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DOT1L in Osteoarthritis

Final Report Summary - DOTOS (DOT1L in Osteoarthritis)

The DOTOS (DOT1L in Osteoarthritis) project (number 623253) was run in the period from March 2014 to March 2016. This project aimed at deciphering the role of DOT1L in the biology and pathology of synovial joints with specific attention towards osteoarthritis (OA), the most common chronic joint disease. Disruptor of telomeric silencing 1-like (DOT1L) is the main histone-modifying enzyme that catalyzes the methylation on lysine-79 of histone 3 (H3K79). The general function of DOT1L is to methylate H3K79 as a member of a large protein complex, which can influence the transcriptional state. DOT1L’s histone methyltransferase activity has been linked to active transcription, and plays a role in many biological processes including the DNA damage response, the cell cycle, embryonic development and cell reprogramming. In cancer biology, DOT1L is involved in MLL-rearranged leukemia and in other tumors. Previously, we reported a strong genetic association between polymorphisms in the human DOT1L gene and hip cartilage thickness as well as OA. Variations in the DOT1L gene are also associated with human height. Furthermore, we earlier demonstrated that silencing of Dot1L inhibited chondrogenic differentiation of murine progenitor cells. Interestingly, DOT1L-associated H3K79 methylation has been linked to the Wingless-type (Wnt) cascade, a fundamental signaling pathway in cartilage biology that requires a tight modulation. This could specifically identify DOT1L as an attractive target for joint diseases, more particularly for OA. DOT1L is an enzyme and, thus, can be pharmacologically modulated. We therefore hypothesized that DOT1L plays a major role in cartilage homeostasis by tightly regulating gene expression patterns of key signaling pathways, such as the Wnt cascade.
Thus, our specific objectives were to decipher the protein and transcriptional network of DOT1L in cartilage, to study the role of DOT1L in vivo in joint development, homeostasis and disease, and to do a translational validation of the previous insights in clinically relevant samples from patients.
Since the beginning of the project, we managed to fulfill the objectives set at the start of the project. Indeed, the in vitro part was perform in primary human chondrocytes extracted from femoral heads. We managed to characterize the DOT1L partners in human chondrocytes in basal conditions or when the Wnt pathway is activated. Moreover, we have gone beyond what was planned, and have found and validated new DOT1L interactors by mass spectrometry. We also performed a microarray in human articular chondrocytes, with provided us in depth insights into the DOT1L molecular network in articular cartilage. The microarray data were analysed with different bioinformatics tools resulting in novel insights into the DOT1L molecular network. We successfully validated all the results obtained in clinically relevant samples.
To study the role of DOT1L in vivo in cartilage development, cartilage specific Dot1l knockout mice were successfully generated, in a series of complex matings combining a Col2 CRE transgenic mice with a mouse strain carrying floxed Dot1l alleles. We managed to arrive at the final genetic model, and the analysis have been performed as planned but in a straightforward way taking into account the results obtained in the in vitro experiments described above, which provided extremely relevant and novel information about the role of DOT1L in cartilage biology. To study the role of DOT1L in vivo in cartilage homeostasis and disease, we performed an alternative strategy to the one that was planned, based in our in vitro success in the optimization for the use of DOT1L inhibitors in chondrocytes. We injected DOT1L inhibitor intra-articularly in the knees of mice, and studied the development of spontaneous osteoarthritis. This change in the workplan allowed us to finish and submit a manuscript to a high-impact journal. However, we are still performing the strategy that was planned to study the role of DOT1L postnatally in cartilage, by generating tamoxifen induced cartilage-specific knockout of Dot1l. We anticipate that this will allow us to obtain further translational insights into DOT1L biology.
Part of the in vitro data were presented in the 35th European Workshop for Rheumatology Research (EWRR) (Budapest, 5-7 March, 2015), and in the Osteoarthritis Research Society International (OARSI) World Congress (Seattle, Washington, from April 30- May 3, 2015). The in vivo data have recently been presented in the 36th EWRR (York, 25-27 February, 2016). We have obtaining an abstract award in two of these conferences (EWRR 2015 and 2016) and the 2015 Celgene Prize in Rheumatology. This highlight the impact of our findings.