Abstract 1484: USP36-governed p21 homeostasis confers radioresistance in pancreatic cancer

Abstract Purpose: Pancreatic cancers are highly aggressive tumors that carry a poor prognosis. More than 90% of pancreatic ductal adenocarcinoma (PDAC) harbors mutationally activated KRAS, and oncogenic KRAS signaling has been implicated in contributing to replication stress and PDAC radioresistance. Cell cycle inhibitor p21waf1/cip1 (p21) can be transcriptionally induced by oncogenic KRAS and p53 in response to DNA damage. DNA damage can also induce p53-independent p21 nucleolus accumulation. Several studies revealed the antagonistic duality of p21 in oncogenesis depending mainly on its intracellular localization, with a tumor suppressor role in the nucleus and oncogenic role in the cytoplasm. The nucleolar deubiquitinase USP36 is pivotal in some cellular processes such as cell cycle maintenance, ribotoxic stress, and DNA replication stress. However, whether and how USP36 regulates p21 in the nucleolus in response to irradiation-induced DNA double-strand breaks remain unclear. Experimental Design: We examined p21 protein level in HEK 293T and PDAC cell lines (MIA PaCa-2, PANC-1) by overexpressing wild-type USP36 and its catalytic-inactive mutant (C131A). We monitored the colocalization of GFP-p21 and mcherry-USP36 in both HEK 293T and MIA PaCa-2 cells. We assessed the association of p21 and USP36 by Co-IP. We determined the residues of USP36 that are phosphorylated in response to DNA damage by point mutation, and analyzed cell cycle distribution by flow cytometry. We examined the roles of USP36 and p21 in radiosensitivity with clonogenic assay. β-galactosidase staining was performed to assessed the cell senescence after radiotherapy. Results: Overexpression of USP36, but not its catalytic-inactive mutant (C131A) induced protein levels of p21 in HEK 293T, MIA PaCa2 and PANC-1 cells. Mcherry-USP36 induced the accumulation of GFP-p21 in nucleolus of HEK 293T cells. In response to DNA damage, p21 translocated to the nucleolus and colocalized with USP36 in cells. Through domain mapping, we determined that the N-terminal of USP36 interacts with p21. Additionally, ATM and ATR phosphorylate USP36 at Ser356 and Ser519. USP36 depleted cells exhibited G1 release and G2/M arrest following DNA damage, and loss of USP36 induced cell senescence in cells following DNA damage. Finally, loss of USP36 sensitizes pancreatic cancer cells to radiation in vitro. Conclusions: Here we report that loss of USP36 sensitizes pancreatic cancer cells to radiotherapy. Our findings support a model in which, in response to DNA damage, USP36 is hyperphosphorylated and activated by ATM/ATR, resulting in stabilization of p21 through USP36-mediated deubiquitination. Stabilization of p21 alters cell cycle progression, ultimately leading to pancreatic cancer radioresistance through enhanced cell recovery after DNA damage. Together, our data supports targeting USP36 and/or p21 for therapeutic intent. Citation Format: Haihua Feng, Sindhu Nair, Veronica Castro Aceituno, Linlin Yang, Haiqing Li, Tiantian Cui, Sergio Corrales Guerrero, Ling Gui, Nicholas Eustace, Terence M. Williams. USP36-governed p21 homeostasis confers radioresistance in pancreatic cancer [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 1484.