Abstract A154: Mechanisms of cellular resistance to the growth inhibitory and cytotoxic effects of MDM2-p53 binding antagonists

Abstract

Abstract Background: About 50% of solid tumors and more than 80% of haematological malignancies express wild-type p53 at diagnosis. Therefore, activation of the p53 pathway by antagonizing its negative regulator Murine Double Minute 2 (MDM2) may offer a new therapeutic strategy for cancer treatment. Recently, several potent and selective small molecule antagonists of the p53-MDM2 protein-protein interaction have been developed. Studies with these compounds have strengthened the concept that selective, non-genotoxic p53 activation may in some circumstances represent an alternative to current cytotoxic chemotherapy. Aims: To investigate the potential for drug resistance against p53-MDM2 interaction antagonists and to establish therapeutic strategies to circumvent this. Methods: NGP neuroblastoma and SJSA-1 osteosarcoma cell lines have been used to select a series of clonal variants resistant to two potent small molecule inhibitors of the MDM2-p53 interaction Nutlin-3 [Science 2004, 303, 844] and MI63 [J.Med.Chem. 2005, 48, 909]. The drug resistance phenotype of these clones was evaluated by SRB growth inhibition assay, western blotting for activation of p53 and downstream mediators of p53 dependent growth inhibition and cytotoxicity, and caspase-3/7 activation analysis, in response to drug treatment. DNA sequence analysis for p53 mutation detection was also conducted to explore possible resistance mechanisms. Results: Following 60-day exposure to 1-5µM plus 60-day exposure to 5-40µM of MI63 or Nutlin-3 respectively, several clones were selected showing 11.5–43.2 fold increases in GI50 values. Clones selected for resistance to one of the agents showed cross-resistance to the other. This increased resistance was reflected in reduced activation of growth inhibition and apoptosis pathways downstream of p53, although induction of p53 protein itself was little changed compared to parental cell lines. All clones exhibited undetectable accumulation of MDM2 and p21 proteins compared with the marked changes seen with parental cells after treatment with 20 µM of Nutlin-3 or 5 µM MI63 for 4–6 hours, even though the p53 protein accumulated to the same extent as parental cells. Decreased cleavage of caspase-3 and PARP proteins were also observed, consistent with the reduced apoptosis. Caspase-3/7 enzymatic activity in the resistant clones in response to exposure to MI63 or Nutlin-3 for 48 hours was also significantly reduced compared to the response in parental cell lines. Point missense mutations of the p53 gene were observed within the resistant cell clones of both SJSA-1 and NGP cell lines. Conclusions: As with other therapeutic agents, treatment with MDM2-p53 binding antagonists is likely to be subject to the development of drug resistance mechanisms. A panel of clones selected for resistance to Nutlin-3 and MI63 showed impaired activation of growth inhibitory and apoptotic pathways downstream of p53 in response to treatment with these agents compared to parental cells. DNA sequencing and p53 pathway analysis were consistent with a p53 mutation resistance mechanism. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A154.