Abstract B34: Clinical pharmacodynamic assay development for the first small molecule inhibitor of Nedd8-activating enzyme (NAE), MLN4924

Abstract

Abstract NEDD8-Activating Enzyme (NAE) initiates the conjugation of the ubiquitin-like protein NEDD8 to its cellular targets, members of the cullin protein family. NEDD8 conjugation to cullins is known to be essential for the ubiquitination activity of cullin-RING ubiquitin ligases (CRLs). CRLs control the timely ubiquitination and subsequent degradation of many proteins with important roles in cell cycle progression and signal transduction. MLN4924 is a first in class, small molecule inhibitor of NAE. Inhibition of NAE with MLN4924 disrupts the conjugation of NEDD8 to CRLs. This subsequently prevents ubiquitination and proteasomal degradation of CDL substrates involved in cell cycle regulation (p27), signal transduction (pIkBa), DNA replication (Cdt-1), stress response (Nrf-2), and other processes crucial to tumor cell growth and survival. The regulation of these markers of NAE inhibition is well characterized across of a variety of cancer cell types grown in culture and as xenografts implanted in immunocompromised mice. Here we describe the development of clinical pharmacodynamic (PD) assays to evaluate two markers of NAE inhibition in the blood compartment, measurement of neddylated cullins and pIkBa levels in peripheral blood mononuclear cells (PBMCs). In vivo administration of a single dose of MLN4924 to nude mice harboring subcutaneous HCT-116 colon carcinoma xenografts results in a dose-dependent decrease in neddylated cullin (CUL-N8) levels and elevation of the CRL substrate pIkBa. In addition, we demonstrate a dose-dependent inhibition of CUL-N8 levels in PBMCs isolated from the same mice. We expanded our PD analysis to human PBMCs treated ex vivo with MLN4924 to assess CUL-N8 levels by quantitativeWestern blot and pIKBa levels by ELISA. PBMCs isolated from healthy volunteers were used to assess the technical and biological variability of theWestern blot and ELISA assays. In these experiments, whole blood was treated ex vivo with increasing concentrations of MLN4924 and PBMCs were subsequently isolated with VACUTAINER® CPT™ tubes that are often utilized in Phase I trials. Repeated CUL-N8 or pIkBa measurements of replicate samples on different days demonstrated coefficient of variation values of less than 12% for both assays. Biological variability of baseline (i.e. untreated) and MLN4924-regulated levels of CUL-N8 and pIKBa in PBMCs was assessed by performing ex vivo treatment of whole blood obtained from the same donors on three separate occasions. Statistical analysis of this data demonstrated good biological reproducibility for both assays. Plasma concentrations measured from the ex vivo treated blood samples indicate that both the CUL-N8 Western and pIkBa ELISA assays detect MLN4924-induced regulation within the predicted range for human plasma exposures in a clinical setting. The demonstrated PD response and anti-tumor activity of MLN4924 in preclinical models has supported its ongoing evaluation for safety and PD in patients with hematological and solid tumor disease in multiple Phase I clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B34.