Abstract 1413: Dissecting the biology of mutant p53 with the Cellular Thermal Shift Assay (CETSA)

The tumor suppressor protein p53 is one of the most important safeguards against malignant transformation of mammalian cells, but it is also one of the most frequently mutated proteins in cancers. Therefore, p53 constitutes an attractive drug target in oncology. However, there has been little success in finding clinical candidate drugs that directly manipulate the function of p53. The p53 protein offers several challenges for investigators wanting to manipulate its function e.g., the absence of accessible binding pockets, lack of enzymatic activity, and its intracellular location. Moreover, another complication with p53 is the plethora of mutations that alter the protein’s characteristics. Most mutational changes in p53 result in a loss of function, but there are also examples of gain of function mutations in p53. We have applied the Cellular Thermal Shift Assay (CETSA) for screening of new chemical hit matter for binding to p53. As CETSA exploits the compound induced changes in protein melting behavior it is important to know the melting characteristics of the protein under investigation. Since p53 displays a wide variety of mutational variants, we decided to investigate three different variants present in three separate breast cancer cell lines: SK-BR-3 (p53R175H), BT-474 (p53E285K), and MCF-7 (p53WT). The melting behavior of p53 protein was similar irrespective of genomic status of p53 in intact cells. However, in lysed cells there was striking difference in p53 protein melting between MCF-7 cells that carry WT p53 and the two other breast cancer cell lines that carry mutations in the gene coding p53. The melting temperature for WT p53 was nearly the same in both lysate and intact cells, whereas there was 3 to 4°C difference between intact cells and lysates derived from the two cell lines that expressed mutated p53. Both of the mutated forms of p53 found in SK-BR-3 cells and BT-474 cells displayed greater thermal stability in lysate compared to MCF-7. Intrigued by this difference we set out to investigate its cause. It is known that certain mutated forms of p53 are rescued from proteasomal degradation by associating with chaperone proteins like Hsp90. Therefore, the different breast cancer cell lines were treated with inhibitors of Hsp90 function and protein melting of the respective form of p53 was assessed. Hsp90 modulation by Geldanamycin had a much more potent effect on thermal stability of mutant p53 compared to the WT counterpart, which indicates that Hsp90 could be involved in conferring increased thermal stability of mutant p53. Citation Format: Tomas Friman, Merve Kacal, Laurence Arnold, Daniel Martinez Molina. Dissecting the biology of mutant p53 with the Cellular Thermal Shift Assay (CETSA) [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 1413.