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

描述(由申请者提供)：这笔资金资助的基本假设是，在接受辐射或导致DNA损伤的药物治疗后，了解能够抑制胸腺细胞和过度表达bcl-2的癌细胞中的凋亡的基因，将牵涉到相关的和药物靶向的途径，通过这些途径bcl2发挥其抗凋亡活性。作为最初亲本提案的两个目标的一部分，进行了正向遗传筛查，并在识别在DNA损伤后抑制bcl2的新突变体方面取得了显著进展。在这些研究过程中，出人意料和令人兴奋的新发现表明，由内质网(ER)应激引发的较少研究的凋亡途径可以识别一组完全不同的靶点，这些靶点可以介导抑制抗凋亡的bcl2途径。因此，我们提交了这份竞争性修订申请，为我们的拨款增加了一个新的研究目标，即利用有前景的内质网应激诱导的凋亡途径作为一种机制来敏化和杀死过表达bcl2的癌细胞。值得注意的是，我们的初步研究表明，内质网应激可以选择性地杀死斑马鱼幼体中的T细胞，而不依赖于bcl2的状态，我们已经有了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，来剖析内质网应激诱导细胞凋亡的分子机制。我们提出了以下新的目标3：在斑马鱼胚胎发育过程中bcl2过度表达的背景下，以及在T-ALL斑马鱼模型中，识别和分析调控ER应激诱导细胞凋亡的关键基因。由于我们在建立关键分析系统和试剂的初步研究方面取得了重大进展，我们已经准备好通过实验在这一新目标上取得重大进展，这些实验肯定可以在ARRA刺激机制的一年半时间框架内完成。为了实现这一目标，将建立促凋亡BH3-only蛋白在内质网应激中的胚胎作用，对我们已经恢复的两个新的内质网应激增强子突变体进行基因定位和分析，并确定内质网应激增强子突变对内质网应激T-ALL细胞的影响。为了加快这些拟议研究的进行，我们迫切需要资金来留住Ujwal Pyati博士，这位博士后研究员目前得到了NIH将于2009年6月30日到期的培训补助金的支持，他开发了我们关于斑马鱼ER应激模型的初步数据。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这项竞争性续期申请所支持的资金后18个月内提交。 公共卫生相关性：bcl2抑制子突变的发现将确定关键靶点，其被抗体或小分子抑制将恢复正常的细胞死亡途径，从而增强对人类B细胞滤泡性淋巴瘤和其他bcl2过表达的耐药癌症的凋亡诱导治疗的有效性。

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.