Abstract 3514: RANBP9 presence affects levels of Tip60 and activated p53 in lung cancer cells in response to DNA damage (2019)

Abstract Lung cancer is the leading cause of cancer-related deaths. Despite recent success with targeted and immune-therapies, DNA-damaging agents, such as platinum-based drugs, still represent the first-line of treatment. However, side effects and drug resistance limit their efficacy and use. We recently demonstrated that RAN Binding Protein 9 (RANBP9) plays a critical role in the DNA-damage response (DDR) of lung cancer cells. However, how it affects the mechanisms and outcome of the DDR is not known. In this regard, p53 activity is crucial for cell cycle arrest or programmed cell death. In particular, acetylation of p53 at lysine 120 by the Tat-Interacting Protein of 60 KDa (Tip60) is dispensable for p53-mediated growth arrest but is essential for p21 induction and p53-dependent apoptosis. It has been reported that RANBP9 and Tip60 co-localize. Here, we investigate whether RANBP9 affects Tip60-dependent activation of p53 and its relevance in deciding cell faith in response to DNA damage. For our study we used H1299, A549 and HeLa cells. By CRISPR/Cas9, we generated RANBP9 wild type (WT) and knockout (KO) A549 clones. We also established KO clones stably re-expressing RANBP9-FLAG. Cell response to DNA damage was assessed upon treatment with Ionizing Radiation (IR) and/or Cisplatin (CDDP). Interaction between RANBP9 with Tip60 was assessed by immunoprecipitation of transfected HeLa cell lysates. We show that RANBP9 and Tip60 co-immunoprecipitate either by pulling-down RANBP9 or Tip60. Using different deletion mutants of Tip60, we found that a region encompassing the Zinc Finger and part of the Histone Acetyl-Transferase domain is required for the interaction with RANBP9. Further, the enzymatic-dead Tip60 retained its ability to interact with RANBP9. Using p53 null H1299 cells, we established that this interaction is independent of p53. However, RANBP9 depletion affects both total and activated p53 (p-p53s15) levels after DNA damage. Re-expressing RANBP9 in stable KO clones rescued p53 levels. Upon CDDP treatment, p53 protein levels were higher in the clones stably re-expressing RANBP9 compared to the KO ones. Upon IR or CDDP treatment, p21 protein levels were higher in the presence of RANBP9. Finally, we found that Tip60 levels were higher in CDDP-treated and untreated WT cells comparing to the KO ones. Our results indicate that RANBP9 and Tip60 constitutively interact. The decrease of p53 in RANBP9 absence suggests that RANBP9 might favor p53 acetylation by Tip60 upon DNA damage. Further investigations will elucidate how RANBP9 affects the levels and the functions of Tip60. Also, we will measure the levels of p53-AcK120 both in the WT and KO clones. If p53-AcK120 is impaired when RANBP9 is absent, RANBP9 KO cells might undergo cell death instead of cell cycle arrest. Altogether, our results shed light on a potential RANBP9-Tip60-p53 axis at play during the DDR. Citation Format: Shimaa Soliman, Arturo Orlacchio, Anna Tessari, Marina Capece, Rosa Visone, Carlo Croce, Dario Palmieri, Vincenzo Coppola. RANBP9 presence affects levels of Tip60 and activated p53 in lung cancer cells in response to DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3514.