Supplementary Figures S1-S9 from Resistance to CDK2 Inhibitors Is Associated with Selection of Polyploid Cells in <i>CCNE1</i>-Amplified Ovarian Cancer
Authors: Etemadmoghadam Dariush , Au-Yeung George , Wall Meaghan , Mitchell Chris , Kansara Maya , Loehrer Elizabeth , Batzios Crisoula , George Joshy , Ftouni Sarah , Weir Barbara A. , Carter Scott , Gresshoff Irma , Mileshkin Linda , Rischin Danny , Hahn William C. , Waring Paul M. , Getz Gad , Cullinane Carleen , Campbell Lynda J. , Bowtell David D. 
Published: April 2023

<p>Supplementary Figures S1-S9 - PDF file 327K, Supplementary Figure S1: A) Baseline expression of CCNE1 and CDK2 in OVCAR-3 (CCNE1 amplified) and OVCAR-4 (CCNE1 gained) cell lines compared to SK-OV-3 (CCNE1 unamplified). *detects both isoforms of CDK2. B) CCNE1 and CDK2 gene expression by RT-PCR after gene knockdown normalized to no siRNA control cells within each cell line. Representative data shown. *detects both isoforms of CDK2. C) Cell viability normalized to no siRNA control cells after transfection with siRNAs against CCNE1 or CDK2. Statistical significance calculated by comparison to non-silencing (NS) siRNA in the same cell line using data from three independent MTS assays performed in triplicate. Average normalized absorbance at 490 nm and SEM plotted (n = 3), T-test *p-value <0.05, ***p-value <0.01 and ***p-value <0.001; Supplementary Figure S2: A) Relative abundance of shRNAs targeting CCNE1 and CDK2 in shRNA screen of 102 tumor cell lines and B) average normalized abundance of CCNE1 shRNA3 in tumor cell lines stratified by CCNE1 copy number status; Supplementary Figure S3: A) PHA-533533 and B) dinaciclib 72 hour MTS proliferation assay dose-response and average IC50 values. Curves show average absorbance normalized to untreated control cells. Error bars are SEM (n=3). T-test **p-value <0.01, ***p-value <0.001; Supplementary Figure S4: A) Clonogenic survival dose response to PHA-533533 in OVCAR-3 parental and R1 cell line. Mean of replicates and SEM from one representative experiment shown. B) MTS dose response to dinaciclib in OVCAR-3 parental and dinacicilib resistant (RD1) cell line. Mean of replicates and SEM shown. C) IC50 values for Dinaciclib-RD1 over time. Cells were cultured in the presence (solid line) or absence of dinaciclib (dashed line). The IC50 value for parental cells is indicated by the dotted line. D) Proliferation rate of parental and two resistant cell lines (533533-R1 and -R6); Supplementary Figure S5: A) Cycle profile of PI stained cells analyzed by flow cytometry after 48 hr treatment with PHA-533533; Supplementary Figure S6: A) Baseline protein expression in proliferating parental and PHA-533533 resistant cell lines (R1, R3, R5, R6, R7) by western blot. B) Gene expression of targets of the PHA-533533 small molecule inhibitor in parental compared to resistant cell lines (n = 4) after drug selection (selected) and subsequent passage in media (stable); Supplementary Figure S7: A) Cell cycle profile of PI stained cells analyzed by flow cytometry. PHA-53353 resistant cell lines (R3, R5 and R7) and B) dinaciclib resistant line (RD1) are comprised of a hyperpentaploid cell population. G1 and G2 peaks (BG1 and BG2) are labeled; Supplementary Figure S8: A) Proportion of hypotriploid and hyperpentaploid cells in the parental, 533533-R1 and -R6 resistant cell lines estimated by counting 50 metaphase karyotypes. Additional representative hypotriploid and hyperpentaploid karyotypes of B) OVCAR-3 parental, C) 533533-R1 (hyperpentaploid only) and D) 533533-R6 cells; Supplementary Figure S9: A) DNA ploidy estimated by ABSOLUTE in TCGA ovarian primary tumors stratified by CCNE1 log2 copy number ratio; >0.3 for gains and >0.8 for amplifications. Fraction of genome lost (log2 copy number ratio < -0.3) in ovarian tumor samples by B) number of predicted whole genome doublings and C) CCNE1 copy number status. T-test ***p-value < 0.001</p>