Selectivity matters: selective ROCK2 inhibitor ameliorates established liver fibrosis via targeting inflammation, fibrosis, and metabolism.

The pathogenesis of hepatic fibrosis is driven by dysregulated metabolism precipitated by chronic inflammation. Rho-associated coiled-coil-containing protein kinases (ROCKs) have been implicated in these processes, however the ability of selective ROCK2 inhibition to target simultaneously profibrotic, pro-inflammatory and metabolic pathways remains undocumented. Here we show that therapeutic administration of GV101, a selective ROCK2 inhibitor with more than 1000-fold selectivity over ROCK1, attenuates established liver fibrosis induced by thioacetamide (TAA) in combination with high-fat diet in mice. GV101 treatment significantly reduces collagen levels in liver, associated with downregulation of pCofilin, pSTAT3, pAkt, while pSTAT5 and pAMPK levels are increased in tissues of treated mice. In vitro, GV101 inhibits profibrogenic markers expression in fibroblasts, adipogenesis in primary adipocytes and TLR-induced cytokine secretion in innate immune cells via targeting of Akt-mTOR-S6K signaling axis, further uncovering the ROCK2-specific complex mechanism of action and therapeutic potential of highly selective ROCK2 inhibitors in liver fibrosis.

ROCK2, but not ROCK1 interacts with phosphorylated STAT3 and co-occupies TH17/TFH gene promoters in TH17-activated human T cells.

Rho-associated coiled-coil kinase (ROCK)2 targeting down-regulates autoimmune responses in animal models and patients, however the underlying molecular mechanism is still an enigma. We report that ROCK2 binds phosphorylated-STAT3 and its kinase activity controls the formation of ROCK2/STAT3/JAK2 complex and optimal STAT3 phosphorylation in human CD4+ T cells during T helper 17 (TH17)-skewing. Moreover, chromatin-immunoprecipitation sequencing (ChIP-seq) analysis revealed that, genome-wide, about 70% of ROCK2 and STAT3 peaks overlapped and co-localized to several key genes controlling TH17 and T follicular helper (TFH) cell functions. Specifically, the co-occupancy of ROCK2 and STAT3 on the Irf4 and Bcl6 genes was validated by ChIP-qPCR analysis. Furthermore, the binding of ROCK2 to both the Irf4 and Bcl6 promoters was attenuated by STAT3 silencing as well as by selective ROCK2 inhibitor. Thus, the present study demonstrated previously unidentified evidence that ROCK2-mediated signaling in the cytosol provides a positive feed-forward signal for nuclear ROCK2 to be recruited to the chromatin by STAT3 and potentially regulates TH17/TFH gene transcription.

The selective ROCK2 inhibitor KD025 reduces IL-17 secretion in human peripheral blood mononuclear cells independent of IL-1 and IL-6.

Reducing the activities of the pro-inflammatory cytokine IL-17 is an effective treatment strategy for several chronic autoimmune disorders. Rho-associated coiled-coil containing kinase 2 (ROCK2) is a member of the serine-threonine protein kinase family that regulates IL-17 secretion in T cells via signal transducer and activator of transcription 3 (STAT3)-dependent mechanism. We reported here that the selective ROCK2 inhibitor KD025 significantly reduced in vitro production of IL-17 in unfractionated human peripheral blood mononuclear cells (PBMCs) stimulated with the dectin-1 agonist Candida albicans. C. albicans induced IL-17 was reduced by 70% (p < 0.0001); a similar reduction (80%) was observed in PBMC stimulated with the Toll-like receptor 2 agonist Staphylococcus epidermidis (p < 0.0001). Treatment of PBMC with KD025 was not associated with a reduction in IL-1ß, IL-6 or IL-1α levels; in contrast, a 1.5 fold increase in the level of IL-1 receptor antagonist (IL-1Ra) was observed (p < 0.001). KD025 down-regulated C. albicans-induced Myosin Light Chain and STAT3, whereas STAT5 phosphorylation increased. Using anti-CD3/CD28 activation of the TCR, KD025 similarly suppressed IL-17 independent of a reduction in IL-1ß. Thus, ROCK2 directly regulates IL-17 secretion independent of endogenous IL-1 and IL-6 supporting development of selective ROCK2 inhibitors for treatment of IL-17-driven inflammatory diseases.

Cutting Edge: Selective Oral ROCK2 Inhibitor Reduces Clinical Scores in Patients with Psoriasis Vulgaris and Normalizes Skin Pathology via Concurrent Regulation of IL-17 and IL-10.

Targeted inhibition of Rho-associated kinase (ROCK)2 downregulates the proinflammatory T cell response while increasing the regulatory arm of the immune response in animals models of autoimmunity and Th17-skewing human cell culture in vitro. In this study, we report that oral administration of a selective ROCK2 inhibitor, KD025, reduces psoriasis area and severity index scores by 50% from baseline in 46% of patients with psoriasis vulgaris, and it decreases epidermal thickness as well as T cell infiltration in the skin. We observed significant reductions of IL-17 and IL-23, but not IL-6 and TNF-α, whereas IL-10 levels were increased in peripheral blood of clinical responders after 12 wk of treatment with KD025. Collectively, these data demonstrate that an orally available selective ROCK2 inhibitor downregulates the Th17-driven autoimmune response and improved clinical symptoms in psoriatic patients via a defined molecular mechanism that involves concurrent modulation of cytokines without deleterious impact on the rest of the immune system.

ROCK2 signaling is required to induce a subset of T follicular helper cells through opposing effects on STATs in autoimmune settings.

Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) cells, which help to generate antibody-producing B cells under normal and autoimmune conditions. Inhibiting ROCK2 in normal human T cells or peripheral blood mononuclear cells from patients with active systemic lupus erythematosus (SLE) decreased the number and function of TFH cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity decreased the abundance of the transcriptional regulator Bcl6 (B cell lymphoma 6) and increased that of Blimp1 by reducing the binding of signal transducer and activator of transcription 3 (STAT3) and increasing that of STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a twofold reduction in the numbers of TFH cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, which are the sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of TFH cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation.

Isoform-specific targeting of ROCK proteins in immune cells.

Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform plays an exclusive role in controlling of T-cell plasticity and macrophage polarization. Specifically, selective ROCK2 inhibition shifts the balance between pro-inflammatory and regulatory T-cell subsets via concurrent regulation of STAT3 and STAT5 phosphorylation, respectively. Furthermore, the administration of an orally available selective ROCK2 inhibitor effectively ameliorates clinical manifestations in experimental models of autoimmunity and chronic graft-vs.-host disease (cGVHD). Because ROCK2 inhibition results in the suppression of M2-type macrophages while favoring polarization of M1-type macrophages, ROCK2 inhibition can correct the macrophage imbalance seen during age-related macular degeneration (AMD). In summary, the exclusive role of ROCK2 in immune system modulation argues for the development and testing of isoform-specific ROCK2 inhibitors for the treatment of inflammatory disorders.

Targeted Rho-associated kinase 2 inhibition suppresses murine and human chronic GVHD through a Stat3-dependent mechanism.

Chronic graft-versus-host disease (cGVHD) remains a major complication following allogeneic bone marrow transplantation (BMT). The discovery of novel therapeutics is dependent on assessment in preclinical murine models of cGVHD. Rho-associated kinase 2 (ROCK2) recently was shown to be implicated in regulation of interleukin-21 (IL-21) and IL-17 secretion in mice and humans. Here, we report that the selective ROCK2 inhibitor KD025 effectively ameliorates cGVHD in multiple models: a full major histocompatibility complex (MHC) mismatch model of multiorgan system cGVHD with bronchiolitis obliterans syndrome and a minor MHC mismatch model of sclerodermatous GVHD. Treatment with KD025 resulted in normalization of pathogenic pulmonary function, which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. Spleens of mice treated with KD025 had decreased frequency of T follicular helper cells and increased frequency of T follicular regulatory cells, accompanied by a reduction in signal transducer and activator of transcription 3 (STAT3) and concurrent increase in STAT5 phosphorylation. The critical role of STAT3 in this cGVHD model was confirmed by data showing that mice transplanted with inducible STAT3-deficient T cells had pulmonary function comparable to the healthy negative controls. The therapeutic potential of targeted ROCK2 inhibition in the clinic was solidified further by human data demonstrating the KD025 inhibits the secretion of IL-21, IL-17, and interferon γ along with decreasing phosphorylated STAT3 and reduced protein expression of interferon regulatory factor 4 and B-cell lymphoma 6 (BCL6) in human peripheral blood mononuclear cells purified from active cGVHD patients. Together these data highlight the potential of targeted ROCK2 inhibition for clinical cGVHD therapy.

ROCKing cytokine secretion balance in human T cells.

Balanced regulation of cytokine secretion in T cells is critical for maintenance of immune homeostasis and prevention of autoimmunity. The Rho-associated kinase (ROCK) 2 signaling pathway was previously shown to be involved in controlling of cellular movement and shape. However, recent work from our group and others has demonstrated a new and important role of ROCK2 in regulating cytokine secretion in T cells. We found that ROCK2 promotes pro-inflammatory cytokines such as IL-17 and IL-21, whereas IL-2 and IL-10 secretion are negatively regulated by ROCK2 under Th17-skewing activation. Also, in disease, but not in steady state conditions, ROCK2 contributes to regulation of IFN-γ secretion in T cells from rheumatoid arthritis patients. Thus, ROCK2 signaling is a key pathway in modulation of T-cell mediated immune responses underscoring the therapeutic potential of targeted inhibition of ROCK2 in autoimmunity.

Selective oral ROCK2 inhibitor down-regulates IL-21 and IL-17 secretion in human T cells via STAT3-dependent mechanism.

Rho-associated kinase 2 (ROCK2) regulates the secretion of proinflammatory cytokines and the development of autoimmunity in mice. Data from a phase 1 clinical trial demonstrate that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects down-regulates the ability of T cells to secrete IL-21 and IL-17 by 90% and 60%, respectively, but not IFN-γ in response to T-cell receptor stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, significantly diminished STAT3 phosphorylation and binding to IL-17 and IL-21 promoters and reduced IFN regulatory factor 4 and nuclear hormone RAR-related orphan receptor γt protein levels in T cells derived from healthy subjects or rheumatoid arthritis patients. Simultaneously, treatment with KD025 also promotes the suppressive function of regulatory T cells through up-regulation of STAT5 phosphorylation and positive regulation of forkhead box p3 expression. The administration of KD025 in vivo down-regulates the progression of collagen-induced arthritis in mice via targeting of the Th17-mediated pathway. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between proinflammatory and regulatory T-cell subsets. Targeting of ROCK2 in man may therefore restore disrupted immune homeostasis and have a role in the treatment of autoimmunity.