AB0058 CELL-TYPE SPECIFIC REGULATION OF IL-1R SIGNALING BY R835, A DUAL IRAK1/4 INHIBITOR
Authors: S. Braselmann, E. Tai, R. Frances, C. Young, V. Markovtsov, E. Masuda, V. Taylor
Journal: Annals of the Rheumatic Diseases (2020)

Background: Interleukin-1 beta (IL-1b) is a key mediator of the inflammatory response and is known to exacerbate damage during chronic disease and acute tissue injury. Through association with the adaptor protein Myd88, interleukin receptor associated kinases (IRAK)1 and 4 initiate signaling downstream of IL-1Rs resulting in the activation of the NFkB and MAPK pathways and the production of proinflammatory cytokines (1). IL-1Rs are broadly expressed across cell types and little is known about differences in signaling between cell types and the role of IRAK1 and IRAK4 kinase activity. Objectives: We have identified a potent and selective IRAK1/4 inhibitor, R835, that substantially suppressed the elevation of LPS (TLR4 agonist)-induced serum cytokines in healthy human volunteers in a recent phase 1 study. The aim of this study was to evaluate the effect of R835 on IL-1R signaling in primary human fibroblasts and endothelial cells. Methods: Human dermal fibroblasts, lung fibroblasts or endothelial cells were stimulated with IL-1b and the effect of R835 on the signaling pathway was evaluated by western blotting. Human dermal fibroblasts were stimulated with different amounts of IL-1b to evaluate both the signaling pathways activated and the cytokines produced. The ability of R835 to inhibit cytokine production induced by high or low amounts of IL-1b in dermal fibroblasts was assessed. Results: In human endothelial cells, inhibition of IRAK1/4 kinases with R835 resulted in a block of IL-1b-induced IRAK4 phosphorylation, IRAK1 degradation and downstream NFkB, p38 and JNK activation. In contrast, in both human primary dermal and lung fibroblasts stimulated with IL-1b, we observed potent inhibition of IRAK4 phosphorylation, IRAK1 degradation, and downstream JNK phosphorylation, but no inhibition of NFkB pathway proteins and only weak inhibition of p38. Upon titration of IL-1b we observed that dermal fibroblasts produced IL-8 and GRO in response to low levels of IL-1b (20pg/ml), and produced additional cytokines including G-CSF and GM-CSF with higher levels of IL-1b (400pg/ml). In the presence of low levels of IL-1b (20pg/ml), we observed a weak activation of NFkB pathway proteins and p38, compared to a very robust NFkB, p38 and additional JNK activation in the presence of higher levels of IL-1b (400pg/ml). Consistent with these results, in dermal fibroblasts, R835 showed little to no inhibition of IL-8 and GRO induced by low levels of IL-1b, but potently inhibited G-CSF and GM-CSF induced by high levels of IL-1b where JNK was activated. Conclusion: This study has elucidated signaling differences between cell types downstream of the IL-1R. In endothelial cells, as in myeloid cells, the kinase activity of IRAK1 and IRAK4 is required for the activation of all downstream signaling. Unexpectedly, in human fibroblasts, IRAK1/4 kinase activity appears to primarily regulate the JNK pathway, and not the NFkB pathway. Concomitant with that, only the cytokines induced by the additional activation of JNK in fibroblasts are regulated by a dual IRAK1/4 inhibitor. Clinically, an IRAK1/4 inhibitor may show select inhibition of IL-1b-induced cytokines depending on the tissue and cell type involved in inflammation. References: [1]Flannery S, Bowie A G. The interleukin-1 receptor-associated kinases: Critical regulators of innate immune signaling. Biochemical Pharmacology, Volume 80, Issue 12, 15 December 2010, Pages 1981-1991. Disclosure of Interests: Sylvia Braselmann Shareholder of: Shareholder of Rigel Pharmaceuticals, Employee of: Employee of Rigel Pharmaceuticals, Ernest Tai Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Roy Frances Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Chi Young Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vadim Markovtsov Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Esteban Masuda Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vanessa Taylor Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals