Bosutinib is a Substrate of the ABC Efflux Transporter P-Glycoprotein.
Authors: Perini Pietro , Redaelli Sara , Piazza Rocco , Viltadi Michela , Boschelli Frank , Gambacorti Carlo 
Published: October 2019

Abstract
Abstract 3763
Poster Board III-699
Bosutinib (Bos) (Wyeth Pharmaceuticals) is a dual Src-Abl tyrosine kinase inhibitor developed to inhibit the activity of BCR-ABL in chronic myeloid leukemia. P-glycoprotein (P-gp) is an ATP-binding cassette (ABC) transporter responsible of Imatinib (Im) efflux, and its increased expression can be related to Im resistance. The aim of the study was to define if P-gp is also responsible for the cellular efflux of Bos, and if P-gp altered expression can be related to Bos resistance. Four different K562 Ph+ cell lines were used. 1) K562S, Im sensitive; 2) K562DOX, cells resistant to Doxorubicin that overexpress P-gp (kind gift of JP Marie, Université Pierre et Marie Curie, Paris); 3) K562DOX siP-gp, K562DOX cells carrying a stable silencing of P-gp (kind gift of E Gunsilius, Innsbruck Medical University, Austria); 4) K562DOX H1, K562DOX cells carrying a control vector (kind gift of E Gunsilius). Real Time PCR confirmed that K562DOX and K562DOX H1 express higher levels of P-gp than K562DOX siP-gp (approximately 10 fold) and K562S (approximately 10000 fold). Western blot α-P-gp showed similar results. We then assessed Bos IC50 on the cell lines with a proliferation assay. K562DOX (IC50=175.3nM) and K562DOX H1 (IC50=102 nM) are resistant to Bos if compared to K562DOX siP-gp (IC50=9.1nM) and K562S (IC50=8.7nM). This data confirm that the P-gp overexpression is related to Bos resistance. Using C-14 radiolabeled Bosutinib (C-14 Bos) (Wyeth Pharmaceuticals) we set up an Intracellular Uptake and Retention (IUR) assay on K562S, K562DOX and K562DOX siP-gp cells. Cells were pre-treated for an hour with 0-60μM of Verapamil (Ver), a P-gp inhibitor, and then treated for two hours with 1μM C-14 Bos. In presence of Ver, K562DOX showed a significant increased amount of intracellular C-14 Bos ([] C-14 Bos Ver60μM/ [] C-14 Bos Ver0μM=5.8) that was not observed in K562S or K562DOX siP-gp. These data confirm that the intracellular concentration of Bos is strictly related to the expression levels and to the activity of P-gp. To test the biological effect of P-gp inhibition, we evaluated Bos IC50 by proliferation and apoptosis induction in K562DOX and K562S cells pre-treated with increasing concentrations of Ver. K562S did not show a significant decrease of Bos IC50 even at the highest concentration of Ver (22.5μM). In K562DOX the same concentration of Ver led to a 20 fold decrease of Bos IC50 (9nM), down to a level comparable to Bos IC50 in K562S cells (8.7nM). Thus, the inhibition of P-gp seems to restore the efficacy of Bos on proliferation inhibition and apoptosis induction in BCR-ABL+ cells. Finally, we evaluated the effect of P-gp inhibition on the phosphorylation levels of BCR-ABL with an α-phosphoTyrosine western blot. K562DOX and K562S cells were treated with Bos concentrations ranging from 0 to 80nM, and in absence or presence of Ver (5μM). Phosphorylation levels of BCR-ABL in K562S treated with Ver didn't show any difference when compared to untreated samples. In K562DOX, in absence of Ver, BCR-ABL was phosphorylated even in presence of high Bos concentration; the treatment with Ver restored the sensitivity to Bos, thus leading to a phosphorylation pattern similar to the one of K562S cells. This is compatible with increasing intracellular concentration of Bos after P-gp inhibition which results in increased molecular effect of Bos on BCR-ABL tyrosine phosphorylation. All together, these data confirm the relevance of expression levels and activity of P-gp for the intracellular concentration of Bos. The intracellular concentration of Bos is strictly related to its molecular activity on BCR-ABL and to its biological effects on Ph+ cells.

Disclosures:
Boschelli: Wyeth Pharmaceuticals: Employment.