Complete loss of ATM function augments replication catastrophe induced by ATR inhibition and gemcitabine in pancreatic cancer models

Abstract

Background Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC. Methods Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo. Results Complete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts. Conclusions ATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker.

MMB-FOXM1-driven premature mitosis is required for CHK1 inhibitor sensitivity

“…Ataxia Telangiectasia Mutated (ATM), ATR, and DNA-dependent Protein Kinase (DNA-PK) activities have been linked to KAP1 phosphorylation at S824 ( White et al, 2006 ). Subsequent studies predominantly focused on ATM-dependent KAP1 phosphorylation in response to DNA DSBs ( White et al, 2012 ; Ziv et al, 2006 ) or more recently DNA-PK-dependent KAP1 phosphorylation in response to ATR inhibition ( Buisson et al, 2015 ; Dunlop et al, 2020 ). To assess the contribution of ATM and DNA-PK activity to KAP1 phosphorylation after treatment with CHK1i or agents that induce DNA damage, i.e., hydroxyurea (HU) or doxorubicin (DOXO), ATM and DNA-PK autophosphorylation was assessed in relation to pKAP1 levels by immunoblot and by flow cytometry in the case of ATM.…”

Quantifying cell cycle-dependent drug sensitivities in cancer using a high throughput synchronisation and screening approach

“…Previous work from our lab identified synergy between gemcitabine and an ATR inhibitor, AZD6738, in human and mouse models of pancreatic cancer in vitro and in vivo [ 24 , 25 ]. Both gemcitabine and AZD6738 had high CCS scores within the drug library screen (0.53 and 0.60 respectively) indicating high S/G2 specificity, consistent with their known modes of action.…”

“…Both gemcitabine and AZD6738 had high CCS scores within the drug library screen (0.53 and 0.60 respectively) indicating high S/G2 specificity, consistent with their known modes of action. Given the ability of these compounds to synergise and induce replication catastrophe [ 24 , 25 ], we investigated whether exposure to both compounds would affect the CCS score as part of a schedule. To match a typical dosing schedule given for this combination in vivo [ 24 , 25 ], we added AZD6738 continuously at two fixed doses (0.3 and 1 μM) after a 6h gemcitabine pulse in G1 or S/G2 accumulated cells.…”

“…= 2.4 mM), suggesting transiently inhibiting ATR in S/G2 cells accounts for a significant proportion of its associated cytotoxicity. With previous reports of hypersensitivity of ATM-null cell lines to ATR inhibition [25,33,34], it is possible this effect could potentially be boosted in cells without functional ATM. This may explain why others have found AZD6738-induced bone marrow toxicity with such tissue types having an abundance of highly proliferative cells [35].…”

A Novel DNA Replication-Related Signature Predicting Recurrence After R0 Resection of Pancreatic Ductal Adenocarcinoma: Prognostic Value and Clinical Implications

“…Compelling evidence has revealed that DNA replication is closely associated with chemotherapy resistance, and several DNA replication-related genes have been confirmed as potential therapeutic targets in PDAC ( Dunlop et al, 2020 ; Lloyd et al, 2020 ). Nevertheless, it remains unclear as to how DNA replication-related genes affect patient outcome, and this phenomenon has not been well reported.…”