Optimization of GMAK to treat glioblastoma multiforme

DESCRIPTION (provided by applicant): Glial cell proliferation occurs over a spectrum from the benign, low-level cell replacement that occurs over a lifetime, through astrocytosis that occurs in response to neurotrauma and in neurodegenerative diseases to the deranged, uncontrolled growth that occurs in glioblastoma (GBM) and astrocytoma. We have previously demonstrated that contact with the neuronal cell surface is necessary and sufficient to induce astrocytes into mitotic quiescennce. Using a subtractive hybridization approach, we generated cDNA libraries from astrocytes that were cultured either in the presence or the absence of neuronal membrane proteins. We then subtracted the libraries from one-another, sequenced the differentially expressed fragments and cloned the coding cDNAs that were enriched in the "plus neuronal protein" library into a pGEX expression vector. The expressed proteins were bound to a solid substrate and tested for their ability to inhibit astrocyte proliferation. The 9th protein assayed in this was demonstrated a significant inhibition of astrocyte proliferation and was termed GM9 (growth mediator 9). GM9 (previously identified as CD81) is a 4- transmembrane domain receptor with expression restricted to astrocytes and a subset of leukocytes. GM9 is required for transduction of the anti-proliferative signal encoded on the neuronal cell-surface. The identification and characterization of GM9 has allowed us to identify and characterize its cognate ligand on the neuronal cell-surface, termed NrS1. Given the polar growth extreme of glioblastoma cells, it is not surprising that all of the tumor cell lines and primary resection and biopsy glioblastoma/astrocytoma cells examined to date have lost expression of. Genomic analysis of several of these cell lines demonstrated that the gene is intact. Treatment of these cells with a histone deacetylase inhibitor (HDACi) relieved repression of the gene, and allowed the tumor cells to "see" NrS1 when presented either in the context of the neuron or as a recombinant protein. Preliminary Data has shown that in vivo treatment of established glioblastoma with a combination of a unique HDACi and a fragment of NrS1 blocks tumor growth and invasion in vivo. The current application describes experiments intended to characterize and optimize the NrS1 fragment for treatment of GBM.