cdk4 6i  (Selleck Chemicals)

Journal: bioRxiv

Article Title: A genetic signature predicts aggressive paraganglioma sensitivity to dual PI3K-CDK4/6 inhibition therapy

doi: 10.1101/2025.02.18.638668

Figure Lengend Snippet: (A,B) IC 50 curves of PC12 ( A ) or MPC ( B ) cells treated with CDK4/6i, PI3Ki, their combination or DMSO control. Cell proliferation was measured after 72h of treatment. For drug concentrations see Suppl. Table S1. The DMSO control was set to 100% and nonlinear regression was used to calculate the IC 50 . Data shown are the mean±SD from 3 independent experiments with 3 technical replicates each. ( C,D ) PC12 ( C ) and MPC ( D ) cells were treated using the indicated concentrations and Caspase 9 activity was measured 72h later. Shown is the relative apoptosis normalized to the DMSO control. Data shown is the mean ± SD from 3 independent experiments with 3 technical replicates each. Statistics: 2way ANOVA. ( E,F ) PC12 cells were plated in chambers without Matrigel to assess migration ( E ), or containing Matrigel to assess invasion ( F ). After 72h of treatment with IC 50 values of the drugs, chambers were collected, stained and cells counted (3 technical replicates for each condition). Data shown are the mean±SD from 3 independent experiments. Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001. ( G,H ) Expression and quantification of total Akt, phospho-Akt (pAKT), total S6, phosphor-S6 and α-Tubulin (loading control) in PC12 ( G ) or MPC ( H ) cells treated with the indicated drugs for 72h. Shown is one representative immunoblot out of 3 independent experiments. Band quantification was conducted using Image J. Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001. ( I,J ) Realtime qRT-PCR for Ccna and Pcna was conducted on PC12 ( I ) and MPC ( J ) cells with the various drugs/drug combination. RNA was extracted 72h post-treatment. Values were normalized against the DMSO control arbitrarily set to 1. Data are shown as the mean of three experiments ± SD. Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001.

Article Snippet: Buparlisib (PI3Ki) (MedChemExpress) and ribociclib (CDK4/6i) (MedChemExpress) were dissolved in DMSO for in vitro applications and in a mixture of 1/9 v/v 1-methyl-2-pyrrolidone/ PEG300.

Techniques: Control, Activity Assay, Migration, Staining, Expressing, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A genetic signature predicts aggressive paraganglioma sensitivity to dual PI3K-CDK4/6 inhibition therapy

doi: 10.1101/2025.02.18.638668

Figure Lengend Snippet: (A, B) Following spheroid formation, PC12 ( A ) or MPC ( B ) cells were treated with different doses of CDK4/6i, PI3Ki, their combination or DMSO control. Cell viability was assessed 72h later. Reported is the relative cell viability normalized against untreated control (arbitrarily set to 1). Data shown are the mean±SD from 3 independent experiments with 8 technical replicates each. Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001.( C,D ) 3D spheroids of PC12 ( C ) and MPC ( D ) cells were treated with the indicated drugs at the concentration of CDK4/6i: 8 μM; PI3Ki: 2 μM, combination: 8μM CDK4/6i + 2 μM PI3Ki. Spheroid size was measured over time and the values at the 3 Day time point are shown. Data shown are the mean ±SD from 3 independent experiments with 8 technical replicates each. Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001. ( E,F) Primary cells were isolated from fresh MENX-associated PCC fragments (n=7). 3D spheroids were generated and treated with the indicated drugs at the concentrations reported in C . ( E ) Cell viability was measured 72h later and normalized against the initial measurement and the DMSO control. Data shown is the mean ±SD with 5-15 technical replicates each tumor (depending on total amount of cells). Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001. ( F ) Primary cells were isolated from 3 rats, processed and treated as in E . Annexin V signal was measured 30h post-treatment to assess apoptosis. Shown is relative apoptosis normalized to the DMSO control. Data shows the mean ±SD from primary cells of 3 rats with 8 technical replicates each. Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001.( G,H ) Primary cells were isolated from 8 fresh patient-derived PPGL and 3D organotypic cultures generated. Spheroids were treated with CDK4/6i (8μM), PI3Ki (2μM), their combination (CDK4/6i 8μM + PI3Ki 2 μM), or DMSO. Cell viability was measured 72h later. Shown is the relative cell viability normalized to the initial measurement and the DMSO control. In ( G ) the mean ±SD from primary cells of 8 patients with 4-8 technical replicates each (depending on total amount of cells) is shown. Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001. In ( H ) the response of 6 individual patients is illustrated as the mean value.

Article Snippet: Buparlisib (PI3Ki) (MedChemExpress) and ribociclib (CDK4/6i) (MedChemExpress) were dissolved in DMSO for in vitro applications and in a mixture of 1/9 v/v 1-methyl-2-pyrrolidone/ PEG300.

Techniques: Control, Concentration Assay, Isolation, Generated, Derivative Assay

Journal: bioRxiv

Article Title: A genetic signature predicts aggressive paraganglioma sensitivity to dual PI3K-CDK4/6 inhibition therapy

doi: 10.1101/2025.02.18.638668

Figure Lengend Snippet: ( A ) Volcano plots depicting the DGEs for the comparison between PI3Ki-, CDK4/6i-, and combination-treated PC12 cells versus untreated control samples. The number of DGEs is indicated. ( B ) Heatmap showing hierarchal clustering of the DGEs between combination and control samples. ( C ) Venn diagram illustrating the intersection of the downregulated genes in the three treatment groups when compared with control samples. ( D ) Alluvial plot showing the overlap of significantly enriched (p< 0.05; FDR<0.25) upregulated (red) and downregulated (blue) pathways between the PI3Ki and combination groups. The mitotic spindle pathway, enriched exclusively in the combination, is highlighted in red. ( E ) GSEA enrichment plot for the only category exclusively downregulated in combination versus control from Hallmark. ( F ) Bar plot showing the top five enriched processes of downregulated genes unique to the combination on GOBP. In parenthesis is the associated GO term code; below the adjusted p-value is shown. The analysis was conducted using Enrichr. ( G) GSEA plot showing the NES of significantly enriched gene sets in the mitotic spindle processes category (p< 0.05; FDR<0.25) from MSigDB. Each dot represents a gene set and in purple are highlighted the gene lists related to mitotic spindle processes. ( H-I ) Heatmaps showing the gradient of Foxm1 expression ( H ) or Foxm1 activity ( I ) in PC12 cells following the indicated treatments.

Article Snippet: Buparlisib (PI3Ki) (MedChemExpress) and ribociclib (CDK4/6i) (MedChemExpress) were dissolved in DMSO for in vitro applications and in a mixture of 1/9 v/v 1-methyl-2-pyrrolidone/ PEG300.

Techniques: Comparison, Control, Expressing, Activity Assay

Journal: bioRxiv

Article Title: A genetic signature predicts aggressive paraganglioma sensitivity to dual PI3K-CDK4/6 inhibition therapy

doi: 10.1101/2025.02.18.638668

Figure Lengend Snippet: ( A ) PC12 cells were injected in CD-1 foxnu female mice. When tumors reached the size of mm 3 , mice were randomized into the 5 treatment groups, color-coded as in B. Drugs were administered by oral gavage daily for three weeks at the following concentrations: CDK4/6i 75mg/Kg; PI3Ki 25mg/Kg; combination “regular dose”: CDK4/6i 75mg/Kg + PI3Ki 25mg/Kg; combination “low dose”: CDK4/6i 25mg/Kg + PI3Ki 12.5mg/Kg. Tumor volume was measured 2X/week by external caliper. Relative tumor volume compared to the beginning of treatment for mice in each treatment group (n=8/group). Shown is the mean ±SD from tumors of 6-8 mice per group after outlier removal in GraphPad Prism using ROUT method. Statistics:1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001. ( B ) FFPE sections of 4 independent tumorgafts per treatment group were stained with the anti-NuSAP antibody to detect dividing cells. Upon immunofluorescence (20X magnification), the number of positive cells per 10fields of view were counted. Shown is the mean ± SD. Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001. ( C,D ) Automated assessment of necrotic areas in the tumorgrafts. ( C ) Sections of. independent tumorgrafts per treatment group were stained with H&E and scanned with a software to detect necrotic areas. In the exemplary images, necrotic areas are in red. ( D ) Quantification of the necrotic areas in the 5 treatment groups, color-coded as in B . Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001.

Article Snippet: Buparlisib (PI3Ki) (MedChemExpress) and ribociclib (CDK4/6i) (MedChemExpress) were dissolved in DMSO for in vitro applications and in a mixture of 1/9 v/v 1-methyl-2-pyrrolidone/ PEG300.

Techniques: Injection, Staining, Immunofluorescence, Software

Journal: bioRxiv

Article Title: A genetic signature predicts aggressive paraganglioma sensitivity to dual PI3K-CDK4/6 inhibition therapy

doi: 10.1101/2025.02.18.638668

Figure Lengend Snippet: ( A ) Heatmap showing the expression of selected mitotic spindle-related genes in PC12 cells treated with CDK4/6i, PI3Ki, their combination or with DMSO control based on RNASeq. A gradual decrease in expression can be observed, starting from the untreated (control) samples (black), followed by samples treated with CDK4/6i (blue), PI3Ki (green), and the combination (red). ( B ) Selected genes co-expression plot, generated by STRING, illustrates the relationships among genes based on their co-expression scores calculated on Homo sapiens RNA expression patterns, and on protein co-regulation provided by ProteomeHD. The color scale indicates the degree of co-expression. ( C ) Validation of gene expression of Foxm1 and its downstream targets in PC12 cells following drug treatments. Genes from the signature shown in A were amplified by realtime qRT-PCR: Aspm, Cenpf, Cep55, Depdc1, E2F8, Kif14, Ncapg, Nusap1, Troap . RNA was extracted 72h post-treatment. Values were normalized against the DMSO-treated control arbitrarily set to 1. Shown is the mean±SD. Statistics: 1way ANOVA. Only statistically significant comparisons are indicated with lines and asterisks. ( D ) Expression of Mad2l1 by realtime QRT-PCR in samples parallel to C . ( E ) Western blot analysis of MAD2 protein was performed in PC12 cells treated with 1μM PI3Ki, 4μM CDK4/6i, their combination or left untreated for 72h. ( F ) The relative Mad2l1 protein levels from E were quantified as ratios to beta-actin and normalized to the DMSO-treated control. Results represent the average of three independent experiments; bars, SE. Statistics: one way ANOVA. ( G ) Characterization of mitotic spindle defects in PC12 cells following PI3Ki and CDK4/6i treatment in combination. PC12 cells cultured on Poly-L-lysine coated coverslips were either untreated or treated with 25 mg/Kg PI3Ki and 75 mg/Kg CDK4/6i in combination for 24 h. Cells were then fixed, permeabilized and immunostained with an antibody against α-tubulin (Green). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (Blue). Mitotic cells were visualized with Leica TCS SP5 (×63 magnification). Exemplary confocal images are shown. ( H ) Quantification of the number of aberrant mitoses in PC12 cells treated as in G . At least 30 mitotic cells were captured per condition. Statistics: 1way ANOVA. ns, not significant; *, p< 0.05; **, p< 0.01; ***, p<0.001; ****, p< 0.0001.

Article Snippet: Buparlisib (PI3Ki) (MedChemExpress) and ribociclib (CDK4/6i) (MedChemExpress) were dissolved in DMSO for in vitro applications and in a mixture of 1/9 v/v 1-methyl-2-pyrrolidone/ PEG300.

Techniques: Expressing, Control, Generated, RNA Expression, Amplification, Quantitative RT-PCR, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: A genetic signature predicts aggressive paraganglioma sensitivity to dual PI3K-CDK4/6 inhibition therapy

doi: 10.1101/2025.02.18.638668

Figure Lengend Snippet: ( A-B ) Bar plots showing the NES (p<0.05; FDR<0.25) on GSEA Hallmarks in metastatic vs. non-metastatic patients from the CNIO cohort ( A ) or the TCGA cohort ( B ) on GSEA Hallmark. ( C-D ) Dot plot showing the NES of significantly enriched gene sets in metastatic vs. non-metastatic patients from the CNIO cohort ( C ) or the TCGA cohort ( D ) relative to mitotic spindle processes (p<0.05; FDR < 0.05) in GOBP from C5 repository of MSigDB. Each dot represents a gene set and in pink ( C ) or blue ( D ) are highlighted the gene lists related to mitotic spindle processes. ( E-F ) Log2FC expression table ( E ) comparing the expression of mitosis-related genes in combination (CDK4/6i+PI3Ki) treated PC12 cells (relative to control) and metastatic PPGLs from the CNIO cohort (relative to non-metastatic cases). Stacked bar plot ( F) summarizing the expression levels of the selected gene list across different tumor behaviors in PPGL TCGA dataset.

Article Snippet: Buparlisib (PI3Ki) (MedChemExpress) and ribociclib (CDK4/6i) (MedChemExpress) were dissolved in DMSO for in vitro applications and in a mixture of 1/9 v/v 1-methyl-2-pyrrolidone/ PEG300.

Techniques: Expressing, Control

Journal: Frontiers in Oncology

Article Title: RET inhibition overcomes resistance to combined CDK4/6 inhibitor and endocrine therapy in ER+ breast cancer

doi: 10.3389/fonc.2024.1497093

Figure Lengend Snippet: RET is overexpressed in ER+ breast cancer cell lines resistant to combined CDK4/6i and endocrine therapy. Dot plots of Hallmark gene set significantly enriched in (A) MPF-R vs. MF-R and (B) TPF-R vs. TF-R. (C) List of genes enriched in gene set “Hallmark estrogen response early”. (D) Evaluation of RET expression in ER+ breast cancer cell lines resistant to combined palbociclib and fulvestrant (MPF-R and TPF-R), resistant to fulvestrant only (MF-R and TF-R), and parental sensitive cells (M-S and T-S) using RNA sequencing. Statistical comparison is shown relative to double-resistant cells. TPM = transcripts per million. The data represent independent experiments in triplicates ± SEM. (E) Quantitative RT-PCR verifying the gene expression alterations of RET . The expression was normalized using the PUM1 gene and shown as a relative expression in MPF-R vs. M-S and TPF-R vs T-S cells. Data represent three independent experiments ± SEM (*0.01 < p < 0.05). (F) Western blotting analysis of lysates from M-S, MF-R, MPF-R, T-S, TF-R and TPF-R cells. 10 µg and 50 µg of total protein of MCF-7- and T47D-derived cells, respectively, were loaded. β-actin was used as a loading control. A representative for three biological replicates is shown. Asterisk indicate significant differences in students t-test (*0.01 < p < 0.05, **0.001 < p < 0.01 and ***0.0001< p <0.001).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in 96% ethanol, CDK4/6i palbociclib isothiocyanate (HY-A0065, MedChemExpress) was dissolved in water, RET inhibitor selpercatinib (also known as LOXO-292, HY-114370, MedChemExpress) was dissolved in DMSO (Sigma-Aldrich).

Techniques: Expressing, RNA Sequencing Assay, Comparison, Quantitative RT-PCR, Western Blot, Derivative Assay, Control

Journal: Frontiers in Oncology

Article Title: RET inhibition overcomes resistance to combined CDK4/6 inhibitor and endocrine therapy in ER+ breast cancer

doi: 10.3389/fonc.2024.1497093

Figure Lengend Snippet: RET-specific siRNA-mediated knockdown inhibits the growth of MPF-R breast cancer cells. The efficiency of RET silencing in combined CDK4/6i- and fulvestrant-resistant cell lines (MPF-R and TPF-R) and their parental sensitive cell lines (M-S and T-S), respectively, transfected with two different RET -specific siRNAs (RET15 and RET17) or scrambled siRNA (control). (A) RT-qPCR verifying reduction of RET mRNA level 48 h post-transfection with RET -specific siRNA. The expression was normalized using the PUM1 gene. The knockdown efficiency is represented as the average percentage compared to the control (scr) of triplicates (mean ± SEM). (B) Western blot validation of protein levels 96 h post-transfection with RET -specific siRNAs. GAPDH was used as a protein loading control. (C) Cell growth at different time points following RET -specific siRNA transfection as assessed by crystal violet assay. Graph columns show cell growth at days 6 and 10 for MCF7-derived cell lines and T47D-derived cell lines, respectively. Scrambled siRNA: control siRNA; RET15, and RET17: two different RET -specific siRNAs. RET15 + 17: combination of both RET -specific siRNAs. Asterisks indicate significant differences in the one-way ANOVA test (****p < 0.0001).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in 96% ethanol, CDK4/6i palbociclib isothiocyanate (HY-A0065, MedChemExpress) was dissolved in water, RET inhibitor selpercatinib (also known as LOXO-292, HY-114370, MedChemExpress) was dissolved in DMSO (Sigma-Aldrich).

Techniques: Knockdown, Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot, Crystal Violet Assay, Derivative Assay

Journal: Frontiers in Oncology

Article Title: RET inhibition overcomes resistance to combined CDK4/6 inhibitor and endocrine therapy in ER+ breast cancer

doi: 10.3389/fonc.2024.1497093

Figure Lengend Snippet: RET gene knockdown impairs cell growth of combined CDK4/6i- and fulvestrant-resistant breast cancer cells by blocking the G2-M phase progression of the cell cycle. Bar graphs and enrichment plots of Hallmark gene sets significantly enriched in (A) MPF-R RET -siRNA vs. control-siRNA and (B) TPF-R RET -siRNA vs. control-siRNA. RET -siRNA 15 and RET-siRNA 17 pools were used. Statistical significance (nominal P-value) of the enrichment score (ES) is calculated using an empirical phenotype-based permutation test. (C) Western blotting of cell cycle regulators in three biological replicates (BR) of MPF-R RET -siRNA versus control-siRNA. GAPDH was used as a loading control.

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in 96% ethanol, CDK4/6i palbociclib isothiocyanate (HY-A0065, MedChemExpress) was dissolved in water, RET inhibitor selpercatinib (also known as LOXO-292, HY-114370, MedChemExpress) was dissolved in DMSO (Sigma-Aldrich).

Techniques: Knockdown, Blocking Assay, Control, Western Blot

Journal: Frontiers in Oncology

Article Title: RET inhibition overcomes resistance to combined CDK4/6 inhibitor and endocrine therapy in ER+ breast cancer

doi: 10.3389/fonc.2024.1497093

Figure Lengend Snippet: RETi resensitizes combined CDK4/6i- and fulvestrant-resistant ER+ breast cancer cells. Cell growth of (A) MPF-R and M-S cells and (B) TPF-R and T-S cells over six days in the presence of fulvestrant (100nM), CDK4/6i (200 nM) and RETi (5µM) alone or different combinations analyzed by crystal violet assay. Growth at day six is represented by columns. The data represents the mean of three biological replicates ± SEM. Asterisks indicate significant differences in one-way ANOVA tests at day six. Means are compared to the mean of the standard combined CDK4/6i and fulvestrant (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and ****p < 0.0001). (C) Western blotting of cell cycle regulators in MPF-R and TPF-R cells treated with RETi alone or combined with CDK4/6i and/or fulvestrant. GAPDH was used as a loading control.

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in 96% ethanol, CDK4/6i palbociclib isothiocyanate (HY-A0065, MedChemExpress) was dissolved in water, RET inhibitor selpercatinib (also known as LOXO-292, HY-114370, MedChemExpress) was dissolved in DMSO (Sigma-Aldrich).

Techniques: Crystal Violet Assay, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: RET inhibition overcomes resistance to combined CDK4/6 inhibitor and endocrine therapy in ER+ breast cancer

doi: 10.3389/fonc.2024.1497093

Figure Lengend Snippet: Combined CDK4/6i and RETi efficiently reduces viability of breast cancer patient-derived organoids resistant to CDK4/6i. (A) RT-qPCR verifying CDK6 , CDK4 , and CCND1 gene expression alterations upon palbociclib treatment. The expression was normalized using the gene PUM1 and shown as relative expression in control vs. treated with CDK4/6i. (B) Effect of increasing concentrations of CDK4/6i palbociclib and RETi selpercatinib, alone or RETi combined with a fixed concentration of CDK4/6i (1 µM), on the viability of patient-derived breast cancer organoid PDO-P48 for seven days. The results represent the mean ± SEMs of three replicates relative to the control (untreated). (C) Dose-effect curves of each single drug, CDK4/6i and RETi, or RETi combined with a fixed concentration of CDK4/6i (1 µM), during treatment of PDO-P48 for seven days. IC50 were calculated by normalizing the transformed data and using the non-linear curve fitting method “log(inhibitor) vs. normalized response – Variable slope”. (D) Viability of PDO-P48 treated with 10 µM of CDK4/6i and RETi single agents or their combination for seven days. (E) Brightfield images depicting PDO-P48 control (untreated) and treated with combined CDK4/6i and RETi. Scale bars: black 400 µm; blue 100 µm; orange 200 µm.

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in 96% ethanol, CDK4/6i palbociclib isothiocyanate (HY-A0065, MedChemExpress) was dissolved in water, RET inhibitor selpercatinib (also known as LOXO-292, HY-114370, MedChemExpress) was dissolved in DMSO (Sigma-Aldrich).

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Control, Concentration Assay, Transformation Assay

Journal: Frontiers in Oncology

Article Title: RET inhibition overcomes resistance to combined CDK4/6 inhibitor and endocrine therapy in ER+ breast cancer

doi: 10.3389/fonc.2024.1497093

Figure Lengend Snippet: High RET expression correlates with shorter overall and relapse-free survival in patients with ER+ breast cancer receiving endocrine therapy. Kaplan-Meier survival curves for (A) OS and (B) RFS for RET expression by KM plotter analysis. (C, D) Kaplan-Meier survival curves evaluating PFS according to RET intensity score in ER+ metastatic lesions from patients treated with combined CDK4/6i and endocrine therapy. (D) Cut-off values: negative RET: intensity = 0; positive RET: intensity ≥ 1. A two-sided p-value calculated using Log-rank testing is shown.

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in 96% ethanol, CDK4/6i palbociclib isothiocyanate (HY-A0065, MedChemExpress) was dissolved in water, RET inhibitor selpercatinib (also known as LOXO-292, HY-114370, MedChemExpress) was dissolved in DMSO (Sigma-Aldrich).

Techniques: Expressing