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

Abstract

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.

Figure 1

Therapeutic administration of KD025 decreases cGVHD pathology. B10.BR mice transplanted with B6 BM and T cells were treated with varying doses of KD025 or 0.4% methylcellulose from days 28 to 56. (A) Day 60 pulmonary function tests. (B) Pathology scores for lung, liver, colon, and spleen on day 60 for mice treated with 150 mg/kg KD025. (C) Representative images of Masson’s Trichrome stain (left) and total mouse immunoglobulin (right) in the lungs of mice on day 60 and (D) quantifications. *P < .05; **P < .01; ***P < .001; ****P < .0001. Error bars represent standard error of the mean (SEM); data from 4 separate experiments with n = 8 per group.

Figure 2

KD025 inhibits Stat3 phosphorylation and increases Stat5 activation decreasing the germinal center response. Day 60 spleens from B10.BR mice transplanted with B6 BM and T cells were analyzed for the (A) frequency of GCs in situ, (B) T follicular regulatory cell frequency, (C) T follicular helper cell frequency, and (D) germinal center B cell frequency. (E) Western blots of whole splenocytes for phosphorylation of Stat3 or Stat5 normalized to actin and (F) IRF4, RORγt, and Bcl6, which are quantified from 5 blots from 2 independent experiments in G and H, respectively. *P < .05; **P < .01; ***P < .001; ****P < .0001. Error bars represent SEM; data from 3 separate experiments with n = 8 per group.

Figure 3

Stat3 expression in T cells is necessary for the development of cGVHD. B10.BR mice were transplanted with B6 WT BM and either WT or Stat3-deficient T cells. (A) Day 60 pulmonary function tests. (B) Pathology scores from lung, liver, spleen, and colon on mice killed on day 60. (C) Frequency of germinal centers in spleens of mice killed on day 60. (D) Frequency of T follicular helper cells on day 60. *P < .05; **P < .01; ***P < .001; ****P < .0001. Error bars represent SEM; data from 2 separate experiments with n = 8 per group.

Figure 4

Reversal of clinical signs of sclerodermatous cGVHD with KD025 treatment. BALB/c mice were transplanted with B10.D2 BM and T cells and given either 150 mg/kg KD025 or 0.4% methylcellulose vehicle control starting after initial signs of cGVHD and harvested on day 51 following transplantation. (A) Clinical skin scores, (B) skin pathology, (C) highlights of pathology (*epidermal hyperplasia and ^∨keratosis; bar, 500 μM), (D) clinical GVHD scores, and (E) percent weight loss. Whole spleens were assessed for Stat3 phosphorylation normalized to actin and expression of IRF4 (F and quantified in G and H, respectively). *P < .05; **P < .01. Error bars represent SEM; data from 2 separate experiments with n = 8 per group.

Figure 5

Immunologic effects of KD025 on human cGVHD. PBMCs from patients with active cGVHD were analyzed for their ability to produce (A) IL-21, (B) IL-17, and (C) IFNγ when treated with 2.5 μM, 10 μM, or DMSO vehicle for 48 hours. (D) The frequency of T follicular helper cells present and (E) expression of pStat3 normalized to actin, IRF4, and Bcl6 in samples treated with KD025, quantified in F, G, and H, respectively. *P < .05; **P < .01. Error bars represent standard deviation.