P3.02-077 Platin Sensitivity and ATM-Deficiency in Non-Small Cell Lung Cancer

Abstract

Platinum based antineoplastic therapies (platins) are a first line treatment for non-small cell lung cancer (NSCLC) that generate DNA breaks and stimulate DNA damage response pathways. An inability to repair damage generated by these agents leads to cytotoxicity and cell death. A key mediator of the DNA damage response is ataxia telangiectasia mutated (ATM), an activator of downstream targets involved in DNA repair, cell cycle arrest, and apoptosis. Our lab has demonstrated that cell lines lacking ATM show increased sensitivity to platins. We hypothesize that platin exposure will activate ATM and that cells deficient in ATM will be innately sensitive to platins. Here we assess the molecular action of ATM in response to platins to determine if ATM-deficiency is predictive of platin sensitivity. ATM status was determined in five NSCLC cell lines using western blotting and RT-qPCR. Cell lines were treated with varying concentrations cisplatin, carboplatin and oxaliplatin for 18-hours and assessed for ATM phosphorylation by western blot. Additionally, downstream targets of ATM (KAP-1, p53, and gamma-H2AX) were investigated to determine ATM pathway activation. Knockdown cell lines were generated using shRNA to ATM before testing for IR and cisplatin sensitivity using clonogenic assay. ATR and ATM inhibitors were tested on knockdown cell lines to investigate pathway response differences after cisplatin and IR. NSCLC cell lines NCI-H226, NCI-H460, and NCI-H522 were found to be ATM-proficient whereas cell lines NCI-H23 and NCI-H1373 were found to be ATM-deficient. ATM-proficient cell lines demonstrated an increased level of phosphorylated-ATM in response to treatments with cisplatin, carboplatin, and oxaliplatin. ATM knockdown cell lines were found to have increased sensitivity to IR, however analysis of cisplatin sensitivity was inconclusive with only 1 out of 4 showing increased sensitivity. ATR inhibition in combination with cisplatin caused a large increase in DNA damage response from ATM and DNA-PKcs suggesting an avenue for synthetic lethality. It is clear that platin exposure induced an ATM mediated signaling response and that cells lacking ATM showed deficiencies in the phosphorylation of key downstream targets. Cells deficient in ATM may therefore be more susceptible to platin therapy due to an impaired DNA repair response. However, the predictive capabilities of ATM loss for platin sensitivity is still unclear. This data suggests that individuals with low or non-functioning ATM may be candidates for precision low dose therapies that exploit this deficiency.