Overcoming Leukemia Chemoresistance in the Central Nervous System

摘要 中枢神经系统（CNS）复发是急性脑梗死患者治疗失败的主要原因。 淋巴母细胞白血病（ALL）。值得注意的是，孤立的CNS复发发生在约3-8%的ALL儿童中， 在一些临床试验中占初始复发的30-40%。此外，目前的CNS导向疗法是 与严重的毒性有关。因此，迫切需要新的CNS导向的白血病疗法 改善长期结果，同时降低治疗相关的发病率。尽管广泛的研究 证明了骨髓微环境在白血病生物学中的关键作用，CNS的影响， 微环境对白血病细胞存活和化学抗性的影响在很大程度上是未知的。我们开发了一种新的 离体共培养系统和体内异种移植方法来研究CNS的作用 白血病生物学和化疗耐药性的利基。然后，我们使用这些模型系统来确定1） 在体外和体内的脑脊液（CSF）中培养的白血病细胞相对于 血清或培养基，2）白血病细胞主要定位于CNS内的脑膜，和3）白血病 与脑膜细胞共培养或与小鼠脑膜相关的细胞表现出存活率提高， 化学抗性我们随后发现脑膜-白血病直接相互作用促进白血病细胞存活 通过调节凋亡平衡、细胞周期进程和静止。重要的是，白血病 化疗耐药性是可逆的，并通过将白血病细胞从脑膜上分离而克服。然后我们 使用共培养粘附试验来鉴定破坏白血病和 脑膜细胞除了鉴定几种抑制典型细胞粘附靶点的药物外， 包括CXCR 4拮抗剂AMD 3100在内，我们发现Me 6 TREN，一种新的小分子， 造血干细胞（HSC）动员化合物，也破坏白血病和造血干细胞（HSC）之间的相互作用。 脑膜细胞这项工作表明脑膜对白血病有着独特而关键的影响 化疗耐药性，并定义了CNS复发的新机制，超越了血液的作用， 脑屏障基于这项工作，我们的中心假设是白血病与脑膜细胞的相互作用是一种相互作用。 中枢神经系统中白血病细胞存活和化疗耐药性的关键调节因子。此外，从治疗 从这一观点出发，我们假设，在中枢神经系统中，小生境破坏可能比在骨髓中更有效 因为相对于血液或血清，CSF的支持环境较差。在此目标 建议使用我们的CNS白血病体外和体内模型系统来剖析分子 在CNS中介导白血病粘附（Aim 1）和化学抗性（Aim 2）的机制， CNS白血病的临床可转化疗法，包括Me 6 TREN和AMD 3100（Aim 3）。 !

ABSTRACT Central nervous system (CNS) relapse is a major cause of treatment failure among patients with acute lymphoblastic leukemia (ALL). Notably, isolated CNS relapse occurs in ~3-8% of children with ALL and accounts for 30–40% of initial relapses in some clinical trials. Furthermore, current CNS-directed therapies are associated with significant toxicities. As a result, novel CNS-directed leukemia therapies are urgently needed to improve long-term outcomes while decreasing treatment-related morbidity. Although extensive research has demonstrated a critical role of the bone marrow microenvironment in leukemia biology, the impact of the CNS microenvironment on leukemia cell survival and chemoresistance is largely unknown. We developed a novel ex vivo co-culture system and an in vivo xenotransplantation approach to investigate the effects of the CNS niche on leukemia biology and chemoresistance. We then used these model systems to identify that 1) leukemia cells cultured in cerebral spinal fluid (CSF) in vitro and in vivo have diminished survival relative to serum or media, 2) leukemia cells predominantly localize to the meninges within the CNS, and 3) leukemia cells co-cultured with meningeal cells, or associated with the meninges of mice, exhibit enhanced survival and chemoresistance. We then identified that direct meningeal-leukemia interactions promote leukemia cell survival by modulating apoptosis balance, cell cycle progression, and quiescence. Importantly, leukemia chemoresistance was reversible and overcome by detaching the leukemia cells from the meninges. We then used a co-culture adhesion assay to identify drugs that disrupt the interaction between leukemia and meningeal cells. In addition to identifying several drugs that inhibit canonical cell adhesion targets and pathways, including the CXCR4 antagonist AMD3100, we found that Me6TREN, a novel small-molecule hematopoietic stem cell (HSC) mobilizing compound, also disrupts the interaction between leukemia and meningeal cells. This work demonstrates that the meninges exert a unique and critical influence on leukemia chemoresistance and defines novel mechanisms of CNS relapse beyond the well-described role of the blood- brain barrier. Based on this work, our central hypothesis is that the leukemia-meningeal cell interaction is a critical regulator of leukemia cell survival and chemoresistance in the CNS. Moreover, from a therapeutic standpoint, we hypothesize that niche disruption may be more efficacious in the CNS than in the bone marrow because of the less supportive environment of the CSF relative to the blood or serum. The objectives in this proposal are to use our in vitro and in vivo model systems for CNS leukemia to dissect the molecular mechanisms that mediate leukemia adhesion (Aim 1) and chemoresistance (Aim 2) in the CNS and test novel, clinically translatable therapies for CNS leukemia including Me6TREN and AMD3100 (Aim 3). !