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.

项目总结 急性髓系白血病(AML)是最常见的成人急性白血病，占儿童白血病的20% 白血病。FMS样酪氨酸激酶3(Flt3)的内部串联复制(ITD)突变是 AML中最常见的突变，尤其与预后不良有关。Flt3-ITD原因 Flt3的结构性激活和激活的flt3驱动白血病发生的有力证据导致了 几种以Flt3为靶向的抑制剂的开发。其中，最有效和最有选择性的Flt3 抑制剂是AC220(奎扎替尼)，它在AML患者中显示出44%的完全缓解率 Flt3-ITD.然而，AC220取得的缓解是短暂的，AC220治疗产生了很大的效果 外周原始细胞比骨髓原始细胞更有效的清除，牵涉到骨髓 微环境是导致耐药性的重要因素。事实上，已经有证据表明骨髓 基质细胞因子可介导对Flt3抑制的细胞毒作用的抵抗。我们之前的研究 实验室研究表明，AC220抑制Flt3会损害谷胱甘肽(GSH)的代谢并诱导 线粒体活性氧(MitoROS)在Flt3-ITD AML细胞中的积聚，这是导致 细胞凋亡性死亡此外，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 抑制Flt3后，GSH代谢受损，更多的mitoROS积聚，并促进细胞凋亡。 然而，这些代谢变化是否以及如何影响骨髓基质细胞介导的 目前尚不清楚AC220对Flt3-ITD AML细胞的保护作用。我的初步数据显示 当在骨髓基质细胞条件培养液中用AC220处理Flt3-ITD AML细胞时，它们失败 诱导有丝分裂原并保护其免受AC220的杀伤作用，AC220与维持 GSH水平和MYC及其靶向谷氨酰胺转运蛋白ASCT2和LAT1的表达。类似的影响有 在常规培养液中对AC220治疗无效的一小部分细胞中持续观察到。 有趣的是，ATM或G6PD结合AC220的敲击大大逆转了这种保护 由条件培养液介导的细胞死亡。此外，ATM或G6PD的故障与 AC220可显著降低条件培养液中MYC的表达。表演 在体外和体内的研究中，我将确定MYC及其靶向谷氨酰胺转运体的维持， ASCT2和LAT1在维持GSH水平和骨髓基质细胞介导的保护中起关键作用 AC220处理的Flt3-ITD AML细胞(目标1)。为了了解ATM和G6PD是如何参与其中的 保护不受AC220的影响，我将确定哪些间质成分负责激活ATM和 G6PD，以及ATM和G6PD的重要下游效应因子是什么。(目标2)。由此得出的结论 研究将为骨髓基质细胞介导的细胞保护机制提供新的见解 来自Flt3靶向治疗的Flt3-ITD AML，并有可能确定联合治疗的其他靶点 旨在克服骨髓基质细胞保护作用并改善患者状况的治疗 结果。