Abstract 2410: The mutant p53 C-terminal domain assists in DNA interactions and cell cycle promotion

Abstract

Abstract Introduction: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and has the worst prognosis of any breast cancer sub-type. More than 80% of TNBC patients harbor mutated TP53. Some mutant p53 proteins (mtp53) exhibit gain-of function (GOF) oncogenic characteristics, contributing to the decreased prognosis for TNBC patients, but also providing a potential therapeutic target for this aggressive disease. Our group previously found mtp53 R273H interacts with replicating DNA, and increases chromatin-bound replication proteins MCM2-7, PCNA, and PARP1. Mutant p53 retains an intact C-terminal domain (CTD), the domain which facilitates non-specific DNA binding. We hypothesize that the CTD of mtp53 facilitates increased DNA replication processivity. Methods: To test this hypothesis, we have transiently transfected human cells to compare wild-type p53, and R273H mtp53 full-length and CTD-truncated variants. We used CRISPR/Cas9 to generate endogenous CTD truncations of mtp53 R273H in the breast cancer cell line MDA-MB-468. Specifically, clones were used which have mtp53 CTD truncations (R273HΔ381-388 and R273HΔ347-393) and one clone with a frameshift resulting in no detectable mtp53 protein (R273Hfs387). Various western blotting techniques were used to assess protein level and sub-cellular localization, including the iPOND (isolation of proteins on nascent DNA) assay. Several microscopy techniques were used to visualize protein-protein interactions in cells including immunofluorescence, proximity ligation, and DNA fiber assays. Results: Our data show mtp53 R273H and wild-type p53 proteins with CTD truncations tether less to chromatin than their full-length counterparts. Also, our data shows that the truncated R273HΔ381-388 mtp53 is in close proximity to PARylated proteins (and potentially even PARylated itself), but the R273HΔ347-393 truncation is not in close proximity to PARylated proteins. Additionally, R273HΔ381-388 and R273HΔ347-393 demonstrate delayed cell cycle progression and reduced proliferation compared to MDA-MB-468 cells. We observe DNA replication processivity is decreased in 468 R273HΔ381-388 and R273Hfs387 cells. Processivity, but not proliferation rate, is rescued in 468 R273HΔ347-393 cells. Conclusions: The combination of reduced cell proliferation in all CTD-truncated cells, and reduced DNA replication processivity in R273HΔ381-388 and R273Hfs387 cells, but not in R273HΔ347-393 indicates a nuanced role for the CTD in DNA replication and S-phase progression. Citation Format: Devon Lundine, Zafar Syed, Viola Ellison, George Annor, Gu Xiao, Jill Bargonetti. The mutant p53 C-terminal domain assists in DNA interactions and cell cycle promotion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2410.