Abstract 4847: Optimizing the combination of the CDK4/6 inhibitor palbociclib and paclitaxel using cell cycle analysis
Authors: Spencer Jade A. , Race Amanda D. , Cooper Patricia A. , Shnyder Steven D. , Loadman Paul M. , Twelves Christopher J. 
Published: September 2020

Abstract
Background: Palbociclib is a CDK4/6 inhibitor approved for the treatment of ER+ HER2- advanced/metastatic breast cancer (MBC). We explored the hypothesis that palbociclib can enhance the efficacy of cytotoxics by synchronising the cell cycle prior to treatment with a cytotoxic, namely paclitaxel. This may be of particular importance in retinoblastoma protein (Rb) expressing triple negative (TN) BC, where hormone therapy is not an option, and increasing the efficacy of cytotoxics could be of great benefit.
Methods: We studied 4 BC cell lines of different molecular subtypes: MDA-MB-175 (ER+, HER2 -, Rb+), MDA-MB-361 (ER+, HER2+, Rb+), HCC-38 (TN, Rb+) and MDA-MB-468 (TN, Rb- as negative control). Cells were exposed to 100 nM palbociclib for up to 72h, followed by washout for up to 72h, with cell cycling monitored throughout by flow cytometry using propidium iodide (PI) staining to establish optimal timings for combining palbociclib with paclitaxel. Cytotoxic activity of the combination was quantified by cell counting and clonogenic assays.
Results: MDA-MB-361, MDA-MB-175 and HCC38 cells exposed to 100 nM palbociclib are arrested in G0/1 by 24h with up to 2%, 3.9% and 5.2% respectively, continuing into S-phase. MDA-MB-468 is unaffected, with the percentage of cells in S-phase similar to controls after treatment with up to 10 μM palbociclib. After palbociclib withdrawal, cells begin to re-enter the cell cycle before 12h. The highest level of synchronization occurs in MDA-MB-361 with up to 47% of cells in S-phase by 16h. By 24h there is a peak in the number of cells in G2/M, after which they return to a normal unsynchronised cycling pattern (4.9-13.2% cells in S-phase). Precise timing of re-entry into the cell cycle after palbociclib withdrawal varies between cell lines and, as this is important for combination with cytotoxics, warrants further investigation. Treating with higher concentrations of palbociclib slows re-entry of cells into S-phase and reduces synchronization. Combination with paclitaxel 16h after palbociclib withdrawal reduces the IC50 of paclitaxel by up to 72% compared to paclitaxel alone in MDA-MB-361 cells. Mice bearing MDA-MB-361 xenografts are being treated using the optimized schedule determined in vitro and tumors are to be dissociated into single cells for cell cycle analysis by flow cytometry, allowing the comparison of the efficacy of paclitaxel +/- palbociclib in vivo. In addition, tissues will be assayed for palbociclib pharmacokinetics. Results of ongoing in vivo studies results will be presented.
Conclusion: Short-term exposure to low dose palbociclib can synchronize Rb expressing cells in S-phase in vitro and, when combined optimally, increases the efficacy of paclitaxel in these cell lines, as indicated by a reduction in IC50. This is being investigated in in vivo studies, the results of which may help to inform future clinical trial design.
Citation Format: Jade A. Spencer, Amanda D. Race, Patricia A. Cooper, Steven D. Shnyder, Paul M. Loadman, Christopher J. Twelves. Optimizing the combination of the CDK4/6 inhibitor palbociclib and paclitaxel using cell cycle analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4847.