vienna bioactivity crispr score  (Broad Institute Inc)

Journal: Nature Chemical Biology

Article Title: Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP

doi: 10.1038/s41589-024-01614-4

Figure Lengend Snippet: a , Genome-wide CRISPR–Cas9 knockout screen in W7-treated KBM7 cells constitutively expressing Cas9 (KBM7-Cas9). The bubble plot displays median sgRNA enrichment after W7 treatment over DMSO, and the bubble size represents significance. Only genes with FDR P values of ≤0.05 are highlighted. Enrichment of sgRNAs targeting selected hits in comparison to the distribution of all sgRNAs is highlighted separately. See also Supplementary Table . b , Dose-resolved normalized viability after 72 h of treatment of KBM7-Cas9 cells expressing sgRNAs targeting ARMH3 , PITPNB or AAVS1 with W7. Data are mean ± s.e.m.; n = 3 independent treatments; KO, knockout. c , Left, phosphatidylinositol 4-kinases PI4KB and PI4K2A contribute to the functional pool of PI4P in the Golgi. The image illustrates the CRISPR screen hits ARMH3 and PITPNB, along with small-molecule inhibitors of respective phosphatidylinositol 4-kinases. Right, dose-resolved normalized viability of KBM7 cells with W7 in the absence or presence of PI4KB inhibitors MI14 and BF738735 (both 1 μM) or PI4K2A inhibitor PI-273 (1 μM). The cells were cotreated for 72 h. Data are mean ± s.e.m.; n = 3 independent treatments.

Article Snippet: ARMH3 -, PITPNB -, OSBP - and OSBP2 -targeting sgRNAs were designed using the Vienna Bioactivity CRISPR score portal and were cloned into LentiGuide-Puro (Addgene, 52963) or into pLenti-U6-sgRNA1-U6-sgRNA2-EF1αs-eBFP2 (a kind gift from J. Zuber (Research Institute of Molecular Pathology)) following standard protocol , .

Techniques: Genome Wide, CRISPR, Knock-Out, Expressing, Comparison, Functional Assay

Journal: Nature Chemical Biology

Article Title: Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP

doi: 10.1038/s41589-024-01614-4

Figure Lengend Snippet: a , Scanning confocal microscopy analysis of the effects of orpinolide treatment (1 μM) on PC2 trafficking in RCS cells. PC2 secretion was synchronized for 4 h at 40 °C before being released by incubation at 32 °C for the indicated duration. Representative images show PC2 localization (488, green), the Golgi area (giantin, 568, purple) and the nuclei (Hoechst, blue); scale bar, 10 μm. b , Quantification of PC2 localization to the Golgi normalized to total PC2 per cell. The graph shows one point per cell from three independent experiments, with between 42 and 58 cells per condition. The data are log 10 -transformed and were analyzed by two-way ANOVA, with P interaction < 0.0001 and P treatment factor < 0.01. A Šídák multiple comparison test compared DMSO against orpinolide at each time point; **** P < 0.0001; NS, not significant ( P = 0.4783, 30 min; P = 0.9126, 45 min). c , Top, expression levels of endogenous OSBP and V5-2HA-tagged OSBP variants (wild-type or M446W mutant) in both wild-type KBM7 cells and KBM7 cells overexpressing these fusions. Bottom, dose-resolved normalized viability after 72 h of treatment of wild-type KBM7 cells and KBM7 cells overexpressing V5-2HA-tagged OSBP variants (wild-type or M446W mutant) with orpinolide. Data are mean ± s.e.m.; n = 3 independent treatments; WT, wild type. d , CRISPR-based dropout experiment in doxycycline-inducible Cas9 KBM7 (KBM7 iCas9) cells transduced with a BFP-based dual-knockout reporter. The reporter plasmid carries a combination of sgRNAs targeting OSBP , OSBP2 or AAVS1 to yield single or dual OSBP / OSBP2 knockout. The bar plots depict relative BFP + cells over 12 days of doxycycline treatment normalized to day 2 for each individual reporter system.

Article Snippet: ARMH3 -, PITPNB -, OSBP - and OSBP2 -targeting sgRNAs were designed using the Vienna Bioactivity CRISPR score portal and were cloned into LentiGuide-Puro (Addgene, 52963) or into pLenti-U6-sgRNA1-U6-sgRNA2-EF1αs-eBFP2 (a kind gift from J. Zuber (Research Institute of Molecular Pathology)) following standard protocol , .

Techniques: Confocal Microscopy, Incubation, Transformation Assay, Comparison, Expressing, Mutagenesis, CRISPR, Transduction, Knock-Out, Plasmid Preparation

Journal: Nature Chemical Biology

Article Title: Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP

doi: 10.1038/s41589-024-01614-4

Figure Lengend Snippet: ( a ) Dose-resolved, normalized viability after 72 h treatment of wild type KBM7 cells and KBM7 cells overexpressing V5-2HA-tagged OSBP variants (wild type or M446W mutant) with OSW-1 (top) or paclitaxel (bottom). Mean ± s.e.m.; n = 3 independent treatments. ( b ) Comparison of the sensitivity of various cancer cell lines to orpinolide with the expression levels of OSBP and OSBP2 (DepMap 23Q2). The compound sensitivity is represented as area under the curve (A.U.C.) calculated from 10-concentration-point dose-response curves after 72 h drug treatment (see Extended Data Fig. ). ( c ) Top: Distribution of OSBP and ORP4 ( OSBP2 ) deletion effects across 1078 cancer cell lines from the DepMap 22Q4 CRISPR data. Bottom: Distribution of OSBP and ORP4 ( OSBP2 ) deletion effects across myeloid and lymphoid cancer cell lines from the DepMap 22Q4 CRISPR data. Wilcoxon matched-pairs signed ranked test was performed on the data.

Article Snippet: ARMH3 -, PITPNB -, OSBP - and OSBP2 -targeting sgRNAs were designed using the Vienna Bioactivity CRISPR score portal and were cloned into LentiGuide-Puro (Addgene, 52963) or into pLenti-U6-sgRNA1-U6-sgRNA2-EF1αs-eBFP2 (a kind gift from J. Zuber (Research Institute of Molecular Pathology)) following standard protocol , .

Techniques: Mutagenesis, Comparison, Expressing, Concentration Assay, CRISPR

Journal: Nature Chemical Biology

Article Title: Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP

doi: 10.1038/s41589-024-01614-4

Figure Lengend Snippet: ( a ) CRISPR-based dropout screen in Jurkat cells constitutively expressing Cas9, transduced with a BFP-based dual knockout reporter. The reporter plasmid carries a combination of sgRNAs targeting OSBP , OSBP2 or AAVS1 locus to yield single or dual OSBP/ORP4 knockout. The bar plots depict relative BFP + cells over 18 days, normalized to day 3 post transduction for each individual reporter system. ( b ) Top: Effect of orpinolide treatment (5 μM) on p-Akt and Akt protein levels in Jurkat and LOUCY cells. Phosphoinositide 3-kinase (PI3K) inhibitor taselisib (0.5 μM, 8 h treatment) was used as a positive control. Representative image of n = 3 independent experiments. Bottom: Quantification of the relative p-Akt/Akt protein levels normalized to the DMSO controls. Mean ± SD; n = 3 independent experiments. ( c ) Left: Effect of orpinolide treatment (5 μM) on p-Akt and Akt protein levels in Jurkat cells at different time points. Representative image of n = 3 independent experiments. Right: Quantification of the relative p-Akt/Akt protein levels normalized to the DMSO controls. Mean ± SD; n = 3 independent experiments.

Article Snippet: ARMH3 -, PITPNB -, OSBP - and OSBP2 -targeting sgRNAs were designed using the Vienna Bioactivity CRISPR score portal and were cloned into LentiGuide-Puro (Addgene, 52963) or into pLenti-U6-sgRNA1-U6-sgRNA2-EF1αs-eBFP2 (a kind gift from J. Zuber (Research Institute of Molecular Pathology)) following standard protocol , .

Techniques: CRISPR, Expressing, Transduction, Knock-Out, Plasmid Preparation, Positive Control