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PARALLEL IN VIVO AND IN VITRO MELANOMA RNAi DROPOUT SCREENS

ABSTRACT: To identify factors preferentially necessary to drive tumor expansion we performed parallel in vitro and in vivo negative selection shRNA screens. Melanoma cells harboring shRNAs targeting several DNA Damage Response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for identification of pharmacologically tractable targets.

ORGANISM(S): Homo sapiens

PROVIDER: GSE61826 | GEO | 2014/09/30

SECONDARY ACCESSION(S): PRJNA262511

REPOSITORIES: GEO

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PARALLEL IN VIVO AND IN VITRO MELANOMA RNAi DROPOUT SCREENS

Project description:To identify factors preferentially necessary to drive tumor expansion we performed parallel in vitro and in vivo negative selection shRNA screens. Melanoma cells harboring shRNAs targeting several DNA Damage Response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for identification of pharmacologically tractable targets. A lentivirus-based kinome shRNA library (four pools) was used to transduce 888mel cells (MOI<0.2). After puromycin selection (1μg/ml), two reference samples were collected as controls. Next, tumor cells (5x105 per injection) were either injected s.c. into 6 NSG mice or plated into 6 independent plates (5x105) for in vitro culture. Tumors were removed from the mice and genomic DNA used to recover shRNAs by PCR amplification followed by deep sequencing. Three analyses were performed independently in parallel: (1) Tumors versus cultured cells (Log2 Fold Change < -1); (2) Tumors versus references (Log2 Fold Change < -2.5) and (3) Cultured cells versus references (Log2 Fold Change < -2.5). Genes targeted with at least 2 shRNAs in each of the analysis were considered hits with an enhanced in vivo effect.

2014-09-30 | E-GEOD-61826 | biostudies-arrayexpress

Homo sapiens

Project description:PARALLEL IN VIVO AND IN VITRO MELANOMA RNAi DROPOUT SCREENS

| PRJNA262511 | ENA

A genome-wide screen identifies competitive growth advantage for human epidermal stem cells with activated YAP

Project description:We performed genome-wide pooled RNAi screens for genes that confer a clonal growth advantage or disadvantage on epidermal stem- and cutaneous squamous cell carcinoma (SCC) cells What we know from our cell biology is that a shRNA underrepresented at t=14 targets a gene that confers growth advantage.

2017-03-31 | GSE79560 | GEO

Effect of depleting GNB1L on PIKK mRNA levels to explore the regulatory mechanism of GNB1L on PIKK proteins

Project description:Our screens identified GNB1L, a high-confidence hit, as a novel positive regulator of DDR signaling. We therefore generated HEK293A-GNB1L-dTAG cells which the GNB1L-dTAG-HA protein decreased to markedly lower levels in the presence of dTAGv-1 than that in cells treated with dTAG-NEG , we next examined whether depletion of GNB1L affects DDR signaling. Notably, depletion of GNB1L diminished all IR-induced DDR signaling. Moreover, depletion of GNB1L also significantly reduced basal DDR signaling as well as PIKKs. To determine whether GNB1L depletion specifically affects PIKKs instead of other DDR factors, we carried out label-free quantitative proteomics analysis by comparing the proteomes in NEG-treated versus in dTAGv-1-treated GNB1L-dTAG cells. The data showed that GNB1L depletion specifically reduced PIKK kinases and ATRIP rather than other DDR factors. Obviously, PIKK kinases, ATRIP and mTOR are major downregulated substrates affected by GNB1L depletion. Therefore, we performed RNA-seq to rule out the possibility that GNB1L regulates PIKK protein levels via transcriptional regulation. The differentially expressed genes (DEGs) as well as the GO analysis confirmed that depleting GNB1L had no effect on PIKK mRNA levels . These data suggested that GNB1L specifically regulates PIKK protein levels instead of other DDR factors.

2023-06-23 | GSE235200 | GEO

CRISPR screens are feasible in TP53 wild-type cells

Project description:CRISPR-Cas9 genome-wide screens were performed in retinal pigment epithelial cells (RPE1) with either wild-type TP53 gene, or a TP53-null background. Results show wild-type TP53 has minimal impact on the efficiency of CRISPR dropout screens.

2019-04-30 | GSE128210 | GEO

Lentiviral shRNA library screen to identify growth-related genes in malignant melanoma

Project description:Little is known about genes that promote melanoma cell growth and proliferation. siRNAs may be used to address the role of individual genesin these processes. RNAi library screens were used in the past to gain a comprehensive overview of all genes involved in cell growth, proliferation, migration and other cellular processes. A large-scale loss-of-function screen for eight different melanoma cell lines was performed using a pooled lentiviral shRNA library (GeneNet Human 50K lentiviral shRNA Library,cat#SI206B-1, System Biosciences) to identify genes relevant for melanoma cell growth and proliferation. shRNAs that lead to cell death or reduced growth of transduced melanoma cells are negatively selected and thereby underrepresented in the final cellular shRNA pool and vice versa. The shRNAs of the shRNA library (3-5 per gene) have complementary sequences to probes on the custom Affymetrix microarray HG-U133Plus2 and were analysed using this array. Well-known melanoma cell lines SK-Mel-103, A375, SK-Mel-147, SK-Mel-19, SK-Mel-28, SK-Mel-29, SK-Mel-5, WM3523cln6 were transduced with the lentiviral shRNA library and grown for 10 days under puromycin selection (day 10), control cells of respective cell lines were transduced and frozen immediately after transduction and genomic integration of shRNAs (day 0). Totel DNA was extracted and genomically integrated shRNAs were hybridized to Affymetrix microarrays (HG-U133Plus2.0 array).

2014-01-24 | E-GEOD-46675 | biostudies-arrayexpress

Lentiviral shRNA library screen to identify growth-related genes in malignant melanoma

2014-01-24 | GSE46675 | GEO

Hypoxia-driven alterations in the B16 melanoma cell transcriptome account for a higher metastatic potential

Project description:Hypoxia-driven alterations in the B16 melanoma cell transcriptome account for a higher metastatic potential: evidence for a role of Ero1L We analyzed transcriptomic adaptations to hypoxia/reoxygenation in B16 melanoma cells. By Ero1L over- and down-expression in vivo, we identified this ER oxidase as an actor of tumor growth and metastasis take. In vitro culture of B16 submitted to hypoxia (oxygen rate less than 1%)

2013-08-01 | E-GEOD-14727 | biostudies-arrayexpress

Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress

Project description:The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR and DNA-PK. ATM is missing or inactivated in the genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the double-strand break-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine-tuned dynamics between the PIKKs following genotoxic stress, such as DNA-PK-dependent attenuation of ATM. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics. Our results highlight the intricate relationships between these PIKKs in regulating the DDR.

2020-11-25 | PXD020884 | Pride

Identification of critical therapeutic nodes to overcome Asparagine deprivation resistance in melanoma

Project description:Asparagine deprivation by L-Asparaginase is a successful therapeutic strategy in Acute Lymphoblastic Leukemia, with resistance occurring due to upregulation of ASNS. L-Asparaginase efficacy in solid tumors is hampered by dose-related toxicities. Large scale loss of function genetic screens identified ASNS, the only human enzyme synthetizing asparagine, as a cancer dependency in several solid malignancies, including melanoma. We here evaluate the therapeutic potential of targeting ASNS in melanoma cells in-vitro and in-vivo. Using ex-vivo quantitative proteome and transcriptome profiling, we observed that concomitant ASNS deletion and asparagine deprivation elicit a compensatory mechanism allowing tumor growth. Genome wide CRISPR screens upon manipulation of aminoacid levels identifies MAPK and GCN2 as critical nodes mediating the observed resistance mechanism. Importantly MEK and GCN2 inhibitor synergize with L-Asparaginase suggesting novel potential therapeutic strategy in melanoma.

2022-02-15 | PXD015152 | Pride

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