Mechanisms of imatinib-resistance in clinical patients and imatinib-resistant cell line showing over-expression of Lyn in chronic myelogenous leukemia (CML)

(1)我们分析了广岛地区142例CML患者的临床资料和样本。在新鲜病例中，60个月时细胞遗传学缓解率为CCyR达到的94%。在新鲜病例中，12个月时MMR为64%，MMR为90%，60个月时CMR为35%。对前6个月的给药着装进行分析。与接受超过400 mg/天剂量的患者相比，接受低于400 mg/天剂量的患者显示出较差的反应。接受超过500 mg/d的患者在实现0拷贝BCR-ABL mRNA方面表现出更好的反应。在快速3个月内BCR-ABL mRNA的下降呈现出三个阶段，这证实了BCR-ABL-FISH数据。在总共42例患者中分析了ABL基因突变，但在伊马替尼治疗过程中，一些CML患者中林恩mRNA升高。(2)使用细胞系的分析：我们建立了伊马替尼敏感（MYL）和耐药（MYL-R）细胞系。伊马替尼刺激引起两者中CrkL的磷酸化，并且引起MYL中的凋亡，而不是MYL-R中的凋亡，这表明MYL-R中的耐药机制是伊马替尼非依赖性的。在MYL-R中观察到林恩mRNA和蛋白的过表达，但在MYL中未观察到。观察到MYL-R而不是MYL中JNK、ERK、STAT 5和Bim的磷酸化。Src家族激酶抑制剂和唑来膦酸均抑制MYL-R中的细胞数量。伊马替尼敏感性部分恢复-IFNα、唑来膦酸、PP 2、CGP 76030、FK 228与伊马替尼有协同作用。达沙替尼还抑制MYL-R细胞中BCR-ABL的磷酸化。TKI-737还抑制MYL的细胞磷酸化。这些结果表明，这些新试剂具有克服CML中伊马替尼耐药性的潜力。

(1) Analyses using clinical data and samples We analyzed total 142 CML patients in Hiroshima area Cytogenetic response was 94% of CCyR achievement at 60 months in fresh cases. Molecular responses were; 64% of MMR at 12 months, 90% of MMR, and 35% of CMR at 60 months in fresh cases. Administration dress in the first 6 months were analyzed. Patients receiving dose of less than 400mg/day revealed poorer response as compared to the patients who received more than 400mg/day. Patients receiving mom than 500mg/day showed better response in terms of achievement of 0 copy of BCR-ABL mRNA The decline of the BCR-ABL mRNA in the fast 3 months showed three-phase, which paralleled the BCR-ABL-FISH data Mutation of ABL gene was analyzed in total 42 patients, but mutation was absent Lyn mRNA was elevated in some of the CML patients during the course of imatinib treatment.(2) Analyses using cell lines: We established imatinib-sensitive (MYL) and resistant-(MYL-R) cell lines. ABL mutations were absent Imatinib stimulation caused phosphorylation of CrkL in the both, and caused apoptosis in MYL but not in MYL-R, suggesting that the mechanisms of resistance in MYL-R was imatinib-independent. Over-expressions of Lyn mRNA and protein were observed in MYL-R, but not in MYL Phosphorylations of JNK, ERK, STAT5, and Bim were observed MYL-R rather than MYL Both Src-family-kinase inhibitor and zoledronic acid suppressed the cell number in MYL-R Transfection of Lyn-siRNA in MYL-R demonstrated decrease of the cell number, decrease of the viable cells, increase of apoptotic cells, and partial recovery of imatinib-sensitivity-IFNα, zoledronic acid, PP2, CGP76030, FK228 showed synergistic effects with imatinib. Dasatinib also suppressed the phosphorylation of BCR-ABL in MYL-R cells. TKI-737 also suppressed the cell phosphorylation of MYL These results demonstrate that these new reagents have potential to overcome imatinib-resistance in CML.