p53 genetic sensor system to identify novel mutant p53 reactivators

Authors

Mrinalini Bhosale, Children's Mercy Kansas CityFollow
Alejandro Parrales, Children's Mercy Kansas CityFollow
Mohamed A.A. Alalem, Children's Mercy HospitalFollow
Atul Ranjan, Children's Mercy Kansas CityFollow
Elizabeth Thoenen, Children's Mercy Kansas CityFollow
Tomoo Iwakuma, Children's Mercy HospitalFollow

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Publication Date

5-2023

Abstract

Tumor suppressor Tp53 is the most common mutated gene in human cancers. Missense mutations on the DNA binding domain of p53 frequently abrogate its tumor suppressor activities. However, wild type p53 (wtp53) activity can be restored of mutant p53 (mutp53) proteins by lowering temperature, insertion of second mutations, and small molecules or peptides. To identify novel compounds that can reactivate wtp53 activities from mutp53, we generated H1299 (p53-null) cells expressing a genetic sensor for wtp53 transcriptional activity. This genetic sensor system consists of two constructs: one containing the GFP gene controlled by SCD1 promoter, which is transcriptionally repressed by wtp53, while the second construct uses the p53-regulated p21 promoter to drive expression of GFP shRNA. Using this sensor, we performed a high-throughput screening of 29,440 compounds and identified two top candidates which specifically reduce GFP expression in p53R175H-expressing cells but not in control p53-null cells. Moreover, these compounds restore wtp53-like conformation of p53R175H and p53R156P proteins and reduce viable cell proliferation on cells with p53R175H when compare with those having wtp53 or p53-null status. These compounds increase the levels of p21, Puma, Bax2 and many more downstream targets of p53 contributing to increase in the transcriptional activity. Since p53 is mutated in more than 50% of human tumors, completion of this study could contribute to develop novel therapies for mutp53-expressing cancers. P53 shows transcriptional activity in its dimeric and tetrameric form. Upon reactivation due to accumulated mutant p53, the wild type converted p53 has lower probability to form a homo-tetramer or homodimer hence its transcriptional activity is hindered. This phenomenon is called dominant negative effect. To address this effect a combination of degrading agent and reactivator should be employed for efficient transcriptional activity. Degrading agents like HSP90 inhibitors or statins can be combined with promising reactivators. We investigated a efficient combination of degrading agent and reactivators for higher transcriptional activity.

Notes

Accessed Restricted by Presenter.

Document Type

Poster-Restricted Access

Recommended Citation

Bhosale, Mrinalini; Parrales, Alejandro; Alalem, Mohamed A.A.; Ranjan, Atul; Thoenen, Elizabeth; and Iwakuma, Tomoo, "p53 genetic sensor system to identify novel mutant p53 reactivators" (2023). Research at Children's Mercy Month 2023. 9.
https://scholarlyexchange.childrensmercy.org/research_month2023/9