Peripheral neuroblastic tumors (PNTs) share a common origin in the sympathetic nervous system, but manifest variable differentiation and growth potential. Malignant neuroblastoma (NB) and benign ganglioneuroma (GN) stand at opposite ends of the clinical spectrum. We hypothesize that a common PNT progenitor is driven to variable differentiation by specific developmental signaling pathways. To elucidate developmental pathways that direct PNTs along the differentiation spectrum, we compared the expression of genes related to neural crest development in GN and NB. In GNs, we found relatively low expression of sympathetic markers including adrenergic biosynthesis enzymes, indicating divergence from sympathetic fate. In contrast, GNs expressed relatively high levels of enteric neuropeptides and key constituents of the Hedgehog (HH) signaling pathway, including Dhh, Gli1 and Gli3. Predicted HH targets were also differentially expressed in GN, consistent with transcriptional response to HH signaling. These findings indicate that HH signaling is specifically active in GN. Together with the known role of HH activity in enteric neural development, these findings further suggested a role for HH activity in directing PNTs away from the sympathetic lineage toward a benign GN phenotype resembling enteric ganglia. We tested the potential for HH signaling to advance differentiation in PNTs by transducing NB cell lines with Gli1 and determining phenotypic and transcriptional response. Gli1 inhibited proliferation of NB cells, and induced a pattern of gene expression that resembled the differential pattern of gene expression of GN, compared to NB (p<0.00001). Moreover, the transcriptional response of SY5Y cells to Gli1 transduction closely resembled the transcriptional response to the differentiation agent retinoic acid (p<0.00001). Notably, Gli1 did not induce N-MYC expression in neuroblastoma cells, but strongly induced RET, a known mediator of RA effect. The decrease in NB cell proliferation induced by Gli1, and the similarity in the patterns of gene expression induced by Gli1 and by RA, corroborated by closely matched gene sets in GN tumors, all support a model in which HH signaling suppresses PNT growth by promoting differentiation along alternative neural crest pathways.

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2077 Background: Cerebellar granule cell precursors (CGCPs) are neuroblasts that proliferate in early postnatal life and may become transformed, giving rise to medulloblastoma. The proliferation of CGCPs is driven by mitogenic signals including Sonic Hedgehog (SHH), and growth factors that activate the protein mTOR. Dysregulation of these intercellular signals can promote medulloblastoma formation. We propose that microenvironmental signals that down-regulate the response of CGCPs to mitogens may inhibit medulloblastoma growth. Retinoic acid (RA) is an endogenous signaling molecule with potent anti-neoplastic effects. We investigated whether SHH, mTOR, and RA signaling pathways interact to regulate CGCP and medulloblastoma proliferation. Methods: We measured proliferation in cultured CGCP explants and the CGCP-derived murine medulloblastoma cell line PZp53 using quantitative phosphohistone-H3 immunocytochemistry. We examined the effects of adding to culture medium SHH, the mTOR inhibitor rapamycin, and all trans-RA (ATRA) in specific combinations. We compared CGCPs from wild type animals to CGCPs from mice with constitutive mTOR activation due to TSC2 mutation. Results: A minimum concentration of 1uM ATRA inhibited SHH-driven CGCP proliferation measurably but incompletely, while 10uM ATRA caused widespread necrosis. CGCPs from TSC2 mutant animals, in which mTOR was constitutively active, were 50% less effected by 1uM ATRA than wild type CGCPs. PZp53 medulloblastoma cells were relatively resistant to ATRA, tolerating 10uM ATRA with reduced but persistent proliferation. 10nM rapamycin decreased but did not eliminate PZp53 proliferation. The combination of rapamycin and ATRA, however, acted synergistically, suppressing proliferation >90%. This suppression persisted at 10-fold lower drug concentrations. Conclusions: CGCPs and CGCP-derived medulloblastoma cells integrate signals transduced by SHH, mTOR, and RA pathways. These signaling pathways can be manipulated by pharmacologic agents in combinations that confer dramatically enhanced antineoplastic effect. We are investigating the molecular basis of the synergy of rapamycin and ATRA. We plan to test the combination in xenografts and ultimately in patients with medulloblastoma. No significant financial relationships to disclose.