Abstract GS2-01: High levels of interferon-response gene signatures are associated with de novo and acquired resistance to CDK4/6 inhibitors in ER+ breast cancer
Authors: De Angelis Carmine , Fu Xiaoyong , Cataldo Maria Letizia , Nardone Agostina , Jansen Valerie M. , Veeraraghavan Jamunarani , Nanda Sarmistha , Qin Lanfang , Sethunath Vidyalakshmi , Pereira Resel , Benelli Matteo , Migliaccio Ilenia , Malorni Luca , Donaldson Joshua , Selenica Pier , Brown David N. , Weigelt Britta , Reis-Filho Jorge S. , Park Ben H. , Hurvitz Sara A. , Slamon Dennis J. , Rimawi Mothaffar F. , Jeselsohn Rinath , Osborne Kent , Schiff Rachel 
Published: March 2020

Abstract
Background: The CDK4/6 inhibitors (i) palbociclib (Palbo), ribociclib, and abemaciclib remarkably improved the outcome of patients with metastatic ER+/HER2- breast cancer (BC) and are now under clinical investigation in early BC. Despite high efficacy, de novo and acquired resistance to CDK4/6i is common. Elucidating the molecular basis for sensitivity and resistance to CDK4/6i is crucial to identify predictive biomarkers and novel therapeutic targets to improve patient outcome. Materials and Methods: MCF7, T47D and ZR75-1 parental (P) BC cells and their derivatives made resistant to tamoxifen, estrogen deprivation (EDR), or fulvestrant were used. The P and EDR models of MCF7 and T47D cells were chronically exposed to increasing concentrations of Palbo to generate derivatives with acquired resistance to Palbo (PalboR). The transcriptomic profiles of P, endocrine-resistant (EndoR) and PalboR models were determined by RNA-seq. IC50s were determined by exposing MCF7, T47D, and ZR75-1 P and EndoR lines (n=12) to increasing concentrations of Palbo. Cell growth was assessed by methylene blue staining, and changes in the mRNA and protein levels of key cell cycle molecules were assessed by RT-PCR and Western blot, respectively. Gene expression data from the Cancer Dependency Map (DepMap), baseline tumors from the NeoPalAna (NCT01723774) and neoMONARCH (NCT02441946) neoadjuvant trials, as well as the TCGA and METABRIC datasets were interrogated for correlations of gene signatures and patient outcome (by KMPlot). Results: Palbo treatment resulted in a dose-dependent inhibition of the growth of P and EndoR BC cell lines, with varying degree of sensitivity among the models. GSEA analysis comparing the least sensitive (IC50&gt;350nM) vs. the most sensitive (IC50&lt;100) cell lines identified the ‘interferon gamma response’ (IFNg) and ‘interferon alpha response’ (IFNa) as the top-ranked hallmark enriched signatures. Likewise, DepMap analysis of ER+/HER2- BC cell lines (n=11) revealed that cells with low CDK4 dependency scores displayed high IFN-signaling. We derived a 35-gene signature (termed ‘IFN-Related Palbociclib-Resistance Signature’, IRPS) comprised of genes belonging to the INFg and INFa gene sets that positively correlated with the Palbo IC50 values of our collection of P and EndoR lines. To extend these findings to primary ER+ BC, we interrogated transcriptomic data from the NeoPalAna and neoMONARCH trials that evaluated neoadjuvant CDK4/6i with endocrine therapy. In both trials, the IFNg, IFNa, and IRPS gene signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. We next investigated the underlying molecular changes and their association with IFN-signaling in our acquired resistant PalboR cell lines. Compared to the untreated cells, the PalboR models commonly displayed alterations in several components of the cyclin D-CDK4/6-Rb axis, including elevated expression of cyclin-D1, -E1, and CDK6, and reduced levels of Rb. Notably, the PalboR derivatives commonly displayed a dramatic activation of IFN/STAT1-signaling compared to their short-term treated or untreated counterparts. In primary ER+/HER2- tumors, the IRPS score was significantly higher in lumB vs. lumA subtype and correlated with increased gene expression of immune checkpoints (PD-1, PD-L1, CTLA-4), endocrine-resistance, and poor prognosis. Conclusion: Aberrant IFN-signaling predicts resistance to CDK4/6i in both ER+/HER2- BC cell lines and in primary BCs from neoadjuvant clinical trials. Experimentally, acquired resistance to Palbo is associated with activation of the IFN-pathway suggesting its involvement in resistance to CDK4/6i. Future studies are warranted to provide mechanistic insights into the association of IFN-signaling with response to CDK4/6i.
Citation Format: Carmine De Angelis, Xiaoyong Fu, Maria Letizia Cataldo, Agostina Nardone, Valerie M. Jansen, Jamunarani Veeraraghavan, Sarmistha Nanda, Lanfang Qin, Vidyalakshmi Sethunath, Resel Pereira, Matteo Benelli, Ilenia Migliaccio, Luca Malorni, Joshua Donaldson, Pier Selenica, David N. Brown, Britta Weigelt, Jorge S. Reis-Filho, Ben H. Park, Sara A. Hurvitz, Dennis J. Slamon, Mothaffar F. Rimawi, Rinath Jeselsohn, Kent Osborne, Rachel Schiff. High levels of interferon-response gene signatures are associated with de novo and acquired 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 GS2-01.