Oral Presentation Advances in Neuroblastoma Research Congress 2016

GPC2 is a putative oncogene and candidate immunotherapeutic target in high-risk neuroblastoma (#26)

Kristopher R Bosse 1 2 , Pichai Raman 1 3 , Robyn T Sussman 1 , Michael Randall 1 , Dan Martinez 4 , Zhongyu Zhu 5 , Jo Lynne Harenza 1 , Bruce Pawel 4 , Tricia Bhatti 4 , Javed Khan 6 , Dimiter S Dimitrov 5 , Crystal Mackall 7 , John M Maris 1 2
  1. Division of Oncology and Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia, PA, USA
  2. Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
  3. Department of Biomedical and Health Informatics, Children's Hospital of Philadelphia, Philadelphia, PA, USA
  4. Department of Pathology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA
  5. Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA
  6. Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
  7. Stanford Cancer Institute, Stanford University, Stanford, CA, USA

Background: Immunotherapeutic strategies have improved outcomes in high-risk neuroblastoma; however a major challenge remains in identifying cell surface molecules that meet the criteria for modern immune-based therapies.

Methods: Potential cell surface immunotherapeutic targets were identified by comparing high-risk neuroblastoma (N=126 primary tumors) and normal tissue RNA sequencing data (GTEx; N=25 normal tissues, 1-313 replicates/tissue). Genes were filtered by cell surface prediction, absolute RNA expression, and tumor DNA copy number. Candidate gene protein expression and cellular localization were confirmed in neuroblastoma primary tumors, patient-derived xenografts (PDXs), and cell lines. Genes were further surveyed for MYCN-mediated transcriptional control with chromatin immunoprecipitation (ChIP) sequencing. Finally, neuroblastoma cell lines were subjected to candidate gene gain and loss of function studies.

Results: The transcriptome-based discovery identified 86 differentially expressed predicted cell surface molecules. We prioritized the 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<3 x 10-10), potential tumor-specific epitope expression, high-level absolute RNA expression (median FPKM=57) and frequent DNA copy number gain (43% of tumors; N=170) associated with higher GPC2 expression (p<0.001). MYCN amplification was also correlated with higher GPC2 expression (p<0.01), MYCN was found to bind the GPC2 promoter by ChIP sequencing, and MYCN depletion resulted in significantly decreased GPC2 expression. Immunoblot, immunofluorescence, immunohistochemistry (IHC), and membrane extraction analysis of primary tumors, PDXs, and cell lines confirmed dense plasma membrane GPC2 expression. IHC analysis of 41 pediatric tissues confirmed very limited normal tissue GPC2 expression. GPC2 depletion in neuroblastoma cell lines (N=12) resulted in significant apoptosis and growth inhibition, and GPC2 forced overexpression significantly increased neuroblastoma cell proliferation. Finally, fully human antibodies specifically targeting neuroblastoma-associated GPC2 were created after identification of specific Fab binders from a phage display antibody library.

Conclusions: GPC2 is a neuroblastoma oncogene and candidate immunotherapeutic target.