Abstract P5-09-02: Tetrameric and monomeric gain-of-function mutant p53 interacts with chromatin

Abstract

Abstract In 80% of triple-negative breast cancers (TNBC) the TP53 gene is mutated, with a high occurrence of missense changed in the DNA binding domain. These mutations lead to loss of wild-type p53 (wtp53) function and some also create mutant p53 (mtp53) proteins with novel gain-of-function (GOF) tumor-promoting capabilities. We previously reported that mtp53 R273H interacts with replication-associated proteins including MCM2-7 and poly ADP-ribose polymerase (PARP1). Moreover, mtp53 is found on actively replicating nascent DNA. What remains unclear is which domains of mtp53 R273H are responsible for the protein’s chromatin-based replication activities. In this study, we tested whether the oligomerization state of mtp53 plays a role in oncogenic GOF activities. We destabilized tetramer formation by using site-directed mutagenesis to introduce oligomerization domain (OD) point mutations A347D, R337C, or L344P. The site-directed mutagenesis constructs were transiently transfected into p53-/- mammalian cell lines to evaluate the altered functions. The oligomerization state of mtp53 was assessed via glutaraldehyde crosslinking and western blot analyses. Western blot and qPCR analyses were used to test how OD mutations in plasmids expressing either wtp53 or R273H mtp53 regulated the targets p21, CDC7 and RRM2. We used chromatin fractionation and western blot analyses to compare the chromatin association of the R273H mtp53 (with variable OD mutants), PARP1, and MCM2 to assess how destabilizing tetramer formation influenced chromatin interactions. Compared to the tetrameric versions of wildtype (wt) and mutant (mt) p53, the wtp53-A347D and R273H-A347D were predominantly dimers, while wtp53-R337C, wtp53-L344P, mtp53 R273H-R337C, and mtp53R273H-L344P were predominantly monomers. When wtp53 tetramerization was blocked so was its ability to activate the cyclin-dependent kinase p21/waf. Interestingly, all oligomerization forms of mtp53 localized to the chromatin suggesting GOF does not require tetramer formation for all functions. Strikingly, all oligomerization mutants of R273H mtp53, compared to the tetrameric mtp53, had a stronger interaction with MCM2. When we examined the mtp53 GOF transcriptional target we found that only tetrameric mtp53 R273H activated the target gene RRM2. Our findings suggest that destabilizing tetramer formation reduced certain mtp53 R273H GOF oncogenic activities while activating others. These changes of functions require examination in the context of triple-negative breast cancers. Citation Format: George Kwakye Annor. Tetrameric and monomeric gain-of-function mutant p53 interacts with chromatin [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-02.