Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: (a) IC 50 values for CDK4/6 inhibitor, PD0332991 (palbociclib) and CDK2 inhibitors, SNS032 and dinaciclib in 10 ovarian cancer cell lines. Cell viability was measured using a luminometric assay following 7d (PD0332991) or 72 h (SNS032, dinaciclib) of exposure to increasing concentrations of drug. The IC 50 values displayed represent the mean plus SEM ( n = 2) ranked from top to bottom from the most sensitive (HEY) to the most resistant (OAW28). PD0332991-resistant cell lines have IC 50 values > 2 μM, while PD0332991-sensitive lines have IC 50 values < 0.5 μM. SKOV3 cells display intermediate sensitivity. In contrast, IC 50 values for SNS032 and dinaciclib fall along a continuum (0.05–0.25 μM for SNS032, 0.005–0.015 μM for dinaciclib). (b) CDKi-resistant cells avoid CDKi-mediated cell cycle arrest. Naïve HEY cells were treated with vehicle (control), 0.5 μM PD0332991, 0.2 μM SNS032 or 0.02 μM dinaciclib for 48 h and analyzed by BrdU/PI staining. HEY-PD R and HEY-SNS R cells were treated with 0.5 μM PD0332991 and 0.2 μM SNS032, respectively for 48 h. A representative FACS image is shown for each condition. (c) PD0332991 and SNS032 delay progression of HEY xenografts. HEY cells were injected into nude mice and treated with vehicle (control), 50 mg/kg PD0332991 or 15mg/kg SNS032 on day 8, 12, 15, 19 and 22 after injection. The data shown represent the mean tumor volume plus SEM ( n = 6). * p = 0.04 (SNS032-treated versus control) and p = 0.0002 (PD0332991-treated versus control). (d), (e) Downregulation of E2F target genes and ETS factors by CDKi. HEY cells were treated with vehicle (control), 0.5 μM PD0332991, 0.2 μM SNS032 or 0.02 μM dinaciclib for 48 h with subsequent RNA and protein isolation. Validation of candidate target genes by (d) qPCR and (e) Western blot analysis are shown. (f) Biochemical changes in CDKi-resistant cells. Lysates derived from two HEY-PD R clones (left) and two HEY-SNS R clones (right) were treated in the absence or presence of 0.2 μM SNS032 or 0.5 μM PD0332991, respectively, for 48 h and probed with the antibodies indicated. Naïve HEY cells were included as controls. SNS032 induces TP53 protein in naïve HEY but not in HEY-SNS R . Elevated cyclin E1 and reduced RB protein levels are present in HEY-PD R . (g) CDK2 inhibition sensitizes OVCAR3 cells to PD0332991. OVCAR3 cells were treated with 0.2 μM SNS032 in the absence or presence of 0.5 μM PD0332991. Cell viability was determined by crystal violet staining after 3 months.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Luminescence Assay, Control, Staining, Injection, Isolation, Biomarker Discovery, Western Blot, Derivative Assay, Clone Assay, Inhibition

Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: CDKi-resistant cells restore expression of E2F target genes and ETS family genes Relative expression of known E2F targets ( a from Bracken et al ., 2004 [ 40 ]) and other select genes following exposure to 0.5 μM PD0332991 or 0.2 μM SNS032 in HEY cells for 48 h (left). Relative expression comparing HEY-SNS R to SNS-exposed HEY cells and HEY-PD R to PD0332991-exposed HEY cells (right). NS, not significant.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Expressing

Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: (a) CDKi-resistant OV90, SKOV3 and OAW28 cells were generated by chronic exposure of parental cells to either 0.2 μM SNS032 alone or a combination of 0.2 μM SNS032 and 0.2 μM PD0332991. IC 50 values were determined using a luminometric viability assay. (b) Relative CCNE1 copy number across six ovarian cancer cell lines as determined by genomic qPCR and aCGH. (c) Recurrent gains and losses associated with CDKi resistance. Genomic DNA was isolated from parental and polyclonal populations of CDKi-resistant cells and subjected to aCGH analysis. Segmented log 2 values are shown for select genes. Red indicates de novo copy number gain, green indicates de novo copy number loss. *a focal amplification region close to the MYC locus is present in these cells. (d) Validation of aCGH results by genomic qPCR. Genomic DNA was isolated from OV90, SKOV3 and OAW28 parental and CDKi R cells. Relative DNA copy number for CCNE1 , RB1 , ERBB2 and MYC was determined by qPCR.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Generated, Luminometric Viability Assay, Isolation, Amplification, Biomarker Discovery

Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: (a) HEY cells were stably transduced with retroviral pBABE-CCNE1 (red lines) or empty vector (black lines). Stable polyclonal populations were treated with 0.5 μM PD0332991 or vehicle over a course of 12 days and counted every four days. Ectopic cyclin E1 expression was confirmed by Western blot analysis and had no effect on proliferation in regular culture conditions (solid lines) but conferred partial resistance in the presence of PD0332991 (dotted lines). * p = 0.002 (b) Ectopic cyclin E1 expression rescues soft agar colony formation in PD0332991-treated HEY cells. Cells were seeded into soft agar followed by treatment with 0.2 μM PD0332991 or vehicle. Colonies were stained and counted after 2 weeks. Columns represent the relative mean plus SEM ( n = 3). * p = 0.01 (c) Depletion of cyclin E1 sensitizes OVCAR3 and (d) HEY cells to PD0332991-induced proliferation arrest. Two individual lentiviral shRNAs targeting CCNE1 were used to genetically deplete cyclin E1 in OVCAR3 and HEY cells. A shRNA targeting GFP was used as a control. After puromycin selection, cells were harvested to confirm CCNE1 knockdown by qPCR (relative CCNE1 expression is given as a percentage) and Western blot analysis (c, upper). Cells were seeded for viability using a luminometric assay in the absence or presence of 0.5 μM PD0332991 for 6d. Columns represent the relative mean plus SEM ( n = 3) * p ≤ 0.01 (c, lower). HEY-shGFP and HEY-shCCNE1 cells were seeded into soft agar and treated with vehicle or 0.2 μM PD0332991 (d, upper). Bar graph represents relative colony number compared to vehicle plus SD ( n = 3). * p ≤ 0.006. (e) Ectopic expression of mutant ERBB2 confers resistance to SNS032. OVCAR3 cells were stably transduced with retroviral pBABE-ERBB2 or empty vector. Stable polyclonal populations were treated with increasing concentrations of SNS032, and viability was assessed by a luminometric viability assay.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Expressing, Western Blot, Staining, shRNA, Control, Selection, Knockdown, Luminescence Assay, Mutagenesis, Luminometric Viability Assay

Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: (a) SKOV3-PD/SNS R release cells are hyperproliferative. CDKi-resistant SKOV3 cells were generated by chronic exposure to either 0.2 μM SNS032 alone or a combination of 0.2 μM PD0332991 and 0.2μM SNS032 for up to two years. SKOV3-PD/SNS R release cells were analyzed after 6 months in the absence of CDKi following two years of exposure. Proliferation rates of SKOV3-PD/SNS R , release and parental cells were determined using a luminometric viability assay. * p = 5.36E-06. (b) Western blot analysis of CDKi-resistant SKOV3 cells using antibodies against RB, RTK and PI3K signaling markers. CDKi-resistant cells hyperactivate ERBB2 and its downstream effectors MAPK and AKT, resulting in increased ETV5 protein expression. (c) ETV4 and ETV5 are upregulated in CDKi-resistant SKOV3 cells. Gene expression in SKOV3-PD/SNS R and parental SKOV3 cells was determined by qPCR. (d) Increased binding of E2F1 at the ETV5 promoter in CDKi-resistant cells. Chromatin from parental and CDKi-resistant SKOV3 cells was isolated and used in a ChIP assay with an E2F1 antibody or IgG control. Relative enrichment (E2F1/IgG) at the ETV5, CCND1 (left) and BRCA1 , BRCA2 (right) promoters is shown for naïve, early and late SKOV3-PD/SNS R cells. (e) ETV5 expression correlates with KRAS and CCNE1 DNA copy number in primary HGSOC. Using the TCGA dataset, expression of KRAS , ETV5 , CCNE1 and BRCA1 (y-axis) were plotted relative to KRAS (x-axis) and CCNE1 (z-axis) copy number status. ETV5 expression is positively correlated with KRAS copy number and inversely correlated with CCNE1 copy number.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Generated, Luminometric Viability Assay, Western Blot, Expressing, Gene Expression, Binding Assay, Isolation, Control

Journal: bioRxiv

Article Title: The TRAPP complex mediates secretion arrest induced by stress granule assembly

doi: 10.1101/528380

Figure Lengend Snippet: (A) Cells were treated with 273 kinase inhibitors (10 μM) from the Sellekchem library and with SA, and Sec24C localization in G3BP puncta (% of total G3BP area) was calculated (see Materials and methods) and is reported as a percentage of the control (cells treated with SA alone). Gray dashed line, mean of control; gray box, control standard deviation (± 18.2%); red line, threshold of positive hits; red box: positive hits. (B) Enrichment Analysis of the positive hits; for each class of inhibitor, the enrichment (in percentage) of compounds falling into positive hits and the enrichment (in percentage) in the total number of compounds was calculated and expressed as a ratio. (C) Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300μM, 30 min), Dinaciclib (10 μM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 minutes SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiments out of 3 independent replicates. (D) Drug specificity, from high (++++) to low (+). for the different CDKs as described by Selleckchem, and Sec24C recruitment to SGs as a percentage of control. (E) Analysis of Sec24C recruitment to SGs in control (MOCK), CDK1-KD, CDK2-KD and CDK1+2-KD HeLa cells. Data are expressed as percentage of Sec24C (mean intensity) in SG puncta normalized for cytosolic signal as a percentage of control conditions. Quantification of one representative experiment. N>100, mean ± s.e.m. ****p<0.0001. (F) Representative images of HeLa cells treated with the best three hits (Flavopiridol, SNS-032, Dinaciclib; 1μM 150 min) and then exposed to SA (300 μM, 30 min), followed by immunostaining for Sec24C and G3BP. Scale bar, 10μm. (G) HeLa cells were treated with Flavopiridol, SNS-032, or Dinaciclib for 150 min at the indicated concentrations, subsequently treated with SA (300 μM, 30 min), and then immunostained for Sec24C and G3BP and imaged by OPERA. BS-181, a specific CDK7 inhibitor, was used as negative control. Sec24C localization to SGs is expressed as a percentage of the control (cells treated with SA alone). Dashed red line, mean of control; yellow box, standard deviation of control (± 9.8%). (H) Cells were treated as in (G) and immunostained for TRAPPC2 and eIF3. Scale bar, 10 μm. The graph shows the quantification of TRAPPC2 localization in SG puncta (mean intensity). Data are mean ± s.e.m. expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol, SNS-032 or Dinaciclib treatment compared to the control (cells treated with SA alone). N=3, three independent experiments, n=60-80 cells per experiment. ****p<0.0001. . Evaluation of CDK1 and CDK2 knock down efficiency.

Article Snippet: To identify which specific CDK might be involved in Sec24C recruitment, we compared the potency of selected CDK inhibitors (Flavopiridol hydrochloride, SNS-032, Dinaciclib, AT7519, PHA-793887, ADZ5438, JNJ-7706621, PHA-767491, BMS-265246, PHA-848125, Roscovitine, Palbociclib, BS-181HCl) in the Sec24C recruitment assay with their ability to inhibit different CDK isoforms, as described by Selleckchem ( https://www.selleckchem.com/CDK.html ).

Techniques: Standard Deviation, Activity Assay, Western Blot, Activation Assay, Inhibition, Immunostaining, Negative Control

Journal: bioRxiv

Article Title: The TRAPP complex mediates secretion arrest induced by stress granule assembly

doi: 10.1101/528380

Figure Lengend Snippet: (A) Immunofluorescence images of Sec24C and TRAPPC2 in vehicle-treated and SNS-032-treated (1 μM, 3 h) cells. Scale bar, 10 μm. Graphs show the quantification of Sec24C and TRAPPC2 in the peri-Golgi area (mean intensity) normalized in SNS-032-treated cells relative to vehicle-treated cells (set as 100%). Mean ± s.e.m. of three independent experiments. **p<0.05, ****p<0.0001. (B) HeLa cells were treated with Dinaciclib (1 μM, 3 h) and permeabilized or not with digitonin as described in Materials and methods. A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency. Upper panels, non-permeabilized (NP) control cells; middle panels, permeabilized control cells; lower panels, permeabilized Dinaciclib-treated cells. Scale bar, 10 μm. (C) Quantification of Sec24C membrane association after CDK inhibitor treatment. Digitonin-permeabilized cells treated with vehicle (CTRL) or the indicated CDK inhibitor (1 μM, 3 h) were immunostained for Sec24C. The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug-treated cells is expressed as fold change compared to the control; n= 60-80; mean ± s.e.m. of three independent experiments ****p<0.0001. Scale bars, 10 μm.

Article Snippet: To identify which specific CDK might be involved in Sec24C recruitment, we compared the potency of selected CDK inhibitors (Flavopiridol hydrochloride, SNS-032, Dinaciclib, AT7519, PHA-793887, ADZ5438, JNJ-7706621, PHA-767491, BMS-265246, PHA-848125, Roscovitine, Palbociclib, BS-181HCl) in the Sec24C recruitment assay with their ability to inhibit different CDK isoforms, as described by Selleckchem ( https://www.selleckchem.com/CDK.html ).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: The TRAPP complex mediates secretion arrest induced by stress granule assembly

doi: 10.1101/528380

Figure Lengend Snippet: (A) HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA (30 min, 300 μM), and immunostained for Sec24C or TRAPPC2 and eIF3. Scale bar 10 μm. (B) Analysis of the proliferation status of the cells after 8 h starvation in HBSS. Left, representative images of starved and nonstarved cells using EdU incorporation (see Materials and methods) and, right, quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells. (C) HeLa cells were seeded at different confluency, treated with SA and stained for Sec24C and G3BP as an SG marker. Scale bar, 10 μm. Flow cytometry (FACS) analysis (right panels) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency. The graph shows quantification of Sec24C mean intensity in SG puncta normalized for the cytosolic Sec24C at the indicated cell confluency. Mean ± s.e.m. of a representative experiment out of n=5 biological replicates. n= 50-80. ns, not significant; ****p<0.0001. (D) Growing and differentiated podocytes were treated with SA (300 μM, 30 min) and stained for TRAPPC2 or Sec24C and eIF3. Scale bar, 10μm. The graphs show quantification of TRAPPC2 and Sec24C (mean intensity) in SGs. Mean ± s.e.m. of three independents experiments ****p<0.0001. (E) CDK kinase activity in growing and differentiated podocytes. Western blot using a specific antibody recognizing phosphoSer-CDK substrates was used on total cell lysates. (F) Western blot analysis of phosphorylated retinoblastoma (p-RB) in growing (non-differentiated) versus differentiated podocytes. ß-actin was used as loading control. . Differentiation of podocytes.

Article Snippet: To identify which specific CDK might be involved in Sec24C recruitment, we compared the potency of selected CDK inhibitors (Flavopiridol hydrochloride, SNS-032, Dinaciclib, AT7519, PHA-793887, ADZ5438, JNJ-7706621, PHA-767491, BMS-265246, PHA-848125, Roscovitine, Palbociclib, BS-181HCl) in the Sec24C recruitment assay with their ability to inhibit different CDK isoforms, as described by Selleckchem ( https://www.selleckchem.com/CDK.html ).

Techniques: Staining, Marker, Flow Cytometry, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: The TRAPP complex mediates secretion arrest induced by stress granule assembly

doi: 10.1101/528380

Figure Lengend Snippet: (A) Box plot representing SG area (see Materials and methods) in mock, TRAPPC2- and TRAPPC3-depleted cells treated with SA. Distribution of values from three independent experiments. ****p<0.0001. (B) HeLa cells microinjected with control preimmune IgG or a TRAPPC3-specific antibody (right panels in green) were treated with SA. The TRAPPC3 Ab disrupts the Golgi , monitored using an anti-TGN46 Ab. Anti-G3BP was used to stain SGs. The graph shows quantification of the SG area. Mean ± s.e.m. three independent experiments; n>80. ****p<0.0001. Scale bar, 10 μm. (C) Structured Illumination Microscopy (SIM)-Super resolution (SR) images of endogenous TRAPPC2 and GFP-Sec23 localizing at SGs, stained for G3BP. Right, magnification of boxed area. Scale bar, 0.5 μm (D,E) Localization of Raptor (D) and RACK1 (E) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 μm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock. Mean ± S.D. three independent replicates. *p<0.02; **p<0.009 in (D) , *p<0.05; ****p<0.0001 in (E). (F,G) Localization of Raptor (F) and RACK1 (G) in untreated cells or cells pretreated with the indicated CDK inhibitor (1 μM, 150 min) and then with SA (300 μM, 30 min). G3BP was used to stain SGs. Scale bar, 10 μm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001. (H) Analysis of cell death after overnight recovery of HeLa cells treated or untreated with CDKi (SNS-032; Flavopiridol or Dinaciclib, 1 μM, 150 min) and then treated with SA (500 μM, 3 h). Images were automatically acquired by OPERETTA microscope. Values indicate the percentage of the total number of nuclei (stained with DAPI) positive for BoBo-3 staining. Mean ± s.d. of one representative experiment out of three independent replicates. ns, not significant, ****p<0.0001. . TRAPP depletion does not affect protein translation inhibition caused by SA treatment.

Article Snippet: To identify which specific CDK might be involved in Sec24C recruitment, we compared the potency of selected CDK inhibitors (Flavopiridol hydrochloride, SNS-032, Dinaciclib, AT7519, PHA-793887, ADZ5438, JNJ-7706621, PHA-767491, BMS-265246, PHA-848125, Roscovitine, Palbociclib, BS-181HCl) in the Sec24C recruitment assay with their ability to inhibit different CDK isoforms, as described by Selleckchem ( https://www.selleckchem.com/CDK.html ).

Techniques: Staining, Microscopy, Inhibition

Journal: BMC Cancer

Article Title: Targeting cyclin-dependent kinase 1 (CDK1) but not CDK4/6 or CDK2 is selectively lethal to MYC-dependent human breast cancer cells

doi: 10.1186/1471-2407-14-32

Figure Lengend Snippet: CDK2 inactivation mediates cell cycle arrest by MYC depletion and is not selectively lethal to MYC-dependent breast cancer cells. (A) Cells were transfected with either pooled MYC siRNA (siMYC) or Non-targeting siRNA control (siCON) at 2 nM or 10 nM. Cell lysates were collected at 72 hour post-transfection and then subjected to Western blotting. β-ACTIN was used as loading control for whole-cell lysates. (B) Cells were transfected with either pooled CDK2 siRNA (siCDK2) or Non-targeting siRNA control (siCON) at 10 nM. 72 hours after transfection, cell proliferation was measured by BrdU incorporation. Data are mean ± SEM analysed in triplicate experiments. (C) A panel of 17 human breast cancer cell lines were treated with CDK2 inhibitor SNS-032 at a series of concentrations for 48 hours followed by measurement of BrdU incorporation. The correlation between sensitivity to MYC siRNA and SNS-032 was assessed. Sensitivity to MYC siRNA was determined by inhibition of BrdU incorporation at 10 nM MYC siRNA and sensitivity to SNS-032 determined by the IC 50 value for each cell line shown in Additional file : Table S2 and S3, respectively. R = Pearson correlation coefficient, P = the corresponding P -value.

Article Snippet: The CDK4/6 inhibitor PD0332991 was purchased from Selleck Chemicals (Houston, TX, USA), CDK2 inhibitor SNS-032 from Symansis (Auckland, New Zealand) and CDK1 inhibitors, RO-3306 and CGP74514A, from Calbiochem (San Diego, CA, USA).

Techniques: Transfection, Control, Western Blot, BrdU Incorporation Assay, Inhibition

Journal: Oncotarget

Article Title: Cyclin-dependent kinase 2 is an ideal target for ovary tumors with elevated cyclin E1 expression

doi:

Figure Lengend Snippet: A. Overnight-cultured cells were harvested and cell lysates were subjected to immunoblotting to detect Cdk1 and Cdk2 protein using the respective antibodies. Membrane was stripped and reprobled with GAPDH polyclonal antibody for loading normalization. B, C. Ovarian cancer cells (40,000 cells/well) were plated in 24-well plates for overnight and varying concentrations of JNJ-7706621 B. or SNS-032 C. were added to cells for 4 days. Cell growth was evaluated by MTT assay and IC50 was estimated as the concentration required for reach 50% growth reduction comparing to untreated cells. D, E . OCC1 D. or OVCAR429 cells E. were treated with 0.5 or 2μM SNS-032 for 2 days, then stained with Annexin V-FITC/propidium iodide and analyzed by FACS. Data are means ± SE. n = 4. *, P < 0.005 vs 0 μM of SNS-032.

Article Snippet: Cdk1 inhibitor JNJ-7706621 and Cdk2 inhibitor SNS-032 (BMS-387032) were purchased from Selleckchem (Houston, TX).

Techniques: Cell Culture, Western Blot, Membrane, MTT Assay, Concentration Assay, Staining