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Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening

View Article: PubMed Central - PubMed

ABSTRACT

Background: Genome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge.

Results: To assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RAS pathway in cell viability assays. In addition, we describe other dependencies such as TBK1 in HCC-827 cells and TRIB2 in CHP-212 cells which merit further investigation.

Conclusions: We show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3042-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Depletion of kinases EGFR and MAP2K1 correlates with sensitivity towards EGFR and MEK inhibitors. aleft panel: HCC-827 and CHP-212 cells were treated with indicated concentrations of Gefitinib for 72 h. Then, cell viability was measured by Cell Titer Glo according to the manufacturer’s instructions. Middle panel: Fold change for the three independent sgRNAs for EGFR from the screen are depicted at time point day 14. Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of Gefitinib. Then, cells were lysed and analysed by Western blot. b same as a) but the MEK inhibitor AZD6244 was used instead (left panel) and fold change for sgRNAs for MAP2K1 and MAP2K3 from the screen are depicted (middle panel). Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of AZD6244. Then, cells were lysed and analysed by Western blot
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Fig3: Depletion of kinases EGFR and MAP2K1 correlates with sensitivity towards EGFR and MEK inhibitors. aleft panel: HCC-827 and CHP-212 cells were treated with indicated concentrations of Gefitinib for 72 h. Then, cell viability was measured by Cell Titer Glo according to the manufacturer’s instructions. Middle panel: Fold change for the three independent sgRNAs for EGFR from the screen are depicted at time point day 14. Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of Gefitinib. Then, cells were lysed and analysed by Western blot. b same as a) but the MEK inhibitor AZD6244 was used instead (left panel) and fold change for sgRNAs for MAP2K1 and MAP2K3 from the screen are depicted (middle panel). Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of AZD6244. Then, cells were lysed and analysed by Western blot

Mentions: To validate the findings of the CRISPR-Cas9 screen, we investigated the correlation between the sgRNA fold depletion and the drug sensitivity of the cell lines. The EGFR-mutant HCC-827 cell line was completely refractory towards MEK inhibition but highly sensitive to the EGFR inhibitor Gefitinib [13, 14] (Fig. 3a, left panel). In contrast, the NRAS-mutant cell line CHP-212 was highly sensitive towards MEK inhibition but insensitive towards Gefitinib (Fig. 3b, left panel). As expected, the drug sensitivity of the two cell lines broadly agreed with the sgRNA-based depletion of these genes observed in the screen (Fig. 3a, b, middle panel). We found that the fold changes of sgRNAs strongly correlated with sensitivity towards respective inhibitors of the target (correlation ρ = 0,99). These results were corroborated by the other MEK inhibitor MEK162 and the EGFR inhibitor Erlotinib (Additional file 1: Figure S6). In conclusion, our data show that sgRNA depletion strongly correlates with sensitivity to respective kinase inhibitors of the associated pathways in cell viability assays.Fig. 3


Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening
Depletion of kinases EGFR and MAP2K1 correlates with sensitivity towards EGFR and MEK inhibitors. aleft panel: HCC-827 and CHP-212 cells were treated with indicated concentrations of Gefitinib for 72 h. Then, cell viability was measured by Cell Titer Glo according to the manufacturer’s instructions. Middle panel: Fold change for the three independent sgRNAs for EGFR from the screen are depicted at time point day 14. Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of Gefitinib. Then, cells were lysed and analysed by Western blot. b same as a) but the MEK inhibitor AZD6244 was used instead (left panel) and fold change for sgRNAs for MAP2K1 and MAP2K3 from the screen are depicted (middle panel). Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of AZD6244. Then, cells were lysed and analysed by Western blot
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Fig3: Depletion of kinases EGFR and MAP2K1 correlates with sensitivity towards EGFR and MEK inhibitors. aleft panel: HCC-827 and CHP-212 cells were treated with indicated concentrations of Gefitinib for 72 h. Then, cell viability was measured by Cell Titer Glo according to the manufacturer’s instructions. Middle panel: Fold change for the three independent sgRNAs for EGFR from the screen are depicted at time point day 14. Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of Gefitinib. Then, cells were lysed and analysed by Western blot. b same as a) but the MEK inhibitor AZD6244 was used instead (left panel) and fold change for sgRNAs for MAP2K1 and MAP2K3 from the screen are depicted (middle panel). Right panel: HCC-827 and CHP-212 cells were treated for 2 h with the indicated concentrations of AZD6244. Then, cells were lysed and analysed by Western blot
Mentions: To validate the findings of the CRISPR-Cas9 screen, we investigated the correlation between the sgRNA fold depletion and the drug sensitivity of the cell lines. The EGFR-mutant HCC-827 cell line was completely refractory towards MEK inhibition but highly sensitive to the EGFR inhibitor Gefitinib [13, 14] (Fig. 3a, left panel). In contrast, the NRAS-mutant cell line CHP-212 was highly sensitive towards MEK inhibition but insensitive towards Gefitinib (Fig. 3b, left panel). As expected, the drug sensitivity of the two cell lines broadly agreed with the sgRNA-based depletion of these genes observed in the screen (Fig. 3a, b, middle panel). We found that the fold changes of sgRNAs strongly correlated with sensitivity towards respective inhibitors of the target (correlation ρ = 0,99). These results were corroborated by the other MEK inhibitor MEK162 and the EGFR inhibitor Erlotinib (Additional file 1: Figure S6). In conclusion, our data show that sgRNA depletion strongly correlates with sensitivity to respective kinase inhibitors of the associated pathways in cell viability assays.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Genome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge.

Results: To assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RAS pathway in cell viability assays. In addition, we describe other dependencies such as TBK1 in HCC-827 cells and TRIB2 in CHP-212 cells which merit further investigation.

Conclusions: We show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3042-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus