Intracellular Retention of ABL Kinase Inhibitors Determines Commitment to Apoptosis in CML Cells Daniel B. Lipka1,2., Marie-Christine Wagner1., Marek Dziadosz3, Tina Schno¨ der1, Florian Heidel1, Mirle Schemionek4,5, Junia V. Melo6,7, Thomas Kindler8, Carsten Mu¨ ller-Tidow4, Steffen Koschmieder4,5, Thomas Fischer1* 1 Department of Hematology and Oncology, University Medical Center, Otto-von-Guericke-University, Magdeburg, Germany, 2 Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center, Heidelberg, Germany, 3 Institute of Forensic Medicine, University Medical Center, Otto-von-Guericke-University, Magdeburg, Germany, 4 Department of Medicine A (Hematology, Oncology and Pneumology), University of M u¨nster, Mu¨ nster, Germany, 5 Department of Oncology, Hematology and Stem Cell Transplantation, University Medical Center, Rheinisch-Westfaelische Technische Hochschule, Aachen, Germany, 6 Department of Haematology, Centre for Cancer Biology, University of Adelaide, Adelaide, Australia, 7 Department of Haematology, Imperial College London, London, United Kingdom, 8 Third Department of Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany Abstract Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high- dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs. Citation: Lipka DB, Wagner M-C, Dziadosz M, Schno¨ der T, Heidel F, et al. (2012) Intracellular Retention of ABL Kinase Inhibitors Determines Commitment to Apoptosis in CML Cells. PLoS ONE 7(7): e40853. doi:10.1371/journal.pone.0040853 Editor: Alan P. Fields, Mayo Clinic College of Medicine, United States of America Received April 23, 2012; Accepted June 14, 2012; Published July 16, 2012 Copyright: 2012 Lipka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research funds granted by the ‘‘Wilhelm-Sander-Stiftung’’ (No 2011.079.1 to FH, DL, and TF) and by the ‘‘Deutsche Forschungsgemeinschaft’’ (GRK-1167, P17-2 to TF and SFB-854/1, TP20 to TF), and by SA Pathology (Australia) and the National Institute for Health Research (NIHR) Biomedical Research Centre, UK funding scheme (to JVM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: DBL and SK received honoraria from Novartis and BMS; FH, TK and TF received honoraria from Novartis; MCW, MD, TS, MS and CMT have no relevant conflicts of interest to disclose. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: thomas.fischer@med.ovgu.de . These authors contributed equally to this work. Introduction Chronic myeloid leukemia (CML) is characterized by the constitutively activated tyrosine kinase BCR-ABL. Treatment of CML with the small molecule tyrosine kinase inhibitor (TKI) imatinib stands as a paradigm for clinical efficacy of targeted small molecule therapy in malignant disease. Imatinib inhibits BCR- ABL tyrosine kinase activity and has been shown to effectively target the malignant clone in vitro and in vivo, resulting in a high percentage of long-term remissions in CML patients. [1] Beyond CML, TKIs are currently either approved or evaluated in numerous other hematologic and solid neoplasms and may become cornerstones of novel treatment strategies in the near future. [2–4]. Preclinical and clinical data derived from studies using imatinib and other compounds suggested that candidates for clinical development should exhibit a sufficiently long plasma half-life to facilitate persistent target inhibition: continuous exposure to imatinib concentrations $1 mM for at least 20 h is necessary to induce apoptosis in BCR-ABL transformed cells in vitro, and clinical trials demonstrated a close relationship between imatinib serum trough-levels and clinical response.[5–7] Finally, the extent of BCR-ABL inhibition, as determined by the level of CRKL dephosphorylation, correlated with clinical activity. [8] Therefore, it has been widely accepted that continuous and complete target inhibition is a prerequisite for clinical efficacy of TKI treatment. Recently, this paradigm has been challenged by data obtained in a clinical trial using the second generation BCR-ABL inhibitor dasatinib. [9] Dasatinib (serum half-life: 3–5 h) demonstrated similar clinical activity but less side effects for once daily dosing with 100 mg as compared to twice daily dosing with 70 mg. [10] Interestingly, the once daily dosing schedule apparently resulted in transient inhibition of BCR-ABL kinase activity only, as re- phosphorylation of the BCR-ABL downstream adaptor protein CRKL was observed 8 h post dasatinib-dosing. [10,11] In addition, in vitro and ex vivo studies suggested that high-dose (HD) pulse-exposure to TKI irreversibly commits BCR-ABL positive PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40853