Controlling the BCL-2 Pathway of Mitochondrial Apoptosis

描述(申请人提供)：癌细胞经常而且可能总是在程序性细胞死亡的遗传途径中具有异常。BCL-2蛋白家族在死亡信号的传递和执行中起着关键作用。癌细胞逃避细胞程序性死亡的一种常见机制是通过改变抗凋亡/促凋亡bcl2成员的比例，导致bcl2抗凋亡分子功能过剩。该提案描述了检验以下假设的策略：(A)抗凋亡的bcl2家族成员可以被特异性地靶向，以及(B)bcl2是抗癌治疗的有效靶点。为了解决(A)问题，我们将使用荧光偏振技术来研究抗凋亡的bcl2家族成员与“仅含BH3的”家族成员的BH3结构域之间的结合作用，以确定这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线粒体和细胞凋亡分析来验证。从这些研究中将出现寡肽，它们的功能是作为抗凋亡的bcl2家族成员的原型抑制物。这种方法已经在基于BAD的BH3结构域的BCL-2多肽抑制剂的初步表征中显示出希望。为了解决(B)，描述了一种依赖bcl2维持的白血病小鼠模型。在这个模型中，小鼠发生淋巴细胞性白血病，当bcl2的表达被多西环素治疗消除时，这种白血病就会缓解。为了测试bcl2在其他组织类型的肿瘤维持中的重要性，bcl2的条件表达将扩展到其他组织，如乳房和黑素细胞。描述了在这些模型中促进癌症的方法。当肿瘤发展时，bcl2的表达将由多西环素控制，以确定bcl2是否是维持肿瘤所必需的。最后，我们将使用上面开发的线粒体、细胞和小鼠试验来测试BH3模拟小分子和模拟多肽来源的有效性和特异性。

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.