Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Combined CDK4/6 and ERK1/2 inhibition enhances anti-tumor activity in NF1-associated plexiform neurofibroma

doi: 10.1158/1078-0432.CCR-22-2854

Figure Lengend Snippet: (A) Venn diagram depicting numbers of significantly upregulated differentially expressed genes (DEGs) common between abemaciclib (CDK4/6i), LY3214996 (ERK1/2i), and the combination (Combo) treatment groups compared to the vehicle (Veh) control. The thresholds for the DEGs were defined as log2 fold change ≥ 1 and p-value ≤ 0.05. (B) Venn diagram depicting numbers of significantly downregulated differentially expressed genes (DEGs) common between the abemaciclib, LY3214996, and combination (Combo) treatment groups compared to the vehicle (Veh) control. The thresholds for the DEGs were defined as log2 fold change ≤ −1 and p-value ≤ 0.05. (C) Volcano plot showing log2 fold-change in gene expression for PNF from Nf1flox/flox;PostnCre mice treated with abemaciclib (n=6) vs Vehicle (n=6) plotted against the -log10 p-value (-log10(P)). The dashed horizontal line denotes -log10(P=0.05). The dashed vertical lines denote log2 fold-changes of −1 and 1. Significantly upregulated genes above these thresholds are denoted in red, while significantly downregulated genes are denoted in green. (D) Volcano plot showing log2 fold-change in gene expression for PNF from Nf1flox/flox;PostnCre mice treated with LY3214996 (n=6) vs Vehicle (n=6) plotted against the -log10 p-value (-log10(P)). The dashed horizontal line denotes -log10(P=0.05). The dashed vertical lines denote log2 fold-changes of −1 and 1. Significantly upregulated genes above these thresholds are denoted in red, while significantly downregulated genes are denoted in green. (E) Volcano plot showing log2 fold-change in gene expression for PNF from Nf1flox/flox;PostnCre mice treated with LY3214996 plus abemaciclib combination (n=6) vs Vehicle (n=6) plotted against the -log10 p-value (-log10(P)). The dashed horizontal line denotes -log10(P=0.05). The dashed vertical lines denote log2 fold-changes of −1 and 1. Significantly upregulated genes above these thresholds are denoted in red, while significantly downregulated genes are denoted in green. (F) Box and whisker plots showing Mki67 mRNA log2 counts in PNF from Nf1flox/flox;PostnCre mice treated with vehicle (n=6), abemaciclib (n=6), LY3214996 (n=6), and combination therapy (Combo; n=6). Whiskers extend from the minima to maxima. The center line represents the median. The box spans the 25th to 75th percentiles. Asterisks indicate statistical significance of treatment vs vehicle according to the Wald test performed by DESeq2 (*** = P ≤ 0.001). Non-significant comparisons are not shown. (G) Box and whisker plots showing Ccnb1 mRNA log2 counts in PNF from Nf1flox/flox;PostnCre mice treated with vehicle (n=6), abemaciclib (n=6), LY3214996 (n=6), and combination therapy (n=6). Whiskers extend from the minima to maxima. The center line represents the median. The box spans the 25th to 75th percentiles. Asterisks indicate statistical significance of treatment vs vehicle according to the Wald test performed by DESeq2 (*** = P ≤ 0.001). Non-significant comparisons are not shown.

Article Snippet: IHC of human CDK4 (#BS-0633R, Bioss, RRID:AB_10856091, 1:400 dilution), murine and human phospho-Rb (S807/811) (#CST-8516, Cell Signaling Technology, RRID:AB_11178658, 1:100 dilution), murine CSF1R (#CST-3152, Cell Signaling Technology, RRID:AB_2085233, 1:100 dilution), and murine Ki67 (#ab16667, Abcam, RRID:AB_302459, 1:200 dilution) was performed in house.

Techniques: Control, Expressing, Whisker Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Combined CDK4/6 and ERK1/2 inhibition enhances anti-tumor activity in NF1-associated plexiform neurofibroma

doi: 10.1158/1078-0432.CCR-22-2854

Figure Lengend Snippet: (A) Principal component analysis (PCA) identifies variation in the functional kinome of PNF bearing sciatic nerve tissue from Nf1flox/flox;PostnCre mice (Plexiform; n=4) vs WT control (Normal; n=3). (B) Volcano plot showing the mean log2 fold-change in MIB binding (LFQ intensity) for kinases in PNF bearing sciatic nerve tissue from Nf1flox/flox;PostnCre mice (Plexiform; n=4) vs WT control (Normal; n=3) plotted against the -log10 Benjamini-Hochberg adjusted p-value. The horizontal dashed line denotes a false discovery rate (FDR) of 0.05. Kinases with significantly increased MIB binding (log2 fold-change ≥1 and -log10 adjusted p-value above the FDR threshold) are denoted in red. Kinases with significantly decreased MIB binding (log2 fold-change ≤ −1 and -log10 adjusted p-value above the FDR threshold) are denoted in green. Several of the top kinases with significantly increased or decreased binding in tumor tissue relative to the control are annotated on the plot. (C) Box and whisker plots demonstrating the mean log2 fold-change in MIB binding (LFQ intensity) of the top 20 kinases with increased binding in PNF bearing sciatic nerve tissue from Nf1flox/flox;PostnCre mice (Plexiform; n=4) vs WT control (Normal; n=3). The box color indicates the family to which each kinase belongs as depicted in the legend. (D) “Tree” plot comparing the functionally enriched kinome of PNF bearing sciatic nerve tissue from Nf1flox/flox;PostnCre mice (Plexiform; n=4) vs WT control (Normal; n=3). The log2 fold change in MIB binding is encoded by node color with red denoting kinases that are increased in abundance and blue denoting kinases that are decreased in abundance. The size of each node is proportional to the –log10 Benjamini-Hochberg adjusted p-value as denoted in the figure legend. Kinases with significantly increased or decreased MIB binding in PNF relative to the WT control are annotated alongside their respective kinase families in bold font. (E) Scatter plot showing log2 fold-change in gene expression of kinases (by RNAseq) vs log2 fold-change MIB binding (LFQ intensity) for PNF bearing nerve tissue in Nf1flox/flox;PostnCre mice (Plexiform; n=4 for MIB binding, n=6 for RNAseq) vs WT control nerve tissue (Normal; n=3 for MIB binding, n=6 for RNAseq). Top kinases with increase in MIB binding and RNAseq expression are highlighted in red and labeled. (F) CDK6, CDK4, and GAPDH (loading control) were detected independently by western blot in PNF bearing nerve tissue from Nf1flox/flox;PostnCre mice (Plexiform; n=2) and WT control nerve tissue (Normal; n=2). Bar graph shows arbitrary densitometry units (ADUs) calculated using KwikQuant Image Analyzer for each band normalized to the loading control. Error bars reflect standard error of the mean (SEM). Asterisks indicate statistical significance according to an unpaired, one-sided t-test (* = P ≤ 0.05). (G) Representative photomicrographs of CDK6 immunohistochemistry-stained sections of normal nerve and human NF1-associated PNF. Corresponding 100μm scale bars denote the magnification with inset high-power magnification showing cellular localization. Bar graph shows the percentage of CDK6 positive cells quantified by the HALO Cytonuclear analysis algorithm (Indica Labs). n=11 Normal and n=6 Plexiform samples were analyzed with n=11 and n=24 fields respectively. Error bars reflect standard error of the mean (SEM). Asterisks indicate statistical significance according to an unpaired, two-sided t-test (**** = P ≤ 0.0001). (H) Representative photomicrographs of CDK4 IHC stained sections of normal nerve and human NF1-associated PNF. Corresponding 100μm scale bars denote the magnification with inset high-power magnification showing cellular localization. Bar graph shows the percentage of CDK4 positive cells quantified by manual counting. n=8 Normal and n=6 Plexiform samples were analyzed with n=24 and n=36 fields respectively. Error bars reflect standard error of the mean (SEM). Asterisks indicate statistical significance according to an unpaired, two-sided t-test (** = P ≤ 0.01) (I) Representative photomicrographs of phospho-Rb (S807/811) IHC stained sections of normal nerve and human NF1-associated PNF. Corresponding 100μm scale bars denote the magnification with inset high-power magnification showing nuclear localization. Bar graph shows percentage of phospho-Rb (S807/811) positive nuclei quantified by the HALO Cytonuclear analysis algorithm (Indica Labs). n=14 Normal and n=5 Plexiform samples were analyzed with n=14 and n=30 fields respectively. Error bars reflect standard error of the mean (SEM). Asterisks indicate statistical significance according to an unpaired, two-sided t-test (**** = P ≤ 0.0001).

Article Snippet: IHC of human CDK4 (#BS-0633R, Bioss, RRID:AB_10856091, 1:400 dilution), murine and human phospho-Rb (S807/811) (#CST-8516, Cell Signaling Technology, RRID:AB_11178658, 1:100 dilution), murine CSF1R (#CST-3152, Cell Signaling Technology, RRID:AB_2085233, 1:100 dilution), and murine Ki67 (#ab16667, Abcam, RRID:AB_302459, 1:200 dilution) was performed in house.

Techniques: Functional Assay, Control, Binding Assay, Whisker Assay, Expressing, Labeling, Western Blot, Immunohistochemistry, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Combined CDK4/6 and ERK1/2 inhibition enhances anti-tumor activity in NF1-associated plexiform neurofibroma

doi: 10.1158/1078-0432.CCR-22-2854

Figure Lengend Snippet: (A) Hierarchically-clustered heatmap of MAPK Pathway Activity Score (MPAS) signature genes, curated by Wagle et al., in PNF from Nf1flox/flox;PostnCre mice treated with vehicle (n=6), abemaciclib (n=6), LY3214996 (n=6), or combination therapy (Combo; n=6). 1-Pearson correlation was used to obtain a distance matrix with complete clustering. Gene expression values (rows) were Z-score normalized. (B) Box and whisker plots showing the Wagle et al. MAPK Pathway Activity Score (MPAS) in PNF from Nf1flox/flox;PostnCre mice treated with vehicle (n=6), abemaciclib (n=6), LY3214996 (n=6), or combination therapy (Combo; n=6). Whiskers extend from the minima to maxima. The center line represents the median. The box spans the 25th to 75th percentiles. Asterisks indicate statistical significance according to the Wilcoxon test (** = P ≤ 0.01). Non-significant comparisons are not shown. (C) Hierarchically-clustered heatmap of MEK activation score signature genes, curated by Dry et al., in PNF from Nf1flox/flox;PostnCre mice treated with vehicle (n=6), abemacicilb (n=6), LY3214996 (n=6), or combination therapy (Combo; n=6). 1-Pearson correlation was used to obtain a distance matrix with complete clustering. Gene expression values (rows) were Z-score normalized. (D) Box and whisker plots showing the Dry et al. MEK activation score in PNF from Nf1flox/flox;PostnCre mice treated with vehicle (n=6), abemaciclib (n=6), LY3214996 (n=6), or combination therapy (n=6). Whiskers extend from the minima to maxima. The center line represents the median. The box spans the 25th to 75th percentiles. Asterisks indicate statistical significance according to the Wilcoxon test (* = P ≤ 0.05; ** = P ≤ 0.01). Non-significant comparisons are not shown. (E) Hierarchically-clustered heatmap of ERK dependent signature genes, curated by Pratilas et al., in PNF from Nf1flox/flox;PostnCre mice treated with vehicle (n=6), abemacicilb (n=6), LY3214996 (n=6), or combination therapy (n=6). 1-Pearson correlation was used to obtain a distance matrix with complete clustering. Gene expression values (rows) were Z-score normalized. (F) Box and whisker plots showing the Pratilas et al. ERK dependent signature score in PNF from Nf1flox/flox;PostnCre mice treated with vehicle (n=6), abemaciclib (n=6), LY3214996 (n=6), or combination therapy (n=6). Whiskers extend from the minima to maxima. The center line represents the median. The box spans the 25th to 75th percentiles. Asterisks indicate statistical significance according to the Wilcoxon test (** = P ≤ 0.01). Non-significant comparisons are not shown. (G) ETV4, DUSP6, and Vinculin (loading control) were detected by Western blot in PNF bearing trigeminal nerve tissue from Nf1flox/flox;PostnCre mice treated with vehicle (n=7), abemaciclib (n=6), LY3214996 (n=6), or the combination (n=6) for 7 days. Bar graphs show quantification of arbitrary densitometry units (ADUs) calculated using ImageJ and normalized to loading control and average of vehicle. Analysis includes data generated from two independent animal experiments with western blots performed in triplicate. An additional blot shown in Supplemental Figure S10D demonstrates ETV4 and DUSP6 expression in the sciatic nerve of animals represented in Figure 6G. Asterisks indicate statistical significance for the comparisons according to Dunnett’s multiple comparisons test (* = P ≤ 0.05; ** = P ≤ 0.01) and error bars reflect standard error of the mean (SEM). (H) Schematic depicting molecular synergism of combined ERK1/2 and CDK4/6 pathway inhibition to reduce RAS/MAPK dependent DUSP6 and ETV4 transcriptional output in PNF.

Article Snippet: IHC of human CDK4 (#BS-0633R, Bioss, RRID:AB_10856091, 1:400 dilution), murine and human phospho-Rb (S807/811) (#CST-8516, Cell Signaling Technology, RRID:AB_11178658, 1:100 dilution), murine CSF1R (#CST-3152, Cell Signaling Technology, RRID:AB_2085233, 1:100 dilution), and murine Ki67 (#ab16667, Abcam, RRID:AB_302459, 1:200 dilution) was performed in house.

Techniques: Activity Assay, Expressing, Whisker Assay, Activation Assay, Control, Western Blot, Generated, Inhibition

Journal: Oncology reports

Article Title: Platycodin D, a metabolite of Platycodin grandiflorum, inhibits highly metastatic MDA-MB-231 breast cancer growth in vitro and in vivo by targeting the MDM2 oncogene.

doi: 10.3892/or.2016.4935

Figure Lengend Snippet: Figure 3. PD induced cell cycle arrest in the G0/G1 phase in MDA-MB-231 cells. (A) Cells were exposed to 2.5, 5, 10 or 20 µM of PD for 48 h and the cell cycle progression was assessed by flow cytometry. The 5, 10 and 20 µM concentrations of PD induced significant cell cycle arrest in the G0/G1 phase. (B and C) The expression levels of G0/G1 phase-related proteins was analyzed by western blotting after treatment with various concentrations of PD for 24 h. PD down regulated the expression of CDK2, CDK4, CDK6, and Cyclin E. (*p<0.05).

Article Snippet: The antibodies against human CDK2, CDK4, CDK6, and Cyclin E used in the western blot analyses were obtained from Boster Biotechnology (Wuhan, China).

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Oncology reports

Article Title: Platycodin D, a metabolite of Platycodin grandiflorum, inhibits highly metastatic MDA-MB-231 breast cancer growth in vitro and in vivo by targeting the MDM2 oncogene.

doi: 10.3892/or.2016.4935

Figure Lengend Snippet: Figure 9. The effects of PD on the expression levels of various proteins in MDA-MB-231 xenograft tumors. Western blotting was performed to assess the expression levels of proteins in the MDM2-MDMX-p53 pathway, down- stream proteins and G0/G1 phase-related proteins in mice after treatment with the three concentrations of PD for four weeks. (A) PD downregulated the expression of the MDM2, MDMX, and mutant p53 proteins. (B) PD upregulated the expression of p21 and p27. (C) PD decreased the expression of G0/G1 phase-related proteins (CDK2, CDK4, CDK6 and Cyclin E).

Article Snippet: The antibodies against human CDK2, CDK4, CDK6, and Cyclin E used in the western blot analyses were obtained from Boster Biotechnology (Wuhan, China).

Techniques: Expressing, Western Blot, Mutagenesis

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: Rabbit polyclonal anti phospho-CDK4 (Thr172) , St John’s Laboratory , Cat#STJ29359; RRID:AB_2928079.

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