Targeting SYK Tyrosine Kinase to Overcome Radiation Resistance in ALL

描述(由申请人提供)：我们建议开发一种新的策略来克服B细胞性急性淋巴细胞白血病(ALL)的辐射抗性，使用一种新的SYK底物结合(P)位点抑制剂C-61，用于靶向和破坏白血病B细胞前体中抗凋亡的SYK-STAT3信号网络。我们的中心工作假设是，通过抑制SYK-STAT3分子靶点可以改善复发B系ALL患者的治疗结果。这将通过使用C-61结合全身照射(TBI)来实现。在特定的目标1下，我们将用定量的流式细胞仪凋亡法和克隆形成法检测SYK P位抑制剂C-61对复发性B系ALL患者原代ALL细胞体外辐射抗性的影响。我们假设C-61将显著增强辐射诱导的原代B系ALL细胞的凋亡，并通过增加它们的辐射敏感性和削弱它们修复亚致死辐射损伤的能力来增加它们的克隆形成部分的辐射诱导的死亡。在特定目标2下，我们将使用复发性B系ALL和亚致死性全身照射(TBI)的SCID小鼠异种移植模型，检测C-61对复发性B系ALL患者原代ALL细胞体内辐射抗性的影响。我们的假设是，在用原代B系ALL细胞攻击的SCID小鼠中，C-61加TBI方案将比单独使用TBI方案更有效地改善无事件生存结果。在特定的目标3下，我们将研究复发的B系ALL患者的原代ALL细胞的激酶表达谱与其体外和体内辐射抗性之间的关系。为了确定一个有助于选择最有可能受益于C-61的患者的复合生物标志物，我们将把原代B系ALL细胞的激酶蛋白表达和SYK、BTK和JAK激酶的活性水平与它们的辐射抗性、体外对C-61诱导的放射增敏的敏感性以及C-61诱导的亚致死性脑损伤的抗白血病效力相关联。在特定目标4下，我们将研究C-61单剂量照射方案在亚致死剂量(2GY)和临床应用总剂量(7GY)两种剂量水平下对辐射耐药BCL-1小鼠B系白血病小鼠HSCT模型的疗效和安全性。我们将在同基因骨髓移植的背景下，在接种1x106 bcl1细胞的BALB/c小鼠中评估2-10GyTBI与C-61联合治疗的有效性和安全性。我们假设C-61的加入不会增加TBI的非血液学毒性，同时显著增强其抗白血病效果。我们预计，将C-61合并到复发B系患者的HSCT前TBI方案中将改善他们的治疗反应和生存结果。这项拟议的研究有可能为采用C-61包含新的TBI方案的范式转换HSCT策略的开发提供基础。 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。这项拟议的研究的目的是开发一种有效的治疗方案，使用一种新的抗白血病候选药物来对抗耐化疗和耐放射治疗的儿童白血病。本研究项目的成功完成可能为儿童白血病的治疗创新提供基础。

DESCRIPTION (provided by applicant): We are proposing to develop a new strategy to overcome radiation resistance in B- lineage acute lymphoblastic leukemia (ALL) using C-61, a novel SYK kinase substrate binding (P)-site inhibitor, for targeting and disrupting the anti-apoptotic SYK-STAT3 signaling network in leukemic B-cell precursors. It is our central working hypothesis that the treatment outcome of relapsed B-lineage ALL patients can be improved by inhibition of the SYK-STAT3 molecular target. This would be accomplished by using C-61 in combination with total body irradiation (TBI) in the context of HSCT. Under Specific Aim 1, we will examine the effects of the SYK P-site inhibitor C-61 on in vitro radiation resistance of primary ALL cells from relapsed B-lineage ALL patients using quantitative flow cytometric apoptosis assays and clonogenic assays. We hypothesize that C- 61 will markedly enhance radiation-induced apoptosis of primary B-lineage ALL cells and augment radiation- induced death of their clonogenic fraction by increasing their radiation sensitivity and impairing their capacity to repair sublethal radiation damage. Under Specific Aim 2, we will examine the effects of C-61 on in vivo radiation resistance of primary ALL cells from relapsed B-lineage ALL patients using SCID mouse xenograft models of relapsed B-lineage ALL and sublethal total body irradiation (TBI). Our hypothesis is that C-61 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. Under Specific Aim 3, we will examine the association between the kinase expression profiles of primary ALL cells from relapsed B-lineage ALL patients and their in vitro as well as in vivo radiation resistance. In an effort aimed at identifying a composite biomarker profile that will help select patients most likely to benefit from C-61, we will correlate the kinase protein expression and activity levels of SYK, BTK, and JAK kinases of primary B-lineage ALL cells with their radiation resistance, sensitivity to C-61 induced radiosensitization in vitro, as well as C-61 induced potentiation of the anti-leukemic potency of sublethal TBI in vivo. Under Specific Aim 4, we will study the efficacy and safety of C-61 containing single dose TBI regimens at both sublethal (2 Gy) as well as clinically applied (7 Gy) total radiation dose levels in a syngeneic murine HSCT model of radiation-resistant BCL-1 murine B-lineage leukemia. We will evaluate the efficacy and safety of TBI at doses ranging from 2-10 Gy in combination with C-61 in BALB/c mice inoculated with 1x106 BCL-1 cells in the context of syngeneic BMT. We hypothesize that the addition of C-61 will not increase the non-hematologic toxicity of TBI, while markedly potentiating its anti-leukemic efficacy. We anticipate that the incorporation of C-61 into the pre-HSCT TBI regimens of patients with relapsed B-lineage ALL will improve their treatment response and survival outcome. The proposed research has the potential provide the foundation for the development of paradigm-shifting HSCT strategies that employ C-61 containing novel TBI regimens. 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 program against chemotherapy- and radiation therapy-resistant childhood leukemia employing a new anti-leukemia drug candidate. The successful completion of this research project may provide the foundation for therapeutic innovation against childhood leukemia.