Abstract Background: Pediatric rhabdoid tumors, namely Malignant Rhabdoid Tumors (MRTs) and Atypical Teratoid Rhabdoid Tumors (ATRTs) in the central nervous system (CNS), are aggressive pediatric malignancies characterized by the loss of functional INI1 protein due to SMARCB1 gene mutations. Current treatment options for these tumors are limited and ineffective, necessitating the exploration of novel therapeutic strategies. Rhabdoid cells retain an intact p53 pathway, a critical tumor suppressor pathway involved in regulating cell cycle and apoptosis. Previous studies have shown that the MDM2 inhibitor, Idasanutlin, effectively eliminates flanked rhabdoid tumors, suggesting that targeting the p53 pathway holds promise for developing effective therapies against MRTs and ATRTs. Methods: In this study, we employed a combination approach using the MDM2 inhibitor, Idasanutlin, and the XPO1 inhibitor, Selinexor, to activate the p53 pathway. The impact of this combination treatment on tumor cell viability, proliferation, and apoptosis was assessed both in vitro and in vivo using MRT and ATRT models. Additionally, CRISPR screening and proteomic studies were conducted to identify specific dependencies in refractory models and investigate their influence on treatment response. Results: Treatment with the combination of Idasanutlin and Selinexor activated the p53 signaling pathway, leading to increased levels of MDM2 and p21 proteins, two direct targets of p53, and resulting in apoptosis in MRT and ATRT models. In vitro experiments demonstrated a significant reduction in cell viability and proliferation upon treatment. In vivo studies using MRT and ATRT xenograft models showed a remarkable decrease in tumor growth following the combination treatment. However, variable responses were observed among different models, indicating the presence of dependency patterns influencing treatment sensitivity. Proteomic studies and genetic manipulation provided further insights into the potential dependencies Conclusions: Our findings highlight the potential of the combination of Idasanutlin and Selinexor to activate the p53 pathway and induce apoptosis in MRT and ATRT models. Although the treatment strategy effectively activated p53, we identified dependencies that may contribute to incomplete treatment sensitivity. Further investigation into these dependency patterns is necessary to optimize the therapeutic approach. Overall, targeting the p53 pathway using combination therapies shows promise for the development of effective treatments for pediatric MRTs and ATRTs, warranting further preclinical and clinical investigations. Citation Format: Alaa Refaat, Hyekyung Cho, Jennifer Stripay, Charles Roberts, Christopher Tinkle, Anang Shelat, Martine Roussel. Targeting the p53 pathway to treat pediatric Malignant Rhabdoid and Atypical Teratoid Rhabdoid Tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C096.