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Abstract：
UNASSIGNED: Ruxolitinib has been approved by the US FDA for the treatment of myeloproliferative neoplasms such as polycythemia vera and primary myelofibrosis. Ruxolitinib will remain a main stay in the treatment of MPN patients due to its effective therapeutic benefits. However, there have been instances of ruxolitinib resistance in MPN patients. As JAK2 is a direct target of ruxolitinib, we generated ruxolitinib-resistant clones to find out the mechanism of resistance.
UNASSIGNED: Cell-based screening strategy was used to detect the ruxolitinib-resistant mutations in JAK2. The Sanger sequencing method was used to detect the point mutations in JAK2. Mutations were re-introduced using the site-directed mutagenesis method and stably expressed in Ba/F3 cells. Drug sensitivities against the JAK2 inhibitors were measured using an MTS-based assay. JAK2 and STAT5 activation levels and total proteins were measured using immunoblotting. Computational docking studies were performed using the Glide module of Schrodinger Maestro software.
UNASSIGNED: In this study, we have recovered seven residues in the kinase domain of JAK2 that affect ruxolitinib sensitivity. All these mutations confer cross-resistance across the panel of JAK2 kinase inhibitors except JAK2-L983F. JAK2-L983F reduces the sensitivity towards ruxolitinib. However, it is sensitive towards fedratinib indicating that our screen identifies the drug-specific resistance profiles. All the ruxolitinib-resistant JAK2 variants displayed sensitivity towards type II JAK2 inhibitor CHZ-868. In this study, we also found that JAK1-L1010F (homologous JAK2-L983F) is highly resistant towards ruxolitinib suggesting the possibility of JAK1 escape mutations in JAK2-driven MPNs and JAK1 mutated ALL. Finally, our study also shows that HSP90 inhibitors are potent against ruxolitinib-resistant variants through the JAK2 degradation and provides the rationale for clinical evaluation of potent HSP90 inhibitors in genetic resistance driven by JAK2 inhibitors.
UNASSIGNED: Our study identifies JAK1 and JAK2 resistance variants against the type I JAK2 inhibitors ruxolitinib, fedratinib, and lestaurtinib. The sensitivity of these resistant variants towards the type II JAK2 inhibitor CHZ-868 indicates that this mode of type II JAK2 inhibition is a potential therapeutic approach against ruxolitinib refractory leukemia. This also proposes the development of potent and specific type II JAK2 inhibitors using ruxolitinib-resistance variants as a prototype.
UNASSIGNED: Cell-based screening strategy was used to detect the ruxolitinib-resistant mutations in JAK2. The Sanger sequencing method was used to detect the point mutations in JAK2. Mutations were re-introduced using the site-directed mutagenesis method and stably expressed in Ba/F3 cells. Drug sensitivities against the JAK2 inhibitors were measured using an MTS-based assay. JAK2 and STAT5 activation levels and total proteins were measured using immunoblotting. Computational docking studies were performed using the Glide module of Schrodinger Maestro software.
UNASSIGNED: In this study, we have recovered seven residues in the kinase domain of JAK2 that affect ruxolitinib sensitivity. All these mutations confer cross-resistance across the panel of JAK2 kinase inhibitors except JAK2-L983F. JAK2-L983F reduces the sensitivity towards ruxolitinib. However, it is sensitive towards fedratinib indicating that our screen identifies the drug-specific resistance profiles. All the ruxolitinib-resistant JAK2 variants displayed sensitivity towards type II JAK2 inhibitor CHZ-868. In this study, we also found that JAK1-L1010F (homologous JAK2-L983F) is highly resistant towards ruxolitinib suggesting the possibility of JAK1 escape mutations in JAK2-driven MPNs and JAK1 mutated ALL. Finally, our study also shows that HSP90 inhibitors are potent against ruxolitinib-resistant variants through the JAK2 degradation and provides the rationale for clinical evaluation of potent HSP90 inhibitors in genetic resistance driven by JAK2 inhibitors.
UNASSIGNED: Our study identifies JAK1 and JAK2 resistance variants against the type I JAK2 inhibitors ruxolitinib, fedratinib, and lestaurtinib. The sensitivity of these resistant variants towards the type II JAK2 inhibitor CHZ-868 indicates that this mode of type II JAK2 inhibition is a potential therapeutic approach against ruxolitinib refractory leukemia. This also proposes the development of potent and specific type II JAK2 inhibitors using ruxolitinib-resistance variants as a prototype.
摘要：
■Ruxolitinib已被美国FDA批准用于治疗骨髓增殖性肿瘤，如真性红细胞增多症和原发性骨髓纤维化。由于其有效的治疗益处，鲁索替尼仍将是MPN患者治疗的主要药物。然而,MPN患者中曾出现鲁索利替尼耐药.由于JAK2是鲁索替尼的直接靶标，我们产生了鲁索替尼耐药克隆,以找出耐药机制.
■基于细胞的筛选策略用于检测JAK2中鲁索替尼耐药突变。采用Sanger测序法检测JAK2中的点突变。使用定点诱变方法重新引入突变并在Ba/F3细胞中稳定表达。使用基于MTS的测定法测量针对JAK2抑制剂的药物敏感性。使用免疫印迹法测量JAK2和STAT5活化水平和总蛋白。使用SchrodingerMaestro软件的Glide模块进行计算对接研究。
■在这项研究中，我们在JAK2激酶结构域中回收了影响鲁索利替尼敏感性的7个残基.所有这些突变赋予除JAK2-L983F之外的JAK2激酶抑制剂组的交叉抗性。JAK2-L983F降低对鲁索替尼的敏感性。然而,它对fedratinib敏感,表明我们的筛查可识别药物特异性耐药谱.所有耐鲁索替尼的JAK2变体都显示出对II型JAK2抑制剂CHZ-868的敏感性。在这项研究中,我们还发现JAK1-L1010F(同源JAK2-L983F)对鲁索替尼具有高度抗性,提示在JAK2驱动的MPN和JAK1突变的ALL中存在JAK1逃逸突变的可能性.最后,我们的研究还表明,HSP90抑制剂通过JAK2降解对鲁索替尼耐药变体有效,并为临床评估由JAK2抑制剂驱动的遗传耐药中的有效HSP90抑制剂提供了理论基础.
■我们的研究确定了针对I型JAK2抑制剂ruxolitinib的JAK1和JAK2抗性变体，费地替尼,和列妥替尼.这些抗性变体对II型JAK2抑制剂CHZ-868的敏感性表明II型JAK2抑制的这种模式是针对鲁索替尼难治性白血病的潜在治疗方法。这也提出了使用鲁索替尼抗性变体作为原型开发有效且特异性的II型JAK2抑制剂。
■基于细胞的筛选策略用于检测JAK2中鲁索替尼耐药突变。采用Sanger测序法检测JAK2中的点突变。使用定点诱变方法重新引入突变并在Ba/F3细胞中稳定表达。使用基于MTS的测定法测量针对JAK2抑制剂的药物敏感性。使用免疫印迹法测量JAK2和STAT5活化水平和总蛋白。使用SchrodingerMaestro软件的Glide模块进行计算对接研究。
■在这项研究中，我们在JAK2激酶结构域中回收了影响鲁索利替尼敏感性的7个残基.所有这些突变赋予除JAK2-L983F之外的JAK2激酶抑制剂组的交叉抗性。JAK2-L983F降低对鲁索替尼的敏感性。然而,它对fedratinib敏感,表明我们的筛查可识别药物特异性耐药谱.所有耐鲁索替尼的JAK2变体都显示出对II型JAK2抑制剂CHZ-868的敏感性。在这项研究中,我们还发现JAK1-L1010F(同源JAK2-L983F)对鲁索替尼具有高度抗性,提示在JAK2驱动的MPN和JAK1突变的ALL中存在JAK1逃逸突变的可能性.最后,我们的研究还表明,HSP90抑制剂通过JAK2降解对鲁索替尼耐药变体有效,并为临床评估由JAK2抑制剂驱动的遗传耐药中的有效HSP90抑制剂提供了理论基础.
■我们的研究确定了针对I型JAK2抑制剂ruxolitinib的JAK1和JAK2抗性变体，费地替尼,和列妥替尼.这些抗性变体对II型JAK2抑制剂CHZ-868的敏感性表明II型JAK2抑制的这种模式是针对鲁索替尼难治性白血病的潜在治疗方法。这也提出了使用鲁索替尼抗性变体作为原型开发有效且特异性的II型JAK2抑制剂。
