Abstract 214: High expression of CKS1B antagonizes MLN4924, a nedd8-activating enzyme inhibitor, that induces myeloma cell growth inhibition through decreased neddylation of SCF complex

Abstract

Abstract Background: MLN4924 binds to the NEDD8-activating enzyme (NAE), and promotes neddylation of SCF (Skp1-Cul1 / Cdc53-F-box protein) complex resulting in inhibition of proteasomal degradation of several substrates. CKS1B acts as an adaptor of SCF complexes involved in cell cycle progression and DNA damage repair in many cancers, including multiple myeloma (MM). In this study, we investigate the functional role of CKS1B expression on MLN4924 in myeloma and determine whether CKS1B can be used as a potential biomarker for sensitivity to MLN4924 treatment in MM. Methods: Three myeloma cell lines, KMS12, KMS28PE, and OCI-MY5 in which CKS1B was artificially over-expressed (CKS1B-OE) as well as their empty vector (EV) controls were treated with MLN4924 at different concentrations. The IC50 and growth inhibition of cells were analyzed via a fluorencent screeing assay. APC-labeled annexinV and PI were assessed by flow cytometry to detect apoptosis and cell cycle changes. The expression of Cullin-NEDD8 and a series of ubiquitined SCF substrates were analyzed by non-reducing western blot. Results: MLN4924 effectively inhibited cell growth and induced cell apoptosis in both CKS1B-OE and EV MM cells. However, MM cells transfected with EV were more sensitive than those transfected with CKS1B-OE as evidenced by the IC50 values (KMS28PE OE vs. EV 99: 65 nM; KMS12 OE vs. EV 125: 72 nM; and OCI-MY5 OE vs. EV 106:89 nM). The cell viabilities at 100 nM were 49.6% vs. 26.7%, 54.5% vs. 34.2%, and 53.6% vs. 36.8% in CKS1B-OE versus EV KMS28PE, KMS12, and OCI-MY5 cells (P<0.05). Western blots showed that over-expression of CKS1B significantly decreased expression of cullin1, nedd8, and the conjugated complex of cullin-nedd8 in MLN4924 treated cells (100 nM, 24h) compared with EV in all tested three cell lines. We also found that high expression of CKS1B was negatively correlated with SCF substrates including p21, CDT-1, p27 and CDT-1. MLN4924 induced a dose-dependent accumulation of these substrates in all three MM cell lines. Conclusion: High expression of CKS1B induces SCF substrate ubiquitination and degradation in MM cells. The NAE inhibitor MLN4924 induces cell apoptosis through decreased neddylation of SCF complex resulting in increase of p27, p21, CDT-1 and p130. CKS1B can be used as a marker of drug resistance to MLN4924, suggesting that a combination of MLN4924 with other drugs will be necessary to treat myeloma patients with high endogenous expression of CKS1B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 214. doi:1538-7445.AM2012-214