Treatment Emergent Mutations in NAEβ Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML and DLBCL Models

Abstract 1413

MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). MLN4924 is a mechanism-based inhibitor of NAE as demonstrated through the formation of a NEDD8-MLN4924 covalent adduct that is a tight-binding inhibitor of NAE. The antitumor activity of MLN4924 has been linked to two mechanisms in pre-clinical models: (1) the induction of DNA re-replication and cell death through dysregulation of Cdt-1 and (2) the inhibition of NF-κB signaling. Importantly, modulation of both pathways has been demonstrated in pharmacodynamic (PD) studies from patients administered MLN4924. To characterize potential mechanisms of resistance to MLN4924 xenograft models of AML and Diffuse Large-B cell Lymphoma (DLBCL) were used. Mice bearing HL-60 and THP-1 (AML) and OCI-Ly10 (DLBCL) xenografts were treated with MLN4924 twice weekly for 95–110 days and most animals achieved a complete tumor regression. However, four of ten HL-60, six of ten THP-1 and one of ten OCI-Ly10 xenografts became refractory and re-grew during the MLN4924 treatment period. These tumors were harvested and subjected to microarray analysis and DNA sequencing of genes required for NEDD8 activation by NAE. Mutations in NAEβ resulting in amino acid substitutions were detected in several xenografts in the ATP binding pocket (A171T) and NEDD8-binding cleft (E204K) suggesting that mutations in the target enzyme may play a role in emergence of refractory tumors. Tumors containing mutations in NAEβ were passaged into new mice and shown to be completely insensitive to MLN4924 confirming resistance. In addition, cell lines derived from the resistant xenografts were shown to be resistant to MLN4924 in vitro but remained sensitive to other chemotherapeutic agents. Biochemical analysis of NAEβ mutants revealed a slower rate of NEDD8-MLN4924 adduct formation and demonstrated that the adduct was no longer bound tightly by the mutant enzyme. Thus, treatment emergent mutations in NAEβ lead to MLN4924 resistance in pre-clinical models through reduced inhibitory potency. These data serve as the framework for understanding mechanisms of resistance to MLN4924, provide rationale for patient selection approaches and offer a strategy to overcome treatment emergent mutations that may arise in clinical studies.