Argininosuccinate Synthetase 1 (ASS1) is silenced in ∼90% of sarcomas. Loss of this urea cycle enzyme causes cells to become dependent upon extracellular arginine for continued cell growth and proliferation. Upon arginine starvation, ASS1(-) sarcoma cells undergo autophagy, increase their glutamine dependence and undergo growth arrest. In order to identify potentially exploitable synthetic lethal targets arising from the induction of autophagy following arginine deprivation, we investigated the metabolic alterations caused by arginine deprivation resulting from treatment with PEGylated arginine deiminase (ADI-PEG20). Mass spectroscopy was performed and revealed a significant increase in the level of serine biosynthesis from glucose which resulted from the up regulation of PHGDH. When paired with ADI-PEG20 treatment, inhibition of serine metabolism results in significant cell death. With recent studies showing the importance of serine biology in cancer, as well as recent generation of a small molecule PHGDH inhibitor to the rate limiting enzyme in serine biosynthesis, this newly identified synthetic lethality proves to be an exploitable therapeutic option for ASS1 deficient sarcomas.
The cell lines were with ADI-PEG20 for three days and subjected to metabolite extraction and capillary electrophoresis mass spectrometry (CE-MS). Similarly, additional samples were cultured for 2 days, with or without ADI-PEG20, and subsequently treated with U-C13 labeled glucose for an additional 24 hours before being subjected to metabolite extraction. Cell death was measured by propidium iodide fluorescent activated cell sorting after inhibition of serine metabolism by the small molecule inhibitor of PHGDH (CBR-5884) or genetic knockdown of key enzymes, with and without ADI-PEG20.
Upon treatment with ADI-PEG20 and subsequent arginine deprivation induced autophagy, the radiolabeled glucose derived component of a vast majority of metabolites decreased significantly. The pathway with the largest increase in metabolic flux was serine biosynthesis, including subsequent conversion into glycine. Significant changes in the enzyme responsible for the rate limiting step of serine biosynthesis, phosphoglycerate dehydrogenase (PHGDH), as well as serine catabolism were induced upon arginine deprivation. Cell death levels were significantly higher in samples when inhibition of PHGDH was paired with ADI-PEG20 treatment.
Starvation mediated by arginine deprivation causes global changes in cellular metabolism. By determining the alterations in the fate of glucose and glutamine upon treatment with ADI-PEG20, we were able to illustrate a significant increase in the level of serine biosynthesis. Inhibition of this escape pathway resulted in cell death. References 1. Bean GR, Kremer JC, Prudner BC, et al: A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas. Cell Death Dis 7:e2406, 2016 2. Kremer JC, Prudner BC, Lange SE, et al: Arginine Deprivation Inhibits the Warburg Effect and Upregulates Glutamine Anaplerosis and Serine Biosynthesis in ASS1-Deficient Cancers. Cell Rep 18:991-1004, 2017
Starvation mediated by arginine deprivation causes global changes in cellular metabolism. By determining the alterations in the fate of glucose and glutamine upon treatment with ADI-PEG20, we were able to illustrate a significant increase in the level of serine biosynthesis. Inhibition of this escape pathway resulted in cell death.
B. Van Tine
Polaris, Inc., Sarcoma Foundation of America, Sarcoma Alliance for Research and Collaboration
B. Van Tine: This work had funding from Polaris, Inc.