Abstract P6-09-02: Effects of palbociclib on thymidine kinase-1 (TK1) in hormone receptor positive (HR+) breast cancer cell lines
Authors: Bonechi M , Migliaccio I , Benelli M , Romagnoli D , Bergqvist M , Mattsson K , Boccalini G , Capaccioli G , De Luca F , Galardi F , Biagioni C , Risi E , McCartney A , Rossi L , Osborne CK , Schiff R , De Angelis C , Guarducci C , Di Leo A , Malorni L 
Published: May 2019

Abstract
Introduction: TK1 plays a crucial role in DNA synthesis and is a well-established marker of cell proliferation. We and others have previously described the potential role of TK1 activity (TKa) as predictive biomarker of response to endocrine therapy in HR+/HER2 negative metastatic breast cancer patients. TK1 synthesis is regulated by the E2F pathway, the target pathway of CDK4/6 inhibitors, and TKa has recently been reported as a potential circulating pharmacodynamic marker of CDK4/6 inhibition in breast cancer. However, modulations of TK1 levels and activity during palbociclib treatment and in the development of treatment resistance are unknown. Here, we report how TK1 expression and TKa are modulated in response to palbociclib in a panel of HR+ breast cancer cell lines: both palbociclib-sensitive (PDS) and with acquired resistance to (PDR).
Material and methods: We used a panel of 7 PDR HR+ breast cancer models previously developed in our lab via chronic exposure of parental cells (MCF7, T47D, ZR75-1, BT474, MDAMB361 and two MCF7 endocrine resistant derivatives) to escalating doses of palbociclib, from a Starting Treatment Concentration (STC) of 50 nM or 350 nM according to the cell line, up to 1 μM. We analyzed gene expression profiles of PDS cells treated with drug vehicle (DMSO) as a control or palbociclib at STC for 3 days, and PDR cells grown with palbociclib 1 μM. Cell proliferation was assessed by methylene blue assay in MCF7 and BT474 PDS and PDR treated for 3, 6 and 9 days with DMSO, palbociclib STC and 1 μM. In parallel, TKa was measured in cell lysates at 3 days of treatment using the DiviTumTM assay (Biovica, Sweden).
Results: Among E2F target genes, gene expression data demonstrated that TK1 was one of the most differentially expressed genes between PDR and PDS treated cells. In PDS cells compared to control, treatment with palbociclib resulted in reduced TK1 expression, with the HER2 positive models (BT474 and MDAMB361) showing the highest reduction. In PDR cells, TK1 expression was higher, but remained slightly inhibited compared to untreated PDS cells. TKa was significantly reduced in PDS cells treated with palbociclib for 3 days compared to vehicle (p&lt;0.05). TKa response to palbociclib was more dramatic in BT474 cells as compared to MCF7. As expected, palbociclib inhibited cell proliferation in PDS models, with a significant reduction observed only after 6 days of treatment, suggesting that TKa may be an early marker of growth inhibition in response to palbociclib. No significant alterations in TKa were observed in PDR cells, at any dose of palbociclib. Similarly, proliferation rate was not affected by palbociclib in PDR cells.
Conclusions: TK1 expression and activity are regulated by palbociclib in HR+ breast cancer cell lines, particularly in HER2 positive models. Ongoing studies of TKa in patients treated with palbociclib will assess the role of TKa as a circulating biomarker for predicting and monitoring response to CDK4/6 inhibitors.
Citation Format: Bonechi M, Migliaccio I, Benelli M, Romagnoli D, Bergqvist M, Mattsson K, Boccalini G, Capaccioli G, De Luca F, Galardi F, Biagioni C, Risi E, McCartney A, Rossi L, Osborne CK, Schiff R, De Angelis C, Guarducci C, Di Leo A, Malorni L. Effects of palbociclib on thymidine kinase-1 (TK1) in hormone receptor positive (HR+) breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-09-02.