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D.G. Bebb



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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.02-003 - Synergetic study of chemotherapeutic drugs according to ATM status in NSCLC cell lines (ID 3441)

      09:30 - 16:30  |  Author(s): D.G. Bebb

      • Abstract

      Background
      The ataxia telangiectasia mutated (ATM) gene produces a protein essential in mechanisms responsible for regulating the controlled growth and proliferation of cells, as well as the DNA damage response. The lack of ATM expression can lead to a predisposition to cancer due to the inability for the protein to actively respond to DNA damage. Previous work in the Bebb lab has shown that the lack of ATM gene expression in non-small cell lung cancer (NSCLC) cells increases sensitivity to ionizing radiation, however little is known about whether ATM status can influence sensitivity to commonly used chemotherapeutic agents. In this study we will explore the chemosensitivity of NSCLC cells in relation to their ATM status, as well as the synergistic effects of combining multiple agents to help develop new treatment strategies for patients with low or absent ATM expression.

      Methods
      Several NSCLC cell lines were screened to identify the ATM and p53 status. Of the cell lines screened, four NSCLC cell lines: NCI-H460, NCI-H226, NCI-H23, and NCI-H1395 were chosen to treat with various chemotherapeutic agents. Optimization of cell proliferation for each cell line was conducted to determine the appropriate concentration of cells that allows for continued proliferation after a 72 hour period. Each of the agents used for cytotoxicity assays target a different mechanism of the cell, including topoisomerase I inhibitors, DNA damaging and alkylating agents, and mitotic and cell cycle inhibitors. The Chou-Talalay method for drug combinations was utilized as the combination index theorem provides a quantitative definition for examining additive, synergistic and antagonistic effects with our primary focus of observing synergy.

      Results
      Several NSCLC cell lines were characterized for their ATM status, and four were selected for cytotoxicity assays: H460 and H226 (ATM normal), and H23 and H1395 (ATM deficient). We observed a noticeable difference in growth rate between cell lines in addition to the differences in ATM and p53 status. Similarly, the cytotoxic response to the chemotherapeutic agents varied greatly, and the relationship of drug sensitivity to ATM status in the cells, as well as the synergistic response to combinational therapy will be presented and discussed.

      Conclusion
      The results of this study have several important implications. Our previous work has shown that resected, early stage NSCLC patients with low ATM expression have worse overall survival, however our demonstration that ATM deficient cell lines are more sensitive to cancer therapy could indicate that these same patients would respond better to certain lines of treatment. Similarly, increased sensitivity to combinational therapy will help mitigate detrimental side effects, leading to better quality of life during treatment periods.

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    P3.02 - Poster Session 3 - Novel Cancer Genes and Pathways (ID 149)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.02-022 - Deep-Sequencing Mutational Analysis of ATM in NSCLC Cell Lines (ID 3446)

      09:30 - 16:30  |  Author(s): D.G. Bebb

      • Abstract

      Background
      Ataxia telangiectasia-mutated (ATM) is a DNA repair protein that is functionally absent in patients with A-T. Individuals with heterozygous or somatic homozygous mutations are predisposed to developing various cancers. Previous attempts to sequence ATM in cancer cell lines or in patient tissue samples have not identified any mutational hot-spots that are linked to the A-T phenotype or cancer predisposition, however much of the sequencing analysis performed to date has used traditional Sanger-based methodology, which is limited in its depth of coverage. Our lab has identified two non-small cell lung cancer (NSCLC) cell lines, NCI-H23 and NCI-H1395, which appear to be deficient for ATM. While H23 cells display sensitivity to ionizing radiation, typical of ATM deficiency, H1395 do not and moreover show downstream activation of several ATM targets. These previous results are important because the hallmark radiation sensitivity, and possible chemosensitivity of ATM-deficient patients may suggest that these patients would be more responsive to low-dose therapies. However, if cells show no detectable ATM but maintain an intact DNA repair mechanism, the usefulness of ATM as a biomarker is questionable. To assess if these observed differences were intrinsic to the ATM gene, we performed next-gen sequencing (NGS) to characterize the mutational spectra of H23 and H1395.

      Methods
      We used the semiconductor-based Ion Torrent PGM to sequence the coding region of ATM from genomic DNA isolated from H23, H1395, and H460 (ATM normal) cells. Variants were identified and mapped to the ATM gene. Additional western blots were performed to confirm mutation analysis.

      Results
      Several missense SNP mutations were identified in both H23 and H1395, however only one was shared by both, c.5948A>G, corresponding to amino acid substitution N1983S, which has previously been identified as a phosphovariant due to its proximity to the activation phosphorylation site, S1981. Interestingly, this SNP was not identified in H1395 in past sequencing attempts, demonstrating the power of NGS. Few of the other SNPs have previously been annotated in the COSMIC database, and it is as yet unknown whether they confer functional disruptions in the protein. Interestingly, the N1983S SNP is located in the same region of ATM used as the epitope for antibodies to both the phosporylated and unphosphorylated forms of ATM, raising the question of whether the antibodies are unable to bind to these variants and thus limiting detection. We performed western blots with antibodies to different epitopes to address this question.

      Conclusion
      We have yet to determine whether the mutations we identified in H23 and H1395 NSCLC cells are the cause of deficiency in these cells. However, use of deep-sequencing methodology has rapidly identified previously unknown mutations in these cell lines, and expansion of these studies to include a cohort of NSCLC patient samples may identify hot-spot mutations that were previously undetected by traditional sequencing methods. Moreover, these results imply that current methods for ATM detection may be insufficient; however supplementary deep-sequencing of these samples could be used to determine the nature of ATM deficiency, and to predict response to therapeutic treatments.

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    P3.05 - Poster Session 3 - Preclinical Models of Therapeutics/Imaging (ID 159)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.05-003 - Role of ATM in response of non-small cell lung cancer cell lines to therapeutic agents (ID 839)

      09:30 - 16:30  |  Author(s): D.G. Bebb

      • Abstract

      Background
      Lung cancer is the leading cause of cancer death worldwide. Non-Small Cell Lung Cancer (NSCLC) which is the predominant type is mostly advanced stage disease at diagnosis. Cytotoxic agents including chemotherapeutic drugs and radiation therapy, the mainstay of advanced NSCLC treatment are neither specific nor curative and are significant causes of morbidity in patients. This poor NSCLC outlook therefore requires a novel therapy as well as predictive markers of treatment response for an improvement. We propose Ataxia Telangiectasia Mutated (ATM), a critical player in the DNA double strand break repair pathway, as a potential predictor of treatment response in NSCLC. Radiation sensitivity is one of the hallmarks of ataxia telangiectasia (A-T), a condition due to ATM mutations. Our lab has previously demonstrated ATM deficiency in about 20% of resected NSCLC tumors and this is associated with poorer survival outcomes; however, no treatment guidelines currently take this into consideration.

      Methods
      We assessed in vitro the altered sensitivity of ATM deficient NSCLC cell lines to both targeted agents (e.g. Poly (ADP) ribose polymerase (PARP) inhibitors) and non-targeted agents (e.g. DNA damaging agents including ionizing radiation (IR) and the commonly used cytotoxic chemotherapies in NSCLC) using the clonogenic survival assay. A panel of NSCLC cell lines (NCI-H23, NCI-H226, NCI-H460, NC1-H522, NCI-H1793, NCI-H1395 and HCC 4006) were characterised for pre-existing ATM deficiency in terms of ATM protein expression levels and functionality using western blot. By examining the cells’ responses to IR, confirmation of ATM deficiency was achieved with clonogenic assay to assess cellular viability and western blot for the expression of IR inducible ATM-dependent phosphorylation sites on target proteins including phosphorylation of serine 1981 of ATM (p-S1981 ATM), serine 15 of p53 (p-S15 p53) and serine 824 of KAP1 (KRAB-associated protein 1).

      Results
      We identified NCI-H23 cells with pre-existing low ATM protein levels (11% relative to BT cells) and undetectable ATM protein in NCI-H1395 cells compare to our positive (BT cells) and negative (L3 cells) control lymphoblastoid cells derived from normal and A-T patients respectively. In addition, H23 and H1395 cells display altered ATM signaling as evidenced by no detectable level of p-S1981 ATM expression post-irradiation. There is increased sensitivity of H23 cells to DNA-damaging agents (such as IR, topotecan and cisplatin) and PARP inhibition compare to ATM proficient NSCLC cell lines.

      Conclusion
      Our results seem to delineate the therapeutic sensitivity of ATM deficient versus ATM proficient cell lines to both non-targeted agents (chemotherapy and radiation therapy) and to targeted agents (PARP inhibitors). ATM could serve as a potential marker in guiding the use of 1) targeted agents such as PARP inhibitors, and 2) conventional chemo-radio therapeutic agents in the treatment of NSCLC. We are in the process of establishing ATM knock-down cells from ATM proficient NSCLC cell lines. Results of the altered sensitivity of established ATM deficient NSCLC cells to our investigative agents will be discussed and presented.