An Investigational Novel Orally Bioavailable Proteasome Inhibitor MLN9708/MLN2238 Triggers Cytotoxicity In Multiple Myeloma Cells Via p21- and Caspase-8-Dependent Signaling Pathway

Therapeutic targeting of Ubiquitin-Proteasome Signaling (UPS) is exemplified by the FDA approval of dipeptidyl boronic acid bortezomib first-in-class proteasome inhibitor for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM). As with other agents, however, dose-limiting toxicities and the development of resistance limit its long-term utility. MLN9708 (Millennium Pharmaceuticals, Inc., Cambridge, MA) is a selective, orally bioavailable proteasome inhibitor currently in phase I clinical development. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form. Similar to bortezomib, MLN2238 is a boronic acid analog; however, it is distinct from bortezomib since preclinical data demonstrates it has a shorter proteasome dissociation half-life, as well as improved pharmacokinetics, pharmacodynamics, and antitumor activity in xenograft models versus bortezomib. In the present study, we examined the anti-tumor activity of MLN2238 in MM cells using in vitro model systems.

We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12BM, OPM2, H929, LP1, 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, CellTiter-Glo, thymidine incorporation, and Annexin V staining. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Statistical significance of data was determined using a Student t test.

We first confirmed the functional specificity of MLN2238 using different experimental strategies: 1) Examination of the proteasome activity using human erythrocyte 20S proteasomes and fluorogenic substrates showed that MLN2238, like bortezomib, primarily inhibits chymotrypsin-like proteasome activity (EC50 = 9 ± 2.3 nM versus bortezomib: 5.5 ± 1.5 nM); 2) Treatment of MM.1S MM cells with MLN2238 induces a time- and dose-dependent accumulation of ubiquitinated proteins; and 3) Washout experiments showed that MLN2238 is a reversible proteasome inhibitor (P < 0.001; n=3). We next examined the effects of MLN2238 in MM cells. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, U266, OPM2, H929, LP1, KMS-12BM, and INA-6) and primary patient cells for 48h significantly decreased their viability (IC50 range 12.5 – 25 nM) (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 for MLN2238. MLN2238-triggered apoptosis was confirmed in MM.1S and H929 cells, evidenced by a marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, MLN2238 induced apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies showed that MLN2238-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of p53, Noxa, PUMA, Bid, E2F, and pAKT; 3) downregulation of MM cell growth and survival proteins: NF-kB, Bcl2, phospho-Rb protein, and cyclin-A; 4) inhibition of migration of MM cells and angiogenesis; and 5) increased expression of CDK inhibitor p21. Importantly, blockade of p21 using both siRNA strategy and p21- knockout HCT116 cells showed significant abrogation of MLN2238-induced cell death (P value < 0.001; n=3). We next examined the requirement for caspase-8 versus caspase-9 during MLN2238-induced apoptosis. Incubation of MM.1S cells with pan-caspase inhibitor (Z-VAD-FMK) markedly abrogates MLN2238-induced apoptosis. Inhibition of caspase-8 (IETD-FMK) led to a significant decrease in MLN2238-triggered cell death, whereas inhibition of caspase-9 (LEHD-FMK) only moderately blocked MLN2238-triggered decreased viability in MM.1S cells (P < 0.001, n=3). These data suggest that MLN2238-induced apoptosis in MM cells is predominantly mediated by caspase-8 extrinsic apoptotic pathway. Finally, combination of MLN2238 with lenalidomide or dexamethasone triggered synergistic anti-MM activity.

Our preclinical study supports further clinical evaluation of MLN9708, either alone or in combination, as a potential MM therapy.

Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. 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.