Journal: EMBO Molecular Medicine

Article Title: Oncogene addiction as a foundational rationale for targeted anti-cancer therapy: promises and perils

doi: 10.1002/emmm.201100176

Figure Lengend Snippet: Targeted therapies in oncogene-addicted cancers

Article Snippet: A Phase 1 trial conducted in 2009 demonstrated the anti-tumour activity of the small-molecule PARP inhibitor olaparib (AstraZeneca) in a population of cancer patients (ovarian, breast and prostate) enriched for BRCA1 or BRCA2 mutations, with BRCA mutation carriers exhibiting radiographic response or meaningful disease stabilization (stable disease for a period of 4 months or more; Fong et al, ).

Techniques:

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Biphasic TRF2 recruitment to non-telomeric damage sites in nuclei of HeLa cells. (A) PAR stimulation by PARG inhibition (PARGi) promotes GFP–TRF2 accumulation at low input-power damage sites (indicated by arrowheads). Box plot shows quantification of the relative increase of GFP signals at damage sites. (B) Time-course analysis of GFP–TRF2 recruitment to laser-induced DNA damage sites (between arrowheads). (C) Quantification of GFP signals at damage sites in B. N=16. (D) Detection of endogenous TRF2 at damage sites. PARP inhibition (PARPi) suppresses phase I, but has no effect on phase II, TRF2 recruitment. (E) Quantification of the effects of PARP inhibitors (NU1025 and olaparib) on immediate (1 min, phase I) and late (30 min, phase II) GFP–TRF2 recruitment. (F) Time course analysis of the effect of IDP depletion on dispersion of TRF2 at damage sites in HeLa cells transfected with control siRNA (siControl) or FET siRNAs (siFET). Left: quantification of signal intensity changes of GFP–TRF2 (blue) and dark line (red). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Scale bar: 10 μm.

Article Snippet: Inhibitor treatment 20 μM PARP inhibitor olaparib (Apexbio Technology) or 1 μM PARG inhibitor (PARGi) 6,9-diamino-2-ethoxyacridine lactate monohydrate (DEA; Trevigen) was added to the cell culture 1 h prior to damage induction.

Techniques: Inhibition, Dispersion, Transfection, Control

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Phase II recruitment is affected by TERT and is dependent on the iDDR region in the hinge domain of TRF2. (A) Box plot shows that TERT depletion using siRNA inhibits phase II recruitment of TRF2 to DNA damage sites in nuclei of HeLa cells. (B) Schematic diagrams of chimeric TRF1/2 mutants (as previously described in Okamoto et al., 2013) used in the experiments represented in panels C–F. (C) Left: representative cell images of the recruitment of chimeric mutants to damage sites (between arrowheads) at ∼1 min (phase I) and ∼30 min (phase II) after damage induction. Right: box plots show quantification of the GFP–TRF2 signal increase at phase I and phase II at damage sites. (D) Comparison of the GFP signal at damage sites in HeLa cells expressing iDDR and TIN2 deletion mutants at 30 min after damage induction. (E) The effect of MRE11 and NBS1 siRNA (siMRE11 and siNBS1) depletion on phase I and II recruitment of GFP–TRF2 was examined comparing to control siRNA (siControl). HeLa cells were fixed and stained with anti-MRE11 antibody (red) to confirm the depletion. Box plot shows quantification of the GFP–TRF2 signal increase at damage sites in control or MRE11 and NBS1 siRNA-treated cells. (F) ChIP-qPCR analysis of GFP–TRF2 binding at I-PpoI cut sites. TRF2 binding was examined in the absence or presence of I-PpoI, and with and without MMS as indicated. Cells were further treated with DMSO or PARP inhibitor (PARPi) (left panel). Cells expressing GFP only were used as a negative control. Alternatively, cells were transfected with control (siCon) or MRE11 and NBS1 siRNA in the presence of I-PpoI with or without MMS (right panel). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Bar graphs show mean±s.d., *P<0.01, **P<0.001, ***P<0.0001. Scale bars: 10 μm.

Article Snippet: Inhibitor treatment 20 μM PARP inhibitor olaparib (Apexbio Technology) or 1 μM PARG inhibitor (PARGi) 6,9-diamino-2-ethoxyacridine lactate monohydrate (DEA; Trevigen) was added to the cell culture 1 h prior to damage induction.

Techniques: Comparison, Expressing, Control, Staining, ChIP-qPCR, Binding Assay, Negative Control, Transfection

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: TRF2 specifically promotes non-sister chromatid HR repair. (A) The effect of TRF2 depletion on DSB repair using the I-SceI HR system. Complementation analysis of TRF2-depleted cells was performed using the wild type and chimeric TRF1/2 mutants. BRCA1 depletion was used as a positive control. Comparable expression levels of the recombinant TRF2 proteins were confirmed using western blot analysis (right). Histone H3 serves as a loading control. (B) The effect of TRF2 depletion on different DSB repair pathways was examined using SCE, classic NHEJ (C-NHEJ) and alternative NHEJ (Alt-NHEJ) assays. (C) Schematic showing similarity between strand invasion in D-loop formation at telomeres and at DSB sites by TRF2. (D) Schematic showing biphasic mechanism of TRF2 recruitment to damage sites. Phase I involves PARP-dependent recruitment through the basic domain. Phase II is mediated by the MYB/SANT domain, which is also dependent on the iDDR region and the Mre11 complex. Bar graphs show mean±s.d.

Article Snippet: Inhibitor treatment 20 μM PARP inhibitor olaparib (Apexbio Technology) or 1 μM PARG inhibitor (PARGi) 6,9-diamino-2-ethoxyacridine lactate monohydrate (DEA; Trevigen) was added to the cell culture 1 h prior to damage induction.

Techniques: Positive Control, Expressing, Recombinant, Western Blot, Control

Journal: Nature Communications

Article Title: DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells

doi: 10.1038/s41467-017-01883-9

Figure Lengend Snippet: Depletion of BRCA2 enhances PD-L1 upregulation after DSBs. a Depletion of BRCA2 enhances the upregulation of PD-L1 after IR. U2OS cells were exposed to BRCA2 siRNA. PD-L1 was examined 48 h after 5 or 10 Gy. Knockdown efficiency of BRCA2 is shown in the bottom panel. b PARP inhibition enhanced PD-L1 upregulation in BRCA2-depleted cells. U2OS cells were exposed to BRCA2 siRNA. PD-L1 was examined 2 and 4 days after the addition of PARPi. Knockdown efficiency of BRCA2 is shown in the bottom panel. Similar results were obtained using a distinct siRNA (Supplementary Fig. ). c The enhancement of PD-L1 expression in BRCA2-depleted cells following PARP inhibition requires Chk1 activity. PD-L1 in U2OS cells was examined 2 and 4 days after the addition of PARPi with or without Chk1i (UCN-01). Similar results were obtained using a distinct siRNA (Supplementary Fig. ). d Depletion of BRCA2 enhanced the upregulation of PD-L1 mRNA after IR. PD-L1 mRNA in U2OS cells with or without BRCA2 siRNA was examined 48 h after 10 Gy. e ATM/Chk1 activity is required for the upregulation of PD-L1 mRNA in BRCA2-depleted cells after IR. PD-L1 mRNA in U2OS cells following BRCA2 siRNA with or without ATMi or Chk1i (UCN-01) was examined 48 h after 10 Gy. Similar results were obtained in H1299 cells (Supplementary Fig. ). f , g Depletion of BRCA2 enhanced the upregulation of cell-surface PD-L1 after IR or PARP inhibition. Cell-surface PD-L1 expression in U2OS ( f ) or H1299 ( g ) cells following BRCA2 depletion was examined by flow cytometry 48 h after 10 Gy. h – k The upregulation of cell-surface PD-L1 requires Chk1 activity. Cell-surface PD-L1 expression in U2OS cells ( h , i ) was examined with or without Chk1 inhibitor after IR or PARPi. Cell-surface PD-L1 expression in H1299 cells was examined with or without Chk1 inhibitor after IR or PARPi ( j , k ). Error bars represent the s.d. of three independent experiments ( d – k ). Statistical significance was determined using Student’s two-tailed t -test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PARP inhibitor (PARPi) (Olaparib; AdooQ BioScience) was also added at 10 μM, and the medium was refreshed with 10 μM PARPi every 48 h until the indicated time point.

Techniques: Inhibition, Expressing, Activity Assay, Flow Cytometry, Two Tailed Test

Journal: Cancers

Article Title: PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma

doi: 10.3390/cancers11060751

Figure Lengend Snippet: PARP protein expression in UM patients (P0) and their corresponding PDXs at various in vivo passages (P1, P4, and P9) as measured by RPPA. Boxplots represent the distribution of data of all PDX models together. Boxes contain 50% of values, upper brackets contain the 25% highest values, lower brackets contain the 25% lowest values. Black lines: median; dots: outliers. P0, patient’s tumor; P1, first in vivo passage; P4, fourth in vivo passage; P9, ninth in vivo passage.

Article Snippet: The PARP inhibitor olaparib, partially provided from Novartis Institutes for Biomedical Research (NIBR, Cambridge, UK) and Astra-Zeneca (Oncology Bioscience, Cambridge, UK), was orally administered at a daily dose of 50 mg/kg or 100 mg/kg, five days a week, depending to the tolerance of tested combinations.

Techniques: Expressing, In Vivo

Journal: Cancers

Article Title: PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma

doi: 10.3390/cancers11060751

Figure Lengend Snippet: Waterfall plots of response to olaparib-based therapies. Upper p values correspond to statistical differences between olaparib compared to each other tested treatment; p values at the bottom correspond to statistical differences between each tested treatment compared to its combination with olaparib (χ 2 test).

Article Snippet: The PARP inhibitor olaparib, partially provided from Novartis Institutes for Biomedical Research (NIBR, Cambridge, UK) and Astra-Zeneca (Oncology Bioscience, Cambridge, UK), was orally administered at a daily dose of 50 mg/kg or 100 mg/kg, five days a week, depending to the tolerance of tested combinations.

Techniques:

Journal: Cancers

Article Title: PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma

doi: 10.3390/cancers11060751

Figure Lengend Snippet: Combination of olaparib and dacarbazine. ( A ) Growth curves of all treated models. ( B–E ) ORR of the four experimental groups, i.e., control (black), olaparib (blue), dacarbazine (dark green), and olaparib + dacarbazine (light green), respectively. RTV: Relative tumor variation. mRTVc: median relative tumor volume of the corresponding control group ( F – G ). Probability of progression of the four experimental groups: doubling time ( F ) and quadrupling time ( G ).

Article Snippet: The PARP inhibitor olaparib, partially provided from Novartis Institutes for Biomedical Research (NIBR, Cambridge, UK) and Astra-Zeneca (Oncology Bioscience, Cambridge, UK), was orally administered at a daily dose of 50 mg/kg or 100 mg/kg, five days a week, depending to the tolerance of tested combinations.

Techniques: Control

Journal: Cancers

Article Title: PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma

doi: 10.3390/cancers11060751

Figure Lengend Snippet: Venn diagram of the proteins significantly ( p < 0.01) correlated to response to DTIC alone (blue) (MP55 + MP77 + MM52 versus MM33), olaparib alone (green) (MM33 versus MP55 + MP77 M MM52), or the DTIC+ olaparib combination (red) (MP55 + MP77 + MM33 versus MM52) (paired t tests assuming equal variance).

Article Snippet: The PARP inhibitor olaparib, partially provided from Novartis Institutes for Biomedical Research (NIBR, Cambridge, UK) and Astra-Zeneca (Oncology Bioscience, Cambridge, UK), was orally administered at a daily dose of 50 mg/kg or 100 mg/kg, five days a week, depending to the tolerance of tested combinations.

Techniques:

Journal: Cancers

Article Title: PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma

doi: 10.3390/cancers11060751

Figure Lengend Snippet: Schematic representation of HR- and NHEJ-related protein expression modifications in the two PDXs MP55 and MM52: Olaparib + DTIC versus DTIC. The intensity of color reflects fold change. Red color: higher (phospho-)protein expression in olaparib + DTIC than in DTIC alone. Green color: lower (phospho-)protein expression in olaparib + DTIC than in DTIC alone.

Article Snippet: The PARP inhibitor olaparib, partially provided from Novartis Institutes for Biomedical Research (NIBR, Cambridge, UK) and Astra-Zeneca (Oncology Bioscience, Cambridge, UK), was orally administered at a daily dose of 50 mg/kg or 100 mg/kg, five days a week, depending to the tolerance of tested combinations.

Techniques: Expressing