The goal of this proposal is to identify the mechanism of N-myc transcriptional downregulation in neuroblastoma cells by retinoic acid (RA). RA exerts its transcriptional effects on N-myc through a roughly defined region of the promoter (RA response region, or RARR). These studies will test the hypothesis that RARR mutations in N-myc expressing cells leads to alteration of the complement or configuration of transcription factors that bind to the promoter, resulting in the inability to downregulate N-myc after RA treatment, the phenotypic resistance of cells to RA-induced differentiation, and the lack of clinical response to RA. In the first specific aim, transient transfections and DNA gel shift, cross linking, and footprinting analyses will be used to identify and characterize the promoter sequences and DNA binding proteins necessary for basal N-myc transcription, its downregulation by RA, and its constitutive expression in RA resistant cells. In the second specific aim, the biological effects of mutations that block the RA induced downregulation of the promoter will be tested using stable transfectants containing N-myc driven by either wild type or mutated promoters. The third specific aim will determine the clinical significance of RARR mutations by comparing their incidence in RA sensitive and resistant cell lines, in tumor pairs obtained at diagnosis and after relapse post-RA therapy, and in high and low risk tumors. The presence of RARR mutations at diagnosis will also be correlated with decreased survival in a population of high-risk neuroblastoma patients treated with RA. RA is currently the only effective biologic modifier for the therapy of neuroblastoma patients with minimal residual disease. Results obtained from these investigations will provide a molecular-based, mechanistic foundation that will allow eventual prognostication of patients with respect to treatment with RA. Such data will aid in identifying RA-resistant patient candidates for trials of other biologically active agents. Ultimately, an understanding of oncogene regulation will facilitate the future rational development of combination differentiation therapy of neuroblastoma, which might be effective even in the induction setting against bulky disease.
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