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细胞凋亡BTK信号，从而显着且选择性地增强辐射诱导的CD 19 + B系ALL细胞的细胞凋亡，并增强辐射诱导的克隆源性部分的死亡。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。 公共卫生相关性：目前，治疗儿童白血病的主要挑战是治愈那些尽管接受了强化化疗但癌症复发的患者。这项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