Characterization of RAS-dependent effector signaling pathways in multiple myeloma

科学背景：致癌性RAS信号传导被认为是多发性骨髓瘤（MM）发病机制的标志，标志着从MGUS前体阶段向MM的转变，并且越来越多地发生在疾病进展阶段。由于RAS目前仍然是一个undruggable治疗靶点，我们的目标是阐明致癌RAS依赖的生存机制在MM细胞，以确定和表征RAS效应候选人作为潜在的治疗targets.Results从第一个资金周期：使用特定的shRNA表达结构，我们可以证明第一次，MM细胞强烈依赖于致癌RAS。进一步的信号转导分析显示，突变的RAS确实介导其致癌作用，而不一定通过经典的和所谓的RAS依赖性途径，如Raf/MAPK和PI 3 K/Akt。然而，尽管Raf/MAPK和PI 3 K/Akt并非主要由致癌RAS激活，但在MM患者亚组中均被组成性激活，并在临床前MM模型中显示有助于恶性肿瘤生长。这表明PI 3 K/Akt和Raf/MAPK至少部分通过突变RAS以外的机制激活。此外，我们有证据表明，Raf介导的效果，而不是强制性地通过经典的MEK/MAPK模块。基于我们的全外显子组测序程序（Z3）的结果，我们假设受体酪氨酸激酶（RTK）的上游突变是MM中组成型PI 3 K/Akt和Raf激活的主要潜在遗传原因。此外，从MM细胞系中的shRNA介导的敲低实验中，我们有初步证据表明Ral途径可能介导MM亚组中RAS的致癌作用。这些观察结果表明，尽管RTK、RAS、Raf和PI 3 K被各种突变不同地激活，但它们在维持恶性生长中协同作用，从而构成MM中的中心致癌信号网络。为了有效地阻断该网络，我们已经开始以系统的方式评价抑制剂的组合。在第一个组合的方法，我们伴随阻断MEK/MAPK和PI 3 K/Akt在一个大的原发性MM样本集，并可以识别敏感和不敏感subgroup.Planned实验第二个资金周期：根据我们以前的工作，这个项目有两个主要的基本假设。第一个假设是Raf/MAPK和PI 3 K/Akt通路可能被上游机制激活，而不是突变的RAS。第二个假设是突变的RAS至少部分通过Raf/MAPK或PI 3 K/Akt以外的途径介导其致癌作用。因此，第一个目标是确定导致Raf和PI 3 K放松管制的上游机制，特别关注在上一个资助期内确定的RTK和其他候选突变的分析。第二个目的是分析Raf和PI 3 K下游的机制，以确定潜在的补救机制和与其他致癌途径的相互作用。第三个目的是检验我们的假设，尚未鉴定的Ral效应蛋白在MM中以RAS依赖的方式发挥关键的促生存作用。最后，由于我们对上述联合治疗的有希望的结果，我们将致力于开发药物治疗方法，以靶向体内RTK/RAS/Raf/PI 3 K网络的关键节点。为此，我们将在不同的MM小鼠模型中系统地测试各种组合方法。

Scientific background: Oncogenic RAS signaling is considered to be a hallmark of multiple myeloma (MM) pathogenesis, marking the transition from the MGUS precursor stage to MM and increasingly occurring in progressed disease stages. Because RAS currently remains an undruggable therapeutic target, we aim to elucidate oncogenic RAS-dependent survival mechanisms in MM cells in order to identify and characterize RAS effector candidates as potential therapeutic targets.Results from the first funding period: Using specific shRNA-expression constructs, we could demonstrate for the first time that MM cells strongly depend on oncogenic RAS. Further signaling analysis revealed that mutated RAS does mediate its oncogenic effect not necessarily via classic and so-called RAS-dependent pathways such as Raf/MAPK and PI3K/Akt. However, although not primarily activated by oncogenic RAS, both Raf/MAPK and PI3K/Akt are constitutively activated in subgroups of MM patients and were shown to contribute to malignant tumor growth in preclinical MM models. This suggests that PI3K/Akt and Raf/MAPK are activated at least in parts by mechanisms other than mutated RAS. In addition, we have evidence that Raf mediates its effect not mandatorily via the classic MEK/MAPK module. Based on results from our whole-exome sequencing program (Z3), we assume that upstream mutations of receptor tyrosine kinases (RTKs) are the major underlying genetic cause of constitutive PI3K/Akt and Raf activation in MM. Furthermore, from shRNA-mediated knockdown experiments in MM cell lines we have tentative evidence that the Ral pathway might mediate the oncogenic effect of RAS in a subgroup of MM. Overall, these observations suggest that RTKs, RAS, Raf and PI3K, although differentially activated by various mutations, co-operate in maintaining malignant growth and thus constitute a central oncogenic signaling network in MM. In order to effectively block this network we have started to evaluate combinations of inhibitors in a systematic manner. In the first combinatorial approach we concomitantly blocked MEK/MAPK and PI3K/Akt in a large primary MM sample set, and could identify sensitive and insensitive subgroups.Planned experiments for the second funding period: Based on our previous work, this project has two main underlying hypotheses. The first hypothesis is that the Raf/MAPK and the PI3K/Akt pathway might be activated by upstream mechanisms other than mutated RAS. The second hypothesis is that mutated RAS mediates its oncogenic effect at least in parts via pathways other than Raf/MAPK or PI3K/Akt. The first goal is therefore to identify the upstream mechanisms that lead to deregulation of Raf and PI3K with a particular focus on the analysis of mutations in RTKs and other candidates identified during the previous funding period. The second aim is to analyze the mechanisms downstream of Raf and PI3K in order to identify potential salvage mechanisms and interactions with other oncogenic pathways. The third aim is to test our hypothesis that yet unidentified Ral-effector proteins play a critical pro-survival role in MM in a RAS-dependent fashion. Finally, sparked by our promising results with the above-mentioned combination treatment, we will aim to develop pharmacological treatment approaches to target critical nodes of the RTK/RAS/Raf/PI3K network in vivo. For this, we will systematically test various combinatorial approaches in different MM mouse models.