Background Systemic Lupus Erythematosis (SLE) is an autoimmune disease characterized by alterations in B-cells, autoantibody production, and elevations in circulating B-cell activating factor (BAFF). Aiolos single nucleotide polymorphisms and elevations in Aiolos mRNA are associated with SLE susceptibility. CC-220 is a Cullin Ring Ligase 4 Cereblon (CRL4CRBN) E3 Ubiquitin Ligase Modulator that induces ubiquitination and degradation of Aiolos and Ikaros transcription factors. We investigated the effects of CC-220 in B-cells from SLE patients to assess its impact on Aiolos protein levels and B-cell proliferation, differentiation, and antibody production.
Objectives Determine the protein levels of the B-cell transcription factors Aiolos and Ikaros in B-cell subtypes of SLE patients and assess the impact of the cereblon modulator CC-220 on Aiolos and Ikaros protein degradation, proliferation, and plasmablast differentiation.
Methods Peripheral blood mononuclear cells (PBMC) were measured for levels of circulating B-cell subtypes, B-cell activation state, and Aiolos and Ikaros protein levels by flow cytometry. Cell types were defined as: CD27-IgD+ naive, CD27+IgD- switched memory, CD27+IgD+ nonswitched memory, and CD27-IgD- double negative B-cells and CD20-CD38+ plasmablasts. Measurements of BAFF, IL-2 and IL-21 from plasma and IgG and IgM were done by ELISA. Plasmablast differentiation was induced by BAFF, IL-2, and IL-21 co-stimulation of B-cell cultures for 5 days in the absence and presence of CC-220.
Results Plasma samples from SLE patients were found to have higher circulating BAFF, similar IL-2 levels, and reduced IL-21 levels relative to healthy individuals. Alterations in circulating B-cell subtypes occur in SLE patients including reduction of memory and elevations of naive and double negative B-cells. Alterations in biomarkers associated with chronic B-cell activation, including elevation of CD95 and reduction of CD21 and CD23 were also observed. Assessment of the B-cell differentiation transcription factors Aiolos and Ikaros in SLE B-cells showed that Aiolos, but not Ikaros is elevated in naive, switched memory, nonswitched memory, and double negative B-cells. CC-220 treatment reduced Aiolos and Ikaros protein levels in both SLE and healthy individuals in all four B-cell subtypes measured. CC-220 treatment was associated with reduced B-cell proliferation, plasmablast differentiation, and secretion of IgG and IgM in cells co-stimulated with BAFF, IL-2 and IL-21. Similarly, CC-220 reduced the expression of genes involved in B-cell differentiation including IRF-4, Xbp-1, Blimp-1, and IgJ indicating an early blockade in B-cell differntiation.
Conclusions Our observations that SLE is associated with alterations of circulating B-cell subtypes and their activation state, Aiolos overexpression in B-cells, and increases in circulating BAFF support the hypothesis that B-cells play a significant role in SLE pathology. Moreover, these observed changes suggest that B-cells may be predisposed towards plasmablast differentiation and antibody production in SLE. CC-220 administration reduced Aiolos and Ikaros protein levels in B-cells and reduced proliferation, plasmablast differentiation and antibody production at clinically obtainable drug concentrations (<10 nM).
Disclosure of Interest G. Ringheim Shareholder of: Celgene Corporation, Employee of: Celgene Corporation, J. Kosek Shareholder of: Celgene Corporation, Employee of: Celgene Corporation, L. Capone Shareholder of: Celgene Corporation, Employee of: Celgene Corporation, P. Schafer Shareholder of: Celgene Corporation, Employee of: Celgene Corporation, Y. Nakayama Shareholder of: Celgene Corporation, Employee of: Celgene Corporation
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