C71-28 Doublecortin-like Kinase 1 Is a Positive Regulator of Fibroblast Activation and Pulmonary Fibrosis

Rationale

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease characterized by aberrant activation of fibroblasts, including excessive proliferation, fibroblast-to-myofibroblast transformation (FMT), survival, migration, tissue invasion, and extracellular matrix (ECM) production. Current FDA-approved therapies slow the decline in lung function but do not halt the progression of fibrosis. The limited understanding of druggable molecular regulators that initiate and maintain fibroblast activation is a major obstacle to developing effective therapies for IPF patients. There is an urgent need for therapies that require a better understanding of the molecular pathways driving fibroblast activation. In this study, we identified Doublecortin-like kinase 1 (DCLK1) as a novel serine/threonine kinase that positively regulates key profibrotic signaling pathways, including TGFα and TGFβ, to promote fibroproliferation, myofibroblast transformation, cell survival, and ECM production in IPF.

Methods

Using the LGRC dataset (GSE47460), we analyzed the correlation between DCLK1 expression and clinical severity (FVC, DLCO) in patients with IPF (N = 160) compared to healthy controls (N = 108). DCLK1 localization in lung fibroblasts was validated via immunostaining of human and preclinical PF tissue and single-nucleus RNA sequencing (snRNA-seq). To identify regulatory mechanisms, we performed promoter analysis and knockdown studies of candidate transcription factors. Finally, the functional role of DCLK1 was assessed in primary IPF fibroblasts using siRNA-mediated knockdown, followed by RT-PCR, western blotting, and assays for proliferation, apoptosis, and TGFβ-induced FMT.

Results

Our results show that DCLK1 is elevated IPF lungs compared with healthy controls. Notably, the increased expression of DCLK1 is associated with a decline in lung function in IPF. This upregulation was also observed in two alternative mouse models of pulmonary fibrosis, including bleomycin- and TGFα-induced pulmonary fibrosis models. SnRNA-seq and immunostaining localized DCLK1 to activated fibroblast subpopulations. Mechanistically, promoter analysis and knockdown experiments identified SOX9 as a critical transcriptional regulator of DCLK1. Functional inhibition of DCLK1 using siRNA attenuated the expression of genes involved in fibroproliferation (AURKB, PLK1), apoptosis resistance (BAX, BCL2), and TGFβ-dependent ECM production (COL1A1, αSMA). In support, DCLK1 knockdown resulted in reduced fibroblast proliferation and increased their apoptotic clearance.

Conclusion

Our findings identify DCLK1 as a novel regulator of pathogenic fibroblast activation, survival, and myofibroblast transition in IPF. These results suggest that targeting the SOX9-DCLK1 axis may offer a therapeutic strategy to inhibit progressive pulmonary fibrosis. Ongoing studies are utilizing fibroblast-specific loss-of-function and gain-of-function models to further validate DCLK1 as a druggable target in IPF.

This abstract is funded by: NIH (1R01 HL134801 and 1R01 HL157176) and University of Cincinnati