Andrew J. Haak

Novel Rho/MRTF/SRF inhibitors block matrix-stiffness and TGF-β-induced fibrogenesis in human colonic myofibroblasts

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.

Targeting the Myofibroblast Genetic Switch: Inhibitors of Myocardin-Related Transcription Factor/Serum Response Factor–Regulated Gene Transcription Prevent Fibrosis in a Murine Model of Skin Injury

Systemic sclerosis (SSc), or scleroderma, similar to many fibrotic disorders, lacks effective therapies. Current trials focus on anti-inflammatory drugs or targeted approaches aimed at one of the many receptor mechanisms initiating fibrosis. In light of evidence that a myocardin-related transcription factor (MRTF)–and serum response factor (SRF)–regulated gene transcriptional program induced by Rho GTPases is essential for myofibroblast activation, we explored the hypothesis that inhibitors of this pathway may represent novel antifibrotics. MRTF/SRF-regulated genes show spontaneously increased expression in primary dermal fibroblasts from patients with diffuse cutaneous SSc. A novel small-molecule inhibitor of MRTF/SRF-regulated transcription (CCG-203971) inhibits expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and collagen 1 (COL1A2) in both SSc fibroblasts and in lysophosphatidic acid (LPA)–and transforming growth factor β (TGFβ)–stimulated fibroblasts. In vivo treatment with CCG-203971 also prevented bleomycin-induced skin thickening and collagen deposition. Thus, targeting the MRTF/SRF gene transcription pathway could provide an efficacious new approach to therapy for SSc and other fibrotic disorders.

Cellular mechanisms of tissue fibrosis. 8. Current and future drug targets in fibrosis: Focus on RHO GTPase-regulated gene transcription

Tissue fibrosis occurs with excessive extracellular matrix deposition from myofibroblasts, resulting in tissue scarring and inflammation. It is driven by multiple mediators, such as the G protein-coupled receptor ligands lysophosphatidic acid and endothelin, as well as signaling by transforming growth factor-β, connective tissue growth factor, and integrins. Fibrosis contributes to 45% of deaths in the developed world. As current therapeutic options for tissue fibrosis are limited and organ transplantation is the only effective treatment for end-stage disease, there is an imminent need for efficacious antifibrotic therapies. This review discusses the various molecular pathways involved in fibrosis. It highlights the Rho GTPase signaling pathway and its downstream gene transcription output through myocardin-related transcription factor and serum response factor as a convergence point for targeting this complex set of diseases.

Optimization of novel nipecotic bis(amide) inhibitors of the Rho/MKL1/SRF transcriptional pathway as potential anti-metastasis agents

CCG-1423 (1) is a novel inhibitor of Rho/MKL1/SRF-mediated gene transcription that inhibits invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. We recently reported the design and synthesis of conformationally restricted analogs (e.g., 2) with improved selectivity for inhibiting invasion versus acute cytotoxicity. In this study we conducted a survey of aromatic substitution with the goal of improving physicochemical parameters (e.g., ClogP, MW) for future efficacy studies in vivo. Two new compounds were identified that attenuated cytotoxicity even further, and were fourfold more potent than 2 at inhibiting PC-3 cell migration in a scratch wound assay. One of these (8a, CCG-203971, IC50=4.2 μM) was well tolerated in mice for 5 days at 100mg/kg/day i.p., and was able to achieve plasma levels exceeding the migration IC50 for up to 3 h.

Design and synthesis of tag-free photoprobes for the identification of the molecular target for CCG-1423, a novel inhibitor of the Rho/MKL1/SRF signaling pathway.

Summary CCG-1423 and related analogues represent a new class of inhibitors of Rho/MKL1/SRF-mediated gene transcription, a pathway that has been implicated in both cancer and fibrosis. The molecular target for these compounds is unknown. To facilitate its identification, a series of tag-free photoaffinity probes was designed and synthesized, each one containing a photoactivatable group and an acetylenic end group for subsequent attachment to a fluorescent tag using click chemistry. All were confirmed to maintain biological activity in a cell-based assay for inhibition of SRE-Luc expression. The functional activity of the most potent probe 24 was further confirmed in an assay for PC-3 cell migration. Photolysis of 24 in intact PC-3 cells followed by cell lysis, click ligation of a fluorescent dye, and gel electrophoresis revealed specific labeling of a single 24 kDa band that could be blocked with an active competitor. Future work will focus on identifying the labeled protein(s).

The Role of HTS in Drug Discovery at the University of Michigan

High throughput screening (HTS) is an integral part of a highly collaborative approach to drug discovery at the University of Michigan. The HTS lab is one of four core centers that provide services to identify, produce, screen and follow-up on biomedical targets for faculty. Key features of this system are: protein cloning and purification, protein crystallography, small molecule and siRNA HTS, medicinal chemistry and pharmacokinetics. Therapeutic areas that have been targeted include anti-bacterial, metabolic, neurodegenerative, cardiovascular, anti-cancer and anti-viral. The centers work in a coordinated, interactive environment to affordably provide academic investigators with the technology, informatics and expertise necessary for successful drug discovery. This review provides an overview of these centers at the University of Michigan, along with case examples of successful collaborations with faculty.

Pharmacological Inhibition of Myocardin-related Transcription Factor Pathway Blocks Lung Metastases of RhoC-Overexpressing Melanoma

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF transcription, we use a previously developed small-molecule inhibitor, CCG-203971, which at low micromolar concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971–mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes G1 cell-cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared with the low RhoC–expressing SK-Mel-19. Furthermore, pharmacologic inhibition of the MRTF pathway reduced both the number and size of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a novel approach to melanoma therapeutics. Mol Cancer Ther; 16(1); 193–204. ©2016 AACR.

Abstract 5030: Serum deprivation induces Ewing Sarcoma cells to adopt a more invasive phenotype and to upregulate RHO-MKL transcriptional activity.

Background: Ewing sarcoma (ES) is an aggressive and metastatic tumor of bone and soft tissue that primarily affects children and adolescents. The primary cause of tumor-related death in patients with ES is metastatic disease. In epithelial-derived cancers metastasis is dependent on phenotypic transition of cells from epithelial- to mesenchymal-like states (EMT). In addition, Rho signaling pathways are primary mediators of cancer cell motility and can induce cell migration and invasion, in part through transcriptional activation of MKL/SRF target genes. The mechanisms of metastasis in non-epithelial tumors such as ES remain to be elucidated. Objective: We sought to determine whether phenotypic plasticity between non-invasive and invasive states is also a feature of ES and to determine the molecular mechanism underlying this transition. Methods: ES cell lines were subjected to serum deprivation and phenotypic differences measured using scratch assays, transwell studies, and the xCelligence system. qRT-PCR, Western blot and immunofluorescence were used to quantitate or visualize changes in gene and protein expression, respectively. Results: Serum deprivation induced ES cells to rapidly adopt a more aggressive phenotype, reflected by changes in cell shape and increased motility, while proliferation was inhibited. Serum replenishment rapidly reverted these phenotypic changes. Mechanistically, markers of EMT were largely unaffected indicating that the observed phenotypic plasticity in ES cells was not mediated by classic EMT-MET transitions. Instead, activation of Rho-MKL signaling was apparent as evidenced by nuclear localization of MKL and upregulation of the metastasis-associated MKL/SRF target gene MYL9 in serum-deprived conditions. Exposure of ES cells to a small molecule antagonist of Rho-MKL signaling inhibited ES cell motility. Conclusions: Serum deprivation causes reversible transformation of ES cells to a more migratory phenotype. This is associated with nuclear translocation of MKL and induction of MYL9, a gene that encodes a regulatory light chain required for myosin stability. We propose a model of ES metastasis in which changes to the microenvironment that result in growth-factor deprivation (e.g. increased tumor size or cytotoxic therapy) induce expression of Rho-MKL transcriptional targets that contribute to cell migration and invasion. The Rho-MKL axis may thus be a novel therapeutic target for prevention of ES metastasis. Citation Format: Merlin Airik, Melanie Krook, Christopher Scannell, Andrew Haak, Richard Neubig, Elizabeth Lawlor. Serum deprivation induces Ewing Sarcoma cells to adopt a more invasive phenotype and to upregulate RHO-MKL transcriptional activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5030. doi:10.1158/1538-7445.AM2013-5030