Erik Koenig

Treatment-Emergent Mutations in NAEβ Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924

MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEβ mutations.

Abstract B156: Regulation of tumor metabolism by the investigational proteasome inhibitor ixazomib in KRAS wild-type and KRAS mutant xenograft tumors.

The investigational proteasome inhibitor ixazomib (MLN2238) inhibits cell growth in a broad panel of solid tumor and hematological cell lines when tested in vitro. In contrast, antitumor activity in xenograft-bearing mice is model-dependent, with some solid tumor models showing no response to ixazomib treatment. A survey of 15 NSCLC and 6 colon xenograft models showed a striking relationship between the degree of antitumor activity of ixazomib and KRAS genotype. Tumors with wild-type (WT) KRAS showed higher sensitivity to ixazomib compared to the tumors harboring a KRAS activating mutation. These xenografts included cell-line-derived subcutaneous xenografts as well as patient derived primary tumors. To confirm that KRAS mutation impacts response to ixazomib, we used SW48 isogenic colon cancer cell lines, in which a KRAS-G13D mutation was introduced into KRAS-WT SW48 cells to generate stable SW48-KRAS-G13D cells. Although in vitro sensitivity to ixazomib was similar between the paired cell lines, in vivo antitumor activity was observed in only SW48 KRAS WT tumors, but not SW48-KRAS-G13D tumors. These data confirm the association of KRAS status and response to ixazomib. Since KRAS activating mutations are known to be associated with metabolic reprogramming, we performed metabolite profiling using in vivo SW48 isogenic tumor pairs treated with or without ixazomib. As expected, prior to treatment there were significant baseline differences between SW48 WT and SW48-KRAS-G13D tumor metabolite profiles in categories such as glutathione levels and glycogen breakdown intermediates, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. After a single dose of ixazomib, significant metabolic regulation was noted in categories including free amino acid pools, and polyamine levels, and some of these changes were more pronounced in the SW48 KRAS WT tumors as compared to the KRAS-G13D tumors. Our data also showed activation of GCN2 mediated signaling following ixazomib treatment, possibly as a result of amino acid deprivation in the tumors. The results suggest additional non-clinical avenues to explore mechanisms of sensitivity and resistance to proteasome inhibitors in solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B156. Citation Format: Nibedita Chattopadhyay, Allison Berger, James Garnsey, Hugues Bernard, Erik Koenig, Eric Lightcap, Ben Amidon. Regulation of tumor metabolism by the investigational proteasome inhibitor ixazomib in KRAS wild-type and KRAS mutant xenograft tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B156.

Abstract A196: Development, validation, and clinical implementation of a peripheral blood RT-PCR pharmacodynamic assay for MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE).

The ubiquitin proteasome system regulates the degradation of intracellular proteins that are involved in many cellular processes. These processes, such as cell cycle, DNA replication, and response to oxidative stress play a key role in tumor growth and survival. The ubiquitination and proteasomal degradation of a subset of proteins involved in these processes is controlled by Cullin-RING ubiquitin ligases (CRLs). NEDD8-activating enzyme (NAE) activates the ubiquitin-like protein NEDD8 for conjugation to CRLs and therefore regulates the proteasomal destruction of CRL substrate proteins. NAE inhibition prevents degradation of CRL substrates (e.g. NRF2, CDT1), leading to their accumulation, subsequent apoptosis and cell death. MLN4924 is an investigational small molecule NAE inhibitor with antitumor activity in preclinical models of several tumor types that is currently in Phase I clinical development. MLN4924 is the first NAE inhibitor to enter clinical development, and evidence of target inhibition and/or downstream pathway modulation were key objectives of the Phase I studies. Here we describe the development, validation and clinical implementation of a pharmacodynamic (PD) RT-PCR assay that quantifies levels of gene transcripts regulated in response to NAE inhibition in human whole blood. To develop a prototype PD assay, Affymetrix gene expression profiling studies on HCT116 cells treated in-vitro with an NAE inhibitor were performed. Genes regulated greater than two-fold by NAE inhibition were identified. The list of genes was observed to be enriched for NRF2 regulated transcripts, consistent with the mechanism of action for MLN4924. Twenty-seven of the most robustly regulated genes were tested by RT-PCR (TaqMan) using RNA samples extracted from MLN4924 treated HCT-116 cells and human PBMCs treated ex-vivo with MLN4924 in order to validate the microarray results and develop a RT-PCR based PD assay in human blood. Transcripts showing a robust response to NAE inhibition in both HCT116 cells and PBMCs were identified for further evaluation in whole blood as a preferred sample matrix for clinical studies. To explore the relationship of MLN4924 regulated transcripts with time and drug concentration, blood samples from multiple healthy volunteers were treated ex-vivo with a range of MLN4924 concentrations for three durations (4, 8, 24 hours). Eight genes (NQO1, SLC7A11, TXNRD1, SRXN1, GSR, GCLM, ATF3, and MAG1) that displayed a robust induction (>3-fold) in whole blood were selected for the PD biomarker panel. Inter- and intra-assay variability was established via testing two pools of cDNA control samples (RNA from MLN4924 ex-vivo treated and untreated blood) in replicates on the same plate, and on multiple plates run separately. All genes had inter- and intra-assay CV values of 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 A196.

Abstract C28: Treatment emergent mutations in NAEbeta confer resistance to the investigational NEDD8-activating enzyme inhibitor MLN4924.

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.