Abstract PD2-05: Differential mechanisms of acquired resistance to abemaciclib versus palbociclib reveal novel therapeutic strategies for CDK4/6 therapy-resistant breast cancers
Authors: Navarro-Yepes Juliana , Chen Xian , Bui Tuyen , Kettner Nicole M , Hunt Kelly K , Keyomarsi Khandan 
Published: March 2020

Abstract
Background: Hormone receptor positive (HR+)/human epidermal growth factor receptor 2 negative (Her2-) is the most prevalent subtype of breast cancer, representing 70% of the cases with metastatic disease. Endocrine therapy plus FDA-approved cyclin-dependent kinases (CDK) 4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have improved the treatment of HR+/Her2- advanced breast cancer patients. However, patients develop clinical resistance (acquired) during the long-term treatment, and in some cases, they do not respond within the first 3 months of treatment (intrinsic resistance), leading to disease progression. Although these inhibitors have the same nominal targets, CDK4/6, abemaciclib can inhibit other kinases that are not inhibited by palbociclib/ribociclib, such as CDK9/7/2/1, GSK3α/β and CAMK2γ/δ. As such, abemaciclib has been shown to inhibit cell growth in Rb deficient cells, in which palbociclib/ribociclib are ineffective. Therefore, we hypothesized that 1) mechanisms driving palbociclib acquired resistance are distinct than those mediating abemaciclib resistance, 2) palbociclib resistant models may be responsive to abemaciclib, and 3) pathways underlying these mechanistic differences can be exploited as alternative therapies to overcome resistance.
Methods: We generated several models to examine mechanisms of resistance to different CDK4/6 inhibitors: 1) MCF7 and T47D (ER+) palbociclib resistant breast cancer cells with increasing concentrations of palbociclib, starting at 1.2 µM (initial IC 50) up to 5 µM (plasma concentrations in patients) in a stepwise manner over a 6 month period, 2) MCF7 abemaciclib resistant cells (0.5, 1 and 1.5 µM), and 3) organoids derived from patient derived xenografts (PDXs) with a similar molecular profile to palbociclib acquired resistance models. Cell cycle changes were evaluated by western blot analysis and flow cytometry.
Results: Western blot analysis revealed a dose dependent downregulation of ERα, Rb, p-Rb and p27, while levels of cyclin E and p-CDK2 increased in a stepwise fashion in palbociclib resistant cells, which were only partially cross resistant to abemaciclib and sensitive to CDK9 inhibitors LDC067 and SNS032. FACS analysis of abemaciclib resistant cells showed an increase in G2/M accompanied by a drastic reduction of p-Rb and a gradual decrease of Rb. ERα levels were slightly decreased in abemaciclib resistant cells compared to the complete loss in palbociclib resistant cells. As in the palbociclib resistant cells, a dose dependent increase in cyclin E and p-CDK2 were also observed. However, Rad51, a key mediator of DNA repair, was downregulated in a stepwise manner only in abemaciclib resistant cells, but not in palbociclib resistant cells. Therefore, we evaluated if a combination therapy targeting DNA repair pathways plus abemaciclib could render palbociclib resistant models more responsive to targets other than cell cycle inhibitors. To this end, we examined the combination of abemaciclib and niraparib in organoid cultures generated from PDX models that are transcriptomic surrogates of palbociclib resistance cells and found a significant reduction of viability, number and density of these organoids. In vivo, using the same PDX models, the combined treatment reduced the tumor growth rate and increased overall survival.
Conclusion: Our results suggest that mechanisms underlaying palbociclib and abemaciclib acquired resistance exhibit differentially altered pathways, such as DNA damage/repair pathways, which can be exploited to overcome CDK4/6 inhibitors therapy resistance in breast cancer patients.
Citation Format: Juliana Navarro-Yepes, Xian Chen, Tuyen Bui, Nicole M Kettner, Kelly K Hunt, Khandan Keyomarsi. Differential mechanisms of acquired resistance to abemaciclib versus palbociclib reveal novel therapeutic strategies for CDK4/6 therapy-resistant breast cancers [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 PD2-05.