Abstract P1-13-21: Loss of CDKN2B expression as a potential marker of resistance to CDK4/6 inhibitor in Luminal Breast Cancer cells
Authors: Cordani Nicoletta , Mologni Luca , Piazza Rocco , Cogliati Viola , Pepe Francesca , Capici Serena , Bella Camillo Di , Jaconi Marta , Cerrito Maria Grazia , Villa Matteo , Tettamanti Pietro , Cavaletti Guido , Lavitrano Marialuisa , Cazzaniga Marina Elena 
Published: March 2023

Abstract
Background Cyclin-Dependent Kinase (CDK) 4/6 inhibitors have significantly improved progression-free survival of Hormone Receptor positive (HR+), Human Epidermal Growth Factor Receptor type 2 negative (HER2-) luminal breast cancers (LBC). Several studies demonstrated that the addition of CDK4/6 inhibitors to endocrine therapy results in a significant prolongation of progression-free survival in patients with endocrine-sensitive or endocrine-resistant LBCs. However, the percentage of patients unresponsive or refractory to these therapies is as high as 40%, and no reliable and reproducible biomarkers able to select a priori responder or resistant patients have been validated till now. The main cause of resistance is the selection of mutant clones in the target oncoprotein. Other mechanisms, like oncogene amplification/overexpression or mutations in other pathways, have been described in several models. Here, we focused on palbociclib, a selective inhibitor of CDK4/6. Methods: We generated and characterized human luminal breast cancer MCF-7 and T47D derived cell lines, able to survive and proliferate at different palbociclib concentrations, which also shows cross-resistance to abemaciclib. The resistant MCF7 cell line was characterized by RNA sequencing. Results: To confirm resistance, we performed cell viability assays in MCF-7 and T47D palbociclib sensitive cells (MCF-7pS and T47pS) versus MCF-7 and T47D palbociclib resistant cells (MCF-7pR5), showing a 10-fold increase of IC50 in MCF-7pR5 compared to parental MCF-7pS cells (16.7 vs 1.8 µM) and a 3-fold increase of IC50 in T47DpR5 vs parental T47DpS. We also confirmed a significant cross resistance using abemaciclib in MCF-7pR5 with an IC50 equal to 6.8 vs 0.35 µM and in T47DpR with an IC50 of 10.72 vs 0.5 µM. RNA sequencing, qRT-qPCR and Western blot results demonstrated a dramatic downregulation of the CDK4 inhibitor CDKN2B in both cell lines and we found upregulation of an miR-31, a putative regulator of CDKN2B. This finding was further validated in a biopsy from a patient progressing on CDK4/6 inhibitor therapy. Conclusions: This study provides new relevant information regarding the mechanism of resistance to CDK4/6 inhibitors and suggests potential new markers to follow up patients during the treatment.
Citation Format: Nicoletta Cordani, Luca Mologni, Rocco Piazza, Viola Cogliati, Francesca Pepe, Serena Capici, Camillo Di Bella, Marta Jaconi, Maria Grazia Cerrito, Matteo Villa, Pietro Tettamanti, Guido Cavaletti, Marialuisa Lavitrano, Marina Elena Cazzaniga. Loss of CDKN2B expression as a potential marker of resistance to CDK4/6 inhibitor in Luminal Breast Cancer cells [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 P1-13-21.