Selective disruption of RORγt-CBFβ interaction by IMU-935 prevents RORγt-dependent Th17 autoimmunity but not thymocyte development
Hongmin Wu, Xiancai Zhong, Ning Ma, Zhiheng He, Guanpeng Wang, Geming Lu, Yate-Ching Yuan, Wencan Zhang, Yun Shi, Nagarajan Vaidehi, Evelyn Peelen, Tanja Wulff, Christian Gege, Hella Kohlhof, Daniel Vitt, Yousang Gwack, Ichiro Taniuchi, Hai-Hui Xue, Zuoming Sun
Hongmin Wu, Xiancai Zhong, Ning Ma, Zhiheng He, Guanpeng Wang, Geming Lu, Yate-Ching Yuan, Wencan Zhang, Yun Shi, Nagarajan Vaidehi, Evelyn Peelen, Tanja Wulff, Christian Gege, Hella Kohlhof, Daniel Vitt, Yousang Gwack, Ichiro Taniuchi, Hai-Hui Xue, Zuoming Sun
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Research Article Autoimmunity Immunology

Selective disruption of RORγt-CBFβ interaction by IMU-935 prevents RORγt-dependent Th17 autoimmunity but not thymocyte development

Abstract

RORγt is a key transcription factor regulating both Th17 differentiation and thymocyte development. Although Th17 cells drive autoimmune diseases, inhibiting RORγt to treat autoimmunity also disrupts thymocyte development and can cause lethal thymic lymphoma. We identified a previously unreported RORγt cofactor, CBFβ, and a highly selective RORγt inhibitor, IMU-935, that preferentially disrupt the RORγt-CBFβ interaction in Th17 cells but not thymocytes. This interaction is essential for RORγt function; mice with a RORγt mutant unable to bind CBFβ had impaired Th17 differentiation, were resistant to experimental autoimmune encephalomyelitis (EAE), and had defective thymocyte development. IMU-935 inhibited Th17 differentiation and reduced EAE severity without affecting thymocyte development by selectively targeting the RORγt-CBFβ interaction in Th17 cells but not in thymocytes. This differential effect arose because different concentrations of IMU-935 were required to disrupt the interaction in Th17 cells versus thymocytes, due to varying levels of RUNX1 that compete with RORγt for CBFβ binding. This study reveals an unreported mechanism for RORγt regulation and a selective RORγt inhibitor that prevents Th17-driven autoimmunity without the risk of lethal lymphoma from thymocyte disruption.

Authors

Hongmin Wu, Xiancai Zhong, Ning Ma, Zhiheng He, Guanpeng Wang, Geming Lu, Yate-Ching Yuan, Wencan Zhang, Yun Shi, Nagarajan Vaidehi, Evelyn Peelen, Tanja Wulff, Christian Gege, Hella Kohlhof, Daniel Vitt, Yousang Gwack, Ichiro Taniuchi, Hai-Hui Xue, Zuoming Sun

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Figure 6

IMU-935 inhibits RORγt’s interaction with cofactor CBFβ in Th17 cells but not in thymocytes.

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IMU-935 inhibits RORγt’s interaction with cofactor CBFβ in Th17 cells bu...
(A) Changes in Th17 differentiation after knockout of indicated genes, relative to nontargeting group (NonT; 100%) in Cas9-expressing CD4+ T cells (n = 3–10/group from 3 pooled independent experiments). (B) Relative luciferase activity from a promoterless control (pGL3) or RORγt reporter (RORBE) in HEK293T cells transfected with indicated expression plasmids for 24 hours. (C) A visualization of the computer-predicted interaction between RORγt and CBFβ (top left) or RUNX1 and CBFβ (top right). Dashed circle highlights the interaction interface between indicated proteins. The cyan fragment indicates the RORγt amino acids 403–413 critical for binding to CBFβ. The black box in the bottom panel indicates the amino acids with >40% contact frequency with CBFβ; the white box indicates <40% contact frequency. (D and E) IP analysis of the RORγt-CBFβ interaction in RORγt–/– CD4+ T cells expressing GFP (EV) or with Flag-RORγt polarized in Th17 conditions (D) in RORγt–/– or WT thymocytes (E) and treated with indicated concentrations of IMU-935. IP with anti–Flag-RORγt (D) or anti-RORγt (E) antibody and immunoblot with anti-CBFβ antibody. Input was analyzed by Western blot (bottom 2 panels). Right: The relative intensity of immunoprecipitated CBFβ band. (F) Flow cytometric analysis of RORγt (top), RUNX1 (middle), and CBFβ (bottom) levels in differentiated Th17 cells, peripheral naive CD4+ T cells, and different subsets of thymocytes. (G and H) IP analysis of the RORγt-CBFβ interaction in control (NonT) or Runx1-deleted Th17 cells (sgRunx1) (G) or thymocytes expressing EV or RUNX1 (H) treated with indicated concentrations of IMU-935, similar to what is described in D and E. Data represent 2 (F), 3 (H), or 4 (B, D, E, and G) independent experiments. Data were assessed by 1-way ANOVA with Dunnett’s (A, D, and E), Tukey’s (B) post hoc test, or 2-tailed Student’s t test (G and H). *P < 0.05; **P < 0.01.