Journal: Cancer Research

Article Title: Inhibition of Karyopherin β1-Mediated Nuclear Import Disrupts Oncogenic Lineage-Defining Transcription Factor Activity in Small Cell Lung Cancer

doi: 10.1158/0008-5472.can-21-3713

Figure Lengend Snippet: Figure 1. KPNB1 is highly expressed across SCLC models and interacts with ASCL1 and NEUROD1 in human SCLC cell lines. A, KPNB1 expression in SCLC-A (green), SCLC-N (blue), SCLC-P (orange), or SCLC-Y (purple) human primary tumors profiled by RNA-seq (5). KPNB1 expression in LUAD, LUSC, and prostate, testis, pancreatic, liver, and breast tumor samples from the TCGA RNA-seq data sets is shown for comparison. B, KPNB1 expression in human SCLC-A, -N, -A/N, -P, or -Y cell lines, LUAD, LUSC, and large cell carcinoma (LCC) cell lines profiled by RNA-seq in the CCLE data set on CellMinerCDB (center line, mean; whiskers, SEM; one-way ANOVA). C, Western blot showing KPNB1 levels in representative cell lines as indicated. D, Strategy for identifying ASCL1 binding partners in SCLC-A cells by LC-MS/MS. E, Proteins identified by mass spectrometry of ASCL1 immunoprecipitates shown ranked by spectral index. F, Coimmunoprecipitation followed by Western blot demonstrating the interaction between KPNB1 and ASCL1 in SCLC-A cell lines NCI-H2107 and NCI-H889 (for NCI-H2107, input is 1% of protein loaded in IPs). G, Coimmunoprecipitation followed by Western blot showing the interaction between NEUROD1 and KPNB1 in the SCLC-N cell lines NCI-H524 and NCI-H2171. C, cytoplasmic extract; N, nuclear extract. Nuclear extracts were used as IP input, nonimmune IgG ¼ IP control. X, indicates wells with no lysate loaded.

Article Snippet: Immunoprecipitates were incubated for 90 minutes (4 C) with 70 mL Pierce Protein G Agarose beads (Thermo Fisher #20397), washed in PBS containing 1%Triton-X100, eluted in Laemmli buffer, and resolved on 4–15% Mini- PROTEAN TGX Stain-Free Protein Gels (Bio-Rad), transferred to PVDF membranes and probed with mouse anti-ASCL1 antibody (1:1,000, BD Biosciences; #556604) or mouse anti-KPNB1 antibody (1:1,000, Santa Cruz #sc-137016), respectively, followed by HRP-conjugated anti-mouse secondary antibody (1:10,000, Invitrogen #31432) or HRP-conjugated Trueblot anti-rabbit secondary antibody (1:1,000, Rockland; #18-8816-33). siRNA transfection Cells were plated at 2 105 cells/mL one day prior to transfection, resuspended, transferred to 12-well plates, and transfected by dropwise addition of a mixture containing 20 mL X-tremeGENE siRNA Transfection Reagent (Sigma-Aldrich; #4476115001), 500 nmol/L siRNA, and 175 mL Opti-MEM (Gibco; #31985062). siRNAs used were: Control siRNA-A (Santa Cruz; #sc-37007), Karyopherin b1 siRNA (h) (Santa Cruz; #sc-35736), and ASCL1 siRNA (Thermo Fisher #HSS100744).

Techniques: Expressing, RNA Sequencing, Comparison, Western Blot, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Control

Journal: Cancer Research

Article Title: Inhibition of Karyopherin β1-Mediated Nuclear Import Disrupts Oncogenic Lineage-Defining Transcription Factor Activity in Small Cell Lung Cancer

doi: 10.1158/0008-5472.can-21-3713

Figure Lengend Snippet: Figure 2. Depletion of KPNB1 reduces nuclear ASCL1 and ASCL1 downstream target gene expression in SCLC-A. A, Western blot showing whole-cell KPNB1 expression in SCLC-A subtype NCI-H2107 cells after transfection with control or KPNB1 siRNAs (siCtrl or siKPNB1). Western blots showing the level of ASCL1 in the nuclear (B) and whole-cell lysate (C) fractions of NCI-H2107 cells at 24 and 48 hours after siRNA transfection. Note, the same membranes were used for A and C as reflected by the GAPDH blots. D, Quantification of relative ASCL1 protein in the nuclear fractions of siCtrl versus siKPNB1-transfected cells across two independent experiments. Lamin B1, nuclear loading control (error bars, SEM; unpaired t test; , P ≤0.05). E, Comparison of ASCL1 downstream target expression by RT- qPCR in siCtrl (black)-, siKPNB1 (red)-, and siASCL1 (green)-transfected NCI-H2107 cells at 72 hours after transfection. Average of three technical replicates is shown (error bars, SEM, unpaired t test; , P ≤0.05; , P ≤0.01; , P ≤0.001; , P ≤0.0001; ns, nonsignificant). F, Diagram of CRISPRi lentiviral construct where the UbC promoter drives expression of catalytically inactive Cas9 (dCas9) fused to the KRAB transcriptional repressor. U6 promoter drives expression of gRNAs targeting the 50UTR of KPNB1 (gKPNB1.1, gKPNB1.2) or ASCL1 (gASCL1.1, gASCL1.2). G, Expression of dCas9-KRAB plus gKPNB1.1 or gKPNB1.2 resulted in an 84% reduction in KPNB1 protein. H,Western blot showing the level of ASCL1 in the nuclearfractionof NCI-H2107 cells expressing gKPNB1.1 or gKPNB1.2 versus control (Ctrl). I,Quantification of nuclear ASCL1 protein. J, Western blot showing undetectable levels of ASCL1 in SCLC-A subtype NCI-H2107 cells expressing dCas9-KRAB plus ASCL1.1 or ASCL1.2 gRNAs. K, Heatmap comparing expression of all DEG identified in CRISPRi_ASCL1 cells between siASCL1 knockdown NCI-H2107 cells previously profiled by RNA-seq (48) and CRISPRi_KPNB1 NCI-H2107 cell lines. Heatmap shows fold change of each gene with respect to each sample’s control. L and M, GSEA from Ctrl versus CRISPRi_KPNB1 cell lines with normalized enrichment scores (NES) and P values for all genes that were downregulated (L) or upregulated (M) following direct CRISPRi-mediated repression of ASCL1.

Article Snippet: Immunoprecipitates were incubated for 90 minutes (4 C) with 70 mL Pierce Protein G Agarose beads (Thermo Fisher #20397), washed in PBS containing 1%Triton-X100, eluted in Laemmli buffer, and resolved on 4–15% Mini- PROTEAN TGX Stain-Free Protein Gels (Bio-Rad), transferred to PVDF membranes and probed with mouse anti-ASCL1 antibody (1:1,000, BD Biosciences; #556604) or mouse anti-KPNB1 antibody (1:1,000, Santa Cruz #sc-137016), respectively, followed by HRP-conjugated anti-mouse secondary antibody (1:10,000, Invitrogen #31432) or HRP-conjugated Trueblot anti-rabbit secondary antibody (1:1,000, Rockland; #18-8816-33). siRNA transfection Cells were plated at 2 105 cells/mL one day prior to transfection, resuspended, transferred to 12-well plates, and transfected by dropwise addition of a mixture containing 20 mL X-tremeGENE siRNA Transfection Reagent (Sigma-Aldrich; #4476115001), 500 nmol/L siRNA, and 175 mL Opti-MEM (Gibco; #31985062). siRNAs used were: Control siRNA-A (Santa Cruz; #sc-37007), Karyopherin b1 siRNA (h) (Santa Cruz; #sc-35736), and ASCL1 siRNA (Thermo Fisher #HSS100744).

Techniques: Targeted Gene Expression, Western Blot, Expressing, Transfection, Control, Comparison, Quantitative RT-PCR, Construct, Knockdown, RNA Sequencing

Journal: Cancer Research

Article Title: Inhibition of Karyopherin β1-Mediated Nuclear Import Disrupts Oncogenic Lineage-Defining Transcription Factor Activity in Small Cell Lung Cancer

doi: 10.1158/0008-5472.can-21-3713

Figure Lengend Snippet: Figure 4. Pharmacologic inhibition of KPNB1 reduces nuclear ASCL1 and NEUROD1 in SCLC-A and SCLC-N. Western blots showing the levels of ASCL1 and NEUROD1 in the nuclear fractions of SCLC-A subtype NCI-H2107 cells (A and C) and SCLC-N subtype NCI- H2171 cells (B and D) treated with their respective IC50 concentrations of the KPNB1 inhibitors IPZ (A and B) or INI-43 (C and D) for 24 hours. Controls include the vehicle DMSO and untreated () cells. Quantification of relative nuclear ASCL1 and NEUROD1 levels is shown next to each blot. Relative nuclear ASCL1 or NEUROD1 was quantified across two independent experiments. Bars, mean SEM; one-way ANOVA; , P ≤0.05; , P ≤0.01; , P ≤0.001.

Article Snippet: Immunoprecipitates were incubated for 90 minutes (4 C) with 70 mL Pierce Protein G Agarose beads (Thermo Fisher #20397), washed in PBS containing 1%Triton-X100, eluted in Laemmli buffer, and resolved on 4–15% Mini- PROTEAN TGX Stain-Free Protein Gels (Bio-Rad), transferred to PVDF membranes and probed with mouse anti-ASCL1 antibody (1:1,000, BD Biosciences; #556604) or mouse anti-KPNB1 antibody (1:1,000, Santa Cruz #sc-137016), respectively, followed by HRP-conjugated anti-mouse secondary antibody (1:10,000, Invitrogen #31432) or HRP-conjugated Trueblot anti-rabbit secondary antibody (1:1,000, Rockland; #18-8816-33). siRNA transfection Cells were plated at 2 105 cells/mL one day prior to transfection, resuspended, transferred to 12-well plates, and transfected by dropwise addition of a mixture containing 20 mL X-tremeGENE siRNA Transfection Reagent (Sigma-Aldrich; #4476115001), 500 nmol/L siRNA, and 175 mL Opti-MEM (Gibco; #31985062). siRNAs used were: Control siRNA-A (Santa Cruz; #sc-37007), Karyopherin b1 siRNA (h) (Santa Cruz; #sc-35736), and ASCL1 siRNA (Thermo Fisher #HSS100744).

Techniques: Inhibition, Western Blot

Journal: Cancer Research

Article Title: Inhibition of Karyopherin β1-Mediated Nuclear Import Disrupts Oncogenic Lineage-Defining Transcription Factor Activity in Small Cell Lung Cancer

doi: 10.1158/0008-5472.can-21-3713

Figure Lengend Snippet: Figure 6. KPNB1-independent nuclear accumulation of ASCL1 rescues SCLC-A growth following KPNB1 inhibition. A, Diagram of rescue constructs. Dox-inducible TRE promoter drives expression of ASCL1 and NEUROD1 variants with mutations in their putative NLS’s () and a KPNB1-independent (KPNB2-specific) PY-NLS added to their N-termini. WT ASCL1 and NEUROD1 constructs served as controls. B, Rescue strategy. In SCLC-A cells, INI- 43 inhibits KPNB1-mediated nuclear import of WT- ASCL1, but not KPNB2-specific PY-NLS- ASCL1. In SCLC-N cells, the respective WT and PY-NLS-NEUROD1 constructs were used. C, Comparison of the fold change in viable cells between the WT-ASCL1 or PY-NLS-ASCL1-expressing SCLC-A subtype NCI-H2107 cell lines at 1 through 4 days after treatment with 0.5 mmol/L (red) and 1.5 mmol/L (green) concentrations of INI-43. D, Comparison of fold change in viable cells between WT-NEUROD1 or PY-NLS-NEUROD1-expressing SCLC-N subtype NCI-H2171 cell lines at 1 through 4 days after treatment with 1.5 mmol/L INI-43. Cell viability was measured by WST-1 assay. Error bars, mean SEM of experiments performed in quintuplicate; unpaired t test; second independent experiment confirmed results; , P ≤0.05; , P ≤0.0001; ns, nonsignificant.

Article Snippet: Immunoprecipitates were incubated for 90 minutes (4 C) with 70 mL Pierce Protein G Agarose beads (Thermo Fisher #20397), washed in PBS containing 1%Triton-X100, eluted in Laemmli buffer, and resolved on 4–15% Mini- PROTEAN TGX Stain-Free Protein Gels (Bio-Rad), transferred to PVDF membranes and probed with mouse anti-ASCL1 antibody (1:1,000, BD Biosciences; #556604) or mouse anti-KPNB1 antibody (1:1,000, Santa Cruz #sc-137016), respectively, followed by HRP-conjugated anti-mouse secondary antibody (1:10,000, Invitrogen #31432) or HRP-conjugated Trueblot anti-rabbit secondary antibody (1:1,000, Rockland; #18-8816-33). siRNA transfection Cells were plated at 2 105 cells/mL one day prior to transfection, resuspended, transferred to 12-well plates, and transfected by dropwise addition of a mixture containing 20 mL X-tremeGENE siRNA Transfection Reagent (Sigma-Aldrich; #4476115001), 500 nmol/L siRNA, and 175 mL Opti-MEM (Gibco; #31985062). siRNAs used were: Control siRNA-A (Santa Cruz; #sc-37007), Karyopherin b1 siRNA (h) (Santa Cruz; #sc-35736), and ASCL1 siRNA (Thermo Fisher #HSS100744).

Techniques: Inhibition, Construct, Expressing, Comparison, WST-1 Assay

Journal: Cancer Research

Article Title: Inhibition of Karyopherin β1-Mediated Nuclear Import Disrupts Oncogenic Lineage-Defining Transcription Factor Activity in Small Cell Lung Cancer

doi: 10.1158/0008-5472.can-21-3713

Figure Lengend Snippet: Figure 7. Pharmacologic inhibition of KPNB1 disrupts SCLC-A PDX growth in vivo. A, FPKM expression values of KPNB1, ASCL1, NEUROD1, POU2F3, and YAP1 in JHU-LX44 and JHU-LX22 PDX samples profiled by RNA-seq. B and C, Comparison of tumor growth in SCLC-A (JHU-LX44; B) and SCLC-N (JHU-LX22; C) PDX-bearing mice dosed with 25 mg/kg INI-43 versus vehicle for 10–16 days (n ¼ 5 per group). D, LC-MS/MS analysis of INI-43 concentrations in plasma versus JHU-LX44 tumor tissue of INI- 43–treated mice at 2 and 24 hours after treatment (n ¼ 2 per group, per time point; error bars, mean SD; , P ≤0.05; ns, nonsignificant). E, Venn diagram showing overlap in upregulated DEG identified following INI-43 treatment in the SCLC-A (pink) versus SCLC-N (green) PDX models. F, In both models, INI-43 treatment leads to extensive upregulation of translation (RPS/RPL/MRP, EEF/EIF), proteasomal (PSM), and ubiquitin (UBA/B/E) genes. G, DEG uniquely identified in the INI-43– treated SCLC-A PDX samples revealed significant depletion of cell-cycle pathways and decreased expression of ASCL1 downstream target genes. The expression of genes encoding proapoptotic proteins and metallothioneins shown in H was also uniquely elevated in the INI-43–treated SCLC-A PDXs. H, Relative expression of select previously identified ASCL1 downstream targets, apoptotic and metallothionein genes in vehicle (n ¼ 2) versus INI-43–treated tumors (n ¼ 3) profiled by RNA- seq. Genes with significantly reduced, or increased expression at the 5% FDR criteria are indicated with a red asterisk, respectively. Average expression of these genes in the vehicle versus INI-43–treated samples is shown on the right.

Article Snippet: Immunoprecipitates were incubated for 90 minutes (4 C) with 70 mL Pierce Protein G Agarose beads (Thermo Fisher #20397), washed in PBS containing 1%Triton-X100, eluted in Laemmli buffer, and resolved on 4–15% Mini- PROTEAN TGX Stain-Free Protein Gels (Bio-Rad), transferred to PVDF membranes and probed with mouse anti-ASCL1 antibody (1:1,000, BD Biosciences; #556604) or mouse anti-KPNB1 antibody (1:1,000, Santa Cruz #sc-137016), respectively, followed by HRP-conjugated anti-mouse secondary antibody (1:10,000, Invitrogen #31432) or HRP-conjugated Trueblot anti-rabbit secondary antibody (1:1,000, Rockland; #18-8816-33). siRNA transfection Cells were plated at 2 105 cells/mL one day prior to transfection, resuspended, transferred to 12-well plates, and transfected by dropwise addition of a mixture containing 20 mL X-tremeGENE siRNA Transfection Reagent (Sigma-Aldrich; #4476115001), 500 nmol/L siRNA, and 175 mL Opti-MEM (Gibco; #31985062). siRNAs used were: Control siRNA-A (Santa Cruz; #sc-37007), Karyopherin b1 siRNA (h) (Santa Cruz; #sc-35736), and ASCL1 siRNA (Thermo Fisher #HSS100744).

Techniques: Inhibition, In Vivo, Expressing, RNA Sequencing, Comparison, Liquid Chromatography with Mass Spectroscopy, Clinical Proteomics, Ubiquitin Proteomics

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Loss of Ephrin B2 Receptor (EPHB2) Sets Lipid Rheostat by Regulating Proteins DGAT1 and ATGL Inducing Lipid Droplet Storage in Prostate Cancer Cells

doi: 10.1038/s41374-021-00583-9

Figure Lengend Snippet: Antibodies used in Western Blot experiments.

Article Snippet: CGI-58/ABHD5 (E-1) , Santa Cruz Biotechnology , sc376931 , 1:250.

Techniques: Western Blot