Cancer Research Center of Toulouse, Inserm Unité Mixte de Recherche 1037, CNRS Equipe de Recherche labellisée 5294, Université de Toulouse, Centre Hospitalier Universitaire Purpan, Toulouse, France.
Institut Cochin, Université Paris Descartes, CNRS UMR 8104, INSERM U 1016, Paris, France.
Leukemia. 2014 Feb;28(2):293-301. doi: 10.1038/leu.2013.168. Epub 2013 Jun 10.
Phosphorylation by Akt on Ser 280 was reported to induce cytoplasmic retention and inactivation of CHK1 with consequent genetic instability in PTEN-/- cells. In acute myeloid leukemia cells carrying the FLT3-internal tandem duplication (ITD) mutation, we observed high rates of FLT3-ITD-dependent CHK1 Ser 280 phosphorylation. Pharmacological inhibition and RNA interference identified Pim1/2, not Akt, as effectors of this phosphorylation. Pim1 catalyzed Ser 280 phosphorylation in vitro and ectopic expression of Pim1/2-induced CHK1 phosphorylation. Ser 280 phosphorylation did not modify CHK1 localization, but facilitated its cell cycle and resistance functions in leukemic cells. FLT3, PIM or CHK1 inhibitors synergized with DNA-damaging agents to induce apoptosis, allowing cells to bypass the etoposide-induced G2/M arrest. Consistently, etoposide-induced CHK1-dependent phosphorylations of CDC25C on Ser 216 and histone H3 on Thr11 were decreased upon FLT3 inhibition. Accordingly, ectopic expression of CHK1 improved the resistance of FLT3-ITD cells and maintained histone H3 phosphorylation in response to DNA damage, whereas expression of unphosphorylated Ser 280Ala mutant did not. Finally, FLT3- and Pim-dependent phosphorylation of CHK1 on Ser 280 was confirmed in primary blasts from patients. These results identify a new pathway involved in the resistance of FLT3-ITD leukemic cells to genotoxic agents, and they constitute the first report of CHK1 Ser 280 regulation in myeloid malignancies.
磷酸化由 Akt 在 Ser 280 被报道诱导 CHK1 的细胞质保留和失活与随之而来的遗传不稳定性在 PTEN-/- 细胞。在携带 FLT3-内部串联重复（ITD）突变的急性髓系白血病细胞中，我们观察到高比例的 FLT3-ITD 依赖性 CHK1 Ser 280 磷酸化。药理学抑制和 RNA 干扰鉴定出 Pim1/2，而不是 Akt，作为这种磷酸化的效应物。Pim1 在体外催化 Ser 280 磷酸化，并且 Pim1/2 的异位表达诱导 CHK1 磷酸化。Ser 280 磷酸化没有改变 CHK1 的定位，但促进了其细胞周期和抵抗功能在白血病细胞中。FLT3、PIM 或 CHK1 抑制剂与 DNA 损伤剂协同作用诱导细胞凋亡，使细胞能够绕过依托泊苷诱导的 G2/M 期阻滞。一致地，FLT3 抑制后，依托泊苷诱导的 CHK1 依赖性 CDC25C Ser 216 和组蛋白 H3 Thr11 磷酸化减少。相应地，CHK1 的异位表达改善了 FLT3-ITD 细胞的耐药性，并维持了对 DNA 损伤的组蛋白 H3 磷酸化，而未磷酸化的 Ser 280Ala 突变体的表达则没有。最后，在患者的原始细胞中证实了 CHK1 的 FLT3 和 Pim 依赖性磷酸化在 Ser 280 上。这些结果确定了一个新的途径涉及到 FLT3-ITD 白血病细胞对遗传毒性药物的耐药性，并且它们构成了 CHK1 Ser 280 在髓样恶性肿瘤中调节的第一个报告。
