Abstract 271: The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells
Authors: Charles R. Dunlop, Yann Wallez, Sandra Bernaldo de Quirós Fernández, Saadia A. Karim, Alan Lau, Frances M. Richards, Duncan I. Jodrell
Journal: Cancer Research (2019)

Abstract Therapeutic targeting of the DNA damage response (DDR) has the potential to improve the poor survival outcomes of pancreatic ductal adenocarcinoma (PDAC). Many common genetic alterations in PDAC augment replication stress, which activates Ataxia-telangiectasia and Rad3-related kinase (ATR). We have demonstrated previously, efficacy of the ATR inhibitor (AZD6738) in PDAC models, particularly when combined with gemcitabine (gem) (Wallez et al, MCT, 2018). This combination strongly induces activation of Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), indicating the induction of double-strand breaks. It has been suggested that ATM-deficiency can sensitize cancer cells to ATR inhibitors. This study sought to assess the relevance of this finding to PDAC and to interrogate the distinct roles of ATM and DNA-PK in response to ATRi/gem in PDAC cell lines. The ATM inhibitor, AZD0156, sensitized human pancreatic cancer cell lines (MIA PaCa-2, HPAF-II, AsPC-1) to AZD6738, as determined by SRB assays (e.g. MIA PaCa-2; AZD6738 GI50 = 3.8 µM, versus 1.3 µM in the presence of 30 nM AZD0156) and in longer term colony forming assays (surviving fraction (SF) (1 µM AZD6738); 78 +/- 0.9%, SF (1 µM AZD6738 + 30nM AZD0156); 11 +/- 4.2%). However, ATM knockdown with siRNA did not sensitize to AZD6738, nor to the combination of AZD6738/gem. This suggests that the presence of kinase-inhibited ATM at sites of DNA damage is more deleterious to PDAC cells than deficiency of ATM protein expression. We established that AZD6738/gem could induce phosphorylation of the canonical targets of ATM (Chk2, KAP1) in MIA PaCa-2 when ATM was knocked down or inhibited. DNA-PK was also activated in response to exposure to ATRi/gem and we postulated that it was responsible for the Chk2/KAP1 activation. We then demonstrated that Chk2/KAP1 activation could be abrogated by DNA-PK inhibition with NU7441. Furthermore, in an ATM-null pancreatic cancer cell line, AZD6738/gem-induced KAP1 phosphorylation was also abrogated by inhibition of DNA-PK. Thus, DNA-PK appears to be responsible for the downstream activation of Chk2 and KAP1, induced by AZD6738/gem in PDAC cells. Therefore, as well as revealing that inhibition of ATM using the novel inhibitor AZD0156 is highly deleterious to ATR inhibited PDAC cells, we have identified a compensatory mechanism via DNA-PK, which may be relevant for maximizing the therapeutic potential of DDR inhibitors in PDAC. Since DNA-PK clearly plays a dominant role in the DDR signaling pathways, we are now assessing whether DNA-PK inhibition synergizes with AZD6738+/- gem. Citation Format: Charles R. Dunlop, Yann Wallez, Sandra Bernaldo de Quirós Fernández, Saadia A. Karim, Alan Lau, Frances M. Richards, Duncan I. Jodrell. The role of ATM and DNA-PK in responding to AZD6738-induced damage in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 271.