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.

Nanotechnology has introduced many exciting new tools for the treatment of human diseases. One of the obstacles in its application to that end is the lack of a fundamental understanding of the interaction that occurs between nanoparticles and living cells. This report describes the quantitative analysis of the kinetics and endocytic pathways involved in the uptake of anatase titanium dioxide (TiO2) nanoparticles into prostate cancer PC-3M cells. The experiments were performed with TiO2 nanoconjugates—6 nm nanoparticles with surface conjugated fluorescent Alizarin Red S (ARS). Results obtained by flow cytometry, fluorescence microscopy, and inductively-coupled plasma mass spectrometry confirmed a complex nanoparticle-cell interaction involving a variety of endocytic mechanisms. The results demonstrated that a temperature, concentration, and time-dependent internalization of the TiO2 nanoparticles and nanoconjugates occurred via clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis.

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Argininosuccinate Synthetase 1 deficiency induces dependence on extracellular arginine for continued cellular growth and survival. Arginine starvation inhibits the Warburg effect and diverts glucose into serine biosynthesis, while simultaneously increasing glutamine metabolism via the tricarboxylic acid cycle. Simultaneous arginine deprivation and inhibition of the subsequent metabolic adaptations induce synthetic lethality.KEYWORDS ADI-PEG20; arginine deiminase; argininosuccinate synthetase 1; ASS1; glutaminase; metabolism; GLS; glucose; glutamine; PHGDH; Warburg Cancer metabolism represents the next wave of cancer therapeutics after immunotherapy. The alterations in cancer cell metabolism that are currently being investigated result from the dramatic metabolic reprogramming that occurs within cancer for the production of biomass. One of the original metabolic hallmarks was the "Warburg Effect," whereby cancer cells preferentially generate energy through glucose fermentation into lactic acid rather than oxidation through the tricarboxylic acid (TCA) cycle and subsequent oxidative phosphorylation. Other common metabolic alterations in cancer include mutations in isocitrate dehydrogenase, overexpression of pyruvate kinase M2 (PKM2), deficiencies in succinate dehydrogenase and fumarate hydratase, and the loss of argininosuccinate synthetase 1 (ASS1) expression. 1 ASS1 is an enzyme involved in clearance of nitrogenous waste via the urea cycle and de novo arginine biosynthesis. Loss of ASS1 expression forces cells to rely on extracellular arginine for continued growth and survival; however, it appears to offer a tumorigenic advantage as loss of ASS1 expression has been shown to be a prognostic biomarker of reduced metastasis-free and overall survival. ASS1-deficient cells undergo autophagy when exposed to the arginine-depleting agent PEGylated arginine deiminase (ADI-PEG20) and undergo cell death upon simultaneous pharmacological or genetic inhibition of autophagy.2,3 Acquired resistance to ADI-PEG20 occurs via a c-Myc-dependent reexpression of ASS1 and renewed cellular arginine biosynthesis capabilities.4,5 Characterization of the cellular consequences of arginine starvation is essential in order to identify and target the metabolic reprogramming that occurs to inhibit the ability of cancer cells to acquire resistance to ADI-PEG20. These findings are being used to develop a biomarker-driven multiagent metabolic therapy for ASS1-deficient cancers.Metabolic characterization of both short-and long-term ADI-PEG20 treatment has shown significant alterations in global metabolism. 5,6 One of the many alterations of metabolic pathways that occur upon short-term ADI-PEG20 treatment was the significant redirection of cellular glucose. While cancer cells typically ferment the majority of cellular glucose into lactic acid and oxidize lesser amounts via the TCA cycle, ADI-PEG20 caused a decrease in the flux of glucose-derived carbons into both lactate and TCA cycle intermediates. The decrease in glucose flux to lactate ...

<p>Supplemental Figure 6. RNA sequencing of tumors.</p>

<p>Supplemental Figure 3. Fibroblasts support growth of Ass1 WT murine and human sarcoma cell lines.</p>

<p>Supplemental Figure 7. Gemcitabine and docetaxel increase toxicity to ASS1 KO tumor cells treated with ADI-PEG20 and either chloroquine or imipramine.</p>