Abstract C28: Treatment emergent mutations in NAEbeta confer resistance to the investigational NEDD8-activating enzyme inhibitor MLN4924.
Authors: Smith Peter G. , Thomas Michael , Traore Tary , Narayanan Usha , Riceberg Jessica , Amidon Ben , Bence Neil , Brownell James , Dick Larry , Manfredi Mark , Sells Todd , Sintchak Mike , Koenig Erik , Gavin James , Milhollen Michael A. 
Published: June 2012

MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that has shown clinical activity in Phase I clinical trials. 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- B 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, cell line and xenograft models of solid tumors were used. HCT-116 (colon), NCI-H460 (lung) and Calu-6 (lung) cells were treated with high concentrations of MLN4924 for four days after which the remaining cells were isolated and cultured in drug free media. Six HCT-116, two NCI-H460 and one Calu-6 clone were isolated and found to be at least 10-fold less sensitive to MLN4924, in most cases the isolated clones were completely insensitive indicating they were now resistant to MLN4924. However, the MLN4924 resistant clones were still sensitive to other chemotherapies including bortezomib (proteasome inhibitor), SN-38 (topoisomerase I inhibitor) and doxorubicin (anthracycline) suggesting a resistance mechanism unique to MLN4924. Co-incubation with Pgp, BCRP or MRP2 transporter inhibitors did not re-sensitize cells to MLN4924 indicating that the resistance mechanism was not related to increased drug efflux. DNA sequencing of NAE pathway genes identified heterozygous mutations in NAEbeta resulting in amino acid substitutions in the ATP binding pocket or NEDD8-binding cleft. Nude rats bearing HCT-116 xenografts were treated with the maximum tolerated dose of MLN4924 on a clinically relevant dosing regimen of Days 1, 4, 8 and 11 of a 21 day schedule. In the first cycle of therapy tumor regressions were observed but ultimately tumors regrew over subsequent cycles and were found to contain heterozygous mutations in NAEbeta. Tumors containing mutations in NAEbeta were transplanted into nude rats and shown to be 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 NAEbeta 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, in pre-clinical models of solid tumors treatment emergent mutations in NAEbeta lead to MLN4924 resistance. DNA sequencing of samples obtained from MLN4924 clinical trials is ongoing. These data may provide rationales for patient selection approaches with MLN4924 and the development of next generation NAE inhibitors that can be designed to overcome treatment emergent mutations found in clinical studies.

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 C28.