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系急性淋巴细胞白血病（ALL）的辐射耐药，使用C-61，一种新的SYK激酶底物结合(P)位点抑制剂，靶向和破坏白血病B细胞前体中的抗凋亡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。这可以通过在HSCT中使用C-61联合全身照射（TBI）来实现。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SYK p位点抑制剂C-61对复发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会通过增加原代b系ALL细胞的辐射敏感性和削弱其修复亚致死辐射损伤的能力，显著增强辐射诱导的ALL细胞凋亡，并增加其克隆源部分的辐射诱导死亡。在特异性目标2中，我们将使用复发b系ALL的SCID小鼠异种移植模型和亚致死全身照射（TBI）来研究C-61对复发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，C-61 + 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。在Specific Aim 3下，我们将研究复发b系ALL患者原发性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。为了确定一种复合生物标志物谱，以帮助选择最有可能从C-61中获益的患者，我们将把初级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放射抗性、体外对C-61诱导的放射增敏的敏感性以及体内C-61诱导的亚致死性TBI抗白血病效力的增强联系起来。在Specific Aim 4下，我们将研究含C-61的单剂量TBI方案在亚致死（2 Gy）和临床应用（7 Gy）总辐射剂量水平下，在抗辐射BCL-1小鼠b系白血病的同基因小鼠HSCT模型中的有效性和安全性。我们将评估2-10 Gy剂量的TBI联合c -61对接种1x106 BCL-1细胞的BALB/c小鼠在同源BMT背景下的疗效和安全性。我们假设C-61的加入不会增加TBI的非血液学毒性，而显著增强其抗白血病功效。我们预计，将C-61纳入复发b系ALL患者的hsct前TBI方案将改善他们的治疗反应和生存结果。拟议的研究有可能为使用含有C-61的新型TBI方案的范式转移HSCT策略的发展提供基础。

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.