Abstract 2317: Preclinical model of Ribociclib resistance in HR-positive breast cancer
Authors: Aske Jennifer Carlson , De Pradip , Dey Nandini 
Published: June 2022

Background: Overactivation of the Cyclin-CDK-RB axis related to uncontrolled cell proliferation is one of the innate characteristics of HR+/HER2- breast cancer. CDK4/6 inhibitors combined with endocrine therapy have become the standard-of-care for patients with HR+/HER2- breast cancer. Although this regime is highly successful in clinics, the acquired resistance to CDK4/6 inhibitors has emerged. Identifying druggable resistance mechanism for CDK4/6 inhibitors is an unmet need.

Aim: To study the convergent mechanisms of early adaptation of acquired resistance to CDK4/6 inhibitors, we have developed two resistant cell lines using both pulse (Holiday ‘H’) and continual selection ‘R’ methods and characterized their response to drugs as compared to parental ‘P’ cells.

Results: Proliferation assays demonstrate a 50 percent reduction in proliferation at 1.5 μM ribociclib in the P cells compared to a 17 percent reduction in R cells. In cell cycle analysis, R cells have an initial decrease in the “S” phase and increase their “G1” following 1 μM ribociclib treatment, similar to P cells up to 24 hours. However, by 48 and through 72 hours, R Cells recover and cycle similarly to the control-treated cells. In contrast, the P cells demonstrate a lasting G1 increase in ribociclib treated cells, leading to apoptosis induction with ribociclib + fulvestrant treatment at 48 and 72 hours. Apoptosis data indicate that R cells treated with ribociclib and fulvestrant or in triplet combination with copanlisib (PI3K alpha/delta inhibitor) are more resistant to treatment than P cells, in which a robust increase in apoptosis is seen. 3D matrigel-based clonogenic assay was performed for 15 days in P, H, and R cells to show the ineffectiveness of ribociclib to block the growth of colonies in R as compared to P cells. Western blot analysis demonstrated RB phosphorylation was abrogated in R cells compared to P cells. The H cells have an intermediary state of resistance as compared to R cells. pMEK, pERK, and pP90RSK were significantly upregulated in R cells compared to P cells. Copanlisib was found to block the AKT and S6RP signal in all three P, H, and R cells.

Conclusion: R cells bear a characteristic signature of alterations in cell signaling involving the PI3K-AKT and RAS-MAPK pathways in response to ribociclib. Compared to P cells, R cells showed significant activation of the RAS-MAPK pathway, while the PI3K-AKT pathway was not altered. Network rewiring of the adaptive resistance using these pair of cell lines will provide an opportunity to study early adaptive response to ribociclib towards understanding and evolving combating strategies in BC following the emergence of resistance to ribociclib. Sequencing genomic alterations in the resistant cell lines are being carried out, and will be presented at the conference.

Citation Format: Jennifer Carlson Aske, Pradip De, Nandini Dey. Preclinical model of Ribociclib resistance in HR-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2317.