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10780432ccr150534-sup-146253_2_supp_3138484_nrfrry.pptx (348.87 kB)

Supplementary Figs from MAPK Activation Predicts Poor Outcome and the MEK Inhibitor, Selumetinib, Reverses Antiestrogen Resistance in ER-Positive High-Grade Serous Ovarian Cancer

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posted on 2023-03-31, 19:08 authored by Karina E. Hew, Philip C. Miller, Dorraya El-Ashry, Jun Sun, Alexandra H. Besser, Tan A. Ince, Mengnan Gu, Zhi Wei, Gao Zhang, Patricia Brafford, Wei Gao, Yiling Lu, Gordon B. Mills, Joyce M. Slingerland, Fiona Simpkins

Supplementary Figure 1. (A) Percent of HGSOCs from the TCGA cohort with mutations in 42 genes associated with the RAS/Raf/MEK/MAPK pathway (N=45/311); (B) Percent of HGSOCs from the TCGA cohort with copy number amplifications or mRNA overexpression of 42 MAPK pathway genes (N=232/311) Supplementary Figure 2. (A); Percent of high-pMAPK and low-pMAPK HGSOCs as defined by RPPA from the TCGA cohort with (A) copy number amplification (B) mRNA overexpression, (C) mutation in which MAPK pathway genes are genetically activated in hMAPK cancers. The numbers with data available for each analysis in TCGA are shown below graphs. Supplementary Figure 3. Dose dependent effects of selumetinib on ER positive, MAPK kinase activated ovarian cancer cells Supplementary Figure 4. In vitro cell cycle effects of MEK and ER inhibition on ER+ BG-1 OVCA cells Supplementary Figure 5: Gene expression changes expressed as log fold change over baseline control untreated cell values after 48 hours of treatment with fulvestrant (1 μM), selumetinib (200 nM) or both, in vitro of representative genes in (A) ER target gene expression (B) Cell signaling pathways (C) Cell cycle pathways. Supplementary Figure 6. Heat map representation of selected triplicate repeat RPPA data from PEO1-R cells treated with either monotherapy fulvestrant (FULV), selumetinib (MEK I) or dual therapy as indicated. Heat map demonstrates inter-sample variability in triplicate repeat assays. Supplementary Table 1. List of genes in the 126 gene MAPK activation profile common to the TCGA and JAPAN cohorts. Supplementary Table 2. Differences in gene mutation, overexpression and amplification between low and high pMAPK HGSOC as defined by RPPA in TCGA cohort Supplementary Table 3.Analysis of xenograft growth rates reveal synergy between fulvestrant and selumetinib therapy on OVCA PEOIR xenograft growth

Funding

Gynecological Cancer Foundation Mary-Jane Welker Ovarian Cancer Research

History

ARTICLE ABSTRACT

Purpose: Although 67% of high-grade serous ovarian cancers (HGSOC) express the estrogen receptor (ER), most fail antiestrogen therapy. Because MAPK activation is frequent in ovarian cancer, we investigated if estrogen regulates MAPK and if MEK inhibition (MEKi) reverses antiestrogen resistance.Experimental Design: Effects of MEKi (selumetinib), antiestrogen (fulvestrant), or both were assayed in ER-positive HGSOC in vitro and in xenografts. Response biomarkers were investigated by gene expression microarray and reverse phase protein array (RPPA). Genes differentially expressed in two independent primary HGSOC datasets with high versus low pMAPK by RPPA were used to generate a “MAPK-activated gene signature.” Gene signature components that were reversed by MEKi were then identified.Results: High intratumor pMAPK independently predicts decreased survival (HR, 1.7; CI > 95%,1.3–2.2; P = 0.0009) in 408 HGSOC from The Cancer Genome Atlas. A differentially expressed “MAPK-activated” gene subset was also prognostic. “MAPK-activated genes” in HGSOC differ from those in breast cancer. Combined MEK and ER blockade showed greater antitumor effects in xenografts than monotherapy. Gene set enrichment analysis and RPPA showed that dual therapy downregulated DNA replication and cell-cycle drivers, and upregulated lysosomal gene sets. Selumetinib reversed expression of a subset of “MAPK-activated genes” in vitro and/or in xenografts. Three of these genes were prognostic for poor survival (P = 0.000265) and warrant testing as a signature predictive of MEKi response.Conclusions: High pMAPK is independently prognostic and may underlie antiestrogen failure. Data support further evaluation of fulvestrant and selumetinib in ER-positive HGSOC. The MAPK-activated HGSOC signature may help identify MEK inhibitor responsive tumors. Clin Cancer Res; 22(4); 935–47. ©2015 AACR.