Novel Dual Notch/PXR Targeting for Colon Cancer Therapy

摘要 结肠癌仍然是美国和世界各地与癌症相关的发病率和死亡率的主要原因 世界。许多治疗药物及其组合正被用来抑制肿瘤的生长和转移 肿瘤。然而，这些策略的几个重大问题是耐药性的发展 药物和药物增加的副作用。耐药性的一个关键原因是直接 或通过不同的激酶途径间接靶向治疗，它们激活孕烷X受体 (PXR)。因此，新的靶向治疗对于抑制特定通路但不会诱发PXR是必不可少的。 在这方面，我们开发了一种新药MRLTHB和水溶性类似物MRLTHBCD，它可以抑制 Noch-1信号转导，不诱导PXR。当前项目的目标是进一步确定该药物的特征 并产生临床前数据作为口服疗法，单独或与5-氟尿嘧啶(5-FU)联合用于 结肠癌。在以前的研究中，我们已经确定下调双皮质激素钙调素样激酶 1(DCLK1)抑制结肠癌移植瘤生长，提示靶向DCLK1将是一种有效的 结肠癌的治疗策略。我们现在已经确定THB和THBCD特异性地抑制DCLK1激酶 CaMKII和CAMKIV的活性与CaMKII和CAMKIV类似，但不影响钙调蛋白的活性。此外，我们 已经确定这些化合物通过一种新的培养方法抑制结肠癌细胞的生长，我们 已经发展出一种名为“盘中肿瘤”(TID)的技术，即癌细胞在三维培养环境中生长 这包括正常的上皮细胞、成纤维细胞和内皮细胞。该模型创造了一种类似于体内的肿瘤 微环境提供了必要的细胞-细胞接触、3D架构和不同 单元类型。在这个系统中观察到的对癌细胞的选择性杀伤表明，这些化合物是高度 具有特效性和良好的效价。从机理上讲，我们已经确定这些化合物抑制了Notch 信号通路和PXR的表达。根据我们的初步研究，THB和DCLK1靶向 THBCD抑制Notch信号和PXR是治疗结肠癌的有效策略 癌症。我们的目标是在目前的应用中继续开发临床前数据。在目标1中，我们建议 确定Notch-1在PXR表达中的作用。在目标2中，我们将进行详细的PK/PD研究 在异种移植和APCmin/+小鼠身上进行临床前研究的最佳剂量 模特们。在目标3中，我们建议继续并确定THB和THBCD的联合效果 用5-氟尿嘧啶抑制结肠癌生长。化合物对Akt磷酸化、Notch-1的影响 DCLK1和PXR在肿瘤中的表达将被测定。这些拟议的研究将 为启动新化合物的临床试验提供令人信服的机械证据 组合。这些研究还将有助于优化靶向化疗方案，并确定新的 未来临床研究的生物标志物。

SUMMARY Colon cancer remains a leading cause of cancer related morbidity and mortality, both in the US and around the world. Many therapeutic agents and their combinations are being used to inhibit the growth and metastasis of the tumor. However, a couple of significant problems with these strategies are the development of resistance to the drugs and the increased side effects of the drugs. A critical reason for the drug resistance is that directly or indirectly targeted therapeutics through various kinase pathways, they activate the pregnane X receptor (PXR). Hence, novel targeted therapeutics is essential that suppress specific pathways but do not induce PXR. In this regard, we have developed a novel drug MRLTHB and water-soluble analog MRLTHBCD, which inhibits Notch-1 signaling and does not induce PXR. The goal of the current project is to further characterize the drug and generate preclinical data as an oral therapeutic both alone and in combination with 5-fluorouracil (5-FU) for colon cancer. In previous studies, we have determined that downregulating Doublecortin calmodulin-like kinase 1 (DCLK1) suppresses colon cancer xenograft growth, suggesting that targeting DCLK1 would be an efficient strategy for colon cancers. We have now determined that THB and THBCD specifically inhibit DCLK1 kinase activity and but do not affect the kinase activity calmodulin like kinases CAMKII and CAMKIV. In addition, we have determined that the compounds inhibit the growth of colon cancer cells in a novel culture method that we have developed termed "Tumor in a Dish" (TiD) where cancer cells are grown in a three-dimensional culture that includes normal epithelial cells, fibroblasts and endothelial cells. The model creates an in vivo-like tumor microenvironment that provides the necessary cell-cell contact, 3D-architecture, and the influence of different cell types. The observed selective killing of cancer cells in this system suggests that the compounds are highly specific and have good potency. Mechanistically, we have determined that the compounds inhibit the Notch signaling pathway and PXR expression. Based on our preliminary studies, DCLK1 targeting by THB and THBCD resulting in suppression of both Notch signaling and PXR is a valid therapeutic strategy for colon cancers. We aim to continue developing preclinical data in the current application. In aim 1, we propose to determine the role of Notch-1 in PXR expression. In aim 2, we will perform detailed PK/PD studies of the compound and find the optimal dose to perform preclinical studies in xenotransplant and APCmin/+ mouse models. In aim 3, we propose then to continue and determine the effect of the combination of THB and THBCD with 5-fluorouracil to inhibit colon cancer growth. Effect of the compounds on Akt phosphorylation, Notch-1 activation, and DCLK1 and PXR expression in the tumor will be determined. These proposed studies would provide compelling mechanistic evidence for initiating clinical trials for the novel compounds alone and in combination. These studies will also aid in optimizing a targeted chemotherapeutic regimen and identify novel biomarkers for the future clinical studies.