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.

Summary: Microphthalmia‐associated transcription factor (MITF) is a member of the basic helix–loop–helix leucine zipper (bHLH‐Zip) family and functions as the master regulator of the melanocytic lineage. MITF‐M is the predominant isoform expressed in melanocytes and melanoma cells, and, unlike other MITF isoforms, it is constitutively nuclear. Mutational analysis revealed three karyophilic signals in the bHLH‐Zip domain of MITF‐M, spanning residues 197–206, 214–217, and 255–265. Structural characterization of the MITF protein showed that basic residues within these signals are exposed for interactions in the absence of DNA. Moreover, our data indicate that neither DNA binding nor dimerization of MITF‐M are required for its nuclear localization. Finally, dimerization‐deficient MITF‐M mutants exhibited a significantly reduced stability in melanoma cells when compared to the wild‐type protein. Taken together, we have shown that, in addition to its well‐established role in DNA binding and dimer formation, the bHLH‐Zip domain of MITF modulates the transcription factor's subcellular localization and stability.

Melphalan flufenamide (hereinafter referred to as “melflufen”) is a peptide‐conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal‐derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA‐MB231. The tumorigenic D492HER2 and MDA‐MB231 cells were more sensitive than normal‐derived D492 cells when treated with melflufen. Compared to the commonly used anti‐cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA‐MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins ƴH2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13^high and CD13^low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti‐neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.