Pharmacokinetics and Metabolism of Melflufen, an Alkylating Peptide-Drug Conjugate, in Patients with Relapsed Refractory Multiple Myeloma.

Melphalan flufenamide (melflufen) is a novel lipophilic peptide-drug conjugate recently approved in the European Union and the United Kingdom for the treatment of relapsed refractory multiple myeloma. Melflufen rapidly crosses the cell membrane, and inside tumor cells, melflufen utilizes peptidases and esterases to release entrapped hydrophilic metabolites with alkylating activity. In vitro, in whole blood, melflufen was rapidly distributed into blood cells and quickly converted to its main metabolite melphalan, with maximum cellular concentrations of noncovalently bound melflufen and melphalan after 1 and 6 minutes, respectively. Melphalan outflow from blood cells was slow, with peak concentrations in plasma after 25 minutes. The pharmacokinetics of melflufen was best described by a 2-compartment model. Following a 30-minutes intravenous infusion of 40 mg in 27 patients with relapsed refactory multiple myeloma, mean half-life in the α phase of the curve was 1.24 minutes, half-life in the ß phase of the curve 26.7 minutes, and clearance 13.4 L/min. Desethyl-melflufen exposure was below 20% compared to melflufen. Based on population analysis (298 patients with relapsed refactory multiple myeloma), the melphalan pharmacokinetics were well characterized by a 3-compartment model with melflufen dosing into a peripheral compartment, assuming instantaneous distribution of melflufen into cells and subsequent rapid metabolism to melphalan. Mean clearance and central and deep peripheral volumes of distribution were 22.4 L/h, 2.70 L, and 51.3 L, respectively. Clearance increased and maximum concentration decreased with increasing body weight and estimated glomerular filtration rate. In conclusion, melflufen administration differs from melphalan administration by a more rapid distribution into cells, which, in conjunction with a rapid intracellular metabolism, allows for higher maximum concentrations of alkylating agents, and by a more extensive distribution of melphalan to peripheral tissues.

Melphalan flufenamide inhibits osteoclastogenesis by suppressing proliferation of monocytes.

Myeloma bone disease is a major complication in multiple myeloma affecting quality of life and survival. It is characterized by increased activity of osteoclasts, bone resorbing cells. Myeloma microenvironment promotes excessive osteoclastogenesis, a process of production of osteoclasts from their precursors, monocytes. The effects of two anti-myeloma drugs, melphalan flufenamide (melflufen) and melphalan, on the activity and proliferation of osteoclasts and their progenitors, monocytes, were assessed in this study. In line with previous research, differentiation of monocytes was associated with increased expression of genes encoding DNA damage repair proteins. Hence monocytes were more sensitive to DNA damage-causing alkylating agents than their differentiated progeny, osteoclasts. In addition, differentiated progeny of monocytes showed increased gene expression of immune checkpoint ligands which may potentially create an immunosuppressive microenvironment. Melflufen was ten-fold more active than melphalan in inhibiting proliferation of osteoclast progenitors. Furthermore, melflufen was also superior to melphalan in inhibition of osteoclastogenesis and bone resorption. These results demonstrate that melflufen may exert beneficial effects in patients with multiple myeloma such as reducing bone resorption and immunosuppressive milieu by inhibiting osteoclastogenesis.

Melflufen for relapsed and refractory multiple myeloma.

INTRODUCTION: The overall survival of patients with multiple myeloma has improved with the advent of novel agents; however, multiple myeloma remains incurable. Combinations of standard-of-care agents such as immunomodulators, proteasome inhibitors, and anti-CD38 monoclonal antibodies are increasingly used in earlier lines of therapy. Patients with disease that is refractory to multiple novel agents represent a population with high unmet medical need and for whom therapies with new mechanisms of action could be beneficial. Melphalan flufenamide (melflufen) has demonstrated encouraging activity in patients with relapsed and refractory multiple myeloma. AREAS COVERED: This review provides an overview of the mechanism of action of melflufen, a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly delivers alkylating agents into tumor cells. It reviews key Phase I and II clinical trial data for melflufen in combination with dexamethasone as well as in triplet combinations with daratumumab or bortezomib. The safety profile of melflufen, which is characterized primarily by clinically manageable hematologic adverse events, is described. EXPERT OPINION: Melflufen has potential to fill a gap in the myeloma treatment landscape by providing a new mechanism of action with clinically meaningful efficacy and a favorable safety profile in patients refractory to multiple novel agents.

Melflufen: A Peptide-Drug Conjugate for the Treatment of Multiple Myeloma.

Despite the availability of new therapies that have led to improved outcomes for patients with multiple myeloma, most patients will eventually relapse. With triplet and even quadruplet combination therapies becoming standard in the first and second line, many patients will have few treatment options after second-line treatment. Melflufen (melphalan flufenamide) is a first-in-class peptide-drug conjugate (PDC) that targets aminopeptidases and rapidly releases alkylating agents into tumor cells. Once inside the tumor cells, melflufen is hydrolyzed by peptidases to release alkylator molecules, which become entrapped. Melflufen showed anti-myeloma activity in myeloma cells that were resistant to bortezomib and the alkylator melphalan. In early phase studies (O-12-M1 and HORIZON [OP-106]), melflufen plus dexamethasone has demonstrated encouraging clinical activity and a manageable safety profile in heavily pretreated patients with relapsed/refractory multiple myeloma, including those with triple-class refractory disease and extramedullary disease. The Phase III OCEAN study (OP-104) is further evaluating melflufen plus dexamethasone in patients with relapsed/refractory multiple myeloma. The safety profile of melflufen is characterized primarily by clinically manageable hematologic adverse events. Melflufen, with its novel mechanism of action, has the potential to provide clinically meaningful benefits to patients with relapsed/refractory multiple myeloma, including those with high unmet needs.

Targeting aggressive osteosarcoma with a peptidase-enhanced cytotoxic melphalan flufenamide.

BACKGROUND: Low survival rates in metastatic high-grade osteosarcoma (HGOS) have remained stagnant for the last three decades. This study aims to investigate the role of aminopeptidase N (ANPEP) in HGOS progression and its targeting with a novel lipophilic peptidase-enhanced cytotoxic compound melphalan flufenamide (melflufen) in HGOS. METHODS: Meta-analysis of publicly available gene expression datasets was performed to determine the impact of ANPEP gene expression on metastasis-free survival of HGOS patients. The efficacy of standard-of-care anti-neoplastic drugs and a lipophilic peptidase-enhanced cytotoxic conjugate melflufen was investigated in patient-derived HGOS ex vivo models and cell lines. The kinetics of apoptosis and necrosis induced by melflufen and doxorubicin were compared. Anti-neoplastic effects of melflufen were investigated in vivo. RESULTS: Elevated ANPEP expression in diagnostic biopsies of HGOS patients was found to significantly reduce metastasis-free survival. In drug sensitivity assays, melflufen has shown an anti-proliferative effect in HGOS ex vivo samples and cell lines, including those resistant to methotrexate, etoposide, doxorubicin, and PARP inhibitors. Further, HGOS cells treated with melflufen displayed a rapid induction of apoptosis and this sensitivity correlated with high expression of ANPEP. In combination treatments, melflufen demonstrated synergy with doxorubicin in killing HGOS cells. Finally, Melflufen displayed anti-tumor growth and anti-metastatic effects in vivo. CONCLUSION: This study may pave the way for use of melflufen as an adjuvant to doxorubicin in improving the therapeutic efficacy for the treatment of metastatic HGOS.

Phase 1 study of the protein deubiquitinase inhibitor VLX1570 in patients with relapsed and/or refractory multiple myeloma.

This phase 1 study sought to characterize the safety, tolerability, and pharmacokinetic behavior of VLX1570, a small molecule inhibitor of the deubiquitinases (DUBs) that remove sterically bulky ubiquitin chains from proteins during processing in the19S regulatory subunit of the proteasome, in patients with relapsed and refractory multiple myeloma (MM). Fourteen patients were treated with escalating doses of VLX1570 ranging from 0.05 to 1.2 mg/kg as a brief intravenous (IV) infusion on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle. Due to its poor aqueous solubility, VLX1570 was formulated in polyethylene glycol, polyoxyethylated castor oil, and polysorbate 80 and administered as a brief intravenous (IV) infusion via a central venous catheter. Anti-myeloma effects were noted at doses at or above 0.6 mg/kg, however, two patients treated at the 1.2 mg/kg dose level experienced severe, abrupt, and progressive respiratory insufficiency, which was associated with diffuse pulmonary infiltrates on imaging studies, similar to those rarely noted with bortezomib and other inhibitors of the 20S proteasome, culminating in death. Although the contribution of VLX1570's formulation to the pulmonary toxicity could not be ruled out, the severity and precipitous nature of the toxicity and the steep relationship between dose and toxicity, the study was discontinued. Despite the severe pulmonary toxicity noted with VLX1570, efforts directed at identifying DUB inhibitors with greater therapeutic indices appear warranted based on the unique mechanism of action, robustness of preclinical antitumor activity, and activity of the DUB inhibitors in MM resistant to PIs targeting the 20S proteasome subunit.

Analysis of determinants for in vitro resistance to the small molecule deubiquitinase inhibitor b-AP15.

BACKGROUND: b-AP15/VLX1570 are small molecule inhibitors of the ubiquitin specific peptidase 14 (USP14) and ubiquitin carboxyl-terminal hydrolase 5 (UCHL5) deubiquitinases (DUBs) of the 19S proteasome. b-AP15/VLX1570 have been shown to be cytotoxic to cells resistant to bortezomib, raising the possibility that this class of drugs can be used as a second-line therapy for treatment-resistant multiple myeloma. Limited information is available with regard to potential resistance mechanisms to b-AP15/VLX1570. RESULTS: We found that b-AP15-induced cell death is cell-cycle dependent and that non-cycling tumor cells may evade b-AP15-induced cell death. Such non-cycling cells may re-enter the proliferative state to form colonies of drug-sensitive cells. Long-term selection of cells with b-AP15 resulted in limited drug resistance (~2-fold) that could be reversed by buthionine sulphoximine, implying altered glutathione (GSH) metabolism as a resistance mechanism. In contrast, drug uptake and overexpression of drug efflux transporters were found not to be associated with b-AP15 resistance. CONCLUSIONS: The proteasome DUB inhibitors b-AP15/VLX1570 are cell cycle-active. The slow and incomplete development of resistance towards these compounds is an attractive feature in view of future clinical use.

A novel tumor spheroid model identifies selective enhancement of radiation by an inhibitor of oxidative phosphorylation.

There is a need for preclinical models that can enable identification of novel radiosensitizing drugs in clinically relevant high-throughput experiments. We used a new high-throughput compatible total cell kill spheroid assay to study the interaction between drugs and radiation in order to identify compounds with radiosensitizing activity. Experimental drugs were compared to known radiosensitizers and cytotoxic drugs clinically used in combination with radiotherapy. VLX600, a novel iron-chelating inhibitor of oxidative phosphorylation, potentiated the effect of radiation in tumor spheroids in a synergistic manner. This effect was specific to spheroids and not observed in monolayer cell cultures. In conclusion, the total cell kill spheroid assay is a feasible high-throughput method in the search for novel radiosensitizers. VLX600 shows encouraging characteristics for development as a novel radiosensitizer.

Towards repositioning of quinacrine for treatment of acute myeloid leukemia - Promising synergies and in vivo effects.

We previously reported that the anti-malarial drug quinacrine has potential to be repositioned for treatment of acute myeloid leukemia (AML). As a next step towards clinical use, we assessed the efficacy of quinacrine in an AML-PS mouse model and investigated possible synergistic effects when combining quinacrine with nine other antileukemic compounds in two AML cell lines. Furthermore, we explored the in vivo activity of quinacrine in combination with the widely used AML agent cytarabine. The in vivo use of quinacrine (100mg/kg three times per week for two consecutive weeks) significantly suppressed circulating blast cells at days 30/31 and increased the median survival time (MST). The in vitro drug combination analysis yielded promising synergistic interactions when combining quinacrine with cytarabine, azacitidine and geldanamycin. Finally, combining quinacrine with cytarabine in vivo showed a significant decrease in circulating leukemic blast cells and increased MST compared to the effect of either drug used alone, thus supporting the findings from the in vitro combination experiments. Taken together, the repositioning potential of quinacrine for treatment of AML is reinforced by demonstrating significant in vivo activity and promising synergies when quinacrine is combined with different agents, including cytarabine, the hypomethylating agent azacitidine and HSP-90 inhibitor geldanamycin.

Synergistic effects of combining proteasome inhibitors with chemotherapeutic drugs in lung cancer cells.

BACKGROUND: The prognosis for patients with disseminated lung cancer is poor and current treatments have limited survival benefit as resistance often occurs, and is often associated with significant toxicity. A possible strategy to improve treatment and evade chemoresistance may be to find new combinations of drugs. The aim of this study was to analyze the potential of combining proteasome inhibitors (PIs) with chemotherapeutic drugs used in the routine treatment for lung cancer patients. RESULTS: The median-effect method was applied to the Fluorometric Microculture Cytotoxicity Assay (FMCA) to evaluate effects of combining two different PIs (bortezomib and b-AP15) with clinically used chemotherapeutic drugs representing different mechanisms of action (cisplatin, gefitinib, gemcitabine and vinorelbine) in two lung cancer cell lines (one sensitive and one resistant). Proteasome inhibition in combination with cisplatin, gemcitabine or vinorelbine had synergistic effects in at least one of the tested cell lines. Furthermore, the effect of gefitinib appeared strongly potentiated by the PI in the least resistant lung cancer cell line, although the level of synergy could not be determined with the median-effect method. CONCLUSIONS: Combining PIs with cisplatin, gefitinib, gemcitabine or vinorelbine show potential as new combination chemotherapy for the treatment of lung cancer.

Melflufen - a peptidase-potentiated alkylating agent in clinical trials.

Aminopeptidases like aminopeptidase N (APN, also known as CD13) play an important role not only in normal cellular functioning but also in the development of cancer, including processes like tumor cell invasion, differentiation, proliferation, apoptosis, motility, and angiogenesis. An increased expression of APN has been described in several types of human malignancies, especially those characterized by fast-growing and aggressive phenotypes, suggesting APN as a potential therapeutic target. Melphalan flufenamide ethyl ester (melflufen, previously denoted J1) is a peptidase-potentiated alkylating agent. Melflufen readily penetrates membranes and an equilibrium is rapidly achieved, followed by enzymatic cleavage in aminopeptidase positive cells, which results in trapping of less lipophilic metabolites. This targeting effect results in very high intracellular concentrations of its metabolite melphalan and subsequent apoptotic cell death. This results in a potency increase (melflufen vs melphalan) ranging from 10- to several 100-fold in different in vitro models. Melflufen triggers a rapid, robust, and an irreversible DNA damage which may account for its ability to overcome melphalan-resistance in multiple myeloma cells. Furthermore, anti-angiogenic properties of melflufen have been described. Consequently, it is hypothesized that melflufen could provide better efficacy but no more toxicity than what is achieved with melphalan, an assumption so far supported by experiences from hollow fiber and xenograft studies in rodents as well as by clinical data from patients with solid tumors and multiple myeloma. This review summarizes the current preclinical and clinical knowledge of melflufen.

Mechanistic characterization of a copper containing thiosemicarbazone with potent antitumor activity.

BACKGROUND: The thiosemicarbazone CD 02750 (VLX50) was recently reported as a hit compound in a phenotype-based drug screen in primary cultures of patient tumor cells. We synthesized a copper complex of VLX50, denoted VLX60, and characterized its antitumor and mechanistic properties. MATERIALS AND METHODS: The cytotoxic effects and mechanistic properties of VLX60 were investigated in monolayer cultures of multiple human cell lines, in tumor cells from patients, in a 3-D spheroid cell culture system and in vivo and were compared with those of VLX50. RESULTS: VLX60 showed ≥ 3-fold higher cytotoxic activity than VLX50 in 2-D cultures and, in contrast to VLX50, retained its activity in the presence of additional iron. VLX60 was effective against non-proliferative spheroids and against tumor xenografts in vivo in a murine model. In contrast to VLX50, gene expression analysis demonstrated that genes associated with oxidative stress were considerably enriched in cells exposed to VLX60 as was induction of reactive oxygen. VLX60 compromised the ubiquitin-proteasome system and was more active in BRAF mutated versus BRAF wild-type colon cancer cells. CONCLUSIONS: The cytotoxic effects of the copper thiosemicarbazone VLX60 differ from those of VLX50 and shows interesting features as a potential antitumor drug, notably against BRAF mutated colorectal cancer.

In vitro and in vivo anti-leukemic activity of the peptidase-potentiated alkylator melflufen in acute myeloid leukemia.

The novel aminopeptidase potentiated alkylating agent melflufen, was evaluated for activity in acute myeloid leukemia in a range of in vitro models, as well as in a patient derived xenograft study. All tested AML cell lines were highly sensitive to melflufen while melphalan was considerably less potent. In the HL-60 cell line model, synergy was observed for the combination of melflufen and cytarabine, an interaction that appeared sequence dependent with increased synergy when melflufen was added before cytarabine. Also, in primary cultures of AML cells from patients melflufen was highly active, while normal PBMC cultures appeared less sensitive, indicating a 7-fold in vitro therapeutic index. Melphalan, on the other hand, was only 2-fold more potent in the AML patient samples compared with PBMCs. Melflufen was equally active against non-malignant, immature CD34+ progenitor cells and a more differentiated CD34+ derived cell population (GM14), whereas the stem cell like cells were less sensitive to melphalan. Finally, melflufen treatment showed significant anti-leukemia activity and increased survival in a patient derived xenograft of AML in mice. In conclusion, melflufen demonstrates high and significant preclinical activity in AML and further clinical evaluation seem warranted in this disease.

Expression of possible targets for new proteasome inhibitors in diffuse large B-cell lymphoma.

OBJECTIVES: Investigating expression of possible targets for proteasome inhibitors in patients with diffuse large B-cell lymphoma (DLBCL) and correlating the findings to clinical parameters and outcome. METHODS: Tumour material from 92 patients with DLBCL treated with either R-CHOP like (n = 69) or CHOP like (n = 23) regimens were stained for possible targets of proteasome inhibitors. RESULTS: The primary target molecule of bortezomib, proteasome subunit beta, type 5 (PSMB5), was not detected in the tumour cells in any of the cases but showed an abundant expression in cells in the microenvironment. However, the deubiquitinases (DUBs) of the proteasome, the ubiquitin carboxyl-terminal hydrolase L5 (UCHL5) and the ubiquitin specific peptidase 14 (USP14), were detected in the cytoplasm of the tumour cells in 77% and 74% of the cases, respectively. The adhesion regulating molecule 1 (ADRM1) was detected in 98% of the cases. There was no correlation between the expression of any of the studied markers and clinical outcome or GC/non-GC phenotype. CONCLUSIONS: We suggest that UCHL5 and/or USP14 should be further evaluated as new targets for proteasome inhibitors in DLBCL. The lack of expression of PSMB5 on the tumour cells might provide an explanation of the relatively poor results of bortezomib in DLBCL.

Iron chelators target both proliferating and quiescent cancer cells.

Poorly vascularized areas of solid tumors contain quiescent cell populations that are resistant to cell cycle-active cancer drugs. The compound VLX600 was recently identified to target quiescent tumor cells and to inhibit mitochondrial respiration. We here performed gene expression analysis in order to characterize the cellular response to VLX600. The compound-specific signature of VLX600 revealed a striking similarity to signatures generated by compounds known to chelate iron. Validation experiments including addition of ferrous and ferric iron in excess, EXAFS measurements, and structure activity relationship analyses showed that VLX600 chelates iron and supported the hypothesis that the biological effects of this compound is due to iron chelation. Compounds that chelate iron possess anti-cancer activity, an effect largely attributed to inhibition of ribonucleotide reductase in proliferating cells. Here we show that iron chelators decrease mitochondrial energy production, an effect poorly tolerated by metabolically stressed tumor cells. These pleiotropic features make iron chelators an attractive option for the treatment of solid tumors containing heterogeneous populations of proliferating and quiescent cells.

Preclinical activity of melflufen (J1) in ovarian cancer.

Ovarian cancer carries a significant mortality. Since symptoms tend to be minimal, the disease is often diagnosed when peritoneal metastases are already present. The standard of care in advanced ovarian cancer consists of platinum-based chemotherapy combined with cytoreductive surgery. Unfortunately, even after optimal cytoreduction and adjuvant chemotherapy, most patients with stage III disease will develop a recurrence. Intraperitoneal administration of chemotherapy is an alternative treatment for patients with localized disease. The pharmacological and physiochemical properties of melflufen, a peptidase potentiated alkylator, raised the hypothesis that this drug could be useful in ovarian cancer and particularily against peritoneal carcinomatosis. In this study the preclinical effects of melflufen were investigated in different ovarian cancer models. Melflufen was active against ovarian cancer cell lines, primary cultures of patient-derived ovarian cancer cells, and inhibited the growth of subcutaneous A2780 ovarian cancer xenografts alone and when combined with gemcitabine or liposomal doxorubicin when administered intravenously. In addition, an intra- and subperitoneal xenograft model showed activity of intraperitoneal administered melflufen for peritoneal carcinomatosis, with minimal side effects and modest systemic exposure. In conclusion, results from this study support further investigations of melflufen for the treatment of peritoneal carcinomatosis from ovarian cancer, both for intravenous and intraperitoneal administration.

The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells.

Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity currently in clinical trials for relapsed multiple myeloma. Here we show that VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Exposure of multiple myeloma cells to VLX1570 resulted in thermostabilization of USP14 at therapeutically relevant concentrations. Transient knockdown of USP14 or UCHL5 expression by electroporation of siRNA reduced the viability of multiple myeloma cells. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response. Sensitivity to VLX1570 was moderately affected by altered drug uptake, but was unaffected by overexpression of BCL2-family proteins or inhibitors of caspase activity. Finally, treatment with VLX1570 was found to lead to extended survival in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.

In vitro and in vivo activity of melflufen (J1)in lymphoma.

BACKGROUND: Melphalan has been used in the treatment of various hematologic malignancies for almost 60 years. Today it is part of standard therapy for multiple myeloma and also as part of myeloablative regimens in association with autologous allogenic stem cell transplantation. Melflufen (melphalan flufenamide ethyl ester, previously called J1) is an optimized derivative of melphalan providing targeted delivery of active metabolites to cells expressing aminopeptidases. The activity of melflufen has compared favorably with that of melphalan in a series of in vitro and in vivo experiments performed preferentially on different solid tumor models and multiple myeloma. Melflufen is currently being evaluated in a clinical phase I/II trial in relapsed or relapsed and refractory multiple myeloma. METHODS: Cytotoxicity of melflufen was assayed in lymphoma cell lines and in primary tumor cells with the Fluorometric Microculture Cytotoxicity Assay and cell cycle analyses was performed in two of the cell lines. Melflufen was also investigated in a xenograft model with subcutaneous lymphoma cells inoculated in mice. RESULTS: Melflufen showed activity with cytotoxic IC50-values in the submicromolar range (0.011-0.92 µM) in the cell lines, corresponding to a mean of 49-fold superiority (p < 0.001) in potency vs. melphalan. In the primary cultures melflufen yielded slightly lower IC50-values (2.7 nM to 0.55 µM) and an increased ratio vs. melphalan (range 13-455, average 108, p < 0.001). Treated cell lines exhibited a clear accumulation in the G2/M-phase of the cell cycle. Melflufen also showed significant activity and no, or minimal side effects in the xenografted animals. CONCLUSION: This study confirms previous reports of a targeting related potency superiority of melflufen compared to that of melphalan. Melflufen was active in cell lines and primary cultures of lymphoma cells, as well as in a xenograft model in mice and appears to be a candidate for further evaluation in the treatment of this group of malignant diseases.

A novel alkylating agent Melflufen induces irreversible DNA damage and cytotoxicity in multiple myeloma cells.

Our prior study utilized both in vitro and in vivo multiple myeloma (MM) xenograft models to show that a novel alkylator melphalan-flufenamide (Melflufen) is a more potent anti-MM agent than melphalan and overcomes conventional drug resistance. Here we examined whether this potent anti-MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signalling pathways via γ-H2AX/ATR/CHK1/Ku80. Melflufen-induced apoptosis was associated with dose- and time-dependent rapid phosphorylation of γ-H2AX. Melflufen induces γ-H2AX, ATR, and CHK1 as early as after 2 h exposure in both melphalan-sensitive and -resistant cells. However, melphalan induces γ-H2AX in melphalan-sensitive cells at 6 h and 24 h; no γ-H2AX induction was observed in melphalan-resistant cells even after 24 h exposure. Similar kinetics was observed for ATR and CHK1 in meflufen- versus melphalan-treated cells. DNA repair is linked to melphalan-resistance; and importantly, we found that melphalan, but not melflufen, upregulates Ku80 that repairs DNA double-strand breaks. Washout experiments showed that a brief (2 h) exposure of MM cells to melflufen is sufficient to initiate an irreversible DNA damage and cytotoxicity. 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.