Objectives: Wnt pathway upregulation contributes to knee osteoarthritis (OA) through osteoblast differentiation, increased catabolic enzymes, and inflammation. The small-molecule Wnt pathway inhibitor, lorecivivint (SM04690), which previously demonstrated chondrogenesis and cartilage protection in an animal OA model, was evaluated to elucidate its mechanism of action. Design: Biochemical assays measured kinase activity. Western blots measured protein phosphorylation in human mesenchymal stem cells (hMSCs), chondrocytes, and synovial fibroblasts. siRNA knockdown effects in hMSCs and BEAS-2B cells on Wnt pathway, chondrogenic genes, and LPS-induced inflammatory cytokines was measured by qPCR. In vivo anti-inflammation, pain, and function were evaluated following single intra-articular (IA) lorecivivint or vehicle injection in the monosodium iodoacetate (MIA)-induced rat OA model. Results: Lorecivivint inhibited intranuclear kinases CDC-like kinase 2 (CLK2) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Lorecivivint inhibited CLK2-mediated phosphorylation of serine/arginine-rich (SR) splicing factors and DYRK1A-mediated phosphorylation of SIRT1 and FOXO1. siRNA knockdowns identified a role for CLK2 and DYRK1A in Wnt pathway modulation without affecting b-catenin with CLK2 inhibition inducing early chondrogenesis and DYRK1A inhibition enhancing mature chondrocyte function. NF-kB and STAT3 inhibition by lorecivivint reduced inflammation. DYRK1A knockdown was sufficient for anti-inflammatory effects, while combined DYRK1A/CLK2 knockdown enhanced this effect. In the MIA model, lorecivivint inhibited production of inflammatory cytokines and cartilage degradative enzymes, resulting in increased joint cartilage, decreased pain, and improved weight-bearing function. Conclusions: Lorecivivint inhibition of CLK2 and DYRK1A suggested a novel mechanism for Wnt pathway inhibition, enhancing chondrogenesis, chondrocyte function, and anti-inflammation. Lorecivivint shows potential to modify structure and improve symptoms of knee OA.

The Wnt pathway is upregulated in tendinopathy, affecting inflammation and tenocyte differentiation. Given its potential role in tendinopathy, this signaling pathway may be a relevant target for treatment. The current study examined the therapeutic potential of SM04755, a topical, small-molecule Wnt pathway inhibitor, for the treatment of tendinopathy using in vitro assays and animal models. In vitro, SM04755 decreased Wnt pathway activity, induced tenocyte differentiation, and inhibited catabolic enzymes and pro-inflammatory cytokines in human mesenchymal stem cells, rat tendon-derived stem cells, and human peripheral blood mononuclear cells. Evaluation of the mechanism of action of SM04755 by biochemical profiling and computational modeling identified CDC-like kinase 2 (CLK2) and dualspecificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as molecular targets. CLK and DYRK1A inhibition by siRNA knockdown or pharmacological inhibition induced tenocyte differentiation and reduced tenocyte catabolism. In vivo, topically applied SM04755 showed therapeutically relevant exposure in tendons with low systemic exposure and no detectable toxicity in rats. Moreover, SM04755 showed reduced tendon inflammation and evidence of tendon regeneration, decreased pain, and improved weight-bearing function in rat collagenase-induced tendinopathy models compared with vehicle control. Together, these data demonstrate that CLK2 and DYRK1A inhibition by SM04755 resulted in Wnt pathway inhibition, enhanced tenocyte differentiation and protection, and reduced inflammation. SM04755 has the potential to benefit symptoms and modify disease processes in tendinopathy.

Purpose: In the synovial joint, Wnt pathway upregulation contributes to osteoarthritis (OA) through increasing osteocyte differentiation, cartilage thinning, and inflammation. SM04690, a novel small molecule, has previously demonstrated OA disease modifying potential through Wnt pathway inhibition in vitro and in vivo. Further studies haveelucidated a novel mechanism of action for SM04690, leading to Wnt pathway inhibition, chondrocyte differentiation, and anti-inflammatory activity. Methods: Wnt pathway activity was measured using a cell-based luciferase reporter assay controlled by a TCF/LEF promoter in SW480 colon cancer cells, a mutant cell line exhibiting active Wnt signaling. A kinome screen (318 kinases) and biochemical kinase assays were performed. SM04690 effects on protein phosphorylation of serine and arginine rich splicing factor (SRSF) proteins, Sirt1, and FoxO1, in hMSCs, chondrocytes and synovial fibroblasts were measured using Western blot. SM04690 and siRNA knockdown effects on chondrogenic and Wnt pathway gene expression were measured using nCounter® gene expression panels (NanoString Technologies), and effects on LPS-induced inflammatory cytokines (IL-6, IL-8, TNF-a) expression in BEAS-2B cells were measured by qPCR and ELISA. In vivo, SM04690 (0.1 mg, 0.3 mg, 1.0 mg) pharmacodynamic effects were evaluated in rat knee OA models: (1) An inflammatory monosodium iodoacetate (MIA) injection-induced knee OA model and (2) a surgical anterior cruciate ligament transection with partial medial meniscectomy (ACLTþpMMX) model, followed by single intra-articular SM04690 or vehicle injection. Knee cartilage was isolated at Days 10 and 35, and phosphorylation and expression of SRSF proteins, NF-kB, STAT3, and Sirt1 measured by Western blot. Statistical analyses used one-way ANOVA for multiple group comparisons and t-tests for comparison between two groups. Results: SM04690 was a potent (EC 50 ¼11nM) inhibitor of Wnt signaling. Biochemical assays identified cdc-like kinases (CLKs) and dualspecificity tyrosine kinase (DYRK1A) as molecular targets of SM04690. SM04690 potently inhibited CLK-mediated phosphorylation of SRSF proteins compared with DMSO control. Separately, SM04690 inhibited DYRK1A-mediated phosphorylation of Sirt1 and FoxO1 resulting in increased levels of total and nuclear FoxO1 compared with DMSO.

Background:Tendinopathy is an inflammatory and degenerative disorder of tendons caused by injuries and/or overuse. Left untreated, tendinopathy can lead to pain and rupture. Current therapeutic options only treat symptoms. Stem cell- and growth factor-based treatments are under investigation but have not established safety or efficacy, leaving considerable unmet need. The Wnt pathway is upregulated in chronic tendinopathy, affecting inflammation and tenocyte differentiation. SM04755, a novel, topical, small-molecule Wnt pathway inhibitor, has previously been shown to inhibit inflammation, protect tenocytes, and increase tenocyte differentiation in nonclinical models.1Objectives:To identify molecular targets of SM04755 and its associated mechanism of action.Methods:Wnt pathway inhibition was measured using a cell-based luciferase reporter assay controlled by a β-catenin/TCF-responsive promoter in SW480 colon cancer cells. A kinome screen (318 kinases) and kinase assays were performed. Effects of SM04755 on phosphorylation of proteins, including serine/arginine-rich splicing factor (SRSF) proteins in rat tendon-derived stem cells (rTDSCs) and peripheral blood mononuclear cells (PBMCs), were measured using western blot. SiRNA-mediated knockdown of CDC-like kinases (CLKs) and dual-specificity tyrosine kinase (DYRK1A) were performed in human mesenchymal stem cells (hMSCs), rTDSCs, and rat tenocytes. Effects of SM04755 and siRNA knockdowns on Wnt pathway gene expression and catabolic enzymes (MMPs) were measured using qPCR. SM04755 and siRNA effects on tenocyte marker expression were assessed by qPCR and immunostaining. Effects of SM04755 on LPS-induced expression of inflammatory cytokines in PBMCs were measured by MSD-based ELISA. Statistical analyses used one-way ANOVA for multiple group comparisons and t-tests for comparison between two groups.Results:SM04755 was a potent inhibitor (EC50=156 nM) of Wnt signaling. Biochemical assays identified CLKs and DYRK1A as molecular targets of SM04755. SM04755 potently inhibited CLK-mediated phosphorylation of SRSF proteins compared with DMSO controls. Knockdowns of CLKs and DYRK1A led to inhibition of Wnt pathway genes (AXIN2,LEF1,TCF4,TCF7, etc.) compared with siRNA controls (siCtrl). CLK1, 2, and 4 and DYRK1A knockdowns also induced expression of tenocyte markers in rTDSCs and inhibited IL-1β-induced expression of catabolic enzymes (MMP1, 3, 9, 13) in tenocytes compared with siCtrl. SM04755 treatment of LPS-stimulated PBMCs resulted in reduced phosphorylation of NF-kB and STAT3 and inhibited production of inflammatory cytokines compared with DMSO.Conclusion:SM04755 inhibited CLKs and DYRK1A, which led to Wnt pathway modulation. Knockdowns of CLKs and DYRK1A, compared with control siRNA, induced tenocyte differentiation and reduced tendon-destroying proteases in tenocytes. This supports the potential disease modification of tendinopathy with SM04755. Furthermore, the anti-inflammatory effects of SM04755 are mechanistically supported by the decreased phosphorylation of STAT3 and NF-kB. These data support that SM04755, as a single agent, may potentially improve symptoms and provide disease modification in tendinopathy. Human tendinopathy trials are planned.References:[1]Deshmukh et al.Arthritis and Rheum. 2016Acknowledgments:Brian Hofilena for chemistry, Josh Stewart for pharmacokinetics, and Luis Dellamary for formulation.Disclosure of Interests:Vishal Deshmukh Shareholder of: Samumed, LLC, Employee of: Samumed, LLC, Alyssa O’Green Shareholder of: Samumed, LLC, Employee of: Samumed, LLC, Tim Seo Shareholder of: Samumed, LLC, Employee of: Samumed, LLC, Yusuf Yazici Shareholder of: Samumed, LLC, Grant/research support from: Bristol-Myers Squibb, Celgene, and Genentech, Consultant of: Celgene and Sanofi, Employee of: Samumed, LLC

BackgroundIn the synovial joint, Wnt pathway upregulation contributes to osteoarthritis (OA) by increasing osteocyte differentiation, cartilage thinning, and inflammation. SM04690, a novel small molecule, has previously demonstrated potential OA disease-modifying effects through Wnt pathway inhibition in vitro and in vivo.ObjectivesTo elucidate the novel mechanism of action for SM04690 on Wnt pathway inhibition, chondrocyte differentiation, and anti-inflammation.MethodsWnt pathway activity was measured using a cell-based TCF/LEF luciferase reporter in SW480 colon cancer cells. A kinome screen (318 kinases) was performed. The effects of SM04690 on protein phosphorylation of serine and arginine rich splicing factors (SRSF proteins), Sirt1, and FoxO1 in hMSCs, chondrocytes, and synovial fibroblasts were measured by Western blot. The effects of SM04690 and siRNA knockdown (KD) on chondrogenic and Wnt pathway gene expression were measured by NanoString gene expression panels and effects on LPS-induced inflammatory cytokines (IL-6, IL-8, TNF-α) in BEAS-2B cells were measured by qPCR and ELISA. In vivo, the pharmacodynamic effects of SM04690 were evaluated in monosodium iodoacetate injection-induced and anterior cruciate ligament transection with partial medial meniscectomy rat knee OA models in which a single intra-articular SM04690 (0.1 µg, 0.3 µg, 1.0 µg) or vehicle injection was administered. Cartilage was isolated at Day 10 and 35; phosphorylation and expression of SRSF proteins, Sirt1, FoxO1, STAT3, and NF-κB were measured by Western blot.ResultsSM04690 was a potent (EC50=11nM) inhibitor of Wnt signaling. Cdc-like kinases (CLKs) and dual-specificity tyrosine kinase (DYRK1A) were identified as molecular targets of SM04690. In hMSCs and chondrocytes, compared to DMSO, SM04690 potently inhibited CLK-mediated phosphorylation of SRSF proteins. SM04690 also inhibited DYRK1A-mediated Sirt1 and FoxO1 phosphorylation, thus increasing total and nuclear FoxO1 levels. Compared to siRNA control, DYRK1A/CLK2 dual KD increased expression of chondrogenic genes (COL2A1, ACAN, COMP, CD44 [all P<0.05]). CLK2 and DYRK1A KDs each inhibited Wnt pathway genes (AXIN2, TCF7, TCF4, LRP5, FZD6, FZD7, PITX2 [all P<0.05]) with no effects on β-catenin levels, compared to siRNA control. In synovial fibroblasts, compared to DMSO, SM04690 decreased phosphorylation of NF-kB and STAT3. In BEAS-2B cells, compared to siRNA control, DYRK1A KD inhibited inflammatory cytokine production (IL-6, IL-8, TNF-α [all P<0.05]), while DYRK1A/CLK2 dual KD enhanced anti-inflammatory effects of DYRK1A KD. In cartilage from rat OA models, compared to vehicle, SM04690 inhibited phosphorylation of SRSF proteins, Sirt1, FoxO1, and STAT3, as well as expression of NF-κB.ConclusionTo our knowledge, this is the first report of SM04690 inhibition of nuclear kinases CLK2 and DYRK1A, leading to effects on the Wnt pathway, chondrocytes, and inflammation (Figure 1). This dual mechanism of SM04690 potentially modifies OA through increased chondrocyte differentiation and function ...