Journal: Cancers

Article Title: CDK4/6 Inhibitors Suppress RB-Null Triple-Negative Breast Cancer by Inhibiting Mutant P53 Expression via RBM38 RNA-Binding Protein

doi: 10.3390/cancers17203339

Figure Lengend Snippet: Both RB-proficient and RB-deficient breast cancer cells are susceptible to CDK4/6 inhibitors. ( A ) Colony formation assay was performed with MCF7 cells treated with or without Palbociclib (2 and 10 μM) or Ribociclib (2 and 10 μM) for 48 h, after which the drugs were withdrawn to allow for colony formation. The relative fold change in cell survival is shown below each treatment. ( B ) The experiment was performed the same as in ( A ) except that MDA-MB-231 cells were mock-treated or treated with Palbociclib (0.5 and 5 μM) or Ribociclib (0.5 and 5 μM) for 48 h. The relative fold change in cell survival is shown below each treatment. ( C , D ) Colony formation assay was performed with MDA-MB-468 ( C ) and BT549 ( D ) cells mock-treated or treated with Palbociclib (2.5 and 10 μM) or Ribociclib (5 and 20 μM) for 48 h. The relative fold change in cell survival is shown below each treatment.

Article Snippet: Streptavidin magnetic beads (Cat# HY-K0208), Palbociclib (Cat# HY-50767), and Ribociclib (Cat# HY-15777) were purchased from Medchem Express (Monmouth Junction, NJ, USA).

Techniques: Colony Assay

Journal: Cancers

Article Title: CDK4/6 Inhibitors Suppress RB-Null Triple-Negative Breast Cancer by Inhibiting Mutant P53 Expression via RBM38 RNA-Binding Protein

doi: 10.3390/cancers17203339

Figure Lengend Snippet: Suppression of mutant p53 expression is likely responsible for CDK4/6 inhibitors sup-pressing TNBC cell survival. ( A , B ) MDA-MB-231 cells were mock-treated or treated with Palbociclib (0–5 μM) ( A ) or Ribociclib (0–5 μM) ( B ) for 24 h, followed by Western blot to measure p-RB, p53 and GAPDH. ( C , D ) MDA-MB-468 cells were mock-treated or treated with Palbociclib (0–5 μM) ( C ) or Ribociclib (0–5 μM) ( D ) for 24 h, followed by Western blot to measure p53 and actin. ( E , F ) BT549 cells were mock-treated or treated with Palbociclib (0–2 μM) ( E ) or Ribociclib (0–5 μM) ( F ) for 24 h, followed by western blot analysis to measure p53 and actin. ( G , I ) MDA-MB-231 ( G ) and MDA-MB-468 ( I ) cells were transiently transfected with a scrambled siRNA or p53 siRNAs for 3 days, followed by Western blot to measure the expression of p53 and actin. ( H , J ) MDA-MB-231 ( H ) and MDA-MB-468 ( J ) cells were transiently transfected with a scrambled siRNA or p53 siRNAs for 3 days, followed by treatment with or without 5 μM of Palbociclib for 24 h. The cell viability was measured by the CellTiter-Glo luminescent cell viability assay. * indicates p < 0.05 by Student’s t test; n.s. indicates not significant.

Article Snippet: Streptavidin magnetic beads (Cat# HY-K0208), Palbociclib (Cat# HY-50767), and Ribociclib (Cat# HY-15777) were purchased from Medchem Express (Monmouth Junction, NJ, USA).

Techniques: Mutagenesis, Expressing, Western Blot, Transfection, Cell Viability Assay

Journal: Cancers

Article Title: CDK4/6 Inhibitors Suppress RB-Null Triple-Negative Breast Cancer by Inhibiting Mutant P53 Expression via RBM38 RNA-Binding Protein

doi: 10.3390/cancers17203339

Figure Lengend Snippet: CDK4/6 inhibitors suppress mutant p53 expression and cell survival via RBM38 RNA-binding protein. ( A ) RBM38-expressing MCF7 cells were treated with or without 5 mM of Palbociclib for 12 h, followed by Western blot analysis to measure the level of RBM38, RB, and actin. ( B ) RBM38-expressing MCF7 cells were treated with or without 5 μM of Ribociclib for 12 h, followed by Western blot analysis to measure the level of RBM38 and actin. ( C ) MDA-MB-231 cells were mock-treated or treated with Palbociclib (0–5 μM) for 18 h, followed by Western blot analysis to measure p-RBM38, RBM38, p53, and actin. ( D ) BT549 cells were transiently transfected with a control vector or various amounts of Flag-tagged CDK4-exprssing vector, followed by Western blot using antibodies against CDK4, RBM38, and actin. ( E ) Isogenic control and RBM38-KO MIA-PaCa2 cells were mock-treated or treated with Palbociclib for 24 h, followed by Western blot analysis using antibodies against RBM38, p53, and actin. ( F ) Isogenic control and RBM38-KO MIA-PaCa2 cells were transfected with scrambled siRNA or siRNAs against CDK4 for 3 days, followed by Western blot analysis to measure the expression of CDK4, p53, and GAPDH. ( G ) A colony formation assay was performed. Isogenic control and RBM38-KO MIA-PaCa2 cells were mock-treated or treated with Palbociclib (5 μM) for 48 h, after which the drug was withdrawn to allow individual colony growth for 2 weeks.

Article Snippet: Streptavidin magnetic beads (Cat# HY-K0208), Palbociclib (Cat# HY-50767), and Ribociclib (Cat# HY-15777) were purchased from Medchem Express (Monmouth Junction, NJ, USA).

Techniques: Mutagenesis, Expressing, RNA Binding Assay, Western Blot, Transfection, Control, Plasmid Preparation, Colony Assay

Journal: Science advances

Article Title: CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon RB function in malignant peripheral nerve sheath tumors.

doi: 10.1126/sciadv.adg8876

Figure Lengend Snippet: Fig. 4. Responses of NF1-MPNST cells to the CDK4/6i ribociclib. (A) Ten NF1-associated MPNST cell lines were treated with increasing doses of the CDK4/6i ribociclib for 5 days. Cell viability was evaluated by using the CCK-8 assay. (B) Two NF1-MPNST cell lines were treated with DMSO or 1 μM ribociclib over a time course. Signal intermediates in ERK and cell cycle pathways were assessed. (C) JH-2-002 cells were treated with DMSO or 1 μM ribociclib for 24 hours. Seventy-one phosphorylated human RTKs were evaluated using human RTK phosphorylation array C1. Signal intensity from technical duplicates was quantified using densitometry analysis and normalized to ribociclib v. DMSO, and, notably, altered RTKs are shown. (D) Cells as in Fig. 3E were treated with increasing doses of ribociclib for 5 days. Cell viability was evaluated by using the CCK-8 assay.

Article Snippet: NF1-MPNST cells were treated with DMSO, 0.3 μM TNO155, and/ or 1 μM ribociclib for 48 hours, and, then, 500 μg protein of lysates were assessed using human apoptosis antibody array (R&D Systems, #ARY009).

Techniques: CCK-8 Assay, Phospho-proteomics

Journal: Science advances

Article Title: CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon RB function in malignant peripheral nerve sheath tumors.

doi: 10.1126/sciadv.adg8876

Figure Lengend Snippet: Fig. 5. Combined inhibition of SHP2 and CDK4/6 effectively suppresses MPNST cell growth. (A) ST8814 and NF90.8 parental (Par) and trametinib-resistant (Res) lines and eight NF1-MPNST cell lines were treated with DMSO, TNO155 (0.3, 1, and 3 μM), ribociclib (1 and 3 μM), and their combination for 7 to 10 days. Direct cell counting using trypan blue exclusion assay was performed by TC20 automated cell counter. (B) Cells as in (A) were treated with drugs for 2 to 3 weeks, and colony formation was evaluated using crystal violet assay. (C) Area under the curve (AUC) is calculated on the basis of the IncuCyte cell confluence monitoring of the seven cell lines as shown, treated with DMSO, TNO155 (0.3 μM), ribociclib (1 μM), and their combination for 6 days. (D) Two NF1-MPNST cell lines were treated with DMSO, TNO155 (0.3 μM), ribociclib (1 μM), and their combination for 6 days. Cell confluence was monitored using IncuCyte imaging systems. (E) Four NF1-MPNST cell lines were treated with DMSO, 0.3 μM TNO155, and/or 1 μM ribociclib for 72 and 96 hours. ERK signaling and cell cycle regulators were evaluated using immunoblot. (F) Three NF1-MPNST cell lines transduced with shPTPN11 #818 were pretreated with vehicle or Dox (300 ng/ml) for 72 hours, followed by treatment with DMSO or 1 μM ribociclib for additional 72 hours. Cell lysates were assessed for expression of the indicated proteins.

Article Snippet: NF1-MPNST cells were treated with DMSO, 0.3 μM TNO155, and/ or 1 μM ribociclib for 48 hours, and, then, 500 μg protein of lysates were assessed using human apoptosis antibody array (R&D Systems, #ARY009).

Techniques: Inhibition, Cell Counting, Trypan Blue Exclusion Assay, Crystal Violet Assay, Imaging, Western Blot, Transduction, Expressing

Journal: Science advances

Article Title: CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon RB function in malignant peripheral nerve sheath tumors.

doi: 10.1126/sciadv.adg8876

Figure Lengend Snippet: Fig. 6. Combination of TNO155 and ribociclib additively inhibits cell cycle and induces apoptosis. (A) Upset matrix plot derived from RNA-seq analysis, showing the overlapping numbers of significant genes (P adj < 0.05 and |fold change| > 1.5) after 24-hour treatment with 0.3 μM TNO155, 1 μM ribociclib, and their combination, and normalized to DMSO control. Rows, the sets; columns, intersections between these sets. (B and C) Heatmaps demonstrating the more potent transcriptional inhibition of mitotic prometaphase (B) and cell cycle checkpoints (C) by combined TNO155 and ribociclib relative to either single agent alone, highlighting BIRC5, PLK1, CLSPN, and CCNA2 (P adj < 0.05 marked with + and LFC). (D) Five NF1-MPNST cell lines were treated with DMSO, 0.3 μM TNO155, and/or 1 μM ribociclib for 48 hours, following overnight starvation in 0.1% FBS-containing culture medium to synchronize cells. Cells were fixed in ice cold 70% ethanol and stained with propidium iodide/ribonu- clease staining solution (Cell Signaling Technology, no. 4087) and then analyzed by flow cytometry. (E) ST8814 and JH-2-079c were treated with DMSO, 0.3 μM TNO155, and/or 1 μM ribociclib for 48 hours, and, then, protein lysates were assessed using human apoptosis antibody array (R&D Systems, no. ARY009). Signal intensity from technical duplicates was quantified by densitometry analysis using ImageJ and normalized to DMSO. Data represent means ± SEM. (F) Nine NF1-MPNST cell lines were treated as in (E), and the indicated proteins involved in apoptosis and ERK signaling were detected using immunoblot.

Article Snippet: NF1-MPNST cells were treated with DMSO, 0.3 μM TNO155, and/ or 1 μM ribociclib for 48 hours, and, then, 500 μg protein of lysates were assessed using human apoptosis antibody array (R&D Systems, #ARY009).

Techniques: Derivative Assay, RNA Sequencing, Control, Inhibition, Staining, Flow Cytometry, Ab Array, Western Blot

Journal: Science advances

Article Title: CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon RB function in malignant peripheral nerve sheath tumors.

doi: 10.1126/sciadv.adg8876

Figure Lengend Snippet: Fig. 7. The combination of TNO155 and ribociclib is active against MPNST tumor growth in vivo. (A and B) Five-to-6-week-old female NRG mice bearing six individual NF1- MPNST PDXs were treated with vehicle, ribociclib (75 mg/kg, once daily), TNO155 (7.5 mg/kg, twice daily), or their combination by oral gavage for 4 weeks. Average tumor volume of four to five mice per arm was plotted over the time course of treatment days. VS, very sensitive; PS, partially sensitive. (C) Tumors of each arm from WU-386 were harvested 4 hours after last dose of 4 weeks on drugs and fixed in 10% NBF. The Ki-67 expression was assessed using im- munohistochemistry (IHC). (D and E) Tumors of each arm from WU-225, WU-386 were harvested 4 hours after last dose, 4 weeks on drugs (D); or 24 hours after last dose, 3 days on drugs (E). The indicated proteins involved in ERK and cell cycle signaling were de- tected using immunoblot. (F) Onco- print of key driver genes in MPNST and putative others is shown. For NF1 and SUZ12, both germline (G) and somatic (S) mutations are shown. (G) Volcano plot demonstrating LFC of VS/PS as a function of −log10 (P adj). Blue dots represent 227 genes that were significantly altered (P adj < 0.05 and |LFC| > 0.585) when comparing the RNA-seq data of VS (JH-2-031, WU- 225, and JH-2-002) v. PS (WU-545, JH- 2-079c, and WU-386). (H) The mice bearing JH-2-031 were on initial treatment as in (A) for about 4 weeks and left untreated for another 4 weeks before rechallenging with the combi- nation for additional 2 weeks. (I) The mice bearing JH-2-002 as in (A) were continuously on treatment for 10 weeks. (J) Tumors of each arm from JH-2-002 and JH-2-031 were harvest- ed 4 hours after last dose of 10 weeks and fixed in 10% NBF. The Ki-67 ex- pression was assessed using IHC.

Article Snippet: NF1-MPNST cells were treated with DMSO, 0.3 μM TNO155, and/ or 1 μM ribociclib for 48 hours, and, then, 500 μg protein of lysates were assessed using human apoptosis antibody array (R&D Systems, #ARY009).

Techniques: In Vivo, Expressing, Western Blot, RNA Sequencing

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: The effect of fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM in the MCF-7 and T47D cell models; 5 µM in the ZR-75-1 cell model) and AKT inhibitor (AKTi, capivasertib, 500 nM in the MCF-7 cell model; 200 nM in the T47D model; 150 nM in the ZR-75-1 cell model), as single agents or in double and triple combinations, was assessed in all cell lines by crystal violet growth assay ( A , B , E , F , I and J ) and CellTiter-Blue viability assay ( C , D , G , H , K , and L ) performed over 6 days. Outgrowth of resistant colonies was investigated in MCF-7 ( M and N ) and T47D models ( O and P ) by weekly evaluation of the percentage of 48 wells at 50% or greater confluence (positive wells) over 12 weeks. Experiments were conducted in three biological replicates and data are shown as mean ± SEM. Asterisks indicate significant differences in the one-way ANOVA test at day 6 (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Growth Assay, Viability Assay

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: Apoptotic levels were determined by evaluating the presence of cytoplasmic nucleosomes in MCF-7 ( A ), T47D ( B ), and ZR-75-1 ( C ) cells using an ELISA cell death detection assay. Data are shown with error bars representing mean ± SEM of three biological replicates. Asterisks indicate significant differences in the one-way ANOVA test (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001). D Western blot analysis of apoptosis markers in the three fulvestrant-resistant breast cancer models. Densitometry analysis of Western blot bands of cleaved-PARP was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. E Western blot analysis of key signal transduction proteins in the three fulvestrant-resistant breast cancer models. F Western blotting analysis of p-AKT (S473) and total AKT expression in the three fulvestrant-resistant breast cancer models. Densitometry analysis of Western blot bands of p-AKT (S473) and total AKT was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. GAPDH was used as a loading control for Western blotting analysis. For all Western blots, a representative of two biological replicates is shown. Both apoptosis assay and harvesting protein for Western blotting analysis were performed 3 days after treatment with fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM in the MCF-7 and T47D cell models; 5 µM in the ZR-75-1 cell model) and AKT inhibitor (AKTi, capivasertib, 500 nM in the MCF-7 cell model; 200 nM in the T47D model; 150 nM in the ZR-75-1 cell model).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Enzyme-linked Immunosorbent Assay, Detection Assay, Western Blot, Software, Control, Transduction, Expressing, Apoptosis Assay

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: Tumor growth curves of MCF-7 ( A ) and 182R-1 ( B ) tumors following treatment with fulvestrant (Fulv, 100 mg/Kg bodyweight; N = 7) or vehicle (castor oil, N = 8) administered subcutaneously once a week. Treatment was initiated when tumors reached 50 mm 3 and continued for 5 weeks. C Mice were sacrificed on week 5 and MCF-7 and 182R-1 tumors were excised. Tumor growth curves of 182R-1 tumors treated with CDK4/6 inhibitor (CDK4/6i, palbociclib, 50 mg/Kg bodyweight; N = 8) alone ( D ), in combination with fulvestrant (100 mg/Kg bodyweight; N = 7 and N = 10) ( D and E ), or in combination with both AKT inhibitor (AKTi, capivasertib 100 mg/Kg bodyweight) and fulvestrant (100 mg/Kg bodyweight; N = 6 and N = 10) ( D and E ), or vehicle (castor oil and 25% w/v HPB cyclodextrin; N = 8) ( D ). CDK4/6i and AKTi were administered by oral gavage once daily for 5 days a week when tumors reached 50 mm 3 ( D ) or 250 mm 3 ( E ), and treatment was continued for up to 8 weeks. Data are shown as mean tumor volume ± SEM. Asterisks indicate a significant difference in ANOVA one-way test ( D ) or two-tailed t -test ( A , B and E ) at the endpoint (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001 and **** p < 0.0001).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: The effect of fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM) and AKT inhibitor (AKTi, capivasertib, 250–500 nM in the MCF-7 cell model; 100 nM in the T47D cell model), as single agents or in the double and triple combination, was assessed in all cell lines by crystal violet growth assay ( A , B , E , and F ) and CellTiter-Blue viability assay ( C , D , G , and H ) performed over 6 days. Outgrowth of resistant colonies was investigated in MPF-R ( I ) and TPF-R ( J ) cells by weekly evaluation of the percentage of 48 wells at 50% or greater confluence (positive wells) over 12 weeks. Experiments were conducted in three biological replicates and data are shown as mean ± SEM. Asterisks indicate significant differences in one-way ANOVA tests at day 6 (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Growth Assay, Viability Assay

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: Apoptotic levels were determined by evaluating the presence of cytoplasmic nucleosomes in MCF-7 ( A ) and T47D ( B ) cell models using an ELISA cell death detection assay. Data are shown with error bars representing mean ± SEM of three biological replicates. Asterisks indicate significant differences in one-way ANOVA tests (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001). C Western blot analysis of apoptosis markers in both models resistant to combined fulvestrant and CDK4/6i. Densitometry analysis of Western blot bands of cleaved-PARP was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. D Western blot analysis of key signal transduction proteins in breast cancer cell models resistant to combined CDK4/6i and fulvestrant therapy. E Western blotting analysis of p-AKT (S473) and total AKT expression in both breast cancer models resistant to combined fulvestrant and CDK4/6i. Densitometry analysis of Western blot bands of p-AKT (S473) and total AKT was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. GAPDH was used as a loading control for Western blotting analysis. For all Western blots, a representative of two biological replicates is shown. Both apoptosis assay and harvesting protein for Western blotting analysis were performed 3 days after treatment with fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM), and AKT inhibitor (AKTi, capivasertib, 250 nM in the MCF-7 cell model; 100 nM in the T47D cell model).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Enzyme-linked Immunosorbent Assay, Detection Assay, Western Blot, Software, Control, Transduction, Expressing, Apoptosis Assay

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: A Tumor growth curves of orthotopic MPF-R tumors treated with a CDK4/6 inhibitor (CDK4/6i, palbociclib, 50 mg/Kg bodyweight) combined with fulvestrant (Fulv, 100 mg/Kg bodyweight; N = 9) or in combination with both AKT inhibitor (AKTi, capivasertib, 100 mg/Kg body weight) and fulvestrant (Fulv, 100 mg/Kg bodyweight; N = 10), or vehicle (castor oil and 25% w/v HPB cyclodextrin; N = 10). CDK4/6i and AKTi were administered by oral gavage once daily for 5 days a week, whereas fulvestrant was administered subcutaneously once a week. Treatment was initiated when tumors reached 100 mm 3 and continued for up to 7 weeks. Mice from the control group were sacrificed and tumors excised on week 6 due to their large size, while mice from double and triple combination groups were sacrificed and tumors excised on week 7. Data are shown as mean tumor volume ± SEM. Asterisks indicate significant differences in the two-tailed t -test at the endpoint (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001). B Western blot analysis of key signal transduction proteins in 3 tumors of each treatment group excised when mice were sacrificed. GAPDH was used as a loading control. A representative of two independent experiments is shown. C Tumor volumes over time of the parental PDX KCC_P_3837 untreated (blue) and treated with combined CDK4/6i palbociclib (25 mg/kg in 2.5% DMSO, 25% β-cyclodextrin, 5 days per week by oral gavage) and fulvestrant (100 mg/kg in castor oil, once weekly via subcutaneous injection) (orange), and of the derivative PDX KCC_P_3837-FPR resistant to combined palbociclib and fulvestrant (red) at the third passage of continuous exposure to combined palbociclib and fulvestrant. D Kaplan-Meier survival plot of progression of PDX KCC_P_3837-FPR (resistant to combined CDK4/6i and fulvestrant) under treatment with combined CDK4/6i and fulvestrant with or without AKTi capivasertib (100 mg/kg in 2.5% DMSO, 25% β-cyclodextrin, 5 days per week by oral gavage) ( N = 5 and N = 4, respectively). Progression was defined as tumors growing to at least 5 mm in the shortest dimension. A two-sided p- value ( p < 0.05) was calculated using log-rank testing. E , F Evaluation of metastasis area > 2500 µm 2 relative to lung area at the endpoint (6 weeks) using an experimental metastasis model. MPF-R tumors were treated with fulvestrant (100 mg/Kg body weight) combined with CDK4/6i (palbociclib, 25 mg/Kg bodyweight; N = 10), fulvestrant combined with AKTi (capivasertib, 100 mg/Kg bodyweight; N = 10), or triple combination ( N = 9). TPF-R tumors were treated with the same dosage of fulvestrant and CDK4/6i and 50 mg/Kg bodyweight of AKTi ( N = 7 in fulvestrant and CDK4/6i group, N = 10 in fulvestrant and AKTi and N = 10 in triple combination group). CDK4/6i and AKTi were administered by oral gavage once daily for 5 days a week, while fulvestrant was administered subcutaneously once a week. Treatment was initiated 3 days before injection of cells in the tail vein and continued for up to 6 weeks. Data are shown as mean ± SEM. Significant differences were evaluated by the two-tailed Mann-Whitney test. G Representative micrographs of MPF-R and TPF-R tumors in mice lungs of each treatment group showing cytokeratin expression by immunohistochemistry (scale bars, 200 µm).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Control, Two Tailed Test, Western Blot, Transduction, Injection, MANN-WHITNEY, Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: Clinical and pathological characteristics of ER+ breast cancer patients with advanced disease treated with combined CDK4/6i and endocrine treatment from pilot and validation cohorts according to p-AKT levels.

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques:

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: A Kaplan-Meier plots evaluating progression-free survival (PFS) according to p-AKT (S473) levels in ER+ metastatic lesions from a validation cohort of ER+breast cancer patients treated with combined CDK4/6i and endocrine therapy in the advanced setting. A two-sided p -value ( p < 0.05) was calculated using log-rank testing. Representative micrographs of all breast cancer metastasis sections showing low p-AKT expression (H-score < 150, B and C ) or high p-AKT expression (H-score ≥ 150, D and E ; scale bars, 100 µm).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: Co-targeting RANK pathway treats and prevents acquired resistance to CDK4/6 inhibitors in luminal breast cancer

doi: 10.1016/j.xcrm.2023.101120

Figure Lengend Snippet:

Article Snippet: Ribociclib , Santa Cruz Biotechnology , LEE011.

Techniques: Recombinant, Control, shRNA, Blocking Assay, Alamar Blue Assay, Flow Cytometry, Isolation, cDNA Synthesis, Bicinchoninic Acid Protein Assay, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Western Blot, Software

Journal: Cancer chemotherapy and pharmacology

Article Title: CNS penetration of the CDK4/6 inhibitor ribociclib in non-tumor bearing mice and mice bearing pediatric brain tumors

doi: 10.1007/s00280-019-03864-9

Figure Lengend Snippet: Ribociclib mean concentrations in extracellular fluid plotted against time by tumor type.

Article Snippet: Ribociclib was added to mouse plasma (BioIVT, Westbury, NY) to make final concentrations of 500, 1000, 2500, and 5000 ng/ml.

Techniques:

Journal: The BMJ

Article Title: Public sector financial support for late stage discovery of new drugs in the United States: cohort study

doi: 10.1136/bmj.l5766

Figure Lengend Snippet: New drugs with contributions from spin-off companies based on publicly supported research

Article Snippet: 3/13/2017 (#209092) , Ribociclib succinate , Novartis Pharms , Astex Therapeutics , University of Cambridge , Patent.

Techniques: