Abstract PD2-02: Activation of the EGFR/RAS/p42,44 MAPK axis as a convergent mechanism of resistance to CDK4/6 inhibitors in ER+ breast cancer
Authors: De Angelis Carmine , Cataldo Maria Letizia , Nardone Agostina , Veeraraghavan Jamunarani , Fu Xiaoyong , Nanda Sarmistha , Qin Lanfang , Sethunath Vidyalakshmi , Pereira Resel , Weigelt Britta , Reis-Filho Jorge S. , Rimawi Mothaffar F. , Jeselsohn Rinath , Osborne Kent , Schiff Rachel 
Published: March 2020

Abstract
Background: In the PALOMA-3 trial, the combination of the CDK4/6 inhibitor palbociclib (P) with fulvestrant (F) led to a significant improvement in clinical outcomes, compared to F+Placebo, in endocrine-pretreated patients (pts) with ER+/HER2- metastatic breast cancer (BC). Not all pts, however, benefit from F+P, and virtually all ultimately progress. Elucidating the resistance mechanisms would assist in identifying biomarkers to select pts who may most benefit from F+P, as well as novel therapeutic targets for combating resistance. Materials and Methods: The ER+ MCF7 BC cell lines made resistant (R) to long-term estrogen deprivation (EDR) or tamoxifen (TamR), and the 600MPE de novo TamR cells were tested for sensitivity to F, P and F+P. MCF7 EDR and MCF7 TamR cells were exposed to increasing concentrations of F+P to generate R derivatives (FPR, cells grow in the presence of F 100nM + P 100nM). The transcriptomic and proteomic profiles of MCF7 EDR, TamR, EDR/FPR and TamR/FPR were determined by RNA-seq and reverse-phase protein arrays (RPPA), respectively. Pathway analysis was performed by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). mRNA and protein levels of select markers were assessed by RT-PCR and Western blot (WB), respectively. Cell growth, colony formation, and migratory and invasive potential were assessed by methylene blue staining, clonogenic assay, and in vitro migration and invasion assays. Results: Treatment of MCF7 EDR and TamR cells with P resulted in a dose-dependent inhibition of cell growth [IC50 = 149nM and 89nM, respectively] and colony formation [clonogenic inhibition at 100nM &gt;85% and &gt;95%, respectively]. In both MCF7 endocrine-R models, P (100nM) or F (100nM) monotherapy significantly inhibited cell growth and reduced phosphorylated (p)-Rb and the E2F target CDK2 protein levels compared to vehicle control. However, F+P resulted in an enhanced cell growth inhibition (&gt;90%) and a more pronounced reduction in p-Rb and CDK2 expression compared to each agent alone. In the FPR lines, ‘oncogenic signatures’ representative of altered KRAS and MEK signaling were highly enriched compared to their sensitive counterparts, as revealed by GSEA analysis of the transcriptomic profiles. IPA analysis of the proteomic profiles confirmed the activation of the MAPK signaling pathway in FPR cells and identified EGFR as one of the top ranked upstream regulators. Increased EGFR mRNA and protein levels in FPR models were confirmed by RT-PCR and WB analysis. Additionally, gene signatures of cyclin D1 hyperactivation and increased cyclin D1 protein levels were enriched in FPR cell lines. Importantly, the EGFR inhibitor gefitinib (1µM), the pan-HER inhibitor neratinib (250nM), and the MEK1 inhibitor selumetinib (S) (1µM) selectively or more greatly inhibited the cell growth and reduced Cyclin D1 protein levels in FPR cells compared to their sensitive counterparts. Further, we found a significant enrichment of the ‘hallmark’ epithelial-mesenchymal transition gene signature in FPR models, which is in line with their increased migratory and invasive capabilities. Treatment with S reduced the migration and invasion of the FPR cells. Finally, the aggressive 600MPE de novo TamR cells harboring hyperactive MEK/MAPK signaling due to the KRAS and MAP2K4 activating mutations were significantly less sensitive to F, P, and F+P. However, the addition of S to F+P significantly increased the cell growth inhibition in this model. Conclusion: Acquired and intrinsic resistance to F+P is associated with hyperactivation of the EGFR/RAS/MEK signaling pathway, which sustains cell proliferation and promotes an aggressive phenotype. The EGFR/RAS/MEK pathway may serve as a predictive biomarker for F+P resistance and offer therapeutic targets to overcome resistance in pts with ER+/HER2- BC.
Citation Format: Carmine De Angelis, Maria Letizia Cataldo, Agostina Nardone, Jamunarani Veeraraghavan, Xiaoyong Fu, Sarmistha Nanda, Lanfang Qin, Vidyalakshmi Sethunath, Resel Pereira, Britta Weigelt, Jorge S. Reis-Filho, Mothaffar F. Rimawi, Rinath Jeselsohn, Kent Osborne, Rachel Schiff. Activation of the EGFR/RAS/p42,44 MAPK axis as a convergent mechanism of resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-02.