Targeting ATAP-mediated Apoptosis in Treatment of Prostate Cancer

描述（由申请人提供）：通过直接靶向Bcl-2家族蛋白诱导细胞凋亡的新疗法的开发一直是癌症研究的主要焦点。一些研究小组已经开发出从促凋亡Bcl-2家族蛋白衍生的BH3肽，或生产出可以作为BH3类肽的化合物，这些化合物可以通过释放促凋亡Bax和/或Bak蛋白来诱导细胞凋亡，从而抑制抗凋亡蛋白的抑制。BH3肽或其衍生物的应用在治疗癌症方面取得了一些成功的结果。然而，这种方法在应用于抗凋亡Bcl-2蛋白过度表达或Bax和Bak水平降低的癌症时似乎有局限性，因为这些蛋白的异常表达会导致对这种BH3方法的抗性。必须考虑绕过Bcl- 2家族蛋白调控的替代策略。先前我们报道过，Bfl-1的一种新型两性尾锚肽ATAP可引发线粒体依赖性细胞死亡。ATAP可直接在线粒体膜上形成细胞色素c释放孔，ATAP的促凋亡活性不需要也不受抗凋亡或促凋亡蛋白的影响。我们还发现，在许多癌细胞中，ATAP比各种bh3肽更有效地诱导细胞凋亡。我们使用内化RGD肽（iRGD）选择性靶向肿瘤并将ATAP传递到癌细胞中。该ATAP- irgd融合肽可以有效合成，并且与天然ATAP相似，ATAP- irgd可以靶向线粒体通透性诱导细胞凋亡。与不加选择地导致细胞死亡的ATAP相反，ATAP- irgd肽似乎选择性地靶向肿瘤细胞凋亡。此外，ATAP-iRGD在对BH3肽治疗有抗性的DU145人前列腺癌细胞系中有效诱导细胞死亡。初步研究表明，将ATAP-iRGD静脉注射到带有人类癌细胞的异种移植裸鼠体内，可以减小肿瘤大小，同时不会产生明显的脱靶毒性。这些发现使我们相信ATAP可以成为一种有效的治疗药物，用于治疗对现有化疗药物有潜在耐药性的癌细胞。此次sbiri期申请将利用我们在生物药物开发方面的专业知识，为开发atp - irgd作为前列腺癌治疗药物进行必要的研究，努力向FDA提交新药研究（IND）申请。这项努力将包括以下两个具体目标。目的1：在小鼠模型中检测ATAP- iRGD的药代动力学、组织分布和毒理学特征。虽然我们的初步结果表明ATAP-iRGD的毒性有限，但进一步的毒理学研究以评估ATAP-iRGD的安全性对IND申请至关重要。这些评估的完成将为我们体内动物模型研究测试ATAP-iRGD治疗前列腺癌的疗效提供重要的指导。目的2：建立ATAP-iRGD在前列腺癌异种移植小鼠模型中的作用。在这里，我们将测试不同剂量的ATAP-iRGD是否可以影响具有不同人类前列腺癌细胞系的异种移植小鼠模型的肿瘤生长。这些努力将包括BH3肽在减少肿瘤生长方面的功效的比较研究。我们将收集肿瘤大小的数据，并利用组织学对ATAP-iRGD或BH3肽治疗后的肿瘤进展进行分级。我们还将使用异种移植小鼠模型进行额外的分布研究。上述研究的完成将为我们未来开发atp - irgd作为一种新型治疗试剂提供原理证明数据，用于治疗由于Bcl-2家族蛋白异常表达而对现有化疗试剂产生耐药性的癌细胞。

DESCRIPTION (provided by applicant): Development of novel therapeutics that can induce apoptosis by directly targeting the Bcl-2 family proteins has been a major focus of cancer research. Several groups have developed BH3 peptides derived from pro- apoptotic Bcl-2 family proteins or produced chemical compounds that could act as BH3 peptidomimetics, which could induce apoptosis by releasing pro-apoptotic Bax and/or Bak protein from inhibition by the anti-apoptotic proteins. Applications of BH3 peptides or their derivatives produced some successful outcomes in treating cancers. However, this approach seems to have limitations when it is applied to cancers displaying overexpression of anti-apoptotic Bcl-2 proteins or reduced Bax and Bak levels, because aberrant expression of these proteins cause resistance to such BH3 approaches. Alternative strategies that bypass regulation by Bcl- 2 family proteins must be considered. Previously we reported that ATAP, a novel amphipathic tail-anchoring peptide of Bfl-1, triggered potent mitochondria-dependent cell death. ATAP can directly form cytochrome c- releasing pores in mitochondrial membranes, and the pro-apoptotic activity of ATAP does not require and is not influenced by either anti- or pro-apoptotic proteins. We also found that ATAP is more effective in inducing apoptosis than the various BH3-peptides in many cancer cells. We used the internalizing RGD peptide (iRGD) for selective tumor targeting and delivery of ATAP into cancer cells. This ATAP-iRGD fusion peptide can be effectively synthesized, and similar to the native ATAP, ATAP-iRGD can target mitochondria permeability for induction of apoptosis. In contrast to ATAP which causes cell death indiscriminately, the ATAP-iRGD peptide appears to selectively target tumor cells for apoptosis. Moreover, ATAP-iRGD was effective at inducing cell death in the DU145 human prostate cancer cell line that was resistant to the effects of BH3 peptide therapy. Preliminary studies with intravenous injection of ATAP-iRGD into xenograft nude mice with human cancer cells could reduce tumors size while not producing significant off-target toxicity. These findings lead us to believe that ATAP can be an effective therapeutic agent for treatment of cancer cells that are potentially resistant to the existing chemotherapeutic reagents. This SBIR-Phase I application will leverage our expertise in biologic drug development to pursue necessary studies for development of ATAP-iRGD as a treatment for prostate cancer in an effort to move towards the filing of an Investigational New Drug (IND) application with FDA. This effort will contain the following two specific aims. Aim 1, test the pharmacokinetic, tissue distribution and toxicological profile of ATAP- iRGD in a mouse model. While our preliminary results suggest limited toxicity associated with ATAP-iRGD, further toxicology studies to evaluate the safety profile of ATAP-iRGD are essential to enable IND filing. Completion of these evaluations will provide an essential guidance for our in vivo animal model studies testing the efficacy of ATAP-iRGD in treatment of prostate cancer. Aim 2, establish the efficacy of ATAP-iRGD in a xenograft mouse model of prostate cancer. Here we will test if various doses of ATAP-iRGD can affect tumor growth in xenograft mouse model with various human prostate cancer cell lines. These efforts will include comparative studies to the efficacy of BH3 peptides in reducing tumor growth. We will collect data on tumor size as well as use histology to grade the progression of the tumor following treatment with ATAP-iRGD or BH3 peptides. We will also conduct additional distribution studies using the xenograft mouse models. Fulfillment of the above studies will provide proof-of-principle data for our future extensive effot toward developing ATAP-iRGD as a novel therapeutic reagent for treatment of cancer cells that are resistant to the existing chemotherapeutic reagents due to the aberrant expression of Bcl-2 family proteins.