Abstract P4-07-07: Triple targeted combination therapy with tucatinib, palbociclib and fulvestrant is active in hormone receptor and HER2-positive human breast tumor cell lines
Authors: Shagisultanova E , Borakove M , Kabos P , Borges V 
Published: May 2019

Abstract
Hormone receptor and HER2-positive (HR+/HER2+) breast cancer displays increased resistance to anti-hormonal and HER2-targeted agents. HER2 and estrogen receptor signaling converge at cyclin D1 and CDK4/6 complex, resulting in the increased cyclin D1 expression and accelerated progression of the cell cycle. Therefore, triple blockade of the estrogen receptor, HER2 and CDK4/6 is reasonable as a novel approach to treatment of HR+/HER2+ disease. Here for the first time we report high activity of novel combination therapy with HER2-targeted small molecule inhibitor tucatinib, CDK4/6 inhibitor palbociclib, and selective estrogen receptor blocker fulvestrant in human breast tumor cell lines.
Methods: HR+/HER2+ human breast tumor cell lines BT474, MDA-MB-361 and UACC-812 were cultured in standard conditions. The subclones of BT474 and MDA-MB-361 resistant to tucatinib and palbociclib were generated by culturing cells in the increasing concentrations of inhibitors over 6 months. Cell survival assays were performed with Cell Titer Glow (Promega, Madison, WI) after 72 hrs of treatment with vehicle, single agents tucatinib, palbociclib and fulvestrant, and with dual and triple combinations. Tucatinib was provided by Seattle Genetics (Seattle, WA), palbociclib and fulvestrant were purchased from Selleckchem (Houston, TX). Protein lysates for western blot were prepared after 24, 48 or 72 hrs of drug treatment at IC70 using RIPA lysis buffer (Thermo Fisher, Waltham, MA) containing protease/phosphatase inhibitors (Roche Diagnostic, Indianapolis, IN). Signals were quantified with NIH ImageJ Imaging Analysis Software or Odyssey Imager software (Li-Cor Bioscience, Lincoln, NE) and normalized to vinculin or alpha-tubulin.
Results: Dual combinations of tucatinib with fulvestrant and tucatinib with palbociclib were synergistic in all three HR+/HER2+ cell lines. Addition of a constant dose of fulvestrant to a combination of tucatinib and palbociclib further improved tumor cell killing in all three breast tumor cell lines tested. Combination of tucatinib and palbociclib resulted in greater suppression of phospho-AKT and total and phospho-CDK2 comparing to either agent alone. Treatment with single agents tucatinib and palbociclib lead to a significant increase of cyclin E expression at 48 and 72hrs after treatment, however, this compensatory mechanism of cell cycle progression was not observed after treatment with tucatinib and palbociclib combination. Experiments with the resistant BT474 and MDA-MB-361 subclones showed that cross-treatment of cells resistant to palbociclib with tucatinib and vice versa suppressed cell growth and decreased expression of phospho-ERK1/2, phospho-AKT, phospho-CDK2 and cyclin E.
Conclusions: In HR+/HER2+ human breast tumor cell lines combination therapy with tucatinib, palbociclib and fulvestrant demonstrated significant synergy, potentially acting through suppression of phospho-AKT and cyclin E pathways, therefore suggesting a promising novel approach to treatment of HR+/HER2+ breast cancer. Clinical testing of tucatinib, palbociclib and anti-hormonal therapy in patients with HR+/HER2+ metastatic breast cancer is ongoing (phase Ib/II clinical trial NCT03054363).
Citation Format: Shagisultanova E, Borakove M, Kabos P, Borges V. Triple targeted combination therapy with tucatinib, palbociclib and fulvestrant is active in hormone receptor and HER2-positive human breast tumor 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 P4-07-07.