Epigenetic Inactivation Targets the Arginine Biosynthetic Pathway At Two Levels in Multiple Myeloma

Arginine is critical for the growth of certain human cancers and is an intermediary in the synthesis of various bioactive molecules. Argininosuccinate synthetase-1 (ASS1) catalyses the rate-limiting step in arginine biosynthesis, the conversion of citruline to arginine. Argininosuccinate lyase (ASL) is an enzyme that catalyses the reversible breakdown of arginosuccinate producing arginine and fumerate. Down-regulation of ASS1 has been demonstrated in several solid cancers and has been shown to confer sensitivity to arginine deprivation (arginine auxotrophic tumors). Methylation-dependent silencing of the ASS1 promoter may account for ASS1 down-regulation and arginine auxotrophy. Arginine deprivation using arginine deiminase (ADI-PEG20) is currently considered as a novel therapeutic intervention for cancer, and early clinical trials have shown promising results in solid tumors. In this perspective, we investigated for the first time the methylation status of the ASS1 and ASL CpG islands in multiple myeloma (MM) and analyzed for clinical relevance.

Genomic DNA was extracted from bone marrow aspirate samples from 46 MM patients (28 male, 18 female, median age 64 years) obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study the methylation in the ASS1 and ASL CpG islands. DNA was isolated and bisulphite modified using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. Ten bone marrow samples, with no neoplastic cells, from patients with borderline thrombocytopenia served as negative controls. Logistic regression analysis was used to measure the association between gene methylation and sex, age, ISS stage, presence of extramedullary disease, renal failure (eGFR<50 ml/min) and bone disease.

Methylation in the CpG island of ASS1 was detected in 71.7% patients (95% CI 57–82%) and of ASL in 37% patients (95% CI 24–51%), while simultaneous methylation in both genes was present in 10 patients. None of the two genes were detectably methylated in the control group. Patients with ASS1 methylation were less likely to have bone disease (p=0.04, OR=0.22) and extramedullary disease (p=0.05, OR=0.22). There was no statistically significant difference in overall survival by methylation status of the studied genes.

Arginine biosynthesis genes ASS1 and ASL are methylated in MM. Methylation of ASS1 was found to be more frequent and negatively associated with bone or extramedullary disease. Further evaluation of ASS1 and ASL methylation is warranted in MM in the perspective of expanding arginine deprivation trials in these patients

No relevant conflicts of interest to declare.