Background/Purpose: Krüppel-like factors (KLFs) are members of the zinc finger family of transcription factors, which includes 17 members in mammals. We found that expression of KLF4 is dysregulated with progression of osteoarthritis (OA) in human and mouse knee cartilage. Further, we identified KLF4 as a new central transcription factor that regulates protective and regenerative functions in joint tissue cells. We hypothesized that molecules that increase KLF4 expression would be novel therapeutic candidates for OA.

Methods: We performed high throughput drug screening (HTS) using the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library with 11,948 clinical-stage molecules. Hit compounds were tested in joint tissue cells The lead compound was tested in the surgical destabilization of the medial meniscus (DMM) model of OA. As mechanistic studies included transcriptomic and proteomic profiling.

Results: Fifty-one hit compounds were identified in the HTS, and 18 compounds were confirmed in a secondary screen. We validated the 18 hits using SW1353 cells and human chondrocytes, and found that mocetinostat, a class I histone deacetylase (HDAC) inhibitor, had the best profile of biological activities. While mocetinostat upregulated KLF4, KLF2, FOXO1 and cartilage signature genes such as COL2A1, COL11A2, SOX9 and PRG4 in human chondrocytes, meniscal cells and pellet-cultured BMSCs, it down-regulated hypertrophic and catabolic genes, including COL10A1, RUNX2, ADAMTS5 and IL6 (Figure 1 for pellet-cultured BMSCs). Mocetinostat enhanced protective functions in human chondrocytes and synoviocytes through down-regulation of inflammatory and catabolic genes. Intraperitoneal administration of mocetinostat into mice after surgical OA induction significantly improved pain behaviors and reduced the severity of OA histopathological changes in cartilage, meniscus, and synovium (Figure 2 for the summed OARSI scores). We performed RNA-seq and TMT mass spectrometry analysis using mocetinostat-treated TC28a2 cells. While the significantly upregulated genes and proteins showed enrichment of Rap1 signaling pathway and ECM-receptor interaction previously shown to be modulated by KLF4, several unique pathways and terms were also enriched such as PPAR signaling pathway, suggesting novel regulatory mechanisms of HDAC inhibitors.

Conclusion: The class I HDAC inhibitor mocetinostat was identified through the HTS of compounds activating endogenous expression of KLF4. Mocetinostat increased protective and regenerative functions in joint tissue cells and reduced the severity of experimental OA in mice, qualifying it as a novel potential therapeutic candidate for OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pharmacological-modulation-of-kruppel-like-factor-4-reduces-the-severity-of-experimental-osteoarthritis-via-tissue-protection-and-regeneration/