Inhibition of histone demethylases as an approach to restore deficient DOT1L activity in osteoarthritis

Abstract

Purpose: Osteoarthritis (OA), the most common chronic joint disease worldwide, is characterized by progressive damage to the articular cartilage, increased joint-associated bone remodelling, and synovial inflammation. Current OA treatments are limited to pain relief, physiotherapy, or replacement surgeries in severe cases, yet disease-modifying drugs are lacking. A genome wide association study (GWAS) revealed a genetic association between polymorphisms in the DOT1L gene and OA. The Disruptor of telomeric silencing 1-like (DOT1L) gene encodes a histone methyltransferase that methylates Lysine 79 of Histone H3 (H3K79). We identified DOT1L as a key protector of cartilage homeostasis, using both human articular chondrocytes and different Dot1l genetic mouse models. Furthermore, we reported that DOT1L activity, indicated by the levels of H3K79 methylation (H3K79me), is reduced in OA as compared to non-OA cartilage. Therefore, maintaining H3K79me seems to be critical to preserve joint health and prevent the development or progression of OA. Here, we hypothesized that H3K79me could be restored or maintained by inhibiting demethylation at the H3K79 site, via targeting specific histone demethylases. There are two main families of histone demethylases: the lysine-specific demethylases (LSD) and Jumonji C (JmjC) demethylases. The LSD family contains two members: LSD1 and LSD2. The JmjC family is further classified into 6 subfamilies: from KDM2 to KDM7. In this new study, we aimed to investigate which histone demethylases are responsible for H3K79 demethylation and whether their specific targeting can lead to protective effects in OA. Methods: We determined the baseline mRNA expression of 28 reported histone demethylases in primary human articular chondrocytes (hACs) from non-OA patients, using real-time qPCR. We also determined the changes in histone demethylase expression in vitro upon OA-mimicking stimuli, namely proinflammatory cytokine IL-1β and the Wnt signalling activator CHIR99021. To interrogate the role of JmjC demethylase family in H3K79me, human articular chondrocyte C28/I2 cells were treated with the JmjC pan inhibitor JIB-04. In parallel, the LSD family members LSD1 and LSD2 were pharmacologically inhibited using LSD1 inhibitor II or silenced using siRNA against LSD2, respectively. The role of the different JmjC subfamilies on H3K79me was studied using selective pharmacological inhibitors. We assessed the levels of H3K79me by Western blot and immunofluorescence analysis. The translational impact of individual JmjC demethylase targeting was evaluated using a siRNA knockdown strategy in hACs from OA patients. In this setup, gene expression of healthy cartilage markers Collagen2a1 (COL2A1) and Aggrecan, as well as of catabolic markers MMP13 and ADAMTS5, was assessed using real-time qPCR. Results: The baseline expression of histone demethylases was mapped in hACs, showing differences in absolute expression among the different enzymes. Treatment with IL1-β resulted in an increase in KDM6B and KDM7A mRNA expression. Conversely, Wnt signalling activation by CHIR99021 led to a downregulation in most of histone demethylases’ mRNA expression. Interestingly, pharmacological inhibition of the JmjC family using JIB-04 resulted in increased H3K79me levels. However, the blockade of LSD family members did not lead to H3K79me changes in articular chondrocytes. Moreover, inhibition of all JmjC demethylase subfamilies increased H3K79me levels. Individual silencing of specific JmjC demethylases increased the expression of chondrocyte healthy markers while reducing the expression of catabolic markers. Conclusions: The discovery approach performed in this study unravels that histone demethylases from the JmjC family control H3K79me in the articular cartilage. Silencing of individual demethylases from this family exerted protective effects in the articular chondrocyte. Therefore, targeting specific histone demethylases could restore the loss of DOT1L activity in OA and lead to therapeutic benefits in this chronic disease.