Amplifying Radiation Potency Against Leukemic Stem Cells

描述（由申请人提供）：在这个探索性项目中，我们提议开发一种新的策略来克服b系ALL白血病干细胞的辐射耐药，使用一种新的重组生物治疗剂，即cd19配体，选择性地放大辐射诱导的促凋亡信号。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，采用重组生物治疗剂选择性地增加电离辐射的抗白血病效力。我们假设在HSCT背景下，重组cd19配体与TBI联合使用可以改善复发b系ALL患者的治疗结果。在Specific Aim 1下，我们将使用定量流式细胞术细胞凋亡测定和克隆性测定来检测重组CD19-L对耐辐射ALL细胞系以及复发b系ALL患者的原代ALL细胞的体外辐射抗性的影响（项目第一年）。我们假设CD19- l会放大辐射诱导的促凋亡BTK信号，从而显著和选择性地增强CD19+ b系ALL细胞的辐射诱导凋亡，并增加其克隆性部分的辐射诱导死亡。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（CD19/Annexin V染色）和共聚焦（TUNEL）细胞凋亡检测平台进行测量。在Specific Aim 2下，我们将使用SCID小鼠移植模型治疗复发性B系ALL和亚致死性TBI，研究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（项目第2年）。我们预计在放疗前和放疗同时使用CD19-L将显著增强HSCT中TBI的抗白血病能力。同样，顺序给药TBI和TBI后CD19-L预计比单独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TBI （2gy）对复发患者原代骨髓标本中的白血病干细胞的抗白血病效力的影响，以及对辐射耐药的b系ALL细胞系的抗白血病效力。我们的工作假设是CD19-L + TBI方案将比单独TBI更有效地改善原发性b系ALL细胞攻击的SCID小鼠的无事件生存结果。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证明，即利用重组CD19-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放射耐药。

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