Abstract B178: Molecular determinants of response to the investigational small molecule inhibitor of Nedd8-activating enzyme (NAE) MLN4924 in melanoma cell lines and patient-derived tumor explant models.
Authors: Tentler John J. , Micel Lindsey N. , Selby Heather M. , Tan Aik Choon , Brunkow Kelsey L. , Pitts Todd M. , Blakemore Stephen , Smith Peter G. , Eckhardt S. Gail 
Published: June 2012

Abstract
Background: The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to proper functioning of the cell. Recent studies have shown that the UPS is more complex than previously thought, and its role in cell cycle regulation, signal transduction, DNA replication, and thus the evolution of the malignant phenotype of many cancers, is now well appreciated. The proteasome inhibitor bortezomib has provided validation of interfering with the UPS for clinical benefit in cancer patients and has provided the impetus for the development of new drugs that target the UPS more specifically. MLN4924 (Millennium Pharmaceuticals, Inc.) is an investigational first-in-class small molecule inhibitor of the key enzyme in the Nedd8 conjugation pathway, NAE. Inhibition of NAE results in in S-phase arrest, DNA re-replication and apoptosis. In this study, we used both in vitro and in vivo models of melanoma to assess the preclinical efficacy of MLN4924 in conjunction with gene array and gene set enrichment analysis (GSEA) to identify differentially expressed genes and pathways as potential molecular determinants of response to this novel compound.
Methods & Results: A panel of thirty-six melanoma cell lines was exposed to MLN4924 and IC50s were determined using the CellTiter Glo method. The cell lines were then classified as sensitive (S) or resistant (R) based on an IC50&lt;0.03μM or &gt;2.59μM, respectively. The most extreme S and R lines were also grown as subcutaneous flank xenograft tumors in nude mice to confirm in vitro responses to MLN4924. Additionally, twelve patient-derived melanoma tumors were implanted in mice and both the cell line and explant xenograft mice were treated s.c. with MLN4924 at 90mg/kg. The compound was well-tolerated and no significant weight loss or lethality was observed. Tumors were measured three times weekly by caliper and tumor growth inhibition (TGI) was analyzed. Of the explant models, five responded favorably to MLN4924, with TGI&gt;50%, with one model exhibiting tumor regression. Pharmacodynamic studies were also performed on melanoma explant tumors from mice exposed to a single dose of MLN4924 which were then harvested over a seven point time course ranging from 30 min to 24 hr post-drug. Immunoblotting of these tumors revealed rapid increases in the levels of caspase 3 as well as phospho-Chk-1 at 1 hr and decreases in neddylated cullin levels beginning at 8 hr, consistent with the known mode of action of MLN4924. Affymetrix 1.0 ST gene array analysis was performed on 6 S and 6 R cell lines in order to identify differentially expressed genes that could be used as predictive biomarkers. GSEA and KEGG pathway analysis of this data identified several DNA repair pathways as being enriched in the S lines while ABC drug transporter pathways were enriched in the R lines.
Conclusion: MLN4924 demonstrated strong antitumor activity in a subset of melanoma cell lines and patient-derived explants. Based on these preclinical results, MLN4924 demonstrates promising data supportive of further patient-selective clinical development in melanoma.
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 B178.