Abstract PR02: GPC2 is a candidate immunotherapeutic target and putative oncogene in high-risk neuroblastoma and other pediatric cancers
Authors: Bosse Kristopher R. , Raman Pichai , Sussman Robyn T. , Randall Michael , Martinez Dan , Zhu Zhongyu , Pawel Bruce , Bhatti Tricia , Khan Javed , Dimitrov Dimiter S. , Mackall Crystal , Maris John M. 
Published: May 2016

Abstract
Background: Children with high-risk neuroblastoma have a poor prognosis despite intensive multimodal chemoradiotherapy. While monoclonal antibodies targeting the disialoganglioside GD2 improve outcomes in neuroblastoma, this therapy is associated with significant “on target-off tumor” toxicities. Thus, a major challenge remains in identifying novel cell surface molecules that meet the stringent criteria for modern immunotherapeutics, including unique tumor expression compared to normal childhood tissues, and preferably that these cell surface molecules be required for tumor sustenance and thus may be less susceptible to immune escape mechanisms.
Methods: Differentially expressed genes that represent putative cell surface immunotherapeutic targets were initially identified by comparing high-risk neuroblastoma RNA sequencing data (N=126 primary tumors) to paired normal tissue data (GTEx; N=25 unique normal tissues; N=1-313 replicate samples/tissue type). For further prioritization, gene sets were filtered by in silico cell surface prediction and by absolute RNA expression, and then by assessing primary tumor DNA copy number via SNP genotyping data (N=177). For prioritized genes, protein expression and cellular localization was confirmed by Western blot, immunohistiochemistry (IHC), immunofluroescence (IF) and membrane extraction techniques in neuroblastoma cell lines and primary tumors. For functional characterization, neuroblastoma cell lines (N=12) were subjected to both gain and loss of function studies for each candidate gene.
Results: The initial transcriptome-based discovery effort identified 649 significantly differentially expressed genes (log-fold change tumor vs. normal &gt;1 for each tissue; adjusted p&lt;0.05), 86 (13%) of which were predicted to be potential cell surface molecules. Through our analytic pipeline, we prioritized the extracellular glycosylphosphatidylinositol (GPI) anchored, signaling co-receptor Glypican-2 (GPC2) for validation given robust differential RNA expression (log-fold change tumor vs. normal tissue = 2.1-8.2; p&lt;3 x 10-10), high-level absolute RNA expression (median FPKM=57; 85% of tumors with FPKM &gt;25) and consistent DNA copy number gain (31% of primary neuroblastomas; N=177) associated with significantly higher GPC2 expression (p&lt;0.0001). Immunoblot analysis confirmed ubiquitous GPC2 protein expression (N=8 high-risk neuroblastomas and 23 cell lines) and membrane extraction, IF, and IHC confirmed dense plasma membrane associated GPC2 protein expression in neuroblastoma cell lines. IHC analysis of primary neuroblastoma tumors (N=165) compared to a parallel array of pediatric normal tissues (N=41) further confirmed GPC2 protein expression to be membrane associated and tumor specific with very limited normal tissue expression. Lentiviral mediated RNAi induced GPC2 depletion in a panel of 12 neuroblastoma cell lines resulted in significant apoptosis and growth inhibition both in transient CellTiter-Glo and Caspase-Glo assays (20-87% decreased growth and 1.5-18.4-fold increased caspase 3/7 level vs. control) and with longer term real-time monitoring of cell growth (RT-CES). GPC2 overexpression resulted in significantly increased cellular proliferation (2.7-fold growth increase vs. control, p&lt;0.0001). Finally, GPC2 was also found to be significantly differentially overexpressed in other embryonal cancers, most notably medulloblastoma.
Conclusions: GPC2 is a candidate cell surface immunotherapeutic target and putative oncogene in high-risk neuroblastoma. More globally, these data show that genome-wide transcriptome analysis integrated with genomic and functional validation can identify differentially expressed cell surface oncogenes that may be attractive immunotherapeutic targets. Development of a GPC2 directed chimeric antigen receptor is ongoing, and progress will be reported.
This abstract is also presented as Poster A13.
Citation Format: Kristopher R. Bosse, Pichai Raman, Robyn T. Sussman, Michael Randall, Dan Martinez, Zhongyu Zhu, Bruce Pawel, Tricia Bhatti, Javed Khan, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is a candidate immunotherapeutic target and putative oncogene in high-risk neuroblastoma and other pediatric cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR02.