Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Olaparib and TRAIL synergize in cancer cells independently of BRCA2 status. A-B Proliferation assays of the effects of olaparib, TRAIL, or their combination on BRCA2 deficient CAPAN1 ( A ), and the respective syngenic BRCA2/CIN cell line ( B ) complemented to express wild-type BRCA2. C-D Proliferation assays of the effects of olaparib, TRAIL or their combination on BRCA2 deficient HCT116 ( C ), and BRCA2 proficient SW620 ( D ). E-F Proliferation assays of the effects of olaparib, TRAIL or their combination on wild-type DLD1 cells ( E ) vs. the homozygous BRCA2 knockout colorectal cancer cell line DLD1 termed DLD1 BRCA2 KO ( F ). Cells were treated for 6 days with the indicated agents and subsequently analyzed via SYBR green proliferation assay. All experiments were performed in triplicate with error bars representing SEM from three independent experiments. Drug interactions were analyzed using the Chou–Talalay method with a combination index (CI) of <1, 1, and >1 indicating synergistic, additive, and antagonistic drug effects, respectively. In the combination index graphs, dots depict the CI at the respective fraction of cancer cells affected (x-axis, 0.0 = no cells dead, 1.0 = all cells dead).

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: Knock-Out, SYBR Green Assay, Proliferation Assay

Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Combined olaparib and the clinically tested TRAIL targeting compound dulanermin synergize in pancreatic cancer cells independently of BRCA2 status. Proliferation assays of the effects of olaparib, dulanermin, or their combination on BRCA2 deficient CAPAN1 ( A ) and BRCA2/CIN ( B ). Cells were treated for 6 days with the indicated agents and subsequently analyzed via SYBR green proliferation assay. All experiments were performed in triplicate with error bars representing SEM from three independent experiments. Drug interactions were analyzed using the Chou–Talalay method with a combination index (CI) of <1, 1, and >1 indicating synergistic, additive, and antagonistic drug effects, respectively. In the combination index graphs, dots depict the CI at the respective fraction of cancer cells affected (x-axis, 0.0 = no cells dead, 1.0 = all cells dead).

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: SYBR Green Assay, Proliferation Assay

Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Combined olaparib and TRAIL enhance S/G2 phase cell cycle arrest and apoptosis. CAPAN1 ( A – C ), and the wild-type BRCA2 expressing BRCA2/CIN treated with various doses of olaparib alone ( A , D ) or in combination with TRAIL (10 ng/mL) ( B , C , E , F ) for 48 h. A,B,D,E depict the percentage of cells in the respective cell cycle phase from all viable cells, i.e., non-sub-G1 cells. C,F depict the percentage of cells in sub-G1 from all cells. Figure ( G ): Representative flow cytometry histograms of CAPAN1 and BRCA2/CIN cells after incubation with control, olaparib (10 µM), TRAIL (10 mg/mL), or its combination. Error bars represent mean ± SEM from at least three experiments. * = statistical significance, p < 0.05.

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: Expressing, Flow Cytometry, Incubation, Control

Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Olaparib influences death receptor expression and caspase activation. ( A – C ): Western blot analysis to assess the expression levels of the indicated regulators of apoptosis in BRCA2 deficient CAPAN1 vs. wild-type BRCA2 expressing BRCA2/CIN treated with various doses of olaparib alone or in combination with TRAIL (10 ng/mL) for 48 h. Data show representative results from three experiments. GAPDH served as a loading control. ( D ): Flow cytometry of TRAIL surface receptor 2 (death receptor 5, DR5) in BRCA2 deficient CAPAN1 vs. wild-type BRCA2 expressing BRCA2/CIN treated with 1 μM olaparib for 6 days. Error bars represent mean ± SEM from at least three experiments. * = statistical significance, p < 0.05, NS = non-significant, MFI = median fluorescence intensity.

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: Expressing, Activation Assay, Western Blot, Control, Flow Cytometry, Fluorescence

Journal: Cancers

Article Title: TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status

doi: 10.3390/cancers14215240

Figure Lengend Snippet: Influence of Olaparib and TRAIL on DNA damage repair. ( A , B ): Western blot analysis to assess the expression levels of the indicated DNA damage proteins in BRCA2 -deficient CAPAN1 vs. wild-type BRCA2 expressing BRCA2/CIN treated with various doses of olaparib alone or in combination with TRAIL for 48 h. Data show representative results from three experiments. GAPDH served as a loading control. ( C , D ): 53BP1 immunostaining of BRCA2 deficient CAPAN1 vs. wild-type BRCA2 expressing BRCA2/CIN treated with 10 μM olaparib +/− 10 ng/mL TRAIL or dimethyl sulfoxide (DMSO) control for 48 h. Quantification of 53BP1 foci per nucleus was based on at least 10 nuclei per sample. Error bars represent mean ± SD. * = statistical significance, p < 0.05.

Article Snippet: DLD1 BRCA2 KO cells were generated by Thomas Hucl and Eike Gallmeier in the laboratory of Scott Kern (Johns Hopkins University, Baltimore, MD, USA) [ ].

Techniques: Western Blot, Expressing, Control, Immunostaining

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: The role of eEF2K in cancer cell survival under chronic extracellular acidosis. A549 (A) or HCT116 (B) cells were cultured in pH 6.7 (6.7EXT) or pH 7.4 (7.4EXT) buffered medium for about 3 months. Cells were then cultured in pH 6.7 or 7.4-buffered media for 48 h with/without 1 mM IPTG and/or 30 μM A484954, before lysis and Western blot analysis. A549 (C) or HCT116 (D, G) cells were also subjected to flow cytometry to determine subG1 population. (E) HCT116 cells were cultured at pH 7.4 in the presence or absence of 1 mM IPTG and 30 μM A484954 for 48 h, and G1 cell size was then determined by flow cytometry. Results are shown as forward scatter-height (FSC-H). (F) HCT116 cells were cultured as described for panel E and then subjected to flow cytometry analysis; the percentage of sub-G1 population is shown. (H) HCT116 cells cultured at pH 6.7 (6.7EXT) or 7.4 (7.4EXT) for approximately 3 months were transfected with GFP-spectrin alone or GFP-spectrin plus FLAG-tagged eEF2K as indicated. Cells were lysed 8 h after transfection, and eEF2K levels were analyzed by Western blotting. Data are expressed as mean ± SE from 3 independent experiments. P values were obtained by two-way ANOVA. *, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001. For panels A and B, data shown are representative of three independent experiments.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Cell Culture, Lysis, Western Blot, Flow Cytometry, Transfection

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: Acidosis activates eEF2K in cancer cells. (A) Validation of the P-eEF2 Thr56 antibody for immunohistochemistry. eEF2K WT and knockout (KO) MEFs were stained with P-eEF2 Thr56. Scale bar, 50 μm. (B) Representative sequential sections of human lung cancer to support expression of NHE-1, GLUT-1, and P-eEF2 Thr56. Nuclei were counterstained with hematoxylin. Ten lung cancer carcinoma samples were stained in total, 7 of which showed strong areas of GLUT-1 expression; of these, 6 codisplayed NHE-1 and P-eEF2 Thr56. Scale bar, 100 μm. A549 (C) or HCT116 (D) cells were cultured in medium buffered at pH 6.4, 6.8, or 7.4 for the indicated times. IPTG (1 μM) was added to A549 (E) or HCT116 (F) cells 5 days before the experiment. Cells were then incubated in medium buffered at different pH values for 48 h with/without 1 mM IPTG, 30 μM A484954, or 25 μM etoposide, followed by lysis and Western blot analysis. (G) A549 cells were treated as described for panel E, and Western blot analysis was then performed to study the phosphorylation of p70S6K1 at Thr389 and S6 at Ser240/Ser244. For panels C to G, data shown are representative of three independent experiments.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Biomarker Discovery, Immunohistochemistry, Knock-Out, Staining, Expressing, Cell Culture, Incubation, Lysis, Western Blot, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: Activation of eEF2K is important in cancer cell survival under acute acidic conditions. A549 (A) or HCT116 (B) cells were treated as described for Fig. 9E and ​andF,F, respectively, and then subjected to cell ATP assay using the CellTitre-Glo kit. A549 (C) or HCT116 (E) cells were treated as described for panels A and B, respectively, and then subjected to cytotoxicity assay using CellTox Green kit. A549 (D) or HCT116 (F) cells were cultured as described for panels A and B, respectively, and then subjected to flow cytometry analysis. The percentage of sub-G1 population is presented. For panels A, B, C, and E, results are expressed as means ± SE from 3 independent experiments in duplicate. For panel D, results are means ± SE from 3 independent experiments. For panel E, results are means ± SE from 2 independent experiments and a third one in duplicate. P values were obtained either by two-way ANOVA (*, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001) or by one-way ANOVA followed by Dunnett's test (@, 0.01 ≤ P < 0.05; #, 0.01 < P ≤ 0.001; $, P < 0.001).

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Activation Assay, ATP Assay, Cytotoxicity Assay, CellTox Assay, Cell Culture, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: The role of eEF2K in protein synthesis under acidosis. (A) IPTG (1 μM) was added to A549 cells 5 days before experiment. A549 cells were then transferred to media at different pH values for 1 h with/without 1 μM IPTG. Rates of protein synthesis were determined, and results are presented as counts per minute (cpm) per microgram protein and expressed as means ± SE from 4 independent experiments. (B) Quantification of IPTG-treated cells as described for panel A expressed as a percentage of control (no IPTG treatment). For panels A and B, P values were obtained either by two-way ANOVA (*, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001) or by one-way ANOVA followed by Dunnett's test (@, 0.01 ≤ P < 0.05; #, 0.01 < P ≤ 0.001; $, P < 0.001). (C) A549 cells were incubated at pH 6.7 or 7.4 for 1 h. Lysates were fractionated on sucrose density gradients. Positions of the 40S, 60S, and 80S ribosomal particles and polysome fractions are shown. Absorbance values (254 nm) are in arbitrary units and on the same scale for each panel. Representative data from 4 independent experiments are shown.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Control, Incubation

Journal: iScience

Article Title: Frequent loss of FAM126A expression in colorectal cancer results in selective FAM126B dependency

doi: 10.1016/j.isci.2024.109646

Figure Lengend Snippet: Loss of FAM126A expression causes FAM126B dependency in CRC cell lines (A) Scatterplot depicting the correlation between FAM126A expression and FAM126B gene effect. TPM stands for transcripts per million clean reads. Pearson correlation coefficient (r) and p value were indicated on the plot. Linear regression was represented by the red line. (B) Detection of FAM126A and FAM126A-V5 in indicated cell lines by western blotting. (C) Competitive cell growth assay after inactivation of FAM126B or POLD3 in RKO-Cas9 and SW48-Cas9 cells expressing vector or FAM126A-V5. Data are the mean ± SD. from three technical replicates and normalized to control (sgChr2-4). (D) Verification of FAM126A knock out clones from DLD1 and HCT116. (E) Competitive cell growth assay after inactivation of FAM126B or POLD3 in FAM126A knock out clones relative to control cells expressing non-targeting control (NTC) sgRNA. Data are the mean ± SD from three technical replicates and normalized to control (sgChr2-4).

Article Snippet: DLD1 , Dr. Deepak Nijhawan’s lab at University of Texas Southwestern Medical Center , N/A.

Techniques: Expressing, Western Blot, Growth Assay, Plasmid Preparation, Control, Knock-Out, Clone Assay

Journal: iScience

Article Title: Frequent loss of FAM126A expression in colorectal cancer results in selective FAM126B dependency

doi: 10.1016/j.isci.2024.109646

Figure Lengend Snippet:

Article Snippet: DLD1 , Dr. Deepak Nijhawan’s lab at University of Texas Southwestern Medical Center , N/A.

Techniques: Recombinant, CRISPR, Software

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A) Immunofluorescence was performed on ovarian B2MUT cells transfected with the indicated siRNAs, using an antibody to γH2AX, to evaluate foci formation. (B) Quantification of the number γH2AX foci formed in (G) in 30 representative cells transfected with siRNA to the indicated genes. (C) Examination of DDR signaling in extracts of WT and BRCA2 mutant cells transfected with the indicated siRNAs and immunoblotted with the indicated antibodies. (D) Quantitation of phospho-Chk1, phospho-H2AX and phospho-RPA32 from the BRCA2 mutant cell extracts in experiment (C).

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: Immunofluorescence, Transfection, Mutagenesis, Quantitation Assay

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A) Extracts from B2MUT ovarian cells expressing Cas9 and the indicated gRNAs were immunoblotted with the indicated antibodies. (B) Quantification of signal detected Western blotting in (A), normalized to vinculin. (C-D) MCA assays in which the indicated GFP-labeled B2MUT cells and E2-Crimson-labeled B2WT cells were mixed and co-infected with 3 individual gRNAs to FEN1. The cell mixture was cultured for 7 d, and the change in percent GFP+ cells was quantified by FACS and normalized to the average of the negative control Grna-expressing cells shown in Supplemental Figure 3A. Error bars reflect the variability of biological triplicates, and the number of asterisks indicates the statistical significance for the corresponding experiment (*=p<0.05,**=p<0.01,***=p<0.001). (E) MCA assay in which colonic GFP-labeled BRCA2 MUT cells and E2-Crimson-labeled BRCA2 WT cells were mixed and co-treated with the indicated doses of a FEN1 inhibitor. The cell mixture was cultured for 7 d, and the change in percent GFP+ cells was quantified by FACS and normalized to negative control gRNA-expressing cells. Error bars reflect the variability of biological triplicates, and asterisks indicate statistical significance as described above. (F) Dependency of BRCA1/2 MUT or BRCA1/2 WT cell lines on FEN1, determined from genome-scale CRISPR-Cas9 essentiality screens across 324 cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) (Meyers et al., 2017). (G) List of gRNA-resistant ORFs tested for complementation in BRCA2 MUT cells expressing Cas9 and a FEN1 gRNA. (H) Examination of the ability of the ORFs listed in (G) to rescue the growth defect caused by expression of Cas9 and a FEN1 gRNA. BRCA2 MUT ovarian cells were co-infected with lentivirus expressing a FEN1 gRNA and the indicated gRNA-resistant FEN1 ORF or negative control peptide. After selection and growth for 8 d, survival was quantified with a FACS-based cell counting method. (I) Extracts from BRCA2 WT cells expressing Cas9, a validated FEN1 gRNA, and the indicated gRNA-resistant ORF were immunoblotted with the indicated antibodies; all panels are derived from the same blot.

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: Expressing, Western Blot, Labeling, Infection, Cell Culture, Negative Control, MCA Assay, CRISPR, Selection, Cell Counting, Derivative Assay

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A) Extracts from the indicated cell lines, untreated or treated with the indicated siRNAs, were immunoblotted with antibodies to BRCA2 and GAPDH. Left and right panels were run as separate gels. (B) Immunofluorescence was performed on cells of the indicated genotypes, with antibodies to γH2AX and Rad51 protein, to evaluate foci formation after 10 Gy IR. (C) The indicated cell lines were passaged in the presence of the indicated dose of olaparib or DMSO. After 3 d, cell survival was quantified using CellTiter-Blue. Error bars reflect the variability of biological triplicates. (D) Schematic diagram depicting the experimental procedure for BRCA2 SL screening in isogenic BRCA2 cell lines. (E) Schematic of wild-type BRCA2 structure, depicted with its functional domains and sites of interaction with key binding partners. Brca2 truncation mutant proteins possessed by the colonic and ovarian BRCA2 MUT cell lines used in this study are shown for comparison.

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: Immunofluorescence, Functional Assay, Binding Assay, Mutagenesis, Comparison

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A-C) Volcano plots for the shRNA colonic, CRISPR colonic, and CRISPR ovarian B2SL screens. Significance (-log10FDR) is plotted against the genetic interaction (GI) score (average log2 fold-change for each gene in BRCA2 MUT versus BRCA2 WT cells). Genes that met a significance threshold of -log10FDR>1 are color-coded as green for relative dropout or red for relative enrichment in BRCA2 MUT vs BRCA2 WT cells. (D-F) Results from the colonic shRNA, colonic CRISPR, and ovarian CRISPR B2SL screens, plotted as the log2 fold-change in BRCA2 MUT cells against the log2 fold-change in BRCA2 WT cells. (G-H) Comparison of GI score (average log2 fold-change for each gene in BRCA2 MUT versus BRCA2 WT cells) in the ovarian CRISPR screen versus the colonic CRISPR screen (G), and the ovarian CRISPR screen versus primary shRNA screen (H).

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: shRNA, CRISPR, Comparison

Journal: Molecular cell

Article Title: Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

doi: 10.1016/j.molcel.2018.12.008

Figure Lengend Snippet: (A) Performance of genes in several key pathways, plotted on a color scale for genetic interaction (GI) score: the normalized average log2 fold-change across both colonic and ovarian CRISPR screens. The asterisk indicates reporting of GI score from the shRNA screen instead of combined CRISPR screens. (B) Schematic of the base excision repair (BER) pathway, showing the strength of the GI score for each gene in the pathway, plotted on the same color scale as in (A). (C-F) MCA assays in which colonic GFP-labeled BRCA2 MUT and E2-Crimson-labeled BRCA2 WT cells were mixed and co-treated with the indicated drugs. The change in percent GFP+ cells was measured by FACS after 12 d and normalized to vehicle control. Error bars reflect the variability of biological triplicates.

Article Snippet: Generation of isogenic cell lines We obtained a BRCA2 mutant cell line ( BRCA2 −/− DLD-1) from Horizon Pharma.

Techniques: CRISPR, shRNA, Labeling, Control