Ramsay Khadeir

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.

Abstract B23: 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)

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.

Optimizing arginine deprivation for hard-to-treat cancers

Arginine-dependent cancers represent a significant fraction of malignancies characterized by loss of ureacycle enzymes, especially argininosuccinate synthetase (ASS1) and argininosuccinate lyase (ASL). As a tumor suppressor ASS1 catalyzes the condensation of aspartate and citrulline into argininosuccinate, and impacts multiple biological pathways involving arginine either directly, for instance via nitric oxide or mTOR, or indirectly, such as modulation of nucleotide synthesis [1, 2]. Loss of ASS1 promotes increased tumor cell proliferation and invasion, and is immunosuppressive, all attributes of a highly tumorigenic cancer [3, 4]. ASS1 deficiency has been identified across the spectrum of haematological, epithelial and mesenchymal tumors, however regulation and expression of the enzyme displays significant variability and is tissue-specific. This is most evident under arginine withdrawal, which has been explored for several decades as a novel metabolic anticancer therapy targeting the arginine-dependent phenotype. Methylation-dependent silencing of the ASS1 promoter reported in mesothelioma and bladder cancer cell lines confers exquisite sensitivity to the arginine-lowering agents, arginine deiminase or arginase. In contrast, ASS1 is induced rapidly in tumor cell lines without ASS1 promoter methylation limiting the applicability of arginine deprivation under these circumstances, particularly as a monotherapy. ASL, which is downstream of ASS1 and converts argininosuccinate into arginine and fumarate, has a secondary role in modulating tumoral arginine auxotrophy and sensitivity to arginine depletors in cancers including glioblastoma multiforme [5]. Arginine deprivation entered the clinic over a decade ago with pegylated arginine deiminase (ADIPEG 20), which catalyzes the conversion of arginine into citrulline and ammonia, thereby recycling the former into arginine in ASS1 competent cells. Several monotherapy cancer studies of ADI-PEG 20 revealed safety and promising early activity despite the antigenic properties of a mycoplasma-derived enzyme. However, a recent phase 3 study of ADI-PEG 20 versus placebo in patients with post-sorafenib relapse in liver cancer was negative for overall survival. Post-hoc analyses revealed that ASS1 was upregulated by sorafenib and may have influenced patient outcome (clinicaltrial.gov identifier NCT01287585). In contrast, a modest improvement in progression-free survival in a randomized phase 2 study in patients with ASS1-deficient mesothelioma versus best supportive care alone was reported in the ADAM trial highlighting a need for patient selection in future studies [6]. Early phase clinical studies of several non-antigenic pegylatedarginases are underway and further testing will reveal how the differential catalysis of arginine into ornithine and urea will impact tumorigenesis. In addition to ASS1 selection several groups have used molecular imaging to investigate responses to ADIPEG 20. The increased glycolytic activity of neoplastic cells (Warburg effect) led to the development of fluoro2-deoxy-D-glucose positron emission tomography (FDG-PET/CT). Early metabolic responses with FDGPET/CT to arginine depletion have been evaluated in the ADAM trial for mesothelioma using EORTC based recommendations for change in standardized uptake value (SUVmax) to define the response categories (progression (PD)> 25% increase and partial metabolic response (PMR) >15% decrease in SUVmax). Whilst no modified RECIST partial or complete responses were observed during the study, FDG-PET/CT revealed a PMR in 46%; stable disease in 31%; a mixed response in 8% and progressive disease (PD) in 15% of patients [6]. However, ADI-PEG 20-induced arginine deprivation is known to increase serine biosynthesis, glutamine anaplerosis, oxidative Editorial

Abstract 5569: The impact of ADI-PEG20 on PDL1 expression in ASS1 deficient uveal melanoma

Uveal melanoma (UM), involving the iris, choroid and ciliary body, is the commonest intraocular tumor in adults. Half of patients develop metastasis with a high mortality despite currently available systemic therapies including immune checkpoint blockade. Studies show that while CTLA4 antagonism has a modest effect in UM, PD1/PDL1 blockade is largely ineffective whereas trials of combination checkpoint blockade are yet to report. We confirmed previously that deficiency of argininosuccinate synthetase 1 (ASS1), a key enzyme involved in arginine synthesis, sensitizes UM cells to arginine deprivation using pegylated arginine deiminase or ADI-PEG20. Early trials of ADI-PEG20 in UM have shown safety and efficacy in the clinic and thus we tested the potential for a combined immunometabolic strategy. ASS1-deficient UM cell lines (OMM1, OMM2.5, MEL270) were analyzed for the immune checkpoint protein PDL1 along with their sensitivity to arginine depletion. We also tested metastatic UM for ASS1 and PDL1 expression, and the impact of ADI-PEG20 treatment using samples from a current clinical trial (NCT02029690). We showed that ASS1 and PDL1 protein expression were absent in the 3 UM cell lines and in a majority of primary tumors (75/102 for ASS1 and 83/102 for PDL1; 5% threshold of expression) and all metastatic tumor biopises (n=16/16 for both). Transfection of ASS1 in OMM1, OMM2.5, MEL270 led to an increase in PDL1 expression by qPCR, western blotting and FACS, which was reversible following knockdown of ASS1. Induction of PDL1 expression by ASS1 was accompanied by interferon (IFN)-alpha and beta (but not IFN-gamma) release into the cell supernatant, and was abrogated using the pan-STAT inhibitor ruxolitinib. Next, PDL1 expression was significantly increased in the 3 ASS1-negative UM cell lines with ADI-PEG20 treatment by 24hrs and was associated with Type I IFN signaling which waned along with PDL1 expression by 48hrs. The ADI-PEG 20 induction of PDL1 was abolished using ruxolitinib, indicating that the upregulation of Type I IFNs is critical for regulation of the PDL1 checkpoint protein. While analysis of UM biopsies of patients progressing on ADI-PEG20 revealed upregulation of ASS1 (n=2/2; and thus resistance to ADI-PEG20) a concomitant increase in PDL1 was not observed (n=0/2). Collectively, our data show that ASS1 is absent in a majority of patient biopsies of primary and metastatic UM tumors and is tightly correlated with PDL1 expression. UM cells lines displayed sensitivity to ADI-PEG20, which upregulated levels of PDL1 expression via Type 1 interferon signaling that may enhance the currently limited efficacy of checkpoint blockade in UM. Further studies are ongoing of the IFN-mediated signaling between ASS1 and PDL1 in UM in response to arginine deprivation with ADI-PEG20. Citation Format: Ramsay S. Khadeir, Melissa M. Phillips, Mandeep Sagoo, Victoria Cohen, Caroline Thuang, Peter W. Szlosarek. The impact of ADI-PEG20 on PDL1 expression in ASS1 deficient uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5569. doi:10.1158/1538-7445.AM2017-5569

Abstract 1156: Widespread deficiency of ASS1 in uveal melanoma and sensitivity to pegylated arginine deiminase

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Uveal melanoma, involving the iris, choroid and ciliary body, is the commonest intraocular tumor in adults. Approximately half of these patients will develop metastasis with a high mortality despite currently available systemic therapies including immune checkpoint blockade. The urea cycle enzyme argininosuccinate synthetase 1 (ASS1), responsible for arginine synthesis, is downregulated in melanoma and other cancers and these are therefore sensitive to arginine deprivation therapy. In early phase trials of the arginine depletor pegylated arginine deiminase, ADI-PEG20, uveal melanoma, in particular, was identified with potential for further therapeutic development. Here, we tested uveal melanoma cell lines for sensitivity to ADI-PEG20 and analyzed enucleated tumors for ASS1 expression to assess the extent of ASS1 deficiency. Methods: ASS1 gene and protein expression were assessed in three uveal melanoma cell lines (OMM1, OMM2.5 and Mel270) by real-time quantitative PCR (qPCR) and western blot analysis, respectively. Sensitivity to ADI-PEG20 was performed using the cell viability MTS assay. We screened 102 enucleated choroidal and ciliary body melanomas for ASS1 protein using red chromogen for the immunohistochemistry (IHC) and selected the most positive area for scoring. Results: The uveal melanoma cell lines expressed negligible ASS1 mRNA with a complete absence of ASS1 protein. All three ASS1 negative uveal melanoma cell lines were sensitive to ADI-PEG20 by day 6 of the MTS assay, whereas an ASS1-expressing positive control cell line was resistant. There was a convincing lack of ASS1 expression (<5% staining) in the majority of uveal melanomas (75/102; 74%) and where ASS1 was present (5-30% staining) in the remainder (27/102; 26%) this was due to a mixture of melanoma cells and intratumoral macrophages, the latter being confirmed with CD68 (IHC) in the cases with the highest ASS1 expression. Conclusion: ASS1 is absent in the tested uveal melanoma cell lines and predisposes to arginine sensitivity with ADI-PEG20 in vitro. Most primary uveal melanomas have a marked deficiency of ASS1, representing a good target for exploring arginine deprivation further in the clinic, either alone or in combination with rationally selected agents. A trial of ADI-PEG20 combined with carboplatin and paclitaxel is planned in metastatic malignant melanoma with an expanded cohort in patients with uveal disease. Citation Format: Ramsay S. Khadeir, Melissa M. Phillips, Mandeep S. Sgoo, Amit Arora, Victoria Cohen, Caroline Thaung, Peter W. Szlosarek. Widespread deficiency of ASS1 in uveal melanoma and sensitivity to pegylated arginine deiminase. [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 1156. doi:10.1158/1538-7445.AM2015-1156

Abstract 3360: Macrophages promote resistance to pegylated arginine deiminase in malignant pleural mesothelioma

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