ATR Inhibition Potentiates PARP Inhibitor Cytotoxicity in High Risk Neuroblastoma Cell Lines by Multiple Mechanisms

Abstract

Background: High risk neuroblastoma (HR-NB) is one the most difficult childhood cancers to cure. These tumours frequently present with DNA damage response (DDR) defects including loss or mutation of key DDR genes, oncogene-induced replication stress (RS) and cell cycle checkpoint dysfunction. Aim: To identify biomarkers of sensitivity to inhibition of Ataxia telangiectasia and Rad3 related (ATR), a DNA damage sensor, and poly (ADP-ribose) polymerase (PARP), which is required for single strand break repair. We also hypothesise that combining ATR and PARP inhibition is synergistic. Methods: Single agent sensitivity to VE-821 (ATR inhibitor) and olaparib (PARP inhibitor), and the combination, was determined using cell proliferation and clonogenic assays, in HR-NB cell lines. Basal expression of DDR proteins, including ataxia telangiectasia mutated (ATM) and ATR, was assessed using Western blotting. CHK1S345 and H2AXS129 phosphorylation was assessed using Western blotting to determine ATR activity and RS, respectively. RS and homologous recombination repair (HRR) activity was also measured by γH2AX and Rad51 foci formation using immunofluorescence. Results: MYCN amplification and/or low ATM protein expression were associated with sensitivity to VE-821 (p < 0.05). VE-821 was synergistic with olaparib (CI value 0.04–0.89) independent of MYCN or ATM status. Olaparib increased H2AXS129 phosphorylation which was further increased by VE-821. Olaparib-induced Rad51 foci formation was reduced by VE-821 suggesting inhibition of HRR. Conclusion: RS associated with MYCN amplification, ATR loss or PARP inhibition increases sensitivity to the ATR inhibitor VE-821. These findings suggest a potential therapeutic strategy for the treatment of HR-NB.

Recent advancements in PARP inhibitors-based targeted cancer therapy

“… 81 Therefore, combination treatment of PARPi with ATR or CHK inhibitors can potentially overcome acquired PARPi-resistance. 108–110 Similar to ATR, ATM is another key mediator that resolves DNA replication stress and maintains genomic stability. 111 , 112 ATM inhibition potentially enhances PARPi sensitivity or reverses acquired PARPi-resistance by preventing replication fork protection or by enhancing replication stress.…”

DNA Damage Response during Replication Correlates with CIN70 Score and Determines Survival in HNSCC Patients

“…Rather, the function of CHK1 in preventing degradation appears to be of paramount importance here, (a) through direct association with the DNA replication fork [ 15 ], (b) control of dormant replication origins, mediated by ATR activation of the intra-s phase checkpoint [ 20 ], (c) control of the nuclease activity of Mus81 [ 47 ]. Together, our selective radiosensitizing effect of CHK1 inhibition of radioresistant HNSCC cells ( Figure 5 ) and the observation that most proteins informative of HNSCC patient survival were directly or indirectly associated with replication ( Figure 3 ), suggest that inhibitors to generally inactivate the S-phase DNA damage response, such as CHK1 or ATR [ 16 ], are promising options for future advancement of existing therapies.…”

“…We and others further observed that PARP1, in addition to its direct involvement in repair processes in HR-deficient HNSCC cell lines, also plays a role in controlling replication [ 14 , 15 ]. Other potential inhibitors of candidate genes with a function in replication, such as CHK1 and ATR [ 16 ], have been the subject of preclinical and clinical investigation in combination with irradiation and also showed promise for specific radiosensitization of genomically unstable HNSCC tumor cell lines [ 4 , 17 ]. Therefore, our goal is to analyze the prognostic significance of the CIN70 score in HNSCC and the impact of DNA repair protein expression using the open-access Cancer Genome Atlas (TCGA) dataset and to further characterize the impact of the identified DNA repair processes after DNA damage by ionizing and UV irradiation, Mitomycin C and topotecan using preclinical models.…”

Molecular Mechanisms of MYCN Dysregulation in Cancers

“…These results indicate targeting DNA repair system or drugs causing DNA damage could be synthetic lethal in MYCN -driven tumors. Recent studies reveal various strategies based on N-MYC-mediated synthetic lethality, including glutaminase inhibition or glutamine deprivation ( 157 ), BCL2 inhibition ( 125 ), eliminating SKP2 complexes ( 158 ), kinesin spindle protein (KSP) inhibition ( 159 ), G9a inhibition ( 160 ), poly (ADP-ribose) polymerase (PARP) inhibition ( 161 , 162 ).…”