1.01 Potential Cdk5 Targeted Preclinical Therapeutics in Pheochromocytoma

K. Strange1, P. Gupta1, A. Carter1, W. Howse1, C. Tan2, H. Ghayee3, K. Pacak4, A. Natarajan5, L. Meijer6, S. Reddy1, J. Bibb1  1University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 2University Of Texas Southwestern Medical Center,Department Of Psychiatry,Dallas, TX, USA 3University Of Florida & Malcom Randall VA Medical Center,Department Of Medicine, Division Of Endocrinology,Gainesville, FL, USA 4National Institute Of Health,National Institute For Child Health And Human Development,New York, NY, USA 5University Of Nebraska College Of Medicine,Omaha, NE, USA 6ManRos Therapeutics,Roscoff, FRANCE, France

Pheochromocytomas (PCC) are catecholamine-producing tumors arising from chromaffin cells in the adrenal medulla. Approximately 10% of PCC develop metastatic disease having 5-year survival rate <40%. No histopathological criteria exist to predict clinical behavior and current treatments for the malignant form of PCC are ineffective. Cdk5 is a non-traditional CDK family member activated by interaction with non-cyclin co-activators p35/p39. Emerging evidences indicates Cdk5 contributes to the pathophysiology of neuroendocrine (NE) cancers. However, the exact molecular mechanisms by which CDK5 causes tumorigenesis remains elusive. We discovered Cdk5 as a biomarker in different types of NE cancers including PCC. These findings gave rationale to test next generation Cdk5 inhibitors in cells derived from human PCC tumors and explore the mechanisms by which Cdk5 drives PCC cell neoplasia. Moreover, we have begun to model Cdk5-driven PCC in transgenic mice.

Well characterized NE cell lines were used including hPheo1, PC12, TT, MTC-SK, SinJ, H1184 and H146. TCGA transcriptome data annotated in UALCAN portal was used to evaluate CDK5 transcript levels. The bitransgenic tetOp system was used to drive p25 expression and induce aberrant Cdk5 activity in mouse NE cells.

PCC were found to express significantly higher Cdk5 transcript levels compared to its normal counterparts. Protein expression analysis showed similarly elevated Cdk5/p35 in human progenitor PCC cell line (hPheo1) and positive immuno-histochemical staining in human derived tumor tissues. We tested four new analogues that selectively target Cdk5 across panel of human NE cancer cells and assayed the effects on cell viability. All derivatives arrested PCC cell growth with median effective concentrations of 0.02, 2.5, 0.3 and 0.11µM for the respective agents. IC50 values were several-fold lower than the non-selective Cdk5 inhibitor, Roscovitine (26 µM), indicating improved potency. To validate that the growth-inhibitory effects were Cdk5-dependent, we assessed the effect of these agents on novel phosphorylation sites on targets downstream of Cdk5 including pSUV, pH1B, pLARP6 and pRBL1. Quantitative immunoblotting with phosphorylation state-specific antibodies showed attenuation of all phosphosites in hPheo1 cells. Finally, we find that PCC arises in transgenic mice overexpressing aberrant Cdk5 activity, further supporting its role in this disease and suggesting new animal models of PCC will be available to use for the preclinical testing of drugs such as those used here.

CDK5/p35 is overexpressed in human PCC. Novel Cdk5 inhibitors arrested PPC cell growth and reduced Cdk5-dependent phosphorylation of tumorigenic signaling mechanisms. Thus, these signaling mechanisms that may be critical to PCC initiation/progression.