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2589Background: Argininosuccinate synthetase (ASS1) loss is a biomarker to select for tumors sensitive to arginine deprivation therapy. In a phase 1 dose-escalation study of ASS1-deficient thoracic...

4085Background: Arginine depletion interferes with pyrimidine metabolism as well as DNA damage repair pathways, and preclinical data indicate that pairing pegylated arginine deaminase (ADI-PEG 20) ...

e14085Background: Patients (pts) with recurrent HGGs are usually managed with alkylating chemotherapy +/- bevacizumab. However, prognosis remains poor with an overall survival (OS) of 7-9 months. P...

2556Background: Arginine deprivation with pegylated arginine deiminase (ADI-PEG 20) has been shown to upregulate tumor programmed death-ligand 1 (PD-L1) expression and T cell infiltration. Current ...

Loss of argininosuccinate synthetase 1 (ASS1), a key enzyme for arginine synthesis, occurs in many cancers, making cells dependent on extracellular arginine and targetable by the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20). We evaluated ASS1 expression and effects of ASS1 loss in bladder cancer which, despite affecting >70,000 people in the United States annually, has limited therapies. ASS1 loss was identified in conventional and micropapillary urothelial carcinoma, small cell, and squamous cell carcinoma subtypes of invasive bladder cancer, as well as in T24, J82, and UM-UC-3 but not in 5637, RT112, and RT4 cell lines. ASS1-deficient cells showed preferential sensitivity to ADI-PEG 20, evidenced by decreased colony formation, reduced cell viability, and increased sub-G1 fractions. ADI-PEG 20 induced general control nonderepressible 2–dependent eukaryotic initiation factor 2α phosphorylation and activating transcription factor 4 and C/EBP homologous protein up-regulation, associated with caspase-independent apoptosis and autophagy. These effects were ablated with selective siRNA silencing of these proteins. ASS1 overexpression in UM-UC-3 or ASS1 silencing in RT112 cells reversed these effects. ADI-PEG 20 treatment of mice bearing contralateral flank UM-UC-3 and RT112 xenografts selectively arrested tumor growth in UM-UC-3 xenografts, which had reduced tumor size, reduced Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. This suggests that ASS1 loss occurs in invasive bladder cancer and is targetable by ADI-PEG 20.

Background: The arginine-degrading enzyme, arginine deiminase conjugated to polyethylene glycol (ADI-SS PEG 20 000 mw), reduces extracellular arginine, has minimal toxicity, decreases tumor burden and improves liver function in patients with chronic hepatitis C virus infection (HCV) and inoperable hepatocellular carcinoma (HCC). Reduced extracellular arginine inhibits viral replication through unknown mechanisms. It is hypothesized that ADI-SS PEG 20 000 mw reduces HCV viral titers through nitric oxide (NO)-dependent effects. Methods: The effects of ADI-SS PEG 20 000 mw (dose, 160 IU/m2; three cycles of four once-weekly i.m. injections) on HCV titers, serum NO and plasma arginine, were evaluated using archived plasma from patients with HCC and HCV and in vitro cell model measurements of HCV replication. Results: ADI-SS PEG 20 000 mw selectively inhibited HCV replication in vitro (IC50 = 0.027 IU/mL). Fifteen HCC/HCV patients completed treatment. The HCV titers were reduced by up to 99% in five out of 10 (50%) HCV-serotype 1b patients (P = 0.0093). These patients also experienced significant improvements in liver function (P = 0.0091). There were concomitant reductions of plasma arginine and serum NO levels. The HCV titer was not reduced in HCV-type 2c patients. Conclusion: Reduction of extracellular arginine by ADI-SS PEG 20 000 mw in HCC patients reduces HCV viral titers and improves liver function, possibly through suppression of NO.

Purpose: This phase I study examined the toxicity and tolerability of pegylated arginine deiminase (ADI-PEG 20) in combination with docetaxel in patients with advanced solid malignancies. Experimental Design: Eligible patients had histologically proven advanced solid malignancies, with any number of prior therapies, Zubrod performance status 0–2, and adequate organ function. Patients received ADI-PEG 20 weekly intramuscular injection ranging from 4.5 to 36 mg/m2 and up to 10 doses of docetaxel (75 mg/m2) every 3 weeks. Primary endpoints were safety, toxicity, and a recommended phase II dose. Circulating arginine levels were measured before each cycle. Tumor response was measured as a secondary endpoint every 6 weeks on study. Results: Eighteen patients received a total of 116 cycles of therapy through four dose levels of ADI-PEG 20. A single dose-limiting toxicity (grade 3 urticarial rash) was observed at the 1st dose level, with no additional dose-limiting toxicities observed. Hematologic toxicities were common with 14 patients experiencing at least one grade 3 to 4 leukopenia. Fatigue was the most prevalent toxicity reported by 16 patients. Arginine was variably suppressed with 10 patients achieving at least a 50% reduction in baseline values. In 14 patients with evaluable disease, four partial responses (including 2 patients with PSA response) were documented, and 7 patients had stable disease. Conclusions: ADI-PEG 20 demonstrated reasonable toxicity in combination with docetaxel. Promising clinical activity was noted, and expansion cohorts are now accruing for both castrate-resistant prostate cancer and non–small cell lung cancer at a recommended phase II dose of 36 mg/m2. Clin Cancer Res; 21(11); 2480–6. ©2015 AACR.

Although allopurinol has been available for approximately 50 years, hyperuricemia and its sequelae are not only prevalent, but the incidence and costs associated with this disorder continue to increase. However, several new therapies have been developed. Recombinant urate oxidase has been useful in the treatment of tumor lysis hyperuricemia, and pegylated urate oxidase shows promise in patients with hyperuricemia and gout. Febuxostat and Y-700 are new oral xanthine oxidase inhibitors that are in human clinical trials. Tailoring of antilipid therapy in selected hyperuricemic and hyperlipidemic patients with fenofibrate may be of benefit in lowering blood cholesterol and uric acid levels. Similarly, treatment of selected hyperuricemic patients who also are hypertensive with losartan or amlodipine may be beneficial in lowering blood pressure and hyperuricemia. Despite these advances, new treatments for hyperuricemia are needed.

Purpose Recently, we reported that a large number of human hepatocellular cancer (HCC) cell lines were auxotrophic for arginine. Here we report the results obtained with the amino acid–degrading enzyme arginine deiminase (ADI) conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw) as a means of lowering plasma arginine to treat HCC. The study was a cohort dose-escalation phase I/II study. Patients and Methods Pharmacodynamic studies indicated an ADI-SS PEG 20,000 mw dose level of 160 U/m2 was sufficient to lower plasma arginine from a resting level of approximately 130 μmol/L to below the level of detection (< 2 μmol/L) for more than 7 days, a dose later defined as the optimal biologic dose. All patients were to receive three cycles at the optimum biologic dose. Results This therapy was well tolerated, even in patients who had no detectable plasma arginine for 3 continuous months of therapy. Of the 19 patients enrolled, two had a complete response, seven had a partial response, seven had stable disease, and three had progressive disease. The median survival for the 19 patients enrolled on this study was 410 days, with four patients still alive at present (> 680 days). Conclusion Elimination of all detectable plasma arginine in patients with HCC was well tolerated and seemed to be effective in the treatment of some patients with HCC. Further testing of ADI-SS PEG 20,000 mw in a larger population of individuals with HCC as well as other human tumors auxotrophic for arginine is warranted.

BACKGROUND Argininosuccinate synthetase (ASS) was the first of two enzymes to convert citrulline to arginine. This pathway allowed cells to synthesize arginine from citrulline, making this amino acid nonessential for the growth of most mammalian cells. Previous studies demonstrated that several human tumor cell lines were auxotrophic for arginine due to an inability to express ASS. Selective elimination of arginine from the circulation of animals with these tumors is a potentially effective anticancer treatment. The purpose of these experiments was to determine the frequency of ASS deficiency and arginine auxotrophy in a variety of human malignant tumors. METHODS The authors analyzed the expression of ASS by immunohistochemistry with a monoclonal antibody in a variety of human tumor biopsies. They found that the incidence of ASS deficiency varied greatly with the tumor type and tissue of origin. RESULTS Melanoma, hepatocellular carcinoma, and prostate carcinoma were most frequently deficient in ASS. Some human cancers were almost always positive for ASS (e.g., lung and colon carcinomas). However, other human cancers, including sarcomas, invasive breast carcinoma, and renal cell carcinoma, also were sometimes ASS deficient. CONCLUSIONS These data indicated that immunohistochemical detection of ASS may prove an effective means for determining ASS deficiency in malignant human tumors and for identifying patients most likely to respond to arginine deprivation therapy. Based on these results, human clinical trials using arginine-degrading enzyme therapy to treat patients with advanced melanoma or hepatocellular carcinoma have been initiated. Cancer 2004;100:826–33. © 2004 American Cancer Society.

Septic shock is mediated in part by nitric oxide (NO) and tumour necrosis factor α (TNFα). NO is synthesized primarily from extracellular arginine. We tested the ability of an arginine-degrading enzyme to inhibit NO production in mice and to protect mice from the hypotension and lethality that occur after the administration of TNFα or endotoxin. Treatment of BALB/c mice with arginine deiminase (ADI) formulated with succinimidyl succinimide polyethylene glycol of Mr 20000 (ADI-SS PEG20000) eliminated all measurable plasma arginine (from normal levels of ∼155μM arginine to 2μM). In addition, ADI-SS PEG20000 also inhibited the production of NO, as quantified by plasma nitrate+nitrite. Treatment of mice with TNFα or endotoxin resulted in a dose-dependent increase in NO production and lethality. Pretreatment of mice with ADI-SS PEG20000 resulted in increased resistance to the lethal effects of TNFα and endotoxin. These observations are consistent with NO production resulting, to some extent, from the metabolism of extracellular arginine. The toxic effects of TNFα and endotoxin may be partially inhibited by enzymic degradation of plasma arginine by ADI-SS PEG20000. Interestingly, pretreatment with ADI-SS PEG20000 did not inhibit the anti-tumour activity of TNFα in vitro or in vivo. This treatment may allow greater amounts of TNFα, as well as other cytokines, to be administered while abrogating side effects such as hypotension and death.

Some tumors, such as melanomas and hepatocellular carcinomas, have a unique nutritional requirement for arginine. Thus, enzymatic degradation of extracellular arginine is one possible means for inhibiting these tumors. Arginine deiminase is an arginine degrading enzyme (ADI) that has been studied as an anti-cancer enzyme. However, ADI has a short serum half-life and, as a microbial enzyme, is highly immunogenic. Formulation of other therapeutic proteins with poly(ethylene glycol) (PEG) has overcome these problems. Here, ADI-PEGs were synthesized using PEGs of varying size, structure (linear or branched chain) and linker chemistries. All ADI-PEGs retained approximately 50% of enzyme activity when PEG was covalently attached to approximately 40% of the primary amines irrespective of the PEG molecular weight or attachment chemistry used. However, it was observed that, as the PEG size increases to 20 kDa, there was a corresponding increase in the pharmacokinetic (pK) and pharmacodynamic (pD) properties of the formulation. Variation in PEG linker or structure, or the use of PEGs >20,000 mw, did not affect the pK or pD. As has been shown with other therapeutic proteins, repeated injection of ADI-PEG into experimental animals resulted in significantly lower titers of antibodies against this protein than unmodified ADI. These data suggest that formulation of ADI with PEG of 20,000 mw results is the optimal method for formulating this promising therapeutic agent.

TPS8582 Background: Argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM) is sensitive to arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20), which also enhances the cytotoxicity of pemetrexed. The TRAP Phase 1 trial (NCT02029690) of ADI-PEG 20 combined with 1st-line pemetrexed (PEM) and cisplatin (CDDP) chemotherapy revealed a 94% disease control rate in non-epithelioid (biphasic and sarcomatoid) MPM subtypes characterized by a 75% rate of ASS1 loss. Thus, we plan to assess the efficacy of ADI-PEG20 or placebo combined with PEM and CDDP in patients (pts) with poor prognosis MPM in a randomized, placebo-controlled, double-blind phase 2/3 global trial. Methods: Up to 386 good performance (ECOG 0-1) pts with non-epithelioid malignant pleural mesothelioma will be enrolled in a phase 2/3 adaptive, biomarker-driven study design. Biopsies will be required prior to randomization: ASS1-agnostic pts will be enrolled initially (phase 2 stage) with an option to restrict enrolment to ASS1-deficient MPM (phase 3 stage). Pts will be randomized to receive weekly ADI-PEG20 (36 mg/m2 IM) or placebo with standard doses of PEM and CDDP for a maximum of 18 weeks (6 cycles) of treatment. Pts who develop CDDP toxicity may be switched to carboplatin. Pts will be assessed every 6 weeks using modified RECIST (RECIST 1.1 allowed for pts with significant extrathoracic disease). The primary endpoint for the phase 2 stage will be overall response rate (ORR) with secondary endpoints of overall survival (OS), safety and toxicity. The phase 2 will test ORR proportions with the placebo triplet set at 15% vs. 35% for the ADI-PEG 20 triplet, with a 1:1 randomization, 80% power. After recruitment of 176 pts, the phase 2 will convert to a phase 3 study with the primary endpoint of OS. In summary, ATOMIC-Meso is the first triplet chemotherapy study to assess the role of targeted arginine deprivation in aggressive subtypes of mesothelioma. Pt accrual has commenced across the US and Asia, with enrolment due in Europe and Australia by 2nd quarter of 2017. [Trial sponsored by Polaris Group]. Clinical trial information: NCT02709512.

8553 Background: Argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM) cells are sensitive to arginine deprivation with pegylated arginine deiminase (ADI-PEG20), which also potentiates the cytotoxic effect of pemetrexed (PEM). In the phase I dose-escalation TRAP study (NCT02029690) we showed that ADI-PEG20 with first-line PEM and cisplatin (CIS) chemotherapy (ADIPEMCIS) produced a 100% disease control rate (DCR) in patients (pts; n = 9) with ASS1-deficient thoracic cancers, with no additional toxicity (Beddowes et al 2017). Here, we present the TRAP expansion cohort experience in MPM. Methods: Good performance (ECOG 0-1) MPM pts with non-resectable disease and measurable by modified RECIST, were enrolled in a phase I TRAP expansion cohort at the maximum tolerated dose (MTD) of ADIPEMCIS, using tumoral ASS1 loss as a selection biomarker. PEM (500mg/m2) and CIS (75mg/m2) were given every 3 weeks with weekly IM ADI-PEG20 (36mg/m2) for a maximum of 6 cycles with maintenance ADI-PEG20 in responding pts. Primary endpoint was tumor response rate (modified RECIST), with secondary endpoints including progression-free survival (PFS), overall survival (OS), and toxicity. We measured plasma arginine and citrulline concentrations, ADI-PEG20 antibodies, and biopsied patients on progression to explore resistance mechanisms. Results: 31 ASS1-deficient MPM pts (median age 67) were enrolled (11 epithelioid, 10 biphasic and 10 sarcomatoid) out of 92 screened pts. Plasma arginine decreased with a reciprocal increase in plasma citrulline. The partial response rate was 35.5% (95% CI 19.2%-54.6%) with a DCR of 93.5% (95% CI 78.6%-99.2%). Median PFS was 5.6 months (95% CI 4-6) and median OS was 10.1 months (95% CI 6.7-17.7). 10/31 pts (32.3%) experienced grade 3/4 treatment-related toxicities, the most common being neutropenia (16.1%). Upregulation of ASS1 expression was observed in 2/3 biopsies on progression. Conclusions: The ADIPEMCIS regimen is active in ASS1-deficient MPM pts, including non-epithelioid disease. Based on these data the ATOMIC-meso phase 2/3 trial has opened comparing ADIPEMCIS versus PEMCISPlacebo, focusing on pts with non-epithelioid MPM. Clinical trial information: NCT02029690.

Arginine depletion via myeloid cell arginase is critically involved in suppression of the adaptive immune system during cancer or chronic inflammation. On the other hand, arginine depletion is being developed as a novel anti-tumor metabolic strategy to deprive arginine-auxotrophic cancer cells of this amino acid. In human immune cells, arginase is mainly expressed constitutively in PMNs. We therefore purified human primary PMNs from healthy donors and analyzed PMN function as the main innate effector cell and arginase producer in the context of arginine deficiency. We demonstrate that human PMN viability, activation-induced IL-8 synthesis, chemotaxis, phagocytosis, generation of ROS, and fungicidal activity are not impaired by the absence of arginine in vitro. Also, profound pharmacological arginine depletion in vivo via ADI-PEG20 did not inhibit PMN functions in a mouse model of pulmonary invasive aspergillosis; PMN invasion into the lung, activation, and successful PMN-dependent clearance of Aspergillus fumigatus and survival of mice were not impaired. These novel findings add to a better understanding of immunity during inflammation-associated arginine depletion and are also important for the development of therapeutic arginine depletion as anti-metabolic tumor therapy.

The current standard of care for epithelial ovarian cancer does not discriminate between different histologic subtypes (serous, clear cell, endometrioid and mucinous) despite the knowledge that ovarian carcinoma subtypes do not respond uniformly to conventional platinum/taxane‐based chemotherapy. Exploiting addictions and vulnerabilities in cancers with distinguishable molecular features presents an opportunity to develop individualized therapies that may be more effective than the current ‘one size fits all' approach. One such opportunity is arginine depletion therapy with pegylated arginine deiminase, which has shown promise in several cancer types that exhibit low levels of argininosuccinate synthetase including hepatocellular and prostate carcinoma and melanoma. Based on the high levels of argininosuccinate synthetase previously observed in ovarian cancers, these tumours have been considered unlikely candidates for arginine depletion therapy. However, argininosuccinate synthetase levels have not been evaluated in the individual histologic subtypes of ovarian carcinoma. The current study is the first to examine the expression of argininosuccinate synthetase at the mRNA and protein levels in large cohorts of primary and recurrent ovarian carcinomas and ovarian cancer cell lines. We show that the normal fallopian tube fimbria and the majority of primary high‐grade and low‐grade serous ovarian carcinomas express high levels of argininosuccinate synthetase, which tend to further increase in recurrent tumours. In contrast to the serous subtype, non‐serous ovarian carcinoma subtypes (clear cell, endometrioid and mucinous) frequently lack detectable argininosuccinate synthetase expression. The in vitro sensitivity of ovarian cancer cell lines to arginine depletion with pegylated arginine deiminase was inversely correlated with argininosuccinate synthetase expression. Our data suggest that the majority of serous ovarian carcinomas are not susceptible to therapeutic intervention with arginine deiminase while a subset of non‐serous ovarian carcinoma subtypes are auxotrophic for arginine and should be considered for clinical trials with pegylated arginine deiminase.

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 [18F]-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. Cancer Res; 74(3); 896–907. ©2013 AACR.

This study aims to develop a molecular imaging strategy for response assessment of arginine deiminase (ADI) treatment in melanoma xenografts using 3'-[(18)F]fluoro-3'-deoxythymidine ([(18)F]-FLT) positron emission tomography (PET).F-FLT response to ADI therapy was studied in preclinical models of melanoma in vitro and in vivo. The molecular mechanism of response to ADI therapy was investigated, with a particular emphasis on biological pathways known to regulate (18)F-FLT metabolism.Proliferation of SK-MEL-28 melanoma tumors was potently inhibited by ADI treatment. However, no metabolic response was observed in FLT PET, presumably based on the known ADI-induced degradation of PTEN, followed by instability of the tumor suppressor p53 and a relative overexpression of thymidine kinase 1, the enzyme mainly responsible for intracellular FLT processing.The specific pharmacological properties of ADI preclude using (18)F-FLT to evaluate clinical response in melanoma and argue for further studies to explore the use of other clinically applicable PET tracers in ADI treatment.

The mechanism of cholera toxin (CT)-stimulated arachidonate metabolism was evaluated. CT caused rapid in vitro synthesis of prostaglandin E2 (PGE2) in murine smooth muscle-like cells (BC3H1), reaching maximal levels within 3 to 4 min. In comparison, cyclic AMP (cAMP) levels were unchanged, and addition of dibutyryl cAMP did not affect PGE2 synthesis. CT-induced PGE2 synthesis was prevented by actinomycin D or cycloheximide, indicating a need for de novo protein synthesis. Northern blot analysis of total RNA from BC3H1 cells revealed that exposure to CT resulted in an increase in abundance of mRNA encoding phospholipase A2 (PLA2)-activating protein (PLAP). PLAP is a regulatory protein that increases the enzymatic activity of cellular PLA(2), which in turn causes increased hydrolysis of arachidonate from membrane phospholipids. Furthermore, CT evoked the accumulation of PLAP mRNA in J774 (murine monocyte/macrophage) and Caco-2 (human intestinal epithelial) cells in vitro, but the responses were more delayed than that of BC3H1 cells. A protein band of approximately 35 kDa, which corresponded to the size of PLAP, was observed in sodium dodecyl sulfate extracts of Caco-2 cells by Western blot (immunoblot) analysis using affinity-purified antibodies to PLAP synthetic peptides. Synthesis of PLAP protein was increased after 2 h of exposure to CT. Exposure of mouse intestinal loops to either CT or live Salmonella typhimurium for 3 h increased mucosal PLAP mRNA levels. The role of PLAP in CT-induced PGE2 synthesis provides an attractive explanation for the reported suppression of CT-induced intestinal secretion by inhibitors of protein synthesis.

The 1987 American College of Rheumatology (ACR) criteria for the classification of rheumatoid arthritis (RA) were clinically assessed. These criteria do not include findings of synovial fluid (SF) analysis and require no exclusion criteria. We have studied sequential patients with arthritis seen in four rheumatology centers in the Philadelphia area. Classifications by the ACR criteria were compared with our clinical diagnoses. Two hundred ninety eight patients were evaluated, 113 with RA and 185 with other diagnoses. Classifications as RA by the ACR criteria corresponded to our clinical diagnosis in 95% of the cases, corroborating the high sensitivity previously reported. However, we found a lower specificity (73%) than that reported (89%). False positive classifications as RA were found in 71% of patients with psoriatic arthritis, 48% of patients with SLE, and 31% of patients with gout. The specificity could be improved to 89% by excluding disorders with obvious distinguishing extraarticular features such as psoriasis or by SF findings of monosodium urate crystals. Awareness of these possible sources of confusion will further increase the teaching and epidemiologic value of these useful simplified criteria.

295 Background: ADI-PEG 20 (A) is a pegylated form of arginine depleting enzyme, arginine deaminase. Normal cells synthesise arginine using the enzyme argininosuccinate synthetase (ASS). A selectively targets malignant cells which lack ASS. A has shown encouraging pre clinical activity as monotherapy in PC, and synergistic activity with gem and taxanes. Methods: Eligibility: Metastatic PC, up to one line of prior treatment (dose escalation cohort:ct) no prior treatment (expansion ct), ECOG 0-1.Treatment: gem 1,000 mg/m2, nab-P 125mg/m2 3 out of 4 weeks, A 18 mg IM weekly (ct 1), A 36mg weekly (ct 2 and expansion ct). Primary endpoint: determine MTD of A in combination with nab-P and gem. Statistical plan: Single arm, non-randomized, open-label, phase IB, standard 3 + 3 dose escalation with expansion ct of 9 patients at MTD. Secondary endpoints: progression-free survival (PFS), response rate, overall survival (OS), safety, tolerability, blood and archival tumor analyses for ASS expression and immunogenicity. Pts treated at MTD and on expansion cohort were eligible for primary and secondary endpoints. Results: Between 11/14 and 09/15, 18 pts enrolled. 4 pts (22%) had 1 prior line of therapy. Toxicity attributable to A was limited to grade 1 rash/erythema at injection site (n = 3). Cohort 2 was expanded to 6 pts due to DLT of elevated LFTs, no further DLTs observed. ≥ grade 3 toxicity attributable to gem and/or nab-P included elevation in AST/ALT, hematologic toxicity, fatigue, diarrhea, neutropenic fever, alopecia as expected. No A related grade 3-5 toxicities were observed. Response: 6 pts (40%) PR, including one previously treated pt, 8 pts (53%) stable disease (SD), 1 pt inevaluable. Two pts continue on study, median PFS 6.5 months (95% CI 5.3 – 15.2), median OS 11.3 months (95% CI 4.9- . ). Eight of 15 pts treated at MTD received 2nd-line chemotherapy at progression. Five of 11 archival tumors samples tested for ASS1 expression by IHC were deficient. Conclusions: A was well tolerated in combination with gem and nab-P. Activity was observed in treated and untreated pts, and in pts with ASS deficient and proficient tumors; synergy of A with cytotoxics may be independent of ASS status and future investigation in an unselected population is warranted. Clinical trial information: NCT02101580.

384 Background: ADI-PEG-20 (pegylated arginine deiminase), an arginine degrading enzyme, and FOLFOX each exhibit clinical activity in a HCC pt subset. Preclinical data indicate that arginine depletion interferes with folate and pyrimidine metabolism as well as DNA damage repair pathways, suggesting a role for pairing AGI-PEG-20 with cytotoxic chemotherapy. Methods: This is a single center phase 1 trial of mFOLFOX-6 and ADI-PEG-20 in treatment-refractory advanced GI tumors to assess safety and tolerability, and to identify the recommended phase 2 dose (R2PD). mFOLFOX6 was administered intravenously biweekly at standard fixed doses and ADI-PEG-20 intramuscularly weekly at 18 or 36 mg/m2. Toxicity was assessed by CTCAE v4.03 and efficacy by RECIST v1.1. A HCC RP2D expansion was completed to explore efficacy. Serum arginine, citrulline, and anti-ADI-PEG-20 antibodies were monitored every other week. Results: 17 pts were treated—7 pts with GI tumors in dose escalation and 10 HCC pts in RP2D dose expansion. Histologies included HCC (13), pancreatic (3) and fibrolamellar carcinoma (1). All HCC pts had Child-Pugh A liver function and had progressed on sorafenib. Treatment-related grade ≥ 3 laboratory adverse events (AEs) occurred in 47% of pts, including neutropenia (4), thrombocytopenia (3), anemia (2), lymphocytopenia (2) and hyponatremia (1). Treatment-related grade ≥ 3 clinical AEs occurred in 2 pts, notably grade 3 fatigue and a grade 3 cardiac event not otherwise specified. No dose limiting toxicities, treatment-related deaths, or cases of hepatic failure were observed. Efficacy data for the 13 HCC pts revealed 3 (23%) PR, 6 (46%) SD, 3 PD and 1 NE. Arginine levels were depleted with therapy (mean ± SEM: baseline 85.3 ± 13.0 uM, 8 wks 4 ± 3.0 uM, 16 wk 1.1 ± 0.8uM). Anti-ADI-PEG-20 antibodies were not detected at 8 weeks (mean ± SEM: baseline 0.7 ± 0.5, 8 wks 1.5 ± 1). Conclusions: Concurrent mFOLFOX6 plus ADI-PEG-20 intramuscularly at 36 mg/m2 weekly shows an acceptable safety profile with rapid and durable reduction in serum arginine. Further evaluation of this combination is warranted in advanced HCC pts. Clinical trial information: NCT02102022.

Sarcomas comprise a large heterogeneous group of mesenchymal cancers with limited therapeutic options. When treated with standard cytotoxic chemotherapies, many sarcomas fail to respond completely and rapidly become treatment resistant. A major problem in the investigation and treatment of sarcomas is the fact that no single gene mutation or alteration has been identified among the diverse histologic subtypes. We searched for therapeutically druggable targets that are common to a wide range of histologies and hence could provide alternatives to the conventional chemotherapy. Seven hundred samples comprising 45 separate histologies were examined. We found that almost 90% were arginine auxotrophs, as the expression of argininosuccinate synthetase 1 was lost or significantly reduced. Arginine auxotrophy confers sensitivity to arginine deprivation, leading temporarily to starvation and ultimately to cell survival or death under different circumstances. We showed that, in sarcoma, arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20) maintains a prolonged state of arginine starvation without causing cell death. However, when starvation was simultaneously prolonged by ADI-PEG20 while inhibited by the clinically available drug chloroquine, sarcoma cells died via necroptosis and apoptosis. These results have revealed a novel metabolic vulnerability in sarcomas and provided the basis for a well-tolerated alternative treatment strategy, potentially applicable to up to 90% of the tumors, regardless of histology.

4017Background: Arginine (Ar) depletion is a putative target in HCC, which lacks the citrulline (Ci) to Ar repleting enzyme argininosuccinate synthetase. A is an Ar degrading enzyme cloned from M. ...

11567Background: Loss of argininosuccinate synthetase 1 (ASS1) sensitizes malignant pleural mesothelioma (MPM) and lung carcinoma (NSCLC) cells to apoptosis following arginine (L-Arg) deprivation. ...

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.

The role of the phospholipase A2 (PLA2) stimulating protein PLAP in the regulation of PLA2 activity was assessed by determination of the effects of PLAP on two purified PLA2s. An approx. 14 kDa enzyme was purified from mouse thymoma cells, EL-4 cells, by cation ion exchange HPLC and immunoaffinity HPLC (with antiserum to the N-terminal sequence of an inflammatory exudate PLA2). An approx. 110 kDa enzyme was purified from mouse mammary carcinoma derived cells by sequential hydrophobic, anion exchange, hydroxyapatite and gel filtration HPLC. Neither PLAP nor melittin, an immunologically related PLA2 stimulating peptide from bee venom, increased the activity of the high molecular weight enzyme. In contrast, there was more than a 20-fold stimulation of the low molecular weight PLA2 by PLAP and an approx. 5-fold stimulation by melittin. The stimulation of enzyme activity by PLAP was observed at a protein to phospholipid ratio of 1:10(6) while the ratio of melittin to phospholipid was 1:3. Thus, PLAP mediated stimulation of PLA2 activity may include an interaction between PLAP and the enzyme, in contrast to melittin stimulation, which involves interactions between melittin and phospholipid.

Over the last 30 years, interest in PLA2 has grown beyond its enzymatic capacity to cleave phospholipids. It has been recognized as the rate-limiting step in the release of arachidonic acid and subsequent formation of prostaglandins, leukotrienes, and other bioactive lipids. Subsequently, PLA2 has not only been found to be present in high concentrations in inflammatory arthritis, but also to induce inflammation when injected into animals. At the same time, investigators into mechanisms of signal transduction demonstrated that a variety of cytokines including IL-1 and TNF, which are found in high concentrations in synovial fluid from patients with RA, stimulate PLA2 activity. These investigations demonstrated further the central role for PLA2 in inflammatory events, especially inflammatory arthritis. Numerous other PLA2 proteins, in addition to the low molecular weight synovial fluid/platelet enzyme, also have been characterized. Their clinical role in arthritis is yet to be elucidated. Human proteins which either inhibit or stimulate PLA2 have also been identified, characterized, and cloned. More recently, exciting investigations, primarily from biotechnology and pharmaceutical companies, into inhibitors of PLA2 have been reported. New PLA2-regulating compounds, which will hopefully move from the laboratory and through clinical trials and then be used to treat patients with arthritis, are on the horizon.

A variety of rheumatologic disorders affect the elderly. Some of these problems are seen almost exclusively in the elderly, such as temporal arteritis and pseudogout. Because of underlying chronic diseases, these patients are also at increased risk for joint infection and resultant sepsis. Evaluation of synovial fluid from the inflamed joint is important. Light microscopy evaluation with a red polarizing compensator can help diagnose crystal-mediated disease, such as gout or pseudogout. Examination of Gram stains can help diagnose infectious arthritis. Thus, appropriate processing of synovial fluid is mandatory for the diagnosis of many rheumatologic disorders that occur in the elderly. A variety of metabolic disorders are associated with pseudogout and should be searched for on laboratory evaluation. Appropriate laboratory evaluation and follow-up following the acute episode are important in the care of these patients. For example, temporal arteritis with resultant blindness is a feared disorder in the elderly. Transient blindness, headaches, jaw claudication, and an elevated Westergren sedimentation rate suggest this diagnosis. Aches and pain in the neck and shoulder area, especially in the morning, are typical of polymyalgia rheumatica. Polymyalgia rheumatica may also be a symptom of temporal arteritis.

Introduction Starvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far. Methods Human myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry. Results 5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group. Conclusion Myeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Background: Loss of the metabolic tumor suppressor, argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, sensitizes mesothelioma and lung carcinoma cells to apoptosis following arginine withdrawal. We have reported treatment with the arginine depletor pegylated arginine deiminase (ADI-PEG 20) in ASS1-negative tumor cells potentiates the cytotoxic effect of pemetrexed, accompanied by suppression of de novo pyrimidine synthesis and the pyrimidine salvage pathway (Allen et al, Cancer Res 2014. We have undertaken a phase I study (NCT02029690) of ADI-PEG 20 combined with first-line pemetrexed and cisplatin chemotherapy in patients (pt) with ASS1-deficient mesothelioma or non-squamous non-small cell lung cancer (NSCLC), primary objective was to recommend a dose for future study (RP2D). Methods: Main inclusion criteria: ≥ 18 years, PS ≤ 1, tumour ASS1 loss (≤ 50% ASS expression by IHC) with adequate organ function and written, informed consent. Exclusion criteria: symptomatic CNS metastases, significant concurrent morbidity, therapeutic anticoagulation, history of seizures, or allergy to trial medication(s). A 3+3+3 phase 1 dose escalation design was used with increasing doses of weekly ADI-PEG 20 (18, 27 and 36 mg/m2 IM) in each of three cohorts plus pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 both given every 3 weeks (1 cycle), maximum 18 weeks. ADI-PEG 20 alone was allowed after 18 weeks if there was stable disease or better. Adverse events were graded using CTCAE v4.03 and dose limiting toxicities (DLT) were defined as: Grade 4 neutropenia (> 7 days), febrile neutropenia, Grade 4 anaemia or thrombocytopenia, other clinically significant Grade 3/4 non haematological toxicity that occurred during cycle 1. Radiological disease response was assessed every 6 weeks (modified RECIST for mesothelioma and RECIST 1.1 for NSCLC) and peripheral blood samples were collected to measure plasma arginine and citrulline levels and antibodies to ADI-PEG 20. Results: 42 pt were screened for tumor ASS1 expression and 15 (36%) were ASS1-deficient. Subsequently, 9 pt were eligible for DLT assessment. Demographics - 6 M:3 F, Age range 62 - 77, NSCLC (4): Mesothelioma (5), Prior therapy EGFRi (1 pt), External beam radiotherapy (2 pt) No DLT were observed at any dose level. Grade ≤ 3, AE related to pemetrexed and cisplatin were (5 pt): nausea (5), fatigue (2) and vomiting, peripheral neuropathy, dry mouth, mouth ulcers, dehydration and neutropenia (1 each). No AE were reported related to ADI PEG 20. Mean cycles of treatment: 9 (range 4 - 14) 1 pt was dose reduced after cisplatin toxicity. Arginine was depleted for ≥3 weeks (3-18 weeks in all treated patients. 7/9 pt had partial response (PR) and 2/9 had stable disease (SD) as best response. Conclusions: The combination was well tolerated, the RP2D is 36mg/m2 ADI-PEG 20 weekly with pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 every 3 weeks. Robust clinical activity has been observed with 78% pt having PR as best response on CT scan, as well as PR and SD in2 pt with sarcomatoid mesothelioma. The tolerability and response / disease control rate suggest that this combination may have clinical utility as first line treatment for these malignancies in ASS1-deficient pt. RP2D expansion cohorts are ongoing for pt with pleural mesothelioma or non-squamous NSCLC. Citation Format: Simon Pacey, James F. Spicer, Pui Ying Chan, Mirela Hategan, Dimitra Repana, Jeremy Peter Steele, Peter Schmid, Gary J R Cook, Monica Diaz, Amanda Johnston, Richard Baird, Adelberto Barba, Ramsay Khadeir, Michael Sheaff, Jose Roca, Teresa Szyszko, John Bomalaski, Peter Wojciech Szlosarek. A phase 1 study in patients with mesothelioma or non small cell lung tumours requiring arginine to assess ADI-PEG 20 with pemetrexed and cisplatin (TRAP study). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B23.

Background Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are therefore sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20) which showed a halving in the risk of disease progression in a randomized phase II study (Szlosarek et al, ASCO 2014). However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, we studied the effect of macrophages, which are abundant in mesothelioma, on the tumor cytotoxicity of ADI-PEG20. Methods ASS1 negative MPM cell lines treated with ADI-PEG20 were analysed using the Affymetrix Human Genome U133 Plus 2.0 array, with validation of genes involved in stromal-tumor cell communication. Additional studies involved using tumor-macrophage co-culture experiments, mass spectrometry and an MPM xenograft model. Results A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified in the ADI-PEG20-treated ASS1 negative MPM cell lines. Notably, a significant increase in ASS1 negative MPM cell viability was seen upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumor cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumor cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumor volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 monotherapy. Conclusion Collectively, our data indicate that as a result of metabolic ‘cross-talk’ between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. Our studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma. Citation Format: Melissa M. Phillips, Ramsay Khadeir, Laura Tookman, Fiona McCarthy, Jeremy Steele, John Bomalaski, Essam Ghazaly, Peter W. Szlosarek. Macrophages promote resistance to pegylated arginine deiminase in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2015-3360

TPS2612 Background: Loss of the metabolic tumor suppressor, argininosuccinate synthetase (ASS1), a rate-limiting enzyme in arginine biosynthesis, sensitizes mesothelioma and lung carcinoma cells to...

e16116 Background: ADI-PEG20 is a pegylated enzyme that degrades arginine, a required amino acid in argininosuccinate synthetase (ASS) deficient tumors. Immunohistochemical staining suggests most p...

The intracellular regulation and kinetics of prostaglandin (PG)E(2) synthesis in human airway epithelial (NCI-H292) cells was investigated. Interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) all induced PGE(2) synthesis (p0.001) and transient (5-15 min) phosphorylation of extracellular signal-regulated kinase (ERK). Phorbol myristate acetate (PMA) and calcium ionophore, A23187 further enhanced PGE(2) synthesis (p0.001) and caused phosphorylation of ERK that was sustained for up to 16 h. COX-2 protein expression and PGE(2) synthesis were increased following exposure to combinations of stimuli that increased intracellular Ca(2+), and activated protein kinase C as well as ERK. Inhibition of ERK almost completely abrogated PGE(2) synthesis in response to all stimuli. Sustained, maximum PGE(2) synthesis was observed when cells were stimulated such that ERK phosphorylation was concomitant with increased COX-2 protein expression. These results argue against redundancy in pathways for PGE(2) synthesis, and suggest that at various stages of inflammation different stimuli may influence ERK activation and COX-2 expression, so as to tightly regulate the kinetics and amount of PGE(2) produced by airway epithelial cells in response to lung inflammation.

Purpose Individuals with metastatic melanoma have a poor prognosis. Many human melanomas are auxotrophic for arginine, and arginine is not an essential amino acid in humans. We hypothesized that this auxotrophy may be therapeutically exploited. A novel amino acid–degrading enzyme (arginine deiminase) conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw) was used to lower plasma arginine in individuals with metastatic melanoma. Patients and Methods Two cohort dose-escalation studies were performed. A phase I study in the United States enrolled 15 patients, and a phase I to II study in Italy enrolled 24 patients. The Italian patients also received two subsequent cycles of treatment, each consisting of four once-weekly injections of 160 U/m2. The goals of these studies were to determine pharmacokinetics (PK), pharmacodynamics (PD), safety, and the antitumor activity of ADI-SS PEG 20,000 mw. Results PK and PD studies indicated that a dose of 160 U/m2 lowered plasma arginine from a resting level of approximately 130 μmol/L to less than 2 μmol/L for at least 7 days; nitric oxide levels also were lowered. There were no grade 3 or 4 toxicities directly attributable to the drug. Six of 24 phase I to II patients responded to treatment (five partial responses and one complete response; 25% response rate) and also had prolonged survival. Conclusion Elimination of all detectable plasma arginine in patients with metastatic melanoma was well tolerated and may be effective in the treatment of this cancer. Further testing of ADI-SS PEG 20,000 mw in a larger population of individuals with metastatic melanoma is warranted.

We report the first pharmacokinetic and clinical response data from a patient with unresectable hepatocellular cancer treated with a new drug, ADI PEG20,000 mw (arginine deiminase-polyethylene glycol 20,000 molecular weight). A single patient with idiopathic cirrhosis and unresectable hepatocellular cancer was treated with escalating dosages of ADI-PEG20,000 mw. Human hepatocellular cancer has been found to be arginine-dependent for growth because of loss of expression or arginosuccinate synthetase, the rate-limiting enzyme in the conversion of citrulline to arginine. Thus, an arginine-degrading enzyme like ADI-PEG20,000 mw should produce cell death in hepatocellular cancer cells without significantly affecting normal cells. There was a dose-dependent reduction of plasma arginine levels after weekly intramuscular administration of ADI-PEG20,000 mw. Successive treatment cycles at the optimal biologic dose of 160 IU/m2 led to reduction in tumor size and serum alpha-fetoprotein levels. Sufficient tumor cytoreduction was achieved with ADI-PEG20,000 mw treatment to permit surgical treatment. The patient developed no toxicities or side effects related to ADI-PEG20,000 mw treatment. The results in a single patient with unresectable hepatocellular cancer treated with ADI-PEG20,000 mw suggests this may be a promising, low-toxicity treatment. Full-scale clinical trials have been initiated.

Some murine melanomas and hepatocellular carcinomas (HCCs) have been shown to be auxotrophic for arginine. Arginine deiminase (ADI; EC 3.5.3.6.), an arginine-degrading enzyme isolated from Mycoplasma, can inhibit growth of these tumors. We found that ADI was specific for arginine and did not degrade other amino acids. Although arginine is not an essential amino acid for most cells, all human melanomas and HCCs tested were found to be inhibited by ADI in vitro. Arginine is synthesized from citrulline in two steps by argininosuccinate synthetase and argininosuccinate lyase. Melanomas and HCCs did not express argininosuccinate synthetase mRNA but did express argininosuccinate lyase mRNA, suggesting that the arginine auxotrophy of these cells was a result of an inability to produce argininosuccinate synthetase. Human melanomas and HCCs were transfected with an expression plasmid containing argininosuccinate synthetase cDNA. The transfected cells were much more resistant to ADI than the parental cells in vitro and in vivo. Initial attempts to use ADI in vivo indicated that this enzyme had little efficacy, consistent with its short circulation half-life. Formulation of ADI with polyethylene glycol to produce ADI-SS PEG(20,000 mw) resulted in an enzyme with a much longer circulation half-life that, and although equally effective in vitro, was more efficacious in the treatment of mice implanted with human melanomas and HCCs. These data indicate that sensitivity of melanoma and HCC is due to the absence of argininosuccinate synthetase in these cells and that an effective formulation of ADI, which causes a sustained decrease in arginine, may be a useful treatment for arginine auxotrophic tumors including melanoma and HCC.

Humans have a non-sense codon inserted into the 5 prime end of the open reading frame of urate oxidase, and thus express an enzymatically inactive fragment of this enzyme; and consequently are unable to metabolize uric acid into allantoin and are prone to develop hyperuricemia and gout. Various urate oxidases (uricase) from mammals and microorganisms have been administered to humans with hyperuricemia and gout. Although successful in lowering plasma uric acid, these therapies have had limited application due to undesirable biochemical properties of the enzymes used, the short circulating half-life, and inherent antigenicity of these preparations.We compared urate oxidase from a variety of sources for specific enzyme activity, pH optimum, affinity, and retention of enzyme activity under physiological conditions. A variety of polyethylene glycols (PEG) were tested to formulate uricase.Urate oxidase from Candida utilis had more favorable enzymatic properties and PEG of 20,000 MW (termed uricase-PEG 20) had greatly reduced antigenicity and increased circulating half-life as compared to those previously described.It is anticipated that uricase-PEG 20 may have utility as a treatment for hyperuricemia and gout.

Nitric oxide (NO) is a biomediator believed to be synthesized primarily from extracellular arginine. Various methodologies have been used to inhibit NO synthesis so as to elucidate its physiological and pathophysiological functions. Several investigators have utilized various argin ine degrading enzymes as a means of lowering extracellular arginine. Arginase, most commonly derived from mammalian sources, has been most often used. However, arginase has failed to inhibit NO synthesis. Therefore, a systematic biochemical characterization of arginase and arginine deiminase (ADI) derived from M. Hominus was undertaken.The murine macrophage cell line N-9 was treated with either arginase or arginine deiminase to determine the effect on intracellular and extracellular arginine and nitric oxide production.Arginase was found to have an alkaline pH optima(approximately 9.5) with little enzyme activity at physiological pH. In contrast, the pH optima of ADI was approximately 6.5, retaining70% of its activity at physiological pH. ADI had more than 1000 fold higher affinity for arginine (Km approximately 30 KM for ADI vs approximately 45 mM for arginase), and was able to lower arginine levels to a much greater extent than arginase. ADI, unlike arginase, was effective in lowering extracellular arginine in tissue culture media and inhibit NO production by the murine macrophage cell line N-9 in response to gamma interferon and LPS stimulation.These data suggest that ADI may be useful for delineating the role of NO in a variety of biological systems as well as determining the role of extracellular arginine in its synthesis.

2563 Background: Besides its selective activity to induce autophagy and caspase-independent apoptosis in argininosuccinate synthetase (ASS)-deficient cancer cells, ADI-PEG20 (pegylated arginine dei...

7507 Background: Argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM) cells are sensitive to arginine deprivation. We examined the efficacy and safety of the arginin...

221 Background: The benefits of chemoradiation therapy in patients with locally advanced pancreatic cancer (LAPC) are limited due to the inherent radioresistance of pancreatic cancer (PC) and high systemic toxicity of current radiosensitizers (e.g., gemcitabine). Hence, the search for newer radiosensitizers with unique anticancer properties continues. Single amino acid arginine starvation is a new promising therapeutic approach for solid tumors (e.g., PC), that are auxotrophic for non-essential amino acids. Arginine degrading enzyme, arginine deiminase (ADI), deprives cells of arginine and thereby exerts its anti-proliferative effects, especially in cancer cells deficient in enzyme argininosuccinate synthase (ASS1). Here we evaluate the effects of ADI-polyethylene glycol formulation (ADI-PEG20) as a radiosensitizer in PC. Methods: The toxicity of ADI-PEG20 in vitro was evaluated using XTT. Effect of ADI-PEG20 as radiosensitizer was determined by clonogenic cell survival. For in vivo, mice with PC tumor xenografts (Panc1), randomized into four groups, were treated with vehicle (PBS), ADI-PEG20 (5 IU/mouse; twice weekly), radiation (IR; 2 Gy × 5 times), and ADI-PEG20 with IR. Tumors were measured following treatment and the tumor re-growth delay time for each group was calculated. Immunohistochemical analysis of Ki-67 and VEGF was done on tumor tissues (paraffin sections) by routine immunofluorescence. Results: ADI-PEG20 selectively sensitized ASS1 deficient PC cells to IR at low, non-toxic concentrations (0.04 and 0.08 μg/mL for 72 h; DER at 10% SF for Panc1 was 1.39 and 1.52; for Miapaca-2, 1.09 and 1.25 respectively), but not ASS1 positive cells (L3.6pl). In vivo, ADI-PEG20 profoundly sensitized PC cells to IR. IR treatment alone delayed the tumor doubling time (7.6 ± 1.7 days compared to the non-treated controls); however, combining ADI-PEG20 with IR delayed the tumor growth by an additional 10 ± 1.3 days (p

Introduction Malignant cells require amino acids for a wide range of core functions. Amino acid deprivation using enzymatic degradation has been used to induce remission in acute lymphoblastic leukemia for decades. Amino acid deprivation may also benefit patients with acute myeloid leukemia (AML). We have previously shown that AML cells lack of argininosuccinate synthetase 1(ASS1), a key enzyme in the pathway that produces arginine (1). Here we tested the effect of an arginine depleting agent, pegylated arginine deiminase (ADI-PEG 20) on primary AML cells in a xenograft model of AML. Methods NOD/SCID/interleukin 2 gamma chain null (NSG) mice were transplanted with 6 primary AML samples. 12 weeks after transplantation of AML mice received either ADI-PEG 20, cytarabine, ADI-PEG 20 plus cytarabine or vehicle. ADI was administered weekly for 4 doses and cytarabine was given for 10 consecutive days (0.2mg per day, roughly equivalent to 40mg in humans). Five weeks after treatment started, mice were killed and the percentage of AML in the bone marrow was determined by flow cytometry. Blood was collected to quantify plasma arginine using liquid chromatography-mass spectrometry/mass spectrometry. Results Plasma arginine levels were depressed following ADI-PEG 20 administration confirming that arginine depletion was achieved in vivo. In all six experiments the combination of ADI-PEG 20 and cytarabine induced a significant reduction in levels of AML compared to control (Figure 1). Critically the combination of ADI-PEG 20 and cytarabine was significantly better than cytarabine alone in three of six experiments. Conclusion Our experiments show that arginine deprivation by ADI-PEG 20 can decrease the leukemic burden in mice transplanted with primary AML cells. The combination of ADI with cytarabine had a greater effect than cytarabine alone in half the experiments. These results provide the rationale to test ADI-PEG 20 with cytarabine in clinical trials. 1. Peter W. Szlosarek, Fiona Luong, Andrew Clear, David Taussig, Simon Joel, Maria Calaminici, Silvana Debernardi, Jude Fitzgibbon, John S. Bomalaski, Arthur E. Frankel, and Dominique Bonnet. Pegylated arginine deiminase (ADI-PEG 20) as a potential novel therapy for argininosuccinate synthetase-deficient acute myeloid leukemia. Cancer Research: April 15, 2011. AACR 102nd Annual Meeting 2011, (AACR Abstract # 467) F. M-M and L. A-M contributed equally D.B., P.W.S. and D.C.T contributed equally Disclosures: Bomalaski: Polaris Group: Employment, Equity Ownership. Szlosarek:Polaris Group: Research Funding.

2569 Background: ADI-PEG20 is an enzyme that degrades arginine (Arg), an amino acid relevant to biosynthetic pathways of normal and malignant cells. It has shown tolerability and activity in several solid tumors. Preclinical studies have shown that Arg deprivation by ADI-PEG 20 in cancer cells induces autophagy, caspase-independent apoptosis, and potentiates DOC-induced cytotoxicity in prostate cancer (PC) models. A phase I trial (standard 3+3 design) of ADI-PEG20 (IM weekly) plus DOC (IV on day 1 q 3 weeks) was conducted to assess feasibility and safety of the combination. Methods: Eligible pts were >18 years of age, had advanced malignant solid tumors, adequate end organ function, and performance status (PS) 0-2. ADI-PEG 20 was escalated over 4 dose levels (4.5, 9, 18, 36 mg/m2). DOC dose was 75 mg/m2. Dose limiting toxicity (DLT) was defined as any of the following in cycle 1: thrombocytopenia [grade (Gr) 3 with bleeding/transfusion, or Gr 4]; neutropenia with fever or documented infection attributable to ADI PEG20; or any ≥ Gr 3 non-heme toxicity related to study drug except alopecia. Allergic reaction associated with DOC was not considered a DLT. Serum levels of Arg were serially measured. Results: 18 pts were accrued: median age, 64.5 yrs; male, 83%; PS 0, 72%. Most common tumors were NSCLC (8), PC (3), and tongue cancer (TC) (2). Median number of prior systemic therapies was 3. One DLT was seen in dose level 1 (urticarial rash) requiring expansion of that dose level to 6 pts. No additional DLTs attributable to ADI PEG20 were seen. Serious adverse events (all expected and attributed to DOC) were recorded in 11/18 pts, including Gr IV neutropenia (6, 33%) and Gr IV anemia (2, 11%). There were 2 on-study deaths unrelated to protocol therapy. In 11 pts with evaluable disease, 1 with TC had a partial response (PR), 6 had stable disease (SD) (3 NSCLC, 2 PC, 1 TC). Arg levels decreased in the 1st cycle for 6/11 pts with available data, including 2 with SD, and 1 with PR. Conclusions: The combination of ADI PEG20 and DOC is feasible with reasonable tolerability in this heavily pre-treated cohort. Full doses of both agents were achievable: ADI-PEG 20 at 36mg/m2 with DOC 75 mg/m2. An expansion cohort of castration resistant PC pts is now accruing at this recommended dose. Clinical trial information: NCT01497925.

10526 Background: To better understand the metabolism of sarcomas, we explored the role of argininosuccinate synthase 1 (ASS1), the rate-limiting enzyme in the conversion of citrulline to arginine in the urea cycle. When ASS1 is not expressed the amino acid arginine becomes an essential amino acid that must be delivered from the diet. The potential for therapeutic manipulation of this pathway with pegylated arginine deiminase (ADI-PEG20) was explored using metabolomic mass spectroscopy. Methods: Immunohistochemistry (IHC) of ASS1 expression on 701 patient specimens representing 45 subtypes of sarcoma was performed. Proliferation and cell death were analyzed in a panel of cell lines. Clonogenic assays were performed using ADI-PEG20 and chloroquine. Autophagy induction and inhibition was assessed. Xenograft models were tested. Metabolomic mass spectroscopy was performed in the presence and absence of ADI-PEG20. Results: lHC analysis demonstrated that ASS1 is not expressed in over 85% of sarcomas (619 of 701 patient samples). Treatment of a panel of LMS, synovial, ASPS, GIST, MPNST, chondrosarcoma, osteosarcoma, and Ewings sarcoma cell lines with ADI-PEG20 resulted in cell cycle arrest but not apoptosis when ASS1 expression was low (13/15 lines). Xenografts of the ASS1 low MNNG/HOS cells and SKLMS1 cells into nude mice followed by treatment with ADI-PEG20 +/- chloroquine demonstrated significantly slower tumor growth as compared to PBS or chloroquine treated controls. Treatment of sarcomas that lack ASS1 with ADI-PEG20 induced autophagy. Global analysis of metabolomic mass spectroscopy demonstrated alterations in PKM2, glutamine dependence, the Warburg effect, the TCA cycle and glutathione biology. Conclusions: This suggests that sarcomas may be sensitive to the arginine depletion therapy using ADI-PEG20. Autophagy induction has global metabolic consequences for sarcoma. Successful control of tumor burden by using arginine deprivation therapy and autophagy inhibition in our preclinical studies will help to orient clinical trials for this innovative treatment in patients with sarcoma that lack ASS1 expression.

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