Contributions
Abstract: PB1899
Type: Publication Only
Background
In chronic myeloid leukemia (CML), the most relevant mechanisms associated with the acquisition of resistance to tyrosine kinase inhibitors (TKIs) are those dependent on the therapeutic target, the BCR-ABL oncoprotein. Point mutations and overexpression are the mechanisms take into considerations for therapeutic selection according to guidelines. However, intracellular drug concentration has proved to be very important in therapy response and in the acquisition of drug resistance. Imatinib (IMA), as other TKI, to achieving the intracellular compartment need influx transporters like OCT1 and OCTN2. In opposition, the presence of efflux transporters, like P-gP and BCRP, remove the TKIs from the cell conditioning the achievement of the therapeutic dose. Based on this, the modulation/inhibition of efflux transporters may contribute to the higher efficacy of TKIs.
Aims
The objective of this study was to evaluate the therapeutic potential of Elacridar (P-gP and BCRP inhibitor) in monotherapy and in combination with Imatinib, trying to overcome resistance in in vitro models of CML.
Methods
To achieve this goal, we used three CML cell lines: K562 cells (sensitive to Imatinib), K562-RC (8x resistant to IMA) and K562 RD (18x resistant to IMA). P-gP and BCRP activity was evaluated by flow cytometry (FC). The therapeutic potential of Elacridar was assessed in cells incubated in the absence and presence of Elacridar, in monotherapy and in combination with increasing doses of Imatinib, by the resazurin method. Cell death was evaluated by optical microscopy (May-Grünwald-Giemsa staining) and by FC (Annexin V/7-AAD). The Apoptosis, DNA Damage, and Cell Proliferation Kit was used to analyze the mechanism of cell death and proliferation. The cell cycle was evaluated by FC (PI/RNAse). The data were analyzed statistically, and the differences were considered significant when p <0.05.
Results
Resistant cell lines show higher expression and activity of P-gP and BCRP compared to the sensitive one. Elacridar in monotherapy, in tested concentrations, did not reach the IC50 in any cell line. However, the association of 250 nM of Elacridar with Imatinib modulated the resistance and re-sensitized resistant cells to Imatinib. In mechanistic terms, Elacridar in monotherapy induced cell death by apoptosis/necrosis, showing no effect in cell cycle progression. In combination with Imatinib was observed cell death by apoptosis, accompanied by increased caspase-3 activation, cleaved PARP, and DNA damage (phosphorylated H2AX). This effect was accompanied by a cell cycle arrest in S phase.
Conclusion
In conclusion, our results suggest that Elacridar in therapeutic combination with Imatinib re-sensitize resistant cell lines to Imatinib, namely in cell lines which the main mechanism of drug resistant was associated with efflux transporters. These results, if translated into clinical practice, may contribute to therapy response improvement in patients resistant to Imatinib.
This work was supported by Center of Investigation in Environment, Genetics, and Oncobiology (CIMAGO) and FCT (SFRH/BD/51994/2012).
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): ABC transporter, Chronic myeloid leukemia, Imatinib resistance
Abstract: PB1899
Type: Publication Only
Background
In chronic myeloid leukemia (CML), the most relevant mechanisms associated with the acquisition of resistance to tyrosine kinase inhibitors (TKIs) are those dependent on the therapeutic target, the BCR-ABL oncoprotein. Point mutations and overexpression are the mechanisms take into considerations for therapeutic selection according to guidelines. However, intracellular drug concentration has proved to be very important in therapy response and in the acquisition of drug resistance. Imatinib (IMA), as other TKI, to achieving the intracellular compartment need influx transporters like OCT1 and OCTN2. In opposition, the presence of efflux transporters, like P-gP and BCRP, remove the TKIs from the cell conditioning the achievement of the therapeutic dose. Based on this, the modulation/inhibition of efflux transporters may contribute to the higher efficacy of TKIs.
Aims
The objective of this study was to evaluate the therapeutic potential of Elacridar (P-gP and BCRP inhibitor) in monotherapy and in combination with Imatinib, trying to overcome resistance in in vitro models of CML.
Methods
To achieve this goal, we used three CML cell lines: K562 cells (sensitive to Imatinib), K562-RC (8x resistant to IMA) and K562 RD (18x resistant to IMA). P-gP and BCRP activity was evaluated by flow cytometry (FC). The therapeutic potential of Elacridar was assessed in cells incubated in the absence and presence of Elacridar, in monotherapy and in combination with increasing doses of Imatinib, by the resazurin method. Cell death was evaluated by optical microscopy (May-Grünwald-Giemsa staining) and by FC (Annexin V/7-AAD). The Apoptosis, DNA Damage, and Cell Proliferation Kit was used to analyze the mechanism of cell death and proliferation. The cell cycle was evaluated by FC (PI/RNAse). The data were analyzed statistically, and the differences were considered significant when p <0.05.
Results
Resistant cell lines show higher expression and activity of P-gP and BCRP compared to the sensitive one. Elacridar in monotherapy, in tested concentrations, did not reach the IC50 in any cell line. However, the association of 250 nM of Elacridar with Imatinib modulated the resistance and re-sensitized resistant cells to Imatinib. In mechanistic terms, Elacridar in monotherapy induced cell death by apoptosis/necrosis, showing no effect in cell cycle progression. In combination with Imatinib was observed cell death by apoptosis, accompanied by increased caspase-3 activation, cleaved PARP, and DNA damage (phosphorylated H2AX). This effect was accompanied by a cell cycle arrest in S phase.
Conclusion
In conclusion, our results suggest that Elacridar in therapeutic combination with Imatinib re-sensitize resistant cell lines to Imatinib, namely in cell lines which the main mechanism of drug resistant was associated with efflux transporters. These results, if translated into clinical practice, may contribute to therapy response improvement in patients resistant to Imatinib.
This work was supported by Center of Investigation in Environment, Genetics, and Oncobiology (CIMAGO) and FCT (SFRH/BD/51994/2012).
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): ABC transporter, Chronic myeloid leukemia, Imatinib resistance