Melflufen Shows Efficacy Against Bortezomib-Resistant Multiple Myeloma Models

Introduction of proteasome inhibitors (PI), such as bortezomib (BTZ) has dramatically improved the clinical outcomes in multiple myeloma (MM). Nevertheless, MM patients become refractory to PI-based therapies. Acquisition of resistance to BTZ is associated with metabolic reprogramming. PI-resistant myeloma cells present with large mitochondria and show increased levels of oxidative phosphorylation. Previously, PI-resistance has been associated with the plasma cell precursor phenotype. BTZ-refractory MM patients have a poor prognosis and need novel treatment approaches. Melflufen (melphalan flufenamide, melflufen) is a first-in-class lipophilic peptide-drug conjugate that targets aminopeptidases and releases alkylating agents into tumor cells. This novel drug is showing promising results in clinical trials involving triple-refractory MM patients. Proteomics analysis showed that acquired BTZ-resistance promotes expression of aminopeptidase B (ApB). In this work we evaluate the potency of melflufen in several models of BTZ-resistant MM including BTZ-adapted myeloma cell lines and chemoresistant MM stem cells from bone marrows of relapsed/refractory MM (RRMM) patients.

The BTZ-adapted derivative of AMO-1 cell line shows higher susceptibility to melflufen as well as an increased expression of ApB on the mRNA (RNPEP) and protein level when compared to parental AMO-1 cells (Fig. 1A). Moreover, several other BTZ-adapted MM cell lines are more susceptible to melflufen than their parental origins (Fig. 1B). Melflufen (at 50 nM) and melphalan (5000 nM) are able to completely abrogate clonogenic outgrowth of malignant plasma cells from bone marrows of RRMM patients, whereas BTZ (5 and 10 nM), lenalidomide (2000 nM), pomalidomide (100 nM), and bendamustine (5000 nM) display only little suppressive effect on RRMM clonogenicity. Intriguingly, melflufen is at least 100-fold more potent than melphalan in suppressing RRMM clonal outgrowth (Fig. 1C). MM patients with high RNPEP mRNA expression performed significantly worse on BTZ-containing Total Therapy 3 according to the regression analysis of GSE2658 (Fig. 1D). In addition, RNPEP mRNA is also upregulated in plasma cell precursors as shown by gene expression analysis of plasma cell differentiation (GSE141005). Melflufen successfully abrogates tumor growth of both BTZ-naïve and BTZ-resistant myeloma when engrafted on chick embryo chorioallantoic membrane whereas BTZ can only prevent the tumor growth of BTZ-naïve myeloma in this model (Fig. 1E). Melflufen is also more active in stopping metastasis of myeloma than bortezomib in this model.

Presence of highly clonogenic cells in MM has suggested that MM is organized in hierarchical manner that parallels normal tissue development similar to AML and brain tumors in which cancer stem cells phenotypically resembling their normal counterparts give rise to differentiated progeny. The ability of anti-myeloma drugs such as steroids, proteasome inhibitors, and immunomodulatory agents to induce initial clinical responses in MM patients seen as decreased bone marrow plasmacytosis and monoclonal immunoglobulin levels reflects the activity of these agents against mature malignant plasma cells. However, the inability of these drugs to produce sustained remissions suggests that clonogenic cells responsible for tumor regrowth are insensitive to these agents. Our data support these observations as bortezomib, lenalidomide, and pomalidomide only mildly affect clonal outgrowth of RRMM samples. The only drugs in our assay which have shown 100% suppression of RRMM clonal outgrowth are melphalan and melflufen. Noteworthy, melflufen is at least 100-fold more active than melphalan in suppression of RRMM suggesting a strong anti-neoplastic effect against relapse-seeding myeloma stem cells. BTZ-resistant MM cells have also shown a higher susceptibility to melflufen in vitro and in vivo. Elevated potency of melflufen in BTZ-resistant MM models might be explained by the over expression of aminopeptidase B encoded by RNPEP gene upon adaptation of MM cells to BTZ. Noteworthy, RNPEP mRNA is up-regulated in the relapsed samples of MM patients that underwent a BTZ-based regimen as well as in early plasma cell progenitors. Thus, melflufen-mediated eradication of BTZ-resistant MM cells including MM stem cells may represent a promising therapeutic option for patients with RRMM.