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CD19+B系ALL细胞的凋亡，并增强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。

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.