Abstract 1618: Blocking MDM2 induces downregulation of PARP and enhances apoptosis in SK-N-SH neuroblastoma cells

Abstract Introduction: Neuroblastoma is a pediatric cancer originating from neural crest cells often characterized by poor prognosis and resistance to standard therapies. RG-7388 is a potent MDM2 inhibitor that disrupts the interaction between p53-MDM2. However, the impact of RG-7388 on cellular pathways, particularly its role in post-translational modifications of cancer cells, remains unexplored. PARP is a critical protein in DNA repair and apoptosis, is often targeted for degradation in cancer therapies. However, the mechanisms underlying PARP degradation in response to RG-7388 treatment in neuroblastoma cells largely remain unclear. We hypothesize that MDM2 re-activation by the rebound mechanism of RG-7388 induces PARP degradation in SK-N-SH neuroblastoma cells, possibly through a E3 ligase mediated ubiquitination mechanism that is eventually contributing to enhanced apoptosis. Methods: To test this hypothesis, SK-N-SH cells were treated with RG-7388 (2 µM), MDM2 specific E3 ligase inhibitor (10 µM), and iRucaparib-AP6 (10 µM), alone or in combination with RG-7388. Cell viability was evaluated using the Trypan Blue Dye Exclusion (TBDE) assay method, while Caspase-3/7 activation was quantified through cleavage of DEVD-Amc substrate. qRT-PCR, Western blot, and Immunofluorescence analyses were conducted to assess expression levels of the cell cycle and apoptotic markers, such as MDM2, p53, p21, and PARP. Results: Treatment with RG-7388 significantly reduced the viability of SK-N-SH cells compared to the control. The decrease in PARP levels seen after RG-7388 treatment is more significant than the levels observed in iRucaparib-AP6. In addition, the Caspase-3/7 activity, that was measured via the DEVD-Amc substrate cleavage assay, was markedly higher in RG-7388 treated cells compared to control. qRT-PCR analysis revealed upregulation of p53 and p21 in RG-7388 treated cells and a significant downregulation in PARP expression. Western blot analysis also confirmed a reduction of PARP levels in RG7388-treated cells, which was greater than the decrease seen with iRucaparib-AP6 treatment. Reversal of the RG-7388 and iRupacarib-AP6 induced downregulation of PARP by E3 Ligase inhibitor suggested that degradation may be major contributor to the downregulation in SK-N-SH cells. Conclusion: Our study results indicate that RG-7388 induces significant downregulation of PARP in the SK-N-SH cells, which was leading to enhanced apoptosis. Furthermore, the results of RG-7388 treatment obtained from SK-N-SH cells confirmed its ability to induce cell death through both p53 activation and downregulation of PARP. Acknowledgment: This project was supported by the National Pediatric Cancer Foundation (NPCF) and also by the Royal Dames of Cancer Research Inc. Ft. Lauderdale, Florida Citation Format: Shyam Sundar Jaganathan, Umamaheswari Natarajan, Appu Rathinavelu. Blocking MDM2 induces downregulation of PARP and enhances apoptosis in SK-N-SH neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1618.