Novel Dual Notch/PXR Targeting for Colon Cancer Therapy

描述(申请人提供)：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。许多治疗药物及其组合正被用于抑制肿瘤的生长和转移。然而，这些策略的几个重大问题是对药物的抗药性的发展和药物副作用的增加。这种药物的一个关键原因 耐药是直接或间接靶向治疗药物，通过不同的激酶途径，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，它抑制Notch-1信号转导，不诱导PXR。目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，作为一种单独和联合5-氟尿嘧啶(5-FU)治疗结肠癌的口服疗法。在以前的研究中，我们已经确定下调双皮质钙调素样激酶1(DCLK1)可以抑制结肠癌异种移植瘤的生长，这表明靶向DCLK1将是治疗结肠癌的有效策略。我们现在已经确定THB和THBCD特异性地抑制DCLK1激酶的活性，但不影响CaMKII和CAMKIV一样的钙调蛋白的活性。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，我们开发了一种名为“盘中肿瘤”(TID)的新培养方法，在这种方法中，癌细胞在包括正常上皮细胞、成纤维细胞和内皮细胞的三维培养中生长。该模型创建了一个类似于体内的肿瘤微环境，提供了必要的细胞-细胞接触、3D结构和不同细胞类型的影响。观察到的选择性杀戮 这一系统中癌细胞的数量表明，这些化合物具有高度的特异性和良好的效力。从机制上讲，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THBCD靶向DCLK1导致抑制Notch信号和PXR是一种有效的结肠癌治疗策略。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，我们将对该化合物进行详细的PK/PD研究，并找到在异种移植和APCmin/+小鼠模型中进行临床前研究的最佳剂量。在目标3中，我们建议继续并确定THB和THBCD与5-氟尿嘧啶联合应用抑制结肠癌生长的效果。这些化合物对Akt磷酸化、Notch-1激活以及DCLK1和PXR在肿瘤中表达的影响将被确定。这些拟议的研究将为启动新化合物单独和联合临床试验提供令人信服的机制证据。这些研究还将有助于优化靶向化疗方案，并为未来的临床研究确定新的生物标志物。

DESCRIPTION (provided by applicant): 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 cell. 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.