Myeloproliferative neoplasms (MPNs) are associated with significant alterations in the bone marrow microenvironment that include decreased expression of key niche factors and myelofibrosis. Here, we explored the contribution of TGF- β to these alterations by abrogating TGF- β signaling in bone marrow mesenchymal stromal cells. Loss of TGF- β signaling in Osx-Cre –targeted MSCs prevented the development of myelofibrosis in both MPL W515L and Jak2 V617F models of MPNs. In contrast, despite the absence of myelofibrosis, loss of TGF- β signaling in mesenchymal stromal cells did not rescue the defective hematopoietic niche induced by MPL W515L , as evidenced by decreased bone marrow cellularity , hematopoietic stem/progenitor cell number, and Cxcl12 and Kitlg expression, and the presence of splenic extramedullary hematopoiesis. Induction of myelofibrosis by MPL W515L was intact in Osx-Cre Smad4 fl/fl recipients, demonstrating that SMAD4-independent TGF- β signaling mediates the myelofibrosis phenotype. Indeed, treatment with a c-Jun N-terminal kinase (JNK) inhibitor prevented the development of myelofibrosis induced by MPL W515L . Together, these data show that JNK-dependent TGF- β signaling in mesenchymal stromal cells is responsible for the development of myelofibrosis but not hematopoietic niche disruption in MPNs, suggesting that the signals that regulate niche gene expression in bone marrow mesenchymal stromal cells are distinct from those that induce a fibrogenic program.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease characterized by aberrant fibroblast/myofibroblast accumulation and excessive collagen matrix deposition in the alveolar areas of lungs. As the first approved IPF medication, pirfenidone (PFD) significantly decelerates lung function decline while its underlying anti-fibrotic mechanism remains elusive. In this study, using transcriptomic and immunofluorescence analyses of primary human IPF tissues, we showed that myocardin related transcription factor (MRTF) signaling is activated in myofibroblasts accumulated in IPF lungs. Furthermore, we showed that PFD inhibits MRTF activation in primary human lung fibroblasts at the clinically achievable concentrations (half-maximal inhibitory concentration (IC50)=50–150 µM, maximal inhibition>90%, maximal concentration of PFD in patients<100 µM). Mechanistically, PFD appears to exert its inhibitory effects by promoting the interaction between MRTF and actin indirectly. Finally, PFD-treated IPF lungs exhibit significantly less MRTF activation in FF areas than naïve IPF lungs. Our results suggest MRTF signaling as a direct target for PFD and implicate that some of the anti-fibrotic effects of PFD may be due to MRTF inhibition in lung fibroblasts.

TGF-β expression is elevated in most cases of myeloproliferative neoplasms (MPNs). However, the contribution of TGF-β to disease pathogenesis is not well understood. Prior studies have shown that TGF-β regulates hematopoietic stem cell (HSC) quiescence. There also is published evidence that increased TGF-β may contribute to myelofibrosis. However, this is controversial, as recent studies have implicated other inflammatory cytokines in the development of myelofibrosis. Here, we test two specific hypotheses. First, we hypothesize that increased TGF-β signaling in mesenchymal stromal cells (MSCs) is required for the development of myelofibrosis. Second, we hypothesize that Jak2 mutated HSCs are resistant to the growth suppressive effect of TGF-β, resulting in a competitive advantage that contributes to their clonal expansion in MPN and clonal hematopoiesis. To test the first hypothesis, we abrogated TGF-β signaling in mesenchymal stem/progenitor cells by deleting Tgfbr2 using a doxycycline-repressible Osterix-Cre transgene (Osx-Cre), which targets all mesenchymal stromal cells in the bone marrow. Osx-Cre was induced at birth (by removal of doxycycline), since we recently reported that the post-natal loss of TGF-β signaling in mesenchymal stromal cells has no discernible effect on basal hematopoiesis or the stem cell niche. We transplanted bone marrow cells from UBC-CreERT2; Jak2V617F mice or c-kit+ cells transduced with MPLW515L retrovirus into irradiated wildtype or Osx-Cre; Tgfbr2f/fmice. For the Jak2V617F model, mice were treated with tamoxifen 6 weeks post transplantation to induce mutant Jak2 expression. Of note, elevated Tgfb1 was present in both MPN models. MPLW515L induced a rapidly fatal MPN with reticulin fibrosis in the bone marrow. A similar hematopoietic phenotype was observed in Osx-Cre; Tgfbr2f/f recipients. However, myelofibrosis, as measured by reticulin staining and Collagen III (Col3a1) mRNA expression, was reduced, but not completely abrogated in Osx-Cre; Tgfbr2f/fmice. Likewise, in the Jak2V617F MPN model, the hematopoietic phenotype was similar in wildtype and Osx-Cre; Tgfbr2f/fmice. Although overt myelofibrosis was not observed in this MPN model, increased RNA and protein expression of Collagen III were detected in the bone marrow. Although still above baseline, Col3a1 expression was significantly reduced in Osx-Cre; Tgfbr2f/frecipient mice. To examine the role of canonical TGF-β signaling in the induction of myelofibrosis, we cultured bone marrow derived MSCs from wildtype and Osx-Cre; Smad4f/f mice, in which canonical TGF-β signaling is abrogated. Treatment of wildtype MSC cultures with TGF-β1 induced the expression of fibrosis associated genes, including Col1a1 and Acta2, and down-regulated expression of key niche factors, including Cxcl12, Scf, and Bglap. Surprisingly, TGF-β1-induced expression of Col1a1 was intact in Smad4-deleted MSC cultures. Conversely, treatment of wildtype MSC cultures with the JNK inhibitor SP600125 abrogated TGF-β1-induced expression of Col1a1. Collectively, these data suggest that TGF-β signaling in MSCs contributes to the development of myelofibrosis in MPN through activation of the non-canonical JNK pathway. To test the second hypothesis, we set up a competitive transplantation assays using wildtype and Jak2V617F hematopoietic cells in which Tgfbr2 had been deleted to abrogate TGF-β signaling in HSCs. Specifically, we transplanted UBC-CreERT2; Jak2V617F; Tgfbr2f/f and UBC-CreERT2; Tgfbr2f/f bone marrow cells at 1:5 ratio into lethally irradiated wild type recipients. For control, we transplanted a 1:5 ratio of UBC-CreERT2; Jak2V617F to wild type cells. Mice were treated with tamoxifen to activate Cre expression 6 weeks post transplantation. Complete blood counts and donor chimerism were measured 6, 10, 14 and 18 weeks after tamoxifen. As expected, in the control group, donor chimerism with Jak2 mutated cells increased over time in myeloid lineages. In contrast, in the Tgfbr2-deleted group, no expansion of Jak2 mutated cells was observed. These data support our hypothesis that Jak2 mutated HSCs are resistant to the growth suppressive effect of TGF-β, providing a selective advantage that contributes to their clonal expansion in MPN and possibly clonal hematopoiesis. Disclosures No relevant conflicts of interest to declare.