Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes.

Rationale: One of the most common metabolic defects in cancers is the deficiency in arginine synthesis, which has been exploited therapeutically. Yet, challenges remain, and the mechanisms of arginine-starvation induced killing are largely unclear. Here, we sought to demonstrate the underlying mechanisms by which arginine starvation-induced cell death and to develop a dietary arginine-restriction xenograft model to study the in vivo effects. Methods: Multiple castration-resistant prostate cancer cell lines were treated with arginine starvation followed by comprehensive analysis of microarray, RNA-seq and ChIP-seq were to identify the molecular and epigenetic pathways affected by arginine starvation. Metabolomics and Seahorse Flux analyses were used to determine the metabolic profiles. A dietary arginine-restriction xenograft mouse model was developed to assess the effects of arginine starvation on tumor growth and inflammatory responses. Results: We showed that arginine starvation coordinately and epigenetically suppressed gene expressions, including those involved in oxidative phosphorylation and DNA repair, resulting in DNA damage, chromatin-leakage and cGAS-STING activation, accompanied by the upregulation of type I interferon response. We further demonstrated that arginine starvation-caused depletion of α-ketoglutarate and inactivation of histone demethylases are the underlying causes of epigenetic silencing. Significantly, our dietary arginine-restriction model showed that arginine starvation suppressed prostate cancer growth in vivo, with evidence of enhanced interferon responses and recruitment of immune cells. Conclusions: Arginine-starvation induces tumor cell killing by metabolite depletion and epigenetic silencing of metabolic genes, leading to DNA damage and chromatin leakage. The resulting cGAS-STING activation may further enhance these killing effects.

Genome-wide CRISPR/Cas9 knockout screening uncovers a novel inflammatory pathway critical for resistance to arginine-deprivation therapy.

Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo, and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.

Arginine deiminase expressed in vivo, driven by human telomerase reverse transcriptase promoter, displays high hepatoma targeting and oncolytic efficiency.

Arginine starvation has the potential to selectively treat both primary tumor and (micro) metastatic tissue with very low side effects. Arginine deiminase (ADI; EC 3.5.3.6), an arginine-degrading enzyme, has been studied as a potential anti-tumor drug for the treatment of arginine-auxotrophic tumors. Though ADI-PEG20 (pegylated ADI by PEG 20,000) already passed the phase I/II clinical trials [1], it is just used as adjuvant therapy because of its low efficiency and less targeting. Then, this paper discussed the efficiency of arginine starvation mediated by ADI expressed in cytoplasm for liver cancers. In order to guarantee the tumor targeting, human telomerase reverse transcriptase (hTERT) promoter was used to drive the expression of ADI in vivo. To access the anti-tumor efficiency of ADI, p53 gene was used as the positive control. Thus, ADI displayed obvious cytotoxicity to BEL7402 and HUH7 cell lines in cytoplasm. The apoptosis rates rose from 15% to nearly 60% after changing the expression vectors from pcDNA4 plasmid to adenovirus. Compared with p53-adenovirus, ADI-adenovirus showed the higher oncolytic activity in the intratumoral injection model of mice. Tumor disappeared after the treatment of ADI-adenovirus for two weeks, and the mice pulled through all. Therefore, ADI is an ideal anti-tumor gene for caner targeting therapy with the help of hTERT promoter.

Argininosuccinate synthetase 1 (ASS1) is a common metabolic marker of chemosensitivity for targeted arginine- and glutamine-starvation therapy.

Argininosuccinate synthetase 1 (ASS1) is the rate-limiting enzyme that catalyzes the biosynthesis of arginine (Arg). Many malignant human tumors are auxotrophic for Arg because ASS1 is silenced. ASS1 has been established as a sensor of Arg auxotrophic response and a chemosensitivity marker for Arg starvation therapy. Here, we report that ASS1 is also a sensor for glutamine (Gln)-deprivation response, and that upregulation of ASS1 expression is associated with resistance to Gln-starvation treatments. Knockdown of ASS1 expression resulted in increased sensitivity to both Arg- and Gln-starvation, whereas increased ASS1 expression by ectopic transfection is associated with resistance to both Arg- and Gln-starvation. The addition of permeable fumarate, a metabolite that bridges the tricarboxylic acid and urea cycles, resulted in downregulation of ASS1 expression and increased sensitivity to both Arg- and Gln-deprivation treatments. Mechanistically, the Gln-deprivation response, like the arginine-auxotrophic response, downregulates HIF-1α resulting in de-silencing of ASS1. Our results demonstrate that ASS1 is a common biosensor for Arg and Gln deprivation response and a shared target for Arg- and Gln-starvation therapies which have been in several current clinical trials.

Cisplatin-induced synthetic lethality to arginine-starvation therapy by transcriptional suppression of ASS1 is regulated by DEC1, HIF-1α, and c-Myc transcription network and is independent of ASS1 promoter DNA methylation.

Many human tumors require extracellular arginine (Arg) for growth because the key enzyme for de novo biosynthesis of Arg, argininosuccinate synthetase 1 (ASS1), is silenced. These tumors are sensitive to Arg-starvation therapy using pegylated arginine deiminase (ADI-PEG20) which digests extracellular Arg. Many previous studies reported that ASS1 silencing is due to epigenetic inactivation of ASS1 expression by DNA methylation, and that the demethylation agent 5-aza-deoxycytidine (Aza-dC) can induce ASS1 expression. Moreover, it was reported that cisplatin suppresses ASS1 expression through ASS1 promoter methylation, leading to synthetic lethality to ADI-PEG20 treatment. We report here that cisplatin supppresses ASS1 expression is due to upregulation of HIF-1α and downregulation of c-Myc, which function as negative and positive regulators of ASS1 expression, respectively, by reciprocal bindings to the ASS1 promoter. In contrast, we found that Aza-dC induces ASS1 expression by downregulation of HIF-1α but upregulation of c-Myc. We further demonstrated that the clock protein DEC1 is the master regulator of HIF-1α and c-Myc that regulate ASS1. cDDP upregulates DEC1, whereas Aza-dC suppresses its expression. Using two proteasomal inhibitors bortezomib and carfilzomib which induce HIF-1α accumulation, we further demonstrated that HIF-1α is involved in ASS1 silencing for the maintenance of Arg auxotrophy for targeted Arg-starvation therapy.

Arginine starvation in colorectal carcinoma cells: Sensing, impact on translation control and cell cycle distribution.

Tumor cells rely on a continued exogenous nutrient supply in order to maintain a high proliferative activity. Although a strong dependence of some tumor types on exogenous arginine sources has been reported, the mechanisms of arginine sensing by tumor cells and the impact of changes in arginine availability on translation and cell cycle regulation are not fully understood. The results presented herein state that human colorectal carcinoma cells rapidly exhaust the internal arginine sources in the absence of exogenous arginine and repress global translation by activation of the GCN2-mediated pathway and inhibition of mTOR signaling. Tumor suppressor protein p53 activation and G1/G0 cell cycle arrest support cell survival upon prolonged arginine starvation. Cells with the mutant or deleted TP53 fail to stop cell cycle progression at defined cell cycle checkpoints which appears to be associated with reduced recovery after durable metabolic stress triggered by arginine withdrawal.