Phuong Luong

Prognostic and Therapeutic Impact of Argininosuccinate Synthetase 1 Control in Bladder Cancer as Monitored Longitudinally by PET Imaging

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [(18)F]-fluoro-L-thymidine (FLT)-positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response.

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.

c-Abl phosphorylation of ΔNp63α is critical for cell viability.

The p53 family member p63 has been shown to be critical for growth, proliferation and chemosensitivity. Here we demonstrate that the c-Abl tyrosine kinase phosphorylates the widely expressed ΔNp63α isoform and identify multiple sites by mass spectrometry in vitro and in vivo. Phopshorylation by c-Abl results in greater protein stability of both ectopically expressed and endogenous ΔNp63α. c-Abl phosphorylation of ΔNp63α induces its binding to Yes-associated protein (YAP) and silencing of YAP by siRNA reduces the c-Abl-induced increase of ΔNp63α levels. We further show that cisplatin induces c-Abl phosphorylation of ΔNp63α and its binding to YAP. Overexpression of ΔNp63α, but not the c-Abl phosphosites mutant, protects cells from cisplatin treatment. Finally, we demonstrate the rescue of p63 siRNA-mediated loss of viability with p63siRNA insensitive construct of ΔNp63α but not the phosphosites mutant. These results demonstrate that c-Abl phosphorylation of ΔNp63α regulates its protein stability, by inducing binding of YAP, and is critical for cell viability.

Arginine Deprivation With Pegylated Arginine Deiminase in Patients With Argininosuccinate Synthetase 1-Deficient Malignant Pleural Mesothelioma: A Randomized Clinical Trial

Importance Preclinical studies show that arginine deprivation is synthetically lethal in argininosuccinate synthetase 1 (ASS1)-negative cancers, including mesothelioma. The role of the arginine-lowering agent pegylated arginine deiminase (ADI-PEG20) has not been evaluated in a randomized and biomarker-driven study among patients with cancer. Objective To assess the clinical impact of arginine depletion in patients with ASS1-deficient malignant pleural mesothelioma. Design, Setting, and Participants A multicenter phase 2 randomized clinical trial, the Arginine Deiminase and Mesothelioma (ADAM) study, was conducted between March 2, 2011, and May 21, 2013, at 8 academic cancer centers. Immunohistochemical screening of 201 patients (2011-2013) identified 68 with advanced ASS1-deficient malignant pleural mesothelioma. Interventions Randomization 2:1 to arginine deprivation (ADI-PEG20, 36.8 mg/m2, weekly intramuscular) plus best supportive care (BSC) or BSC alone. Main Outcomes and Measures The primary end point was progression-free survival (PFS) assessed by modified Response Evaluation Criteria in Solid Tumors (RECIST) (target hazard ratio, 0.60). Secondary end points were overall survival (OS), tumor response rate, safety, and quality of life, analyzed by intention to treat. We measured plasma arginine and citrulline levels, anti–ADI-PEG20 antibody titer, ASS1 methylation status, and metabolic response by 18F-fluorodeoxyglucose positron-emission tomography. Results Median (range) follow-up in 68 adults (median [range] age, 66 [48-83] years; 19% female) was 38 (2.5-39) months. The PFS hazard ratio was 0.56 (95% CI, 0.33-0.96), with a median of 3.2 months in the ADI-PEG20 group vs 2.0 months in the BSC group (P = .03) (absolute risk, 18% vs 0% at 6 months). Best response at 4 months (modified RECIST) was stable disease: 12 of 23 (52%) in the ADI-PEG20 group vs 2 of 9 (22%) in the BSC group (P = .23). The OS curves crossed, so life expectancy was used: 15.7 months in the ADI-PEG20 group vs 12.1 months in the BSC group (difference of 3.6 [95% CI, −1.0 to 8.1] months; P = .13). The incidence of symptomatic adverse events of grade at least 3 was 11 of 44 (25%) in the ADI-PEG20 group vs 4 of 24 (17%) in the BSC group (P = .43), the most common being immune related, nonfebrile neutropenia, gastrointestinal events, and fatigue. Differential ASS1 gene-body methylation correlated with ASS1 immunohistochemistry, and longer arginine deprivation correlated with improved PFS. Conclusions and Relevance In this trial, arginine deprivation with ADI-PEG20 improved PFS in patients with ASS1-deficient mesothelioma. Targeting arginine is safe and warrants further clinical investigation in arginine-dependent cancers. Trial Registration clinicaltrials.gov Identifier: NCT01279967

Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia

One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis.

Abstract 1431: Gene expression analysis of argininosuccinate synthetase loss and the effects of pegylated arginine deiminase in malignant pleural mesothelioma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Malignant pleural mesothelioma (MPM) is a devastating asbestos-related malignancy that is increasing in many countries worldwide with few systemic treatment options beyond platinum and antifolate chemotherapy. Deficiency of the arginine biosynthetic enzyme argininosuccinate synthetase (ASS1) occurs in up to 50% of MPM cell lines and primary tumors and is being validated as a biomarker in patients treated with the arginine-depleting agent, pegylated arginine deiminase (ADI-PEG20). To understand the role of ASS1 loss and the effect of the ADI-PEG20 in MPM we used pathway analysis tools on microarray gene expression data from a representative panel of MPM cell lines. First, we identified that ASS1 loss was linked to several protumorigenic functions including increased cell invasiveness and migration, which were confirmed subsequently using invasion assays and organotypic modelling, respectively. We also detected a large number of enriched pathways connected to the immune response including communication between innate and adaptive immune cells and interferon signalling. Furthermore, ASS1 deficiency was linked to worse outcome with a median survival of 6 months in ASS1 low expressors compared to 12 months for ASS1 high expressors using a retrospective dataset (n=41; p=0.003). Second, ADI-PEG20 treatment modulated numerous pathways in ASS1-deficient cells including suppression of mTOR and folate metabolism, while promoting stress, oxidant and amino acid signalling. Third, bioinformatics analyses of drug interactions using Connectivity map revealed that arginine deprivation using ADI-PEG20 may potentiate several chemotherapeutic and targeted agents in the clinic. Taken together, our bioinformatics approach links loss of ASS1 in MPM cells to a more aggressive phenotype and identifies several potential combination strategies with ADI-PEG20 for further clinical investigation. Citation Format: Rosalind Cutts, Puthen V. Jithesh, Barbara Delage, Phuong Luong, Gareth Thomas, Claude Chelala, Peter W. Szlosarek. Gene expression analysis of argininosuccinate synthetase loss and the effects of pegylated arginine deiminase in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1431. doi:10.1158/1538-7445.AM2014-1431

Abstract 1885: A comprehensive untargeted UPLC-MS based metabolomic analysis ofASS1-deficient solid tumor cell lines treated with arginine deiminase.

Background Arginine is an important amino acid for tumor cell growth and development. Argininosuccinate synthetase 1 (ASS1) is a key enzyme required for biosynthesis of arginine. Preclinically, ASS1-deficient tumor cells are particularly sensitive to arginine depletion, and randomised trials exploring this strategy are in progress in hepatocellular carcinoma and mesothelioma using the drug pegylated arginine deiminase (ADI-PEG20). In this study, we determined the metabolic changes induced by ADI-PEG20 treatment in a panel of bladder cancer and mesothelioma cell lines with promoter methylation-dependent silencing of ASS1. We used two cancer cell lines expressing ASS1 as a control. Methods Malignant mesothelioma, ASS1-negative (H2591, MSTO and JU77) and ASS1- positive (H28) cell lines were used. For bladder cancer, ASS1-negative (T24, 253J, UMUC-3) and ASS1-positive (RT112) cell lines were also used. All cells were treated with 750 ng/ml of ADI-PEG20 for 24 hours which induces up to 90% killing of arginine-dependent cell lines. Then, ultra performance liquid chromatography-mass spectrometry (UPLC-MS) technique was employed for untargeted quantitation of the metabolomic changes induced by ADI-PEG20. Results: All cell lines treated with ADI-PEG20 could be clearly discriminated from their untreated control pair when using PCA multivariate analysis. Arginine depletion was noted in all treated cell lines irrespective of ASS1 expression, however the reduction was at least one-log-fold greater in the ASS1-negative tumor cells. Citrulline, n-a-acetylcitrulline, and glutamine were upregulated specifically in ASS1-negative tumor cell lines. The main impact of ADI-PEG20 treatment was on pyrimidine metabolism in the ASS1-deficient tumor cells with upregulation of thymine and downregulation of thymidine, ureidosuccinic acid, uridine monophosphate and 5-hydroxymethyluracil. Notably, we identified that the reduction of the thymidine nucleotide pool was linked to suppression of thymidylate synthetase and dihydrofolate reductase, and paradoxically, reduced uptake of 3H-FLT. Finally, ADI-PEG20 caused variable effects on cytidine, uridine and ornithine levels in different cancer cell lines. Conclusion This study provides an insight into possible metabolic pathways affected by ADI-PEG20. The impact of ADI-PEG20 on thymidine metabolism, in particular, may be employed as a potential biomarker for optimizing the efficacy of ADI-PEG20 in the treatment of arginine auxotophic cancers. Citation Format: Essam A. Ghazaly, Phuong Luong, Malgorzata Chmielewska-Kassasir, Chantelle Hudson, John S. Bomalaski, L Wozniak, Norbert E. Avril, Simon P. Joel, Peter W. Szlosarek. A comprehensive untargeted UPLC-MS based metabolomic analysis of ASS1-deficient solid tumor cell lines treated with arginine deiminase. [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 1885. doi:10.1158/1538-7445.AM2013-1885

Abstract B139: Arginine deprivation with pegylated arginine deiminase regulates key metabolic genes in RNA and DNA synthesis in ASS1-deficient malignant mesothelioma cells: Clinical implications.

Tumors deficient in argininosuccinate synthetase-1 (ASS1), a key enzyme involved in arginine synthesis, are auxotrophic for arginine and sensitive to amino acid deprivation. Currently, several trials are testing the arginine-depleting agent, pegylated arginine deiminase (ADI-PEG20) in patients with cancer, including a randomised phase II multicenter UK study in patients with ASS1-deficient mesothelioma (NCT01279967). Here, we sought to identify key pathways involved in the mechanism of action of ADI-PEG20 using a panel of ASS1-deficient malignant mesothelioma (MPM) cell lines as our model. Epithelioid (2591), biphasic (MSTO) and sarcomatoid (JU77) mesothelioma cells were exposed to ADI-PEG20 and analysed using Affymetrix gene expression profiling with validation of candidate genes by qPCR and western blotting, and metabolic studies by LC-MS and 1H-NMR. ADI-PEG20 modulated several metabolic genes involved in nucleotide synthesis in the MPM cell lines. We identified suppression of ribonucleotide reductase (M1 and M2 isoforms), thymidylate synthase and dihydrofolate reductase by 24hrs, the latter enzymes being known targets of pemetrexed, a multitargeted antifolate used in the first-line therapy of mesothelioma. Moreover, metabolic profiling revealed upregulation of several modified nucleosides, including ribothymidine and downregulation of the nucleotide, guanosine monophosphate. Lastly, in vitro studies confirmed potentiation between ADI-PEG20 and pemetrexed and sequential therapy led to reduced tumor growth in vivo. In conclusion, arginine depletion modulates several key enzymes involved in nucleotide synthesis, providing a rationale for combining ADI-PEG20 with pemetrexed-containing regimens in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B139.