SUMMARYTargeting defects in metabolism is an underutilized strategy for the treatment of cancer. Arginine auxotrophy resulting from the silencing of argininosuccinate synthetase 1 (ASS1) is a common metabolic alteration reported in a broad range of aggressive cancers. To assess the metabolic effects that arise from acute and chronic arginine starvation in ASS1-deficient cell lines, we performed metabolite profiling. We found that pharmacologically induced arginine depletion causes increased serine biosynthesis, glutamine anaplerosis, oxidative phosphorylation, and decreased aerobic glycolysis, effectively inhibiting the Warburg effect. The reduction of glycolysis in cells otherwise dependent on aerobic glycolysis is correlated with reduced PKM2 expression and phosphorylation and upregulation of PHGDH. Concurrent arginine deprivation and glutaminase inhibition was found to be synthetic lethal across a spectrum of ASS1-deficient tumor cell lines and is sufficient to cause in vivo tumor regression in mice. These results identify two synthetic lethal therapeutic strategies exploiting metabolic vulnerabilities of ASS1-negative cancers.

Sarcomas comprise a large heterogeneous group of mesenchymal cancers with limited therapeutic options. When treated with standard cytotoxic chemotherapies, many sarcomas fail to respond completely and rapidly become treatment resistant. A major problem in the investigation and treatment of sarcomas is the fact that no single gene mutation or alteration has been identified among the diverse histologic subtypes. We searched for therapeutically druggable targets that are common to a wide range of histologies and hence could provide alternatives to the conventional chemotherapy. Seven hundred samples comprising 45 separate histologies were examined. We found that almost 90% were arginine auxotrophs, as the expression of argininosuccinate synthetase 1 was lost or significantly reduced. Arginine auxotrophy confers sensitivity to arginine deprivation, leading temporarily to starvation and ultimately to cell survival or death under different circumstances. We showed that, in sarcoma, arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20) maintains a prolonged state of arginine starvation without causing cell death. However, when starvation was simultaneously prolonged by ADI-PEG20 while inhibited by the clinically available drug chloroquine, sarcoma cells died via necroptosis and apoptosis. These results have revealed a novel metabolic vulnerability in sarcomas and provided the basis for a well-tolerated alternative treatment strategy, potentially applicable to up to 90% of the tumors, regardless of histology.

Arginine auxotrophy due to the silencing of argininosuccinate synthetase 1 (ASS1) occurs in many carcinomas and in the majority of sarcomas. Arginine deiminase (ADI-PEG20) therapy exploits this metabolic vulnerability by depleting extracellular arginine, causing arginine starvation. ASS1-negative cells develop resistance to ADI-PEG20 through a metabolic adaptation that includes re-expressing ASS1. As arginine-based multiagent therapies are being developed, further characterization of the changes induced by arginine starvation is needed. In order to develop a systems-level understanding of these changes, activity-based proteomic profiling (ABPP) and phosphoproteomic profiling were performed before and after ADI-PEG20 treatment in ADI-PEG20-sensitive and resistant sarcoma cells. When integrated with metabolomic profiling, this multi-omic analysis reveals that cellular response to arginine starvation is mediated by adaptive ERK signaling and activation of the Myc-Max transcriptional network. Concomitantly, these data elucidate proteomic changes that facilitate oxaloacetate production by enhancing glutamine and pyruvate anaplerosis and altering lipid metabolism to recycle citrate for oxidative glutaminolysis. Based on the complexity of metabolic and cellular signaling interactions, these multi-omic approaches could provide valuable tools for evaluating response to metabolically targeted therapies.

22Arginine auxotrophy due to the silencing of argininosuccinate synthetase 1 (ASS1) occurs in many 23 cancers, especially sarcomas. Arginine deiminase (ADI-PEG20) therapy exploits this metabolic 24 vulnerability by depleting extracellular arginine, causing arginine starvation. ASS1-negative cells 25 develop resistance to ADI-PEG20 through a metabolic adaptation that includes re-expressing 26 ASS1. As arginine-based multiagent therapies are being developed, further characterization of 27 the changes induced by arginine starvation is needed. In order to develop a systems-level 28 understanding of these changes, activity-based proteomic profiling (ABPP) and 29 phosphoproteomic profiling were performed before and after ADI-PEG20 treatment in ADI-30 PEG20-sensitive and resistant sarcoma cells. When integrated with previous metabolomic 31 profiling (Kremer et al, 2017a), this multi-omic analysis reveals that cellular response to arginine 32 starvation is mediated by adaptive ERK signaling, driving a Myc-Max transcriptional network. 33Concomitantly, these data elucidate proteomic changes that facilitate oxaloacetate production by 34 enhancing glutamine and pyruvate anaplerosis, and altering lipid metabolism to recycle citrate for 35 oxidative glutaminolysis. Based on the complexity of metabolic and cellular signaling interactions, 36 these multi-omic approaches could provide valuable tools for evaluating response to metabolically 37 targeted therapies. 65invariably contribute to ABPP as well (Wolfe et al, 2013; Piazza et al, 2018a; Veyel et al, 2018). 66Ultimately, ABPP integrates multiple informative proteomic parameters and provides a broad view 67 of proteomic regulation. For example, ABPP can identify adaptive kinomic changes based on 68 either altered kinase expression or activity (Duncan et al, 2012). 69The mechanisms of developing resistance to arginine starvation in sarcomas have been 70 partially defined, and include stabilization of nuclear cMyc (Prudner et al, 2019b), and increased 71 glutamine anaplerosis in order to produce aspartate (Kremer et al, 2017a). In addition, others 72 have examined mechanisms of ASS1 re-expression (Tsai et al, 2017; Long et al, 2017) and 73Deptor regulation (Ohshima et al, 2017). However, the underlying proteomic changes that initiate 74 these events and coordinate metabolic reprogramming remain unknown. We pursued systems 75 biology profiling to understand resistance to arginine starvation, as these approaches have proven 76 effective in delineating the adaptive changes involved in highly pleiotropic phenotypes such as 77 drug resistance (Zecena et al, 2018; Galluzzi et al, 2014), Myc activation, and various metabolic 78 changes (Tomita & Kami, 2012; Schaub et al, 2018). 79To understand ADI-PEG20-resistance of ASS1-negative sarcomas at a systems level, we 80 performed multi-omic profiling using phosphoproteomics and activity-based proteomics, and 81 coupled these data with existing metabolomic analyses (Kremer et al, 2017a). ADI-PEG20-82 senstive leiomyosarcoma cells (SKLMS1) h...