463. Conditionally Replicative Adenovirus Expressing Degradation-Resistant p53 for Enhanced Oncolysis of MDM2 Overexpressing Cancers

Abstract

Conditionally replicative adenoviruses (CRAds) are novel anti-cancer agents, designed to selectively replicate in and to destroy cancer cells. A critical determinant for the oncolytic potency of CRAds is their capacity to induce cancer cell death. In this respect, dysfunctional cell death pathways in cancer cells reduce CRAd efficacy. Previously, we showed that expressing p53 tumor suppressor protein from the genome of the CRAd AdΔ24 (with a deletion in the pRb-binding E1A-CR2 domain) resulted in more effective cytotoxicity on most cancer cell lines (Cancer Res., 62:6165–6171, 2002). More recently, we confirmed this finding on primary tumor cells in vitro and in vivo. However, in approximately 20% of tested cancer cell lines AdΔ24-p53 was not more effective than AdΔ24. In search for molecular determinants of resistance against the oncolysis enhancing effect of exogenous p53 we analyzed these cell lines for expression of the major negative p53 regulator MDM2. Western analysis on 5 resistant cell lines revealed strong MDM2 expression in 4 cases, suggesting that MDM2-mediated inhibition and degradation of exogenous p53 could have limited the oncolysis of these cells by AdΔ24-p53. To develop a CRAd for more effective treatment of MDM2 overexpressing cancers, we made the new AdΔ24-derivative AdΔ24-p53(14/19). This CRAd expresses a variant of p53 with amino acid substitutions L14Q and F19S that abrogate p53 binding to MDM2 (Genes Dev., 8:1235–1246, 1994). AdΔ24-p53 and AdΔ24-p53(14/19) expressed similar levels of p53 protein, which peaked at 24 hours after infection, as tested by Western analysis on infected p53-null SaOs-2 cells. Transactivation of a p53-dependent reporter construct transfected into SaOs-2 cells by AdΔ24-p53(14/19) was approximately 40% of transactivation by AdΔ24-p53. The oncolytic potency of AdΔ24, AdΔ24-p53 and AdΔ24-p53(14/19) was compared on MDM2-overexpressing MKN45 gastric carcinoma cells and MG-63, MNNG-HOS and CAL-72 osteosarcoma cells. To this end, cells were infected at a range of MOI and cultured to allow replication and lateral spread. After culture, remaining viable cells were stained with crystal violet. Consistent with previous observations, AdΔ24 and AdΔ24-p53 were similarly oncolytic. In contrast, AdΔ24-p53(14/19) was on average 10-times more effective than AdΔ24 and AdΔ24-p53. Hence, abrogating binding of exogenous p53 to MDM2 overcame resistance to p53-mediated oncolysis enhancement in MDM2 overexpressing cancer cells, confirming our hypothesis that CRAd oncolysis enhancement by exogenous p53 can be improved by preventing its degradation. Furthermore, our findings suggest that knowledge on molecular defects in cancer cells can be used to design tailored CRAds with enhanced oncolytic potency.