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信号传导的一个主要方面。第一个尝试解决与NF 1基因有关的重要问题，NF 1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影响全球3500人中有1人的疾病。使用生物化学方法以及基于细胞的屏幕，我们将尝试和确定蛋白质或其他细胞因子，调节神经纤维蛋白的能力，调节Ras。最近，我们确定了 第一种是Ras以外的真正的神经纤维蛋白结合蛋白，Ras是神经纤维蛋白活性所必需的Spred蛋白。我们将建立在这一发现，以确定神经纤维蛋白/Spred复合物是如何组装和调节，此外，我们将进行无偏筛选的因素，通过神经纤维蛋白调节Ras，或调节Ras独立的功能，这种高度保守的蛋白质。其次，我们将分析K-Ras的单个突变体对上游信号传导的需求，以及下游途径的特异性激活。与前一节一样，我们将通过结合蛋白质本身的生化特性分析和下游途径分析来做到这一点。我们将专注于已知的途径，明确激活不同的致癌形式的Ras，如Raf MAPK途径，和无偏见的分析信号差异，揭示了通过分析单个蛋白质在一个干净的，同基因的背景。最后，我们发现K-Ras促进癌细胞中的干细胞样表型，使它们能够从少量细胞非常有效地形成肿瘤，转移并产生耐药性。这些表型是由K-Ras与钙调蛋白结合并抑制非经典wnt信号传导，同时增加β-连环蛋白和其他参与干细胞信号传导的因子的转录引起的。由K-Ras癌症产生并在干细胞表型中起主要作用的这些因子之一是白血病抑制因子（LIF）。我们已经发现，中和LIF可以防止体内肿瘤的发生，并使胰腺肿瘤细胞敏感 到吉西他滨，这样建立的肿瘤可以完全根除。我们建议使用几种胰腺癌模型进行人源化抗LIF单克隆抗体的临床前分析，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与钙调蛋白的结合被PKC在丝氨酸-181上的磷酸化所阻止。非肿瘤促进天然产物prostratin在体内激活PKC并破坏K-Ras-钙调蛋白相互作用。口服prostratin可预防小鼠模型中胰腺癌的发生，且无明显副作用。已经合成了几种prostratin的类似物：我们将测试所有这些，并制造更多。我们将确定哪种形式的PKC参与K-Ras磷酸化，并对最有前途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，我们将推进其中一种进行临床测试。

 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.