Additional file 2 of MDM2 inhibition in combination with endocrine therapy and CDK4/6 inhibition for the treatment of ER-positive breast cancer

Additional file 2: Fig. S1 MDM2 inhibition activates p53 and reduces tumour proliferation in vitro and in vivo. A. Full gel and blot scans for the Western blots shown in Fig. 1b. Total protein was visualised using BioRad stain-free imaging technology according to the manufacturer’s instructions. B. Analysis of cell cycle phase using flow cytometry to quantify propidium iodide staining of genomic DNA shows significant alterations to cell cycle phase distribution in p53wt models consistent with arrest in both G1 and G2 after incubation for 48 hours with 1μM NVP-CGM097. Red = G1 (bottom), blue = S (middle), green = G2/M (top). Statistical significance from χ2 test using the vehicle treated profile as the expected value is indicated. C. NVP-CGM097 (50mg/kg daily, red) significantly inhibited tumour volumes compared to vehicle (2% DMSO daily, green) at endpoint. Final tumour volumes were compared using two-tailed T test to determine significance. D. Representative images of Ki-67 quantification of endpoint tumours in Qupath software showing the classification of different tissue compartments: tumour (red and blue), stroma (green), and necrosis (black); and detection of Ki-67 negative and positive tumour cells. A single classifier was applied to all tumour sections. Fig. S2. NVP-CGM097 treatment causes gene expression changes in cell cycle and p53 pathways in vitro. A. Multidimensional scaling (MDS) plot showing the level of sample similarity between MCF-7 cell lines treated with vehicle, NVP-CGM097, fulvestrant and combination therapy (NVP-CGM097 plus fulvestrant). B. Venn diagram showing the overlap between differentially expressed genes (adjusted p-value < 0.05- and 2-fold change) induced by treatment in MCF-7 cell lines. C. KEGG pathway analysis using RNA-Seq transcriptomic data shows a significant negative enrichment of Cell Cycle regulation in MCF-7 cell lines following 48 hours of treatment with 1μM NVP-CGM097 and positive enrichment of p53 Signalling Pathway. D. KEGG enrichment analysis showing p53 Signalling Pathway activation in MCF-7 cell lines following treatment with NVP-CGM097. Fig. S3 Enrichment analysis of significant differentially expressed genes against curated gene sets. Fig. S4 MDM2 inhibition induces changes in gene and protein expression associated with the cell cycle in endocrine resistant models in vitro and in vivo. A. Incucyte analysis of cell numbers of parental MCF-7 cells (blue) and MCF-7 FasR cells (red) over time after treatment with 100 nM fulvestrant. Vehicle treated parental MCF-7 cell growth (grey) is shown for comparison. B. Full gel images for the Western blots shown in Fig. 4c. C. Quantification of ER staining in the nucleus and cytoplasm of three independent replicates per arm from PDX model Gar15-13 following 10 days of treatment with vehicle (2% DMSO daily, green), NVP-CGM097 (50mg/kg daily, red), fulvestrant (5 mg weekly, blue) or the combination of fulvestrant and NVP-CGM097 (purple). Tumours treated with fulvestrant alone or in combination had significantly reduced ER staining in both cellular compartments compared to tumours not treated with fulvestrant. Tumour cell identification and ER quantification were performed in QuPath software. D. Heat map of significant downregulated genes from the E2F Targets and G2/M Checkpoint gene sets in the Gar1513 PDX model. E. Normalised log2 expression levels of significant p53 target genes in in the Gar1513 PDX model induced by NVP-CGM097 (red), fulvestrant (blue) and the combination of NVP-CGM097 and fulvestrant (purple), averaged by condition, associated with cell cycle, apoptosis and senescence. Fig. S5. MDM2 inhibition in combination with CDK4/6 inhibition synergistically downregulates cell cycle markers in vitro. A. MDS plot of MCF-7 cell lines treated for 48 h with Vehicle (0.01% DMSO), 1 μM NVP-CGM097, 500 nM palbociclib, or the combination of NVP-CGM097 and palbociclib. B. Venn diagram showing the overlap between differentially expressed genes (adjusted p-value