Understanding the mechanism of bone marrow stromal cell-mediated protection of FLT3-ITD AML from FLT3-targeted therapy

了解骨髓基质细胞介导的 FLT3-ITD AML 免受 FLT3 靶向治疗影响的机制

• 批准号: 10317091
• 负责人: Hae J Park
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10317091
• 关键词: AKT Signaling Pathway; ATM Signaling Pathway; ATM activation; Acute Myelocytic Leukemia; Acute leukemia; Address; Adult; Antioxidants; Apoptosis; Apoptotic; Bone Marrow; Cell Death; Cells; Childhood Leukemia; Cytoprotection; Data; Development; Disease remission; Drug Targeting; Drug resistance; FLT3 gene; Gene Expression; Genes; Genetic; Glucosephosphate Dehydrogenase; Glutamine; Glutathione; Granulocyte-Macrophage Colony-Stimulating Factor; Human; Impairment; In Vitro; Interleukin-6; Investigation; Maintenance; Mediating; Metabolic; Metabolism; Mitochondria; Molecular; Mutation; Outcome; PI3K/AKT; Patient-Focused Outcomes; Patients; Peripheral; Pharmacology; Phenocopy; Phosphorylation; Play; Population; Production; Prognosis; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Receptor Protein-Tyrosine Kinases; Refractory; Research; Resistance; Signal Transduction; Stromal Cells; Testing; Tyrosine Kinase Inhibitor; Xenograft procedure; acute myeloid leukemia cell; adult leukemia; ataxia telangiectasia mutated protein; combinatorial; cytotoxic; experimental study; improved; in vivo; inhibitor; insight; knock-down; leukemia; leukemogenesis; mRNA Expression; mouse model; neutralizing antibody; novel; overexpression; protective effect; resistance mechanism; targeted treatment; therapy design

PROJECT SUMMARY Acute myeloid leukemia (AML) is the most common adult acute leukemia and accounts for 20% of childhood leukemias. Internal tandem duplication (ITD) mutations in FMS-like tyrosine kinase 3 (FLT3) are among the most common mutations in AML and are particularly associated with a poor prognosis. FLT3-ITD causes constitutive activation of FLT3 and the strong evidence that activated FLT3 drives leukemogenesis has led to the development of several FLT3-targeted inhibitors. Among these, the most potent and selective FLT3 inhibitor is AC220 (Quizartinib), which demonstrated a 44 % complete remission rate in AML patients harboring FLT3-ITD. However, remissions achieved by AC220 were short-lived, and AC220 treatment resulted in much more effective clearing of peripheral blasts than bone marrow blasts, implicating the bone marrow microenvironment as an important contributor to drug resistance. Indeed, it has been shown that bone marrow stromal cell factors can mediate resistance to the cytotoxic effects of FLT3 inhibition. Previous studies from our lab have shown that FLT3 inhibition by AC220 impairs glutathione (GSH) metabolism and induces mitochondrial reactive oxygen species (mitoROS) accumulation in FLT3-ITD AML cells, which is causative in apoptotic cell death. In addition, genetic knockdown or pharmacological inhibition of Ataxia Telangiectasia Mutated (ATM) or its downstream target, Glucose-6-Phosphate Dehydrogenase (G6PD), resulted in further impairment of GSH metabolism, more mitoROS accumulation, and enhanced apoptosis upon FLT3 inhibition. However, whether and how these metabolic alterations influence bone marrow stromal cell-mediated protection of FLT3-ITD AML cells from AC220 treatment is not understood. My preliminary data suggest that when FLT3-ITD AML cells are treated with AC220 in conditioned media of bone marrow stromal cells, they fail to induce mitoROS and are protected from the killing effect of AC220, which is associated with maintenance of GSH levels and expression of MYC and its target glutamine transporters, ASCT2 and LAT1. Similar effects are consistently observed in a small subset of cells that are refractory to AC220 treatment in regular media. Interestingly, knockdown of ATM or G6PD in combination with AC220 substantially reverses the protection from cell death mediated by conditioned media. Furthermore, knockdown of ATM or G6PD in combination with AC220 results in significant reduction of MYC expression in cells cultured in conditioned media. Performing both in vitro and in vivo studies, I will determine if maintenance of MYC and its target glutamine transporters, ASCT2 and LAT1 play a key role in maintaining GSH levels and bone marrow stromal cell-mediated protection of FLT3-ITD AML cells from AC220 treatment (Aim 1). To understand how ATM and G6PD are involved in this protection from AC220, I will determine what stromal components are responsible for activation of ATM and G6PD, and what the essential downstream effectors of ATM and G6PD are. (Aim 2). Findings from this research will provide new insights into the mechanism of bone marrow stromal cell-mediated protection of FLT3-ITD AML from FLT3-targeted therapy, and potentially identify additional targets for combinatorial therapies designed to overcome the protective effects of bone marrow stromal cells and improve patient outcomes.

项目概要 急性髓系白血病 (AML) 是最常见的成人急性白血病，占儿童期的 20% 白血病。 FMS 样酪氨酸激酶 3 (FLT3) 的内部串联重复 (ITD) 突变属于 AML 中最常见的突变，尤其与不良预后相关。 FLT3-ITD 原因 FLT3 的组成型激活以及激活的 FLT3 驱动白血病发生的有力证据导致 几种FLT3靶向抑制剂的开发。其中，最有效和选择性最强的FLT3 抑制剂是 AC220（Quizartinib），在患有 AML 的患者中显示出 44% 的完全缓解率 FLT3-ITD。然而，AC220 实现的缓解是短暂的，AC220 治疗导致了很多 比骨髓原始细胞更有效地清除外周原始细胞，这表明骨髓 微环境是耐药性的重要影响因素。事实上，已有研究表明，骨髓 基质细胞因子可以介导对 FLT3 抑制的细胞毒性作用的抵抗。我们之前的研究 实验室表明 AC220 抑制 FLT3 会损害谷胱甘肽 (GSH) 代谢并诱导 FLT3-ITD AML 细胞中线粒体活性氧 (mitoROS) 积累，这是导致 细胞凋亡。此外，共济失调毛细血管扩张症的基因敲除或药物抑制 突变 (ATM) 或其下游靶标葡萄糖-6-磷酸脱氢酶 (G6PD) 导致进一步 FLT3 抑制后，GSH 代谢受损、mitoROS 积累更多、细胞凋亡增强。 然而，这些代谢改变是否以及如何影响骨髓基质细胞介导的 FLT3-ITD AML 细胞免受 AC220 治疗的保护作用尚不清楚。我的初步数据表明 当 FLT3-ITD AML 细胞在骨髓基质细胞条件培养基中用 AC220 处理时，它们会失败 诱导 mitoROS 并免受 AC220 的杀伤作用，这与维持 GSH 水平以及 MYC 及其目标谷氨酰胺转运蛋白 ASCT2 和 LAT1 的表达。类似的效果还有 在常规培养基中对 AC220 处理无效的一小部分细胞中始终观察到这一现象。 有趣的是，ATM 或 G6PD 与 AC220 组合的击倒大大逆转了保护 来自条件培养基介导的细胞死亡。此外，ATM 或 G6PD 的敲低与 AC220 导致在条件培养基中培养的细胞中 MYC 表达显着降低。表演 在体外和体内研究中，我将确定是否维持 MYC 及其目标谷氨酰胺转运蛋白， ASCT2 和 LAT1 在维持 GSH 水平和骨髓基质细胞介导的保护中发挥关键作用 来自 AC220 治疗的 FLT3-ITD AML 细胞（目标 1）。了解 ATM 和 G6PD 如何参与其中 AC220 的保护，我将确定哪些基质成分负责激活 ATM 和 G6PD，以及 ATM 和 G6PD 的重要下游效应器是什么。 （目标 2）。由此得出的结论 研究将为骨髓基质细胞介导的保护机制提供新的见解 FLT3-ITD AML 来自 FLT3 靶向治疗，并可能确定组合的其他靶点 旨在克服骨髓基质细胞的保护作用并改善患者状况的疗法 结果。