MTOR INHIBITION IN CML: A NEW THERAPEUTIC OPTION
(Abstract release date: 05/19/16)
EHA Library. Bela Sarmento-Ribeiro A. 06/09/16; 134719; PB1819
Dr. Ana Bela Sarmento-Ribeiro
Contributions
Contributions
Abstract
Abstract: PB1819
Type: Publication Only
Background
BCR-ABL1 fusion gene, which encodes an oncoprotein with a deregulated tyrosine kinase activity, is the hallmark of chronic myeloid leukemia (CML) – a myeloproliferative disorder. Despite the high rate of response to treatment with Imatinib and other tyrosine kinase inhibitors (TKI), the number of patients with suboptimal response or resistance to TKI has been increased. The BCR-ABL oncoprotein activates multiples signaling pathways responsible for tumor cells characteristic, namely the high cellular proliferation rate and resistance to apoptosis. One of these pathways is the PI3K/AKT/mTOR pathway, which is correlated with the increase in cell survival and resistance to apoptosis. Leukemic stem cells use these pathways as a mechanism of defense against treatment and as tumor maintenance.
Aims
The aim of this study was to evaluate the therapeutic potential of the mTOR inhibitor, Everolimus, in CML cell lines sensitive and resistant to TKI and in primary cultures of CML patients.
Methods
For this purpose, we used a CML cell line sensitive to Imatinib, the K562 cells, and established two sub-cell lines resistant to Imatinib, the K562-RC and K562-RD cells. Cell lines were treated in the absence and presence of different concentrations of Everolimus and the effect in cell viability was analyzed by the resazurin assay. Cell death was determined by flow cytometry (FC) using the Annexin V/Propidium Iodide staining. The cell cycle analysis was accessed using Propidium Iodide incorporation by FC. Ex-vivo studies were performed in peripheral blood samples of 56 patients under TKI treatment. In our cohort, 52 (93%) patients presented molecular response and 4 (7%) cytogenetic response. Moreover, 39 (74%) patients were treated with Imatinib, and 14 (26%) with 2nd or 3rd generation TKI. Patients’ samples were culture with increasing concentrations of Everolimus during 48h. Cytotoxic effect was evaluated by FC in different cell populations using Annexin V staining.
Results
Our results show that Everolimus induced a reduction in cell lines viability, with an IC50 of 20µM for sensitive cells and 25µM for Imatinib resistant cell lines. The cell death was induced by apoptosis and this drug has also an antiproliferative effect through an arrest in cell cycle progression in G0/G1.In ex-vivo studies, Everolimus reduced cell viability by increasing apoptosis of hematopoietic stem cells (CD34+ cells) without cytotoxicity to lymphocytes. In the the dose of 25 µM this mTOR inhibitor induced in CD34 cells an increase of 19% of these cells positive to annexin V compared with control, and only 8% of lymphocytes are in apoptosis. Comparing the response to TKI treatment with sensibility to Everolimus, we observed a tendency to higher efficacy in patients with cytogenetic response (CR) comparing with patients under molecular response (MR) (24,5% vs 18,7% of CD34 cells positive to annexin V). When compared patients under Imatinib treatment versus patients treated with 2nd or 3rd generation TKI, we observed a better response to Everolimus in the second group, also associated with lower toxicity to lymphocytes.
Conclusion
Our results reveal the efficacy of Everolimus in inducing cell death in CML cells, without cytotoxicity to normal cells, suggesting that Everolimus could be an alternative targeted therapeutic approach in CML patients. However, it is important to increase the number of patients in the study to confirm our results.This work was support by CIMAGO (Project 18/12) and R.A. was supported by FCT with a PhD grant (SFRH/BD/51994/2012).
Session topic: E-poster
Keyword(s): Apoptosis, CD34+ cells, Chronic myeloid leukemia, MTOR
Type: Publication Only
Background
BCR-ABL1 fusion gene, which encodes an oncoprotein with a deregulated tyrosine kinase activity, is the hallmark of chronic myeloid leukemia (CML) – a myeloproliferative disorder. Despite the high rate of response to treatment with Imatinib and other tyrosine kinase inhibitors (TKI), the number of patients with suboptimal response or resistance to TKI has been increased. The BCR-ABL oncoprotein activates multiples signaling pathways responsible for tumor cells characteristic, namely the high cellular proliferation rate and resistance to apoptosis. One of these pathways is the PI3K/AKT/mTOR pathway, which is correlated with the increase in cell survival and resistance to apoptosis. Leukemic stem cells use these pathways as a mechanism of defense against treatment and as tumor maintenance.
Aims
The aim of this study was to evaluate the therapeutic potential of the mTOR inhibitor, Everolimus, in CML cell lines sensitive and resistant to TKI and in primary cultures of CML patients.
Methods
For this purpose, we used a CML cell line sensitive to Imatinib, the K562 cells, and established two sub-cell lines resistant to Imatinib, the K562-RC and K562-RD cells. Cell lines were treated in the absence and presence of different concentrations of Everolimus and the effect in cell viability was analyzed by the resazurin assay. Cell death was determined by flow cytometry (FC) using the Annexin V/Propidium Iodide staining. The cell cycle analysis was accessed using Propidium Iodide incorporation by FC. Ex-vivo studies were performed in peripheral blood samples of 56 patients under TKI treatment. In our cohort, 52 (93%) patients presented molecular response and 4 (7%) cytogenetic response. Moreover, 39 (74%) patients were treated with Imatinib, and 14 (26%) with 2nd or 3rd generation TKI. Patients’ samples were culture with increasing concentrations of Everolimus during 48h. Cytotoxic effect was evaluated by FC in different cell populations using Annexin V staining.
Results
Our results show that Everolimus induced a reduction in cell lines viability, with an IC50 of 20µM for sensitive cells and 25µM for Imatinib resistant cell lines. The cell death was induced by apoptosis and this drug has also an antiproliferative effect through an arrest in cell cycle progression in G0/G1.In ex-vivo studies, Everolimus reduced cell viability by increasing apoptosis of hematopoietic stem cells (CD34+ cells) without cytotoxicity to lymphocytes. In the the dose of 25 µM this mTOR inhibitor induced in CD34 cells an increase of 19% of these cells positive to annexin V compared with control, and only 8% of lymphocytes are in apoptosis. Comparing the response to TKI treatment with sensibility to Everolimus, we observed a tendency to higher efficacy in patients with cytogenetic response (CR) comparing with patients under molecular response (MR) (24,5% vs 18,7% of CD34 cells positive to annexin V). When compared patients under Imatinib treatment versus patients treated with 2nd or 3rd generation TKI, we observed a better response to Everolimus in the second group, also associated with lower toxicity to lymphocytes.
Conclusion
Our results reveal the efficacy of Everolimus in inducing cell death in CML cells, without cytotoxicity to normal cells, suggesting that Everolimus could be an alternative targeted therapeutic approach in CML patients. However, it is important to increase the number of patients in the study to confirm our results.This work was support by CIMAGO (Project 18/12) and R.A. was supported by FCT with a PhD grant (SFRH/BD/51994/2012).
Session topic: E-poster
Keyword(s): Apoptosis, CD34+ cells, Chronic myeloid leukemia, MTOR
Abstract: PB1819
Type: Publication Only
Background
BCR-ABL1 fusion gene, which encodes an oncoprotein with a deregulated tyrosine kinase activity, is the hallmark of chronic myeloid leukemia (CML) – a myeloproliferative disorder. Despite the high rate of response to treatment with Imatinib and other tyrosine kinase inhibitors (TKI), the number of patients with suboptimal response or resistance to TKI has been increased. The BCR-ABL oncoprotein activates multiples signaling pathways responsible for tumor cells characteristic, namely the high cellular proliferation rate and resistance to apoptosis. One of these pathways is the PI3K/AKT/mTOR pathway, which is correlated with the increase in cell survival and resistance to apoptosis. Leukemic stem cells use these pathways as a mechanism of defense against treatment and as tumor maintenance.
Aims
The aim of this study was to evaluate the therapeutic potential of the mTOR inhibitor, Everolimus, in CML cell lines sensitive and resistant to TKI and in primary cultures of CML patients.
Methods
For this purpose, we used a CML cell line sensitive to Imatinib, the K562 cells, and established two sub-cell lines resistant to Imatinib, the K562-RC and K562-RD cells. Cell lines were treated in the absence and presence of different concentrations of Everolimus and the effect in cell viability was analyzed by the resazurin assay. Cell death was determined by flow cytometry (FC) using the Annexin V/Propidium Iodide staining. The cell cycle analysis was accessed using Propidium Iodide incorporation by FC. Ex-vivo studies were performed in peripheral blood samples of 56 patients under TKI treatment. In our cohort, 52 (93%) patients presented molecular response and 4 (7%) cytogenetic response. Moreover, 39 (74%) patients were treated with Imatinib, and 14 (26%) with 2nd or 3rd generation TKI. Patients’ samples were culture with increasing concentrations of Everolimus during 48h. Cytotoxic effect was evaluated by FC in different cell populations using Annexin V staining.
Results
Our results show that Everolimus induced a reduction in cell lines viability, with an IC50 of 20µM for sensitive cells and 25µM for Imatinib resistant cell lines. The cell death was induced by apoptosis and this drug has also an antiproliferative effect through an arrest in cell cycle progression in G0/G1.In ex-vivo studies, Everolimus reduced cell viability by increasing apoptosis of hematopoietic stem cells (CD34+ cells) without cytotoxicity to lymphocytes. In the the dose of 25 µM this mTOR inhibitor induced in CD34 cells an increase of 19% of these cells positive to annexin V compared with control, and only 8% of lymphocytes are in apoptosis. Comparing the response to TKI treatment with sensibility to Everolimus, we observed a tendency to higher efficacy in patients with cytogenetic response (CR) comparing with patients under molecular response (MR) (24,5% vs 18,7% of CD34 cells positive to annexin V). When compared patients under Imatinib treatment versus patients treated with 2nd or 3rd generation TKI, we observed a better response to Everolimus in the second group, also associated with lower toxicity to lymphocytes.
Conclusion
Our results reveal the efficacy of Everolimus in inducing cell death in CML cells, without cytotoxicity to normal cells, suggesting that Everolimus could be an alternative targeted therapeutic approach in CML patients. However, it is important to increase the number of patients in the study to confirm our results.This work was support by CIMAGO (Project 18/12) and R.A. was supported by FCT with a PhD grant (SFRH/BD/51994/2012).
Session topic: E-poster
Keyword(s): Apoptosis, CD34+ cells, Chronic myeloid leukemia, MTOR
Type: Publication Only
Background
BCR-ABL1 fusion gene, which encodes an oncoprotein with a deregulated tyrosine kinase activity, is the hallmark of chronic myeloid leukemia (CML) – a myeloproliferative disorder. Despite the high rate of response to treatment with Imatinib and other tyrosine kinase inhibitors (TKI), the number of patients with suboptimal response or resistance to TKI has been increased. The BCR-ABL oncoprotein activates multiples signaling pathways responsible for tumor cells characteristic, namely the high cellular proliferation rate and resistance to apoptosis. One of these pathways is the PI3K/AKT/mTOR pathway, which is correlated with the increase in cell survival and resistance to apoptosis. Leukemic stem cells use these pathways as a mechanism of defense against treatment and as tumor maintenance.
Aims
The aim of this study was to evaluate the therapeutic potential of the mTOR inhibitor, Everolimus, in CML cell lines sensitive and resistant to TKI and in primary cultures of CML patients.
Methods
For this purpose, we used a CML cell line sensitive to Imatinib, the K562 cells, and established two sub-cell lines resistant to Imatinib, the K562-RC and K562-RD cells. Cell lines were treated in the absence and presence of different concentrations of Everolimus and the effect in cell viability was analyzed by the resazurin assay. Cell death was determined by flow cytometry (FC) using the Annexin V/Propidium Iodide staining. The cell cycle analysis was accessed using Propidium Iodide incorporation by FC. Ex-vivo studies were performed in peripheral blood samples of 56 patients under TKI treatment. In our cohort, 52 (93%) patients presented molecular response and 4 (7%) cytogenetic response. Moreover, 39 (74%) patients were treated with Imatinib, and 14 (26%) with 2nd or 3rd generation TKI. Patients’ samples were culture with increasing concentrations of Everolimus during 48h. Cytotoxic effect was evaluated by FC in different cell populations using Annexin V staining.
Results
Our results show that Everolimus induced a reduction in cell lines viability, with an IC50 of 20µM for sensitive cells and 25µM for Imatinib resistant cell lines. The cell death was induced by apoptosis and this drug has also an antiproliferative effect through an arrest in cell cycle progression in G0/G1.In ex-vivo studies, Everolimus reduced cell viability by increasing apoptosis of hematopoietic stem cells (CD34+ cells) without cytotoxicity to lymphocytes. In the the dose of 25 µM this mTOR inhibitor induced in CD34 cells an increase of 19% of these cells positive to annexin V compared with control, and only 8% of lymphocytes are in apoptosis. Comparing the response to TKI treatment with sensibility to Everolimus, we observed a tendency to higher efficacy in patients with cytogenetic response (CR) comparing with patients under molecular response (MR) (24,5% vs 18,7% of CD34 cells positive to annexin V). When compared patients under Imatinib treatment versus patients treated with 2nd or 3rd generation TKI, we observed a better response to Everolimus in the second group, also associated with lower toxicity to lymphocytes.
Conclusion
Our results reveal the efficacy of Everolimus in inducing cell death in CML cells, without cytotoxicity to normal cells, suggesting that Everolimus could be an alternative targeted therapeutic approach in CML patients. However, it is important to increase the number of patients in the study to confirm our results.This work was support by CIMAGO (Project 18/12) and R.A. was supported by FCT with a PhD grant (SFRH/BD/51994/2012).
Session topic: E-poster
Keyword(s): Apoptosis, CD34+ cells, Chronic myeloid leukemia, MTOR
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