Abstract| Volume 30, SUPPLEMENT 1, S40-S41, April 2022


      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 unique histone methyltransferase that methylates Lysine 79 of Histone H3 (H3K79). We previously 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 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 a panel of histone demethylases in primary human articular chondrocytes (hACs) from non-OA patients, and mapped their expression upon OA-mimicking stimuli (IL-1β and Wnt signalling activator CHIR99021), using real-time qPCR. 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. Furthermore, we used a 3D micromass model of C28/I2 cells to evaluate changes in glycosaminoglycan content by Alcian blue staining upon histone demethylase targeting. siRNA silencing was used to dissect the individual role of selective JmjC histone demethylases on H3K79me in C28/I2 cells. This was followed by a translational assessment of the individual targeting 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 evaluated using real-time qPCR. Intra-articular injection of daminozide (inhibitor of KDM2/7 subfamily) was performed in an in vivo OA mouse model induced by destabilization of the medial meniscus (DMM), and histological analyses were performed.
      Results: We found striking differences in the baseline expression of the different histone demethylases in hACs. Treatment with proinflammatory cytokine 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 in human articular chondrocytes. However, blockade of LSD family members did not lead to H3K79me changes. Inhibition of all JmjC demethylase subfamilies increased H3K79me levels, but only targeting of KDM2/7 and KDM6 subfamilies led to an increase in glycosaminoglycan content. Individual silencing of KDM2B, KDM7A, and KDM6B increased H3K79me. Interestingly, specific knockdown of KDM7A resulted in increased expression of chondrocyte healthy markers while reducing the expression of catabolic markers. Histological assessments after intra-articular injection of daminozide (inhibitor of the KDM2/7 subfamily) in DMM mouse model increased H3K79me and led to protective effects by reducing cartilage damage and osteophyte formation, as well as downregulating the chondrocyte hypertrophy marker collagen type 10 (COLX).
      Conclusions: The discovery approach performed in this study unravels that histone demethylases from the JmjC family control H3K79me status in articular cartilage. Silencing of individual JmjC demethylases exerts protective effects in human articular chondrocytes. We provide evidence of the therapeutic potential of targeting KDM2/7 subfamily in the DMM mouse model of OA. Thus, targeting specific histone demethylases can restore the loss of DOT1L activity in OA and lead to therapeutic benefits in this chronic disease.