PARP inhibition increases sensitivity to cisplatin in non-small cell lung carcinoma via induction of TET-dependent hydroxymethylation

Nevena Grdovic¹Anja Tolić¹Mirna Jovanovic²Jovana Rajic¹Marija Đorđević¹Marijana Stojanovic¹Stefan Markovic Hadzic¹Tijana Markovic¹Ana Saric¹Svetlana Dinic¹Jelena Arambasic Jovanovic¹Mirjana Mihailovic¹Ana Podolski-Renic²Milica Pešić^3*Melita Vidakovic^1*Aleksandra Slobodan Uskokovic¹

• ¹Molecular Biology Department, Siniša Stanković Institute for Biological Research, University of Belgrade, Belgrade, Serbia
• ²Neurobiology Department, Siniša Stanković Institute for Biological Research, University of Belgrade, Belgrade, Serbia
• ³Neurobiology Department, Siniša Stanković Institute for Biological Research, University of Belgrade, Belgrade, Serbia

Introduction: Platinum-based chemopotentiation by poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) has been confirmed in some non-small cell lung carcinoma (NSCLC) models but the molecular mechanisms of PARPi synergy with chemotherapeutics remains poorly clarified. This study aimed to evaluate the efficacy of PARPi niraparib in mitigating resistance to cisplatin through increased Ten-eleven translocations (TET) enzyme activity and 5-hydroxymethylcytosine (5hmC) level in human NSCLC lung carcinoma model. Methods: Chemosensitivity of human NSCLC, sensitive and multidrug-resistant (NCI-H460 and NCI-H460/R, respectively), lung adenocarcinoma cell line (A549) and normal embryonic lung fibroblasts (MRC-5) to increasing concentration of cisplatin was studied by the MTT assay. The MTT and the median effect analyses was used to evaluate the efficacy of niraparib combinatorial synergy with cisplatin, dimethyloxalylglycine (DMOG) and vitamin C. The inhibition of PARP activity was determined by PARP activity assay. The global level of 5hmC was examined by confocal microscopy, slot-blot and flow cytometry which was used for monitoring cell death at different stages. The protein level of lamin B, PARP1, procaspase-3 and TETs was assessed by Western blotting. The expression of TET enzyme was analyzed by real-time quantitative PCR. Results: The chemosensitivity to cisplatin was found to be correlated with level of 5hmC in NSCLC and MRC-5 cell lines. The A549 was characterized with the lowest sensitivity to cisplatin and 5hmC level and selected for chemosensitisation via PARPi dependent restoration of 5hmC level. Treatment of A549 cells with niraparib resulted in decreased PARP activity, increased 5hmC level and synergism with cisplatin by promoting cisplatin-induced apoptosis. The treatment with niraparib and cisplatin resulted in synergistic effect in the hydroxylase activity of TET2 in terms of the increased 5hmC production in A549 cells. Conclusion: The improved responsiveness to cisplatin following PARPi–mediated sensitisation of A549 cells, accompanied by restoration of 5hmC levels, provides additional insights into the epigenetic mechanisms underlying the synergy between PARPi and cisplatin. The use of PARPi in clinical settings could increase the number of patients enriched in 5hmC who might benefit from platinum-based therapy. Additionally, targeting epigenetic marks could provide a molecular basis for personalized cancer therapy directed to overcome tumor resistance.