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Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho-signaling where GSK3 inhibition inactivates energy-sensing by AMP-kinase through dephosphorylation of the AMP-kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti-Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

Background Aberrantly activated neuronal kinase CDK5 invoke alternations in mitochondrial dynamics and metabolic dysfunction associated with neurological disorders. Although pro‐tumorigenic role of CDK5 has been recently defined but there is insufficient data to understand the mechanistic role of CDK5 in cancer associated mitochondrial structure and function. Hypothesis In this study we intend to investigate putative signalling hotspots downstream of aberrantly activated CDK5 in neuroendocrine tumors of adrenal glands called Pheochromocytoma (PC). Approach Our preliminary data for the first time showed deregulation of Ca2+ homeostasis and overexpression of CDK5 and its co‐activators‐p35/25 in malignant PC tumors inflicted with loss‐of‐function mutations in mitochondrial enzyme‐Succinate dehydrogenase B (SDHB). To elucidate role of CDK5 in tumorigenesis, we created a novel bi‐transgenic mouse model where doxycycline inducible overexpression of CDK5/p25 (p25OE) developed organ specific PC lesions. Results Phopshoproteomic analysis on p25OE PC tumors unraveled a tumor suppressive mechanism modulated by novel phosphorylation site on metabolic protein‐PRKAG2 (S‐65). PRKAG2 is a non‐catalytic regulatory subunit of AMP‐activated protein kinase (AMPK). S‐65 site on PRKAG2 were found downregulated in human PC tumors compared to normal adrenal medulla. Further biochemical characterization uncovered CDK5‐GSK3β‐AMPK crosstalk underlying human PC cellular proliferation. Picking CDK5 pocket with a novel CDK5 inhibitor ‘MRT3‐007’ induced anti‐proliferative effects with simultaneous increase in phosphorylation states of PRKAG2(S‐65) / AMPK(T‐172) sites followed by excessive mitochondrial fission. Conclusion These intriguing initial findings set the ground to discover therapeutic vulnerabilities associated with CDK5/p25/PRKAG2/AMPK signaling cascade and Mitochondrial Dynamics in Neuroendocrine tumors. Schematic depiction of working model demonstrating a novel crosstalk between a metabolic sensor ‘PRKAG2/AMPK’ and ‘Mitochondrial Fission’ synapsed via neuronal kinase ‘CDK5/p25’ in Pheochromocytoma (PC tumors) inflicted with mutations in SDHB gene.

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