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

项目摘要 急性髓细胞白血病（AML）是最常见的成人急性白血病，占儿童的20% 白血病FMS样酪氨酸激酶3（FLT 3）中的内部串联重复（ITD）突变是其中之一。 AML中最常见的突变，尤其与预后不良相关。FLT 3-ITD原因 FLT 3的组成性激活和激活的FLT 3驱动白血病发生的有力证据已经导致 几种FLT 3靶向抑制剂的开发。其中，最有效和选择性的FLT 3 抑制剂是AC 220（Quizartinib），其在具有以下特征的AML患者中表现出44%的完全缓解率： FLT3-ITD。然而，AC 220实现的缓解是短暂的，并且AC 220治疗导致了很大的 比骨髓原始细胞更有效地清除外周原始细胞， 微环境作为耐药性的重要贡献者。事实上，已经证明骨髓 基质细胞因子可以介导对FLT 3抑制的细胞毒性作用的抗性。我们以前的研究 实验室已经表明AC 220抑制FLT 3损害谷胱甘肽（GSH）代谢并诱导 FLT 3-ITD AML细胞中的线粒体活性氧（mitoROS）积累，这是FLT 3-ITD AML细胞中的致病因素。 细胞凋亡此外，遗传敲除或药物抑制共济失调毛细血管扩张症 突变（ATM）或其下游靶标葡萄糖-6-磷酸脱氢酶（G6 PD），导致进一步的 FLT 3抑制后GSH代谢受损、更多的线粒体ROS积累和增强的细胞凋亡。 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 FLT 3-ITD AML细胞对AC 220处理的保护作用尚不清楚。我的初步数据显示 当在骨髓基质细胞的条件培养基中用AC 220处理FLT 3-ITD AML细胞时， 诱导mitoROS，并保护免受AC 220的杀伤作用，这与维持 GSH水平和MYC及其靶谷氨酰胺转运蛋白ASCT 2和LAT 1的表达。类似的效果 在常规培养基中对AC 220处理难治的一小部分细胞中一致观察到。 有趣的是，ATM或G6 PD与AC 220的组合的敲除基本上逆转了保护作用。 由条件培养基介导的细胞死亡。此外，ATM或G6 PD的敲除与 AC 220导致在条件培养基中培养的细胞中MYC表达的显著降低。执行 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， ASCT 2和LAT 1在维持GSH水平和骨髓基质细胞介导的保护中起关键作用 来自AC 220处理的FLT 3-ITD AML细胞（目的1）。要了解ATM和G6 PD如何参与其中， 保护免受AC 220，我将确定哪些基质成分负责激活ATM， G6 PD，以及ATM和G6 PD的重要下游效应物是什么。(Aim 2）。时发现的问题 研究将为骨髓基质细胞介导的保护机制提供新的见解。 FLT 3-ITD AML从FLT 3靶向治疗，并可能确定其他目标的组合 设计用于克服骨髓基质细胞的保护作用并改善患者 结果。

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.