The Combination of Bortezomib and NPI-0052 Exerts Anti-Tumor Activity in Waldenstrom Macroglobulinemia (WM).

Background: WM is an incurable low-grade lymphoplasmacytic lymphoma. Bortezomib has recently demonstrated about 50% ORR in patients with relapsed WM. We therefore investigated the in vitro effect of the new proteasome inhibitor NPI-0052 (N) alone and in combination with Bortezomib (B).

Methods: WM cell lines (BCWM1,WSU-WM) and IgM secreting cell lines (MEK1, Namalwa) were used. Bone marrow primary CD19⁺ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured using MTT assay and [³H]-thymidine uptake. Determination of the synergistic effect [combination index (CI)] of combination was calculated using the CalcuSyn software. Cell signaling and apoptotic pathways were determined by Western Blot. We also tested the effect of N on WM cells in the co-culture with BMSCs. Activity of the 20S proteasome was determined by detecting the release of the fluorophore AMC, after cleavage from the labeled substrates specific for each enzymatic activity.

Results: N induced cytotoxicity and inhibition of DNA synthesis (IC₅₀ 15nM) in BCWM.1 (48 h). Similar effects were demonstrated in IgM secreting cell lines and primary CD19⁺ WM cells (IC₅₀ 18–30nM). No cytotoxicity was observed on peripheral blood mononuclear cells. The combination of N+B significantly inhibited BCWM.1 proliferation compared to each agent alone: B (5nM) induced cytotoxicity in 8.5%, which increased to 26%, 40% and 53% in the presence of N 2.5nM (CI:0.83), 5nM (CI:0.72) and 10nM (CI:0.7) respectively, indicating synergism. To determine the mechanism of synergy, we investigated the effect of the two agents and their combination on proteasome activity and on signaling pathways, specficially the Akt pathway. Both N and B inhibited the three proteasome activities: the combination of N+B was increased compared to the effect of each agent alone on the caspase-like (C-l) activity of the proteasome. B and N used as single agents induced 29% and 34% inhibition of the C-l activity, respectively, compared to 60% when B and N were used in combination. The C-l activity preferentially cleaves substrates with an aspartic residue in P1 position, like the substrates of the caspases. N induced caspase-8, PARP cleavage and increase of Smac as well as down-modulation of the anti-apoptotic proteins c-IAP, XIAP, survivin, Bcl-2, Mcl-1. The combination of N+B induced a stronger and more significant induction of caspase-8, -PARP cleavage, as well as caspase-3 and -9, which were not affected by using N alone. Similarly, Smac modulation resulted in a more significant induction when cells were exposed to both proteasome inhibitors. N inhibited Akt phosphorylation in BCWM.1 cells (6h) in a dose-dependent manner. GSK3 phosphorylation and ribosomal protein-S6, Akt-downstream target proteins, were also markedly inhibited. Importantly, N inhibited Akt phosphorylation and Akt activity in BCWM.1, even when combined with B, which induced increase of Akt phosphorylation. Lastly, neither exposure to IL-6 nor adherence to BMSCs conferred protection to WM cells against NPI-induced cytotoxicity.

Conclusion: NPI-0052 has significant antitumor activity in WM in vitro especially in combination with Bortezomib. These results provide the framework for clinical trials in WM.