MLN4924, a Novel Small Molecule Inhibitor of Nedd8-Activating Enzyme, Demonstrates Potent Anti-Tumor Activity in Diffuse Large B-Cell Lymphoma

MLN4924 is a first-in-class, small molecule inhibitor of the Nedd8 Activating Enzyme (NAE) in Phase I clinical trials in hematological malignancies. Inhibition of NAE by MLN4924 leads to decreased neddylation and inhibition of cullin-dependent ubiquitin ligase (CDL) activity. CDLs are enzyme complexes which control the ubiquitination and degradation of proteins with important roles in cell cycle progression and cell survival. CDL-mediated degradation of pIkBa regulates NF-kB signaling by freeing cytoplasmic NF-kB transcription factors to translocate to the nucleus promoting cell proliferation and survival. In tumors dependent on the NF-kB pathway for growth and survival, we hypothesized that MLN4924 inhibition of CDL activity would prevent pIkBa degradation and inhibit NF-kB signaling.

We utilized models of ABC-like Diffuse Large B-cell Lymphoma (ABC-like DLBCL, OCI-Ly10 and OCI-Ly3 cells) dependent on NF-kB signaling for survival and Germinal Center B-cell like DLBCL (GCB-like DLBCL, OCI-Ly19 and OCI-Ly7 cells) that are not dependent on NF-kB signaling for survival. In vitro, we show that NAE inhibition by MLN4924 in ABC-like DLBCL produces marked stabilization of pIkBa, inhibits p65 nuclear translocation and NF-KB gene transcription demonstrating an inhibition of NF-kB signaling. The inhibition of NF-KB signaling in Ly10 cells results in a G1 phenotype and an acute induction of apoptosis. In contrast, in GCB-like DLBCL we observed an elevation of multiple substrates of the CDLs, an accumulation of cells with increased DNA content (>4N) followed by a DNA damage response and induction of cell death. This mechanism of action in GCB-like DLBCL cells is observed in other tumor cell lines that are not dependent on NF-kB signaling for survival.

In vivo administration of MLN4924 to mice bearing xenograft tumors of OCI-Ly10 and OCI-Ly19 resulted in a pharmacodynamic response of NAE pathway inhibition. In both models, a single dose of MLN4924 resulted in time and dose-dependent inhibition of total neddylated cullin levels and stabilization of CDL substrates including the CDL3^Keap1 substrate, Nrf-2. Notably, in the OCI-Ly10 model, a single dose of MLN4924 resulted in a marked elevation of pIkBa levels, indicative of NF-kB pathway inhibition, and induction of apoptosis. In both OCI-Ly10 and OCI-Ly19 xenograft models, inhibition of the NAE pathway following repeated daily and intermittent dosing of MLN4924 translated into significant tumor growth inhibition. In the OCI-Ly10 model tumor regressions were observed showing this model to be particularly sensitive to MLN4924 treatment, reflecting the addiction of these tumors to NF-kB signaling. Additionally we demonstrate an inhibition of the NAE pathway and NF-KB signaling in a primary human tumor DLBCL xenograft model (PHTX-22L) resulting in tumor regressions following MLN4924 treatment.

In summary, in tumors dependent on NF-kB signaling for growth and survival, MLN4924 inhibition of CDL activity provides a novel mechanism for targeted NF-kB pathway modulation and therapeutic intervention. In addition, these data demonstrate that MLN4924 is a novel agent that has broad activity in pre-clinical models of lymphoma.