MLN4924, a Potent and Novel Small Molecule Inhibitor of Nedd8 Activating Enzyme, Induces DNA Re-Replication and Apoptosis in Cultured Human Tumor Cells

Abstract Successful therapeutic intervention in the ubiquitin-proteasome system (UPS) in multiple myeloma and mantle cell lymphoma with proteasome inhibitors has led to the pursuit of additional targets within the UPS. Through this effort we have identified MLN4924, a novel, first-in-class small molecule inhibitor of Nedd8 activating enzyme (NAE) currently in Phase I trials in hematological and non-hematological malignancies. The initial step in the pathway for conjugation of the ubiquitin-like protein Nedd8 to its cellular targets requires the activity of NAE. Nedd8 conjugation is required for the proper function of mammalian cullin-dependent ubiquitin ligases (CDLs). These CDLs in turn control the timely ubiquitination and subsequent degradation of many proteins with important roles in cell cycle progression and signal transduction. Inhibition of NAE leads to decreased activity of the CDLs impacting cellular processes relevant to tumor cell growth and survival thereby providing a rationale for targeting NAE as an anti-cancer strategy. MLN4924 was used to explore the consequences of inhibiting the Nedd8 pathway in cultured human cancer cells. Here we show that MLN4924 has broad in vitro potency against several myeloma, lymphoma and leukemia cell lines including; RPMI-8226, NCI-H929, WSU-DLCL2, Ly10, Ly19, HL-60 and MOLT-4 respectively, as well as multiple non-hematological cell lines. MLN4924 specifically inhibits Nedd8-cullin formation leading to increased steady state levels of direct CDL substrates by preventing their ubiquitination and degradation through the proteasome. Many of these CDL substrates are involved in cell cycle progression. One such CDL substrate is the critical DNA replication licensing factor Cdt1. Over-expression of Cdt1 has been shown to induce DNA re-replication in cells resulting in DNA damage, cell cycle arrest and genomic instability. Here we show that MLN4924 dramatically affects the cell cycle distribution of HCT-116 and WSU-DLCL2 cells resulting in S-phase accumulation and apparent increase in nuclear size and DNA content. HCT-116 cells treated with MLN4924 showed an increased and prolonged ability to incorporate BrdU demonstrating active DNA synthesis in S-phase consistent with over replication of the DNA. Aphidicolin synchronized HCT-116 cells released into MLN4924 did not progress into mitosis as exhibited by the absence of the mitotic marker pH3 (S10) suggesting that the observed phenotype was occurring within the same cell cycle. Immunoflourescence and western blot analysis of HCT-116 cells treated with MLN4924 also showed an increase in the nuclear localization and stabilization of Cdt1 consistent with the hypothesis that MLN4924 disrupts the normal cell cycle regulation and turnover of this CDL substrate. The aberrant re-replication phenotype observed following MLN4924 treatment in HCT-116 and WSU-DLCL2 cells was associated with the activation of a DNA damage checkpoint response through the ATM/ATR pathways assessed by the expression of elevated levels of phospho-p53(S15), phospho-Chk1(S317) and phospho-H2AX(S139). Western blot analysis of aphidicolin synchronized HCT-116 cells released into MLN4924 demonstrated the sequential nature of the DNA damage response consistent with the re-replication phenotype. The initial appearance of ssDNA breaks (phosph-Chk1 and phospho-Rad17) presumably resulting from stalled replication forks, illustrated early ATR activation followed by the induction of the ATM pathway as a result of dsDNA breaks ( phospho-Chk2 and phospho-NBS1). Finally the cells inability to recover from the dsDNA damage resulted in apoptosis as demonstrated by the appearance of cleaved caspase-3 and cleaved PARP. The gross accumulation of over replicated DNA and DNA-damage following NAE inhibition resulting in cell cycle arrest and apoptosis is consistent with DNA re-replication and demonstrates a novel mechanism of action for MLN4924 in cultured human tumor cells. These data have important implications for the use of MLN4924 in hematological malignancies as a single agent, and potentially in combination with other therapies.