P. Gupta1, A. Carter1, K. Strange1, D. W. House1, H. Ghayee2, K. Pacak3, J. Bibb1, S. Reddy1 2University Of Florida,Endocrinology,Gainesville, FL, USA 3National Institutes of Health,Medical Neuroendocrinology,Bethesda, MD, USA 1University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA
Introduction: Pheochromocytomas (PC) are a rare and potentially devastating neuroendocrine neoplasm. Surgical resection can be curative, but 22% of these tumors will behave aggressively. Current systemic therapy options are limited. We have previously demonstrated CDK5's role pathogenesis of PC, and CDK5 inhibitors' efficacy treating PC cell lines in vitro. CDK5 inhibitors have not been studied sufficiently in animal PC models, and the downstream targets of CDK5 have not been identified for further therapies. Hypoxic inducing factor 1α (HIF1α) has been previously described as a potential area of study in PC. In normoxic conditions, HIF1α is rapidly degraded through the ubiquitin protease pathway after hydroxylation of proline-402. In hypoxia and some malignancies, HIF1α is stabilized, leading to activation of several downstream processes including neovascularization. In this study we sought to test the efficacy of CDK5 inhibitors in vivo, to define the importance of HIF1α in human PC tumors, and to study the potential relationship between CDK5 and HIF1α in PC models.
Methods: Murine metastatic PC models were generated through tail vein injections of MTT luciferase cells into Nu/Nu mice. After development of metastatic liver tumors, the mice were treated with CDK5 inhibitors. Lesions were quantified by bioluminescence (BLI). Human PC tumors and normal adrenal medulla were examined for phosphorylated HIF1α (p-HIF1α) at serine-687 through Western blots. PHEO cells were interrogated for the relationships between CDK5 and HIF1α. Direct phosphorylation reactions were performed in vitro between CDK5 and HIF1α.
Results: In vivo BLI demonstrated that MRT3-007 is more effective than roscovitine in metastatic murine PC models (Figure A). p-HIF1α is present in sporadic and and succinate dehydrogenase mutations (SDHB and SDHD) PC, but not in von Hipple Lindau PC or in normal adrenal medualla (FIGURE B). In normoxia, CDK5 inibition causes loss of HIF1α (FIGURE C). This was also true in hypoxia (highlighted in the box FIGURE D), consistent with the hypothesis that aberrant CDK5 activity stabilizes HIF1α. Finally, direct phosphorylation reactions demonstrate increased levels of p-HIF1α in the presence of CDK5. The addition of a CDK5 inhibitor reversed this (FIGURE E). Therefore, CDK5 directly phosphorylates and stabilizes HIF1α.
Conclusion: CDK5 inhibitors effectively treat advanced PC in murine models, serving as the basis of future CDK5 inhibitor human trials for PC patients. HIF1α phosphorylation/stabilization is the result of aberrant CDK5 activity in PC. This process can be further examined as potential therapy for PC patients.
