Purpose: Multiple developmental pathways including Notch, Hedgehog, and Wnt are active in malignant brain tumors such as medulloblastoma and glioblastoma (GBM). This raises the possibility that tumors might compensate for therapy directed against one pathway by upregulating a different one. We investigated whether brain tumors show resistance to therapies against Notch, and whether targeting multiple pathways simultaneously would kill brain tumor cells more effectively than monotherapy.Experimental Design: We used GBM neurosphere lines to investigate the effects of a gamma-secretase inhibitor (MRK-003) on tumor growth, and chromatin immunoprecipitation to study the regulation of other genes by Notch targets. We also evaluated the effect of combined therapy with a Hedgehog inhibitor (cyclopamine) in GBM and medulloblastoma lines, and in primary human GBM cultures.

Medulloblastoma is a malignant pediatric brain tumor. Current treatment following patient stratification into standard and high-risk groups using clinical features, has improved survival. However, a subset of patients with standard-risk features have unanticipated aggressive disease, underscoring the need for a better understanding of tumor biology and development of novel treatments. Poor differentiation, a hallmark of medulloblastomas is associated with elevated expression levels of the repressor of neuronal differentiation REST. Here, we assessed if elevated REST expression levels had prognostic significance and if its pharmacological manipulation would promote neurogenesis and block tumor cell growth. REST levels in patient tumors were measured by immunohistochemistry (IHC) and stratified into low/moderate- (+/++/+++) and high-REST (+++++) groups. Kaplan-Meier curves revealed that patients with high-REST tumors had worse overall and event-free survival compared to patients with REST-negative or REST-low tumors. Since histone deacetylases (HDACs), are required for REST-dependent repression of neurogenesis, we evaluated a panel of HDAC inhibitors (HDACIs) for their effects on growth and differentiation of established and primary REST-positive cell lines. MS-275, trichostatin-A (TSA), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) upregulated expression of the REST-target neuronal differentiation gene, Syn1, suggesting a potential effect of these HDACIs on REST function. Interestingly, VPA and TSA substantially increased histone acetylation at the REST promoter and activated its transcription, whereas SAHA unexpectedly promoted its proteasomal degradation. A REST-dependent decrease in cell growth was also observed following SAHA treatment. Thus, our studies suggest that HDACIs may have therapeutic potential for patients with REST-positive tumors. This warrants further investigation.