Abstract P4-09-11: Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors
Authors: Kobayashi Yoshie , Motoi Yusuke , Fujimoto Mutsumi , Shigematsu Hideo 
Published: March 2023

Abstract
Background CDK4/6 inhibitors are the latest and highly specific CDK inhibitors approved for metastatic and early estrogen receptor (ER)-positive breast cancer patients, improving the survival outcomes. Prior preclinical research has indicated various aberrations of cell cycle regulation and activated oncogenic signaling pathway as the mechanisms of resistance to CDK4/6 inhibitors. However, there is currently no available biomarker except ER and human epidermal growth factor receptor 2 (HER2) in clinical practice. The aim of our study is to elucidate diverse mechanisms of acquired resistance of ER-positive breast cancers to CDK4/6 inhibitors and develop personalized medicine in ER-positive breast cancer patients.
Materials and methods To generate resistant lines, the ER-positive human breast cancer cell lines MCF7 and T47D were treated individually with increasing concentrations of the CDK4/6 inhibitors palbociclib and ribociclib until a target concentration of 3μM for six months. Cells were maintained in RPMI 1640 containing 10% fetal bovine serum, 100 U/ml penicillin/streptomycin at 37°C in a 5% CO2 incubator. Western blot analysis was performed for whole cell extract proteins from resistant cells to CDK4/6 inhibitors and parental cells. Colony formation assay was done to observe cell viability.
Results Western blot analysis showed elevated FGFR2, CDK6, pS6RP, and cyclin E1 in resistant cells compared to parental cells. Specifically, cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, while FGFR2 and CDK6 in T47D palbociclib- and ribociclib-resistant cells. Notably, CDK6 was upregulated in MCF7 ribociclib-resistant cells, whereas increased cyclin E1 was detected in T47D palbociclib-resistant cells, respectively. Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in generated resistant cell lines.
Conclusions The tumors harboring resistance to CDK4/6 inhibitors have heterogenous mechanisms of therapeutic resistance to these agents. The novel combination treatment of targeted molecular therapy and CDK4/6 inhibition will lead development of personalized medicine for ER-positive breast cancer based on elucidation of diverse resistant mechanisms to CDK4/6 inhibition.
Western blot analysis of lysates from MCF-7 cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo)

Cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, whereas CDK6 was upregulated especially in MCF7 ribociclib-resistant cells.
Western blot analysis of lysates from T47D cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo)

FGFR2 and CDK6 were increased in T47D palbociclib- and ribociclib-resistant cells, whereas increased cyclin E1 was detected especially in T47D palbociclib-resistant cells.
Viability in MCF7 ribociclib-resistant and parental cells by colony formation assay

Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in MCF7 resistant-cells.
Citation Format: Yoshie Kobayashi, Yusuke Motoi, Mutsumi Fujimoto, Hideo Shigematsu. Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-11.