Toman O, Kabickova T, Vit O, Fiser R, Polakova K Machova, Zach J, Linhartova J, Vyoral D, Petrak J
Institute of Hematology and Blood Transfusion, CZ-12820 Prague 2, Czech Republic.
BIOCEV, First Faculty of Medicine, Charles University in Prague, CZ-25250 Vestec, Czech Republic.
Oncol Rep. 2016 Sep;36(3):1258-68. doi: 10.3892/or.2016.4945. Epub 2016 Jul 18.
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.
慢性髓性白血病（CML）的治疗在甲磺酸伊马替尼用于临床后显著改善了患者的预后。然而，一部分患者会对伊马替尼和其他酪氨酸激酶抑制剂（TKIs）产生耐药性，主要原因是融合的BCR-ABL癌基因激酶结构域编码区域的点突变。为了确定伊马替尼耐药CML细胞中的潜在治疗靶点，我们通过在生长培养基中长时间暴露于伊马替尼，获得了伊马替尼耐药的CML-T1人细胞系克隆（CML-T1/IR）。突变分析表明，BCR-ABL中的Y235H突变可能是CML-T1/IR对伊马替尼耐药的主要原因。为了确定选择性消除伊马替尼耐药细胞的替代治疗靶点，我们使用二维电泳-质谱（2-DE-MS）比较了CML-T1和CML-T1/IR细胞的蛋白质组图谱。我们鉴定出8种差异表达蛋白，耐药细胞中Na+/H+交换调节因子1（NHERF1）强烈上调，这表明该蛋白可能影响CML-T1/IR细胞的胞质pH、Ca2+浓度或Wnt等信号通路。我们测试了几种化合物，包括临床使用的干扰上述过程的药物，并测试了它们对CML-T1和CML-T1/IR细胞的相对毒性。钙通道阻滞剂、钙信号拮抗剂和钙稳态调节剂，即毒胡萝卜素、离子霉素、维拉帕米、羧基酰胺三唑以及免疫抑制药物环孢素A和他克莫司（FK-506）对CML-T1/IR细胞具有选择性毒性。本研究推测了这些化合物在CML-T1/IR细胞中的假定细胞靶点。我们提出Ca2+稳态可能是对TKIs耐药的CML细胞的潜在治疗靶点。我们证明蛋白质组学方法可用于表征CML细胞的TKI耐药群体，为患者提供未来的个体化治疗选择。
