Targeting oncogene-driven translation and integrated stress response signaling in Cancer

结直肠癌和肝细胞癌(分别为结直肠癌和肝细胞癌)的特点是固有的高水平的蛋白质翻译。这构成了旨在干扰蛋白稳定机制的治疗的预敏化状态。基于我们之前专注于eIF2B5在结肠癌中的作用以及CDH1在肝癌中的作用的工作，我们将针对结直肠癌和肝癌中蛋白质翻译机制的组件。这两种途径都证实了真核细胞翻译起始因子2α(eIF2α)的磷酸化调控对于结直肠癌和肝癌的增殖和生存是必不可少的。我们现在将更详细地讨论整合应激反应网络的分子调控的几个方面，以进一步开发这些新的治疗方案。在一个聚焦的shRNA筛选中，我们已经确定eIF2鸟嘌呤交换因子(α)eIF2B5对于具有致癌的wnt信号的结直肠癌细胞的生存是必不可少的。我们还可以进一步证明，当eIF2B5丢失时，MYC的诱导会导致这些细胞产生压倒性的蛋白毒性。此外，在结直肠癌小鼠模型中，当eIF2ECF-α的表达降低时，APCmin小鼠的存活时间显著延长。由于eIF2B5功能的结构性抑制可能导致救援通路的代偿性激活，我们旨在研究急性eIF2B5基因敲除在体内对结直肠癌的潜在治疗作用。我们还将进一步确定在结直肠癌中四种eIF2B5激酶中的哪一种占主导地位，以及抑制该激酶是否可以模仿eIF2B5基因敲除的效果。使用有机培养物，将调查是否存在WNT途径的额外突变，使eIF2B5丢失敏感，或者致癌的MYC水平是否是激活的WNT信号的主要下游靶点，并促进这种脆弱性。这些结果将与对生物库的遗传和表型研究相关联，该生物库目前是从多达100种人类CRC有机培养物中建立的。在酵母-2-杂交筛选中，我们已经鉴定了促进复合共激活子CDH1与Gadd34相互作用的后期，Gadd34是胁迫下eif2α去磷酸化的主要调节因子。在哺乳动物细胞中，CDH1促进Gadd34的泛素化。在体外和异种移植小鼠模型中，CDH1的缺失导致Gadd34周转减少，蛋白毒性增加，以及对内质网应激诱导的细胞死亡的超敏反应。我们发现，在NRAS驱动的肝癌中诱导了CDH1，抑制CDH1诱导显著延长了动物的生存时间。在此，我们旨在研究癌基因NRAS和其他肝细胞癌驱动因子对CDH1的调控机制。利用体内筛选，我们将调查ISR的哪些效应分子主要参与肝癌的发生。最后，我们的目标是研究eIF2B5和CDH1缺失在结直肠癌和肝细胞癌中的作用机制是否存在协同作用。

Colorectal and hepatocellular carcinomas (CRC and HCC, respectively) are characterized by high intrinsic levels of protein translation. This constitutes a pre-sensitized state for therapies which aim at interfering with mechanisms of proteostasis. Based on our previous work focusing on the role of eIF2B5 in colon- and on CDH1 in liver carcinomas, we will target components of the protein translation machinery in CRC and HCC. Both avenues identified the regulation of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation to be essential for the proliferation and survival of CRC and HCC. We will now address several aspects of the molecular regulation of the integrated stress response (ISR) network in greater detail to further the development of these novel treatment options.In a focused shRNA screen we have identified the eIF2α guanine exchange factor (GEF) eIF2B5 as essential for CRC cell survival with oncogenic WNT signaling. We could further show that the induction of MYC leads to overwhelming proteotoxicity in these cells upon loss of eIF2B5. Furthermore, in a CRC mouse model, APCmin mice survived significantly longer when the eIF2α-GEF expression was reduced. As constitutive suppression of eIF2B5 function may result in compensatory activation of rescue pathways, we aim to investigate the potentially therapeutic effects of acute eIF2B5 knockdown in CRC in vivo. We will furthermore determine which of the four eIF2α kinases is dominant in CRC, and whether the inhibition of this kinase can mimick the effects of eIF2B5 knockdown. Using organoid cultures, it will be investigated if there are additional mutations of the WNT pathway which sensitize to loss of eIF2B5, or if oncogenic MYC levels are the major downstream target of activated WNT signaling and facilitate this vulnerability. These results will be correlated to genetic and phenotypic investigations on a biobank that is currently established from up to 100 human CRC organoid cultures. In a yeast-2-hybrid screen, we have identified the anaphase promoting complex co-activator CDH1 to interact with GADD34, the major regulator of eIF2α dephosphorylation under stress. In mammalian cells, CDH1 facilitates the ubiquitylation of GADD34. Loss of CDH1 led to a reduction in GADD34 turnover, increased proteotoxicity, and hypersensitivity towards endoplamatic reticulum stress induced cell death, both in vitro and in a xenograft mouse model. We found that CDH1 is induced in NRAS-driven liver cancer, and that inhibition of CDH1 induction significantly prolonged the survival of the animals. Here we aim to investigate the mechanisms of regulation of CDH1 by oncogenic NRAS and other HCC drivers. Using an in vivo screen, we will investigate which effectors of the ISR are majorily involved in liver carcinogenesis. Finally, we aim to investigate if there is a synergism in the mechanism of action of loss of eIF2B5 and CDH1, both in CRC and HCC.