Abstract To cope with constant challenges toward the fidelity of genome by DNA damaging insults (both endogenous and exogenous), activation of the DNA damage response (DDR) is an essential step in preventing genomic instability. Studies of mammalian mechanisms have revealed that there are comprehensive, coordinated pathways that, when activated, serve to regulate the DDR. Recently, we have shown that hyperactive DDR mediated by the Ataxia-Telangiectasia Mutated (ATM) kinase is linked to activation of the Zinc finger protein SNAI1 (Snail). Activated Snail in metastasis is well-characterized by its role as transcriptional repressor of E-cadherin thereby inducing an epithelial-to-mesenchymal transition (EMT). Here we report that, in response to DNA damage, activation of the ATM-Snail pathway, mediated by Snail Serine 100 phosphorylation, also contributes to the regulation of the cellular sensitivity to ionizing radiation. Expression of a hyperactive Snail led to radioresistance. More interestingly, the Serine 100 to Glutamic Acid mutant variant of Snail displayed an enhanced invasion index after low dose radiation, independent of survival. Functional genomics and proteomics studies revealed downstream targets and interacting partners of Snail in the DDR. Together, we highlight the functional significance of the ATM-Snail pathway in the DDR. Citation Format: Rebecca J. Boohaker, Joshua Fried, Xiaoli Cui, Bo Xu. Delineation of the ATM-Snail pathway in the DNA damage response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3939. doi:10.1158/1538-7445.AM2014-3939

The spindle assembly checkpoint (SAC), which blocks anaphase onset until all chromosomes have bi-oriented, is one of the key self-monitoring systems of the eukaryotic cell cycle for genome stability. The mitotic arrest-deficient protein 1 (Mad1), a critical component of the SAC, is hyperphosphorylated in mitosis. However, the kinases responsible for Mad1 phosphorylation and its functional significance are not fully understood. Here we report that Mad1 is phosphorylated on Serine 214 by the Ataxia-Telangiectasia Mutated (ATM) kinase, a critical DNA damage response protein also activated in mitosis and required for the SAC. We demonstrate that Mad1 Serine 214 phosphorylation promotes the formation of homodimerization of Mad1 and its heterodimerization with Mad2. Further we show that Mad1 Serine 214 phosphorylation contribute to activation of the SAC and the maintenance of chromosomal stability. Together, these findings reveal an important role of ATM-mediated Mad1 Serine 214 phosphorylation in mitosis.

PURPOSE:Activation of the DNA damage responsive protein kinase ATM is a critical step for cellular survival in response to ionizing irradiation (IR). Direct targets of ATM regulating radiosensitivity remain to be fully investigated. We have recently reported that ATM phosphorylates the transcriptional repressor Snail on Serine 100. We aimed to further study the functional significance of ATM-mediated Snail phosphorylation in response to IR.MATERIAL AND METHODS:We transfected vector-only, wild-type, the Serine 100 to alanine (S100A) or to glutamic acid (S100E) substitution of Snail into various cell lines. We assessed colony formation, γ-H2AX focus formation and the invasion index in the cells treated with or without IR.RESULTS:We found that over-expression of the S100A mutant Snail in HeLa cells significantly increased radiosensitivity. Meanwhile the expression of S100E, a phospho-mimicking mutation, resulted in enhanced radio-resistance. Interestingly, S100E could rescue the radiosensitive phenotype in ATM-deficient cells. We also found that expression of S100E increased γ-H2AX focus formation and compromised inhibition of invasion in response to IR independent of cell survival.CONCLUSION:ATM-mediated Snail Serine 100 phosphorylation in response to IR plays an important part in the regulation of radiosensitivity.

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia-telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO Developing novel sensitizers for cancer radiotherapy has been accelerated by recent advances of our understanding on how human cells respond to DNA damaging agents. One central element in determining the cellular response to irradiation (IR) is the ATM kinase, mutation of which causes the human autosomal recessive disorder ataxia-telangiectasia (A-T). A-T is characterized by progressive neuro-degeneration, a variable immunodeficiency, an extremely high predisposition to the development of lymphoid malignancies and hypersensitivity to radiation. Because ATM is central to cellular responses to IR, blocking its activity could make tumors much more sensitive to radiation. Although specific ATM inhibitors have shown promising preliminary results, several obstacles of applying these methods in the clinical setting exist. For example, due to the pleiotropic effects of the mutation, such as neuro-degeneration, immunodeficiency and cancer predisposition, the outcome of directly inhibiting ATM kinase activity can be complicated, as it is unclear whether the only effect of these reagents will be to confer radiosensitization. Instead of directly inhibiting ATM kinase activity to increase radiosensitivity, an alternative approach is to target IR-induced ATM activation, as this may directly lead to an increase in radiosensitivity without interfering with other important functions of ATM. Activation of ATM in response to IR requires interaction with the extreme C-terminus of NBS1. We have recently made proof-of-principle discoveries showing that targeting NBS1-ATM interactions confers radiosensitization. In order to identify small molecules that can specifically inhibit NBS1-ATM interactions, we have developed a fluorescence polarization (FP) assay feasible for high throughput screening (HTS). The in vitro binding assay is carried out using Texas Red-labeled NBS1 small peptides with GST-conjugated ATM Heat-Repeat domains. Several Texas Red-labeled peptides containing the wild-type or mutant NBS1 C-terminal sequences were synthesized. For the GST ATM production, we subcloned ATM Heat-repeat domain cDNA into a pGEX-2T bacterial expression vector and purified GST-ATM protein. The dynamic range of the assay is defined as the polarization difference between the bound peptide and the free peptide. The stability of the FP assay to variables such as dose responses, incubation time, total fluorescence, and dimethyl sulfoxide (DMSO) tolerance was assessed. The assay tolerates 5% of DMSO without significant loss of signals, which allows for testing of wide ranges of compound concentrations and enables screening poorly soluble compounds. Under optimized conditions, a Z\#8242; factor of 0.71 was achieved in a 384-well format for high throughput screening, indicating a feasible and robust assay for HTS. This assay has been enrolled in the NIH Roadmap Initiative Molecular Libraries Program for large scale HTS. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2303.

4205 The Ataxia-Telangiectasia Mutated kinase, ATM, plays a crucial role in the cellular response to DNA damage. Loss of ATM functions in humans results in Ataxia-Telangiectasia (A-T), an autosomal recessive disease manifested by progressive neuro-degeneration, immunodeficiency, cancer predisposition and hyper-radiosensitivity. Upon DNA damage, ATM binds strongly to damaged sites and activated ATM in turn phosphorylates a list of substrates in pathways that together ensure cellular survival and recovery. We previously reported that ATM was required for down-regulation of Histone H3 phosphorylation and the G2/M checkpoint in response to ionizing radiation (IR). However, the detailed mechanism of the processes remains unknown. It was known that Protein Phosphatase 1 (PP1), the major phosphatase responsible for Histone H3 dephosphorylation, was activated in an ATM-dependent manner after IR. In this study, we report that ATM is required for the IR-induced rapid dissociation of PP1 from its regulatory subunit Inhibitor-2 (I-2). We also find that ATM phosphorylates I-2 at Serine 43 in response to IR. ATM phosphorylation of I-2 leads to dissociation of the PP1/I-2 complex and activation of PP1. Furthermore, ATM-mediated I-2 phosphorylation results in activation of the G2/M checkpoint. These studies have demonstrated a novel pathway that links ATM, PP1 and I-2 in cellular responses to DNA damage.

Protein phosphatase 1 (PP1), a major protein phosphatase important for a variety of cellular responses, is activated in response to ionizing irradiation (IR)-induced DNA damage. Here, we report that IR induces the rapid dissociation of PP1 from its regulatory subunit inhibitor-2 (I-2) and that the process requires ataxia-telangiectasia mutated (ATM), a protein kinase central to DNA damage responses. In response to IR, ATM phosphorylates I-2 on serine 43, leading to the dissociation of the PP1-I-2 complex and the activation of PP1. Furthermore, ATM-mediated I-2 phosphorylation results in the inhibition of the Aurora-B kinase, the down-regulation of histone H3 serine 10 phosphorylation, and the activation of the G(2)/M checkpoint. Collectively, the results of these studies demonstrate a novel pathway that links ATM, PP1, and I-2 in the cellular response to DNA damage.

ATM and NBS1, mutation of which lead to the human autosomal recessive diseases ataxia telangiectasia and Nijmegen breakage syndrome (NBS), respectively, are essential elements in the cellular response to DNA damage induced by ionizing radiation (IR). ATM is a member of the phosphatidylinositol 3-kinase family and is activated by IR in an NBS1-dependent manner. The extreme C terminus of NBS1 contains an evolutionarily conserved sequence motif that is critical for binding to and activation of ATM after IR. ATM phosphorylates a series of targets to initiate cell cycle arrest and promote cell survival in response to DNA damage. Therefore, targeting the NBS1-ATM interaction may lead to a novel approach for specific ATM inhibition and radiosensitization. We developed small peptides containing the conserved C-terminal sequence of NBS1 to investigate whether these peptides can interfere with the DNA damage pathway. We found that wild-type NBS1 inhibitory peptides (wtNIP) can abrogate NBS1-ATM association in the presence or absence of IR. We also found that cells exposed to wtNIP displayed a significant reduction in radiation-induced gamma-H2AX and NBS1 focus formation compared with cells treated with control peptides, demonstrating that wtNIP possesses a strong inhibitory effect on ATM. The inhibitory effect of wtNIP also leads to a significant decrease in clonogenic survival in response to IR. Furthermore, wtNIP does not radiosensitize cells with defective ATM, suggesting a specific inhibition of ATM. Together, these data provide a proof of principle for the use of NBS1 C-terminal small peptides as specific ATM inhibitors and radiosensitizers.

PURPOSE:Combination treatment with radiotherapy and chemotherapy has emerged as the dominant form of cancer adjuvant regimens in recent years. Clofarabine, a newly approved drug for pediatric leukemia, is a second-generation purine nucleoside analogue that can block DNA synthesis and inhibit DNA repair. Therefore, we hypothesized that clofarabine could work synergistically with radiotherapy to increase the tumor cell response. METHODS AND MATERIALS:The effects of clofarabine on radiosensitivity have been established in several tumor cell lines in vitro and in vivo using colony-forming assays and tumor xenografts. The effect of clofarabine on the DNA damage response was also studied in vitro by measuring gamma-H2AX focus formation. RESULTS:Clonogenic survival was significantly reduced in irradiated cells treated with clofarabine, demonstrating the strong radiosensitizing effect of clofarabine. Furthermore, clofarabine displayed a radiosensitizing effect that was greater than gemcitabine or 5-fluorouracil. We also found that low doses of clofarabine can prolong the presence of radiation-induced gamma-H2AX nuclear focus formation, and high doses of clofarabine can induce DNA double-strand breaks, suggesting that clofarabine can interfere with DNA damage response pathways. In addition, clofarabine-induced radiosensitization was also established in vivo using a colorectal cancer model, DLD-1, in athymic nude mice. When combined with fractionated radiotherapy, a moderate dose of clofarabine led to a significant increase in tumor growth inhibition. CONCLUSION:Clofarabine acts as a powerful radiosensitizer both in vitro and in vivo by interfering with the DNA damage response.