P6.10 - Identifying ATMic non-small cell lung cancer cell lines: Implications for designing clinical trials

Abstract

ABSTRACT Introduction: We have shown that mantle cell lymphoma and gastric cancer cell lines deficient in the DNA repair protein ATM display increased sensitivity to ionizing radiation and a greater susceptibility to PARP inhibitor-induced synthetic lethality–a phenotype we have labeled “ATMic”. Since this has potential clinical application, it will be important to be able to reliably identify ATMic tumors so that appropriate precision therapy can be offered to patients. We compared the utility of various assays to identify ATMicity in a series of Non-small cell lung cancer (NSCLC) cell lines. Methodology: A series of 6 NSCLC cell lines were studied. Known ATM competent (BT) and ATM deficient (L3) cell lines were used as controls. Each was assessed for its ATM status (a) molecularly, by sequencing, RT PCR and western blotting, and (b) functionally, by DNA damage-induced signaling and radiation sensitivity and PARP inhibitor-induced synthetic lethality using the cytotoxicity or clonogenic assay. ATMic cell lines were defined as those that displayed increased sensitivity to ionizing radiation and PARP inhibitor-induced synthetic lethality using the PARP inhibitor Olaparib. Results: Two NSCLC cell lines, H23 and H1373 exhibited ATMicity. Conclusion: ATMic NSCLC cell lines can be best identified by assessing ATM protein expression rather than sequencing or mRNA expression. NSCLC patients whose tumors display the ATMic phenotype would be candidates for PARP inhibitor treatment and lower doses of radiation. Our results suggest that such patients can be identified by measuring ATM protein levels within the tumor. Clinical trials of low dose radiation and PARP inhibition where patients are stratified according to tumour ATM protein expression are warranted. Table 1 . comparison of in vitro methodologies for assessing ATM status of cell lines § quantified by ImageJ 0.5hr after cell irradiation after normalization to the loading control and expressed as relative to ATM in BT (ATM positive control) cells. * quantified by ImageJ 0.5hr after cell irradiation after normalization to KAP1 for each cell line. # by cytotoxicity assay using wst-1 in non adherent cell lines BT and L3 by clonogenic assay of adherent cells H23, H226, H460, H522, H1373, H1395 Western Blot RT-qPCR N.G. Sequencing Sensitivity #¶ Cell Lines ATM§ pS824-KAP1* ATM ATM mutations 3 µM Olaparib 4Gy IR BT 1.0 0.22 + N/A - - L3 0.0 0.0 – N/A + + H23 0.1 0.0 + 3 SNP + + H460 4.4 0.30 + Wild-type - - H522 6.6 0.26 N/A Wild-type - - H1373 0.06 0.09 N/A 1 nonsense + + H1395 0.4 0.44 - 2 SNP - - H226 1.3 0.34 ++ Wild-type - -