Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents

Abstract

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3rRITA10 μM to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.

Dual Targeting of Wild-Type and Mutant p53 by Small Molecule RITA Results in the Inhibition of N-Myc and Key Survival Oncogenes and Kills Neuroblastoma Cells In Vivo and In Vitro

“…This type of compounds should reduce the chance of emergence of de novo resistance and enhance clinical success. Indeed, in line with our data on the ability of RITA to reactivate mutant p53 in neuroblastoma, recent study using UKF-NB-3 neuroblastoma cells as a model does not suggest p53 mutations being the mechanism of acquired resistance to RITA, in contrast to nutlin3a (41). Interestingly, several p53-binding molecules that rescue mutant p53 have been shown to activate the function of wild-type p53 as well.…”

Resistance acquisition to MDM2 inhibitors

“…Notably, resistance acquisition to RITA, a compound that was described to interfere with the interaction of p53 and MDM2 by binding to p53 [10], was not associated with the formation of p53 loss-of-function mutations in our model system [46]. Among ten UKF-NB-3 neuroblastoma sub-lines adapted to RITA, only one sub-line harboured a (A76T) p53 mutation.…”

“…p53 wild-type nutlin-3-resistant cell lines appeared to have inactivated p53 signalling by alternative mechanisms and displayed similar characteristic cross-resistance profiles to anti-cancer drugs and radiation as p53-mutated cells [46]. Moreover, p53-mutated and p53 wild-type nutlin-3-resistant neuroblastoma cells could not be discriminated by hierarchical cluster analyses based on transcriptomics data [46].…”

“…Nutlin-3-resistant cells were established within six to 13 passages, and p53 mutations became detectable after three to five passages [42]. In addition, nutlin-3-resistant cells displayed not only resistance to nutlin-3, but also to a broad range of different cytotoxic anti-cancer drugs and radiation [42,46]. In addition, nutlin-3-resistant cells displayed not only resistance to nutlin-3, but also to a broad range of different cytotoxic anti-cancer drugs and radiation [42,46].…”

Molecular dynamics of the full-length p53 monomer

“…143 Understanding the structural restrain of its structure is pivotal to understand the function of p53 [144][145][146] as well as its potential therapeutic exploitation. 147,148 The regulation of p53 protein half-life is crucial to his function 149,150 and, consequently, for cancer progression. [151][152][153] This proteosomal degradation is indeed a powerful therapeutic target.…”