Journal: The Journal of Biological Chemistry

Article Title: Whole-genome CRISPR screening identifies PI3K/AKT as a downstream component of the oncogenic GNAQ–focal adhesion kinase signaling circuitry

doi: 10.1016/j.jbc.2022.102866

Figure Lengend Snippet: PI3K/AKT pathway activation drives resistance to FAKi in GNAQ-mutant UM. A , schematic of whole-genome CRISPR screen experimental design. Created with Biorender.com . B , cell viability represented by beta score where a positive beta score indicates positive selection (resistance) (beta >0.5, indicated by dotted line ), and a negative beta score indicates negative selection (sensitivity) (beta <−0.5, indicated by dotted line ) under FAKi treatment. C , over-representation analysis of top sgRNAs (FDR <0.015) with positive beta score using KEGG and Biocarta gene sets. Color intensity of bars fade by decreasing −Log 10 p value. D , overall survival analysis of UM The Cancer Genome Atlas patient cohort with high ( top 25%) or low ( bottom 25%) expression of PI3K/AKT/mTOR Hallmark gene signature. Dotted lines indicate 95% confidence interval. E , cell viability of 92.1 UM cells after siRNA knockdown of PTEN or TSC2 compared with control siRNA in response to VS-4718 (FAKi) treatment for 72 h, percent viability is normalized to vehicle treatment (mean ± SD, n = 3). F , phosphorylation of FAK, AKT, and S6 after siRNA-mediated knockdown of CRISPR top hits (PTEN and TSC2) in 92.1 UM cells. Representative immunoblots are shown from n = 3 independent experiments. FAKi, focal adhesion kinase inhibitor; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog; sgRNA, single-gide RNA; TSC2, tuberous sclerosis complex 2; UM, uveal melanoma.

Article Snippet: The human Brunello whole-genome CRISPR pooled library was a gift from David Root and John Doench (Addgene #73178).

Techniques: Activation Assay, Mutagenesis, CRISPR, Selection, Expressing, Knockdown, Control, Phospho-proteomics, Western Blot

Journal: Science Advances

Article Title: Nuclear NAD + homeostasis governed by NMNAT1 prevents apoptosis of acute myeloid leukemia stem cells

doi: 10.1126/sciadv.abf3895

Figure Lengend Snippet: ( A ) A schematic outline of the genome-wide CRISPR-Cas9 screen. ( B ) A volcano plot showing both positively and negatively selected genes (FDR < 0.25). ( C ) A schematic showing the three NAD + biosynthesis pathways, with metabolites and enzymes in normal and italic font, respectively. ( D ) The read numbers of four sgRNAs against NAMPT (left) and NMNAT1 (right) at day 25 compared to input. ( E ) Cancer cell line dependency scores (CERES) of genes involved in NAD + biosynthesis ( n = 563). The box plot presents interquartile range, and the whiskers show 95% confidence interval. ( F ) CERES of NAMPT and NMNAT1 in leukemia ( n = 26) and other cancer cell lines ( n = 505). Box plots are annotated with the lower quartile, median, and upper quartile for each category’s z scores. ( G ) Scatter plot showing the correlation between the dependency score of NAMPT and NMNAT1 ( n = 563). ( H ) Immunoblots of NAMPT (left) and NMNAT1 (right) in MOLM13 cells at day 4 after deleting these genes. ( I ) Competitive growth assay in Cas9-expressing MOLM13 cells that express sgRNAs against negative control (NC) or genes in the NAD + biosynthesis pathway. The percentages of sgRNA-expressing cells were normalized to those on day 4 after transduction. ( J ) Measurement of intracellular NAD + amounts (left) and NAD + /NADH (reduced form of NAD + ) ratio (right) in GMP and MLL-AF9–driven L-GMPs ( n = 4). ( K ) Intracellular NAD + amounts (left) and NAD + /NADH ratio (right) in human cord blood CD34 + HSPCs and PDX-derived AML samples ( n = 4). All data represent mean ± SD; * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired Student’s t test unless otherwise noted. See also fig. S1.

Article Snippet: 67979 for negative control and 67980 for targeting GFP expression), and a whole-genome sgRNA library (Addgene no. 67989) were generated by transfecting 293T cells with 5.4 μg of transfer plasmid, 5.4 μg of psPAX2 (Addgene no. 12260) packaging plasmid, and 1.2 μg of pMD2.G (Addgene no. 12259) envelope plasmid in a 10-cm culture plate using a polyethyenimine (PEI) transfection protocol.

Techniques: Genome Wide, CRISPR, Western Blot, Growth Assay, Expressing, Negative Control, Transduction, Derivative Assay

Journal: Science Advances

Article Title: Nuclear NAD + homeostasis governed by NMNAT1 prevents apoptosis of acute myeloid leukemia stem cells

doi: 10.1126/sciadv.abf3895

Figure Lengend Snippet: ( A ) Viability of MOLM13 and OCI-AML2 cells treated with FK866 (10 μM) with or without NAD + precursors (NMN, NA, NR, and NAM; 1 mM). ( B ) Viability of MOLM13 and OCI-AML2 cells transduced with NMNAT1_sg (+) or negative control sgRNA [NC_sg (−)] with or without NAD + precursors (NMN, NA, NR, and NAM; 1 mM). ( C ) Immunofluorescence images of NMNAT1 in MOLM13 cells. ( D ) Nuclear NAD + levels in MOLM13 cells after deleting NMNAT1 or a negative control (NC) locus. ( E ) CRISPR domain screening using sgRNAs targeting different exons of NMNAT1 in Cas9-expressing MOLM13 cells. ( F ) Relative expansion of MOLM13 cells transduced with sgRNA against NMNAT1 or NC with or without the indicated mouse Nmnat1 constructs. ( G ) Survival curves of NSG-SGM3 mice transplanted with control or NMNAT1 -deleted MOLM13 cells with the indicated murine Nmnat1 constructs. ( H and I ) Cell cycle analysis by BrdU incorporation (H) and annexin V staining (I) of MOLM13 cells after deleting NMNAT1 or NC. ( J ) Immunoblotting showing phospho-p53, γH2AX, and NMNAT1 in MOLM13 cells after lentiviral deleting NMNAT1 . ( K and L ) Annexin V staining (K) or relative expansion (L) of MOLM13 cells after deleting NMNAT1 with or without consecutive targeting of TP53 . ( M ) Relative expression of p53 target genes after deleting NMNAT1 . ( N ) Immunoprecipitation assay showing increased acetylated lysine of p53 in NMNAT1 -deleted MOLM13 cells. ( O ) Relative expansion of NMNAT1 -deleted MOLM13 cells expressing WT or K320/373/381R- TP53 constructs. ( P ) Competitive growth assay after deleting the indicated genes. All data represent mean ± SD; * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired Student’s t test unless otherwise noted. See also fig. S2.

Article Snippet: 67979 for negative control and 67980 for targeting GFP expression), and a whole-genome sgRNA library (Addgene no. 67989) were generated by transfecting 293T cells with 5.4 μg of transfer plasmid, 5.4 μg of psPAX2 (Addgene no. 12260) packaging plasmid, and 1.2 μg of pMD2.G (Addgene no. 12259) envelope plasmid in a 10-cm culture plate using a polyethyenimine (PEI) transfection protocol.

Techniques: Transduction, Negative Control, Immunofluorescence, CRISPR, Expressing, Construct, Control, Cell Cycle Assay, BrdU Incorporation Assay, Staining, Western Blot, Immunoprecipitation, Growth Assay

Journal: Nature Communications

Article Title: Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics

doi: 10.1038/s41467-018-05190-9

Figure Lengend Snippet: EAC-derived organoids retain intra-tumor heterogeneity. M-FISH spectral karyotype of 10 cells from organoid culture of a CAM277 and e CAM388. Each chromosome has a unique color. Organoid cultures of b , c , d CAM277 and f , g , h CAM338 were continually cultured and sequenced at different passages to study clonal dynamics. b , f Segmented Venn diagram represents clonal composition of tumor cells at each time point. The compositions of each clone at different time points are listed in Supplementary Table . c , g Number of total mutations and mutated cancer-related genes from individual subclones. d , h The percentage contribution for each of the 6 mutational signatures across successive passages. The passage number is depicted as the Org_P value

Article Snippet: Five human chromosome pools were labeled with ATTO 425-, ATTO 488-, CY3-, CY5-, and Texas Red-dUTPs (Jena Bioscience), respectively, using WGA 3 re-amplification kit (Sigma-Aldrich) and home-made dNTP mixtures optimized for the incorporation of the aforementioned labeled dUTPs by Taq polymerase.

Techniques: Derivative Assay, Cell Culture