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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
  • Moderators:
  • Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 12408

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    P3.02-022 - Deep-Sequencing Mutational Analysis of ATM in NSCLC Cell Lines (ID 3446)

    09:30 - 09:30  |  Author(s): L.F. Petersen
    • 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.