Investigation of pegargiminase targeting the Fanconi anemia pathway and its role in DNA double-strand breaks induced by melphalan in uveal melanoma.

Abstract

e15114 Background: Uveal melanoma (UM) is a hard-to-treat argininosuccinate synthetase 1 (ASS1)-deficient cancer with approximately 50% of patients succumbing to metastases predominantly to the liver. Arginine deprivation with pegylated arginine deiminase (ADI-PEG20; pegargiminase) is a novel anti-metabolite therapy with proven activity in extending overall survival in a recent phase 3 double-blind randomized study in mesothelioma (Szlosarek et al, JAMA Oncol 2024; NCT02709512), and with favorable activity reported in early trials in uveal melanoma (Chan et al, PCMR 2022; NCT02029690). Here, we investigated the preclinical rationale for combining pegargiminase with melphalan, an alkylating agent approved for the treatment of hepatic-centric UM disease. Methods: Drug sensitivity of ASS1-deficient UM cell lines was performed in 2D culture using proliferation and cytotoxicity assays, followed by studies of cell death and cell cycle, DNA double-strand breaks (DSBs; intoDNA) and interrogation of the molecular mechanism of action by RNA seq. Results: Two (92.1 and MP41) of four UM cell lines were ASS1-deficient and analyzed for drug sensitivity. Both ADI-PEG20 and melphalan suppressed 92.1 and MP41 cell line proliferation and triggered cytotoxicity, effects which were enhanced with the drug combination specifically in the 92.1 cell line (p < 0.0001 vs control; p = 0.0024 vs ADI-PEG20; p = 0.0004 vs melphalan). Moreover, PARP cleavage increased with the drug combination compared with either drug alone in the 92.1 cell line with a higher fraction of annexin V positivity detected with the combination (p < 0.0001 vs control; p < 0.0001 vs ADI-PEG20; p = 0.001 vs melphalan). ADI-PEG20 synergized with melphalan to increase DNA DSBs in the nucleus (5.0-fold vs control), that was accompanied by downregulation of multiple genes of the Fanconi anemia pathway by ADI-PEG20, including FANCD2 (p < 0.0001), UBE2T (p < 0.0001), EME1 (p < 0.01), BRIP1 (p < 0.0001), FANCA (p < 0.001), FANCE (p < 0.05), FANCI (p < 0.001) and FANCB (p < 0.01). Cell cycle analysis revealed distinct arrest in G2/S phase with ADI-PEG20 and melphalan. Conclusions: Melphalan and arginine deprivation therapy is a rational new synthetically lethal drug combination that has the potential to improve outcomes in patients with ASS1-deficient uveal melanoma. Based on the good tolerability and safety of both drugs in patients, a clinical trial of melphalan with pegargiminase is planned (ATOMIC-UM).