P2.01-090 Platin Induced Phosphorylation of ATM and ATM-Deficiency as a Predictive Marker of Platin Sensitivity 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 adducts leading to the formation of both single and double stranded DNA breaks. Effective DNA damage response pathways contribute to cell survival and resistance to these agents. Ataxia telangiectasia mutated (ATM) is an important mediator of the DNA damage response involved in the activation of key components of 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 sensitivity to platins due to an impaired DNA damage response. 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 g-H2AX) were investigated to determine ATM pathway activation. Finally, transient and stable ATM knockdowns were generated using siATM and shATM. These cells were then tested for platin sensitivity by trypan blue viability or clonogenic assays. 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 platins. In addition, KAP-1, a downstream target of ATM showed increased phosphorylation in response to these treatments when compared to non-treated controls. In contrast, ATM-deficient cell lines showed no increased levels of phosphorylated ATM or KAP-1 in response to platins. Preliminary analysis of siATM transient knockdowns in NCI-H226 shows an increased sensitivity to cisplatin. It is clear that platin exposure activated an ATM mediated signalling response and that cells lacking ATM showed deficiencies in the phosphorylation of key downstream targets of this pathway. Cells deficient in ATM may therefore be more susceptible to platin therapy due to an impaired DNA damage response. This data suggests that individuals with low or non-functioning ATM may be candidates for precision low does therapies that exploit this deficiency.