A Novel Alkylating Agent Melphalan Flufenamide Ethyl Ester Induces an Irreversible DNA Damage in Multiple Myeloma Cells

Background and Rationale The alkylating agent melphalan is actively used in multiple myeloma therapy; however, dose-limiting toxicities and development of resistance limits its use. Melphalan flufenamide ethyl ester (melflufen) is an enzyme-activated analogue of melphalan which allows for a more rapid and higher intracellular accumulation of melphalan in tumor cells than is achievable by direct exposure to equimolar doses of melphalan. Our earlier study showed that melflufen is a more potent anti-MM agent than melphalan, that can overcome conventional drug resistance and induce synergistic anti-MM activity in combination with bortezomib, lenalidomide, or dexamethasone (Chauhan et al, Clinical Cancer Res 2013, 19(11): 3019-3031). These preclinical studies provided the basis for an ongoing phase 1 clinical trial of melflufen in MM. Here we examined whether the potent anti-MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signaling pathways.

Material and Methods We utilized melphalan-sensitive (MM.1S, RPMI-8226) and melphalan-resistant (LR-5) human MM cell lines. Immunoblot analysis was performed using antibodies specific against γ-H2AX, ATR, CHK1, Ku80, or GAPDH. Cell viability, cell cycle analysis were performed using MTT and propidium iodide staining. Cell viability, cell cycle and immunoblot studies were performed using equimolar concentrations of melflufen to melphalan, as in our prior study (Chauhan et al, Clinical Cancer Res 2013, 19(11): 3019-3031). Statistical significance was determined from Student’s t test. Melflufen was obtained from Oncopeptides AB, and melphalan was purchased from Sigma Chemical Company.

Results Melflufen triggered cytotoxicity in MM cell lines including, melphalan-resistant LR-5 cells. An early event in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX (γ-H2AX) at the sites in proximity to DNA breaks. Melflufen-induced apoptosis was associated with dose- and time- dependent rapid phosphorylation of γ-H2AX. We next compared the kinetics of induction of γ-H2AX/ATR/CHK1/Ku80 in melflufen- versus melphalan-treated MM cells. Cells were treated with equimolar concentrations of melflufen or melphalan for 2h, 4h, 6h, and 24h; protein lysates were prepared and subjected to immunoblot analysis with antibodies specific against γ-H2AX, ATR, CHK1, Ku80, GAPDH. Melflufen, but not melphalan, triggered induction of γ-H2AX, ATR, and CHK1 as early as after 2h exposure in both melphalan-sensitive and –resistant cells. Melphalan triggered induction of γ-H2AX in MM.1S and RPMI-8826 cells at 6h and 24h, respectively; and as expected, no induction of γ-H2AX was observed in LR-5 melphalan-resistant cells even after 24h treatment. Similar kinetics were observed for ATR and CHK1. We next examined the induction of DNA repair pathway in response to melflufen and melphalan using Ku80 as a marker protein. Melphalan, but not melflufen, triggered induction of Ku80. These data suggest that 1) melflufen triggers an early, and rapid DNA damage versus melphalan; 2) while melphalan induces DNA repair, no mechanism of DNA repair process is activated in response to melflufen treatment. Reports that the mechanisms for melphalan-resistance includes activation of DNA repair pathways, coupled with our findings showing lack of DNA repair pathway in melflufen-treated cells, suggest that melflufen overcomes melphalan-resistance, at least in part, by triggering an irreversible DNA damage. To further validate these data, we performed drug washout experiments and analysis of cell viability. Cells were treated with equimolar doses of melflufen or melphalan for 1h; cells were then washed with plain medium to remove drugs, cultured in fresh medium for additional 48h, and then analyzed for viability. One hour treatment of cells with melflufen, but not melphalan, triggers cytotoxicity in MM cells. Similar findings were observed using cell cycle analysis: % Sub G0/G1 after melflufen treatment was 81.1% in MM.1S; 80% in RPMI-8226; and 77% in LR-5 cells. Of note, melphalan-treated cells triggered only minimal (10%) accumulation of cells in Sub-G0/G1 phase.

Conclusion Our data therefore suggest that melflufen triggers a rapid, robust and an irreversible DNA damage, which may account for its ability to overcome melphalan-resistance in MM cells.