TGF-β1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis
Lilian Varricchio, Camelia Iancu-Rubin, Bhaskar Upadhyaya, Maria Zingariello, Fabrizio Martelli, Paola Verachi, Cara Clementelli, Jean-Francois Denis, Adeeb H. Rahman, Gilles Tremblay, John Mascarenhas, Ruben A. Mesa, Maureen O’Connor-McCourt, Anna Rita Migliaccio, Ronald Hoffman
Lilian Varricchio, Camelia Iancu-Rubin, Bhaskar Upadhyaya, Maria Zingariello, Fabrizio Martelli, Paola Verachi, Cara Clementelli, Jean-Francois Denis, Adeeb H. Rahman, Gilles Tremblay, John Mascarenhas, Ruben A. Mesa, Maureen O’Connor-McCourt, Anna Rita Migliaccio, Ronald Hoffman
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Research Article Hematology

TGF-β1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis

Abstract

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-β plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-β1 than TGF-β2 and TGF-β3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-β1/TGF-β3 protein trap, to block the excessive TGF-β signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-β1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-β1–induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-β1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

Authors

Lilian Varricchio, Camelia Iancu-Rubin, Bhaskar Upadhyaya, Maria Zingariello, Fabrizio Martelli, Paola Verachi, Cara Clementelli, Jean-Francois Denis, Adeeb H. Rahman, Gilles Tremblay, John Mascarenhas, Ruben A. Mesa, Maureen O’Connor-McCourt, Anna Rita Migliaccio, Ronald Hoffman

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Figure 3

AVID200 reverses the G0-G1 blockade caused by rTGF-β1 and increases the clonogenicity of ND MNC cells.

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AVID200 reverses the G0-G1 blockade caused by rTGF-β1 and increases the ...
(A) The bar graph shows the percentages SD of ND MNCs in G0, G1, SG2M after treatment with 10 ng/mL rTGF-β1 alone or in combination with 50 nM AVID200 for 48 hours. The cells were stained with Hoechst 33342 and Pyronin Y. n = 3 different donors (** P < 0.01, *** P < 0.001, by ANOVA). (B) Fold increase of ND MNCs cultured for 6 days with cytokines alone (CTR) or in the presence of 10 ng/mL rTGF-β1 with or without 50 nM AVID200. Results are presented as the fold increase with respect to day 0 and are presented as mean ± SD of results from experiments performed with 3 different donors. (C) Number of colonies assayed in duplicate from cultures 25 × 104 ND MNCs containing a cytokine combination (CTR) and 10 ng/ml rTGF-β1 alone or in combination with 50 nM AVID200. Results are presented as mean ± SD obtained in 2 separate experiments, using 2 different donors. (D) Molecular networks targeted by TGF-β in MNCs obtained from NDs. Representative WB analyses of pSMAD2, SMAD2/SMAD3, GATA2, p57Kip2, p53, p21, caspase 3, and pp38 of total extracts derived from ND treated with rTGF-β1 and AVID200 alone or in combination for 48 hours as indicated. GAPDH was used as loading control (n = 3 different donors). (E) CyTOF viSNE analysis for downstream effectors of TGF-β signaling, pSMAD2/pSMAD3, and (F) pp38 levels in CD34+, CD14+, and CD19+ gated cell populations from healthy donors (cord and adult blood; n = 2 different donors) previously treated with 10 or 100 ng/mL rTGF-β1 alone or in combination with 50 or 100 nM AVID200 for 30 minutes. ND, normal donor; MNC, mononuclear cell; viSNE, visualization of stochastic neighbor embedding.