Purpose: Radical cystectomy (RC), remains the “standard” treatment for Muscle Invasive Bladder Cancer (MIBC), but is associated with major impact on quality of life. Radiation therapy (RT) is an alternate treatment as it preserves the bladder. However, lack of local control remains problematic. Therefore, the use of a radio-sensitizer could overcome those challenges and limit side effects. In this sense, inhibiting the mTOR signaling pathway in combination with radiation is a promising therapeutic strategy. As the cyclin dependent kinase inhibitor p21 WAF1/Cip1 has been found to be associated with DNA damage repair and autophagy, which are two major pathways associated with radiation resistance, we sought to investigate the role of p21 in response to combination therapy in bladder cancer. Methods: Expression of p21 was characterized in different bladder cancer cell lines via Western Blotting. Correlation of p21 expression and response to mTOR inhibitor was done using proliferation assay. Knockdown of p21 was carried out via RNAi silencing. To assess the effect of the p21 expression in response to combination therapy, Clonogenic assay was done. Western blot was utilized to assess the effect of mTOR inhibition in p21 expression. In vivo, athymic mice were subcutaneously injected with bladder cancer cell lines. Treatment consisted of either placebo or combined RAD001 (12 hours prior to radiation) and fractionated IR. Tumor volume was measured at endpoint. Immunohistochemistry was used to detect the levels of p21 in paraffin-embedded mouse xenograft bladder cancer tissues treated with placebo, IR, RAD001 and in combination. Autophagy was investigate by LC3 cytoplasmic localization by immunofluoresecence and LC3 I/II levels were detected by Western Blot. Clevage of caspase-3 was used to determine apoptosis by Western Blot. Results: There was a significant correlation between the levels of p21 and sensitivity to mTOR inhibitor (P value <005). RAD001 induced a dose response decreased in the level of p21 expression. Addition of the mTOR inhibitor, along with knocking down p21 significantly inhibited colony formation compared to untreated p21-scramble cells. A significant effect in tumor volume was observed in vivo in the group treated with the combination arm of compared to all other groups (P value <005). Levels of p21 increased following the treatment with IR alone and in combination with RAD001, whereas a slight decrease was observed following the treatment with RAD001 alone. Addition of RAD001 induces autophagy, and point to the functional role of p21 in inhibiting autophagy as its knock-down was successful at increasing the levels of LC3 I/II even without RAD001 addition. Conclusion: Our in vitro and in vivo results support a functional role for p21 in mediating the response to combination therapy, thus promoting p21 as a potential player that can be modulated in the treatment of bladder cancer. Citation Format: Jose J. Mansure, Shraddha Solanki, Wael Alamjed, Roland Nassim, Fabio Cury, Simone Chevalier, Wassim Kassouf. p21 WAF1/Cip1 -mediated radiosensitization of bladder cancer cells by mTOR inhibitor, RAD001 disrupts the balance between autophagy and apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1654.