A Novel Paradigm In Pharmacodynamics of Tyrosine Kinase Inhibitors: Pulse Treatment Induced Apoptosis Is Mediated by Intracellular Retention
Authors: Wagner Marie-Christine , Dziadosz Marek , Schnoeder Tina , Heidel Florian H. , Fischer Thomas , Lipka Daniel B. 
Published: October 2019

Abstract
Abstract 1828
Small molecule tyrosine kinase inhibitors (TKI) have become a valuable therapeutic tool in a variety of malignancies. It has been widely accepted that continuous target inhibition is a prerequisite for clinically effective TKI treatment. This paradigm has been questioned by evidence obtained in a clinical trial for chronic myeloid leukemia (CML) using the second generation BCR-ABL inhibitor dasatinib: Once daily dosing (QD) resulted in only transient inhibition of BCR-ABL kinase activity. Nevertheless, similar clinical activity was demonstrated for the QD schedule as compared to twice daily dosing (BID). Recent in vitro data demonstrated that pulse treatment with 50–100 nM dasatinib for 20 min to 4h induced apoptosis in BCR-ABL positive cells. Pulse treatment with imatinib using standard concentrations (1-7,5 μM) was not effective. However, if pulses with high doses of imatinib (32,5 μM) are applied, induction of apoptosis was observed. So far, the underlying molecular mechanism for this effect remains elusive.
Our study aims to elucidate the underlying molecular mechanism of induction of apoptosis upon TKI pulse treatment in a variety of hematopoietic cells dependent on oncogenic kinases.
We employed the human K562, and MV4-11 cell lines, as well as cellular reconstitution models of murine hematopoietic Ba/F3 cells stably transfected with BCR-ABL(p210), JAK2-V617F, or FLT3-ITD, respectively. Human primary cells from healthy controls and CML patients were obtained after written informed consent. Cells were incubated either with dasatinib or imatinib (BCR-ABL inhibition), JAK-Inhibitor I (JAK2-V617F inhibition) or with PKC412 (FLT3-ITD inhibition). Concentrations used were 100 nM dasatinb, 25 μM imatinib, 10 μM Jak-Inhibitor I or 3,5 μM PKC412, respectively, for 2–24h, followed by thorough washing steps. Cells were then incubated in medium without TKI. Upon a total incubation period of 24h, apoptosis was measured using flow cytometry and Western blotting (caspase 3 cleavage). In parallel, inhibition of intracellular signaling pathways (pSTAT5, pERK, pCrkl) was determined by Western blotting. To test for intracellular retention of TKI, untreated cells were incubated with cell culture supernatant obtained after incubation of previously washed TKI treated cells for 2h in TKI-free media. Again, apoptosis was measured at 24h and inhibition of signaling pathways was analyzed at various time points. Drug levels of imatinib and dasatinib in the cell culture supernatants before and after the washing procedures were quantified by HPLC.
TKI pulse treatment with each inhibitor for a period of 2h was sufficient to effectively induce apoptosis independent of the transforming oncogene and of cellular origin (mouse or human). To test for possible cellular retention upon TKI pulse treatment, we introduced additional washing steps and applied the supernatants to previously untreated cells. This analysis revealed: 1) Cells were almost completely rescued from apoptosis when additional washing steps were applied and 2) supernatants induced high levels of apoptosis in previously untreated cells. To confirm that the nature of this effect is indeed cellular TKI retention, imatinib and dasatinib concentrations in cellular supernatants were monitored at various time points (0, 30, 60, and 120min). This revealed a dramatic, time-dependent increase in either imatinib or dasatinib concentrations reaching levels well above the IC50 concentration of each inhibitor. These results were confirmed using normal primary human CD34+ cells and peripheral blood CML-MNCs. To further confirm that the TKI is being stored intracellularly upon TKI pulse treatment, we lysed pulse-treated cells immediately after completion of the first washing procedure. By HPLC we were able to detect relevant TKI concentrations in the cell lysate, thus confirming that TKIs are indeed retained intracellularly.
Taken together, using three different oncogene-dependent cellular reconstitution models, our data demonstrate that cellular TKI retention and prolonged inhibition of signal transduction is the common molecular mechanism in induction of apoptosis upon TKI pulse treatment. This suggests that both in-vitro and in-vivo analysis of pharmacokinetics and pharmacodynamics are required to determine optimal dosing schedules in future clinical trials.

Disclosures:
No relevant conflicts of interest to declare.