Evaluating the Antitumor Activity of MLN9708 in a Disseminated Mouse Model of Double Transgenic iMyc ^Ca/Bcl-X_L Plasma Cell Malignancy.

The first generation proteasome inhibitor VELCADE^® (bortezomib) is indicated for the treatment of patients with multiple myeloma (MM), a form of plasma cell malignancy (PCM). MLN9708 is our novel proteasome inhibitor that selectively and reversibly binds to, and potently inhibits the b5 site of the 20s proteasome in preclinical studies. We have recently demonstrated that MLN9708 significantly prolongs tumor-free survival of double transgenic iMyc^Ca/Bcl-X_L mice, a genetically-engineered mouse model of de novo PCM. Here we describe the in vivo evaluation of cell lines derived from double transgenic iMyc^Ca/Bcl-X_L mice and the antitumor activity of MLN9708 in a disseminated mouse model of iMyc^Ca/Bcl-X_L PCM.

MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Double transgenic iMyc^Ca/Bcl-X_L mice develop de novo PCM, in which neoplastic plasma cell development is driven by the targeted expression of the oncoprotein Myc and anti-apoptotic Bcl-X_L (J. Clin. Invest. 113:1763-1773, 2004). DP54 and DP42 are plasma cell tumor cell lines isolated from the bone marrow and lymph nodes, respectively, of syngeneic mice previously inoculated with iMyc^Ca/Bcl-X_L tumors (Cancer Res. 67:4069-4078, 2007). In vitro, DP54 and DP42 cells express both the Myc and Bcl-X_L transgenes, various plasma cell and B-cell markers including CD38, CD138 and B220, and have gene expression profiles very similar to human MM.

Cell viability studies were performed to determine the antiproliferative effects of MLN2238 in DP54 and DP42 cells in vitro. To evaluate DP54 and DP42 cells in vivo, these cells were aseptically inoculated into the tail vein of NOD-SCID mice. Progressions of the resultant PCM were monitored and tumor burdens were evaluated by magnetic resonance imaging (MRI), ex vivo mCT imaging, and histopathology. Mouse plasma samples were collected at the end of the studies and levels of immunoglobulin were assessed. To establish a preclinical disseminated mouse model of iMyc^Ca/Bcl-X_L PCM, freshly dissociated DP54-Luc cells (constitutively expressing firefly luciferase under a mouse Ig-k promoter) were aseptically inoculated into the tail vein of NOD-SCID mice. Once tumor growth has been established, mice were randomized into treatment groups and then treated with vehicle, bortezomib (at 0.7mg/kg intravenously [IV] twice weekly [BIW]) or MLN2238 (at 11 mg/kg IV BIW) for 3 consecutive weeks. Tumor burden was measured by bioluminescent imaging.

In vitro, both DP54 and DP42 cells were sensitive to MLN2238 treatment (LD₅₀ values of 14 and 25 nM, respectively). In vivo, NOD-SCID mice rapidly succumbed to PCM after being inoculated with DP54 and DP42 cells (25 and 14 days post-inoculation, respectively), where the disease was accompanied by marked elevation of plasma immunoglobulins. MRI scans revealed the presence of multiple lesions and several abnormalities were found including: cranial deformation, bowel distortion, splenomegaly and renal edema. Tumor infiltrates, ranging from minor to extensive, were identified in multiple organ compartments (brain<kidney<liver<lymph nodes<spleen<bone marrow) by histopathological analysis. Ex vivo mCT imaging has also revealed signs of bone erosion in the cranial sagittal sutures. Dissemination of DP54-Luc cells after tail vein inoculations was detected by in vivo bioluminescent and confirmed by ex vivo imaging where luminescent tumor nodules were identified in the spleen, kidneys, liver, intestine, lymph nodes, spinal bone and cranium. To assess the antitumor activity of MLN2238, an efficacy study was performed using the DP54-Luc disseminated model. Tumor burden (bioluminescence), skeletal malformation (mCT) and overall survival after treatment with bortezomib and MLN2238 will be presented.

The DP54-Luc disseminated mouse model of double transgenic iMyc^Ca/Bcl-X_L PCM recapitulated several key features of human MM and provided real-time assessment of novel MM therapy preclinically. MLN9708 is currently in human clinical development for both hematological and solid tumor indications.

Cao:Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Li:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Silverman:Milllennium: Employment. Janz:Milllennium: Research Funding. Van Ness:Milllennium: Research Funding. Kupperman:Milllennium: Employment. Manfredi:Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership.