Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus Dresden der TU Dresden, Fetscherstr. 74, 01307 Dresden, Germany.
Ann Hematol. 2010 Jul;89(7):653-62. doi: 10.1007/s00277-009-0889-1. Epub 2010 Jan 30.
Treatment of acute myeloid leukemia (AML) remains challenging with many patients harboring unfavorable prognostic parameters such as FLT3 internal tandem duplication (FLT3-ITD) mutations leading to a constitutively activated FLT3-receptor tyrosine kinase (RTK). Activation of proteins by phosphorylation of tyrosine residues is a common mechanism in leukemia development. Therefore, specific tyrosine kinase inhibitors (TKI) have been developed for AML therapy and are currently under investigation. The staurosporine derivate PKC412 (Midostaurin) was found to be an effective inhibitor of the FLT3-RTK and is currently undergoing clinical trials for FLT3-mutated AML patients. Since resistance towards TKIs has been observed in vitro and in clinical trials, we have generated a PKC412-resistant clone (MV4-11r) of the human myelomonoblastic cell line MV4-11, which carries a homozygous FLT3-ITD mutation. MV4-11r displayed higher vitality after addition of PKC412 compared with MV4-11 with a pronounced reduction of apoptotic cells. Cytogenetic characterization revealed the acquisition of additional aberrations in the resistant cell line such as clonal alterations at chromosome 13q with additional FLT3 signals. Microarray analysis revealed significant expression changes in several genes prior to and after incubation with PKC412. The expression status of candidate genes being regulated by FLT-ITD like JAG1, p53, MCL-1, C-KIT, and FLT3/-L was confirmed by real-time PCR. In summary, resistance against PKC412 appears to be mediated by up-regulation of anti-apoptotic genes and down-regulation of proapoptotic signals as well as genes that are involved in normal and malignant hematopoiesis.
急性髓细胞白血病（AML）的治疗仍然具有挑战性，许多患者存在不良预后因素，如 FLT3 内部串联重复（FLT3-ITD）突变，导致 FLT3-受体酪氨酸激酶（RTK）持续激活。磷酸化酪氨酸残基激活蛋白是白血病发生的常见机制。因此，已经开发了针对 AML 治疗的特异性酪氨酸激酶抑制剂（TKI），并正在进行研究。发现 staurosporine 衍生物 PKC412（Midostaurin）是 FLT3-RTK 的有效抑制剂，目前正在进行针对 FLT3 突变 AML 患者的临床试验。由于已经在体外和临床试验中观察到对 TKI 的耐药性，我们生成了人髓单核细胞白血病细胞系 MV4-11 的 PKC412 耐药克隆（MV4-11r），该细胞系携带纯合 FLT3-ITD 突变。与 MV4-11 相比，添加 PKC412 后 MV4-11r 的活力更高，凋亡细胞明显减少。细胞遗传学特征显示，耐药细胞系中获得了额外的异常，例如染色体 13q 上的克隆改变，并伴有额外的 FLT3 信号。微阵列分析显示，在与 PKC412 孵育前后，几个基因的表达发生了显著变化。通过实时 PCR 证实了受 FLT-ITD 调节的候选基因（如 JAG1、p53、MCL-1、C-KIT 和 FLT3/-L）的表达状态。总之，对 PKC412 的耐药性似乎是通过上调抗凋亡基因和下调促凋亡信号以及参与正常和恶性造血的基因来介导的。
