Increased a5β1 integrin adhesion to fibronectin contributes to megakaryocytosis in primary myelofibrosis models

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm, characterized by megakaryocytosis, extramedullary hematopoiesis, and bone marrow fibrosis.¹ Amongst other potential etiologies, the bone marrow (BM) microenvironment has also been investigated for its contribution to PMF pathology. Specifically, it has been suggested that abnormal interactions between hematopoietic stem cells and the BM microenvironment may contribute to PMF progression.¹ Extracellular matrix (ECM) proteins, key components of the BM microenvironment, are excessively expressed in patients with PMF.^1,2 More specifically, the BM secretion of the ECM component, fibronectin, seems to correlate with PMF severity and BM fibrosis.²

To further explore the role of BM ECM in PMF pathology, Shinobu Matsuura and colleagues² investigated the role of fibronectin and its main megakaryocyte receptor, a5β1 integrin, in preclinical PMF models. The results of this study were published in Blood and have been summarized below.

Study design

Primary cells

• Mouse megakaryocytes were differentiated in vitro from the BM of Vav1-hJAK2-V617F transgenic (JAK2^V617F+) mice using pegylated megakaryocyte growth and development factor (PEG-MGDF, 25 ng/ml)
• Human megakaryocytes from PMF patients (n = 7) or healthy individuals (n = 5) were cultured from BM samples
  • All PMF patients were JAK2-V617F positive and presented with BM fibrosis (Grade 1, n = 1; Grade 2, n = 3; Grade 3, n = 3) 

Murine PMF model

• The JAK2^V617F+ homozygous transgenic PMF model used was backcrossed to C57BL/6J mice. Animals were age- and gender-matched to wildtype (wt) mice for all experiments

Anti-a5 antibody treatment of JAK2^V617F+ mice

• Three doses (1mg/kg) of the rat anti-a5 subunit antibody (5H10-27) or rat isotype control antibody were administered intravenously in JAK2^V617F+ mice, every 48 hours (5-day protocol)
• Sample collection and analysis was performed 24 hours following the last antibody dose

Results

Overexpression of the a5 integrin subunit in JAK2^V617F+ megakaryocytes

• BM immunohistochemistry revealed an increase in fibronectin deposition in the BM of JAK2^V617F+ mice when compared with wt mice
• Moreover, the numbers of both total (CD41⁺) and mature (CD42d⁺) megakaryocytes were significantly higher in the BM of JAK2^V617F+ mice than wt mice
• The a5 subunit, but not β1, expression was significantly higher in primary BM megakaryocytes and megakaryocyte progenitors from JAK2^V617F+ mice, when compared with those from wt mice
• Consistent with the in vivo data, the expression of the a5 subunit of the a5β1 integrin (fibronectin receptor), but not the β1, was significantly higher in in vitro differentiated JAK2^V617F+ mouse megakaryocytes
• JAK2^V617F+ mouse megakaryocytes were able to bind fibronectin stronger than their wt counterparts in vitro:
  • This binding was abolished by treatment with an anti-a5 antibody (HMα5-1), indicating that the a5 integrin subunit mediates the interaction with fibronectin

Treatment with anti-a5 antibody reduces megakaryocyte differentiation in JAK2^V617F+mice

• To further explore this result, JAK2^V617F+ or wt mouse megakaryocyte differentiation from BM samples was performed in fibronectin-coated plates with or without an anti-a5 antibody (HMα5-1):
  • Treatment with the anti-a5 antibody led to a significant reduction in total and mature (CD41⁺, CD42d⁺) JAK2^V617F+ megakaryocytes but not wt ones
• To validate the in vitro results further, the authors used JAK2^V617F+ transgenic mice:
  • Intravenous administration of an anti-a5 antibody (5H10-27) in JAK2^V617F+ mice significantly lowered the number of total megakaryocytes (CD41⁺, CD42d^-) but not of mature megakaryocytes, when compared with control antibody-injected animals

In vitro results in megakaryocytes from patients with PMF

• Primary human megakaryocytes from BM samples of patients with PMF showed similar results to the above results, with
  • significantly higher a5 subunit, but not β1, levels when compared with healthy individuals, and;
  • anti-a5 antibody neutralization leading to significantly reduced megakaryocyte adhesion and differentiation from CD34⁺ cells when compared with controls

Conclusions

The preclinical results of this study indicate that the a5 subunit of the a5β1 integrin is upregulated in PMF megakaryocyte progenitors leading to an elevated adhesion to its ECM target, fibronectin. Inhibition of a5 binding, both in vitro and in vivo, led to significantly reduced megakaryocytes in a JAK2^V617F+ mouse model but not in wt mice, bringing them close to normal wt megakaryocyte levels. These data suggest that the a5 integrin subunit might be a potential therapeutic target for PMF. Nevertheless, further clinical data are needed to validate the significance of these findings.