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

Article Title: Synthetic Lethal Screens Reveal Co-Targeting FAK and MEK as a Multimodal Precision Therapy for GNAQ -Driven Uveal Melanoma

doi: 10.1158/1078-0432.CCR-20-3363

Figure Lengend Snippet: (A) OMM 1.5 cells expressing Cas9 were infected with the Brunello Human Kinome CRISPR sgRNA KO library at a MOI of 0.3. After selection, cells were treated with vehicle or 0.5μM VS-4718 (FAKi) for 10 days. (B) Left, Cell viability represented as fold change in FAKi-treated cells compared to control. Highlighted significant hits represent synthetic lethal genes with FAKi treatment. Right, KEGG pathways analysis for the top depleted sgRNAs (n=200). (C) 92.1 cell viability after 72h treatment with vehicle, 1μM Go-6983 (PKCi), 1μM VS-4718 or combination of both. (D) Time-course analysis of FAK and ERK phosphorylation in 92.1 cells treated with VS-4718 (1μM), Go-6983 (1μM) or trametinib (MEKi, 10nM). (E) Quantification of pFAK/FAK and pERK/ERK ratios in 92.1 cells treated with 1μM VS-4718 or vehicle for 1h. (F) Left, Cell viability after 72h treatment with VS-4718 (1μM) in 92.1 cells expressing or not MEK-DD (S218/222D). Right, Immunoblot showing pERK levels in 92.1 cells expressing or not MEK-DD (S218/222D). (C, E and F) Data shown represent the mean ± SEM of three independent experiments. ***p<0.001; **p< 0.01; n.s. not significant.

Article Snippet: FAK, phospho-FAK, ERK, phospho-ERK, cleaved PARP1, cleaved Caspase 3, YAP, Cas9, and GAPDH antibodies were purchased from Cell Signaling.

Techniques: Expressing, Infection, CRISPR, Selection, Western Blot

Journal: mBio

Article Title: CRISPR-Cas9 Screening of Kaposi’s Sarcoma-Associated Herpesvirus-Transformed Cells Identifies XPO1 as a Vulnerable Target of Cancer Cells

doi: 10.1128/mBio.00866-19

Figure Lengend Snippet: Identification of XPO1 as a vulnerable gene of KSHV-transformed KMM cells and of cell lines of gastric cancer AGS and liver cancer HUH7 cells. (A and B) XPO1 expression in MM cells and in MM cells infected by KSHV (KMM) and by mutant viruses with a deletion of vFLIP (ΔvFLIP), vCyclin (ΔvCyclin), or a cluster of 10 pre-miRNAs (ΔmiR) analyzed by reverse transcription-quantitative PCR (qRT-PCR) (A) and Western blotting (B). (C) Expression of XPO1 analyzed by qRT-PCR in MM cells, KMM cells, ΔmiR cells, or ΔmiR cells complemented with individual KSHV pre-miRNAs (K1 to K12). (D) Expression of XPO1 following siRNA knockdown analyzed by Western blotting in MM cells and KMM cells. (E) Expression of XPO1 following siRNA knockdown analyzed by Western blotting in AGS and HUH7 cells. (F) Analysis of cell proliferation following siRNA knockdown of XPO1 in MM, KMM, AGS, and HUH7 cell lines. (G) Analysis of cell proliferation following treatment with DMSO or KPT-8602 at different concentrations for 3 days in MM, KMM, AGS, and HUH7 cells. (H) Formation of colonies in soft agar following treatment with DMSO or KPT-8602 at 1 µM in KMM, AGS, and HUH7 cells. Representative fields are shown, and efficiencies of colony formation are presented.

Article Snippet: Primary antibodies to β-actin (Santa Cruz), p62 (CST), SIRT1 (CST), Cas9-hemagglutinin (Cas9-HA; Santa Cruz), PML (CST), XPO1 (CST), p53 (CST), and phospho-p53 (CST) were used.

Techniques: Transformation Assay, Expressing, Infection, Mutagenesis, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: mBio

Article Title: CRISPR-Cas9 Screening of Kaposi’s Sarcoma-Associated Herpesvirus-Transformed Cells Identifies XPO1 as a Vulnerable Target of Cancer Cells

doi: 10.1128/mBio.00866-19

Figure Lengend Snippet: Inhibition of XPO1 induces PML-mediated p53 activation and cell cycle arrest. (A) Effect of KPT-8602 treatment on cell cycle progression of MM, KMM, AGS, and HUH7 cells. Cells treated with 1 µM KPT-8602 for 48 h were analyzed by flow cytometry after BrdU and propidium iodide staining. (B) Analysis of phospho-p53 (p-p53) and total p53 in MM, KMM, AGS, and HUH7 cells after treatment with 1 µM KPT-8602 by Western blotting. (C) Examination of PML in MM, KMM, AGS, and HUH7 cells after treatment with 1 µM KPT-8602 for 24 h by immunofluorescence assay. The slides were counterstained with DAPI, and pictures were taken with a confocal microscopy (magnification, ×600). (D) Expression of PML in AGS and HUH7 cells following siRNA knockdown analyzed by Western blotting. (E) Analysis of p-p53 in AGS and HUH7 cells following siRNA knockdown of PML analyzed by Western blotting.

Article Snippet: Primary antibodies to β-actin (Santa Cruz), p62 (CST), SIRT1 (CST), Cas9-hemagglutinin (Cas9-HA; Santa Cruz), PML (CST), XPO1 (CST), p53 (CST), and phospho-p53 (CST) were used.

Techniques: Inhibition, Activation Assay, Flow Cytometry, Staining, Western Blot, Immunofluorescence, Confocal Microscopy, Expressing

Journal: mBio

Article Title: CRISPR-Cas9 Screening of Kaposi’s Sarcoma-Associated Herpesvirus-Transformed Cells Identifies XPO1 as a Vulnerable Target of Cancer Cells

doi: 10.1128/mBio.00866-19

Figure Lengend Snippet: Induction of p62 nuclear accumulation and colocalization with p53 following XPO1 inhibition in AGS and HUH7 cells. (A) Expression of p62 in MM, KMM, AGS, and HUH7 cells after treatment with 1 µM KPT-8602 for 24 h analyzed by Western blotting. (B) Expression of p53 and p62 in AGS, HUH7, MM, and KMM cells after treatment with 1 µM KPT-8602 for 24 h analyzed by immunofluorescence assay. The sections were counterstained with DAPI, and pictures were taken with a confocal microscopy (magnification, ×600).

Article Snippet: Primary antibodies to β-actin (Santa Cruz), p62 (CST), SIRT1 (CST), Cas9-hemagglutinin (Cas9-HA; Santa Cruz), PML (CST), XPO1 (CST), p53 (CST), and phospho-p53 (CST) were used.

Techniques: Inhibition, Expressing, Western Blot, Immunofluorescence, Confocal Microscopy

Journal: mBio

Article Title: CRISPR-Cas9 Screening of Kaposi’s Sarcoma-Associated Herpesvirus-Transformed Cells Identifies XPO1 as a Vulnerable Target of Cancer Cells

doi: 10.1128/mBio.00866-19

Figure Lengend Snippet: XPO1 inhibition-induced p53 activation in PML-NBs depends on p62 nuclear accumulation in AGS and HUH7 cells. (A) Expression of PML and p62 in AGS, HUH7, MM, and KMM cells after treatment with 1 µM KPT-8602 for 24 h analyzed by immunofluorescence assay. The sections were counterstained with DAPI, and pictures were taken with a confocal microscopy (magnification, ×600). (B) Expression of p62 in AGS and HUH7 cells following siRNA knockdown analyzed by Western blotting. (C) Analysis of p-p53 in AGS and HUH7 cells after treatment with 1 µM KPT-8602 for 24 h following siRNA knockdown of p62 by Western blotting.

Article Snippet: Primary antibodies to β-actin (Santa Cruz), p62 (CST), SIRT1 (CST), Cas9-hemagglutinin (Cas9-HA; Santa Cruz), PML (CST), XPO1 (CST), p53 (CST), and phospho-p53 (CST) were used.

Techniques: Inhibition, Activation Assay, Expressing, Immunofluorescence, Confocal Microscopy, Western Blot