A novel regimen to target both pancreatic cancer K-ras and antiapoptotic proteins

描述（由申请人提供）：本R03试点项目的目标是证明一种创新的联合治疗方案的概念证明，该方案旨在针对具有K-ras突变和一种或多种抗凋亡蛋白异常表达的胰腺癌。胰腺癌中K-ras癌基因突变和主要抗凋亡蛋白（如survivin、Mcl-1、XIAP、cIAP2）的异常表达在很大程度上促进了胰腺癌的发展和侵袭性（治疗抵抗、转移和复发）。在90%的胰腺癌患者中观察到k -ras功能突变。基因沉默突变的K-ras诱导细胞凋亡，抑制胰腺癌细胞生长、侵袭性、恶性肿瘤形成和异种移植肿瘤生长。然而，目前还没有针对胰腺癌K-ras突变的有效靶向治疗方法。利用K-ras突变体细胞与正常细胞的对比筛选方法，鉴定了药用植物阿莫罗拉15种化学成分，并利用半合成方法从这15种化学成分中获得了50多种衍生物。然后用K-ras突变细胞和正常细胞对这些化合物进行重新筛选。AMR-Me和AMR-MeOAc被鉴定为对K-ras突变细胞选择性抑制最有效的化合物。我们之前的研究表明，AMR-Me靶向K-ras通路，每天3mg /kg治疗小鼠28天无明显毒性，但延长了白血病小鼠的生存期。我们计划将AMR-MeOAc（最佳选择性）与我们的新化合物FL118（选择性抑制survivin、Mcl-1、XIAP和cIAP2）联合使用，以测试这种新的联合靶向治疗方案。先前的研究表明，与单独使用任何一种药物相比，K-ras沉默与吉西他滨联合使用可显著减少小鼠的肿瘤体积。因此，我们假设用AMR-MeOAc和FL118抑制突变的K-ras和主要的抗凋亡蛋白，比单独使用任何一种药物都能显著增强对胰腺癌细胞凋亡的诱导和对肿瘤生长的抑制。本项目提出以下三个具体目标。目的1：确定在低剂量FL118存在或不存在的情况下，AMR-MeOAc对胰腺癌细胞生长、凋亡和相关信号通路中蛋白质调节的影响。目的2：利用人胰腺癌细胞系来源的异种移植模型，确定在存在或不存在低剂量FL118的情况下AMR-MeOAc的疗效。目的3：确定在存在或不存在低剂量FL118的情况下，AMR-MeOAc对直接来源于患者胰腺癌组织的异种移植物的疗效。具有K-ras功能获得突变和一种或多种抗凋亡蛋白（survivin, Mcl-1， XIAP, cIAP2）异常表达的胰腺癌很难治疗。该项目可能会开发出一种新颖的靶向组合策略，以有效控制这种具有挑战性和难以治疗的癌症。

DESCRIPTION (provided by applicant): The goal of this R03 pilot project is to demonstrate a proof of concept for an innovative combination treatment regimen designed to target pancreatic cancer with K-ras mutations and aberrant expression of one or more antiapoptotic proteins. K-ras oncogenic mutations and aberrant expression of major antiapoptotic proteins (e.g. survivin, Mcl-1, XIAP, cIAP2) in pancreatic cancer heavily contribute to pancreatic cancer development and aggressiveness (treatment resistance, metastasis, and relapse). Gain-of-K-ras-function mutations is observed in >90% of pancreatic cancer patients. Genetic silencing of mutated K-ras induces apoptosis and inhibits pancreatic cancer cell growth, invasiveness, malignant tumor formation, and xenograft tumor growth. However, there are no effective targeted therapies available for pancreatic cancer K-ras mutations. Using a novel screening approach with K-ras mutant cells versus normal cells, fifteen chemical constituents from the medicinal plant Amoora rohituka were identified, and over 50 derivatives were generated using semi-synthetic approaches from the 15 hits. These compounds were then rescreened using K-ras mutant cells versus normal cells. AMR-Me and AMR-MeOAc were identified as the most potent compounds selectively against the K-ras mutant cells. Our previous studies indicated that AMR-Me targets the K-ras pathway, and that 3 mg/kg daily for 28-day treatment of mice shows no clear toxicity, while it extends leukemia mouse survival. We plan to combine AMR-MeOAc (best selectivity) with our novel compound, FL118, which selectively inhibits survivin, Mcl-1, XIAP, and cIAP2, for testing this novel combinational-targeted treatment regimen. It has previously been shown that combination of K-ras silencing with gemcitabine dramatically reduces tumor volumes in mice compared with either single agent alone. Therefore, we hypothesize that inhibition of both mutated K-ras and the major antiapoptotic proteins with the novel agents AMR-MeOAc and FL118 would lead to a strikingly enhanced induction of apoptosis and inhibition of pancreatic cancer cell and tumor growth than the inhibition from either agent alone. The following three specific aims are proposed in this project. Aim 1: Determine the efficacy of AMR-MeOAc in the presence or absence of low dose FL118 on pancreatic cancer cell growth, apoptosis, and modulation of proteins in the relevant signaling pathways. Aim 2: Determine the efficacy of AMR-MeOAc in the presence or absence of low dose FL118 using human pancreatic cancer cell line-derived xenograft models. Aim 3: Determine the efficacy of AMR-MeOAc in the presence or absence of low dose FL118 against xenografts directly derived from patient pancreatic cancer tissues. Pancreatic cancer with K-ras gain-of-function mutations and aberrant expression of one or more antiapoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) is hard to treat. This project may develop a novel and targeted combination strategy to effectively control this challenging and difficult-to-treat cancer.