Developmental therapy for selectively targeting MEK-ERK pathway in cancer cells and tumor stromal compartment

项目概要/摘要 RAS-RAF-MEK-ERK通路中的致癌突变存在于46%的人类癌症中。这 启发了许多小分子BRAF和MEK通路抑制剂的成功开发。这些试剂 目前被FDA批准用于治疗含有BRAF突变的转移性黑素瘤。虽然 BRAF和MEK抑制剂已经显示出对患者的一些益处，这些“靶向”治疗具有非常低的耐受性。 治疗指数，因为这些小分子也靶向正常细胞，导致不良的，甚至致命的，副作用， 方面的影响.此外，无论这些抑制剂的临床疗效是通过对肿瘤的直接作用还是通过对肿瘤的抑制作用， 肿瘤间质区室的调节仍然难以捉摸。在本申请中，我们解决了上述关键问题。 未满足的需求，并将开发一种有效的和高度选择性的肿瘤MEK灭活剂，通过工程炭疽 基于毒素的蛋白质递送系统。通过调控特定肿瘤上毒素受体的表达， 基质细胞类型，我们也将描绘如何基质MEK抑制调节肿瘤的发展。 我们提出了两个平行的，但独立的具体目标，以实现这些目标。在目标1中，我们将生成一个 炭疽毒素为基础的，高肿瘤选择性MEK灭活剂，并评估其在各种肿瘤中的抗肿瘤活性。 肿瘤模型这种MEK灭活剂特异性结合主要毒素受体CMG 2（毛细血管形态发生 蛋白-2）对肿瘤细胞和肿瘤基质细胞的作用。通过严格依赖 同时存在两种不同的肿瘤相关蛋白酶，MMPs和尿激酶，以进入肿瘤 细胞和肿瘤基质细胞。一旦进入，它就会使MEK-ERK信号转导失活，实现有效的选择性靶向。 在目标2中，我们将利用我们独特的肿瘤-宿主-毒素系统来确定MEK-ERK信号转导在肿瘤细胞中的作用。 肿瘤基质细胞在肿瘤发展和毒素的肿瘤靶向。我们以前建立了一个 允许CMG 2在肿瘤中的各种特定细胞类型中获得功能或丧失功能的遗传系统 微环境因此，我们假设对癌细胞上CMG 2表达的遗传操作， CMG 2-/-小鼠全身的各种肿瘤基质细胞将提供第一个易于处理的遗传系统， 描述特定细胞类型在肿瘤微环境中的作用。因此，在目标2中，我们将确定 选择的肿瘤基质细胞类型，包括肿瘤内皮细胞（EC）、肿瘤相关的细胞因子（TNF-α）和肿瘤相关的细胞因子（TNF-α）， 巨噬细胞（TAM）、癌症相关成纤维细胞（CAF）、B细胞和调节性T细胞（Treg），以及 MEK-ERK抑制这些细胞在肿瘤发展中的分子机制。在完成这些 研究中，我们希望开发出一种高效的，肿瘤选择性MEK灭活剂作为一种新的肿瘤靶向 有治疗作用的这些研究也将明确确定MEK-ERK信号在肿瘤和细胞凋亡中的作用。 和间质，从而验证和鉴定特定的肿瘤间质细胞类型作为未来肿瘤治疗的靶点。 治疗学发展。

Project Summary/Abstract Carcinogenic mutations in the RAS-RAF-MEK-ERK pathway are present in 46% of all human cancers. This has inspired the successful development of many small molecule BRAF and MEK pathway inhibitors. These agents are currently FDA approved for the treatment of metastatic melanomas containing BRAF mutations. Although BRAF and MEK inhibitors have shown some benefits to patients, these “targeted” therapeutics have a very low therapeutic index, since these small molecules also target normal cells, causing undesirable, even fatal, side effects. Moreover, whether the clinical efficacy of these inhibitors is via direct effects on the tumor or through modulation of the tumor stromal compartment remains elusive. In this application, we address the above critical unmet needs and will develop a potent and highly tumor-selective MEK inactivator by engineering an anthrax toxin-based protein delivery system. Through manipulating the expression of the toxin receptor on specific tumor stromal cell types, we will also delineate how stromal MEK inhibition regulates tumor development. We propose two parallel, but independent Specific Aims to achieve these goals. In Aim 1, we will generate an anthrax toxin-based, highly tumor-selective MEK inactivator and evaluate its anti-tumor activity in a variety of tumor models. This MEK inactivator specifically binds to the major toxin receptor CMG2 (capillary morphogenesis protein-2) on tumor cells and tumor stromal cells. Specificity is ensured through strict reliance on the simultaneous presence of two distinct tumor-associated proteases, MMPs and urokinase, to gain entry into tumor cells and tumor stromal cells. Once inside, it inactivates MEK-ERK signaling, achieving potent selective targeting. In Aim 2, we will employ our unique tumor-host-toxin system to determine the roles of MEK-ERK signaling in tumor stromal cells in tumor development and in the toxin’s tumor targeting. We have previously established a genetic system allowing CMG2 gain-of-function or loss-of-function in various specific cell types in the tumor microenvironment. Thus, we hypothesize that a genetic manipulation of CMG2 expression on cancer cells and various tumor stromal cells in the whole body of CMG2-/- mice will provide the first tractable genetic system to delineate the role of specific cell types in the tumor microenvironment. Therefore, in this Aim 2, we will determine the roles of selected tumor stromal cell types including tumor endothelial cells (EC), tumor-associated macrophages (TAM), cancer-associated fibroblasts (CAF), B cells, and regulatory T cells (Treg), and the molecular mechanisms of MEK-ERK inhibition in these cells in tumor development. Upon completion of these studies, we expect to have developed a highly potent, tumor-selective MEK inactivator as a novel tumor-targeted therapeutic. These studies will also unambiguously determine the role of MEK-ERK signaling in both the tumor and the stroma, thereby validating and identifying specific tumor stromal cell types as targets for future therapeutics development.