Bret Bannerman

Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, several groups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications.

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

Purpose: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Experimental Design: Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease. Results: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Conclusions: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. Clin Cancer Res; 17(23); 7313–23. ©2011 AACR.

Abstract C99: Beyond bortezomib: Development of Millennium's next-generation proteasome inhibitors.

Bortezomib (Velcade®) is a proteasome inhibitor that has been approved by the U.S. Food and Drug Administration for the treatment of patients with multiple myeloma or relapsed mantle cell lymphoma. It is currently available in more than 90 countries worldwide. The molecule is an N-capped dipeptidyl boronic acid and its molecular mechanism involves slow-tight binding to the chymotrypsin-like (β5) sites of the 26S proteasome. In the development of Millennium9s next generation inhibitors, we have investigated several parameters affecting the drug9s activity in biological systems. These include tissue proteasome concentration, blood/plasma partitioning and the kinetics of proteasome inhibition in cultured cells. We find that the abundance of the proteasome (approx. 1–5 μM β5 active site concentration) in cells and tissues together with the slow rate of dissociation of bortezomib from the proteasome (110 min. half-life) led to partitioning of the inhibitor in red blood cells, thereby limiting its distribution to potential sites of therapeutic action. Based on the hypothesis that more rapid dissociation from the proteasome should improve tissue distribution, we have developed an investigational, dipeptidyl boronic acid proteasome inhibitor (MLN9708). MLN9708 displays comparable potency (i.e. similar Ki) to bortezomib for the β5 site of the proteasome but a shorter (18 min.) half-life of dissociation. This affects rapid recovery of proteasome activity in tissue culture cells upon washout of the drug as well as reduced blood/plasma partitioning in mice, supporting the hypothesis that a more rapid equilibrium proteasome inhibitor can improve tissue distribution. MLN9708 is currently in phase 1 clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C99.

Abstract 2828: Antitumor activity of the investigational proteasome inhibitor MLN9708 in preclinical models of NSCLC

The successful development of VELCADE® (bortezomib) for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for hematological malignancies. The investigational drug MLN9708 is a modified dipeptidyl boronic acid and a potent, reversible and specific inhibitor of the proteasome. MLN9708 is currently being evaluated in Phase I trials for hematologic malignancies as well as solid tumors. In preclinical models, MLN9708 showed better antitumor activity and PD response compared to bortezomib.Upon exposure to aqueous solutions or plasma, MLN9708 immediately hydrolyzes to MLN2238, the biologically active form. MLN2238 was used for all preclinical studies described in this report. We evaluated the antitumor activity of MLN2238 in several non small cell lung cancer (NSCLC) xenografts. More than 70% tumor growth inhibition was observed in the HCC-827 xenograft model with 13 mg/kg, IV, twice weekly (BIW) dosing. In the H1650 NSCLC xenograft model, 40-50% tumor growth inhibition was found at 8 and 11 mg/kg, IV, BIW dosing. However, a synergistic effect on H1650 tumor growth inhibition was found when MLN2238 was combined with chemotherapeutic agents including doxorubicin 8 mg/kg IV, weekly (QW) dosing (90% tumor growth inhibition) and taxotere 5 and 10 mg/kg, IV, QW (80-100% tumor growth inhibition). Furthermore, the combination of MLN2238 (8 and 11 mg/kg, IV, BIW) and taxotere (10 mg/kg, IV, QW) resulted in tumor regression in the H1650 xenograft model, whereas no regressions were observed with either drug as a single agent. Similar single agent activity and combination effect with taxotere was observed We also evaluated single agent activity and combination with taxotere in an early-passage primary human tumor xenograft model, PHTX-132Lu. In an effort to understand the factors that may determinecontributing sensitivity vs resistance to proteasome inhibitors, we tested the ability of MLN2238 to inhibit anchorage independent growth and colony formation of NSCLC tumor cells in semi-solid medium. For these studies, tumor cells are derived from primary human tumor explants which have been maintained by serial passage in immunocompromised mice. Initial experiments show a range of sensitivity to MLN2238 among the primary tumors, Comparisons of genomic data, including gene expression profiles, copy number variation, and sequencing of commonly mutated genes, may illuminate factors contributing to sensitivity and resistance. In addition, in vivo studies will be performed on selected models to examine the value of this in vitro colony formation assay in predicting in vivo sensitivity. Data suggest that MLN2238 has antitumor activity alone and in combination with cytotoxic agents in preclinical models of NSCLC, and these results support the ongoing Phase I clinical investigation of MLN9708 in solid tumor types including NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2828. doi:10.1158/1538-7445.AM2011-2828

Abstract 397: The antitumor activity of the investigational drug MLN9708 in genetically engineered mouse models of plasma cell malignancy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: The proteasome inhibitor VELCADE® (bortezomib) is an important component of the chemotherapeutic strategy in treating multiple myeloma, a plasma cell malignancy (PCM). While genetically engineered mouse models (GEMMs) of cancer often accurately recapitulate their human disease counterparts, their usage in drug discovery settings has been very limited. Here we describe the antitumor activity of a second generation proteasome inhibitor, the investigational drug MLN9708, in the iMycCα/Bcl-XL GEMMs of PCM, in which neoplastic plasma cell development is driven by enforced expression of the Myc and Bcl-XL transgenes. Materials: MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma; MLN2238 was used for all studies below. We previously demonstrated that double transgenic iMycCα/Bcl-XL (C57BL6/FVB) mice develop de novo PCM with short onset (135 days) and full penetrance (100%). We derived a plasma cell tumor (PCT) cell line, DP54, from the bone marrow of a syngeneic mouse previously inoculated with an iMycCα/Bcl-XL tumor. DP54 PCT cells were stably transfected with the firefly luciferase gene, clonally isolated, and designated as DP54-Luc cells. Methods: Nine-week-old iMycCα/Bcl-XL (C57BL6/FVB) mice were untreated or treated with bortezomib (1.2 mg/kg intravenously [IV] twice weekly [BIW]) or MLN2238 (18 mg/kg IV BIW) for 6 consecutive weeks and monitored for tumor-free survival for an additional 25 weeks. To establish disseminated and intraosseous mouse models of iMycCα/Bcl-XL PCM, freshly dissociated DP54-Luc cells were aseptically injected into the lateral tail veins and the bone marrow space of the upper right tibia, respectively, of immunocompromised mice. Once tumor growth was established, mice were randomized and treated with vehicle (5%HPbCD), bortezomib (0.8 mg/kg IV BIW) or MLN2238 (13 mg/kg IV BIW) for 3-4 consecutive weeks. The doses used represent the maximum tolerated dose for each drug in each mouse strain. Results: In the iMycCα/Bcl-XL GEMM of de novo PCM, treatment with bortezomib or MLN2238 significantly prolonged tumor-free survival (+27 and +36 days, respectively; p<0.0001) and decreased plasma immunoglobulin levels compared to untreated controls. In the models of iMycCα/Bcl-XL PCM, treatment with bortezomib and MLN2238 significantly reduced disease burden as measured by IVIS® bioluminescent imaging. Conclusion: GEMMs of cancer have often been viewed as promising alternatives to traditional subcutaneous xenograft models, yet data to support their wider use in drug discovery settings are sparse. Here we demonstrated that carefully implemented GEMM studies can be integrated as an important part of the drug-discovery paradigm. MLN9708 is currently in clinical development for both hematologic and solid tumor indications. Safety and efficacy have not been established. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 397.