Abstract 1161: A new, therapeutically actionable target for the VHL E3 ubiquitin ligase in renal cell carcinoma

Abstract

Abstract Background: Loss of function of the von Hippel Lindau (VHL) tumor suppressor is a nearly ubiquitous feature of clear cell renal cell carcinoma (ccRCC). The current therapeutic strategies in RCC were developed around knowledge that the VHL E3 ubiquitin ligase targets hypoxia inducible factors (HIFs) for degradation. AURKA is a well-established mitotic kinase and an emerging target in numerous cancers arising from its critical roles in tumor growth and survival. More recently AURKA was discovered to also have important non-mitotic functions in G1/G0 cells, where it activates HDAC6 to regulate microtubule stability. Here we report that AURKA is a novel target for the VHL E3 ligase, which plays an important role in modulating the non-mitotic activity of AURKA. Methods: In vitro and in vivo ubiquitination assays were performed to establish AURKA as a direct target of VHL. Immunoblot analyses were used to determine AURKA levels in human RCC patient material and VHL deficient cell lines. Immunofluorescence was utilized to assess the impact of VHL-mediated AURKA ubiquitination on microtubule targets using primary cilia. Results: In this study, we show that AURKA is a novel target for VHL's E3-ligase activity. VHL directly regulates AURKA expression in non-mitotic, quiescent cells by promoting AURKA degradation via the 26S proteasome. The ubiquitination assays showed enhanced AURKA ubiquitination in the presence of VHL. We found that VHL mediates AURKA degradation via a multi-monoubiquitin chain linkage, in contrast to the more traditional and abundant K48-linkage of proteins targeted for proteasome-mediated degradation. Biochemical studies revealed that unlike HIFα recognition by VHL, which requires proline hydroxylation, VHL interacted with, and degraded AURKA independent of its hydroxylation status, suggesting an alternate mechanism for recognition of AURKA. Importantly, using primary cilia as a biomarker for stabile microtubules and activity of AURKA/HDAC6, we showed that the loss of primary cilia observed in VHL-null cells was rescued by alisertib (AURKA inhibitor) and rocilinostat (HDAC6 inhibitor). Conclusion: In conclusion, the biochemical evidence presented here identifies AURKA as a novel target of VHL's E3 ubiquitin ligase activity. We demonstrate a previously unknown mechanism for VHL mediated multi-mono ubiquitination of AURKA in regulating its non-mitotic functions, important in enabling the G0/G1 transition of cells and maintaining the cilia-centrosome cycle. Importantly, as an alternative to the current HIF related anti-angiogenic therapies, identifying AURKA as a novel VHL target lays the foundation for new therapeutic avenues in RCC, targeted specifically at the epithelial defects associated with VHL-null renal cell carcinoma. Citation Format: Elshad Hasanov, Guang Chen, Pratim Chowdhury, Justin Weldon, Eric Jonasch, Subrata Sen, Cheryl Lyn Walker, Ruhee Dere. A new, therapeutically actionable target for the VHL E3 ubiquitin ligase in renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1161.