Nutlin‐3a selects for cells harbouring TP53 mutations

Abstract

TP53 mutations occur in half of all human tumours. Mutagen‐induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock‐in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen‐treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2–5 months) and much effort is expended maintaining TP53‐WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin‐3a, an MDM2 inhibitor that leads to stabilisation and activation of wild‐type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin‐3a to examine the effect on cell growth and p53 activation. Nutlin‐3a induced the p53 pathway in TP53‐WT HUFs and inhibited cell growth, whereas most TP53‐mutated HUFs were resistant to Nutlin‐3a. We then assessed whether Nutlin‐3a treatment could discriminate between TP53‐WT and TP53‐mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin‐3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin‐3a‐resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin‐3a‐sensitive clones were TP53‐WT. These data suggest that including a Nutlin‐3a counter‐screen significantly improves the specificity and efficiency of the HIMA, whereby TP53‐mutated clones are selected prior to sequencing and TP53‐WT clones can be discarded.

Characterising Mutational Spectra of Carcinogens in the Tumour Suppressor Gene TP53 Using Human TP53 Knock-in (Hupki) Mouse Embryo Fibroblasts

“…It is very common for a sensitive culture to fully recover after 3-7 days. Additionally, very rarely some sensitive clones carry TP53 mutations [22]. Resistant cultures show the same morphology and a similar growth rate to untreated cultures.…”

“…Over the past few years, we have developed and validated modifications to the original HIMA protocol to improve the sensitivity and applicability of the assay [13,15,22], and the updated experimental approach is illustrated in Figure 3. The two major changes are explained below.…”

“…Nutlin-3a blocks the interaction between p53 and its negative regulator MDM2 by binding to the latter [23]. When cells with WT-TP53 are treated with Nutlin-3a, p53 is stabilised and activated, leading to growth arrest [22]. Cells harbouring mutations in TP53, on the other hand, are resistant to Nutlin-3a and continue to grow normally after treatment.…”

“…Therefore, because there is no selection specifically for TP53 mutants in the original HIMA protocol, a lot of time and effort is spent culturing clones that have WT-TP53. Our group has demonstrated that a Nutlin-3a counter-screen, as shown in Figure 3, can be used to select for mutant-TP53 HUFs and against WT-TP53 HUFs once clones emerge from senescence [22]. Most mutant-TP53 HUFs, including heterozygous TP53 mutants that retain one WT allele, are identified by the Nutlin-3a counter-screen within 2.5 months of initiating the HIMA.…”

Bimodality of gene expression in cancer patient tumors as interpretable biomarkers for drug sensitivity

“…Mutation and CNV features were found to be the best in predicting sensitivity in 11.2% of the drugs (Fig 4 E and F). An example of these drugs is Nutlin-3A, an MDM2 inhibitor that activates wild-type p53 mutation 59,60 . TP53 wild-type mutation was predicted by our approach to indicate sensitivity to Nutlin-3A ( Fig 4E).…”