Results of a high-throughput screen to identify compounds that modulate VHL proteostasis.
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369 Background: Mutated von Hippel Lindau (VHL) protein causes disease in sporadic renal cell carcinoma (RCC), and in the eponymous hereditary VHL disease. We developed and tested a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point mutated VHL, and potentially reverse the disease phenotype. Methods: The 786-0 cell line was infected with full-length W117A mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against the known proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of downstream functional readouts, including HIF and GLUT1 levels, were performed. Results: The proteasome inhibitors bortezomib and MG132, and the Prestwick compounds 8-azaguanine, thiostrepton and thioguanosine were found to reliably upregulate VHL-W117A-Venus in 786-0 cells. Thiostrepton has also been recognized as having proteasome inhibitory effects. 8-azaguanine was found to downregulate HIF2 levels, and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate post-translational processing. In addition, nuclear-cytoplasmic localization of pVHL varied amongst the different compounds, additionally suggesting unique effects of each of the agents on VHL homeostasis. Conclusions: We have successfully identified compounds that stabilize mutated VHL, including several proteasome inhibitors. It is possible that proteasome inhibition will have a specific clinical effect on patients with point-mutated pVHL. Further work is currently underway to further test this hypothesis in animal xenograft studies using cell lines containing various mutated VHL isoforms, and clinical trials using next-generation proteasome inhibitors are being explored.
