Characterization of RAS-dependent effector signaling pathways in multiple myeloma

科学背景：致癌RAS信号被认为是多发性骨髓瘤（MM）发病机制的标志，标志着从MGUS前体阶段过渡到MM，并且越来越多地出现在疾病进展阶段。由于RAS目前仍然是一个不可药物的治疗靶点，我们的目标是阐明癌性RAS依赖于MM细胞的生存机制，以识别和表征RAS效应候选物作为潜在的治疗靶点。第一个资助期的结果：使用特异性shrna表达构建体，我们首次证明MM细胞强烈依赖致癌RAS。进一步的信号分析表明，突变的RAS确实介导其致癌作用，而不一定是通过经典的和所谓的RAS依赖通路，如Raf/MAPK和PI3K/Akt。然而，尽管不是主要由致癌RAS激活，Raf/MAPK和PI3K/Akt在MM患者亚组中都被组成性激活，并且在临床前MM模型中被证明有助于恶性肿瘤的生长。这表明PI3K/Akt和Raf/MAPK至少在一定程度上是由RAS突变以外的机制激活的。此外，我们有证据表明，Raf不是通过经典的MEK/MAPK模块强制调节其作用。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（RTKs）的上游突变是MM中组成PI3K/Akt和Raf激活的主要潜在遗传原因。此外，从MM细胞系中shrna介导的敲低实验中，我们初步证明Ral途径可能介导RAS在MM亚群中的致癌作用。总的来说，这些观察结果表明RTKs， RAS， Raf和PI3K，尽管不同的突变不同地激活，但它们合作维持恶性生长，从而构成了MM的中心致癌信号网络。为了有效阻断该网络，我们已经开始系统地评估抑制剂的组合。在第一种组合方法中，我们在一个大的初级MM样本集中同时阻断了MEK/MAPK和PI3K/Akt，并且可以识别敏感和不敏感的亚群。第二个资助期的计划实验：基于我们之前的工作，这个项目有两个主要的潜在假设。第一种假设是，Raf/MAPK和PI3K/Akt通路可能是由上游机制激活的，而不是突变的RAS。第二种假设是，突变的RAS至少部分通过Raf/MAPK或PI3K/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PI3K解除管制的上游机制，并特别关注分析在上一个资助期间确定的rtk和其他候选基因的突变。第二个目的是分析Raf和PI3K下游的机制，以确定潜在的挽救机制以及与其他致癌途径的相互作用。第三个目的是验证我们的假设，即尚未确定的ral效应蛋白以ras依赖的方式在MM中发挥关键的促生存作用。最后，基于上述联合治疗的令人鼓舞的结果，我们将致力于开发针对体内RTK/RAS/Raf/PI3K网络关键节点的药物治疗方法。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.