modulating factor

Multiple Roles of BRIT1/MCPH1 in DNA Damage Response, DNA Repair, and Cancer Suppression

Mammalian cells are frequently at risk of DNA damage from both endogenous and exogenous sources. Accordingly, cells have evolved the DNA damage response (DDR) pathways to monitor and assure the integrity of their genome. In cells, the intact and effective DDR is essential for the maintenance of genomic stability and it acts as a critical barrier to suppress the development of cancer in humans. Two central kinases for the DDR pathway are ATM and ATR, which can phosphorylate and activate many downstream proteins for cell cycle arrest, DNA repair, or apoptosis if the damages are irreparable. In the last several years, we and others have made significant progress to this field by identifying BRIT1 (also known as MCPH1) as a novel key regulator in the DDR pathway. BRIT1 protein contains 3 breast cancer carboxyl terminal (BRCT) domains which are conserved in BRCA1, MDC1, 53BP1, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. Our in vitro studies revealed BRIT1 to be a chromatin-binding protein required for recruitment of many important DDR proteins (ATM, MDC1, NBS1, RAD51, BRCA2) to the DNA damage sites. We recently also generated the BRIT1 knockout mice and demonstrated its essential roles in homologous recombination DNA repair and in maintaining genomic stability in vivo. In humans, BRIT1 is located on chromosome 8p23.1, where loss of hetero-zigosity is very common in many types of cancer. In this review, we will summarize the novel roles of BRIT1 in DDR, describe the relationship of BRIT1 deficiency with cancer development, and also discuss the use of synthetic lethality approach to target cancers with HR defects due to BRIT1 deficiency

The Mre11-Rad50-Nbs1 complex mediates activation of TopBP1 by ATM

The activation of ATR-ATRIP in response to double-stranded DNA breaks (DSBs) depends upon ATM in human cells and Xenopus egg extracts. One important aspect of this dependency involves regulation of TopBP1 by ATM. In Xenopus egg extracts, ATM associates with TopBP1 and thereupon phosphorylates it on S1131. This phosphorylation enhances the capacity of TopBP1 to activate the ATR-ATRIP complex. We show that TopBP1 also interacts with the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. This interaction involves the Nbs1 subunit of the complex. ATM can no longer interact with TopBP1 in Nbs1-depleted egg extracts, which suggests that the MRN complex helps to bridge ATM and TopBP1 together. The association between TopBP1 and Nbs1 involves the first pair of BRCT repeats in TopBP1. In addition, the two tandem BRCT repeats of Nbs1 are required for this binding. Functional studies with mutated forms of TopBP1 and Nbs1 suggested that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. These findings suggest that the MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs

Cyclin E overexpression as a biomarker for combination treatment strategies in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a virulent form of breast cancer, and novel treatment strategies are urgently needed. Immunohistochemical analysis of tumors from women with a clinical diagnosis of IBC (n = 147) and those with non-IBC breast cancer (n = 2510) revealed that, whereas in non-IBC cases cytoplasmic cyclin E was highly correlated with poor prognosis (P < 0.001), in IBC cases both nuclear and cytoplasmic cyclin E were indicative of poor prognosis. These results underscored the utility of the cyclin E/CDK2 complex as a novel target for treatment. Because IBC cell lines were highly sensitive to the CDK2 inhibitors dinaciclib and meriolin 5, we developed a high-throughput survival assay (HTSA) to design novel sequential combination strategies based on the presence of cyclin E and CDK2. Using a 14-cell-line panel, we found that dinaciclib potentiated the activity of DNA-damaging chemotherapies treated in a sequence of dinaciclib followed by chemotherapy, whereas this was not true for paclitaxel. We also identified a signature of DNA repair–related genes that are downregulated by dinaciclib, suggesting that global DNA repair is inhibited and that prolonged DNA damage leads to apoptosis. Taken together, our findings argue that CDK2-targeted combinations may be viable strategies in IBC worthy of future clinical investigation

Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was dose-dependently decreased by SchB in vitro (IC50: 7.25 μM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer therapy

The Telomeric Protein TRF2 Binds the ATM Kinase and Can Inhibit the ATM-Dependent DNA Damage Response

The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Here we show that overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogated the cell cycle arrest after ionizing radiation and diminished several other readouts of the DNA damage response, including phosphorylation of Nbs1, induction of p53, and upregulation of p53 targets. TRF2 inhibited autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 was found to directly interact with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. We propose that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage