ABSTRACT
Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.
Subject(s)
BRCA1 Protein , BRCA2 Protein , DNA Replication , Drug Resistance, Neoplasm , Histones , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Female , Humans , Mice , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , BRCA1 Protein/metabolism , BRCA1 Protein/deficiency , BRCA1 Protein/genetics , BRCA2 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/deficiency , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/drug therapy , Carrier Proteins/metabolism , Carrier Proteins/genetics , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Damage , DNA Repair , DNA Replication/drug effects , Drug Resistance, Neoplasm/genetics , Histones/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Mice, NudeABSTRACT
The inhibition of ataxia-telangiectasia mutated (ATM) has been shown to chemo- and radio-sensitize human glioma cells in vitro and therefore might provide an exciting new paradigm in the treatment of glioblastoma multiforme (GBM). The effective treatment of GBM will likely require a compound with the potential to efficiently cross the blood-brain barrier (BBB). Starting from clinical candidate AZD0156, 4, we investigated the imidazoquinolin-2-one scaffold with the goal of improving likely CNS exposure in humans. Strategies aimed at reducing hydrogen bonding, basicity, and flexibility of the molecule were explored alongside modulating lipophilicity. These studies identified compound 24 (AZD1390) as an exceptionally potent and selective inhibitor of ATM with a good preclinical pharmacokinetic profile. 24 showed an absence of human transporter efflux in MDCKII-MDR1-BCRP studies (efflux ratio <2), significant BBB penetrance in nonhuman primate PET studies (Kp,uu 0.33) and was deemed suitable for development as a clinical candidate to explore the radiosensitizing effects of ATM in intracranial malignancies.