Abstract A196: Development, validation, and clinical implementation of a peripheral blood RT-PCR pharmacodynamic assay for MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE).

Abstract

Abstract The ubiquitin proteasome system regulates the degradation of intracellular proteins that are involved in many cellular processes. These processes, such as cell cycle, DNA replication, and response to oxidative stress play a key role in tumor growth and survival. The ubiquitination and proteasomal degradation of a subset of proteins involved in these processes is controlled by Cullin-RING ubiquitin ligases (CRLs). NEDD8-activating enzyme (NAE) activates the ubiquitin-like protein NEDD8 for conjugation to CRLs and therefore regulates the proteasomal destruction of CRL substrate proteins. NAE inhibition prevents degradation of CRL substrates (e.g. NRF2, CDT1), leading to their accumulation, subsequent apoptosis and cell death. MLN4924 is an investigational small molecule NAE inhibitor with antitumor activity in preclinical models of several tumor types that is currently in Phase I clinical development. MLN4924 is the first NAE inhibitor to enter clinical development, and evidence of target inhibition and/or downstream pathway modulation were key objectives of the Phase I studies. Here we describe the development, validation and clinical implementation of a pharmacodynamic (PD) RT-PCR assay that quantifies levels of gene transcripts regulated in response to NAE inhibition in human whole blood. To develop a prototype PD assay, Affymetrix gene expression profiling studies on HCT116 cells treated in-vitro with an NAE inhibitor were performed. Genes regulated greater than two-fold by NAE inhibition were identified. The list of genes was observed to be enriched for NRF2 regulated transcripts, consistent with the mechanism of action for MLN4924. Twenty-seven of the most robustly regulated genes were tested by RT-PCR (TaqMan) using RNA samples extracted from MLN4924 treated HCT-116 cells and human PBMCs treated ex-vivo with MLN4924 in order to validate the microarray results and develop a RT-PCR based PD assay in human blood. Transcripts showing a robust response to NAE inhibition in both HCT116 cells and PBMCs were identified for further evaluation in whole blood as a preferred sample matrix for clinical studies. To explore the relationship of MLN4924 regulated transcripts with time and drug concentration, blood samples from multiple healthy volunteers were treated ex-vivo with a range of MLN4924 concentrations for three durations (4, 8, 24 hours). Eight genes (NQO1, SLC7A11, TXNRD1, SRXN1, GSR, GCLM, ATF3, and MAG1) that displayed a robust induction (>3-fold) in whole blood were selected for the PD biomarker panel. Inter- and intra-assay variability was established via testing two pools of cDNA control samples (RNA from MLN4924 ex-vivo treated and untreated blood) in replicates on the same plate, and on multiple plates run separately. All genes had inter- and intra-assay CV values of <5%. This RT-PCR panel has been implemented in four Phase I clinical trials with MLN4924 in both solid tumor and hematological malignancies. Each study demonstrates induction of the NAE-regulated transcripts following MLN4924 administration starting at drug doses below the maximum tolerated dose. These results demonstrate the successful development and implementation of a RT-PCR assay that detects downstream effects of NAE inhibition in human whole blood. 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 A196.