MLN4924, a Novel Investigational NEDD8 Activating Enzyme Inhibitor, Exhibits Preclinical Activity In Multiple Myeloma and Waldenström's Macroglobulinemia through Mechanism Distinct From Existing Proteasome Inhibitors

Multiple myeloma (MM) and Waldenström Macroglobulinemia (WM) have both shown clinical responses to Bortezomib therapy which blocks the elimination of ubiquitin tagged regulatory proteins by the proteasome. The NEDD8 activating enzyme (NAE)-inhibitor MLN4924 is a novel agent which demonstrates selective inhibition of the proteins for degradation in the ubiquitin pathway and may offer benefits to MM and WM patients through the more targeted approach.

A panel of human MM and WM cell lines were tested for their in vitro response to MLN4924 using MTT colorimetric survival assays. MM and WM cell lines tested exhibited dose and time dependent decrease of their viability upon exposure to MLN4924 (IC₅₀=25-150 nM). In addition, miRNA and gene expression studies in response to MLN4924 were compared to treatment of the same cells with bortezomib. In vivo safety studies were performed in mice and animal efficacy studies are ongoing in both MM and WM engrafted mice.

A panel of MM and WM cells were treated with MLN4924 for 72hrs and compared to the colon carcinoma line HCT116 and normal cell lines HS-5 (stroma) and THLE-3 (hepatocytes). In addition, a longitudinal assessment of viability of MM1S (MM) and BCWM1 (WM) cells during a 72hr incubation with MLN4924 (500nM) showed commitment to death <48hrs. This result, coupled with the observation that normal donor peripheral blood mononuclear cells (PBMCs) and HS-5 stromal cells were less sensitive (IC₅₀ >1000 nM) than the MM or WM cell lines tested, suggest that this compound exhibits a rapid, tumor-selective effect at clinically relevant conditions. We also evaluated primary MM (CD138+) and WM (CD19+) patient bone marrow cells and observed sub-μ M activity by MLN4924. In addition, we tested a series of combinations of MLN4924 with dexamethasone, doxorubicin and bortezomib in both MM1S and BCWM1 cells lines and observed additive activity or greater with MLN4924. Gene expression profiling revealed distinct signatures, in MM1S and BCWM1 lines, as well as distinct patterns of gene expression changes which were induced by MLN4924 vs. bortezomib. For instance, while bortezomib potently induces a compensatory upregulation of transcripts for ubiquitin/proteasome and heat shock protein genes which, in MM1S or BCWM1 cells, were not observed in response to MLN4924 treatment. Additional studies with the proteasome inhibitor MLN9708 revealed similar patterns of expression as bortezomib. These results indicate that MLN4924 does not induce pronounced proteotoxic stress in MM or WM cells, highlighting the distinct effect of MLN4924 on the ubiquitin/proteasome pathway compared to inhibitors which target the 20S proteasome subunit. Longitudinal miRNA profiling revealed a distinct pattern of miRNA expression in MLN4924-treated vs. bortezomib-treated MM and WM cells. Lastly, animal safety studies showed that MLN4924 was tolerated at doses up to 60mg/kg 2x daily for 1 week. Efficacy studies in MM and WM are ongoing.

MLN4924 induces cell killing at sub-μ M concentrations for both MM and WM cells with higher sensitivity of tumor cells compared to normal tissues, exhibits selective gene expression and miRNA regulation and can be safely administered to mice. These studies provide the framework for the clinical investigation of MLN4924 in MM and WM.

McMillin:Axios Biosciences: Equity Ownership. Smith:Millennium: Employment. Birner:Millennium: Employment. 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. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.