Purpose: Many cancers lack argininosuccinate synthetase 1 cells grew robustly through arginine starvation in vivo, while ADI(ASS1), the rate-limiting enzyme of arginine biosynthesis. This PEG20 remained completely lethal in vitro, evidence that pointed deficiency causes arginine auxotrophy, targetable by extracellular toward a novel mechanism of resistance mediated by the micro-arginine-degrading enzymes such as ADI-PEG20. Long-term environment. Coculture with Ass1-competent fibroblasts rescued tumor resistance has thus far been attributed solely to ASS1 growth through macropinocytosis of vesicles and/or cell fragments, reexpression. This study examines the role of ASS1 silencing on followed by recycling of protein-bound arginine through autophtumor growth and initiation and identifies a noncanonical mechagy/lysosomal degradation. Inhibition of either macropinocytosis anism of resistance, aiming to improve clinical responses to ADI- or autophagy/lysosomal degradation abrogated this growth support PEG20. effect in vitro and in vivo. Experimental Design: Tumor initiation and growth rates were Conclusions: Noncanonical, ASS1-independent tumor resismeasured for a spontaneous Ass1 knockout (KO) murine sarcoma tance to ADI-PEG20 is driven by the microenvironment. This model. Tumor cell lines were generated, and resistance to arginine mechanism can be targeted by either the macropinocytosis inhibitor deprivation therapy was studied in vitro and in vivo. imipramine or the autophagy inhibitor chloroquine. These safe, Results: Conditional Ass1 KO affected neither tumor initiation widely available drugs should be added to current clinical trials to nor growth rates in a sarcoma model, contradicting the prevalent overcome microenvironmental arginine support of tumors and idea that ASS1 silencing confers a proliferative advantage. Ass1 KO improve patient outcomes.