Journal: Cellular & molecular immunology

Article Title: Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library.

doi: 10.1038/cmi.2016.38

Figure Lengend Snippet: Figure 1 Computer-aided homology modeling. (a) The ribbon structure of the human PD-1 and PD-L1 complex based on the computer- guided modeling and docking methods. (b) The fine structure of the binding mode between human PD-1 and PD-L1. The purple sticks denote the key amino-acid residues in human PD-L1, whereas the green balls and sticks denote the key amino-acid residues in human PD-1. (c) The 3-D theoretical structure of IgHV3-33*01, where the balls and sticks denote H28, H31 and H32. (d) The 3-D theoretical structure of IgHK1-11*01, where the balls and sticks denote L27, L50 and L91.

Article Snippet: The CD274 (NM_014143) Human cDNA ORF Clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Binding Assay

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Representative fluorescent images of MOC1 and MOC2 cells after 48 h of incubation with increasing titers of GFP-expressing RP1-15. GFP shown in gray scale. (B) MOC1 and MOC2 cell viability after 48 h with increasing RP1 titers in vitro, assessed via CellTiter-Glo assay. Significance determined using 1-way ANOVA with Dunnett test. RLU = relative light units.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Incubation, Expressing, In Vitro, Glo Assay

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 promotes Chk2 content. a . Immunoblot from whole cell lysates of CTRL and PDL1 KO B16 cells. Vinculin (VINC), loading control. γH2AX Ser139 blot is shown as γH2AX. b . Immunoblot from whole cell lysates of CTRL and PDL1 KO ES2 cells. VINC, loading control. c . Immunoblot of CTRL and PDL1 KO ES2 cells transfected with empty vector (EV, gray) and/or PDL1 plasmid (black) at depicted μg. d . Immunoblot of CTRL versus PDL1 KO T24 cells after 72-h incubation with vehicle or gemcitabine (5 ng/mL). e . Quantification of relative intracellular PDL1 and Chk2 expression of 12 human high-grade serous ovarian tumours at upfront treatment. Statistical analysis by linear regression

Article Snippet: For PDL1 mem expression constructs, a mouse PDL1-tGFP fusion protein construct (OriGene MG203953) was PCR amplified and subcloned into the pDisplay vector (Invitrogen) at the BglII and PstI sites, resulting in N-terminal fusion to the murine Igκ-chain leader sequence and C-terminal fusion to the platelet derived growth factor receptor transmembrane domain, anchoring PDL1 to the extracellular plasma membrane (PDL1 mem ).

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Incubation, Expressing

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 deficiency promotes Chk2-dependent synthetic lethality to Chk1i and ATRi in vitro. a . In vitro cell viability of CTRL versus PDL1 KO B16, T24, and ID8agg cells or CTRL versus PDL1 lo 4T1 and MDA-MB-231 cells incubated with the selective Chk1i rabusertib for 5 days at indicated doses (mean ± SD, n = 3 independent replicates, P values by two-way ANOVA). Viability normalised to vehicle controls. b . Flow cytometry analysis of Annexin/PI-stained CTRL versus PDL1 KO T24 cells and c . CTRL versus PDL1 lo 4T1 cells after treatment with vehicle (DMSO) or 1 µM rabusertib. Q1 (% dead cells) quantified. P, two-way ANOVA. d . Clonogenic crystal violet stain of PDL1 KO T24 or PDL1 lo 4T1 and respective CTRL cells treated with indicated rabusertib (Chk1i) concentrations for 5 days. e . Cell viability of indicated CTRL versus PDL1-deficient cell lines after ATRi (AZD6738) or f . ATMi (AZD0156) incubation. P , two-way ANOVA. g . IC 50 for the Chk1i prexasertib and h . ATMi AZD0156 in 44 human SCLC-A cell lines segregated by low (lo) or high (hi) PDL1 expression. P, two-sided t -test. Grubb’s test was used to identify outliers. i . Immunoblot after Chk2 re-expression plasmids (Chk2 RE ) or empty vector controls were transiently introduced into CTRL or PDL1 lo 4T1 cells and treated with rabusertib (Chk1i, 2.5 µM) or vehicle (DMSO) for 72 h. j . PDL1 lo 4T1 viability with transient Chk2 RE as in i , treated with 2.5 µM rabusertib for 5 days in vitro versus CTRL and PDL1 lo with empty vector. Data are mean ± SD normalised to vehicle. P, unpaired t -test

Article Snippet: For PDL1 mem expression constructs, a mouse PDL1-tGFP fusion protein construct (OriGene MG203953) was PCR amplified and subcloned into the pDisplay vector (Invitrogen) at the BglII and PstI sites, resulting in N-terminal fusion to the murine Igκ-chain leader sequence and C-terminal fusion to the platelet derived growth factor receptor transmembrane domain, anchoring PDL1 to the extracellular plasma membrane (PDL1 mem ).

Techniques: In Vitro, Incubation, Flow Cytometry, Staining, Expressing, Western Blot, Plasmid Preparation

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 depletion promotes Chk1i synthetic lethality in vivo that requires adaptive immunity in distinct models. Growth in mammary fat pad of a . CTRL and b . PDL1 lo 4T1 tumours in immune deficient NSG mice treated with vehicle or rabusertib (Chk1i). Data represented as mean ± SEM. P by two-way ANOVA. c. Tumour end-point weights from a and b . P, unpaired t -test. d-e . Tumour growth curves of NSG mice challenged with d . CTRL and e . PDL1 KO B16 cells and treated with rabusertib daily. P , two-way ANOVA. f . NSG mice bearing B16 tumours as in d-e were sacrificed day 21 post-challenge and tumours were weighed. P , unpaired t -test. g - h . Growth of subcutaneous g . CTRL versus h . PDL1 KO B16 tumours in WT mice treated with vehicle or rabusertib. Data represented as mean ± SEM. P, two-way ANOVA. i . Tumour end-point weights from g and h . P, unpaired t- test. j - l . Flow cytometry immune analyses of indicated populations in CTRL versus PDL1 KO B16 tumours treated with vehicle or rabusertib in vivo. Mice were sacrificed day 17 post-challenge. P, unpaired t -test. m . Tumour growth curves of Rag2 KO mice bearing PDL1 KO B16 tumours treated with vehicle or rabusertib daily. P , two-way ANOVA. n . Rag2 KO mice as in m were sacrificed day 20 post-challenge. P , unpaired t -test

Article Snippet: For PDL1 mem expression constructs, a mouse PDL1-tGFP fusion protein construct (OriGene MG203953) was PCR amplified and subcloned into the pDisplay vector (Invitrogen) at the BglII and PstI sites, resulting in N-terminal fusion to the murine Igκ-chain leader sequence and C-terminal fusion to the platelet derived growth factor receptor transmembrane domain, anchoring PDL1 to the extracellular plasma membrane (PDL1 mem ).

Techniques: In Vivo, Flow Cytometry

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Autochthonous PDL1-deficient tumour cells phenocopy genetic PDL1 silencing by inducing Chk2 deficiency and Chk1i synthetic lethality in vitro and in vivo. a . Immunoblot validation of PDL1 expression in NRAS Q61R -mutant melanoma cells derived from CTRL TN 61R (NCH1; PDL1 + ) or PDL1 − TN 61R (NFH1; PDL1 − ) mice. Cells were treated in vitro with 0.1 ng/mL IFNγ for 48 h. b . Immunoblot of NCH1 (PDL1 + ) or NFH1 (PDL1 − ) cells treated in vitro with the Chk1i rabusertib or vehicle (veh) for indicated proteins. c-d . Cell viability of PDL1 + NCH1 versus PDL1 − NFH1 melanoma cells treated with the Chk1i c . rabusertib or d . prexasertib for 72 h in vitro. Viability normalised to vehicle. P, two-way ANOVA. NSG mice were challenged with e. PDL1 + NCH1 cells or f . PDL1 − NFH1 cells and treated with rabusertib daily. P , two-way ANOVA. g . Tumour-bearing NSG mice were sacrificed day 18 post-challenge. Tumours were excised and weighed. P , unpaired t -test. WT mice were challenged with h . NCH1 or i . NFH1 cells and treated with vehicle or rabusertib daily. P , two-way ANOVA. j . Tumour-bearing WT mice were sacrificed, and tumours were excised and weighed. P , unpaired t -test. k - m . WT mice bearing NCH1 or NFH1 tumours were sacrificed day 28 post-challenge and profiled for indicated immune populations by flow cytometry. MFI, mean fluorescence intensity. P , unpaired t -test

Article Snippet: For PDL1 mem expression constructs, a mouse PDL1-tGFP fusion protein construct (OriGene MG203953) was PCR amplified and subcloned into the pDisplay vector (Invitrogen) at the BglII and PstI sites, resulting in N-terminal fusion to the murine Igκ-chain leader sequence and C-terminal fusion to the platelet derived growth factor receptor transmembrane domain, anchoring PDL1 to the extracellular plasma membrane (PDL1 mem ).

Techniques: In Vitro, In Vivo, Western Blot, Biomarker Discovery, Expressing, Mutagenesis, Derivative Assay, Flow Cytometry, Fluorescence

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 control of Chk2 is through intracellular PDL1 and independent of PD1 and immunity. a . Immunoblots of PDL1 KO and PD1 KO B16 cells versus CTRL B16 cells for indicated proteins. Vinculin (VINC) loading control. b . In vitro MTT cell viability of CTRL T24 cells treated with the αPD1 antibodies pembrolizumab (50 µg/mL), balstilimab (50 µg/mL), or genetic shRNA PD1 knockdown (PD1 lo ) with increasing doses of the Chk1i rabusertib for 5 days at indicated doses. P by two-way ANOVA. Viability shown as relative values following normalisation to respective isotype or vehicle-treated controls. c . Schematic of membrane (mem)-localising versus intracellular (cyto)-localising PDL1 expression constructs (see methods). d . RNA-seq analyses of PDL1 mem versus PDL1 cyto B16 cells using gene set enrichment analysis. Top 10 altered hallmark pathways ( p < 0.01) are shown by PDL1 subcellular location. e . Immunoblot comparing ATM, Chk2, γH2AX, and vimentin expression of indicated B16 cells. Vinculin (VINC) as loading control. f . MTT viability assay of PDL1 mem versus PDL1 cyto cells after 96-h of in vitro treatment with the Chk1i rabusertib at indicated concentrations. P, two-way ANOVA. g . Viability analysis of CTRL versus PDL1 KO T24 cells treated with indicated rabusertib concentrations ± 1 ng/mL IFNγ, 10 ng/mL TNFα, or vehicle. P, two-way ANOVA. h . Immunoblot of cell lysates of membrane and cytosolic fractions of CTRL T24 cells treated with vehicle (PBS), IFNγ, or TNFα as in g . Na + /K + pump as membrane loading control and GAPDH as cytosolic loading control.

Article Snippet: For PDL1 mem expression constructs, a mouse PDL1-tGFP fusion protein construct (OriGene MG203953) was PCR amplified and subcloned into the pDisplay vector (Invitrogen) at the BglII and PstI sites, resulting in N-terminal fusion to the murine Igκ-chain leader sequence and C-terminal fusion to the platelet derived growth factor receptor transmembrane domain, anchoring PDL1 to the extracellular plasma membrane (PDL1 mem ).

Techniques: Control, Western Blot, In Vitro, shRNA, Knockdown, Membrane, Expressing, Construct, RNA Sequencing, MTT Viability Assay

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: PDL1 protects Chk2 from ubiquitination in distinct tumours. a . Volcano plot of RNA-seq data depicting PDL1 KO effect on select mRNA levels versus CTRL B16. b . qRT-PCR of Chek2 mRNA in CTRL versus PDL1 KO B16 cells treated with DMSO or rabusertib. P , unpaired t -test. c . Immunoblot of CTRL versus PDL1 KO B16 cells treated with cycloheximide (CHX). Summary graph depicts normalised total Chk2 protein levels quantified using ImageJ. d . Immunoblot of CTRL versus PDL1 KO B16 and e . ES2 cells treated with MG132 (2 µM) for 12-h. f. Immunoblot of immunoprecipitated Chk2 from CTRL, PDL1 KO , and stable PDL1 knock-in (PDL1 KI ) B16 cells and g . CTRL and PDL1 KO ES2 cells after MG132 treatment. h . CTRL and PDL1 KO T24 cells treated with MG132 for 16-h plus vehicle (H 2 O) or 10 ng/mL gemcitabine (gem). Endogenous Chk2 was immunoprecipitated and Chk2 and ubiquitination (Ub) were detected by immunoblot

Article Snippet: For PDL1 mem expression constructs, a mouse PDL1-tGFP fusion protein construct (OriGene MG203953) was PCR amplified and subcloned into the pDisplay vector (Invitrogen) at the BglII and PstI sites, resulting in N-terminal fusion to the murine Igκ-chain leader sequence and C-terminal fusion to the platelet derived growth factor receptor transmembrane domain, anchoring PDL1 to the extracellular plasma membrane (PDL1 mem ).

Techniques: Ubiquitin Proteomics, RNA Sequencing, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Knock-In

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Tumour PDL1 protects Chk2 from PIRH2 E3 ligase-mediated degradation in distinct tumours through its immunoglobulin-like domains. a . Non-targeting or PIRH2-specific siRNA vectors transfected into CTRL or PDL1 KO B16 cells or b . PDL1 + NCH1 cells and PDL1 − NFH1 cells. c . Interaction among PDL1, Chk2 and PIRH2 in B16 cells stably expressing vector control or turbo-GFP (tGFP)-tagged PDL1 (PDL1 tGFP ) treated with MG132 and co-immunoprecipitated with tGFP trap beads followed by immunoblotting. d . Endogenous PIRH2 co-immunoprecipitated in CTRL, PDL1 KO , and PDL1 KI B16 cells. e . Left: Flag-tagged human PDL1 and Streptavidin-tagged human Chk2 were co-expressed in Tni cells and affinity pull-downs of lysates with Flag resin and eluate fractions (1,2,3,4) followed by SDS-PAGE and Coomassie blue staining. Right: eluates were pooled for reciprocal Chk2 affinity pulldown using Strep-tactin resin. L, lysates; FT, flow-through; I, input. f . Viability of PDL1 truncation mutant-expressing B16 cells 72-h post rabusertib (2.5 μM) treatment. P, unpaired t -test. g . Immunoblot of domain-expressing B16 cells immunoprecipitated using anti-mCherry beads

Article Snippet: For PDL1 mem expression constructs, a mouse PDL1-tGFP fusion protein construct (OriGene MG203953) was PCR amplified and subcloned into the pDisplay vector (Invitrogen) at the BglII and PstI sites, resulting in N-terminal fusion to the murine Igκ-chain leader sequence and C-terminal fusion to the platelet derived growth factor receptor transmembrane domain, anchoring PDL1 to the extracellular plasma membrane (PDL1 mem ).

Techniques: Transfection, Stable Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, SDS Page, Staining, Mutagenesis

Journal: Molecular Cancer

Article Title: Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors

doi: 10.1186/s12943-024-02147-z

Figure Lengend Snippet: Pharmacologic tumour PDL1 depletion replicates genetic tumour PDL1 KO by depleting Chk2 and inducing Chk1i synthetic lethality. a . Immunoblot of CTRL T24 cells for indicated proteins after 72 h of treatment with vehicle (DMSO), 1 µM rabusertib (Chk1i), 80 μM cefepime, or combination. b . Immunoblot of CTRL, PDL1 KO , and stable PDL1 KI B16 cells treated with 80 μM cefepime for indicated time points. c-e . Cell viability of c. CTRL MDA-MB-231, d . CTRL T24, and e. PDL1 KO T24 cells treated with indicated concentrations of rabusertib ± 80 μM cefepime or vehicle (DMSO) for 96 h. P, two-way ANOVA. f . Immunoblot of CTRL T24 cells treated with vehicle, 1 μM rabusertib, 80 μM ceftazidime, or both for 72 h. g . Immunoblot of CTRL, PDL1 KO and stable PDL1 KI B16 cells treated with 80 μM ceftazidime for indicated time points. h - j . Cell viability of h . CTRL MDA-MB-231, i . CTRL T24, and j . PDL1 KO T24 cells treated with indicated concentrations of rabusertib ± 80 μM ceftazidime or vehicle for 96 h. P, two-way ANOVA. k . MTT viability analysis of CTRL T24 cells treated for 96 h with 80 μM of indicated β-lactams with rabusertib. P, two-way ANOVA. l . Growth curves of PDL1 + NCH1 tumours implanted subcutaneously in WT mice treated with vehicle, ceftazidime, rabusertib, or both. P, two-way ANOVA. m . Tumour weights of NCH1 tumours from l . at endpoint (day 18). P, unpaired t -test. Vehicle (veh), ceftazidime (ctz), rabusertib (rab), and ceftazidime + rabusertib (combo) groups are indicated

Article Snippet: For PDL1 mem expression constructs, a mouse PDL1-tGFP fusion protein construct (OriGene MG203953) was PCR amplified and subcloned into the pDisplay vector (Invitrogen) at the BglII and PstI sites, resulting in N-terminal fusion to the murine Igκ-chain leader sequence and C-terminal fusion to the platelet derived growth factor receptor transmembrane domain, anchoring PDL1 to the extracellular plasma membrane (PDL1 mem ).

Techniques: Western Blot