Systems level profiling of arginine starvation reveals MYC and ERK adaptive metabolic reprogramming

Abstract

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...