Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl − clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl − clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl − clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl − clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl − clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of "cells-of-origin" in epithelial cancers, namely their propensity for switch to primitive cell states.

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain cancer and has a very poor prognosis. A subpopulation of cells known as GBM stem-like cells (GBM-SC) have the capacity to initiate and sustain tumor growth and possess molecular characteristics similar to the parental tumor. GBM-SCs are known to be enriched in hypoxic niches and may contribute to therapeutic resistance. Therefore, to identify genetic determinants important for the proliferation and survival of GBM stem cells, an unbiased pooled shRNA screen of 10,000 genes was conducted under normoxic as well as hypoxic conditions. A number of essential genes were identified that are required for GBM-SC growth, under either or both oxygen conditions, in two different GBM-SC lines. Interestingly, only about a third of the essential genes were common to both cell lines. The oxygen environment significantly impacts the cellular genetic dependencies as 30% of the genes required under hypoxia were not required under normoxic conditions. In addition to identifying essential genes already implicated in GBM such as , and, the screen also identified new genes that have not been previously implicated in GBM stem cell biology. The importance of the serum and glucocorticoid-regulated kinase 1 (SGK1) for cellular survival was validated in multiple patient-derived GBM stem cell lines using shRNA, CRISPR, and pharmacologic inhibitors. However, SGK1 depletion and inhibition has little effect on traditional serum grown glioma lines and on differentiated GBM-SCs indicating its specific importance in GBM stem cell survival. This study identifies genes required for the growth and survival of GBM stem cells under both normoxic and hypoxic conditions and finds SGK1 as a novel potential drug target for GBM. .

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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

Glioblastoma stem like cells (GSC) are known to have gene expression patterns and growth phenotypes more similar to the tumor of origin than do traditional glioma cell lines grown in serum. They also exhibit increased resistance to drugs and radiation and are likely to be responsible for tumor recurrence following therapy. As a result, it is important to identify therapeutic targets for these cells. Toward this end, we have conducted RNAi kinome screens in GSCs to identify genes that are required for their growth and survival. In addition, glioblastomas (GBM) often have significant regions of hypoxia and GSCs tend to be resistant to hypoxia. We have therefore conducted shRNA screens in both normoxic and hypoxic conditions and screened three independent patient derived GSC lines. Of 501 kinases screened, 8-11% were scored as hits with at least 2 shRNAs per kinase in each cell line, but only ∼2% were common hits for all the cell lines in both oxygen conditions. The majority of the hits were shared in both oxygen conditions, but about one sixth were specific to hypoxia in each of the cell lines. However, none of the hypoxia specific hits were common to all three cell lines. Several genes well known to have a role in GBM such as EGFR, CDK4 and KDR were scored as common hits. The non-receptor associated tyrosine kinase YES1 was scored as a novel hit common to all the three cell lines in both the oxygen conditions. YES1 is overexpressed in GBM and several other solid tumors. Depletion of YES1 expression by shRNA and CRISPR was found to be strongly growth inhibitory in GSCs suggesting that it could be a therapeutic target for GBM. Ongoing experiments include studying the mechanism of YES1 associated cell death. Citation Format: Surbhi Goel- Bhattacharya, Sejuti Sengupta, Brent H. Cochran. An RNAi kinome screen identifies YES1 as a potential target for glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C160.