Late-breaker at AACR on IMiD mechanism of action:
Proceedings of the 1999 AACR · NCI · EORTC International Conference]
Copyright © 1999 by the American Association for Cancer Research
#0753 Differential Effects of a Thalidomide Analogue on NF-B Mediated Transcription in Monocytic and T cell lines. Payvandi, F., Haley, M., Schafer, P., Muller, W.G., Chen, R., and Stirling, D. Celgene Corporation, 7 Powder Horn Drive, Warren, NJ 07059.
Celgene recently reported on a novel series of thalidomide analogs (IMiDs) that potently inhibit TNF- production in activated hPBMCs and stimulate activated T cell proliferation. The molecular mechanism of action of this series of compounds has not yet been determined. Herein, we report on initial studies to determine their mechanism of action. We have begun studies to investigate the effects of one of these IMiDs on NF-B activation in Jurkat T cells and in the U937 promonocytic cell line. Jurkat cells were transfected with pNF-B-luciferase and stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of the IMiD. The IMiD increased NF-B luciferase activity in antibody-stimulated cells (1.7-2.1 fold at 250 nM), but not unstimulated cells. The IMiD also increased NF-B luciferase activity to a similar degree when Jurkat cells were stimulated with Raji B cells plus the superantigen Staphylococcal Enterotoxin E. IL-2 production by transfected Jurkat cells, as assessed by ELISA, also showed the same pattern of enhancement by the IMiD. These results indicated that the IMiD may increase IL-2 production at the transcriptional level. We have also investigated the effects of the IMiD on IL-2 mRNA stability. In a gel shift assay using nuclear extracts from antibody-stimulated Jurkat cells, the IMiD increased NF-B DNA-binding activity (approximately 2 fold at 250 nM). OCT-1 binding activity was not affected by the IMiD. Supershift experiments showed that p50/Rel (p65) hetrodimers were the primary NF-B complex in the stimulated Jurkat cells. Cotransfection of the super-repressor IBa-flag (SRIB-flag) (Ser32Ala, Ser36Ala) along with pNF-B-luciferase cells completely eliminated the increase in NF-B transcriptional activity caused by the IMiD, confirming that the increased NF-B activity was dependent upon IB. Neutralizing anti-IL-2 antibody did not block the enhancement of NF-B transcriptional activity by the IMiD, indicating that the increase in NF-B activity was not due to IL-2 autocrine effects. U937 were also transfected with pNF-B-luciferase and stimulated with LPS in the presence or absence the IMiD. Unlike the effect in Jurkat cells, the IMiD inhibited LPS-stimulated NF-B-luciferase activity in U937 cells (55% inhibition at 250 nM). TNF- production by transfected U937 cells showed the same pattern of inhibition by the IMiD. These experiments demonstrate that the effects of this IMiD are cell type dependent. Furthermore, this IMiD exert its effects at least in part by acting on NF-B pathway in both T cells and monocytes. Further experiments are in progress to determine the biochemical mechanism of action of this series of thalidomide analogues.
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