Inhibitory Effects Of The Btk Inhibitor, Ibrutinib, On Her2 Amplified Breast Cancer Growth, Cell Cycle Progression And Clonogenicity

Introduction: Ibrutinib is a Btk inhibitor developed for treatment of B cell malignancies. It binds covalently to cys-481 in Btk9s kinase domain, which contributes to the drug9s selectivity and enables once daily dosing. A homologous kinase domain cysteine is conserved in 9 other inhibitable kinases including those of the ErbB family. Ibrutinib thereby inhibits the in vitro growth of HER2 amplified breast cancer (BrCa) cells at pharmacologically relevant concentrations, and in vivo, in MB-453 xenografts, at dosages that model clinical exposure. To further characterize these effects we determined the effect of ibrutinib on growth, cell cycle progression, apoptosis and clonogenicity and stem cell-like subpopulations following treatment for short durations. Methods: BT-474 and SK-BR-3 BrCa cells were treated with ibrutinib at various times and concentrations, and culture continued after washing with fresh media. Cell growth was determined by alamar blue assay or direct cell counting. Cell cycle analysis was performed with PI staining and flow cytometry. Apoptosis was quantitated using flow cytometry with annexin-V/PI staining. Clonogenic assays were performed in 6-well plates with crystal violet staining. Putative “stem-like” cells were assessed by quantitating the aldehyde dehydrogenase 1 (ALDH1) population. Results: After drug washout, persistence of inhibition of ErbB kinase9s phosphorylation and down-stream signaling was observed on a time and exposure dependent basis: while substantial washout-resistant inhibition occurred after 15 min treatment with 0.5 µM ibrutinib, over 1 hour was required for irreversibility following 0.1 µM treatment. Growth inhibition was sustained for 6 days following 1 hr exposure to 0.1 - 0.5 µM of ibrutinib. BrCa cells with short exposure were arrested in G1 with decreased S phase 24h later. ErbB kinases9 activation and downstream signaling were also inhibited for up to 6 days. Although growth inhibition was largely cytostatic, 17% and 30% for BT-474 cells and SK-BR-3 cells, respectively, were apoptotic after 6 days at 0.5uM. Continuous treatment induced higher proportions of apoptotic cells. One hour washout or continuous treatment with ibrutinib decreased the putative “stem-like” subpopulation in BrCa cells as measured by Aldefluor assay. A short exposure of BrCa cells to ibrutinib also reduced their in vitro clonogenicity. Conclusions: Brief exposure to the Btk inhibitor, Ibrutinib, causes irreversible inhibition of ErbB kinases with consequent growth inhibition of HER2 amplified lines characterized by impaired cell cycle progression, evidence of apoptosis, and reductions of the Aldefluor population and clonogenicity. The unique combination of inhibitory properties of ibrutinib for both ErbB and TEC family kinases could prove to be advantageous in selected clinical settings. Citation Format: Jun Chen, Betty Y. Chang, Laurence Elias. Inhibitory effects of the BTK inhibitor, ibrutinib, on Her2 amplified breast cancer growth, cell cycle progression and clonogenicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4564. doi:10.1158/1538-7445.AM2014-4564