Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib‑resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients.

• MeSH
• Fusion Proteins, bcr-abl metabolism   MeSH
• Drug Resistance, Neoplasm drug effects   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy   metabolism   MeSH
• Homeostasis drug effects   MeSH
• Imatinib Mesylate pharmacology   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• Mutation drug effects   MeSH
• Cell Line, Tumor MeSH
• Proteome metabolism   MeSH
• Proteomics methods   MeSH
• Signal Transduction drug effects   MeSH
• Calcium metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fusion Proteins, bcr-abl MeSH
• Imatinib Mesylate MeSH
• Protein Kinase Inhibitors MeSH
• Proteome MeSH
• Calcium MeSH

BACKGROUND AND OBJECTIVE: The availability of different tyrosine kinase inhibitors (TKIs) with distinct anti-leukemic potency enables optimization of current therapeutic regimens; however, some patients lose their therapy response and acquire TKI resistance. In this study, we describe a single-center experience of monitoring BCR-ABL1 kinase domain (KD) mutations and discuss the impact of treatment on mutation selection. METHODS: Chronic myelogenous leukemia (CML) patients treated with TKIs at the Department of Internal Medicine-Hematology and Oncology, Masaryk University and University Hospital Brno during 2003-2011 were included in this study. A total number of 100 patients who did not achieve an optimal therapy response or who lost their therapy response were screened for the presence of BCR-ABL1 KD mutations, using direct sequencing. RESULTS: Our data show that pretreatment with non-specific non-TKI drugs prior to TKI therapy does not preferentially select for initial BCR-ABL1 KD mutations, in contrast to first-line imatinib therapy, which shows a clear predominance of T315I or P-loop mutations compared with mutations located in other KD regions. In addition, the median time to detection of P-loop mutations was substantially shorter in patients treated with first-line imatinib than in those pretreated with non-TKI drugs. Furthermore, analysis of CML patients who had recurrent resistance to TKI therapy revealed possible therapy-driven selection of BCR-ABL1 KD mutations. Finally, we confirm the previously described poor prognosis of CML patients with mutations in the BCR-ABL1 KD, since 40.0% of our CML patients who harbored a BCR-ABL1 KD mutation died from CML while receiving TKI treatment. Moreover, among the patients who are still on treatment, 27.8% have already progressed. Our data also confirm the unique position of the T315I mutation with respect to its strong resistance to currently approved TKIs. CONCLUSION: On the basis of the 'real-life' data described in this study, it is possible that the therapy itself results in its failure and selects the most resistant mutations under the selective pressure of the applied therapy regimen in some CML patients who harbor BCR-ABL1 KD mutations.

• MeSH
• Fusion Proteins, bcr-abl genetics   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy   genetics   MeSH
• Adult MeSH
• Protein Kinase Inhibitors therapeutic use   MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Aged MeSH
• Protein-Tyrosine Kinases genetics   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fusion Proteins, bcr-abl MeSH
• Protein Kinase Inhibitors MeSH
• Antineoplastic Agents MeSH
• Protein-Tyrosine Kinases MeSH

BACKGROUND AND OBJECTIVE: It has been shown that the occurrence of the BCR-ABL1 T315I mutation leads to a very poor therapeutic outcome in chronic myelogenous leukemia (CML) patients treated with tyrosine kinase inhibitors. Therefore, early detection of this mutation could potentially lead to early therapeutic intervention and a better prognosis with the ongoing treatment regimen. METHODS: The detection of BCR-ABL1 kinase domain (KD) mutations was performed by direct sequencing of peripheral blood (PB), total bone marrow (BM), and BM CD34+ cells from a reported CML patient. RESULTS: In this patient, the T315I mutation was detected in BM CD34+ cells 6 months prior to its emergence in PB, suggesting evolution and expansion of the T315I mutation clone, which most likely originated from more primitive CML cells. CONCLUSION: Our finding reflects the natural development of a T315I mutation within the hematopoietic system of the reported patient and indicates the importance of BCR-ABL1 mutation monitoring in more primitive cell populations. Considering the natural history of T315I development in this reported CML case, we hypothesize that BCR-ABL1 KD mutations may be pre-concentrated in more primitive CML cells, which subsequently expand into the PB. These findings may have future implications for the strategy used for detecting BCR-ABL1 mutations.

• MeSH
• Antigens, CD34 analysis   MeSH
• Fusion Proteins, bcr-abl genetics   MeSH
• Benzamides MeSH
• Bone Marrow Cells cytology   enzymology   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive blood   drug therapy   genetics   MeSH
• Hydroxyurea therapeutic use   MeSH
• Imatinib Mesylate MeSH
• Humans MeSH
• Mutation MeSH
• Piperazines therapeutic use   MeSH
• Disease Progression MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Pyrimidines therapeutic use   MeSH
• Aged MeSH
• Protein-Tyrosine Kinases genetics   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, CD34 MeSH
• Fusion Proteins, bcr-abl MeSH
• Benzamides MeSH
• Hydroxyurea MeSH
• Imatinib Mesylate MeSH
• nilotinib MeSH Browser
• Piperazines MeSH
• Antineoplastic Agents MeSH
• Pyrimidines MeSH
• Protein-Tyrosine Kinases MeSH