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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. 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Oscar Krijgsman

PROVIDER: E-GEOD-61826 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

2014-09-30 | GSE61826 | GEO

RAN-seq analysis comparing gene expression difference between control and Decr2 knock-down B16.SIY tumors

Project description:Purpose: To undstand the mechanism by which Decr2 knock-down tumors were resistant to CD8+ T cell killing in vitro and anti-PD-L1 therapy in vivo, we performed RNA-seq analysis comparing Decr2-knockdown versus control B16.SIY cells Methods: mRNA was isolated from Decr2 shRNAs (2 shRNA) and Control shRNA infected B16.SIY cells. mRNA profiles were generated by deep sequencing, in triplicate, using the Illumina HiSeq platform. Alignment was performed using the Kallisto software. All submitted samples passed quality control using the R package FastQC. Raw read counts were processed by TMM normalization followed by log2 transformation. Limma voom was used to identify differentially expressed genes between DECR2 knockdown and control cells.

2022-02-11 | GSE196123 | GEO

Identification of Zdhhc20 knock-down cell palmitome

Project description:An shRNA screen to search for the mediators of pancreatic cancer metastasis identified palmitoyl transferase ZDHHC20 as a protein that mediates this process by promoting the outgrowth of metastatic cells in vivo. We sought to identify the changes in the palmitome of the cells expessing scrambled shRNA or shRNAs targeting Zdhhc20.

2023-02-09 | PXD038048 | Pride

Immunotherapy Targets in Tumors

Project description:Recent work has shown that cytotoxic T cells play a central role in immune-mediated control of cancers1-3, and monoclonal antibodies that target inhibitory receptors on T cells can induce significant clinical benefit in patients despite advanced disease4-6. However, many of the regulatory pathways that result in loss of T cell function within immunosuppressive tumors remain unknown. Here we show that such regulatory mechanisms can be systematically discovered in vivo in the tumor microenvironment. We devised a pool shRNA screening approach aimed at identifying genes that block the function of tumor-infiltrating CD8 T cells. We postulated that shRNAs targeting key inhibitors would enable robust T cell infiltration and proliferation in tumors, despite multiple inhibitory signals. Candidate shRNAs were discovered by transfer of shRNA-transduced T cells into tumor-bearing mice, followed by deep sequencing to quantify the representation of all hairpins in tumors and lymphoid organs. A subset of shRNAs induced T cell accumulation in tumors but not the spleen, demonstrating feasibility of discovering shRNAs with differential action across tissues. One of the targets was Ppp2r2d, a regulatory subunit of the family of PP2A phosphatases7. Control shRNA-transduced T cells underwent apoptosis upon recognition of melanoma cells, while Ppp2r2d shRNA-transduced T cells accumulated in tumors due to enhanced proliferation and reduced apoptosis. Ppp2r2d shRNAexpressing T cells also significantly delayed tumor growth. This in vivo approach has wide applications to dissect complex immune functions in relevant tissue microenvironments.

2014-03-14 | GSE53388 | GEO

Immunotherapy Targets in Tumors

2014-03-14 | E-GEOD-53388 | biostudies-arrayexpress

Sub-genomewide shRNAs constructed using an optimized selection algorithm and microRNA backbone provide stronger evidence for follow-up studies

Project description:Sub-genomewide shRNA libraries were constructed using the current RNAi consortium constructs as well as using the DSIR (siRNA algoirthm) and a novel shRNA specific algorithm (shERWOOD). All libraries were placed into mir30 expression vectors. The shERWOOD libraries were also placed in a vector harboring an optimized mir cassette (ultramir). Each library was screened using the pancreatic cell line A385. A concensus set of essential genes identified as the set for which two shRNAs depleted in each of the libries. For these genes, a great percentage of shERWOOD seletected shRNA depleted. In addition the placement of shERWOOD selected constructs into ultramir scaffoled increased the rate of shRNA depletion for essential genes further. Purpose: shRNA screens were carried out using various library construction strategies to identify the strategy that provides the best shRNA screening results. Method: Libraries were constructed using the TRC shRNA set as well as shRNAs identified using the DSIR and shERWOOD algorithms. shRNA libraries were cloned into mir30 expression vectors. shERWOOD shRNAs were also cloned into an expression vector harboring an optimized microRNA scaffold termed ultramir. Each resultant library was screened using the pancreatic cell line A385. Each library was analyzed separately to identify a set of genes where at least two shRNAs depleted. These gene sets were intersected to develop a set of essential genes. Results: The shERWOOD shRNA libraries provided the highest number depleting shRNAs for each essential gene. Further these shRNAs depleted to a greater extent than did the shRNAs from the other libraries. When shERWOOD libraries were placed into the ultramir cassette a greater number of shRNAs per essential gene depleted.

2014-11-24 | E-GEOD-62185 | biostudies-arrayexpress

shRNA profiling of human breast carcinoma cells expressing over 300 shRNA constructs using barcode tiling arrays - screen 1

Project description:RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct.. In this part of the study pooled shRNAs were transduced into MDA-MB-231 breast carcinoma cell lines and their inhibitory effects four weeks post transduction were analyzed via microarray hybridization.

2009-12-08 | E-MEXP-2476 | biostudies-arrayexpress

shRNA profiling of human breast carcinoma cells expressing over 300 shRNA constructs using barcode tiling arrays - screens 2 and 3

2009-12-08 | E-MEXP-2477 | biostudies-arrayexpress

shRNA cassette sequencing from disomic and trisomic fibroblasts cultured in the presence of shRNA kinome library for 14 days

Project description:Kinome-wide shRNA screen to identify kinases differentially required for survival of cells disomic and trisomic for chromosome 21 Cells were transduced in triplicate and propagated for 14 days to allow for enrichement or depletion of shRNAs from the population

2016-07-29 | E-GEOD-79840 | biostudies-arrayexpress

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

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