Continuous treatment with abemaciclib leads to sustained and efficient inhibition of breast cancer cell proliferation

Abstract

Abemaciclib is an oral, selective cyclin-dependent kinase 4 & 6 inhibitor (CDK4 & 6i), approved for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC) as monotherapy for endocrine refractory disease, and with endocrine therapy (ET) for initial treatment and after progression on ET. Abemaciclib has also shown clinical activity in combination with ET in patients with high risk early BC (EBC). Here, we examined the preclinical attributes of abemaciclib and other CDK4 & 6i using biochemical and cell-based assays. In vitro, abemaciclib preferentially inhibited CDK4 kinase activity versus CDK6, resulting in inhibition of cell proliferation in a panel of BC cell lines with higher average potency than palbociclib or ribociclib. Abemaciclib showed activity regardless of HER2 amplification and phosphatidylinositol 3-kinase (PI3KCA) gene mutation status. In human bone marrow progenitor cells, abemaciclib showed lower impact on myeloid maturation than other CDK4 & 6i when tested at unbound concentrations similar to those observed in clinical trials. Continuous abemaciclib treatment provided profound inhibition of cell proliferation, and triggered senescence and apoptosis. These preclinical results support the unique efficacy and safety profile of abemaciclib observed in clinical trials.

Keywords: CDK4/6; abemaciclib; breast cancer; cell lines; continuous dosing.

Figure 1. Abemaciclib is a more potent inhibitor of CDK4 than CDK6 in both biochemical and in vitro cell-based assays.

In a head-to-head comparison, abemaciclib showed higher potency to inhibit CDK4 than CDK6 with a broader margin of selectivity than other CDK4 and CDK6 inhibitors. This was confirmed in biochemical assays and in cellular systems. Biochemical potency (KiATP) and selectivity ratio CDK6/CDK4 (additional IC₅₀ values, FB assay and TR-FRET in Supplementary Materials) as cell assays for abemaciclib and palbociclib are included in Table A). (B) represents the Western Blot showing the expression of CDK4 and CDK6 in different cell models. MDA-MB-453 and NCI-H1568 cells (highlighted) showed preferential expression of CDK4 or CDK6 respectively. (C) Dose-response curves of abemaciclib, palbociclib, and ribociclib showing inhibition of Rb (retinoblastoma) ser780 phosphorylation, quantitated intracellularly by high content imaging in CDK4 and CDK6 dependent cell models (MDA-MB-453 and NCI-H1568) Data reported as an average of four independent determinations (n = 4) ± standard deviation (SD; error bars). Abbreviations: TR-FRET: Time-Resolved Foerster Resonance Energy Transfer; FB: Filter Binding.

Figure 2. Abemaciclib shows greater potency than palbociclib & ribociclib in breast cancer cells.

In vitro drug response waterfall plots for (A) abemaciclib, (B) palbociclib, (C) or ribociclib in a panel composed of 40 cell lines, either ER– (blue) or ER+ (yellow). Bar graph of log IC₅₀ values (uM) and cell type. Cell lines are color coded by subtype: yellow is luminal ER+; blue is ER–. Waterfall plots were generated using the geometric mean for each cell line and treatment.

Figure 3. Impact on neutrophils maturation is lower upon abemaciclib treatment comparing with others CDK4 & 6 inhibitors in preclinical models.

(A and B) Viability of isolated mature neutrophils from human whole blood at Cmax, fu. (C and D) Myeloid maturation assay, measuring cells per mL on Day 13; (D) shows normalized data. Data are plotted as the mean +/– SD of more than two independent replicates. Abbreviations: HWB: Human Whole Blood; hBM: human Bone Marrow; PI: Propidium Iodide; Cmax; fu: maximum unbound concentration in plasma. ^*** p-value < 0.0001; ns. or no ^*: non-significant. One way ANOVA among three groups. In a pairwise comparison there is a size effect of 18.54 for abemaciclib versus palbociclib and 61.99 for abemaciclib versus ribociclib with p-values of 0.0035 and < 0.0001 respectively.

Figure 4. Prolonged treatment of breast cancer cells with a CDK4 & 6 inhibitor is necessary to sustain cell growth inhibition and promote apoptosis.

T47D cells were treated with DMSO, 50, 100 or 250 nM of the CDK4 & 6i abemaciclib or palbociclib for 2, 6, and 9 days. (A–C) percentage of apoptotic cells are monitored by Annexin V and PI. The data are plotted as the mean (+/– SD) of three experiments for CDK4 & 6i treatment, and the mean of three experiments (+/– SD) for untreated samples. ANOVA analysis of Day 6 data show that % cell in apoptosis (late, total, or dead) is significantly different between groups treated with abemaciclib or not treated (FC 2.9, 3.1 and 3.3 respectively); % cell in late apoptosis is significantly different between groups treated with palbociclib or not treated (FC 1.45), although there is not significant change in the case of total apoptosis or dead cells in groups treated with palbociclib or not treated (FC 1.51 and 1.48 respectively). Abbreviations: ANOVA: Analysis of Variance; SD: Standard deviation; FC: Fold Change. ^*** p-value ≤ 0.0001, ^** p-value ≤ 0.001; if no p-value presented, the differences between groups were not statistically different.

Figure 5. Washout studies demonstrated durable effects after abemaciclib removal.

Cells treated with either abemaciclib or palbociclib + 5 nM tamoxifen. (A) Diagram of study experiment. (B) Cell proliferation inhibition. (C) Percentages of senescent cells were monitored by cell even green kit; data normalized. (D) Percentage of apoptotic cells, monitored by Annexin V and PI. The data are plotted as the mean (+/− SD) of three experiments. Abbreviation: NT: non-treated. ^* p-value ≤ 0.05; ^** p-value ≤ 0.01; ^*** p-value ≤ 0.001; ^**** p-value ≤ 0.0001; ns. or no ^*: non-significant.