Efficacy of ALK5 inhibition in myelofibrosis
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
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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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 3

Galunisertib suppresses collagen production by MSCs derived from MPLW515L mice in vitro.

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Galunisertib suppresses collagen production by MSCs derived from MPLW515...
(A) Photographs of excised spleens from MPLWT (n = 3) and MPLW515L (n = 3) mice to demonstrate the exemplary increase in size of MPLW515L mice consistent with the presence of an myeloproliferative neoplasm–like (MPN-like) disease. (B) Reticulin staining was performed with bones isolated from MPLWT and MPLW515L mice. Scale bars: 10 μm. (C and D) Col1A1 and Col3A1 mRNA levels were assessed by qRT-PCR in mesenchymal stromal cells (MSCs) derived from MPLWT and MPLW515L mice. Results are representative of 3 independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 by ANOVA, followed by Dunnett’s multiple comparison test. (E) Representative immunofluorescence images of collagen I (red), collagen III (green), and DAPI (blue) in MSCs derived from MPLW515L mice and treated with galunisertib (Galu) in vitro. A merged image is shown on the right. Scale bar: 75 μm. (F) Mean pixel fluorescent intensity was acquired for the images in D (n = 5) to compare collagen deposition. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA, followed by Dunnett’s multiple comparison test. In C, D, F, and G, graphs represent the mean and 95% confidence interval represented as a box-and-whisker blot of 5 individual mice or images per group. (G) Signaling pathways including p-Smad3 and p-STAT3 were detected by Western blot in MSCs. See complete unedited blots in the supplemental material.