Disrupting NEDD8-Mediated Protein Turnover with MLN4924 Significantly Augments the Efficacy of Cytarabine
Authors: Nawrocki Steffan T , Kelly Kevin R , Oberheu Kelli , Mahalingam Devalingam , Smith Peter G , Carew Jennifer S 
Published: October 2019

Abstract
Abstract 3255
Cytarabine-based therapy has been utilized in acute myeloid leukemia (AML) therapy for more than 30 years. However, the complete response (CR) rates are markedly inferior in older compared to younger patients with AML (45% versus 75%, respectively) due, in part, to the reduced ability of elderly patients to tolerate intensive therapy. Improving the outcomes for patients treated with cytarabine-based regimens represents a major clinical challenge in this disease. A randomized study of elderly patients with AML demonstrated that low dose cytarabine (LDAC) is superior to best supportive care. However, this regimen was not associated with any CRs in patients with adverse karyotype disease and/or poor baseline performance scores. Novel approaches are urgently needed to increase the efficacy of LDAC therapy for these patients. Timed protein destruction plays a crucial role in cellular homeostasis and is essential for many critical functions including cell cycle progression, signal transduction, and apoptosis. The processes that govern protein degradation frequently become dysregulated in cancer cells. Aberrant protein turnover contributes to disease progression, metastasis, and therapeutic resistance and therefore is an attractive target for selective pharmacological inhibition. The cullin-RING ubiquitin ligases (CRLs) are a subset of E3 ubiquitin ligases whose activity is regulated by modification with the ubiquitin-like molecule NEDD8. The CRLs control the ubiquitination and subsequent degradation of many proteins with important roles in cell cycle progression, DNA damage, stress responses, and signal transduction. MLN4924 is a potent and selective small molecule inhibitor of NEDD8 activating enzyme (NAE), the proximal regulator of the NEDD8 conjugation pathway, and has entered Phase I clinical trials for AML and other forms of cancer. Our earlier preclinical studies demonstrated that MLN4924 induced cell death in AML cell lines and primary patient specimens independent of FLT3 expression and stromal-mediated survival signaling and led to the stabilization of key NAE targets, inhibition of NF-kB activity, DNA damage, and reactive oxygen species generation. Notably, administration of MLN4924 to mice bearing AML xenografts was very well tolerated, led to stable disease regression and inhibition of NEDDylated cullins. Based on the high tolerability, potency, and multifaceted mechanism of action of MLN4924, we hypothesized that it may significantly augment the efficacy of the standard agent cytarabine. To test our hypothesis, we first investigated the effects of this therapeutic combination on cell viability, clonogenic survival, and apoptosis induction in a panel of AML cell lines. MLN4924 cooperated with cytarabine to significantly reduce cell viability, inhibit clonogenic survival, and induce mitochondrial-dependent apoptosis. The addition of MLN4924 did not significantly alter the sensitivity of normal peripheral blood mononuclear cells from healthy donors to cytarabine, indicating that this combination may have therapeutic selectivity. Immunoblotting analyses revealed that MLN4924 enhanced cytarabine-induced stabilization of the NEDD8 target and cell cycle regulator, p27. The MLN4924/cytarabine combination also promoted increased phosphorylation of the DNA damage response regulator Chk1. Targeted knockdown of Chk1 demonstrated a critical role for Chk1 as a mediator of the pro-apoptotic effects of this combination. In vivo examining the combination is in progress and will be presented. Our collective findings suggest that combining the novel NAE inhibitor MLN4924 with cytarabine is a promising strategy for AML therapy that warrants further investigation.

Disclosures:
Smith: Millennium Pharmaceuticals, Inc.: Employment.