Amplifying Radiation Potency Against Leukemic Stem Cells

描述（由申请方提供）：在该探索性项目中，我们提议开发一种新策略，使用新型重组生物抑制剂（即CD 19-配体）选择性扩增辐射诱导的促凋亡信号，以克服B系ALL中白血病干细胞的辐射抗性。拟议研究的长期目标是建立针对复发性B系ALL的“个性化”放射治疗方案，采用重组生物素制剂选择性地增加电离辐射的抗白血病效力。我们假设，在HSCT的背景下，使用重组CD 19-配体联合TBI可以改善复发性B系ALL患者的治疗结果。在特定目标1下，我们将使用定量流式细胞术凋亡测定和克隆形成测定来检查重组CD 19-L对耐辐射ALL细胞系以及复发性B系ALL患者的原代ALL细胞的体外辐射抗性的影响（项目第1年）。我们假设，CD 19-L将放大辐射诱导的促凋亡BTK信号，从而显著地和选择性地增强辐射诱导的CD 19 + B系ALL细胞的凋亡，以及增强辐射诱导的其克隆形成部分的死亡。白血病细胞的辐射抗性将使用我们的标准定量流式细胞术（CD 19/膜联蛋白V染色）和共聚焦（TUNEL）凋亡测定平台来测量。在特定目标2下，我们将使用复发性B系ALL和亚致死性TBI的SCID小鼠异种移植模型，检查CD 19-L对复发性B系ALL患者的原代骨髓标本中白血病干细胞的体内辐射抗性的影响（项目第2年）。我们预计，在HSCT背景下，在放疗前和放疗同时使用CD 19-L将显著增强TBI的抗白血病效力。同样，TBI和TBI后CD 19-L的顺序施用预期比单独的TBI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CD 19-L的无毒剂量水平，然后检查CD 19-L对亚致死TBI（2戈伊）针对来自复发患者的原代骨髓样本中的白血病干细胞以及SCID小鼠异种移植模型系统中的辐射抗性B系ALL细胞系的抗白血病效力的影响。我们的工作假设是，CD 19-L加TBI方案将比单独TBI更有效地改善用原代B系ALL细胞攻击的SCID小鼠的无事件生存结局。我们预计，这一探索性研究项目的成功完成将为针对复发性B系ALL的潜在范式转移治疗创新提供第一个临床前原理验证，从而使用重组CD 19-L作为选择性放射增敏剂，在辐射后放大促凋亡信号，克服白血病干细胞的辐射抗性。

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.