Abstract 1104: Identification of genes required for glioblastoma stem cell growth and survival using pooled RNAi screening with next generation sequencing

Glioblastoma Multiforme (GBM) is the most common type of malignant brain cancer. Due to its aggressive and invasive phenotype, GBMs are classified as a grade IV tumor, and have a very poor prognosis. The GBM tumor cell population is heterogeneous and a small percentage of the tumor population, known as GBM stem cells, has the capacity to initiate and sustain tumor growth. These GBM stem cells (GBM-SCs) possess properties of self-renewal, high proliferation, radio- and chemo-resistant and have the ability to survive in hypoxic niches. In order to more effectively treat the tumor and prevent relapse, it is important to target the GBM-SC population within the tumor in addition to the bulk tumor population, which is not achieved by the conventional therapy. We are using an in vitro pooled RNAi screening approach to identify novel genetic targets for the development of drugs targeting the GBM stem cell population. In this approach, we will knockdown 10,000 genes in the GBM stem cells and perform a loss-of-function screen in order to identify the shRNAs that are inhibitory to the cellular growth in hypoxic as well as under normoxic conditions by quantifying the shRNA depletion between the conditions and a reference sample. Using this approach, we have not only identified genes which have been previously characterized to be important in GBM-SC biology such as CDK4, KIF11 and Ran, we have also identified genes, which have not been well studied in the context of GBM stem cell biology. Serum and Glucorticoid regulated Kinase -1 (SGK1) is a AGC Kinase family member which was scored as a hit in two different GBM-SC lines in the pooled screening, as well as in a separate kinase specific arrayed shRNA screen performed in the lab under both normoxic and hypoxic conditions. SGK1 is a known modulator in the PI-3 Kinase pathway and is thought to have a complementary role to Akt signaling. We have validated SGK1 as a gene important in GBM-SC survival using shRNA mediated knockdown as well as using pharmacological inhibition of the kinase in 4 different patient derived GBM stem cell lines. Furthermore, depletion of SGK1 protein results in an increase in cleaved PARP protein, which is a known marker of apoptosis induction, indicating SGK1s role as a pro-survival kinase. Further experiments are being performed to elucidate the important of this kinase in vivo using mouse xenograft experiments, as well as the pathway mediators through which SGK1 signaling occurs in GBM. Using this non-biased screening approach, we identified several genes important in GBM stem cell survival in hypoxic and normoxic conditions. Further, pathway analysis of the putative hits in will also provide us with an insight into the biochemical pathways that regulate these cells leading to development to better therapeutics for GBM. Citation Format: Shreya Kulkarni, Surbhi Goel-Bhattacharya, Sejuti Sengupta, Brent Cochran. Identification of genes required for glioblastoma stem cell growth and survival using pooled RNAi screening with next generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1104. doi:10.1158/1538-7445.AM2015-1104