Efficacy of ALK5 inhibition in myelofibrosis
Lanzhu Yue, … , Amit Verma, Pearlie K. Epling-Burnette
Lanzhu Yue, … , Amit Verma, Pearlie K. Epling-Burnette
Published April 6, 2017
Citation Information: JCI Insight. 2017;2(7):e90932. https://doi.org/10.1172/jci.insight.90932.
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Research Article Hematology

Efficacy of ALK5 inhibition in myelofibrosis

Abstract

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

Authors

Lanzhu Yue, Matthias Bartenstein, Wanke Zhao, Wanting Tina Ho, Ying Han, Cem Murdun, Adam W. Mailloux, Ling Zhang, Xuefeng Wang, Anjali Budhathoki, Kith Pradhan, Franck Rapaport, Huaquan Wang, Zonghong Shao, Xiubao Ren, Ulrich Steidl, Ross L. Levine, Zhizhuang Joe Zhao, Amit Verma, Pearlie K. Epling-Burnette

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Figure 2

Galunisertib antagonizes the stimulatory effect of TGF-β1 on collagen production by normal murine and human MSCs in vitro.

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Galunisertib antagonizes the stimulatory effect of TGF-β1 on collagen pr...
(A and B) Mesenchymal stromal cells (MSCs) derived from MPLWT mice (n = 6) were cultured in the presence of TGF-β1 and different concentrations of galunisertib (Galu) in vitro for 72 hours, following which collagen I (Col1A1) and collagen III (Col3A1) mRNA levels were assessed by qRT-PCR with TATA sequence–binding protein (TBP), as reference gene (70). *P < 0.05, **P < 0.01, ****P < 0.0001 by ANOVA, multiple comparisons using 20 ng/ml TGF-β group as control. (C and D) Human COL1A1 and COL3A1 mRNA levels were measured by qRT-PCR in MSCs that were derived from normal human bone marrow (n = 3) and treated with human TGF-β (hTGF-β) and galunisertib for 72 hours. **P < 0.01, ***P < 0.001, ****P < 0.0001 by ANOVA, multiple comparisons using 10 ng/ml hTGF-β group as control. Results are representative of 3 independent experiments. (E) Representative immunofluorescence images showing collagen I (red), collagen III (green), and DAPI (white) in MSCs derived from MPLWT mice with different treatments in vitro. A merged image is shown on the right. Scale bar: 75 μm. (F) Mean pixel intensity was acquired from the immunofluorescence images (n = 3–6) to quantify collagen I and collagen III deposition. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA, followed by Dunnett’s multiple comparison test. All dot plots represent randomly taken images with means indicated. (G) Western blot showing p-Smad3 and p-STAT3 in MSCs derived from MPLWT mice under indicated treatments, which represents 1 of 3 independent experiments conducted. See complete unedited blots in the supplemental material.