Comparative biological activity of palbociclib and ribociclib in hormone receptor-positive breast cancer

Abstract

This study examines the biological effects of palbociclib and ribociclib in hormone receptor-positive breast cancer, pivotal to the HARMONIA prospective phase III clinical trial. We explore the downstream impacts of these CDK4/6 inhibitors, focusing on cell lines and patient-derived tumor samples. We treated HR+ breast cancer cell lines (T47D, MCF7, and BT474) with palbociclib or ribociclib (100 nM or 500 nM), alone or combined with fulvestrant (1 nM), over periods of 24, 72, or 144 h. Our assessments included PAM50 gene expression, RB1 phosphorylation, Lamin-B1 protein levels, and senescence-associated β-galactosidase activity. We further analyzed PAM50 gene signatures from the CORALLEEN and NeoPalAna phase II trials. Both CDK4/6 inhibitors similarly inhibited proliferation across the cell lines. At 100 nM, both drugs partially reduced p-RB1, with further decreases at 500 nM over 144 h. Treatment led to reduced Lamin-B1 expression and increased senescence-associated β-galactosidase activity. Both drugs enhanced Luminal A and reduced Luminal B and proliferation signatures at both doses. However, the HER2-enriched signature significantly diminished only at the higher dose of 500 nM. Corresponding changes were observed in tumor samples from the CORALLEEN and NeoPalAna studies. At 2 weeks of treatment, both drugs significantly reduced the HER2-enriched signature, but at surgery, this reduction was consistent only with ribociclib. Our findings suggest that while both CDK4/6 inhibitors effectively modulate key biological pathways in HR+/HER2- breast cancer, nuances in their impact, particularly on the HER2-enriched signature, are dose-dependent, influenced by the addition of fulvestrant and warrant further investigation.

Figure 1

Biological changes during CDK4/6 inhibition in vitro. (A) T47D and MCF7 cells were treated with increasing doses of palbociclib or ribociclib +/− fulvestrant (1 nM) for 72 h. Shown are representative graphs of cell viability redouts determined by Hoechst 33342. Data was normalised to untreated cells and three independent experiments were performed. Mean values ± SEM are shown. (B) T47D and MCF7 cells were treated with palbociclib or ribociclib (100 or 500 nM) for 24, 72 or 144 h and expression of p-RB1 and Lamin-B1 was assessed by western blot. Actin was used as a loading control. (C) T47D and MCF7 cells were treated with palbociclib or ribociclib (100 or 500 nM) for 24, 72 or 144 h and SA-β-gal activity was determined by flow cytometry. Data was normalised to untreated cells and three independent experiments were performed. Mean values ± SEM are shown.

Figure 2

Changes in the HER2-enriched signature upon treatment with CDK4/6 inhibitors +/− fulvestrant in vitro. (A) Heatmap of a multiclass SAM representing the PAM50 molecular subtypes, proliferation score and genes that are differentially expressed (FDR < 5%) in T47D, MCF7, and BT474 cells treated with CDK4/6 inhibitors (100 or 500 nM) +/− fulvestrant (1 nM). Three independent mRNA extractions per cell line were performed. (B) Paired samples t-test analyses showing changes in the HER2-enriched signature following treatment of T47D, MCF7, and BT474 cells with CDK4/6 inhibitors (100 or 500 nM) +/− fulvestrant (1 nM). Three independent mRNA extractions and gene expression analyses were performed for each cell line.

Figure 3

Changes in the PAM50 signatures in the CORALLEEN and NeoPalAna studies. Schematic summaries of the samples analyzed from (A) the CORALLEEN trial design and (B) the NeoPalAna trial design. (C) Paired samples t-test analyses showing changes in the PAM50 signatures at cycle 1 day 15 (C1D15) in tumor samples from CORALLEEN and (D) NeoPalAna phase II studies. (E) Changes in the PAM50 signatures at surgery in tumor samples from CORALLEEN and (F) NeoPalAna. (G) Changes in the HER2-enriched signature in tumor samples from CORALLEEN and (H) NeoPalAna from patients who underwent surgery ≤ 8 or > 8 days from the last dose of CDK4/6 inhibitors + endocrine therapy.