Role of Redox Dependent Signaling in Leukemia

描述（由申请人提供）：CML（慢性粒细胞白血病）的独特之处在于BCR/ABL癌基因驱动疾病的发作和进展。抑制BCR/ABL激酶的治疗方法，如甲磺酸伊马替尼，已经极大地改变了慢性期疾病的治疗，但在急变期和针对BCR/ABL中特定点突变的治疗效果较差。急变期是疾病的一个非常具有侵袭性的阶段，其特征在于基因组不稳定和继发性突变。这些特征与活性氧（ROS）引起的DNA损伤有关。我们和其他人发现p210 BCR/ABL单独过表达会导致活性氧水平增加。我们的初步数据显示，ROS水平增加的结果是Src激酶家族成员Fyn的上调。我们假设ROS依赖性信号转导通过Fyn有助于发展到急变。BCR/ABL过表达细胞显示Fyn蛋白上调4倍。这种上调被抗氧化剂阻断，从而将增加的ROS水平和Fyn表达联系起来。BCR/ABL中的点突变赋予对甲磺酸伊马替尼的抗性，其保留了Fyn的上调，这表明Fyn可能是难治性患者的适当治疗靶点。使用shRNA敲低Fyn在体外和体内减缓白血病细胞生长，抑制45%的克隆生长，并导致对伊马替尼的敏感性增加。这些显著的变化表明Fyn对于白血病细胞增殖和对伊马替尼的敏感性是重要的。阻断Fyn还降低了细胞内过氧化物的总体水平，表明Fyn可以放大ROS水平，从而导致随后的DNA损伤和继发性突变。这些初步数据将Fyn置于增加的ROS的下游和上游，表明存在正反馈回路，由此Fyn通过BCR/ABL激活导致增加的ROS，可能在爆炸危机中发挥重要作用。该提案将阐明氧化剂依赖性Fyn上调的机制和后果，并在小鼠模型和临床标本中验证这些发现。该提案的具体目标是：#1：确定BCR/ABL和ROS水平上调Fyn的机制。#2：评估Fyn上调对ROS依赖性增殖信号和DNA损伤的影响。#3：测试BCR/ABL启动的ROS和Fyn上调对CML鼠模型和患者标本进展的相对贡献。这些数据将提供对BCR/ABL介导的ROS改变的一个重要后果的洞察：Fyn上调。我们的长期目标是了解白血病中的氧化剂依赖性信号传导，以制定预防进展的策略。在含有BCR/ABL的细胞中抑制ROS产生或阻断通过Fyn的下游信号传导的策略可以提供有希望的治疗方式。

DESCRIPTION (provided by applicant): CML (chronic myeloid leukemia) is unique in that the BCR/ABL oncogene drives disease onset and progression. Therapies inhibiting the BCR/ABL kinase, such as imatinib mesylate, have dramatically altered the treatment of chronic phase disease, but are less effective in blast crisis and against specific point mutations in BCR/ABL. Blast crisis is a very aggressive phase of the disease characterized by genomic instability and secondary mutations. These features have been linked to DNA damage caused by reactive oxygen species (ROS). We and others find that overexpression of p210 BCR/ABL alone causes an increase in levels of ROS. Our preliminary data reveal that a consequence of this increased level of ROS is upregulation of Fyn, a Src kinase family member. We hypothesize that ROS dependent signaling transduced through Fyn contributes to progression to blast crisis. BCR/ABL overexpressing cells display a four-fold upregulation of Fyn protein. This upregulation is blocked by antioxidants, thus linking increased ROS levels and Fyn expression. Point mutations in BCR/ABL that confer resistance to imatinib mesylate retain upregulation of Fyn, suggesting that Fyn may be an appropriate therapeutic target in refractory patients. Knockdown of Fyn using shRNA slows leukemia cell growth in vitro and in vivo, inhibits clonogenic growth by 45% and causes increased sensitivity to imatinib. These striking changes indicate that Fyn is important for leukemia cell proliferation and sensitivity to imatinib. Blockade of Fyn also lowers the overall levels of intracellular peroxides, suggesting that Fyn can amplify ROS levels, thus contributing to subsequent DNA damage and secondary mutations. These preliminary data place Fyn both downstream and upstream of increased ROS, suggesting the presence of a positive feedback loop, whereby Fyn activation through BCR/ABL leads to increased ROS potentially playing an important role in blast crisis. This proposal will elucidate the mechanism(s) and consequences of oxidant-dependent Fyn upregulation and validate these findings in murine models and in clinical specimens. The specific aims of this proposal are to: #1: Determine the mechanism by which BCR/ABL and ROS levels upregulate Fyn. # 2: Evaluate the effects of Fyn upregulation on ROS dependent proliferative signals and DNA damage. #3: Test the relative contributions of BCR/ABL initiated ROS and Fyn upregulation on progression in CML murine models and patient specimens. These data will provide insight into one significant consequence of BCR/ABL-mediated ROS alterations: Fyn upregulation. Our long-term goal is to understand oxidant-dependent signaling in leukemia in order to devise strategies to prevent progression. Tactics that quelch ROS production, or block downstream signaling through Fyn in BCR/ABL containing cells may provide promising therapeutic modalities.