Journal: Cancer Discovery

Article Title: A Covalent Allosteric Molecular Glue Suppresses NRF2-Dependent Cancer Growth

doi: 10.1158/2159-8290.CD-25-1187

Figure Lengend Snippet: KEAP1_Cys 151 liganding by VVD-065 induces NRF2 degradation and inhibits NRF2 target gene expression. A, Progression of KEAP1_Cys 151 ligands from the initial hit VVD-325 to more advanced VVD-860 and VVD-065. Bardoxolone methyl and PSTC served as reference compounds. (*, Bardoxolone methyl is a covalent reversible compound, and therefore the value is not directly comparable with the other values.) B, ARE-luciferase reporter activity in NRF2 W24C KYSE70 cells treated for 18 hours with PSTC, bardoxolone methyl, and VVD KEAP1_Cys 151 ligands. C, Chemoproteomic determination of KEAP1_Cys 151 engagement by VVD-065 in KYSE70 lysates. D, Simple Western analysis of NRF2 and actin in KYSE70 cells treated for 18 hours with VVD-064 and VVD-065 at indicated concentrations. E, qPCR-based expression analysis of NRF2 target genes in KYSE70 cells treated for 18 hours with VVD-065 at indicated concentrations. N.D. = Not Determined ( B, Created with BioRender, BioRender.com .)

Article Snippet: First, the RNP was complexed by mixing 3 μL of Synthego NRF2 Gene Knockdown Kit V2 (pooled single-guide RNAs) with 2 μL Cas9 (IDT; #1081059).

Techniques: Targeted Gene Expression, Luciferase, Activity Assay, Simple Western, Expressing

Journal: Cancer Discovery

Article Title: A Covalent Allosteric Molecular Glue Suppresses NRF2-Dependent Cancer Growth

doi: 10.1158/2159-8290.CD-25-1187

Figure Lengend Snippet: VVD-065 is a highly selective ligand of KEAP1_Cys 151 . A, Cysteine-directed selectivity analysis of VVD-065 in MDA-MB-468 cells (>24,000 sites were surveyed). B, Left, chemical structure of the alkyne probe; right, protein-directed selectivity analysis of VVD-065 in KYSE70 cells following click-chemistry and pull-down of proteins with VVD-369. C, MS analysis of KYSE70 parental cells treated with 100 nmol/L VVD-065 for 24 hours. D, NRF2 expression change in VVD-065–treated KYSE70 parental, KEAP1 KO, and NFE2L2 KO cells as measured by MS.

Article Snippet: First, the RNP was complexed by mixing 3 μL of Synthego NRF2 Gene Knockdown Kit V2 (pooled single-guide RNAs) with 2 μL Cas9 (IDT; #1081059).

Techniques: Expressing

Journal: Cancer Discovery

Article Title: A Covalent Allosteric Molecular Glue Suppresses NRF2-Dependent Cancer Growth

doi: 10.1158/2159-8290.CD-25-1187

Figure Lengend Snippet: VVD-065 stabilizes KEAP1–CUL3 complex formation. A, Binding interactions of VVD-065. VVD-065 binds in a hydrophobic cleft and forms a hydrogen bond with the side chain of R135 and a water-mediated interaction with the backbone amide of His129. B, Model of the KEAP1–CUL3 complex highlighting the distance between the KEAP1–NRF2 interface (yellow), the KEAP1–CUL3 interface, and the VVD-065 binding site (red). KEAP1 (light and dark blue) forms a dimer through the BTB domain, and each copy independently binds to CUL3 (gray). VVD-065 binding site in the KEAP1 monomer. C, Superposition of bardoxolone-bound KEAP1 (orange, 4CXT) with VVD-065–bound KEAP1 (blue), highlighting the concerted movement of residues C151, M147, F174, and F52. D, Superposition of APO–KEAP1 (dark red, 4CXI), KEAP1–CUL3 complex (purple and gray, 5NLB), KEAP1–bardoxolone (orange, 4CXT), and KEAP1–VVD-065 (blue). E, Effects of bardoxolone methyl and VVD-065 on the KEAP1–CUL3 interaction measured by HTRF assay. F, MS analysis of KEAP1-interacting proteins in the presence and absence of VVD-065 using a miniTurbo-based proximity labeling assay. G, Viability levels of KYSE70 cells treated with VVD-065 under either the adherent (2D monolayer assay) or nonadherent (3D sphere assay) culture condition. H, Correlation analysis of NRF2 degradation and growth inhibition in cells treated with VVD-065. NRF2 expression was measured using Simple Western. Growth inhibition at a concentration of 0.2 μmol/L was plotted for all cell lines, except for NCI-H23 and KYSE180, for which a concentration of 0.3 μmol/L was used. Mutation status of each cell line is indicated by different colors. Cell lines showing >50% growth inhibition with genetic depletion of NRF2 (Supplementary Fig. S10C) are indicated by filled square. Cell lines with <50% growth inhibition in Supplementary Fig. S10C are indicated by filled circles.

Article Snippet: First, the RNP was complexed by mixing 3 μL of Synthego NRF2 Gene Knockdown Kit V2 (pooled single-guide RNAs) with 2 μL Cas9 (IDT; #1081059).

Techniques: Binding Assay, HTRF Assay, Labeling, Inhibition, Expressing, Simple Western, Concentration Assay, Mutagenesis

Journal: Cancer Discovery

Article Title: A Covalent Allosteric Molecular Glue Suppresses NRF2-Dependent Cancer Growth

doi: 10.1158/2159-8290.CD-25-1187

Figure Lengend Snippet: VVD-065 exhibits antitumorigenic effects in vivo . A–C, Pharmacokinetic (PK) and PD properties of VVD-065 in a KYSE70 xenograft model. KYSE70 tumor-bearing animals were orally dosed at 5, 25, and 50 mg/kg once ( A and B ) or for 7 days ( C ). A, Plasma was collected 30 minutes after the dose for PK analysis, and tumors were collected 24 hours after the dose to measure covalent binding of VVD-065 to KEAP1_Cys 151 . B, Expression of NRF2 target genes in KYSE70 tumors treated with VVD-065 at indicated doses measured by qPCR. C, Expression of NRF2 protein in vehicle-treated or VVD-065–treated tumors. Chemoproteomic determination of KEAP1 Cys151 engagement by VVD-065 in KYSE70 lysates. From left, lanes 1-2 - vehicle, 3-4 5 mg/kg VVD-065 once daily. D, PK–PD time-course analysis in the KYSE70 xenograft model. KYSE70 tumor−bearing animals were orally dosed once (QD × 1) or for 7 days (QD × 7) with VVD-065 at 5 mg/kg. Plasma exposure, KEAP1_Cys 151 target engagement, NRF2 protein expression, and expression of NRF2 targets at RNA ( AKR1B10 , AKR1C1 , ALDH3A1 , CYP4F11 , GPX2 , NR0B1 , and SLC7A11 ) and protein (AKR1B10, AKR1C1, ALDH3A1, CYP4F11, and NR0B1) levels were measured at indicated timepoints. E, Antitumor efficacy of VVD-065 in the KYSE70 xenograft model. Data are shown as mean ± SEM; n = 9–10 animals/group. Mice were dosed orally with VVD-065 at indicated doses. F, Antitumor efficacy of VVD-065 in the HCC95 xenograft model. Data are shown as mean ± SEM; n = 10 animals/group. Mice were dosed orally with VVD-065 at indicated doses. For E and F , statistical significance was calculated by two-way ANOVA (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). BID, twice a day; QD, once a day.

Article Snippet: First, the RNP was complexed by mixing 3 μL of Synthego NRF2 Gene Knockdown Kit V2 (pooled single-guide RNAs) with 2 μL Cas9 (IDT; #1081059).

Techniques: In Vivo, Clinical Proteomics, Binding Assay, Expressing, Drug discovery

Journal: Cancer Discovery

Article Title: A Covalent Allosteric Molecular Glue Suppresses NRF2-Dependent Cancer Growth

doi: 10.1158/2159-8290.CD-25-1187

Figure Lengend Snippet: VVD-065 exhibits antitumorigenic effects in PDX models. A–D, Percent change in tumor volume on response calling day (days 20–28 after treatment initiation) in indicated sqNSCLC ( A ), ESCC ( B ), HNSCC ( C ), and LUAD ( D ) PDX models after VVD-065 administration. VVD-065 was administered orally at 50 mg/kg once a day (QD) or twice a day (BID) to mice bearing the indicated PDX models ( n = 2–3/group). Bottom, amino acid changes in NRF2/KEAP1/CUL3 are shown for PDX models. WT models are indicated by green filled boxes. Dotted line represents the 50% TGI threshold. E, Antitumor efficacy of VVD-065 in LU1258, LU6403, LU6407, and LU6917 PDX models. Data are shown as mean ± SEM; n = 2–3 animals/group. Mice were dosed orally with VVD-065 at indicated doses. F, Antitumor efficacy of VVD-065 in the ES3864 PDX model. Data are shown as mean ± SEM; n = 2 animals/group. Mice were dosed orally with VVD-065 at indicated doses. G, Antitumor efficacy of VVD-065 in ES3882 and LU2639 PDX models. Data are shown as mean ± SEM; n = 2–3 animals/group. Mice were dosed orally with VVD-065 at indicated doses. H, Antitumor efficacy of VVD-065 in the HN0696 PDX model. Data are shown as mean ± SEM; n = 3 animals/group. Mice were dosed orally with VVD-065 at indicated doses. For E–H , statistical significance was calculated by two-way ANOVA (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).

Article Snippet: First, the RNP was complexed by mixing 3 μL of Synthego NRF2 Gene Knockdown Kit V2 (pooled single-guide RNAs) with 2 μL Cas9 (IDT; #1081059).

Techniques:

Journal: Cell Death & Disease

Article Title: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

doi: 10.1038/cddis.2013.541

Figure Lengend Snippet: Identification of UCA1 as an hnRNP I-binding partner. ( a ) Enrichment of UCA1 by hnRNP I antibody, as detected by RIP assay. ( b ) Confirmation of the interaction between UCA1 and hnRNP I by RNA precipitation assay using UCA1 RNA probe. Preparation of UCA1 RNA probe and cellular extract from MCF-7 cells were detailed in the text. ( c ) The 5′ end of UCA1 is responsible for its interaction with hnRNP I, as detected by RNA precipitation. UCA1 RNA probes used in this experiment were indicated in top panel. RoR exon 4 was used as a positive control. ( d ) A putative hnRNP I-binding motif in UCA1 is critical to its interaction with hnRNP I. Description of the mutant UCA1 at the putative hnRNP I-binding site was indicated in left panel where the conserved binding motif was underlined. Y, either C or T. Detection of hnRNP I in the pellets by western blot as indicated in right panel. Error bars represent S.E.M., n =3. ** P <0.01

Article Snippet: UCA1 siRNAs and control siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA). hnRNP I-siRNA mixture was purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Positive Control, Mutagenesis, Western Blot

Journal: Cell Death & Disease

Article Title: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

doi: 10.1038/cddis.2013.541

Figure Lengend Snippet: Effect of hnRNP I on UCA1 expression. ( a ) Ectopic expression of hnRNP I increases UCA1, as detected by qRT–PCR. ( b ) Suppression of UCA1 by hnRNP I-siRNA. ( c ) Effect of hnRNP I-siRNA on UCA1 stability. MCF-7 cells were first transfected with control siRNA or hnRNP I-siRNA overnight and the transfected cells were treated with actinomycin D at 1 μ g/ml. RNA was isolated at 0, 6 and 12 h, respectively. Error bars represent S.E.M., n =3. * P <0.05; ** P <0.01

Article Snippet: UCA1 siRNAs and control siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA). hnRNP I-siRNA mixture was purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Isolation

Journal: Cell Death & Disease

Article Title: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

doi: 10.1038/cddis.2013.541

Figure Lengend Snippet: Induction of UCA1 through interaction with hnRNP I. ( a ) Doxo induces UCA1 independent of p53, as detected by qRT–PCR in MCF-7 and MDA-MB-231 cells. Cells were treated with doxo at 1 μ g/ml for 24 h before they were harvested for RNA extraction. ( b ) Suppression of doxo-induced UCA1 by hnRNP I-siRNA. MCF-7 cells were first transfected with control siRNA or hnRNP I-siRNA and then treated with doxo at 1 μ g/ml for 24 h before they were harvested for RNA extraction. ( c ) The phosphorylated form of hnRNP I is localized in the cytoplasm and is responsible for interaction with UCA1. RNA precipitation was used to detect hnRNP I. The same membrane was probed simultaneously with hnRNP I antibody (mouse origin) and p-hnRNP I antibody (rabbit origin), followed by secondary antibody labeled with either IRDye 680 or IRDye 800. The bottom panel shows the cytoplasmic (cyto) and nuclear (nuc) extracts used for RNA precipitation assays in the top panels. GAPDH serves as a cytoplasmic marker and NPM as a nuclear marker. ( d ) Redistribution of hnRNP I in response to doxo treatment. MCF-7 cells were first transfected with GFP–hnRNP I fusion construct, followed by doxo treatment (1 μ g/ml) for 8 h or UV treatment (20 J/m 2 ) for 3 h before fixation for fluorescent microscopy. ( e ) Colocalization of phosphorylated hnRNP I with UCA1 by IF and fluorescence in situ hybridization. ( f ) Doxo increases UCA1 pulldown by hnRNP I antibody. MCF-7 cells were treated with doxo the same way as in d , and then cytoplasmic fraction was prepared for RIP assay. Error bars represent S.E.M., n =3. ** P <0.01

Article Snippet: UCA1 siRNAs and control siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA). hnRNP I-siRNA mixture was purchased from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, RNA Extraction, Transfection, Control, Membrane, Labeling, Marker, Construct, Microscopy, Fluorescence, In Situ Hybridization

Journal: Cell Death & Disease

Article Title: Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

doi: 10.1038/cddis.2013.541

Figure Lengend Snippet: UCA1 suppresses p27 protein level by competing for hnRNP I. ( a ) Interaction of p27 mRNA with hnRNP I, as detected by RIP assay. ( b ) Although ectopic expression of UCA1 suppresses, UCA1-siRNA increases the p27 protein level. MCF-7 cells were transfected with vector or UCA1; control siRNA or UCA1-siRNA and then harvested for western blotting 24 h after transfection. ( c ) Suppression of the p27 protein level by hnRNP I-siRNA. The procedure was same as in b . ( d ) Effect of UCA1 and UCA1-siRNA on the p27 5′-UTR luciferase reporter activity. MCF-7 cells were transfected with the p27 5′-UTR luciferase reporter along with UCA1 or UCA1-siRNA. Luciferase assay was carried out 24 after transfection. Relative luciferase activity is Renilla luciferase activity relative to Firefly luciferase activity as an internal control. ( e ) Suppression of the interaction of p27 mRNA with hnRNP I by UCA1. MCF-7 cells were transfected with vector control or UCA1, and cellular extract was prepared for RIP assay using hnRNP I antibody 24 h after transfection. Error bars represent S.E.M., n =3. * P <0.05; ** P <0.01

Article Snippet: UCA1 siRNAs and control siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA). hnRNP I-siRNA mixture was purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay

Journal: Nature Communications

Article Title: FcγR engagement reprograms neutrophils into antigen cross-presenting cells that elicit acquired anti-tumor immunity

doi: 10.1038/s41467-021-24591-x

Figure Lengend Snippet: a , b Wild-type (WT) blood neutrophils treated with Ova, anti-Ova, or Ova-IC, cultured with GM-CSF and evaluated 3 days later by flow cytometry for survival and acquisition of CD11c and MHCII on Ly6G + cells (left panel), and CD80, CD86, and CCR7 on Ly6G + CD11c + MHCII + cells (right panel) ( a ). Representative gating strategy for Ova-IC generated nAPCs ( b ). c Neutrophils from CD45.1 (45.1) or CD45.2 (45.2) mice pre-treated with Ova or Ova-IC, or anti-Ova or Ova-IC, co-cultured with GM-CSF and analyzed for percent of Ly6G + cells expressing CD11c and MHCII. d WT bone marrow neutrophils (BMN) pre-treated with SLE patient or normal human (NH) sera, or SLE-IgG+ RNP (SLE-ICs) or normal human sera-IgG+RNP, cultured without GM-CSF and analyzed as in ( a ). Representative FACS plots are shown. RNP alone resulted in 0.38 ± 0.11% conversion. e BMNs from WT, γ −/− , FcγRIIIB(3B)/γ −/− or FcγRIIA(2A)/γ −/− mice treated with SLE or NH sera and analyzed as in ( a ). f Blood neutrophils from CD11c-YFP (yellow fluorescent protein) reporter mice treated with SLE-ICs, labeled with a nuclear stain and imaged at day 0 and 2 of culture for nuclear changes (i, iii) and CD11c induction (YFP positive) (ii, iv). Arrows: YFP + cells with nuclear change. Arrowheads: Unconverted neutrophils. Scale bar = 5 μm; all images are at the same magnification. g Live cell imaging of cells treated as in ( f ). Representative images, taken at indicated hours, of nuclear changes and CD11c-YFP acquisition in tracked cells (left) and quantitation of the same (right). Scale bar = 5 μm; all images are at the same magnification. Data are mean ± s.e.m. a , d One-way analysis of variance and Dunnett’s multiple comparison test, c , e Student t -test for unpaired comparisons with Dunn-Bonferroni ** p < 0.005, * p < 0.05.

Article Snippet: To generate SLE-IgG/RNP immune complexes, solutions containing 2 mg/ml of purified SLE IgG or normal human IgG were mixed with equal volumes of 0.3 mg/ml of Sm/RNP complexes (#SRC-3000, Immunovision) in PBS (without Ca, Mg) and incubated for 4 h at 37 °C.

Techniques: Cell Culture, Flow Cytometry, Generated, Expressing, Labeling, Staining, Live Cell Imaging, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Group II intron as cold sensor for self-preservation and bacterial conjugation

doi: 10.1093/nar/gkaa313

Figure Lengend Snippet: SHAPE profiling reveals intron structure disruptions under cold shock. ( A ) Superimposed SHAPE profile representing overall differences between the 10°C and 30°C samples. SHAPE modifications were analyzed by primer extension on a 6% urea/polyacrylamide sequencing gel . SHAPE profiling was performed on the intron RNA subjected to IEP-assisted in vitro splicing at 10°C or 30°C for 3h. Differences in SHAPE profiles comparing 10°C to 30°C are highlighted in colors (red denotes increased SHAPE reactivity in cold and blue denotes decreases). Nucleotides shown in grey were excluded from SHAPE probing. ( B ) 3D illustration of important tertiary interactions displayed in panel A as shown in cryo-EM structure of the intron RNP (PDB: 5g2x) . Left: DIII locks DV (pink) into DI primarily via the μ–μ′ (purple) interaction. Right: DIa-c (yellow) makes contact with the distal DId (teal) primarily via α–α′ (orange) and β–β′ (blue) interactions, forming the essential DI scaffold. The catalytic triad nucleotides are denoted by enlarged black tubes.

Article Snippet: The intron RNA and the IEP were mixed as described above and incubated in the RNP buffer (100 mM KCl, 5 mM MgCl 2 , 20 mM Tris–HCl (pH 7.5) and 1 U/μl murine RNase-inhibitor) at 10°C or 30°C for 3 h. The mixture was then analyzed on a 4% native polyacrylamide gel run at 4°C followed by SYPRO Ruby staining (Thermo Fisher) according to manufacturer's instructions.

Techniques: Sequencing, In Vitro, Cryo-EM Sample Prep