Abstract 5205: Melflufen, a peptide-conjugated alkylator, shows efficacy in breast cancer cell lines
Authors: Schepsky Alexander , Traustadottir Gunnhildur A. , Joelsson Jon Petur , Ingthorsson Saevar , Kricker Jennifer , Bergthyorsson Jon T. , Petursdottir Katrina B. , Gudjonsson Thorkell , Slipicevic Ana , Lehmann Fredrik , Gudjonsson Thorarinn 
Published: September 2020

Abstract
Introduction: Melflufen is a lipophilic peptide-conjugated alkylator currently in phase 3 trial for the treatment of late stage multiple myeloma. Due to its lipophilic nature it readily enters cells where it is cleaved by peptidases leading to entrapment and enrichment of hydrophilic alkylator payloads. Since peptidases are often upregulated in many cancer types, melflufen represents an attractive candidate for cancer therapy. Here, we have analysed the efficacy of the known alkylator melphalan and melflufen in isogenic normal and cancerous breast epithelial cell lines D492 and D492HER2 as well as the triple-negative MDA-MB-231 cell line using both 2D and 3D cell cultures and in vivo studies.
Methods and results: Cell viability and cell proliferation studies unveiled that all cell lines were affected by melphalan and melflufen in both conditions but to a distinct extent. Melphalan treated cells needed quite high drug dosages to see an effect. In contrast, when treated with melflufen both cancerogenic D492HER2 and MDA-MB-231 lines were more sensitive than the normal-derived D492 line. Immunofluorescence staining showed that the sensitivity was lower in 3D culture compared to 2D culture. Western blot analysis and immunostaining revealed that treatment with melflufen induced profound DNA damage. Through comparison with the commonly used anti-cancer drug doxorubicin in cell viability studies we were able to show that melflufen was significantly more effective in reducing cellular growth. Chicken chorioallantoic membrane (CAM) assays showed comparable effects in vivo. The effect of melflufen in D492HER2 was attenuated if cells were pre-treated with the aminopeptidase inhibitor bestatin confirming the role of aminopeptidases in mediating the effect of melflufen in breast cancer cells. The aminopeptidase CD13 has been shown to facilitate melflufen cleavage. By separating both D492 and D492HER2 cells into CD13high and CD13low subpopulations and subsequently treating them with melflufen, we demonstrated increased melflufen sensitivity in the CD13high compared to CD13low population in D492HER2. Furthermore, siRNA knockdown of CD13 reduced the efficacy of melflufen corroborating its role in melflufen hydrolysis. Interestingly, knockdown of two other aminopeptidases, LAP3 and DPP7, led to similar efficacy reduction, suggesting that other aminopeptidases can facilitate melflufen to melphalan conversion.
Conclusion: In this study, we have shown that melflufen is a highly efficient and selective anti neoplastic agent in breast cancer cell lines in-vitro and in-vivo and its efficacy is facilitated by expression of aminopeptidases including CD13, LAP3 and DPP7 indicating that melflufen could be a potent drug in breast cancer cells expressing high level of aminopeptidases.
Citation Format: Alexander Schepsky, Gunnhildur A. Traustadottir, Jon Petur Joelsson, Saevar Ingthorsson, Jennifer Kricker, Jon T. Bergthyorsson, Katrina B. Petursdottir, Thorkell Gudjonsson, Ana Slipicevic, Fredrik Lehmann, Thorarinn Gudjonsson. Melflufen, a peptide-conjugated alkylator, shows efficacy in breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5205.