Journal: bioRxiv

Article Title: Selenocysteine metabolism is a targetable vulnerability in MYCN -amplified cancers

doi: 10.1101/2022.05.17.492172

Figure Lengend Snippet: (A) Analysis of the depmap portal ( www.depmap.org ) reveals MYCN -amplified SK-N-DZ as hypersensitive cell lines to the GPX4 inhibitors RSL3 and ML210. ( B) Strategy of the genome-wide CRISPR activation (CRISPRa) screen in MYCN- amplified SK-N-DZ cells. ( C) Overexpression phenotypes conferring resistance to 300 nM RSL3 (left) or 1 μM Erastin (right) treatment. Significant hits are marked in blue (FDR ≤ 0.05), while the highest scoring hit, LRP8 , is highlighted in red. ( D) Overexpression phenotypes conferring resistance to RSL3 (100 nM) induced ferroptosis in the pooled validation CRISPRa screen. Significantly enriched genes are marked and labelled in blue (FDR ≤ 0.05). The highest scoring hit from the primary screens, LRP8 , is highlighted in red ( E) Strategy of the single-cell CRISPRa screen to characterize hits from the ferroptosis-resistance screen. Guide RNA labels are recovered alongside the whole transcriptome readout for each cell. (F) Transcriptomic consequences of CRISPRa of 14 scoring hits from the primary and the validation screens. Each row represents one CRISPRa cluster. For each cluster, the top 50 genes with the most significantly differential expression (compared to the non-targeting control cluster) were selected and merged to a signature gene list represented by the columns. Columns and rows were hierarchically clustered based on Pearson correlation.

Article Snippet: The human CRISPR activation pooled library Set A (Addgene plasmid #92379 was a gift from David Root and John Doench) was amplified as described previously ( ).

Techniques: Amplification, Genome Wide, CRISPR, Activation Assay, Over Expression, Biomarker Discovery, Quantitative Proteomics, Control

Journal: bioRxiv

Article Title: Selenocysteine metabolism is a targetable vulnerability in MYCN -amplified cancers

doi: 10.1101/2022.05.17.492172

Figure Lengend Snippet: (A) Top left: number of unique molecular identifiers (UMIs) per cell in the scCRISPRa sample. Top right: number of detected genes per cell. Bottom left: percentage of mitochondrial reads detected. Bottom right: number of genes per UMI (log-transformed) reflecting library complexity. The median values are printed and highlighted by a dot. (B) The number of cells (y-axis, in log-scale) assigned to each scoring hit (indicated on the x-axis) in the single-cell experiment. OE: overexpression, NT ctrl: cells assigned to non-targeting control gRNAs. (C) Pairwise comparison of transcriptome similarities of validated CRISPRa clusters and genes derived from the gene expression signature list (based on Spearman correlation). Color intensities and ratios of the pie-chart indicate relative overlap. Rows and columns are hierarchically ordered and related groups are marked with a blue rectangle. *** P < 0.001, ** P < 0.01, * P < 0.05. (D) Dot plot showing expression of target genes (x-axis) in each CRISPRa cluster (y-axis). The size of the dots reflects the fraction of cells within a particular cluster for which the expression of the gene was detected. OE: overexpression. NT ctrl: cluster of cells assigned to non-targeting control gRNAs. (E) Changes in gene expression following CRISPRa (clusters represented by each row) of genes that were detected as significantly differentially expressed (FDR ≤ 0.05) in at least seven of the 14 scoring CRISPRa clusters. Columns and rows were hierarchically clustered based on Pearson correlation. (F) Changes in expression of ferroptosis related genes (upper) and transcriptional core regulatory circuits maintaining cell state in MYCN -amplified neuroblastoma derived from (lower) that were detected as significantly differentially expressed in at least one of the CRISPRa clusters represented by each row. (G) Gene Ontology (GO) term enrichment analysis conducted on the signature gene list that was derived from the top 50 genes showing the most significant differential expression in each scoring CRISPRa cluster. Selected terms are shown up to FDR ≤ 0.05. BP: biological process, CC: cellular compartment, MF: molecular function. (H) Ridge-plot showing the expression of YBX3 in each scoring CRISPRa cluster. OE: overexpression, NT ctrl: cluster of cells assigned to non-targeting control gRNAs. *** P < 0.001, ** P < 0.01, * P < 0.05, ns: nonsignificant (FDR corrected p-values calculated by Wald tests comparing mean expression values in each CRISPRa cluster versus the NT ctrl cluster).

Article Snippet: The human CRISPR activation pooled library Set A (Addgene plasmid #92379 was a gift from David Root and John Doench) was amplified as described previously ( ).

Techniques: Transformation Assay, Over Expression, Control, Comparison, Derivative Assay, Gene Expression, Expressing, Amplification, Quantitative Proteomics

Journal: bioRxiv

Article Title: Selenocysteine metabolism is a targetable vulnerability in MYCN -amplified cancers

doi: 10.1101/2022.05.17.492172

Figure Lengend Snippet: (A) Schematic representation of the SLC-focused CRISPR knockout screen in the SK-N-DZ cell line grown in the presence of defined selenium sources. (B) CRISPR gene log2 fold change in SK-N-DZ cells grown in defined supplemented media. (C) Comparison of SLC7A11 expression in a panel of 23 neuroblastoma cell lines against 1349 non-neuroblastoma cell lines demonstrating the lineage-specific lower expression of SLC7A11, SLC3A2 and SLC26A6 ( www.depmap.org ). (D) Dot plot depicting the correlation of the dependency of neuroblastoma cell lines on LRP8 and GPX4 (CERES score of −1 means full dependency based on CRISPR–Cas9 knockout screening data) and the expression levels of SLC7A11 in a panel of 27 neuroblastoma cell lines (depmap portal; https://depmap.org/portal/ ). Cell lines with low expression of SLC7A11 were found to be dependent on LRP8 (Pearson correlation r: 0.4858).

Article Snippet: The human CRISPR activation pooled library Set A (Addgene plasmid #92379 was a gift from David Root and John Doench) was amplified as described previously ( ).

Techniques: CRISPR, Knock-Out, Comparison, Expressing

Journal: bioRxiv

Article Title: Selenocysteine metabolism is a targetable vulnerability in MYCN -amplified cancers

doi: 10.1101/2022.05.17.492172

Figure Lengend Snippet: Immunoblot analysis of LRP8, SCLY, GPX4 and Flag in LRP8-deficient HT1080 ( A ) and A375 ( C ) cells overexpressing hLRP8 -Flag or empty vector (Mock). Parental cell lines only expressing Cas9 are shown as control. Dose-dependent toxicity of the ferroptosis inducers iFSP1, Erastin, BSO and RSL3 in LRP8-deficient (LRP8 KO) HT1080 ( B ) and A375 ( D ) cell lines stably transduced with a vector expressing hLRP8 -Flag. Parental cell lines only expressing Cas9 are shown as control. Data are the mean ± s.e.m. of n = 3 wells of a 96-well plate from three independent experiments. (E) Results of a CRISPR deletion screen conducted in H1080 and A375 cell lines displayed as log2 fold change between LRP8-deficient and wild-type cells.

Article Snippet: The human CRISPR activation pooled library Set A (Addgene plasmid #92379 was a gift from David Root and John Doench) was amplified as described previously ( ).

Techniques: Western Blot, Plasmid Preparation, Expressing, Control, Stable Transfection, Transduction, CRISPR