MPN-383: GLI1 Promotes the Pro-Fibrotic Function of Monocyte-Derived Fibrocytes in Myelofibrosis

Abstract

Primary myelofibrosis (MF), post-polycythemia vera (PV) MF, and post-essential thrombocytosis MF are myeloproliferative neoplasms (MPNs) characterized by progressive bone marrow (BM) fibrosis. A recent study suggested that glioma-associated oncogene-1 (GLI1), a downstream effector of the embryonic Hedgehog pathway, is implicated in the pathogenesis of BM fibrosis in MF. Because we and other investigators have found that monocyte-derived fibrocytes, rather than mesenchymal stromal cells (MSCs), induce BM fibrosis in MF, we sought to determine the role of GLI1 in MF fibrocytes. To do this, we analyzed BM biopsy sections of patients with MF using multiplex fluorescence immunohistochemistry and detected high levels of GLI1 in CD45+/CD68+/procollagen I+ fibrocytes and low levels of GLI1 in CD90+/CD105+ MSCs (<i>P</i>=0.009 and <i>P</i>=0.002, respectively). Using immunostaining, RNA <i>in situ</i> hybridization, gene expression, and western immunoblotting analyses of cultured BM fibrocytes or MSCs, we observed significantly higher levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF fibrocytes than in MF MSCs or normal BM-derived fibrocytes (<i>P</i><0.05 each). Next, by transplanting human MF BM cells into NSG mice, we found that the engrafted cells differentiated into GLI1-expressing cells that induced BM fibrosis. Inhibition of GLI using non-specific GLI antagonist 61 (GANT61) decreased the formation of both MF fibrocytes and normal fibrocytes (<i>P</i><0.05). However, GLI1 silencing using small interfering RNA specifically induced apoptosis of MF fibrocytes and fibrocyte-forming cells (<i>P</i>=0.008 and <i>P</i><0.001, respectively) and increased fragmentation of MF fibrocyte DNA. In addition, GLI1 knockdown reduced MMP2, MMP9, SMAD2/3, and procollagen I gene expression (<i>P</i><0.05) and protein levels. Finally, because we recently found that GLI1 expression in leukemic cells is induced by constitutively activated signal transducer and activator of transcription 3 (STAT3), we performed chromatin immunoprecipitation, luciferase, and STAT3-silencing assays on MF fibrocytes and found that STAT3 binds and activates GLI1 gene promoter in these cells. In conclusion, GLI1, triggered by constitutively activated STAT3, activates pro-fibrotic signaling pathways in MF fibrocytes. Downregulation of GLI1 hampers fibrocyte survival and function, suggesting that GLI1 inhibition might alleviate BM fibrosis in patients with MF.