Abstract 11: Arginine deprivation therapy using ADI-PEG20 as a novel therapeutic modality in the treatment of bladder cancer.
Authors: Gupta Sounak , Maria Mir , Bomalaski John , Elson Paul J. , Singh Kamini , Harwalkar Jyoti , Carver Paula , Almasan Alex , Hansel Donna E. 
Published: August 2014

Abstract
Background: Argininosuccinate synthetase (ASS1) catalyzes the rate limiting step in the biosynthesis of arginine. ASS1-deficient tumors are therefore candidates for arginine deprivation therapy, utilizing ADI-PEG20 as it degrades circulating arginine. We therefore evaluated 187 patients with bladder cancer, who underwent radical cystectomy between 1988 and 2008 at our institution for ASS1 expression. ASS1 was evaluated as a predictor of disease outcome, while in-vitro studies were conducted to assess the therapeutic efficacy of ADI-PEG20 in representative cell lines.
Design: ASS-1 expression was assessed by immunostaining and stratified based on moderate to strong expression (2+, 3+) versus absent to weak expression (0 or 1+). Both univariate and multivariate statistical analysis of ASS1 expression, along with various clinical parameters, was carried out to determine the effect on overall (OS) and progression-free (PFS) survival. In addition, multiple bladder cancer cell-lines were screened for ASS1 expression and utilized as an in-vitro model. Downstream signaling changes in these cells, in response to arginine deprivation, were assessed by quantitative PCRs and immunoblotting and this was correlated with changes in cell viability, proliferation and cell death. The latter was assessed by a combination of clonogenic and MTT assays in addition to propidium iodide (PI) staining alone or PI /Annexin V double staining followed by flow cytometry.
Results: 63% had conventional urothelial carcinoma (UCa), while the rest had rarer variants such as squamous cell carcinoma (SCC: 18%), the micropapillary variant (9%), adenocarcinoma (5%) and small cell carcinomas (4%). Micropapillary variants (71%) and adenocarcinomas (90%) tended to show moderate to strong ASS1 expression, while UCa (42%) and small cell carcinomas (38%) demonstrated intermediate ASS1 expression (2+, 3+). In contrast, patients with SCC tended to have weak ASS1 expression (91%, 0 or 1+). In univariate analysis, increased ASS1 expression was associated with poorer OS (p≤.10 in all cases for OS) and PFS (p=.03 for PFS (2+, 3+ vs 0, 1+)). In multivariate analysis, ASS1 expression (3+ vs 0, 1+ or 2+), p=.04, along with positive nodes (p=.0005), surgical margins (p=.007), and higher pT-stage (p=.02) were all seen to be independent predictors of poorer OS. In-vitro studies with UCa derived J82 cells (that were found to be ASS1 deficient) following ADI-PEG20 treatment revealed an activation of GCN2, a kinase known to be activated by amino acid deprivation. Downstream changes included induction of the pro-apoptotic gene CHOP. This was correlated with a reduction in cell viability (IC50=0.24ug/ml) that was attributed to increased apoptosis.
Conclusion: High ASS1 expression is an independent predictor of poor OS and PFS in bladder cancer. Furthermore, ADI-PEG20 represents a promising new therapy for ASS1-deficient bladder cancers.
Citation Format: Sounak Gupta, Mir Maria, John Bomalaski, Paul J. Elson, Kamini Singh, Jyoti Harwalkar, Paula Carver, Alex Almasan, Donna E. Hansel. Arginine deprivation therapy using ADI-PEG20 as a novel therapeutic modality in the treatment of bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 11. doi:10.1158/1538-7445.AM2013-11