New Ways of Targeting K-Ras

 描述(由申请人提供)：我们提出了三个相关的研究领域，每个领域都集中在RAS信号的一个主要方面。首次尝试解决与NF1基因有关的重要问题，NF1基因现在被认为是人类癌症的主要肿瘤抑制因子，也是神经纤维瘤病的遗传基础，神经纤维瘤病是一种全球每3500人中就有一人患病的疾病。使用生化方法和基于细胞的筛选，我们将尝试识别调节神经纤维蛋白调节RAS能力的蛋白质或其他细胞因子。最近，我们确定了 首先是RAS以外的骨化神经纤维蛋白结合蛋白，这是对神经纤维蛋白活性至关重要的Spred蛋白。我们将在这一发现的基础上确定神经纤维蛋白/Spred复合体是如何组装和调节的，此外，我们还将对通过神经纤维蛋白调节RAS的因素进行无偏见的筛选，或调节这种高度保守的蛋白质的RAS非依赖功能。其次，我们将分析K-RAS的单个突变体对上游信号的需求，以及对下游通路的特定激活。我们将像上一节一样，通过结合蛋白质本身的生化特性分析和下游途径的分析来实现这一点。我们将集中在不同致癌形式的RAS明显不同激活的已知途径，如Raf MAPK途径，以及通过分析干净、等基因背景中的单个蛋白质揭示的信号差异的无偏分析。最后，我们发现K-RAS促进癌细胞中的干细胞样表型，使它们能够非常有效地从少量细胞形成肿瘤，转移并产生抗药性。这些表型是由于K-RAS与钙调蛋白结合并抑制非规范的WNT信号，同时增加β-连环蛋白和其他参与干细胞信号传递的因子的转录所致。白血病抑制因子(LIF)是K-RAS癌产生的在干细胞表型中起主要作用的因子之一。我们发现，中和LIF可防止体内肿瘤的发生，并使胰腺肿瘤细胞增敏 吉西他滨，这样既有的肿瘤就可以被彻底根除。我们建议使用几种胰腺癌模型进行人源化抗LIF单抗的临床前分析，目的是根据这些实验的结果将这些抗体转移到临床测试中。K-RAS与钙调蛋白的结合通过丝氨酸-181上的磷酸化和PKC来阻止。非肿瘤促进的天然产物前列腺素在体内激活PKC，并破坏K-RAS-钙调蛋白相互作用。口服前列环素可防止小鼠胰腺癌的发生，且无明显副作用。已经合成了几个前列腺素类似物：我们将对所有这些进行测试，并生产更多。我们将确定哪种形式的PKC参与K-RAS的磷酸化，并对最有希望的类似物的安全性和有效性进行彻底的临床前测试，因此，如果成功，我们将推动其中之一进入临床测试。

 DESCRIPTION (provided by applicant): We propose three related area of research, each focused on a major aspect of Ras signaling. The first attempts to solve an important question relating to the NF1 gene, now recognized as a major tumor suppressor in human cancer as well as genetic basis for neurofibromatosis, a disease that affects 1 in 3500 people worldwide. Using biochemical approaches as well as cell-based screens, we will try and identify proteins or other cellular factors that regulate neurofibromin's ability to regulate Ras. Recently, we identified the first bone fide neurofibromin binding proteins other than Ras, the Spred proteins that are essential for neurofibromin's activity. We will build on this discovery to determine how the neurofibromin/Spred complex is assembled and regulated, and, in addition, we will perform unbiased screens for factors that regulate Ras through neurofibromin, or regulate Ras-independent functions of this highly conserved protein. Second, we will analyze individual mutants of K-Ras for their requirement for upstream signaling, and for the specific activation of downstream pathways. We will do this, as in the previous section, by combining analysis of biochemical properties of the proteins themselves, and analysis of downstream pathways. We will focus on known pathways that are clearly activated differently by different oncogenic forms of Ras, such as the Raf MAPK pathway, and unbiased analysis of signaling differences that are revealed by analysis of individual proteins in a clean, isogenic background. Finally, we have discovered that K-Ras promotes a stem-cell like phenotype in cancer cells that enables them to form tumors very efficiently from small numbers of cells, to metastasize and to become drug resistant. These phenotypes are caused by K-Ras binding to calmodulin and suppressing non-canonical wnt signaling, while increasing transcription from beta-catenin and other factors involved in stem-cell signaling. One of these factors that is produced by K-Ras cancers and plays a major role in the stem-cell phenotype is Leukemia Inhibitory Factor (LIF). We have found that neutralizing LIF prevents tumor initiation in vivo and sensitizes pancreatic tumor cells to gemcitabine, so that established tumors can be completely eradicated. We propose to undertake pre-clinical analysis of humanized anti-LIF monoclonal antibodies, using several models of pancreatic cancer, with the intention of moving these antibodies into clinical testing based on the results of these experiments. Binding of K-Ras to calmodulin is prevented by phosphorylation on serine-181, by PKC. The non-tumor promoting natural product, prostratin, activates PKC in vivo and disrupts K-Ras-calmodulin interaction. Oral administration of prostratin prevents development of pancreatic cancers in mouse models, with no obvious side effects. Several analogs of prostratin have been synthesized: we will test all of these, and make more. We will identify which form of PKC is involved in K-Ras phosphorylation and perform thorough preclinical testing for safety and efficacy of the most promising analogs, so that, if successful, we will advance one of these towards clinical testing.