Genetic and functional characterization of putative Ras/Raf interaction inhibitors in C. elegans and mammalian cells

Abstract Background Activation of the mammalian Ras-Raf-MEK-ERK MAPK signaling cascade promotes cellular proliferation, and activating Ras mutations are implicated in cancer onset and maintenance. This pathway, a therapeutic target of interest, is highly conserved and required for vulval development in C. elegans. Gain-of-function mutations in the Ras ortholog lead to constitutive pathway signaling and a multivulva (Muv) phenotype. MCP compounds were identified in a yeast two-hybrid screen for their ability to disrupt Ras-Raf interactions. However, this had not been confirmed in another system, and conflicting results were reported regarding selective MCP-mediated blockade of Ras- and Raf-mediated biological activities in mammalian cells. Here we used the easily-scored Muv phenotype as an in vivo readout to characterize the selectivity of MCP110 and its analogs, and performed biochemical studies in mammalian cells to determine whether MCP treatment results in impaired interaction between Ras and its effector Raf. Results Our genetic analyses showed significant dose-dependent MCP-mediated reduction of Muv in C. elegans strains with activating mutations in orthologs of Ras (LET-60) or Raf (LIN-45), but not MAP kinases or an Ets-like transcription factor. Thus, these inhibitors selectively impair pathway function downstream of Ras and upstream of or at the level of Raf, consistent with disruption of the Ras/Raf interaction. Our biochemical analyses of MCP110-mediated disruption of Ras-Raf interactions in mammalian cells showed that MCP110 dose-dependently reduced Raf-RBD pulldown of Ras, displaced a fluorescently-tagged Raf-RBD probe from plasma membrane locations of active Ras to the cytosol and other compartments, and decreased active, phosphorylated ERK1/2. Conclusions We have effectively utilized C. elegans as an in vivo genetic system to evaluate the activity and selectivity of inhibitors intended to target the Ras-Raf-MAPK pathway. We demonstrated the ability of MCP110 to disrupt, at the level of Ras/Raf, the Muv phenotype induced by chronic activation of this pathway in C. elegans. In mammalian cells, we not only demonstrated MCP-mediated blockade of the physical interaction between Ras and Raf, but also narrowed the site of interaction on Raf to the RBD, and showed consequent functional impairment of the Ras-Raf-MEK-ERK pathway in both in vivo and cell-based systems

Redefinition of the Yeast Two-Hybrid System in Dialogue with Changing Priorities in Biological Research

Examination of the pattern of reagent creation and application in the two-hybrid system since 1989 reveals the expansion of a simple core technology to address increasingly sophisticated problems in protein interaction. As the technology has matured, its clear suitability for large-scale proteomic projects has made a major focus of its application the generation of global organismal protein interaction networks. In an inversion of emphasis, the increasing availability of such information now provides a master plan with the potential to specify the most promising directions for biological investigations (i.e., by directing the physiological validation of predicted critical protein-protein interactions). Recent derivatives of the two-hybrid system enable the targeting of such key interactions by facilitating the identification of essential amino acids conferring protein interaction specificity and of small molecules that selectively disrupt defined interaction pairs. Finally, the creation of mammalian expression systems based on two-hybrid principles became a new tool to create and probe novel biological systems. Taken in sum, this trajectory emphasizes the point that the creation of tools and the evolution of the idea of what is an interesting biological problem are in intimate dialogue

Anti-tumor effects of ONC201 in combination with VEGF-inhibitors significantly impacts colorectal cancer growth and survival in vivo through complementary non-overlapping mechanisms

Abstract Background Small molecule ONC201 is an investigational anti-tumor agent that upregulates intra-tumoral TRAIL expression and the integrated stress response pathway. A Phase I clinical trial using ONC201 therapy in advanced cancer patients has been completed and the drug has progressed into Phase II trials in several cancer types. Colorectal cancer (CRC) remains one of the leading causes of cancer worldwide and metastatic disease has a poor prognosis. Clinical trials in CRC and other tumor types have demonstrated that therapeutics targeting the vascular endothelial growth factor (VEGF) pathway, such as bevacizumab, are effective in combination with certain chemotherapeutic agents. Methods We investigated the potential combination of VEGF inhibitors such as bevacizumab and its murine-counterpart; along with other anti-angiogenic agents and ONC201 in both CRC xenograft and patient-derived xenograft (PDX) models. We utilized non-invasive imaging and immunohistochemistry to determine potential mechanisms of action. Results Our results demonstrate significant tumor regression or complete tumor ablation in human xenografts with the combination of ONC201 with bevacizumab, and in syngeneic MC38 colorectal cancer xenografts using a murine VEGF-A inhibitor. Imaging demonstrated the impact of this combination on decreasing tumor growth and tumor metastasis. Our results indicate that ONC201 and anti-angiogenic agents act through distinct mechanisms while increasing tumor cell death and inhibiting proliferation. Conclusion With the use of both a murine VEGF inhibitor in syngeneic models, and bevacizumab in human cell line-derived xenografts, we demonstrate that ONC201 in combination with anti-angiogenic therapies such as bevacizumab represents a promising approach for further testing in the clinic for the treatment of CRC

Revue de l'instruction publique en France et dans les pays étrangers

25 octobre 18601860/10/25 (A20,N30)-1860/10/25