Journal: Science signaling

Article Title: A post-translational modification code for CFTR maturation is altered in cystic fibrosis

doi: 10.1126/scisignal.aan7984

Figure Lengend Snippet: A. CX-4945 mediated inhibition of CK2α prevents phosphorylation of wild-type CFTR at Thr421, Ser422 and Ser427 as quantified with spiked-in synthetic, heavy isotope-labeled peptides. Data are means ± S.D. of n=3 experiments; statistical analysis noted below. B. Experimental outline for AHA labeling and enrichment of newly synthesized CFTR by click chemistry. C. Western blot of input lysate for CFTR-IP before click reaction. D. Western blot showing that wild-type CFTR maturation is dependent on CK2α phosphorylation in the RI element. Blots are representative of n=3 experiments. E. Quantification of newly synthesized immature (band B) and mature (band C) CFTR relative to time point 0 (h, hours). F. Western blot of mature CFTR (band C) in FRT cells expressing various CFTR mutants. G. Phosphorylation at Thr421, Ser422 and Ser427 in the various CFTR mutants described in (F) was quantified with spiked-in synthetic heavy isotope-labeled peptides, relative to the amount of mature CFTR present. Data are mean ± S.D. of ≥ n=4 independent measurements per site; statistical analysis noted below. (H to K) AHA pulse-chase assays in primary human bronchial epithelial (NHBE) cells (H) and FRT cells expressing G551D or wild-type CFTR (K) treated with CX-4945 or DMSO. Subsequent quantification of newly synthesized immature CFTR (band B) and mature CFTR (band C) at the time points indicated relative to time 0 (I and K). Top panels show input and loading control. SA-HRP, streptavidin-conjugated horse radish peroxidase. Time of AHA labeling before CFTR immunoprecipitation is indicated. Blots are representative, and the data are means ± S.D. of n=3 independent biological replicates. *P < 0.0332, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001 by unpaired, two-tailed t- tests with Bonferroni-Sidak correction (A) or one-Way ANOVA with Tukey post-test (I and K).

Article Snippet: Treatment with 5 – 10 μM VX809 (Selleck, Houston, TX), 10 μM CX-4945 (Santa Cruz Biotechnologies, Dallas, TX) or vehicle (DMSO), was carried out for 20 hours or as indicated.

Techniques: Inhibition, Labeling, Synthesized, Western Blot, Expressing, Pulse Chase, Immunoprecipitation, Two Tailed Test

Journal: Science signaling

Article Title: A post-translational modification code for CFTR maturation is altered in cystic fibrosis

doi: 10.1126/scisignal.aan7984

Figure Lengend Snippet: Degradation network identified for wild-type CFTR, wild-type CFTR treated with CX-4945, or ΔF508 CFTR in 16HBE41o- and CFBE41o- cells shows recruitment of ΔF508- specific interactions in wild-type CFTR expressing cells treated with CX-4945 (red nodes). Data represent independent biological replicates (wt n=8, ΔF508 n=8, wt+CX-4945 n=3).

Article Snippet: Treatment with 5 – 10 μM VX809 (Selleck, Houston, TX), 10 μM CX-4945 (Santa Cruz Biotechnologies, Dallas, TX) or vehicle (DMSO), was carried out for 20 hours or as indicated.

Techniques: Expressing

Journal: Science signaling

Article Title: A post-translational modification code for CFTR maturation is altered in cystic fibrosis

doi: 10.1126/scisignal.aan7984

Figure Lengend Snippet: A. Quantification of phosphorylated sites Ser422 and Ser427, methylation site Lys442 and ubiquitination site Lys420 at 37°C (red bars) or at permissive temperature of 30°C (blue bars). B. Quantification of ΔF508 CFTR phosphorylation at Thr421, Ser422 and Ser427 by synthetic heavy-labeled peptides shows reduced phosphorylation upon shRNA- mediated knockdown of CSNK2A1. C. Western blot showing that shRNA-mediated knockdown of CSNK2A1 impairs ΔF508 CFTR maturation at permissive temperature. Detection of b-actin was used as loading control. D. Representative traces of forskolin (F) and genistein (G) stimulated ΔF508 CFTR short circuit currents (/sc) showing that knockdown of CSNK2A1 (right) or treatment with 10 μM CX-4945 (left) prevent temperature shift (28°C) induced rescue of ΔF508 CFTR channel activity. Specificity of the current is established by treatment with CFTR inhibitor 172 (I). E. Quantification of Δ/sc currents as log2 fold change relative to control cells. All data are representative or mean ± S.D. of n=3 independent biological replicates. *P < 0.0332, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001 by one-Way ANOVA with Bonferroni post-test.

Article Snippet: Treatment with 5 – 10 μM VX809 (Selleck, Houston, TX), 10 μM CX-4945 (Santa Cruz Biotechnologies, Dallas, TX) or vehicle (DMSO), was carried out for 20 hours or as indicated.

Techniques: Methylation, Labeling, shRNA, Western Blot, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Combined targeting of G protein‐coupled receptor and EGF receptor signaling overcomes resistance to PI 3K pathway inhibitors in PTEN ‐null triple negative breast cancer

doi: 10.15252/emmm.202011987

Figure Lengend Snippet: A Cell viability of cell lines treated for 4 days with 0.25 μM AZD8186, 1 μM GDC0941, or 1 μM MK2206. PTEN‐deficient triple‐negative breast cancer cell lines are represented by black bars, PTEN‐deficient glioblastoma cell lines by black/gray dashed black, PIK3CA‐mutant breast cancer cell lines by white bars, and PIK3CA WT and PTEN WT breast cancer cell lines by gray bars. Each bar represents the mean of three experiments. B Plot of expression data extrapolated from RPKM RNAseq files of Broad Institute Cancer Cell Line Encyclopedia (CCLE, https://portals.broadinstitute.org/ccle ), and specifically showing expression of PIK3CA, PIK3CB, PIK3CD, and PIK3CG across PTEN‐null TNBC cell lines (namely: MDA‐MB‐468, HCC1395, HCC1937, HCC38, HCC70, and BT‐549) and B‐cell acute lymphocytic leukemia (B‐cell ALL) lines (namely: BDCM, JM1, KOPN8, MHHCALL3, MHHCALL4, MUTZ5, NALM6, REH, RS411, SEM, and SUPB15). Mean ± SD. Statistical significance of two‐tailed unpaired t ‐test in PIK3CD expression ** P = 0.0014 and in PIK3CG expression ** P = 0.0015. C Cell viability of MDA‐MB‐468 cells infected with 10 shRNAs pools during the shRNA screen (the library was divided into 10 pools, and each dot represents one pool) and co‐treated as indicated. Viability of cells was normalized to cell numbers in the vehicle‐treated condition within each pool and expressed as mean ± SD. D MDA‐MB‐468 was treated with serial dilutions of erlotinib or cetuximab in the presence of vehicle, AZD8186 (0.25 μM), GDC0941 (0.25 μM), or MK2206 (0.45 μM), as indicated. Cell viability was measured after 4 days (erlotinib) or 6 days (cetuximab) and normalized within each of the PI3K pathway inhibitor‐treated condition to the viability in the absence of erlotinib or cetuximab. Average ± SD of triplicates and representative of two independent experiments. E, F Viability of six PTEN‐null vs five PTEN‐WT TNBC cell lines treated for 6 days with pan‐PI3Ki (GDC0941 1 μM) (E), or AKTi (MK2206 0.45 μM) (F), alone or in combination with EGFRi (gefitinib 3 μM). Mean of three independent experiments ± SD. Statistical significance of two‐tailed unpaired Student's t ‐test in PTEN‐null pan‐PI3Ki vs pan‐PI3Ki + EGFRi ** P = 0.0041, PTEN‐null EGFRi vs pan‐PI3Ki + EGFRi *** P = 0.0008, PTEN‐WT pan‐PI3Ki vs pan‐PI3Ki + EGFRi n.s. P = 0.166, PTEN‐WT EGFRi vs pan‐PI3Ki + EGFRi n.s. P = 0.0897, PTEN‐null AKTi vs AKTi + EGFRi * P = 0.0281, PTEN‐null EGFRi vs AKTi + EGFRi *** P = 0.0059, PTEN‐WT AKTi vs AKTi + EGFRi n.s. P = 0.1936, PTEN‐WT EGFRi vs AKTi + EGFRi n.s. P = 0.2302. G Viability of three PTEN‐null (MDA‐MB‐468, HCC70 and HCC1937), three PTEN‐WT (MDA‐MB‐231, MDA‐MB‐157, and HCC1428) and two PIK3CA‐mutant (SUM159 and BT20) TNBC cell lines treated for 6 days with PI3Kβi (AZD8186 90 nM) (left), or PI3Kαi (BYL719 1.2 μM) (right), alone or in combination with EGFRi (gefitinib 3 μM). Mean of 3–4 independent experiments ± SD. Statistical significance of two‐tailed unpaired Student's t ‐test PI3Kbi + EGFR treatments in PTEN‐null vs PTEN‐WT * P = 0.0158, PI3Kai + EGFR treatments in PTEN‐null vs PTEN‐WT n.s. P = 0.1081. H Dot‐plot showing the fold change (log 2 ) in number of reads between vehicle and AZD8186‐treated conditions vs the P ‐value of the difference between the two treatment conditions for each shRNA. Three out of four shRNAs targeting CSNK2A2 and three out of seven shRNAs targeting CSNK2B showed a P ‐value < 0.2 calculated by two‐sided paired t ‐test and are highlighted in the figure. I, J MDA‐MB‐468 was infected with the indicated shRNAs targeting CSNK2B and selected by puromycin. CSNK2B mRNA was then measured by RT–qPCR (I), and cell viability was measured after 4 days of treatment with serial dilutions of AZD8186 (J) Average ± SD of triplicates and representative of three independent experiments. K MDA‐MB‐468 cells were treated with serial dilutions of AZD8186 in combination with vehicle or with the indicated concentrations of CX4945. Viability was measured after 6 days and normalized within each of the CX4945‐treated condition to the viability in the absence of AZD8186. Mean ± SD of triplicates and representative of three independent experiments. L–N Viability of six PTEN‐null vs five PTEN‐WT TNBC cell lines treated for 6 days with PI3Kβi (AZD8186 10 nM) (L), AKTi (MK2206 0.45 μM) (M), or pan‐PI3Ki (GDC0941 1 μM) (N) alone or in combination with CK2i (CX4945 1 μM). Mean of 3–5 independent experiments ± SD. Statistical significance calculated by two‐tailed unpaired Student's t ‐test. PI3KBi vs PI3KBi + CK2i * P = 0.0265, CK2i vs PI3KBi + CK2i * P = 0.0128; AKTi vs AKTi + CK2i * P = 0.0494, CK2i vs AKTi + CK2i **** P < 0.0001; pan‐PI3Ki vs pan‐PI3Ki + CK2i * P = 0.0235, CK2i vs pan‐PI3Ki + CK2i **** P < 0.0001; n.s. P > 0.05.

Article Snippet: CX‐4945 was obtained from MedChemExpress.

Techniques: Mutagenesis, Expressing, RNA sequencing, Two Tailed Test, Infection, shRNA, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: Combined targeting of G protein‐coupled receptor and EGF receptor signaling overcomes resistance to PI 3K pathway inhibitors in PTEN ‐null triple negative breast cancer

doi: 10.15252/emmm.202011987

Figure Lengend Snippet: A Three cell lines derived and cloned from a mammary tumor arisen in a Wap‐cre:Pten fl/fl :Tp53 fl/fl mouse were treated for 16 h with vehicle, PI3Kβi (AZD8186 250 nM), or EGFRi (gefitinib 3 μM), alone or in combination. The cell lysates were probed with the indicated antibodies. Quantification of the bands was performed by ImageLite software. B Viability of HCC70 or ZR‐75‐1 parental cells (Par) and their respective acquired‐resistant derivatives (AZD8186‐Res or MK2206‐Res) treated for 18–21 days with serial dilutions of AZD8186 or MK2206, as indicated, and stained by crystal violet. The staining was then dissolved in 10% acetic acid and 100 μl transferred in a 96‐well plate for absorbance reading at 595 nm. Average ± SD of triplicates and representative of two independent experiments. C Time‐course analysis of MDA‐MB‐468 cells treated for the indicated times with vehicle, PI3Kβi (AZD8186 250 nM), or CK2i (CX4945 2 μM), alone or in combination. The cell lysates were probed with the indicated antibodies. AKT S129 is a known phosphorylation target of CK2, and the antibody raised against this phosphorylation site was used as a marker of CK2 activity. Quantification of the bands was performed by ImageLite software. D p110β co‐immunoprecipitates with EGFR. Total lysates from MDA‐MB‐468 or HCC70 were incubated with mouse IgG control or anti‐EGFR antibody, as indicated. The immuno‐precipitated complexes or the total lysates where probed with the indicated antibodies. E MDA‐MB‐468 cells were treated for 24 h with vehicle, AKTi (MK2206 1 μM), EGFRi (gefitinib 3 μM), or MEKi (trametinib 50 nM), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.

Article Snippet: CX‐4945 was obtained from MedChemExpress.

Techniques: Derivative Assay, Clone Assay, Software, Staining, Phospho-proteomics, Marker, Activity Assay, Incubation, Control

Journal: Oncotarget

Article Title: CK2 blockade causes MPNST cell apoptosis and promotes degradation of β-catenin

doi: 10.18632/oncotarget.10668

Figure Lengend Snippet: ( A ) MPNST (S462-TY) cells were treated with 2 unique shRNAs targeting CSNK2A1 for 72 h, and western blot analysis confirmed a reduction of CK2α protein. NT = cells treated with a non-targeting shRNA control. ( B ) Three day CK2α shRNA treatment had a cytotoxic effect on MPNST cell lines as measured by MTS; y-axis is O.D. reading at 490 nm. ( C ) Phase contrast photomicrographs show S-462TY cells 3 days after NT or shCK2α #1. ( D ) Increasing concentrations of CX-4945 (72 h) decrease MPNST cell line growth. CX-4945 has less effect on control iHSC cells as measured by MTS; y-axis is O.D. reading at 490 nm. ( E ) CK2α is overexpressed in MPNST cell lines as compared to the iHSC control cell line. ( F ) Phase contrast shows that CX-4945 treatment depletes cell population at 24 h as compared to the DMSO control. ( G ) MPNST cell lines show a decrease in CK2 activity in response to escalating CX-4945 concentrations (24 h) as measured by a western blot analysis using anti-CK2 substrate, and to undergo apoptosis as indicated by increased cleaved PARP. H. CX-4945 causes MPNST cells (S462-TY) to increase the percent of cells in the G2/M phase of the cell cycle as indicated by flow analysis of propidium iodide stained cells (S.D. of DMSO = 1.8% and CX4945 = 3.8%). Asterisks in B indicate differences after Student's t -test (* p < 0.05, ** p < 0.01, *** p < 0.001). CX-4945 cell viability assay, MTS, and qPCR results are the mean of three independent biological replicates each in triplicate, ± S.D.

Article Snippet: = 0.85) suggests that CX4945 with PD0325901 is moderately synergistic as defined by Compusyn ( www.combosyn.com ) (Figure ).

Techniques: Western Blot, shRNA, Control, Activity Assay, Staining, Viability Assay