Controlling the BCL-2 Pathway of Mitochondrial Apoptosis

描述（由申请人提供）：癌细胞在程序性细胞死亡的遗传途径中经常并且可能总是具有畸变。BCL-2蛋白家族在死亡信号的传递和执行中起关键作用。癌细胞逃避程序性细胞死亡的一个常见机制是通过改变抗凋亡/促凋亡BCL-2成员的比例，导致BCL-2抗凋亡分子的功能过剩。本提案描述了验证以下假设的策略：(a)抗凋亡BCL-2家族成员可以被特异性靶向，以及(b) BCL-2是抗癌治疗的有效靶点。为了解决(a)，将使用荧光偏振检查抗凋亡BCL-2家族成员和来自“仅BH3”家族成员的BH3结构域之间的结合相互作用，以确定两类蛋白质之间相互作用的特异性。结合相互作用的相关性将通过功能性线粒体和细胞凋亡测定来验证。从这些研究中，将出现作为抗凋亡BCL-2家族成员原型抑制剂的寡肽。这种方法已经在基于BAD的BH3结构域的BCL-2肽抑制剂的初步表征中显示出前景。为了解决(b)，描述了依赖BCL-2维持的白血病小鼠模型。在该模型中，当强力霉素治疗消除BCL-2的表达时，小鼠发生淋巴性白血病。为了检验BCL-2在其他组织类型肿瘤维持中的重要性，将BCL-2的条件表达扩展到乳腺、黑素细胞等其他组织。在这些模型中描述了促进癌症的方法。当癌症发生时，BCL-2的表达将被强力霉素控制，以确定BCL-2是否需要维持肿瘤。最后，我们将使用上面开发的线粒体、细胞和小鼠实验来测试小分子和肽源BH3模拟物的功效和特异性。

DESCRIPTION (provided by applicant): Cancer cells frequently and perhaps invariably possess aberrations in the genetic pathway of programmed cell death. The BCL-2 family of proteins plays a critical role in the signaling and execution of death signals. A common mechanism by which cancer cells evade programmed cell death is by altering the ratio of antiapoptotic/ pro-apoptotic BCL-2 members, resulting in a functional excess of BCL-2 antiapoptotic molecules. This proposal describes strategies to test the hypotheses that (a) anti-apoptotic BCL-2 family members can be specifically targeted, and (b) BCL-2 is a valid target for anti-cancer therapy. To address (a), binding interactions between anti-apoptotic BCL-2 family members and BH3 domains from "BH3-only" family members will be examined using fluorescence polarization to determine the specificity of interaction between the two classes of proteins. The relevance of the binding interactions will be validated using functional mitochondrial and cellular assays of apoptosis. From these studies will emerge oligo-peptides which function as prototype inhibitors of anti-apoptotic BCL-2 family members. This approach has already shown promise in the preliminary characterization of a peptide inhibitor of BCL-2 based on the BH3 domain of BAD. To address (b), a mouse model of leukemia which is dependent on BCL-2 for maintenance is described. In this model, mice develop a lymphoid leukemia which remits when expression of BCL-2 is eliminated by treatment with doxycycline. To test the importance of BCL-2 in tumor maintenance in other tissue types, the conditional expression of BCL-2 will be extended to other tissues such as breast and melanocytes. Methods are described for the promotion of cancer in these models. When cancer develops, BCL-2 expression will be controlled by doxycycline to determine if BCL-2 is required for tumor maintenance. Finally, we will use the mitochondrial, cellular and mouse assays developed above to test the efficacy and specificity of BH3 mimetics of both small molecule and peptidomimetic origin.