Modulation of the Wnt pathway through inhibition of CLK2 and DYRK1A by lorecivivint as a novel, potentially disease-modifying approach for knee osteoarthritis treatment

Abstract

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.

Review Article: Is Wnt Signaling an Attractive Target for the Treatment of Osteoarthritis?

“…Systemic levels of the drug were extremely low. Intra-articular injection also provided clear protection against osteoarthritis development in rats both in a surgical instability model [42] and in model characterized by inflammation and pain [43], thus paving the way for human trials.…”

“…Further cartilage protection could be conferred by the inhibitory effects of lorecivivint on the expression of tissue destructive enzymes from the matrix metalloproteinase family [42]. Of note, the compound also demonstrated antiinflammatory effects by reducing the expression of cytokines such as interleukin-1b, tumor necrosis factor and interleukin-6 in synovial fibroblasts and in peripheral blood mononuclear cells [43], two groups of cells that can play a role in the inflammatory reaction that is often seen in patients with osteoarthritis. These promising results prompted the use of lorecivivint in in vivo models of osteoarthritis [42].…”

“…4). The compound works as a kinase inhibitor and was demonstrated to mainly affect the activity of CDC-like kinase enzymes (CLKs) [43], in particular CLK2, in nanomolar concentrations, and dual-specificity tyrosine phosphorylation-regulated kinase enzymes (DYRK) [43], in particular DYRK1A, again in nanomolar concentrations. These molecules had not yet been clearly associated with Wnt signaling, thereby requiring further investigations.…”

“…Inhibition of CLK2 affects this process and results in abnormal transcripts that may have dominant negative effects on the signaling cascade. Experimental data suggest that the effects on Wnt signaling transcription factors reduce the levels of Wnt target genes and simultaneously increase the levels of markers associated with the healthy chondrocyte profile such as aggrecan, collagen type 2, and DOT1L [43]. Moreover, negative modulation of CLK2 activity also appears to inhibit NF-kB signaling, a strong pro-inflammatory cascade.…”

“…Interestingly, in addition to inhibition of CLK2, effects on DYRK1A offers an interesting perspective on the mechanism of action. DYRK1A is associated with anti-inflammatory effects as it affects the [43]. Moreover, DYRK1A also affects activity of transcription factor FOXO1 and of epigenetic modulator SIRT1 [43].…”