Anti-Myeloma Activity of Enzymatically Activated Melphalan Prodrug J1
Authors: Chauhan Dharminder , Bandi Madhavi , Singh Ajita V , Podar Klaus , Richardson Paul G , Munshi Nikhil C. , Viktorsson Kristina , Lewensohn Rolf , Spira Jack , Anderson Kenneth C. 
Published: October 2019

Abstract
Abstract 1838

Background and Rationale:
The alkylating drug melphalan is routinely used in clinical protocols for the treatment of multiple myeloma (MM). Importantly, clinical trials in MM have effectively utilized combination of melphalan with proteasome inhibitor bortezomib and prednisolone (VMP regimen) to reduce toxicity, overcome drug resistance and enhance cytotoxicity. These findings highlight the utility of conventional alkylating agent, and importantly, provide impetus to develop conventional agents based prodrugs with a potent cytotoxic activity. In this context, pharmacological screening of alkylating oligopeptides led to the identification of a novel melphalan-containing prodrug J1 (L-melphalanyl-p-L-fluoro phenylalanine ethyl ester) - a new molecular entity with a more distinct activity profile than melphalan (Gullbo J, et al., Anticancer Drugs 2003,14:617–24; Gullbo J, et al., Invest New Drugs 2004, 22:411–20; Wickstrom M, et al., Mol Cancer Ther 2007, 6:2409–17). J1 is rapidly incorporated into the tumor cells cytoplasm, followed by intracellular hydrolysis in part mediated by aminopeptidase N (APN), resulting in a 10-fold greater release of free intracellular melphalan than exposure to melphalan at the same molar concentration (Gullbo J, et al., J Drug Target 2003,11:355–63; Wickstrom et al., Biochem Pharmacol 2010, 79(9):1281-90). In vitro studies showed a greater cytotoxic potency of J1 versus melphalan against different human solid cancers; however, its effect in MM is undefined. In the present study, we examined the anti-tumor activity of J1 in MM cells using both in vitro and in vivo model systems.

Methods and Models:
We utilized MM.1S, MM.1R, RPMI-8226, melphalan-resistant derivative of RPMI-8226 (LR-5), KMS12BM, and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability-, proliferation-, and apoptosis assays were performed using Trypan blue, MTT, thymidine incorporation, and Annexin V/Propidium iodide staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymologic assays. Statistical significance of data was determined using Student t test.

Results:
As aminopeptidase N (APN) has been shown to play a key role in conversion of J1 into melphalan in solid tumors, we first examined both expression and enzymatic activity of APN in MM cells. Immunoblot analysis showed a high expression of APN in various MM cell lines. Similarly, colorimetric analysis of APN enzymatic activity using the APN substrate L-alanine-4-nitro-anilide demonstrated elevated APN activity in MM cells. Moreover, pre-treatment of MM cells with APN inhibitor Bestatin showed a moderate, but significant blockade of J1-induced cytotoxicity in MM cells (P < 0.05; n=3). We next examined the effects of J1 in MM cells. Treatment of MM cell lines and primary patient cells for 24h significantly decreased their viability (IC50 range 0.5 – 1.0 uM; P < 0.001; n=3) without markedly affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index forJ1. Of note, the IC50 range of melphalan for MM cell lines is 10–40 uM. J1-triggered apoptosis was confirmed in MM.1R and RPMI-8226 cells, evidenced by marked increase in Annexin V+ and PI-cell population (P < 0.001, n=3). Importantly, J1induced apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies showed that J1-triggered apoptosis in MM cells is associated with 1) activation of caspase-7, caspase-8, caspase-9, caspase-3, and PARP; 2) induction of phospho-c-Jun and phospho-JNK, p53, and p21; 3) release of mitochondrial apoptogenic protein cytochrome-c; 4) inhibition of VEGF-induced migration of MM cells and angiogenesis; and 5) induction of DNA damage response, evidenced by increase in phospho-histone H2AX. Pre-treatment of MM cells with pan-caspase inhibitor Z-VAD-fmk attenuated J1-triggered MM cell apoptosis (P value < 0.001; n=3). Finally, treatment of tumor-bearing mice with J1 (3 mg/kg, twice a week for 2 weeks), but not vehicle alone, significantly (P < 0.008) inhibits MM tumor growth in these mice.

Conclusions:
Our study provides the rationale for clinical protocols evaluating J1, either alone or in combination, to improve patient outcome in MM.

Disclosures:
Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Spira:Oncopeptide AB: Employment, Equity Ownership. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.