A role for macrophages under cytokine control in mediating resistance to ADI-PEG20 (pegargiminase) in ASS1-deficient mesothelioma

Our plain-English summary of A role for macrophages under cytokine control in mediating resistance to ADI-PEG20 (pegargiminase) in ASS1-deficient mesothelioma, by M.M. Phillips, I. Pavlyk, M. Allen, et al (Pharmacological Reports, 2023).

The study

• Authors: M.M. Phillips, I. Pavlyk, M. Allen, E. Ghazaly, R. Cutts, J. Carpentier, J.S. Berry, C. Nattress, S. Feng, G. Hallden, C. Chelala, J. Bomalaski, J. Steele, M. Sheaff, F. Balkwill, P.W. Szlosarek
• Journal: Pharmacological Reports
• Published: 2023;75(3):570-584
• Cited by: 7 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Pegylated arginine deiminase (ADI-PEG20; pegargiminase) depletes arginine and improves survival outcomes for patients with argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM). Optimisation of ADI-PEG20-based therapy will require a deeper understanding of resistance mechanisms, including those mediated by the tumor microenvironment. Here, we sought to reverse translate increased tumoral macrophage infiltration in patients with ASS1-deficient MPM relapsing on pegargiminase therapy.

Methods: Macrophage-MPM tumor cell line (2591, MSTO, JU77) co-cultures treated with ADI-PEG20 were analyzed by flow cytometry. Microarray experiments of gene expression profiling were performed in ADI-PEG20-treated MPM tumor cells, and macrophage-relevant genetic "hits" were validated by qPCR, ELISA, and LC/MS. Cytokine and argininosuccinate analyses were performed using plasma from pegargiminase-treated patients with MPM.

Results: We identified that ASS1-expressing macrophages promoted viability of ADI-PEG20-treated ASS1-negative MPM cell lines. Microarray gene expression data revealed a dominant CXCR2-dependent chemotactic signature and co-expression of VEGF-A and IL-1α in ADI-PEG20-treated MPM cell lines. We confirmed that ASS1 in macrophages was IL-1α-inducible and that the argininosuccinate concentration doubled in the cell supernatant sufficient to restore MPM cell viability under co-culture conditions with ADI-PEG20. For further validation, we detected elevated plasma VEGF-A and CXCR2-dependent cytokines, and increased argininosuccinate in patients with MPM progressing on ADI-PEG20. Finally, liposomal clodronate depleted ADI-PEG20-driven macrophage infiltration and suppressed growth significantly in the MSTO xenograft murine model.

Conclusions: Collectively, our data indicate that ADI-PEG20-inducible cytokines orchestrate argininosuccinate fuelling of ASS1-deficient mesothelioma by macrophages. This novel stromal-mediated resistance pathway may be leveraged to optimize arginine deprivation therapy for mesothelioma and related arginine-dependent cancers.

© 2023. The Author(s).

In plain English

This laboratory study looked at why malignant pleural mesothelioma lacking the enzyme ASS1 may resist pegargiminase (ADI-PEG20), a drug that removes the amino acid arginine. It did not study asbestos exposure. The authors grew three ASS1-negative mesothelioma cell lines with macrophages, a type of immune cell, from healthy donors. Macrophages protected tumor cells from the drug by up to 30%, even without direct contact. The drug raised inflammatory signalling genes in tumor cells. One of them, IL-1α, increased ASS1 gene activity (measured as mRNA) in macrophages. In co-culture, argininosuccinate, a building block for arginine, doubled in the surrounding fluid, and adding it rescued tumor cells from the drug. In blood from ADAM trial patients (n = 68), the drug was followed by significant rises in plasma VEGF-A, IL-8, CXCL2 and CXCL3, with no change in the comparison group. Patients with early progression had higher VEGF-A, IL-8 and CXCL2 and a significant rise in argininosuccinate. In mice, depleting macrophages added to the drug's effect on tumor growth. The authors concluded their data implicate a macrophage-mediated resistance mechanism. They noted they could not directly test a causal role for macrophage ASS1.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study combining cell culture (tumor cells grown with macrophages), gene expression profiling, analysis of stored blood plasma from patients in earlier clinical trials, and a mouse xenograft experiment
• Who was studied: ASS1-negative malignant pleural mesothelioma cell lines (2591, MSTO, JU77), plus ASS1-positive control lines. Macrophages derived from blood of healthy human donors. Plasma from patients with ASS1-deficient MPM in the ADAM trial (n = 68), given ADI-PEG20 plus best supportive care or best supportive care only. Limited plasma from patients in the TRAP study. Nude mice carrying MSTO tumors, 12 per group in five groups.
• Exposure: No asbestos exposure was studied. The exposure was the arginine-depleting drug ADI-PEG20 (pegargiminase): 750 ng/ml in cell experiments, and 5 IU/100 µl weekly in mice, alone or combined with clodronate-containing liposomes (CLIP) to deplete macrophages.
• Main results:
  • In the three ASS1-negative MPM cell lines, co-culture with macrophages protected tumor cells from ADI-PEG20 by up to 30% compared with tumor cells cultured alone. This was seen with and without direct cell contact.
  • ADI-PEG20 increased expression of IL-1α, IL-8, CXCL2, CXCL3 and VEGF-A in the ASS1-negative cell lines. For example, microarray fold changes for VEGFA were 4.03 (2591), 4.17 (MSTO) and 3.33 (JU77 wild-type), versus 0.37 in JU77 cells overexpressing ASS1. qPCR showed a 5- to 70-fold increase in cytokine mRNA.
  • IL-1α alone increased ASS1 mRNA expression in macrophages.
  • When macrophages and MSTO cells were co-cultured with ADI-PEG20, argininosuccinate in the cell supernatant roughly doubled by 48 h.
  • Adding 0.5 µg/ml argininosuccinate rescued ASS1-negative MPM cells from ADI-PEG20.
  • Silencing the enzyme ASL in MSTO tumor cells removed the macrophage-mediated resistance.
  • In ADAM trial patients given ADI-PEG20 plus best supportive care, plasma VEGF-A, IL-8, CXCL2 and CXCL3 rose significantly. No change was detected in the best supportive care only group, and IL-1α was undetectable in both groups.
  • Among patients on ADI-PEG20 monotherapy, those with early disease progression on CT had significantly higher VEGF-A, IL-8 and CXCL2 than those with disease control.
  • Plasma argininosuccinate rose significantly only in patients whose disease progressed by week 8 of ADI-PEG20 plus best supportive care.
  • In mice with MSTO tumors, there was no re-expression of ASS1 by the tumor cells. Clodronate liposomes reduced ADI-PEG20-directed macrophage infiltration and suppressed tumor growth. ADI-PEG20 had limited single-agent activity by day 10 and was additive with CLIP at later timepoints.
• The authors' conclusion: The authors concluded that their data implicate a macrophage-mediated mechanism of resistance to ADI-PEG20 in ASS1-negative MPM, in which macrophages under cytokine control deliver argininosuccinate to the tumor. They suggested that targeting macrophages alongside ADI-PEG20 may potentially benefit other arginine-dependent cancers.
• Limitations the authors state:
  • A causal role for macrophage-derived ASS1 was not tested in the co-culture model because specific ASS1 antagonists were lacking.
  • The cytokine findings need further testing in the co-culture model using specific cytokine and chemokine antagonists.
  • The authors could not show that argininosuccinate is directly released by IL-1α-stimulated macrophages cultured alone.
  • Cytokine release measured by ELISA in cell supernatants was not always significant compared to controls.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Macrophage, Cytokine, Pegargiminase (ADI-PEG20), Mesothelioma, Pleural Mesothelioma, Tumor Microenvironment.

Related studies

• Expansion Phase 1 Study of Pegargiminase Plus Pemetrexed and Cisplatin in Patients With Argininosuccinate Synthetase 1-Deficient Mesothelioma: Safety, Efficacy, and Resistance Mechanisms — P.W. Szlosarek, M.M. Phillips, et al. (2020)
• Phase 1 Dose-Escalation Study of Pegylated Arginine Deiminase, Cisplatin, and Pemetrexed in Patients With Argininosuccinate Synthetase 1-Deficient Thoracic Cancers — E. Beddowes, J. Spicer, et al. (2017)
• Inhibition of the Polyamine Synthesis Pathway Is Synthetically Lethal with Loss of Argininosuccinate Synthase 1 — M. Locke, E. Ghazaly, et al. (2016)
• Tumor-associated macrophage-derived inflammatory cytokine enhances malignant potential of malignant pleural mesothelioma — D. Horio, T. Minami, et al. (2020)
• Role of 3'-Deoxy-3'-[18F] Fluorothymidine Positron Emission Tomography-Computed Tomography as a Predictive Biomarker in Argininosuccinate Synthetase 1-Deficient Thoracic Cancers Treated With Pegargiminase — T.A. Szyszko, J.T. Dunn, et al. (2022)
• Fibrocyte-like cells mediate acquired resistance to anti-angiogenic therapy with bevacizumab — A. Mitsuhashi, H. Goto, et al. (2015)

Browse return to medical research.