Melanomas and other cancers that do not express argininosuccinate synthetase (AS), the rate-limiting enzyme for arginine biosynthesis, are sensitive to arginine depletion with pegylated arginine deiminase (ADI-PEG20). However, ADI resistance eventually develops in tumors due to AS upregulation. Although it has been shown that AS upregulation involves c-Myc, the underlying mechanisms remain unknown. Here we show that ADI-PEG20 activates Ras signaling and the effector ERK and PI3K/AKT/GSK-3β kinase cascades, resulting in phosphorylation and stabilization of c-Myc by attenuation of its ubiquitin-mediated protein degradation mechanism. Inhibition of the induced cell signaling pathways using PI3K/AKT inhibitors suppressed c-Myc induction and enhanced ADI-mediated cell killing. Notably, in an animal model of AS-negative melanoma, combination therapy using a PI3K inhibitor plus ADI-PEG20 yielded additive anti-tumor effects as compared with either agent alone. Taken together, our findings offer mechanistic insight into arginine deprivation metabolism and ADI resistance, and they illustrate how combining inhibitors of the Ras/ERK and PI3K/AKT signaling pathways may improve ADI-PEG20 anti-cancer responses.

The maintenance of genomic stability in cells is relentlessly challenged by environmental stresses that induce DNA breaks, which activate the DNA-damage pathway mediated by ataxia-telangiectasia mutated (ATM) and its downstream mediators to control damage-induced cell-cycle checkpoints and DNA repair. Here, we show that FOXO3a interacts with ATM to promote phosphorylation of ATM at Ser 1981 and prompting its downstream mediators to form nuclear foci in response to DNA damage. Silencing FOXO3a in cells abrogates the formation of ATM-pS1981 and phospho-histone H2AX foci after DNA damage. Increasing FOXO3a in cells promotes ATM-regulated signalling, the intra-S-phase or G2-M cell-cycle checkpoints, and the repair of damaged DNA, whereas cells lacking FOXO3a did not trigger the DNA-repair mechanism after DNA damage. The carboxy-terminal domain of FOXO3a binds to the FAT domain of ATM, thereby contributing to the activation of ATM. These results suggest that ATM may be regulated directly by FOXO3a in the DNA-damage response.