Myofibroblasts are crucial to the pathogenesis of tissue fibrosis. Their formation of stress fibers results in the release of myocardin-related transcription factor (MRTF), a transcriptional coactivator of serum response factor (SRF). MRTF-A (Mkl1)-deficient mice are protected from lung fibrosis. We hypothesized that the SRF/MRTF pathway inhibitor CCG-203971 would modulate myofibroblast function in vitro and limit lung fibrosis in vivo. Normal and idiopathic pulmonary fibrosis lung fibroblasts were treated with/without CCG-203971 (N-[4-chlorophenyl]-1-[3-(2-furanyl)benzoyl]-3-piperidine carboxamide) and/or Fas-activating antibody in the presence/absence of transforming growth factor (TGF)-β1, and apoptosis was assessed. In vivo studies examined the effect of therapeutically administered CCG-203971 on lung fibrosis in two distinct murine models of fibrosis induced by bleomycin or targeted type II alveolar epithelial injury. In vitro, CCG-203971 prevented nuclear localization of MRTF-A; increased the apoptotic susceptibility of normal and idiopathic pulmonary fibrosis fibroblasts; blocked TGF-β1-induced myofibroblast differentiation; and inhibited TGF-β1-induced expression of fibronectin, X-linked inhibitor of apoptosis, and plasminogen activator inhibitor-1. TGF-β1 did not protect fibroblasts or myofibroblasts from apoptosis in the presence of CCG-203971. In vivo, CCG-203971 significantly reduced lung collagen content in both murine models while decreasing alveolar plasminogen activator inhibitor-1 and promoting myofibroblast apoptosis. These data support a central role of the SRF/MRTF pathway in the pathobiology of lung fibrosis and suggest that its inhibition can help resolve lung fibrosis by promoting fibroblast apoptosis.

Background Ras homolog gene family, member A (RhoA)/Rho-associated coiled-coil forming protein kinase signaling is a key pathway in multiple types of solid organ fibrosis, including intestinal fibrosis. However, the pleiotropic effects of RhoA/Rho-associated coiled-coil forming protein kinase signaling have frustrated targeted drug discovery efforts. Recent recognition of the role of Rho-regulated gene transcription by serum response factor (SRF) and its transcriptional cofactor myocardin-related transcription factor A (MRTF-A) suggest a novel locus for pharmacological intervention. Methods Because RhoA signaling is mediated by both physical and biochemical stimuli, we examined whether pharmacological inhibition of RhoA or the downstream transcription pathway of MRTF-A/SRF could block intestinal fibrogenesis in 2 in vitro models. Results In this study, we demonstrate that inhibition of RhoA signaling blocks both matrix-stiffness and transforming growth factor beta–induced fibrogenesis in human colonic myofibroblasts. Repression of alpha-smooth muscle actin and collagen expression was associated with the inhibition of MRTF-A nuclear localization. CCG-1423, a first-generation Rho/MRTF/SRF pathway inhibitor, repressed fibrogenesis in both models, yet has unacceptable cytotoxicity. Novel second-generation inhibitors (CCG-100602 and CCG-203971) repressed both matrix-stiffness and transforming growth factor beta–mediated fibrogenesis as determined by protein and gene expression in a dose-dependent manner. Conclusions Targeting the Rho/MRTF/SRF mechanism with second-generation Rho/MRTF/SRF inhibitors may represent a novel approach to antifibrotic therapeutics.

Background Crohn’s disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods The stiffness of fresh ex vivo samples from normal human small intestine, Crohn’s disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin (αSMA) staining, and gene expression. Results Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures as well as between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased αSMA protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase and pro-inflammatory gene expression, and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions Matrix stiffness, representative of the pathological stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways suggesting the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to autopropagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease.

Background & Aims Intestinal fibrosis is a critical complication of Crohn’s disease (CD). Current in vitro models of intestinal fibrosis cannot model the complex intestinal architecture, while in vivo rodent models do not fully recapitulate human disease and have limited utility for large-scale screening. Here, we exploit recent advances in stem cell derived human intestinal organoids (HIOs) as a new human model of fibrosis in CD. Methods Human pluripotent stem cells were differentiated into HIOs. We identified myofibroblasts, the key effector cells of fibrosis, by immunofluorescence staining for alpha-smooth muscle actin (αSMA), vimentin, and desmin. We examined the fibrogenic response of HIOs by treatment with transforming growth factor beta (TGFβ) in the presence or absence of the anti-fibrotic drug spironolactone. Fibrotic response was assayed by expression of fibrogenic genes (COL1A1 (collagen, type I, alpha 1), ACTA2 (alpha smooth muscle actin), FN1 (fibronectin 1), MYLK (myosin light chain kinase), and MKL1 (megakaryoblastic leukemia (translocation) 1)) and proteins (αSMA). Results Immunofluorescent staining of organoids identified a population of myofibroblasts within the HIO mesenchyme. TGFβ stimulation of HIOs produced a dose-dependent pro-fibrotic response. Spironolactone treatment blocked the fibrogenic response of HIOs to TGFβ. Conclusions HIOs contain myofibroblasts and respond to a pro-fibrotic stimulus in a manner that is consistent with isolated human myofibroblasts. HIOs are a promising model system that might bridge the gap between current in vitro and in vivo models of intestinal fibrosis in IBD.

Purpose: Lineage plasticity in prostate cancer-most commonly exemplified by loss of androgen receptor (AR) signaling and a switch from a luminal to alternate differentiation program-is now recognized as a treatment resistance mechanism. Lineage plasticity is a spectrum, but neuroendocrine prostate cancer (NEPC) is the most virulent example. Currently there are limited treatments for NEPC. Moreover, the incidence of treatment-emergent NEPC (t-NEPC) is increasing in the era of novel AR inhibitors. In contradistinction to de novo NEPC, t-NEPC tumors often express the AR, but AR's functional role in t-NEPC is unknown.Furthermore, targetable factors that promote t-NEPC lineage plasticity are also unclear.Experimental Design: Using an integrative systems biology approach, we investigated enzalutamide-resistant t-NEPC cell lines and their parental, enzalutamide-sensitive adenocarcinoma cell lines. The AR is still expressed in these t-NEPC cells, enabling us to determine the role of the AR and other key factors in regulating t-NEPC lineage plasticity.Results: AR inhibition accentuates lineage plasticity in t-NEPC cells-an effect not observed in parental enzalutamide-sensitive adenocarcinoma cells. Induction of an AR-repressed, lineage plasticity program is dependent on activation of the transcription factor E2F1 in concert with the BET bromodomain chromatin reader BRD4. BET inhibition (BETi) blocks this E2F1/BRD4regulated program and decreases growth of t-NEPC tumor models and a subset of t-NEPC patient tumors with high activity of this program in a BETi clinical trial.Conclusions: E2F1 and BRD4 are critical for activating an AR-repressed t-NEPC lineage plasticity program. BETi is a promising approach to block this program.Research.

Intestinal fibrosis is one of the major complications of Crohn's disease (CD) for which there are no effective pharmacological therapies. Vitamin D deficiency is common in CD, though it is not known whether this is a contributing factor to fibrosis, or simply a consequence of the disease itself. In CD, fibrosis is mediated mainly by activated intestinal myofibroblasts during remodeling of extracellular matrix in response to wound healing. We investigated the effects of CARD-024 (1-alpha-hydroxyvitamin D5), a vitamin D analog with minimal hypercalcemic effects, on the pro-fibrotic response of intestinal myofibroblasts to two fibrogenic stimuli: TGFβ stimulation and culture on a physiologically stiff matrix. TGFβ stimulated a fibrogenic phenotype in Ccd-18co colonic myofibroblasts, characterized by an increase in actin stress fibers and mature focal adhesions, and increased αSMA protein expression, while CARD-024 repressed αSMA protein expression in a dose-dependent manner. Culture of colonic myofibroblasts on physiological high stiffness substrates induced morphological changes with increased actin stress fibers and focal adhesion staining, induction of αSMA protein expression, FAK phosphorylation, induction of fibrogenic genes, and repression of COX-2 and IL-1β. CARD-024 treatment repressed the stiffness-induced morphological features including stellate cell morphology and the maturation of focal adhesions. CARD-024 repressed the stiffness-mediated induction of αSMA protein expression, FAK phosphorylation, and MLCK and ET-1 gene expression. In addition, CARD-024 partially stimulated members of the COX-2/IL-1β inflammatory pathway. In summary, CARD-024 attenuated the pro-fibrotic response of colonic myofibroblasts to high matrix stiffness, suggesting that vitamin D analogs such as CARD-024 may ameliorate intestinal fibrosis.

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Background Fibrosis is the final common pathway to intestinal failure in Crohn’s disease, but no medical therapies exist to treat intestinal fibrosis. Activated myofibroblasts are key effector cells of fibrosis in multiple organ systems, including the intestine. AXL is a receptor tyrosine kinase that has been implicated in fibrogenic pathways involving myofibroblast activation. We aimed to investigate the AXL pathway as a potential target for the treatment of intestinal fibrosis. Methods To establish proof of concept, we first analyzed AXL gene expression in 2 in vivo models of intestinal fibrosis and 3 in vitro models of intestinal fibrosis. We then tested whether pharmacological inhibition of AXL signaling could reduce fibrogenesis in 3 in vitro models of intestinal fibrosis. In vitro testing included 2 distinct cell culture models of intestinal fibrosis (matrix stiffness and TGF-β1 treatment) and a human intestinal organoid model using TGF-β1 cytokine stimulation. Results Our findings suggest that the AXL pathway is induced in models of intestinal fibrosis. We demonstrate that inhibition of AXL signaling with the small molecule inhibitor BGB324 abrogates both matrix-stiffness and transforming growth factor beta (TGF-β1)–induced fibrogenesis in human colonic myofibroblasts. AXL inhibition with BGB324 sensitizes myofibroblasts to apoptosis. Finally, AXL inhibition with BGB324 blocks TGF-β1-induced fibrogenic gene and protein expression in human intestinal organoids. Conclusions The AXL pathway is active in multiple models of intestinal fibrosis. In vitro experiments suggest that inhibiting AXL signaling could represent a novel approach to antifibrotic therapy for intestinal fibrosis such as in Crohn’s disease.