Abstract B40: Development of a pharmacodynamic assay for the second‐generation proteasome inhibitor MLN9708 in clinical trials of multiple myeloma

The ubiquitin‐proteasome system processes the majority of cellular proteins and is the principal pathway by which cells regulate protein homeostasis. The successful development of VELCADE® (bortezomib) for Injection for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for the treatment of malignancies. MLN9708 is a second‐generation reversible proteasome inhibitor developed to achieve greater oral bioavailability, improved pharmacokinetics and greater antitumor activity than bortezomib. MLN9708 is currently in human clinical development for both hematological and non‐hematological malignancies. MLN9708 immediately hydrolyzes to the biologically active form MLN2238 upon exposure to aqueous solutions or plasma, and MLN2238 was used for all preclinical studies described below. In vitro, MLN2238 inhibited 20S proteasome activity, preferentially binding the 20S 5 site with an IC 50 of 3.4 nM and demonstrated potent activity against cultured cancer cells in cell viability assays. In vivo, MLN2238 achieved exposures that resulted in significant blood and tumor proteasome inhibition in xenograft‐bearing mice and had increased plasma and tumor exposure compared to bortezomib when dosed at their respective maximum tolerated doses (MTD). MLN2238 also elicited a stronger pharmacodynamic (PD) response than bortezomib in xenograft tumors, as measured by tumor 20S 5 site‐specific activity and expression levels of GADD34 and ATF‐3, two genes involved in the unfolded protein response (UPR) pathway shown to be upregulated in response to proteasome inhibition. Here we describe the development of an ATF‐3 IHC assay suitable for use in bone marrow samples isolated from multiple myeloma patients enrolled in Phase I trials of MLN9708. Using both quantitativeWestern blotting and IHC assays, we demonstrated that treatment of cultured myeloma cell lines with MLN2238 results in a dose‐dependent increase in ATF‐3 levels. Antibody specificity was confirmed by IHC analysis of HCT‐116 cells following knockdown of ATF‐3 by siRNA. An ATF‐3 PD response is detectable by IHC in several xenograft models and in selected normal tissues from mice dosed with MLN2238. The elevation in ATF‐3 is delayed by several hours compared to 20S proteasome inhibition in cells and tissues, consistent with it being a downstream effect of proteasome inhibition. To test the use of the ATF‐3 IHC assay on clinical samples from multiple myeloma patients, we developed a dual staining assay with CD38, a marker expressed on multiple myeloma cells. We show baseline staining of ATF‐3 and CD38 in formalin‐fixed paraffin embedded bone marrow aspirates and biopsies from patients who have not received MLN9708. This assay has potential for use in evaluating the levels of CD38 and ATF‐3 in pre‐ and post‐treatment bone marrow samples from patients treated with MLN9708. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B40.