Inhibition of Pim kinases in acute myeloid leukemia

描述(由申请人提供)： 急性髓系白血病(AML)发病率高，治疗反应差，是退伍军人重要的健康问题。值得注意的是，FMS样酪氨酸激酶3(Flt3)受体酪氨酸激酶基因的内部串联重复(ITD)突变存在于三分之一的AML患者的AML细胞中，并与化疗后的快速复发有关。Flt3抑制剂有活性，但反应是有限的和短暂的。PIM-1是一种丝氨酸苏氨酸激酶，在AML细胞中表达，在有这种分子异常的AML细胞中，在Flt3-ITD下游过表达。PIM-1激酶调节多种与细胞增殖、细胞周期、细胞凋亡和耐药有关的蛋白，其中包括c-myc、促凋亡蛋白Bad和抗凋亡蛋白Mcl-1，还参与DNA修复。我们发现在Flt3-ITD AML细胞中，Pim-1也能磷酸化Flt3并在正反馈环中促进其异常信号转导，并且Pim-1能使Flt3-ITD AML细胞对Flt3抑制剂诱导的细胞凋亡敏感。我们随后发现，Pim激酶抑制剂也能使Flt3-ITD AML细胞对用于治疗AML的化疗药物诱导的凋亡敏感。PIM-1底物蛋白也在AML干细胞的关键途径中表达，这些蛋白的PIM-1磷酸化可能保护AML干细胞免受化疗和Flt3抑制剂的影响。PIM激酶抑制剂目前处于临床前和临床开发阶段。1.这项赠款提案的总体假设是，Pim-1在Flt3-ITD AML对现有治疗的耐药中发挥关键作用，抑制Pim-1将通过消除多种耐药机制和疾病进展来改善Flt3-ITD AML的治疗结果。这项工作的具体目的是：1.确定Pim激酶抑制使Flt3-ITD AML细胞对化疗药物和Flt3抑制剂诱导凋亡的敏感性的机制；2.优化Flt3-ITD AML中Pim激酶抑制剂、化疗药物和Flt3抑制剂的给药程序；3.测试Pim激酶抑制剂与化疗药物和Flt3抑制剂在体内给药对Flt3-ITD AML细胞、AML干细胞和正常造血细胞的影响。使用体外培养系统和体内模型，将在AML细胞系和患者样本中研究Pim-1激酶抑制的效果。在目标1中，我们将验证Pim激酶抑制通过抑制Flt3-ITD信号、改变促和抗凋亡蛋白的表达和激活、促进活性氧的产生和/或抑制DNA损伤的修复而使Flt3-ITD细胞对凋亡敏感的假说。在目标1中获得的机械性见解将指导与化疗药物和Flt3抑制剂的暴露相关的Pim激酶抑制计划的设计。日程安排的方法将在体外进行测试，以细胞凋亡为读数。此外，抑制Pim激酶对细胞周期的影响可能会导致对药物的动力学耐药性，特别是阿糖胞苷，并将进行测试。最后，将研究对正常造血细胞的影响，以建立治疗指标。在目标3中，在目标2中进行体外优化的策略将在体内测试免疫缺陷小鼠模型。预计拟议的工作将导致在治疗Flt3-ITD AML中加入Pim激酶抑制剂的方案的开发和优化，目的是改善这一不利的AML亚型的预后。

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is an important health concern for veterans, due to its high incidence and frequent poor response to treatment. Notably, internal tandem duplication (ITD) mutations of the fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase genes are present AML cells in a third of AML patients and are associated with rapid relapse following chemotherapy. FLT3 inhibitors have activity, but responses are limited and transient. Pim-1 kinase is a serine threonine kinase that is expressed in AML cells and is overexpressed downstream of FLT3-ITD in AML cells with this molecular abnormality. Pim-1 kinase regulates diverse proteins involved in proliferation, cell cycle, apoptosis and drug resistance, including, among others, c-MYC, the pro-apoptotic protein Bad and the anti-apoptotic protein Mcl-1, and is also implicated in DNA repair. We demonstrated that Pim-1 also phosphorylates FLT3 and promotes its aberrant signaling in a positive feedback loop in FLT3-ITD AML cells, and that Pym kinase inhibitors sensitize FLT3-ITD AML cells to apoptosis induction by FLT3 inhibitors. We then found that Pim kinase inhibitors also sensitize FLT3-ITD AML cells to apoptosis induction by chemotherapy drugs used to treat AML. Pim-1 substrate proteins are also expressed in key pathways in AML stem cells, and Pim-1 phosphorylation of these proteins may protect AML stem cells from the effects of chemotherapy and of FLT3 inhibitors. Pim kinase inhibitors are in current preclinical and clinical development. 1. The overall hypotheses of this grant proposal is that Pim-1 plays a key role in resistance of FLT3-ITD AML to available treatments, and that inhibition of Pim kinase will improve treatment outcomes in FLT3-ITD AML by abrogating multiple mechanisms of drug resistance and of disease progression. The Specific Aims of the proposed work are: 1. To determine the mechanisms by which Pim kinase inhibition sensitizes FLT3-ITD AML cells to induction of apoptosis by chemotherapy drugs and by FLT3 inhibitors; 2. To optimize scheduling of administration of Pim kinase inhibitors, chemotherapy drugs and by FLT3 inhibitors in FLT3-ITD AML; and 3. To test the effects of in vivo administration of Pim kinase inhibitors with chemotherapy drugs and with FLT3 inhibitors on FLT3-ITD AML cells and AML stem cells and on normal hematopoietic cells. The effects of Pim-1 kinase inhibition will be studied in AML cell lines and patient samples using both in vitro culture systems and an in vivo model. In Aim 1, we will test the hypotheses that Pim kinase inhibition sensitizes FLT3-ITD cells to apoptosis by inhibiting FLT3-ITD signaling, altering expression and activation of pro- and anti-apoptotic proteins, promoting generation of reactive oxygen species and/or inhibiting repair of DNA damage. Mechanistic insights achieved in Aim 1 will guide design of scheduling of Pim kinase inhibition in relation to exposure to chemotherapy drugs and to FLT3 inhibitors. Approaches to scheduling will be tested in vitro, with apoptosis as the readout. In addition, effects of Pim kinase inhibition on cell cycle could result in kinetic resistance to drugs, and notably cytarabine, and will be tested. Finally, effects on normal hematopoietic cells will be studied to establish a therapeutic index. In Aim 3, strategies optimized in vitro in Aim 2 will be tested in vivo an immunodeficient mouse model. It is anticipated that the proposed work will lead to development and optimization of regimens incorporating Pim kinase inhibitors in the treatment of FLT3-ITD AML, with the goal of improved outcomes in this unfavorable AML subset...