Journal: bioRxiv

Article Title: Cyclin A/B RxL Macrocyclic Inhibitors to Treat Cancers with High E2F Activity

doi: 10.1101/2024.08.01.605889

Figure Lengend Snippet: A) Structure of cyclin A (PDB code: 1JSU) with blue indicating basic, red acidic and white neutral surfaces. “HP” indicates the Hydrophobic Patch formed in part by the highly conserved MRAIL motif in cyclins and “S” indicates the smaller adjacent hydrophobic pocket. B) Docked model of CIRc-004 filled-in structure in green bound to cyclin A. Shown next to it is the detailed “stick” structure representation of CIRc-004 docked to cyclin A. C) Biochemical activity of cyclin RxL macrocyclic inhibitors in cyclin A1/Cdk2, cyclinE1/Cdk2 and cyclin B/CDK1 complexes measured by Fluorescence Polarization. D ) GI 50 waterfall plot of 46 human SCLC cell lines panel. E ) Heatmap displays the activity patterns of various Hallmark pathways within a panel of 46 SCLC cell lines. The Gene Set Variation Analysis (GSVA) method, utilizing the MSigDb Hallmark collection of RNA-seq data, was employed to calculate scores for each pathway. Each column represents a distinct SCLC cell line ordered from most sensitive to least sensitive based on GI 50 values for cyclin RxL A/B inhibitor (CIRc-004). p-value <0.05 are shown and calculated by Wilcoxon rank sum exact test. F ) Dose response assays of the indicated human SCLC cell lines (NCI-H1048, NCI-H446, and NCI-H69) and human NSCLC cell lines (A549, HCC4006, and NCI-H1299) treated for 6 days with increasing doses of the cyclin A/B RxL inhibitor (CIRc-004). Average half-maximal effective concentration (EC50) for cyclin A/B RxL inhibitor (CIRc-004) shown next to respective cell line. G ) Representative flow cytometry analysis of cleaved PARP in NCI-H1048 cells treated with CIRc-004 (200 nM) or DMSO (vehicle) for 3 days. H ) Quantitation of cleaved PARP positive cells analyzed by flow cytometric analysis in the different SCLC and NSCLC lung cancer cell lines treated for 3 days with the indicated doses of the cyclin A/B RxL inhibitor (CIRc-004). I ) Cell cycle distribution of the indicated SCLC and NSCLC cell lines treated with CIRc-004 (200 nM) or DMSO (vehicle) for 24 hours and then stained with propidium iodide (PI). For F, H and I , n=3 biological independent experiments and data are mean +/− SD. Arrow in F indicates DMSO-treated sample which was used for normalization. Statistical significance for H was calculated using unpaired, two-tailed students t -test. Where indicated, *=p<0,05, **=p<0.01, ***=p<0.001, ****=p<0.000.

Article Snippet: The primary antibodies and dilutions used were: anti-Myt1 (Fortis Life Sciences A302-424A 1:1000), mouse-anti-cyclin B (Cell Signaling #4135 1:1000), mouse-anti-E2F1 (SCBT #sc-251, 1:1000), mouse-anti-cyclin A (Cell Signaling #4656, 1:1000), mouse anti-CDK2 (Origene TA502935), rabbit anti-Cdc2 (Cell Signaling 28439).

Techniques: Activity Assay, Fluorescence, RNA Sequencing Assay, Concentration Assay, Flow Cytometry, Quantitation Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Cyclin A/B RxL Macrocyclic Inhibitors to Treat Cancers with High E2F Activity

doi: 10.1101/2024.08.01.605889

Figure Lengend Snippet: A ) Schema for the genome-wide CRISPR/Cas9 resistance screen in NCI-H1048 cells infected with the Brunello genome-wide sgRNA library. Following selection (day 10), cells were treated with cyclin A/B RxL inhibitor (CIRc-004, 200 nM); cyclin A/B/E inhibitor (CIRc-001, 200 nM); Cdk2 inhibitor (PF-07104091, 500 nM), or the inactivate enantiomer of CIRc-004 (CIRc-005, 200 nM) used as a negative control and then harvested at day 26 when the sgRNA-infected cells showed resistance. B-C ) Top enriched and depleted hits determined by Apron analysis of the B ) cyclin A/B RxL inhibitor (CIRc-004), or the C ) Cdk2 (PF-07104091) at the end timepoint (day 26) all relative to the CIRc-005 enantiomer negative control at the end timepoint (day 26). For B-C, n=2 biological independent experiments. D ) Venn diagram showing top enriched hits (q-value<0.25) among the different treatment groups indicated. Shown next to it are the protein-protein interaction network analysis ( http://string-db.org/ ) of statistically significant hits shared between cyclin A/B, cyclin A/B/E RxL and Cdk2 inhibitors linked to the biological processes indicated and all involve in the spindle assembly checkpoint in mitosis. E-H ) Dose response assays of NCI-H1048 cells infected with two independent non-targeting sgRNAs (sgCtrl) or two independent sgRNAs against E ) CCNB1 , F ) CDK2 , G ) KNTC1 , H ) MAD1L1 , treated for 6 days with increasing doses of CIRc-004. I ) Quantitation of phospho-histone H3 flow cytometric data of NCI-H1048 cells infected with the sgRNAs indicated and treated with CIRc-004 at 20 nM for 24 hours. Data is plotted as fold change after cyclin A/B RxL inhibitor treatment relative to vehicle. J ) Quantitation of cleaved PARP flow cytometric data of NCI-H1048 cells infected with the sgRNAs indicated and treated with CIRc-004 at 20 nM for 3 days. Data is plotted as fold change after cyclin A/B RxL inhibitor treatment relative to vehicle. K ) Genes with mutations in more than one out of eight CIRc-004 resistant clones from the forward genetic screen in iHCT-116 cells (see Methods). L ) Immunoblot of NCI-H1048 cells stably expressing vector, Flag-CDC20 WT, or Flag-CDC20 R445Q mutant. M ) Dose response assay of NCI-H1048 cells from L treated with increasing doses of CIRc-004. N ) Quantitation of cleaved PARP flow cytometric data of NCI-H1048 cells from L treated with CIRc-004 at 20 nM for 3 days. O ) Quantitation of phospho-histone H3 flow cytometric data of NCI-H1048 cells from L treated with CIRc-004 at 20 nM for 24 hours. For E-J , L-O , n=3 biological independent experiments and data are mean +/– SD. Arrows in E-H indicates DMSO-treated sample which was used for normalization. Statistical significance in E-H , N-O was calculated using unpaired, two-tailed students t -test. Where indicated, *=p<0,05, **=p<0.01, ***=p<0.001, ****=p<0.0001.

Article Snippet: The primary antibodies and dilutions used were: anti-Myt1 (Fortis Life Sciences A302-424A 1:1000), mouse-anti-cyclin B (Cell Signaling #4135 1:1000), mouse-anti-E2F1 (SCBT #sc-251, 1:1000), mouse-anti-cyclin A (Cell Signaling #4656, 1:1000), mouse anti-CDK2 (Origene TA502935), rabbit anti-Cdc2 (Cell Signaling 28439).

Techniques: Genome Wide, CRISPR, Infection, Selection, Negative Control, Quantitation Assay, Clone Assay, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Cyclin A/B RxL Macrocyclic Inhibitors to Treat Cancers with High E2F Activity

doi: 10.1101/2024.08.01.605889

Figure Lengend Snippet: A ) Schema for the base editor CRISPR/Cas9 cyclin A/B RxL inhibitor resistance screen in NCI-H1048 cells. Cells were infected with a custom-made sgRNA library including 2802 sgRNAs tiling CCNB1 , CCNA2 , CDK2 and CDC20 , and positive and negative control sgRNAs (see Methods). At day 10 [early timepoint (ETP)], cells were treated with the cyclin A/B RxL inhibitor (CIRc-004, 200 nM) or the inactivate enantiomer of CIRc-004 (CIRc-005, 200 nM) and then harvested at day 26 [late timepoint (LTP)]. B ) Dot plot of average z-scored log-fold change (LFC) for each sgRNA tiling CCNB1 in cells treated with CIRc-004 vs. CIRc-005 at day 26. The x-axis indicates the position of each sgRNA along CCNB1 protein coding sequence. Variants of interest are labeled with the predicted amino acid position and mutational change. Top graph is the A>G and bottom graph is the C>T base editor screen. n=4 biological replicates. C ) Predicted AlphaFold2 model of the cyclin B1:Cdk1 complex where high confidence interactions of cyclin B’s amino acids 169-177 with Cdk1 are highlighted in yellow. The table below lists the high confidence amino acid residues involved in the interaction between cyclin B1 and Cdk1. D ) Dot plot of fold change variant reads at each nucleotide base position within CCNB1 comparing NCI-H1048 Cas9 base-editor cells transduced with the sgRNA library in A treated with CIRc-004 vs. CIRc-005 at day 26. Variants of interest are labeled with the mutational change. n=2 biological replicates. E ) FACS-based competition assay of GFP-positive NCI-H1048 Cas9 base-editor cells transduced with the indicated CCNB1 sgRNA variants and GFP. GFP-positive CCNB1 sgRNA variant cell lines were mixed with the parental base editor counterpart at a ∼1:4 ratio and then treated with CIRc-004 at the concentrations indicated for 13 days. Fold-change enrichment of GFP-positive cells comparing CIRc-004 vs. DMSO control is shown. Data are mean +/− SD and n=3 biological independent experiments. Statistical significance was calculated using unpaired, two-tailed students t -test. Where indicated, *=p<0,05, **=p<0.01, ***=p<0.001, ****=p<0.0001. F ) Fraction of variant alleles from deep amplicon sequencing of genomic DNA from CCNB1 Tyr170His, CCNB1 Tyr177Cys, and CCNB1 Glu169Lys cell lines from the competition assay in E at the experimental endpoint (Day 13) treated with CIRc-004 (200 nM) or DMSO. Top panel shows the common mutant variants observed for each sample. n=2 biological independent experiments. G ) Volcano plot of IP of cyclin B followed by mass spectrometry (IP-MS) in NCI-H1048 cells first treated with CIRc-004 (50 nM) relative to CIRc-005 (50 nM) for 2 hours. n=3 independent experiments. H ) Immunoblot analysis after IP of endogenous cyclin B1 in NCI-H1048 cells treated with CIRc-004 (300 nM), CIRc-005 (inactive enantiomer of CIRc-004, I.E), or DMSO for 2 hours. n=2 biological independent experiments. I ) Immunoblot analysis after IP of exogenous cyclin B1 using an HA antibody in HEK-293T cells expressing CCNB1 WT-HA, CCNB1 triple mutant-(E169K/Y170H/Y177C)-HA, or a negative control vector, and treated with CIRc-004 (300 nM) or DMSO for 2 hours. n=3 biological independent experiments. J ) Immunoblot analysis of NCI-H1048 cells infected with 2 independent sgRNAs targeting CDK2 or a non-targeting control (sgCtrl) and treated with CIRc-004 at 20 nM or DMSO for 24 hours.

Article Snippet: The primary antibodies and dilutions used were: anti-Myt1 (Fortis Life Sciences A302-424A 1:1000), mouse-anti-cyclin B (Cell Signaling #4135 1:1000), mouse-anti-E2F1 (SCBT #sc-251, 1:1000), mouse-anti-cyclin A (Cell Signaling #4656, 1:1000), mouse anti-CDK2 (Origene TA502935), rabbit anti-Cdc2 (Cell Signaling 28439).

Techniques: CRISPR, Infection, Negative Control, Sequencing, Labeling, Variant Assay, Transduction, Competitive Binding Assay, Control, Two Tailed Test, Amplification, Mutagenesis, Mass Spectrometry, Western Blot, Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: Nuclear Outsourcing of RNA Interference Components to Human Mitochondria

doi: 10.1371/journal.pone.0020746

Figure Lengend Snippet: A. Purity assessment of mitochondrial fraction. Cyclin-dependent kinase 2 (CDK2) was assessed in the mitochondrial fraction by immunoblot analyses to check for nuclear and cytoplasmic contaminants relatively to mitochondrial protein ATP synthase subunit α (ATP5A1). The density of bands was measured using the ImageJ software and is represented as a relative intensity. Values are means±SD of three independent experiments. B. Western blot analysis of AGO2 in subcellular mitochondrial fraction from HeLa cells. AGO2 protein was detected using a rabbit polyclonal antibody anti-AGO2 in mitochondrial protein fraction (Mito) and total protein extracts (Lysate) from HeLa cells. ATP5A1 was used as a mitochondrial marker, actin as a cytosolic marker, and CDK2 as a nuclear/cytosolic marker. Representative image is shown of three independent experiments. C. Western blot analysis of AGO2 at mitochondrial membranous and soluble fraction from U2OS cells. Mitochondria were suspended either in isotonic mitochondrial buffer (MB) or in hypo-osmotic mitochondrial buffer (MB/10) alone or supplemented either with NaCl 1 M or Na 2 CO 3 0.1 M, and fragilized by freeze-thaw cycles and sonication when indicated. All samples were centrifuged (150,000× g) to separate the membrane pellet (p) from the soluble protein supernatants (s). 15 µg of protein extracts of each sample were subjected to Western Blot. The following proteins were immunodecorated on blots: voltage-dependent anion channel 1 (VDAC1) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 9 (NDUFA9) as markers of mitochondrial membranes and cytochrome c (CYCS), as a intermembrane marker. Immunodetection of AGO2 is shown as the lower panel.

Article Snippet: The following primary antibodies were commercially purchased: mouse monoclonal anti-ATP5A1, MS507, Mitosciences, Eugene, USA, 1∶1000 for immunoblotting and 1∶750 for immunofluorescence; mouse monoclonal anti-NDUFA9, MS111, Mitosciences, Eugene, USA, 1∶5000; mouse monoclonal anti-VDAC1, MSA03, Mitosciences, Eugene, USA, 1∶2500; mouse monoclonal anti-CDK2, sc-6248, Santa Cruz Biotechnology, Santa Cruz, USA, 1∶1000; mouse monoclonal anti-actin, VMA1501R, AbCys, Paris, France, 1∶500; rabbit monoclonal anti-cythocrome c, 1896-1, Epitomics, Burlingame, USA, 1∶2000 and rabbit polyclonal anti-SLUG, sc-15391X, Santa Cruz Biotechnology, Santa Cruz, USA, 1∶250.

Techniques: Western Blot, Software, Marker, Sonication, Immunodetection

Journal: iScience

Article Title: Icaritin inhibits CDK2 expression and activity to interfere with tumor progression

doi: 10.1016/j.isci.2022.104991

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-p27 Rabbit polyclonal anti-CDK2 Mouse monoclonal anti-E2F Rabbit polyclonal anti-FOXO1 Rabbit polyclonal anti-Rb , proteintech , 25614-1-AP; RRID: AB_2880161 10122-1-AP: RRID: AB_2078556 66515-1-Ig; RRID: AB_2881878 18592-1-AP; RRID: AB_10860103 10048-2-Ig; RRID: AB_2177320.

Techniques: Recombinant, Control

Journal: Bioscience Reports

Article Title: USP52 inhibits cell proliferation by stabilizing PTEN protein in non-small cell lung cancer

doi: 10.1042/BSR20210486

Figure Lengend Snippet: ( A ) H292 and H460 cells were stably infected with lentiviral LV-NC or LV- USP52 , followed by Western blot against USP52. GAPDH was used as a loading control. ( B ) H292 and H460 cells stably infected with LV-NC or LV- USP52 were cultured for indicated time, followed by CCK-8 assay at days 0, 1, 3 or 5. ( C ) H292 and H460 cells were stably infected with l LV-NC or LV- USP52 , followed by Western blot against CCND1, CDK2, p53 and GAPDH. ( D ) The relative abundance of CCND1, CDK2 and p53 in H292 and H460 from (C) were quantified by gray scanning after LV- USP52 plasmids transformed at 0, 1, 2, 4 μg. These experiments were repeated for three times. One-way ANOVA, * P <0.05, ** P <0.01. NS, not significant.

Article Snippet: Blots were probed with the following antibodies: anti-USP52 mouse monoclonal (Santa Cruz Biotechnology, Inc., Dallas, TX, U.S.A., 1:1000), anti-GAPDH mouse monoclonal (Invitrogen, Carlsbad, CA, U.S.A., 1:3000), anti-cyclinD1 mouse monoclonal (Invitrogen, Carlsbad, CA, U.S.A., 1:1000), anti-CDK2 mouse monoclonal (Invitrogen, Carlsbad, CA, U.S.A., 1:1000), anti-p53 mouse monoclonal (Invitrogen, Carlsbad, CA, U.S.A., 1:1000), anti-HA mouse monoclonal (Invitrogen, Carlsbad, CA, U.S.A., 1:2000), anti-p-AKT mouse monoclonal (Invitrogen, Carlsbad, CA, U.S.A., 1:1000), anti-AKT mouse monoclonal (Invitrogen, Carlsbad, CA, U.S.A., 1:1000), anti-p-mTOR mouse monoclonal (Invitrogen, Carlsbad, CA, U.S.A., 1:1000), anti-mTOR mouse monoclonal (Invitrogen, Carlsbad, CA, U.S.A., 1:1000) and mouse monoclonal anti-PTEN antibody (Cell Signaling Technology, Inc., Danvers, MA, U.S.A., 1:1000).

Techniques: Stable Transfection, Infection, Western Blot, Cell Culture, CCK-8 Assay, Transformation Assay