Amplifying Radiation Potency Against Leukemic Stem Cells

描述(申请人提供)：在这个探索性项目中，我们建议开发一种新的策略来克服B系白血病干细胞的辐射抗性，所有这些都使用一种新的重组生物治疗剂，即CD19-配体，用于选择性地放大辐射诱导的促凋亡信号。这项拟议研究的长期目标是建立针对复发B系的“个性化”放射治疗方案，所有方案都使用一种重组生物治疗剂，以选择性地增加电离辐射的抗白血病效力。我们假设，在HSCT中使用重组CD19-配体联合TBI可以改善复发B系ALL患者的治疗结果。在特定目标1下，我们将采用流式细胞术和克隆形成实验检测重组CD19-L对辐射耐药ALL细胞系以及复发B系ALL患者原代ALL细胞体外辐射抗性的影响(项目第一年)。我们假设CD19-L将放大辐射诱导的促凋亡的BTK信号，从而显着和选择性地增强辐射诱导的CD19B系ALL细胞的凋亡，并增加其克隆形成部分的辐射诱导的死亡。白血病细胞的辐射抗性将使用我们的标准定量流式细胞仪(CD19/Annexin V染色)和共聚焦(TUNEL)凋亡分析平台进行测量。在特定目标2下，我们将使用复发性B系ALL和亚致死性脑损伤SCID小鼠异种移植模型(项目第二年)，检测CD19-L对复发性B系ALL患者原代骨髓标本中白血病干细胞体内辐射抗性的影响。我们预期CD19-L在HSCT中的联合应用及放疗前应用将显著增强其抗白血病效应。同样，颅脑损伤和脑损伤后CD19-L的序贯应用有望比单纯脑损伤更有效。我们将首先进行小鼠毒性和药代动力学实验，以确定CD19-L的无毒剂量水平，然后在SCID小鼠异种移植模型系统中检测CD19-L对亚致死量(2GyTBI)抗白血病效果的影响。我们的工作假设是，CD19-L联合脑损伤方案在改善以原代B系ALL细胞攻击的SCID小鼠的无事件生存结果方面将比单用脑损伤更有效。我们预计，这一探索性研究项目的成功完成将为针对复发B系ALL的潜在范式转换治疗创新提供第一个临床前原理证明，根据该创新，白血病干细胞的辐射抵抗可以通过使用重组CD19-L作为选择性放射增敏剂来克服，这种放射增敏剂可以在辐射后放大促凋亡信号。 公共卫生相关性：目前，儿童白血病治疗中的主要挑战是治愈那些尽管进行了密集化疗但癌症复发的患者。建议研究的目的是开发一种有效的治疗方法

DESCRIPTION (provided by applicant): In this exploratory project, we are proposing to develop a new strategy to overcome radiation resistance of leukemic stem cells in B-lineage ALL using a novel recombinant biotherapeutic agent, namely CD19-Ligand, for selectively amplifying radiation-induced pro-apoptotic signaling. The long-term goal of the proposed research is to establish "personalized" radiation therapy regimens against relapsed B-lineage ALL employing a recombinant biotherapeutic agent to selectively increase the anti-leukemic potency of ionizing radiation. We hypothesize that the treatment outcome of relapsed B-lineage ALL patients can be improved by using recombinant CD19-Ligand in combination with TBI in the context of HSCT. Under Specific Aim 1, we will examine the effects of recombinant CD19-L on in vitro radiation resistance of radiation-resistant ALL cell lines as well as primary ALL cells from relapsed B-lineage ALL patients using quantitative flow cytometric apoptosis assays and clonogenic assays (Year 1 of the Project). We hypothesize that CD19-L will amplify radiation- induced pro-apoptotic BTK signals thereby markedly and selectively enhancing radiation-induced apoptosis of CD19+ B-lineage ALL cells as well as augmenting radiation-induced death of their clonogenic fraction. The radiation resistance of leukemic cells will be measured using our standard quantitative flow cytometric (CD19/Annexin V staining) and confocal (TUNEL) apoptosis assay platforms. Under Specific Aim 2, we will examine the effects of the CD19-L on in vivo radiation resistance of leukemic stem cells in primary bone marrow specimens from relapsed B-lineage ALL patients using SCID mouse xenograft models of relapsed B- lineage ALL and sublethal TBI (Year 2 of the Project). We anticipate that the use of CD19-L before and concomitant with radiation will markedly enhance the anti-leukemic potency of TBI in the context of HSCT. Likewise, the sequential administration of TBI and post-TBI CD19-L is expected to be more effective than TBI alone. We will first perform mouse toxicity and pharmacokinetics experiments to determine non-toxic dose levels of CD19-L and then examine the effects of CD19-L on the anti-leukemic potency of sublethal TBI (2 Gy) against leukemic stem cells in primary bone marrow specimens from relapsed patients as well as radiation- resistant B-lineage ALL cell lines in a SCID mouse xenograft model system. Our working hypothesis is that CD19-L plus TBI regimens will be more effective than TBI alone in improving the event-free survival outcome of SCID mice challenged with primary B-lineage ALL cells. We anticipate that the successful completion of this exploratory research project will provide the first preclinical proof-of-principle for a potentially paradigm-shifting therapeutic innovation against relapsed B-lineage ALL, whereby the radiation resistance of leukemic stem cells is overcome using recombinant CD19-L as a selective radiosensitizer that amplifies pro-apoptotic signaling after radiation. PUBLIC HEALTH RELEVANCE: Currently, the major challenge in the treatment of childhood leukemia is to cure patients who experience a recurrence of their cancer despite intensive chemotherapy. The purpose of the proposed research is the development of an effective treatment