Aminopeptidase Expression in Multiple Myeloma Associates with Disease Progression and Sensitivity to Melflufen

SIMPLE SUMMARY: The aims of this study were to investigate aminopeptidase expression in multiple myeloma and to identify the aminopeptidases responsible for the activation of the peptide–drug conjugate melflufen in multiple myeloma. We observed a differential expression of aminopeptidases between relapsed/refractory and newly diagnosed multiple myeloma patients. A higher expression of the aminopeptidase genes XPNPEP1, RNPEP, DPP3, and BLMH in multiple myeloma plasma cells was associated with shorter patient overall survival. The peptide–drug conjugate melflufen was particularly active towards plasma cells from relapsed/refractory multiple myeloma patients. Melflufen could be hydrolyzed to its active form by the aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP, all of which are expressed in multiple myeloma. These results indicate critical roles for aminopeptidases in disease progression and the activity of melflufen in multiple myeloma. ABSTRACT: Multiple myeloma (MM) is characterized by extensive immunoglobulin production leading to an excessive load on protein homeostasis in tumor cells. Aminopeptidases contribute to proteolysis by catalyzing the hydrolysis of amino acids from proteins or peptides and function downstream of the ubiquitin–proteasome pathway. Notably, aminopeptidases can be utilized in the delivery of antibody and peptide-conjugated drugs, such as melflufen, currently in clinical trials. We analyzed the expression of 39 aminopeptidase genes in MM samples from 122 patients treated at Finnish cancer centers and 892 patients from the CoMMpass database. Based on ranked abundance, LAP3, ERAP2, METAP2, TTP2, and DPP7 were highly expressed in MM. ERAP2, XPNPEP1, DPP3, RNPEP, and CTSV were differentially expressed between relapsed/refractory and newly diagnosed MM samples (p < 0.05). Sensitivity to melflufen was detected ex vivo in 11/15 MM patient samples, and high sensitivity was observed, especially in relapsed/refractory samples. Survival analysis revealed that high expression of XPNPEP1, RNPEP, DPP3, and BLMH (p < 0.05) was associated with shorter overall survival. Hydrolysis analysis demonstrated that melflufen is a substrate for aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP. The sensitivity of MM cell lines to melflufen was reduced by aminopeptidase inhibitors. These results indicate critical roles of aminopeptidases in disease progression and the activity of melflufen in MM.