snp6 array data  (Thermo Fisher)

Journal: Nature Communications

Article Title: Germline variation contributes to false negatives in CRISPR-based experiments with varying burden across ancestries

doi: 10.1038/s41467-024-48957-z

Figure Lengend Snippet: a The association between all genetic variants (from SNP6 genotyping arrays) and all putative ancestry-associated genetic dependencies was computed by linear regression with correction for cancer lineage as a covariate. Nominal p -values were adjusted for multiple hypothesis testing with the Benjamini–Yekutieli procedure. Genes with at least one significant association (d-QTL) are indicated in red ( n = 33). Raw data are described in Source Data 2AB. b The minor allele frequency of the most significant variant from a is indicated for the ancestry group with the lowest and highest frequency in the cell line collection. Cell lines of predominantly American or South Asian ancestry were not included in this analysis because the sample sizes for these ancestry groups are too low. Raw data are described in Source Data 2AB. c Position of the most significant germline variant from a relative to the Transcription Start Site (TSS) of the dependency gene in question. Variants that are within 1 megabase of the TSS are indicated in red ( n = 29) and those that are greater than 1 megabase or are on a different chromosome are black ( n = 20). Raw data are described in Source Data 2C. d Association between each d-QTL Single Nucleotide Variant (SNV) and expression of the dependency gene in question. Genes in this analysis were filtered to include only those with at least one significant d-QTL association (indicated in red in a ). CDKN2B was excluded from this analysis because the association between the d-QTL variant and CDKN2B expression resulted from a technical artifact (Supplementary Fig. ). Raw data are described in Source Data 2D. e Median expression across cell lines for all genes (top), all genes classified as common essential in The Cancer Dependency Map 22Q1 release (middle), and all ancestry-associated genetic dependencies (bottom). Raw data are described in Source Data 2E.

Article Snippet: We therefore systematically cataloged local ancestral haplotypes across the genomes of the 994 (out of 1829 total) cell line models in the Cancer Cell Line Encyclopedia collection for which publicly available Affymetrix SNP6 germline variant data have been analyzed , leveraging germline variants from 10,345,968 SNPs genome-wide to infer local ancestry.

Techniques: Variant Assay, Expressing

Journal: Nature Communications

Article Title: Germline variation contributes to false negatives in CRISPR-based experiments with varying burden across ancestries

doi: 10.1038/s41467-024-48957-z

Figure Lengend Snippet: a Association between East Asian (EAS) or European (EUR) ancestry and guide depletion scores for all guides targeting ancestry-associated dependencies. Guides with a Single Nucleotide Variant (SNV) in the targeting sequence are indicated in red. Raw data are described in Source Data 3A. b For all sgRNA sequences in the Avana library, the fraction of cell lines with a SNV in its targeting sequence. Guides which are affected in >10 cell lines ( n = 3209) are indicated in red to the right of the dashed line, whereas those that are affected in <10 cell lines are not shown. Raw data are described in Source Data 3B. c Variants (from WES/WGS) for all cell lines were mapped to the targeting sequence of all Avana guides (see Data Availability). Genes are stratified by the number of guides with a mismatch in at least one cell line. Raw data are described in Source Data 3C. d Germline (red) and somatic (gray) variants from 32 tumor types profiled in TCGA were mapped to targeting sequences for guides in the Avana library. The total number of variants in each sample that map to Avana guides is plotted on the x-axis. In the boxplot, the box includes the second and third data quartiles divided by a median line, and whiskers represent the first and fourth quartiles. Boxplot summary values are described in Source Data 3Db. Raw data are described in Source Data 3D. e Guides were stratified by the position of mismatches within the sgRNA targeting sequence and the association between the SNV and the guide depletion score was computed for each sgRNA in the Avana library (black boxes). P -values were computed with two-sided t-tests between cell lines with and without each SNV. The impact of mismatches on guide activity from Doench et al. is indicated in blue circles. In the boxplot, the box includes the second and third data quartiles divided by a median line, and whiskers represent the first and fourth quartiles. Boxplot summary values are described in Source Data 3Eb. Raw data are described in Source Data 3E.

Article Snippet: We therefore systematically cataloged local ancestral haplotypes across the genomes of the 994 (out of 1829 total) cell line models in the Cancer Cell Line Encyclopedia collection for which publicly available Affymetrix SNP6 germline variant data have been analyzed , leveraging germline variants from 10,345,968 SNPs genome-wide to infer local ancestry.

Techniques: Variant Assay, Sequencing, Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: NRF2 activation promotes aggressive lung cancer and associates with poor clinical outcomes

doi: 10.1158/1078-0432.CCR-20-1985

Figure Lengend Snippet: (A) Representative high-magnification images of murine lungs at 7 weeks post adeno-Cre infection for each genotype. Blue Arrow: Atypical adenomatous hyperplasia; Blue Star: Solid adenoma; Red Arrow: Acinar carcinoma with mucous containing neoplastic cells; Yellow Star: Mucous present within gland lumen and airways; Green Cross: Papillary carcinoma. By 6–12 weeks, KrasLSL-G12D mice had only developed coalescing areas of AAH (blue arrow), while Keap1fl/flKrasLSL-G12D mice developed more extensive AAH, with a single Keap1fl/flKrasLSL-G12D mouse developing an adenoma (blue star) and 4/6 (66%) developing non-mucinous BEH. Stk11fl/flKrasLSL-G12D mice showed evidence of mucinous differentiation with large goblet cells (red arrow) and abundant wispy basophilic mucinous material in the alveolar spaces (yellow star). Unique among triple mutant lungs was the presence of intra-bronchiolar papillary carcinomas comprised of a fibrovascular core, long papillary fronds extending into the bronchiole lumen and loaded with abundant viscous mucus. (B) Representative images from 6–7-week old KrasLSL-G12 , Keapfl/flKrasLSL-G12D, Stk11fl/flKrasLSL-G12D and Keapfl/flStk11fl/flKrasLSL-G12D lungs stained with Alcian Blue/PAS to assess mucin production. The KrasLSL-G12D and Keapfl/flKrasLSL-G12D images display non-mucinous atypical epithelial hyperplasia, the Stk11fl/flKrasLSL-G12D image is of a carcinoma with mucinous differentiation and the Keapfl/flStk11fl/flKrasLSL-G12D image displays marked bronchiolar epithelial hyperplasia with mucinous differentiation. (C) Expression of Nkx2–1 and mucinous markers in murine lungs. High expression levels are shown in red; low levels in blue. The 16 murine lung samples are annotated by their Keap1 and Stk11 status. (D) The average expression of mucinous genes MUC5AC, MUC5B and AGR2 (mucinous signature score) in function of genotype in TCGA LUAD tumors. Tumors are grouped by STK11 and/or KEAP1/NRF2 alteration, independent of KRAS status. According to a multivariate linear model of mucinous marker expression as a function of STK11, KEAP1/NRF2, and KRAS status, both KEAP1/NRF2 and KRAS alterations are significantly associated with mucinous marker expression (p = 0.033 and 0.014, respectively), but the effect of STK11 loss is dominant (p = 5e-5). (E) NKX2–1 expression in function of genotype in TCGA LUAD tumors. Tumors are grouped by STK11 and/or KEAP1/NRF2 alteration, independent of KRAS status. (F) Flow cytometry-based assessment of ROS levels in dissociated mouse lung epithelium tumor cells (CD45−EpCAM+) using carboxy-H2DCFDA dye, graphed as percent of CD45−EpCAM+ (n=5). (G) Determination of oxidative stress levels in lungs from different genotypes. The TBARS (Thiobarbituric Acid Reactive Substances) assay was used to measure malondialdehyde levels, a marker of lipid peroxidation and resultant oxidative stress (n=7–8). (H) 4-hydroxynonenal (4HNE), a common byproduct of lipid peroxidation during oxidative stress, was measured in lung tissue homogenates by ELISA (n=7–8). (I) Estimation of total GSH levels in lungs from mice with different genotypes (n=6–8). Statistical analyses represent One-way ANOVA: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: TCGA RNA-sequencing, Affymetrix SNP6 copy number, and whole exome-sequencing data for 439 lung adenocarcinoma (LUAD) ( 7 ) and 306 lung squamous cell carcinoma (LUSC) ( 24 ) tumors are from the National Cancer Institute Genomic Data Commons ( https://gdc.cancer.gov ).

Techniques: Infection, Mutagenesis, Staining, Expressing, Marker, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: NRF2 activation promotes aggressive lung cancer and associates with poor clinical outcomes

doi: 10.1158/1078-0432.CCR-20-1985

Figure Lengend Snippet: (A) Overlap of KRAS, STK11, and KEAP1/NRF2 alterations in 439 human lung adenocarcinoma (LUAD) tumors from the TCGA dataset. (B) Expression of 96 genes consistently induced by KEAP1/NRF2 independently of histology, in 439 LUAD tumors. High expression levels are shown in red; low levels in blue. Tumors are annotated by KRAS, STK11, KEAP1/NRF2 genomic status, and NRF2 signature score, and ordered by increasing score. (C) KEGG pathways significantly enriched within the 96-gene signature, with adjusted p-value <0.05. The full list of KEGG pathways is available in Table S2. (D) Volcano plot of genes for the comparison of KEAP1-mutant vs. -wildtype HCC515 cells, independent of STK11 status and media condition. (E) Volcano plot of genes for the comparison of Keap1-altered vs. -wildtype murine lung samples (independent of Stk11). Highlighted in red are 77/96 signature genes with mouse orthologs. (F) Kaplan-Meier curve of overall survival (OS) in 428 LUAD patients. High signature is significantly associated with worse OS in a univariate model (HR 1.79, 95% CI 1.25–2.56, p-value 0.0014), and in a multivariate model when accounting for KRAS and STK11 alteration status (HR 1.82, 95% CI 1.27–2.60, p-value 0.0011). (G) STK11 expression by NRF2 signature-low/high in LUAD. STK11-mutant tumors are colored in green. Denoted p-value compares NRF2 signature-high vs. low LUAD tumors, using the Mann-Whitney-Wilcoxon test. The association of STK11 expression with the NRF2 signature is independent of STK11 mutation status: multivariate linear model predicting STK11 expression based on signature status and STK11 status; p-value signature = 6e-11, p-value STK11 0.8.

Article Snippet: TCGA RNA-sequencing, Affymetrix SNP6 copy number, and whole exome-sequencing data for 439 lung adenocarcinoma (LUAD) ( 7 ) and 306 lung squamous cell carcinoma (LUSC) ( 24 ) tumors are from the National Cancer Institute Genomic Data Commons ( https://gdc.cancer.gov ).

Techniques: Expressing, Comparison, Mutagenesis, MANN-WHITNEY