Genetic Modifiers of the Anti-apoptotic Functions of Bcl-2

描述（由申请人提供）：该资助的基本假设是，在用辐射或诱导DNA损伤的药物治疗后，可以抑制过度表达BCL-2的胸腺细胞和癌细胞凋亡的基因的知识将涉及BCL-2发挥其抗凋亡活性的相关和药物靶向途径。作为原始亲本提案的两个目标的一部分，进行了正向遗传筛选，并在鉴定DNA损伤后抑制BCL-2的新型突变体方面取得了显著进展。在这些研究过程中，取得了意想不到和令人兴奋的新发现，表明由内质网（ER）应激触发的较少研究的凋亡途径可以鉴定出一组完全不同的靶点，这些靶点可以介导抗凋亡BCL-2途径的抑制。因此，我们提交了这项竞争性修订申请，为我们的资助增加了一个新的研究目标，即利用有前途的ER应激诱导的凋亡途径作为敏化和杀死BCL-2过表达癌细胞的机制。值得注意的是，我们的初步研究表明，ER应激可以选择性地杀死T细胞在斑马鱼幼虫的BCL-2状态无关，我们已经有令人信服的初步数据，建立了斑马鱼作为一个强大的新的脊椎动物模型系统解剖ER应激诱导细胞凋亡的分子机制。我们提出了以下新目标3：确定和分析在斑马鱼胚胎发育过程中过度表达BCL-2和T-ALL斑马鱼模型中调节ER应激诱导的细胞凋亡的关键基因。由于我们在建立关键检测系统和试剂的初步研究中取得了重大进展，我们已经做好了“铲准备”，通过在ARRA刺激机制的一年半时间内完成的实验，在这个新的目标上取得重大进展。在实现这一目标，胚胎的促凋亡BH 3蛋白在ER应激中的作用将被建立，我们已经恢复的两个新的ER应激增强子突变体将被遗传作图和分析，ER应激增强子突变对ER应激T-ALL细胞的影响将被确定。为了加速这些拟议的研究的进行，我们迫切需要资金来留住Ujwal Pyati博士，他是一名博士后研究员，目前获得NIH培训补助金的支持，该补助金将于2009年6月30日到期，他开发了我们关于斑马鱼ER压力模型的初步数据。我们还将立即聘请一名合格的研究技术人员，并订购我们新实验所需的用品和试剂。这些实验将完成，描述这些结果的论文将在收到本竞争性更新申请支持的资金后18个月内提交。 公共卫生相关性：BCL-2抑制突变的发现将确定关键靶点，其通过抗体或小分子的抑制将恢复正常的细胞死亡途径，从而增强B细胞滤泡性淋巴瘤和其他BCL-2过表达的抗凋亡癌症的凋亡诱导疗法的有效性。

DESCRIPTION (provided by applicant): The underlying hypothesis of this funded grant is that knowledge of the genes that can suppress apoptosis in thymocytes and cancer cells overexpressing BCL-2, after treatment with radiation or drugs that induce DNA damage, will implicate relevant, and drug targetable, pathways through which BCL-2 exerts its anti-apoptotic activity. As part of the two aims of the original parent proposal, a forward genetic screen was conducted and outstanding progress has been made identifying novel mutants that suppress BCL-2 following DNA damage. During the course of these studies, unexpected and exciting new findings were made indicating that a less studied apoptotic pathway triggered by Endoplasmic Reticulum (ER) stress can identify a completely different set of targets that can mediate suppression of the anti-apoptotic BCL-2 pathway. Thus, we are submitting this competitive revision application to add a new research aim for our grant to exploit the promising ER stress-induced apoptotic pathway as a mechanism to sensitize and kill BCL-2-overexpressing cancer cells. Remarkably, our preliminary studies show that ER stress can selectively kill T-cells in zebrafish larvae independent of BCL-2 status and we already have convincing preliminary data that establishes the zebrafish as a powerful new vertebrate model system for dissecting the molecular mechanisms of ER stress-induced apoptosis. We propose the following New Aim 3: To identify and analyze critical genes that modulate ER stress-induced apoptosis in the context of overexpressed BCL-2 during zebrafish embryogenesis and in zebrafish models of T-ALL. Due to our significant progress in preliminary studies establishing critical assay systems and reagents, we are 'shovel ready' to make major advances on this new Aim through experiments that can definitely be completed within the one and one-half year time-frame of this ARRA stimulus mechanism. In accomplishing this aim, the embryonic role of pro-apoptotic BH3-only proteins in ER stress will be established, two novel ER stress enhancer mutants that we already have recovered will be genetically mapped and analyzed, and the effects of ER stress enhancer mutations on ER-stressed T-ALL cells will be determined. In order to accelerate the conduct of these proposed studies we urgently need funding to retain Dr. Ujwal Pyati, a postdoctoral fellow currently supported on an NIH training grant that expires June 30, 2009, who developed our preliminary data on the zebrafish ER stress model. We also will immediately hire a qualified research technologist and order the supplies and reagents needed for our new experiments. These experiments will be completed and the papers describing these results will be submitted within 18 months of receipt of the funds supported by this competitive renewal application. PUBLIC HEALTH RELEVANCE: The discovery of BCL-2 suppressor mutations will identify pivotal targets whose inhibition by antibodies or small molecules will restore normal cell death pathways, thus enhancing the effectiveness of apoptosis-inducing therapies for B-cell follicular lymphoma and other BCL-2-overexpressing apoptosis-resistant cancers in humans.