Abstract LB-C04: Phosphoproteomics unravels dynamic regulation of energy sensor by CDK5 in Neuroendocrine Tumor

Abstract

Background: Pheochromocytoma (PC) is a rare Neuroendocrine (NE) tumor usually originates from adrenal gland’s chromaffin cells. These tumors are often benign but can become malignant causing excessive secretion of catecholamines leading to life threatening situation. Pro-tumorigenic role of CDK5, a proline-directed serine/threonine neuronal kinase has been implicated in tumor development. However, the broader downstream signalling events contributing to the function of CDK5 are largely unknown in NE tumors, particularly Pheochromocytoma. Results-Here we showed for the first time that aberrant CDK5/p25 signaling attributes human PC lesions both sporadic and in tumours’ incorporating mutations in key susceptibility genes such as SDHx and VHL. Unique doxycycline regulated bi-transgenic (TG) mouse model system was designed to drive CDK5/p25 overexpression (OE) in a NE cell specific manner localized within adrenal medulla. PC lesions developed in the TG mouse model accurately recapitulate human PC pathobiology and thus rendered a platform for discovering precision use of novel CDK5 inhibitors. Picking CDK5 pocket with a novel CDK5 inhibitor ‘MRT3-007’ induced significant tumor regression in TG/ SDHB knockdown xenograft/ & metastatic PC models. Next, to identify putative CDK5 substrates, we analysed phosphoproteomic profile of [p25‘turned On’/ Proliferating] versus [p25‘turned Off’/ Arrested] PC lesions. The analyses identified dynamic downregulation of 122 phosphosites in proliferating tumors. Further interrogation of biological relevance of top 5 downregulated phosphorylation sites unleashed anti-proliferative function of Phospho-Ser-65 (pS65) on PRKAG2 which is a non-catalytic regulatory gamma subunit of metabolic regulator- AMP-activated protein kinase (AMPK). Biochemical characterization uncovered mysterious CDK5-GSK3β-AMPK crosstalk regulating proliferation of human PC (hPheo1) cells. Preliminary findings show that our hit compound inhibits CDK5 with simultaneous elevation of novel phosphorylation on pS65-PRKAG2 embarking catalytic activation of AMPK kinase. Moreover, in vitro kinase data suggests that CDK5 influences activation of AMPK enzymatic components via regulation of pS9/21-GSK3β activation dephosphorylation. Once activated, AMPK invokes anti-proliferative response by promoting excessive mitochondrial fission in PC cells. In Conclusion, this study illuminates comprehensive view of phosphorylation events downstream of complex CDK5 signaling in human PC. Identification of novel CDK5/p25-GSK3-PRKAG2-AMPK signalling cascade may provide new insights into target-directed Neuroendocrine Cancer Therapeutics. Citation Format: PRIYANKA GUPTA, KEEHN STRANGE, RAHUL TELENGE, LAURENT MEIJER, HANS GHAYEE, KAREL PACAK, SUSHANTH REDDY, JAMES BIBB. Phosphoproteomics unravels dynamic regulation of energy sensor by CDK5 in Neuroendocrine Tumor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C04. doi:10.1158/1535-7163.TARG-19-LB-C04