Molecular and cellular effects of NEDD8-activating enzyme inhibition in myeloma

Abstract

The NEDD8-activating enzyme is upstream of the 20S proteasome in the ubiquitin/proteasome pathway and catalyzes the first step in the neddylation pathway. NEDD8 modification of cullins is required for ubiquitination of cullin-ring ligases that regulate degradation of a distinct subset of proteins. The more targeted impact of NEDD8-activating enzyme on protein degradation prompted us to study MLN4924, an investigational NEDD8-activating enzyme inhibitor, in preclinical multiple myeloma models. In vitro treatment with MLN4924 led to dose-dependent decrease of viability (EC(50) = 25-150 nmol/L) in a panel of human multiple myeloma cell lines. MLN4924 was similarly active against a bortezomib-resistant ANBL-6 subline and its bortezomib-sensitive parental cells. MLN4924 had submicromolar activity (EC(50) values <500 nmol/L) against primary CD138(+) multiple myeloma patient cells and exhibited at least additive effect when combined with dexamethasone, doxorubicin, and bortezomib against MM.1S cells. The bortezomib-induced compensatory upregulation of transcripts for ubiquitin/proteasome was not observed with MLN4924 treatment, suggesting distinct functional roles of NEDD8-activating enzyme versus 20S proteasome. MLN4924 was well tolerated at doses up to 60 mg/kg 2× daily and significantly reduced tumor burden in both a subcutaneous and an orthotopic mouse model of multiple myeloma. These studies provide the framework for the clinical investigation of MLN4924 in multiple myeloma.

Figure 1. Levels of NEDD8 transcript in primary MM tumor cells correlate with clinical outcome

Bort and MNL4924 structures are shown for these studies (A). Kaplan-Meier curves of progression-free survival for Bort-treated MM patients with low vs. high levels of NEDD8 transcript in the phase 2 SUMMIT (B, P=0.0173, log-rank test) and the phase 3 APEX trials (C, P=0.0482, log-rank test). Kaplan-Meier curves of progression-free survival for Bort- vs. dexamethasone-treated MM patients with low (D, P=0.0012, log-rank test) vs. high (E, P=0.6149, log-rank test) levels of NEDD8 transcript in the phase 3 APEX trial.

Figure 2. Activity of MLN4924 on MM cells

MM (OPM2, INA-6, KMS-34, H929, JJN3, MM.1S, KMS-18 and Dox40), WM (BCWM1, WSU-WM) and colon carcinoma (HCT116) cell lines were treated with MLN4924 (0–1000nM for 72 hrs) and viability was assessed by MTT assay. The most sensitive cell lines (OPM2, INA-6) have EC50 values < 40 nM, whereas the most resistant MM cell line (Dox40) has an EC50 value > 2 μM (A). Bone marrow aspirates from MM patients were processed using Miltenyi anti-CD138 microbeads for purification of MM cells, which were then cultured in RPMI1640 media containing 20% serum. CD138⁺ selected cells were treated with increasing doses of MLN4924 for 72 hrs and viability was assessed by CellTiterGlo (B). To access the sensitivity of non-malignant cells, PBMCs were isolated from normal donors and screened in the presence and absence of PHA stimulation (5ng/mL) for 72hrs. The number of viable cells in each condition is expressed as percent of the non-PHA-stimulated drug-free control (C). The MM cell line ANBL6 and its sub-line ANBL6-V5R were tested for sensitivity to Bort and MLN4924. ANBL6-V5R was more resistant to Bort, but not MLN4924, compared to the parental cell line (D).

Figure 3. MLN4924 overcomes the protective effect of nonmalignant accessory cells of the bone microenvironment

MM.1S and OPM2 MM cells were cultured in the presence and absence of HS-5 stromal cells (A, B) or differentiated osteoclasts (C, D) and treated with MLN4924 for 72 hrs. The resulting cell viability was normalized to each respective no drug control (n=4). Comparable activity was observed against MM.1S and OPM2 cells both in the presence or absence of these bone microenvironment cells with treatment with MLN4924.

Figure 4. In vivo anti-MM activity of MLN4924

RPMI8226/S cells were injected subcutaneously in CB.17-SCID mice and treatment was initiated once tumors had reached approximately 200mm³. MLN4924 was dosed subcutaneously at 10, 30 and 60 mg/kg BID for 21 days and tumor volume monitored (N=12 per group). There was a statistically significant difference in tumor burden only when comparing the control with 60mg/kg group (p<0.05; 1-way ANOVA; A). In addition, an orthotopic model of MM was evaluated. Sublethally irradiated NOD/SCID mice injected I.V. with MM1.S MM cells were randomly assigned to receive MLN4924 treatment (60 mg/kg BID i.p.) or vehicle control. Tumor burden was evaluated by whole-body bioluminescence imaging weekly and tumor burden calculated using Living Image software (B). Treated mice had lower tumor burden compared to vehicle treated controls (p=0.1186 day 33; p=0.0269 day 38; N=6 per group).

Figure 5. Immunoblotting analysis and transcriptional profiles of MLN4924-treated MM cells

MM.1S cells treated with MLN4924 show upregulation (as early as 8hrs) of p27, CTD1 and NRF2, which are known targets of the NEDD8 pathway, followed by markers of cell death, such as cleavage of PARP and caspase-3 at later time-points (as early as 32 hours after initiation of treatment) (A). The gene expression profiling changes triggered in MM.1S cells by MLN4924 vs. Bort were evaluated using the HG-U133plus2 oligonucleotide microarray. The profile of MLN4924-triggered transcriptional changes was distinct from the one induced by Bort exposure (B). Known transcriptional responses to 20S proteasome inhibition, such as upregulation of proteasome subunits (C) minimal or absent in the MLN4924-treated cells.

Figure 6. Combinations of MLN4924 with established anti-MM agents

MLN4924 treatment was combined with agents used for clinical management of MM, namely Bort (A), Doxo (B), and Dex (C). Activity was observed with these combinations at drug doses relevant to those achieved clinically in the treatment of patients.