Abstract PD7-08: Igf1r mediates cdk4/6 inhibitor (cdk4/6i) resistance in tumor samples and in cellular models
Authors: Wander Seth A. , Mao Pingping , Lloyd Maxwell R. , Johnson Gabriela N. , Kowalski Kailey , Nayar Utthara , Guenther Lillian M. , Stegmaier Kimberly , Winer Eric P. , Lin Nancy U. , Wagle Nikhil 
Published: February 2021

Abstract
Background: Deciphering the molecular landscape of resistance to the CDK4/6 inhibitors represents a critically important question for patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC). Emerging insights from sequencing efforts suggest that inactivating alterations in the RB1 tumor suppressor occur in a small minority of patients and that a variety of heterogeneous mediators provoke resistance in patient samples. Proteins implicated in CDK4/6i resistance include cell cycle regulators such as cyclin E1/2, CDK6, and aurora kinase as well as known oncogenic signal transduction mediators involved in activation of the RAS-MEK and AKT-mTOR pathways. The insulin-like growth factor 1 receptor (IGF1R) has been implicated in modulating anti-estrogen resistance, and IGF1R inhibitors are currently in various stages of pre-clinical and clinical development.
Methods: We identified patients with amplification events in IGF1R from a database containing targeted sequencing of solid tumor samples obtained from patients with HR+ MBC enrolled on a research biopsy protocol. Tumor biopsies may have been obtained at various points during each patient’s clinical treatment course. HR+ T47D cells were modified to over-express IGF1R via lentiviral infection and selection. Derivative cell lines were treated with IGF-1 ligand and downstream activation of the PI3K/AKT and RAS/MEK pathways were assessed via western blotting. Control cells (expressing GFP) were mixed with IGF1R-expressing cells 1:1 and cultured in the presence of IGF-1 ligand and palbociclib or other drugs, for 1-3 weeks. At the timepoint of interest, cells were harvested and the relative proportion of GFP or IGF1R-expressing cells were interrogated via flow cytometry.
Results: We identified seven patients with HR+ MBC and IGF1R amplifications via targeted sequencing of tumor biopsies. Five of these patients had exposure to CDK4/6i-based therapy in the metastatic setting. Three patients demonstrated intrinsic resistance to CDK4/6i treatment (with duration &lt;6 months) and biopsies were obtained prior to CDK4/6i exposure or, in one case, while on treatment. In an additional patient, after nine months of CDK4/6i-based therapy, an IGF1R amplification was present at the time of progression. In one counter-example, a baseline biopsy revealed IGF1R amplification and subsequent clinical benefit with CDK4/6i, exceeding 10 months, was noted. T47D cells over-expressing IGF1R demonstrated increased pERK and pAKT activation following introduction of IGF-1 ligand. Control GFP and IGF1R-expressing cells were plated 1:1 and cultured in the presence of IGF-1 ligand and palbociclib. In a flow cytometry-based competition assay, an IGF-1 dose-dependent increase in the relative proportion of IGF1R-expressing cells was noted after one, two, and three weeks of palbociclib treatment. The extent of IGF1R-expressing cell enrichment was attenuated in the presence of either a MEK inhibitor or an IGF1R inhibitor.
Conclusions: IGF1R amplification events were identified in tumor biopsy samples that reflect either intrinsic or acquired resistance to CDK4/6i-based therapy. HR+ breast cancer cells which over-express IGF1R demonstrate enrichment under palbociclib drug selection in a flow cytometry-based competition assay, which was abrogated by concurrent use of a MEK or IGF1R inhibitor. These results suggest that IGF1R may join the increasingly heterogeneous landscape of CDK4/6i resistance mediators. Further exploration of this possibility is warranted. A subset of patients with IGF1R-mediated CDK4/6i resistance could benefit from therapeutic strategies designed to downregulate MEK or IGF1R activity.
Citation Format: Seth A. Wander, Pingping Mao, Maxwell R. Lloyd, Gabriela N. Johnson, Kailey Kowalski, Utthara Nayar, Lillian M. Guenther, Kimberly Stegmaier, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Igf1r mediates cdk4/6 inhibitor (cdk4/6i) resistance in tumor samples and in cellular models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-08.