A Phase 1 study of ADI-PEG20 (pegargiminase) combined with cisplatin and pemetrexed in ASS1-negative metastatic uveal melanoma.

Metastatic uveal melanoma (UM) is a devastating disease with few treatment options. We evaluated the safety, tolerability and preliminary activity of arginine depletion using pegylated arginine deiminase (ADI-PEG20; pegargiminase) combined with pemetrexed (Pem) and cisplatin (Cis) chemotherapy in a phase 1 dose-expansion study of patients with argininosuccinate synthetase (ASS1)-deficient metastatic UM. Eligible patients received up to six cycles of Pem (500 mg/m2 ) and Cis (75 mg/m2 ) every 3 weeks plus weekly intramuscular ADI (36 mg/m2 ), followed by maintenance ADI until progression (NCT02029690). Ten of fourteen ASS1-deficient patients with UM liver metastases and a median of one line of prior immunotherapy received ADIPemCis. Only one ≥ grade 3 adverse event of febrile neutropenia was reported. Seven patients had stable disease with a median progression-free survival of 3.0 months (range, 1.3-8.1) and a median overall survival of 11.5 months (range, 3.2-36.9). Despite anti-ADI-PEG20 antibody emergence, plasma arginine concentrations remained suppressed by 18 weeks with a reciprocal increase in plasma citrulline. Tumour rebiopsies at progression revealed ASS1 re-expression as an escape mechanism. ADIPemCis was well tolerated with modest disease stabilisation in metastatic UM. Further investigation of arginine deprivation is indicated in UM including combinations with immune checkpoint blockade and additional anti-metabolite strategies.

Phase 1, pharmacogenomic, dose-expansion study of pegargiminase plus pemetrexed and cisplatin in patients with ASS1-deficient non-squamous non-small cell lung cancer.

INTRODUCTION: We evaluated the arginine-depleting enzyme pegargiminase (ADI-PEG20; ADI) with pemetrexed (Pem) and cisplatin (Cis) (ADIPemCis) in ASS1-deficient non-squamous non-small cell lung cancer (NSCLC) via a phase 1 dose-expansion trial with exploratory biomarker analysis. METHODS: Sixty-seven chemonaïve patients with advanced non-squamous NSCLC were screened, enrolling 21 ASS1-deficient subjects from March 2015 to July 2017 onto weekly pegargiminase (36 mg/m2 ) with Pem (500 mg/m2 ) and Cis (75 mg/m2 ), every 3 weeks (four cycles maximum), with maintenance Pem or pegargiminase. Safety, pharmacodynamics, immunogenicity, and efficacy were determined; molecular biomarkers were annotated by next-generation sequencing and PD-L1 immunohistochemistry. RESULTS: ADIPemCis was well-tolerated. Plasma arginine and citrulline were differentially modulated; pegargiminase antibodies plateaued by week 10. The disease control rate was 85.7% (n = 18/21; 95% CI 63.7%-97%), with a partial response rate of 47.6% (n = 10/21; 95% CI 25.7%-70.2%). The median progression-free and overall survivals were 4.2 (95% CI 2.9-4.8) and 7.2 (95% CI 5.1-18.4) months, respectively. Two PD-L1-expressing (≥1%) patients are alive following subsequent pembrolizumab immunotherapy (9.5%). Tumoral ASS1 deficiency enriched for p53 (64.7%) mutations, and numerically worse median overall survival as compared to ASS1-proficient disease (10.2 months; n = 29). There was no apparent increase in KRAS mutations (35.3%) and PD-L1 (<1%) expression (55.6%). Re-expression of tumoral ASS1 was detected in one patient at progression (n = 1/3). CONCLUSIONS: ADIPemCis was safe and highly active in patients with ASS1-deficient non-squamous NSCLC, however, survival was poor overall. ASS1 loss was co-associated with p53 mutations. Therapies incorporating pegargiminase merit further evaluation in ASS1-deficient and treatment-refractory NSCLC.

Phase 1b study of pegylated arginine deiminase (ADI-PEG 20) plus Pembrolizumab in advanced solid cancers.

Background: Pegylated arginine deiminase (ADI-PEG 20) is a metabolism-based strategy that depletes arginine, resulting in tumoral stress and cytotoxicity. Preclinically, ADI-PEG 20 modulates T-cell activity and enhances the therapeutic efficacy of programmed death-1 (PD-1) inhibition. Methods: A phase 1b study, including a dose-escalation cohort and an expansion cohort, was undertaken to explore the effects of ADI-PEG 20 in combination with pembrolizumab, an anti-PD-1 antibody, for safety, pharmacodynamics, and response. CD3 levels and programmed death-ligand 1 (PD-L1) expression were assessed in paired biopsies collected prior to and after ADI-PEG 20 treatment but before pembrolizumab. Results: Twenty-five patients, nine in the dose-escalation cohort and sixteen in the expansion cohort, were recruited. Treatment was feasible with adverse events consistent with those known for each agent, except for Grade 3/4 neutropenia which was higher than expected, occurring in 10/25 (40%) patients. Mean arginine levels were suppressed for 1-3 weeks, but increased gradually. CD3+ T cells increased in 10/12 (83.3%) subjects following ADI-PEG 20 treatment, including in three partial responders (p = .02). PD-L1 expression was low and increased in 3/10 (30%) of subjects. Partial responses occurred in 6/25 (24%) heavily pretreated patients, in both argininosuccinate synthetase 1 proficient and deficient subjects. Conclusions: The immunometabolic combination was safe with the caveat that the incidence of neutropenia might be increased compared with either agent alone. ADI-PEG 20 treatment increased T cell infiltration in the low PD-L1 tumor microenvironment. The recommended phase 2 doses are 36 mg/m2 weekly for ADI-PEG 20 and 200 mg every 3 weeks for pembrolizumab.

Phase I study of ADI-PEG20 plus low-dose cytarabine for the treatment of acute myeloid leukemia.

Most acute myeloid leukemia (AML) cells are argininosuccinate synthetase-deficient. Pegylated arginine deiminase (ADI-PEG20) monotherapy depletes circulating arginine, thereby selectively inducing tumor cell death. ADI-PEG20 was shown to induce complete responses in ~10% of relapsed/refractory or poor-risk AML patients. We conducted a phase I, dose-escalation study combining ADI-PEG20 and low-dose cytarabine (LDC) in AML patients. Patients received 20 mg LDC subcutaneously twice daily for 10 days every 28 days and ADI-PEG20 at 18 or 36 mg/m2 (dose levels 1 and 2) intramuscularly weekly. An expansion cohort for the maximal tolerated dose of ADI-PEG20 was planned to further estimate the toxicity and preliminary response of this regimen. The primary endpoints were safety and tolerability. The secondary endpoints were time on treatment, overall survival (OS), overall response rate (ORR), and biomarkers (pharmacodynamics and immunogenicity detection). Twenty-three patients were included in the study, and seventeen patients were in the expansion cohort (dose level 2). No patients developed dose-limiting toxicities. The most common grade III/IV toxicities were thrombocytopenia (61%), anemia (52%), and neutropenia (30%). One had an allergic reaction to ADI-PEG20. The ORR in 18 evaluable patients was 44.4%, with a median OS of 8.0 (4.5-not reached) months. In seven treatment-naïve patients, the ORR was 71.4% and the complete remission rate was 57.1%. The ADI-PEG20 and LDC combination was well-tolerated and resulted in an encouraging ORR. Further combination studies are warranted. (This trial was registered in ClinicalTrials.gov as a Ph1 Study of ADI-PEG20 Plus Low-Dose Cytarabine in Older Patients With AML, NCT02875093).

Expansion Phase 1 Study of Pegargiminase Plus Pemetrexed and Cisplatin in Patients With Argininosuccinate Synthetase 1-Deficient Mesothelioma: Safety, Efficacy, and Resistance Mechanisms.

INTRODUCTION: Pegargiminase (ADI-PEG 20; ADI) degrades arginine and potentiates pemetrexed (Pem) cytotoxicity in argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM). We conducted a phase 1 dose-expansion study at the recommended phase 2 dose of ADI-PEG 20 with Pem and cisplatin (ADIPemCis), to further evaluate arginine-lowering therapy in ASS1-deficient MPM and explore the mechanisms of resistance. METHODS: A total of 32 patients with ASS1-deficient MPM (11 epithelioid; 10 biphasic;11 sarcomatoid) who were chemonaive received weekly intramuscular pegargiminase (36 mg/m2) with Pem (500 mg/m2) and cisplatin (75 mg/m2) intravenously, every 3 weeks (six cycles maximum). Maintenance pegargiminase was permitted until disease progression or withdrawal. Safety, pharmacodynamics, immunogenicity, and efficacy were determined. Biopsies were performed in progressing patients to explore the mechanisms of resistance to pegargiminase. RESULTS: The treatment was well tolerated. Most adverse events were of grade 1/2, whereas four nonhematologic grade 3/4 adverse events related to pegargiminase were reversible. Plasma arginine decreased whereas citrulline increased; this was maintained by 18 weeks of ADIPemCis therapy. The disease control rate in 31 assessed patients was 93.5% (n = 29 of 31; 95% confidence interval [CI]: 78.6%-99.2%), with a partial response rate of 35.5% (n = 11 of 31; 95% CI: 19.2%-54.6%). The median progression-free and overall survivals were 5.6 (95% CI: 4.0-6.0) and 10.1 (95% CI: 6.1-11.1) months, respectively. Progression biopsies on pegargiminase revealed a statistically significant influx of macrophages (n = 6; p = 0.0255) and patchy tumoral ASS1 reexpression (n = 2 of 6). In addition, we observed increased tumoral programmed death-ligand 1-an ADI-PEG 20 inducible gene-and the formation of CD3-positive T lymphocyte aggregates on disease progression (n = 2 of 5). CONCLUSIONS: The dose expansion of ADIPemCis confirmed the high clinical activity and good tolerability in ASS1-deficient poor-prognosis mesothelioma, underpinning an ongoing phase 3 study (ClinicalTrials.govNCT02709512). Notably, resistance to pegargiminase correlated with marked macrophage recruitment and-along with the tumor immune microenvironment-warrants further study to optimize arginine deprivation for the treatment of mesothelioma.

A Phase I Study of Pegylated Arginine Deiminase (Pegargiminase), Cisplatin, and Pemetrexed in Argininosuccinate Synthetase 1-Deficient Recurrent High-grade Glioma.

PURPOSE: Patients with recurrent high-grade gliomas (HGG) are usually managed with alkylating chemotherapy ± bevacizumab. However, prognosis remains very poor. Preclinically, we showed that HGGs are a target for arginine depletion with pegargiminase (ADI-PEG20) due to epimutations of argininosuccinate synthetase (ASS1) and/or argininosuccinate lyase (ASL). Moreover, ADI-PEG20 disrupts pyrimidine pools in ASS1-deficient HGGs, thereby impacting sensitivity to the antifolate, pemetrexed. PATIENTS AND METHODS: We expanded a phase I trial of ADI-PEG20 with pemetrexed and cisplatin (ADIPEMCIS) to patients with ASS1-deficient recurrent HGGs (NCT02029690). Patients were enrolled (01/16-06/17) to receive weekly ADI-PEG20 36 mg/m2 intramuscularly plus pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 intravenously once every 3 weeks for up to 6 cycles. Patients with disease control were allowed ADI-PEG20 maintenance. The primary endpoints were safety, tolerability, and preliminary estimates of efficacy. RESULTS: Ten ASS1-deficient heavily pretreated patients were treated with ADIPEMCIS therapy. Treatment was well tolerated with the majority of adverse events being Common Terminology Criteria for Adverse Events v4.03 grade 1-2. The best overall response was stable disease in 8 patients (80%). Plasma arginine was suppressed significantly below baseline with a reciprocal increase in citrulline during the sampling period. The anti-ADI-PEG20 antibody titer rose during the first 4 weeks of treatment before reaching a plateau. Median progression-free survival (PFS) was 5.2 months (95% confidence interval (CI), 2.5-20.8) and overall survival was 6.3 months (95% CI, 1.8-9.7). CONCLUSIONS: In this recurrent HGG study, ADIPEMCIS was well tolerated and compares favorably to historical controls. Additional trials of ADI-PEG20 in HGG are planned.

A phase 1 study of ADI-PEG 20 and modified FOLFOX6 in patients with advanced hepatocellular carcinoma and other gastrointestinal malignancies.

PURPOSE: Arginine depletion interferes with pyrimidine metabolism as well as DNA damage repair pathways. Preclinical data indicates that pairing pegylated arginine deiminase (ADI-PEG 20) with fluoropyrimidines or platinum enhances cytotoxicity in vitro and in vivo in arginine auxotrophs. METHODS: This is a single-center, open-label, phase 1 trial of ADI-PEG 20 and modified FOLFOX6 (mFOLFOX6) in treatment-refractory hepatocellular carcinoma (HCC) and other advanced gastrointestinal tumors. A 3 + 3 dose escalation design was employed to assess safety, tolerability, and determine the recommended phase 2 dose (RP2D) of ADI-PEG 20. A RP2D expansion cohort for patients with HCC was employed to define the objective response rate (ORR). Secondary objectives were to estimate progression-free survival (PFS), overall survival (OS), and to explore pharmacodynamics and immunogenicity. Eligible patients were treated with mFOLFOX6 intravenously biweekly at standard doses and ADI-PEG-20 intramuscularly weekly at 18 (Cohort 1) or 36 mg/m2 (Cohort 2 and RP2D expansion). RESULTS: Twenty-seven patients enrolled-23 with advanced HCC and 4 with other gastrointestinal tumors. No dose-limiting toxicities were observed in cohort 1 or 2. The RP2D for ADI-PEG 20 was 36 mg/m2 weekly with mFOLFOX6. The most common any grade adverse events (AEs) were thrombocytopenia, neutropenia, leukopenia, anemia, and fatigue. Among the 23 HCC patients, the most frequent treatment-related Grade ≥ 3 AEs were neutropenia (47.8%), thrombocytopenia (34.7%), leukopenia (21.7%), anemia (21.7%), and lymphopenia (17.4%). The ORR for this group was 21% (95% CI 7.5-43.7). Median PFS and OS were 7.3 and 14.5 months, respectively. Arginine levels were depleted with therapy despite the emergence of low levels of anti-ADI-PEG 20 antibodies. Arginine depletion at 4 and 8 weeks and archival tumoral argininosuccinate synthetase-1 levels did not correlate with response. CONCLUSIONS: Concurrent mFOLFOX6 plus ADI-PEG-20 intramuscularly at 36 mg/m2 weekly shows an acceptable safety profile and favorable efficacy compared to historic controls. Further evaluation of this combination is warranted in advanced HCC patients.

Is Vascular Amyloidosis Intertwined with Arterial Aging, Hypertension and Atherosclerosis?

Vascular amyloidosis (VA) is a component of aging, but both VA and aging move forward together. Although, not all age-related molecules are involved with VA, some molecules are involved in a crosstalk between both of them. However, the cellular mechanism by which, vascular cells are phenotypically shifted to arterial remodeling, is not only involved in aging but also linked to VA. Additionally, patients with hypertension and atherosclerosis are susceptible to VA, while amyloidosis alone may provide fertile soil for the initiation and progression of subsequent hypertension and atherosclerosis. It is known that hypertension, atherosclerosis and amyloidosis can be viewed as accelerated aging. This review summarizes the available experimental and clinical evidence to help the reader to understand the advance and underlying mechanisms for VA involvement in and interaction with aging. Taken together, it is clear that VA, hypertension and atherosclerosis are closely intertwined with arterial aging as equal partners.

A phase 1/1B trial of ADI-PEG 20 plus nab-paclitaxel and gemcitabine in patients with advanced pancreatic adenocarcinoma.

BACKGROUND: ADI-PEG 20 is a pegylated form of the arginine-depleting enzyme arginine deiminase. Normal cells synthesize arginine with the enzyme argininosuccinate synthetase (ASS1); ADI-PEG 20 selectively targets malignant cells, which lack ASS1. METHODS: A single-arm, nonrandomized, open-label, phase 1/1B, standard 3 + 3 dose escalation with an expansion cohort of 9 patients at the recommended phase 2 dose (RP2D) was conducted. Patients who had metastatic pancreatic cancer, up to 1 line of prior treatment (the dose-escalation cohort) or no prior treatment (the expansion cohort), and an Eastern Cooperative Oncology Group performance status of 0 to 1 were included. Patients received both gemcitabine (1000 mg/m2 ) and nab-paclitaxel (125 mg/m2 ) for 3 of 4 weeks and intramuscular ADI-PEG 20 at 18 mg/m2 weekly (cohort 1) or at 36 mg/m2 weekly (cohort 2 and the expansion cohort).The primary endpoint was to determine the maximum tolerated dose and RP2D of ADI-PEG 20 in combination with nab-paclitaxel and gemcitabine. RESULTS: Eighteen patients were enrolled. No dose-limiting toxicities (DLTs) were observed in cohort 1; cohort 2 was expanded to 6 patients because of 1 DLT occurrence (a grade 3 elevation in bilirubin, aspartate aminotransferase, and alanine aminotransferase). The most frequent adverse events (AEs) of any grade were neutropenia, thrombocytopenia, leukopenia, anemia, peripheral neuropathy, and fatigue; all 18 patients experienced grade 3/4 AEs. The most frequent grade 3/4 toxicities, regardless of the relation with any drugs, included neutropenia (12 patients or 67%), leukopenia (10 patients or 56%), anemia (8 patients or 44%), and lymphopenia (6 patients or 33%). The RP2D for ADI-PEG 20 was 36 mg/m2 weekly in combination with standard-dose gemcitabine and nab-paclitaxel. The overall response rate among patients treated at the RP2D in the first-line setting was 45.5% (5 of 11).The median progression-free survival time for these patients treated at the RP2D was 6.1 months (95% confidence interval, 5.3-11.2 months), and the median overall survival time was 11.3 months (95% confidence interval, 6.7 months to not reached). CONCLUSIONS: ADI-PEG 20 was well tolerated in combination with gemcitabine and nab-paclitaxel. Activity was observed in previously treated and untreated patients with advanced pancreatic cancer and in patients with ASS1-deficient and -proficient tumors. Cancer 2017;123:4556-4565. © 2017 American Cancer Society.

Phase 1 Dose-Escalation Study of Pegylated Arginine Deiminase, Cisplatin, and Pemetrexed in Patients With Argininosuccinate Synthetase 1-Deficient Thoracic Cancers.

Purpose Pegylated arginine deiminase (ADI-PEG 20) depletes essential amino acid levels in argininosuccinate synthetase 1 (ASS1) -negative tumors by converting arginine to citrulline and ammonia. The main aim of this study was to determine the recommended dose, safety, and tolerability of ADI-PEG 20, cisplatin, and pemetrexed in patients with ASS1-deficient malignant pleural mesothelioma (MPM) or non-small-cell lung cancer (NSCLC). Patients and Methods Using a 3 + 3 + 3 dose-escalation study, nine chemotherapy-naïve patients (five MPM, four NSCLC) received weekly ADI-PEG 20 doses of 18 mg/m2, 27 mg/m2, or 36 mg/m2, together with pemetrexed 500 mg/m2 and cisplatin 75 mg/m2 which were given every three weeks (maximum of six cycles). Patients achieving stable disease or better could continue ADI-PEG 20 monotherapy until disease progression or withdrawal. Adverse events were assessed by Common Terminology Criteria for Adverse Events version 4.03, and pharmacodynamics and immunogenicity were also evaluated. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 for NSCLC and by modified RECIST criteria for MPM. Results No dose-limiting toxicities were reported; nine of 38 reported adverse events (all grade 1 or 2) were related to ADI-PEG 20. Circulating arginine concentrations declined rapidly, and citrulline levels increased; both changes persisted at 18 weeks. Partial responses were observed in seven of nine patients (78%), including three with either sarcomatoid or biphasic MPM. Conclusion Target engagement with depletion of arginine was maintained throughout treatment with no dose-limiting toxicities. In this biomarker-selected group of patients with ASS1-deficient cancers, clinical activity was observed in patients with poor-prognosis tumors. Therefore, we recommend a dose for future studies of weekly ADI-PEG 20 36 mg/m2 plus three-weekly cisplatin 75 mg/m2 and pemetrexed 500 mg/m2.

Enhanced cytotoxicity in triple-negative and estrogen receptor­positive breast adenocarcinoma cells due to inhibition of the transient receptor potential melastatin-2 channel.

We previously demonstrated a unique protective role for the transient receptor potential, melastatin-2 (TRPM2) cation channel in breast cancer cells. In the present study, we investigated the chemotherapeutic effects elicited by inhibiting this protective role in metastatic breast adenocarcinoma cells. TRPM2 inhibition led to dose-dependent increases in MDA-MB-231 breast adenocarcinoma cell death after treatment with doxorubicin or the DNA-methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine. Similar results were observed after RNAi silencing of TRPM2 in these cells after doxorubicin treatment. However, TRPM2 RNAi silencing also led to increased MCF-7 breast adenocarcinoma cell death after tamoxifen treatment, yet not in non-cancerous human mammary epithelial cells. These results thus revealed that TRPM2 inhibition selectively increased cytotoxicity in a triple-negative and an estrogen receptor-positive breast cancer cell line, with minimal deleterious effects in non-cancerous breast cells. Analysis of DNA damage revealed enhanced DNA damage levels in MCF-7 cells treated with doxorubicin due to TRPM2 inhibition. Analysis of cell death demonstrated that inhibition of apoptosis, caspase-independent cell death or autophagy failed to significantly reduce cell death induced by TRPM2 inhibition and chemotherapy. These results indicate that TRPM2 inhibition activates alternative pathways of cell death in breast cancer cells. Taken together, our results provide significant evidence that TRPM2 inhibition is a potential strategy to induce triple-negative and estrogen receptor-positive breast adenocarcinoma cell death via alternative cell death pathways. This is expected to provide a basis for inhibiting TRPM2 for the improved treatment of breast cancer, which potentially includes treating breast tumors that are resistant to chemotherapy due to their evasion of apoptosis.

Inhibition of the transient receptor potential melastatin-2 channel causes increased DNA damage and decreased proliferation in breast adenocarcinoma cells.

Transient receptor potential, melastatin-2 (TRPM2) is a plasma membrane cation channel with important roles in sensory functions and promoting cell death. However, we demonstrated here that TRPM2 was present in the nuclei of MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, and its pharmacologic inhibition or RNAi silencing caused decreased cell proliferation. Neither an effect on proliferation nor a localization of TRPM2 in the nucleus was observed in noncancerous HMEC and MCF-10A human mammary epithelial cells. Investigation of possible effects of TRPM2 function in the nucleus demonstrated that pharmacologic inhibition or RNAi silencing of TRPM2 in MCF-7 and MDA-MB-231 human breast adenocarcinoma cells caused up to 4-fold increases in DNA damage levels, as compared to noncancerous breast cells after equivalent treatments. These results indicate that TRPM2 has a novel nuclear function in human breast adenocarcinoma cells that facilitates the integrity of genomic DNA, a finding that is distinct from its previously reported role as a plasma membrane cation channel in noncancerous cells. In summary, we report here a novel effect promoted by TRPM2, where it functions to minimize DNA damage and thus may have a role in the protection of genomic DNA in breast cancer cells. Our study therefore provides compelling evidence that TRPM2 has a unique role in breast adenocarcinoma cells. Accordingly, these studies suggest that TRPM2 is a potential therapeutic target, where its pharmacologic inhibition may provide an innovative strategy to selectively increase DNA damage levels in breast cancer cells.

Development and validation of LC-MS/MS method for quantification of pseudolaric acid B from the root bark of Pseudolarix kaempferi in rat plasma: application to a pharmacokinetic study.

Pseudolaric acid B (PAB), which is the main biologically active diterpene acid of Pseudolarix kaempferi, has presented anti-fungal, anti-tumor, anti-fertility, and anti-tubulin activities. In this study, a sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method with multiple-reaction monitoring mode was employed for quantification of PAB in rat plasma. The calibration curve was linear over the range of 0.86-288 ng/mL with correlation coefficient (r) greater than 0.995 for PAB, and the lower limit of quantification was 0.86 ng/mL in rat plasma. The accuracy of PAB was between -9.1% and 7.0% relative error, and precision ranged from 1.2 to 10.6% relative standard deviation. This method was successfully applied to the pharmacokinetic studies of PAB in Sprague-Dawley rats. After single intravenous administration of 2.0, 4.0, and 8.0mg/kg PAB to rats, the t1/2 were (16.1 ± 5.6), (30.0 ± 13.7), and (27.4 ± 5.3)min, respectively. The pharmacokinetics (C(2min), AUC) of PAB within the used dosage range were in accordance with linear pharmacokinetic characteristics.

Quantitative proteomic analysis in HCV-induced HCC reveals sets of proteins with potential significance for racial disparity.

BACKGROUND: The incidence and mortality of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) is higher in African Americans (AA) than other racial/ethnic groups in the U.S., but the reasons for this disparity are unknown. There is an urgent need for the discovery of novel molecular signatures for HCV disease progression to understand the underlying biological basis for this cancer rate disparity to improve the clinical outcome. METHODS: We performed differential proteomics with isobaric labeling tags for relative and absolute quantitation (iTRAQ) and MS/MS analysis to identify proteins differentially expressed in cirrhotic (CIR) and HCC as compared to normal tissues of Caucasian American (CA) patients. The raw data were analyzed using the ProteinPilot v3.0. Searches were performed against all known sequences populating the Swiss-Prot, Refseq, and TrEMBL databases. Quality control analyses were accomplished using pairwise correlation plots, boxplots, principal component analysis, and unsupervised hierarchical clustering. Supervised analysis was carried out to identify differentially expressed proteins. Candidates were validated in independent cohorts of CA and AA tissues by qRT-PCR or Western blotting. RESULTS: A total of 238 unique proteins were identified. Of those, around 15% were differentially expressed between normal, CIR & HCC groups. Target validation demonstrates racially distinct alteration in the expression of certain proteins. For example, the mRNA expression levels of transferrin (TF) were 2 and18-fold higher in CIR and HCC in AA as compared to CA. Similarly; the expression of Apolipoprotein A1 (APOA1) was 7-fold higher in HCC of AA. This increase was mirrored in the protein expression levels. Interestingly, the level of hepatocyte nuclear factor4a (HNF4a) protein was down regulated in AA, whereas repression of transcription is seen more in CA compared to AA. These data suggest that racial disparities in HCC could be a consequence of differential dysregulation of HNF4a transcriptional activity. CONCLUSION: This study identifies novel molecular signatures in HCV-induced HCC using iTRAQ-based tissue proteomics. The proteins identified will further enhance a molecular explanation to the biochemical mechanism(s) that may play a role in HCC racial disparities.

Roles of poly(ADP-ribose) glycohydrolase in DNA damage and apoptosis.

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme that catalyzes the hydrolysis of poly(ADP-ribose) (PAR), an essential biopolymer that is synthesized by poly(ADP-ribose) polymerases (PARPs) in the cell. By regulating the hydrolytic arm of poly(ADP-ribosyl)ation, PARG participates in a number of biological processes, including the repair of DNA damage, chromatin dynamics, transcriptional regulation, and cell death. Collectively, the research investigating the roles of PARG in the cell has identified the importance of PARG and its value as a therapeutic target. However, the biological role of PARG remains less understood than the role of PAR synthesis by the PARPs. Further complicating the study of PARG is the existence of multiple PARG isoforms in the cell, the lack of optimal PARG inhibitors, and the lack of viable PARG-null animals. This review will present our current knowledge of PARG, with a focus on its roles in DNA-damage repair and cell death.

Inhibition of poly(ADP-ribose) polymerase-1 or poly(ADP­ribose) glycohydrolase individually, but not in combination, leads to improved chemotherapeutic efficacy in HeLa cells.

The genome-protecting role of poly(ADP-ribose) (PAR) has identified PAR polymerase-1 (PARP-1) and PAR glycohydrolase (PARG), two enzymes responsible for the synthesis and hydrolysis of PAR, as chemotherapeutic targets. Each has been previously individually evaluated in chemotherapy, but the effects of combination PARP-1 and PARG inhibition in cancer cells are not known. Here we determined the effects of the inhibition of PARP-1 and the absence or RNAi knockdown of PARG on PAR synthesis, cell death after chemotherapy and long-term viability. Using three experimental/clinical PARP-1 inhibitors in PARG-null cells, we show decreased levels of PAR and increased short­term and long­term viability with each inhibitor, with the exception of DPQ. Treatment with the experimental chemotherapeutic agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), led to increased cell death in PARG-null cells, but decreased cell death when pretreated with each PARP-1 inhibitor. Similar results were observed in MNNG-treated HeLa cells, where RNAi knockdown of PARG or pretreatment with ABT-888 led to increased HeLa cell death, whereas combination PARG RNAi knockdown + ABT-888 failed to produce increased cell death. The results demonstrate the ability of the PARP-1 inhibitors to decrease PAR levels, maintain viability and decrease PAR-mediated cell death after chemotherapeutic treatment in the absence of PARG. Further, the results demonstrate that the combination of PARP-1 and PARG inhibition in chemotherapy does not produce increased HeLa cell death. Thus, the results indicate that inhibiting both PARP-1 and PARG, which both are chemotherapeutic targets that increase cancer cell death, does not lead to synergistic cell death in HeLa cells. Therefore, strategies that target PAR metabolism for the improved treatment of cancer may be required to target PARP-1 and PARG individually in order to optimize cancer cell death.

Silencing of Apoptosis-Inducing factor and poly(ADP-ribose) glycohydrolase reveals novel roles in breast cancer cell death after chemotherapy.

BACKGROUND: Cell death induced by poly(ADP-ribose) (PAR) and mediated by apoptosis-inducing factor (AIF) is well-characterized in models of ischemic tissue injury, but their roles in cancer cell death after chemotherapy are less understood. METHODS: Here we investigated the roles of PAR and AIF by RNA interference (RNAi) in MDA-MB-231 and MCF-7 breast adenocarcinoma cells after chemotherapy. Differences in effects were statistically tested by analysis-of-variance and unpaired student's t-test. RESULTS: Silencing of AIF by RNAi led to decreased MDA-MB-231 and MCF-7 breast cancer cell death after chemotherapy, which demonstrates a critical role for AIF. RNAi silencing of PAR glycohydrolase (PARG), the primary enzyme that catalyzes the hydrolysis of PAR, led to increased PAR levels but decreased cell death. Further investigation into the possible role of PAR in apoptosis revealed decreased caspase-3/7/8/9 activity in PARG-null cells. Interestingly, the pharmacologic inhibition of caspase activity in PARG-silenced breast cancer cells led to increased cell death after chemotherapy, which indicates that an alternative cell death pathway is activated due to elevated PAR levels and caspase inhibition. AIF silencing in these cells led to profound protection from chemotherapy, which demonstrates that the increased cell death after PARG silencing and caspase inhibition was mediated by AIF. CONCLUSIONS: The results show a role for AIF in breast cancer cell death after chemotherapy, the ability of PAR to regulate caspase activity, and the ability of AIF to substitute as a primary mediator of breast cancer cell death in the absence of caspases. Thus, the induction of cell death by PAR/AIF may represent a novel strategy to optimize the eradication of breast tumors by activating an alternative cell death pathway.

Synergistic cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine and absence of poly(ADP-ribose) glycohydrolase involves chromatin decondensation.

DNA-alkylating agents in combination with poly (ADP-ribose) (PAR) synthesis inhibitors are a promising treatment for cancer. In search of other efficacious alternatives, we hypothesized that the absence of poly(ADP-ribose) glycohydrolase (PARG), which leads to the inhibition of PAR hydrolysis, would lead to increased DNA alkylation after treatment with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). At a sublethal dose, MNNG shows synergistic cytotoxicity in PARG-null embryonic trophoblast stem (TS) cells. The PAR modifications of histone H1 and histone H2B are much more pronounced in PARG null-TS cells exposed to MNNG, suggesting their relevance in the efficacy of this combination therapy. Because the PAR modification of these chromatin binding proteins leads to chromatin remodeling, a possible mechanism for the observed synergistic effects involves the subsequent decondensation of chromatin, which may cause the genomic DNA to be more accessible to MNNG alkylation. Further analysis demonstrated chromatin decondensation in PARG null-TS cells as visualized by electron microscopy. In addition, treatment with MNNG led to an increase in O6- methylguanine levels in PARG null-TS cells compared to wild-type, which demonstrates increased DNA alkylation in the absence of PARG. Taken together, we provide compelling evidence that the absence of PARG leads to chromatin decondensation, which in turn leads to increased amounts of DNA alkylation and cell death induced by low doses of MNNG. Therefore, combination therapy of PARG inhibition and a DNA- alkylating agent is a potential treatment to induce the death of cancer cells.

Activation of cell death mediated by apoptosis-inducing factor due to the absence of poly(ADP-ribose) glycohydrolase.

We previously demonstrated that the absence of poly(ADP-ribose) glycohydrolase (PARG) led to increased cell death following DNA-damaging treatments. Here, we investigated cell death pathways following UV treatment. Decreased amounts of PARG-null embryonic trophoblast stem (TS) cells were observed following doses of 10-100 J/m2 as compared to wild-type cells. In wild-type cells, caspase-cleaved poly(ADP-ribose) polymerase-1 (PARP-1) and activated caspase-3 were detected 12-24 h after UV treatment. Surprisingly, both were detected at decreased levels only after 24 h in PARG-null TS cells, indicating a decreased level and delayed presence of caspase-mediated events. Further, a time- and dose-dependent accumulation of poly(ADP-ribose) (PAR) levels after UV was observed in PARG-null TS cells and not in wild-type cells. Determination of the levels of nicotinamide adenine dinucleotide (NAD+), the substrate for PAR synthesis and a coenzyme in cellular redox reactions, demonstrated a UV dose-dependent decrease in the level of NAD+ in wild-type cells, while NAD+ levels in PARG-null TS cells remained at higher levels. This indicates no depletion of NAD+ in PARG-null TS cells following increased levels of PAR. Lastly, cell death mediated by apoptosis-inducing factor (AIF) was analyzed because of its dependence on increased PAR levels. The results demonstrate nuclear AIF translocation only in PARG-null TS cells, which demonstrates the presence of AIF-mediated cell death. Herein, we provide compelling evidence that the absence of PARG leads to decreased caspase-3 activity and the specific activation of AIF-mediated cell death. Therefore, the absence of PARG may provide a strategy for specifically inducing an alternative apoptotic pathway.

Enhanced DNA accessibility and increased DNA damage induced by the absence of poly(ADP-ribose) hydrolysis.

Poly(ADP-ribose) (PAR) is a therapeutic target primarily identified through inhibiting its synthesis by PAR polymerase-1 (PARP-1). However, inhibiting its hydrolysis by PAR glycohydrolase (PARG) has therapeutic potential in cancer. Unknown is the effect of elevated PAR levels on cellular processes and if this effect can enhance the therapeutic value of PARG. Here, we demonstrate in PARG null embryonic trophoblast stem (TS) cells that the absence of PAR hydrolysis led to PAR-modified histones H1, H2A, and H2B. To determine if this led to the differential vulnerability of DNA to modification, TS cells were treated with DNA-modifying agents. The results demonstrate increased DNA laddering by micrococcal nuclease and an increased amount of DNA intercalation by acridine orange in PARG null-TS cells. This increased access to PARG null-TS cell DNA was further verified by the detection of increased DNA damage following treatment with UV radiation and a minimal dose of the DNA-alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Further, this DNA damage was predominantly unrepaired 12 h after treatment in PARG null-TS cells. Finally, TS cells were treated with DNA-modifying chemotherapeutic agents. The results demonstrate up to 4-fold increases in cell death in PARG null-TS cells after treatment with epirubicin or sub-IC(50) doses of cisplatin and cyclophosphamide. Taken together, we provide compelling evidence that increased DNA access induced by the absence of PARG enhances the efficacy of DNA-modifying agents. Thus, this study demonstrates that greater DNA accessibility, increased DNA damage, and increased cell death all contribute to the PARG null cell phenotype in response to genotoxic stress.