Cellular Retention of TKI and Apoptosis Commitment Figure 7. Measurement of TKI concentrations in cell culture supernatants using chromatographic methods. Imatinib and dasatinib concentrations were measured in cell culture supernatants using HPLC and LC/MS/MS, respectively. The sampling procedure and time-scale were identical to that described in Figure 5. (A) Ba/F3-BCR-ABL cells (I), K562 cells (II), primary human CML patient MNCs (III) and normal CD34+ enriched cells (IV) were pulse exposed to 25 mM imatinib followed by drug wash-out as described in Figure 1B. (B) Ba/F3-BCR-ABL cells (I) and normal enriched CD34+ cells (II) were pulse exposed to 100 nM dasatinib followed by drug wash-out as described in Figure 1B. The red lines represent the in vitro IC50 for the respective TKI used. Supernatant 1 (‘‘S1’’) was measured and served as a positive control. Measurement of cell culture media taken immediately upon replating served as a control for efficient washing (‘‘0 min’’). Depicted are mean values + SEM; at least 3 independent experiments were performed. For primary material one representative experiment is shown. doi:10.1371/journal.pone.0040853.g007 HD-TKI pulse-exposure was proposed. However, the mechanism of induction of apoptosis upon HD-TKI pulse-exposure remained elusive at the molecular level. [12] This prompted us to investigate the molecular mechanisms of cell death induced by HD-TKI pulse-exposure in more detail. It appeared unlikely that short-term potent kinase inhibition could initiate an irreversible cell death program without altering onset and kinetics of apoptosis. Indeed, the data presented here provide evidence that HD-TKI pulse- exposure does not irreversibly initiate apoptosis, since cells can be completely rescued by drug wash-out. Western blot experiments confirmed persistent target inhibition after HD-TKI pulse- exposure, with no evidence of BCR-ABL (Y412) or STAT5 re- phosphorylation after the first round of media exchange. This indicates substantial residual TKI activity when employing a single drug wash-out procedure. In line with previously published data on HD-TKI pulse- exposure, we observed re-phosphorylation of CRKL in BCR-ABL cells after the first drug wash-out step, while discordantly BCR-ABL (Y412) and STAT5 were still dephosphorylated.[12–14] Interest- ingly, BCR-ABL (Y177)-phosphorylation remained almost unaf- fected upon TKI exposure. This suggested differential kinetics and/ or dynamics of BCR-ABL (Y412)- and STAT5-phosphorylation as compared to CRKL- and BCR-ABL (Y177)-phosphorylation. Employing titration experiments using increasing concentrations of either imatinib or dasatinib, we measured STAT5- and CRKL- phosphorylation after different incubation times. This confirmed different kinetics as well as dynamics of STAT5- versus CRKL- phosphorylation. This difference might translate into a low diagnostic sensitivity for residual TKI activity in vitro, if CRKL- phosphorylation is used as a sole test for BCR-ABL tyrosine kinase activity. [12–14]. The apparent contradiction in our finding, that BCR-ABL (Y177)-phosphorylation does not correlate with BCR-ABL substrate phosphorylation is supported by recent publications. [29] While BCR-ABL (Y177) has been shown to play a crucial role for leukemic transformation capacity of BCR-ABL[30–33], kinase activity of BCR-ABL and downstream signaling is mainly regulated by BCR-ABL (Y412)-phosphorylation.[34–36] Along this line, a recent paper demonstrated that BCR-ABL (Y177) is phosphorylated by JAK2, and not by ABL. [29] It has been proposed that BCR-ABL (Y177)-phosphorylation provides fine- tuning of BCR-ABL downstream signaling rather than switching BCR-ABL signaling on and off. [29] In our hands, STAT5 is a useful surrogate parameter to monitor immediate effects of BCR- ABL kinase activity as its phosphorylation positively correlates with cell survival. Moreover, it has been demonstrated that STAT5 signaling is indispensable for initiation and maintenance of BCR-ABL mediated leukemic transformation.[37–40]. Results obtained by employing successive rounds of drug wash- out suggested prolonged intracellular TKI exposure to be the critical mechanism involved in induction of apoptosis upon HD- TKI pulse-treatment. Dose-dependent intracellular accumulation of TKI upon imatinib exposure has already been reported previously. [41] Along this line, we hypothesized that pronounced intracellular TKI-accumulation might be responsible for the observed results. Indeed, measurements of intracellular imatinib and dasatinib uncovered remarkable intracellular TKI accumu- lation upon HD-TKI pulse-exposure. Moreover, intracellular TKI accumulation is characterized by a slow time-dependent decrease in intracellular TKI levels upon drug wash-out. This was paralleled by a time-dependent increase of TKI concentrations in extracellular media upon washing and re-plating of cells, indicating release of TKI from an intracellular compartment into the cell culture media. Consistent with this, we demonstrated that HD-TKI pulse-exposure with imatinib was ineffective at inducing apoptosis in cells expressing the ABC-family transporters ABCB1 or ABCG2. Of note, sensitivity to HD-TKI pulse-exposure was restored by pharmacological inhibition of ABC-transporters. From a clinical perspective, our findings may prove useful for refinement of effective TKI dosing schedules, especially when applying TKIs with short plasma half-life. Along this line, recently, it was demonstrated that a short plasma half-life of a given compound may not necessarily predict a deficit in terms of clinical efficacy. [10] Our in vitro model of HD-TKI pulse-exposure revealed a previously unrecognized pharmacokinetic interplay between TKI concentrations in the extracellular media and intracellular TKI concentrations when a high-dose TKI pulse is applied. Both, dramatic intracellular TKI accumulation and delayed TKI release strongly argue in favor of an active cellular maintenance and/or uptake mechanism that can prevent a sudden decrease in intracellular TKI concentration. Indeed, recently it has been demonstrated that OCT-1 mediates cellular influx of imatinib, and that transporter activity correlates with effica- cy.[42–44] On the other hand, it has been shown that OCT-1 has less impact on cellular influx of dasatinib and nilotinib. [43,45,46] Therefore, we believe that additional drug-transporter proteins contribute to intracellular accumulation of TKIs. However, the data presented here is consistent with a model where intracellular accumulation and retention of TKIs in vivo also translates into significantly higher intracellular TKI concentrations as compared to the extracellular medium. It is conceivable that in the setting of high-dose pulse therapy this may then result in prolonged intracellular TKI exposure significantly exceeding plasma half- life of a given TKI. In conclusion, we show that dramatic intracellular TKI accumulation and retention result in prolonged target inhibition which appears to be the sole underlying molecular mechanism in HD-TKI pulse-exposure (imatinib, dasatinib) mediated induction of apoptosis in vitro. Moreover, the data illustrate that potent but transient kinase inhibition per se is not sufficient to irreversibly commit oncogene transformed cells to apoptosis. As we have observed intracellular TKI accumulation and retention in other oncogenic kinase models such as FLT3-ITD [47] and JAK2- V617F (data not shown), the mechanism described here may indicate a general pharmacokinetic feature of TKIs. However, this point clearly requires further investigation. Based on our data presented here, monitoring both, plasma and intracellular drug levels of imatinib and dasatinib in vivo will provide pharmacokinetic data which may prove useful to optimize dosing schedules in upcoming clinical trials. We speculate that PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e40853