Abstract B39: Investigation of the effects of alterations in the glutamate receptor, GRIK2, on osteosarcoma tumorigenesis

Osteosarcoma is a primary, malignant bone tumor that mainly occurs in young adults and survival rates (60-70% over five years) have remained steady and novel treatment targets are needed. Osteosarcoma tumors exhibit chromosomal instability and are heterogeneous. In this study we identified recurrent copy number alterations in 44 flash-frozen osteosarcoma tumors obtained at biopsy with 25 blood-matched samples. Copy number losses were observed in 14% of tumors in the 6q16.3 region, specifically in the 5 intergenic space and first intron of the GRIK2 gene. GRIK2 encodes an ionotropic glutamate receptor that has been identified as a tumor-suppressor gene in gastric cancer, but the relationship between alterations in this region and osteosarcoma are still to be determined. We found that the focal deletions do not overlap with any coding sequence of GRIK2; however, there is a region of interest that contains a SETDB1 histone methyltransferase binding site that is shared by several deletions. The 5 intergenic space is enriched with H3K9Me3 histone marks which is indicative of transcriptional repression. SETDB1 is responsible for adding H3K9Me3 histone marks and deletion of this site may alter the expression of GRIK2. The goal of this study is to determine the relationship between noncoding deletions in 6q16.3 and GRIK2 by examining the effects of the deletions on the expression, function, and regulation on osteosarcoma cells in vitro. Gene expression levels of GRIK2 were quantified with RT-qPCR in both osteosarcoma tumors and cell lines. The functional role of GRIK2 was characterized in both U2-OS and SaOS-2 osteosarcoma cells through gain-of-function experiments. Cell viability was measured by the XTT assay and cell migration was quantified with a wound-healing assay. Apoptosis levels were assessed with an annexin V experiment. The effect of noncoding deletions of the SETDB1 binding site was examined using CRISPR/cas9 gene editing. Following transfection of a CRISPR/cas9 vector, U2-OS cells were sorted by fluorescence-activated cell sorting, grown for 7-10 days, and GRIK2 gene expression levels were measured. The expression levels of GRIK2 varied in tumors and were low in cell lines. Several tumors with focal deletions were among a subset of tumors with increased transcript levels relative to cell lines. Due to this observation, GRIK2 was overexpressed in U2-OS and SaOS-2 cells and found to result in decreased cell proliferation, migration, and increased apoptosis levels. Gene editing of the SETDB1 binding site was confirmed following DNA extraction and PCR of the deleted region. Following cell sorting and growth, GRIK2 transcript levels were increased in edited cells relative to untreated cells. Noncoding deletions in the 6q16.3 region were associated with increased expression levels of GRIK2 and overexpression of GRIK2 in vitro may result in a less aggressive phenotype. Gene editing of the SETDB1 binding site with CRISPR/cas9 demonstrated that focal deletions of the region may be involved in epigenetic regulation of GRIK2. Noncoding deletions of GRIK2 are linked to expression and functional changes in GRIK2 and further studies will examine GRIK2 as a potential target for therapeutic intervention. Citation Format: Justin G. Mayers, Nalan Gokgoz, Jay S. Wunder, Irene L. Andrulis. Investigation of the effects of alterations in the glutamate receptor, GRIK2, on osteosarcoma tumorigenesis [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B39.