Ixazomib Inhibits Dendritic Cell Maturation and Function

Targeting T cells alone has yielded limited success in the prevention of graft versus host disease (GvHD) after allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Due to their immune modulatory effects and their inhibition of maturation and function of DC, proteasome inhibitors have prompted investigators to examine their potential role in the prevention of GvHD.  Ixazomib is a specific and reversible proteasome inhibitor with rapid dissociation from 20S proteasome. It is orally bioavailable. We aimed to explore its effect on healthy volunteer DCs. DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with a purity over 90% (STEMCELL Technologies). DCs were treated with ixazomib at different concentrations for 4 hrs and then stimulated with Lipopolysaccharide (LPS) for 16 hrs. After the treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of ixazomib on the production of pro-inflammatory cytokines, DCs were incubated with ixazomib at increasing concentration before or after addition of LPS.  Pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Experiments were done in triplicate at least. Unpaired T test was used for statistical analysis. P < 0.05 was considered significant.

Ixazomib inhibited expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose related. Ixazomib also decreased the percentage of DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10 nM and dropped to 68% and 43% on average with concentrations of 20nM and 40nM. There was no significant change in the cytokine production when ixazomib was added 4 hours after LPS. On the other hand, ixazomib significantly decreased production of IL-6 and IL-23 by DCs at the concentration of 20-30nM. However, there was no significant change of TNF-β and INF-γ upon ixazomib treatments.

In summary, ixazomib inhibits DC maturation with relative preservation of cell viability. On the other hand, ixazomib inhibits pro-inflammatory cytokine production in DCs only when it is added before LPS stimulation. This may have clinical implications and suggests that adequate inhibition of DCs before graft infusion might be essential to the prevention GvHD.