Loss of CDKN2B expression as a potential marker of resistance to CDK4/6 inhibitor in Luminal Breast Cancer cells

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.