Integrin-Mediated Adhesion to Extracellular Matrix Protein Fibronectin Drives Megakaryocytosis in JAK2^V617F+ Primary Myelofibrosis

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in Primary Myelofibrosis (PMF). Myelofibrosis was long regarded as a bystander of disease in PMF. However, as cells have the ability to sense the surrounding ECM through integrin receptors, we examined the hypothesis that abnormal ECM in myelofibrosis, mediated by integrin activation, contributes to BM megakaryocytosis in JAK2^V617F+ PMF.

Due to its complex genetic landscape, modeling PMF in animals is challenging. While there is no ideal model to replicate the human disease, Vav1-hJAK2^V617F transgenic mice (JAK2^V617F+) (Xing S et al. Blood. 2008; 111:5109-5117) are most appropriate for our studies because they express the most frequent mutation in PMF and show megakaryocytosis in BM and a predictable development of myelofibrosis throughout the animal's life.

Fibronectin (FN) is a dimeric ECM glycoprotein produced by a variety of cells in BM. FN secretion by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Analysis of myelofibrotic BM from JAK2^V617F+ mice revealed elevated levels of FN in BM ECM.

Integrins are heterodimeric cell adhesion receptors of α and β subunits that connect the cell cytoskeleton to ECM. Integrin engagement and subsequent signaling affect cell differentiation, proliferation, migration and survival. Integrins α5β1, α4β1, αIIbβ3, and αυβ3 are FN-binding integrins known to be expressed in megakaryocytes. Expression analysis detected significantly elevated expression of α5, αIIb, αυ and β3 subunits in CD41+ megakaryocytes fromJAK2^V617F+ mice in vitro and in vivo. Expression of β1 integrin was unchanged in megakaryocytes fromJAK2^V617F+ mice, but expression of 9EG7, an antibody clone that detects the high-affinity conformation of β1 integrin, was elevated in JAK2^V617F+ CD41+ megakaryocytes. Levels of the high-affinity form of β1 integrin decrease in response to inhibition of α5 integrin function (using Hmα5-1 antibody), indicating a high degree of correlation between affinity to FN and β1 activation in megakaryocytes fromJAK2^V617F+ mice.

As suggested by expression studies, in-vitro differentiated JAK2^V617F+ megakaryocytes showed increased affinity to FN in adhesion assays. To ascertain the contribution of adhesion to FN in development of megakaryocytosis, megakaryocytes were differentiated in vitro on FN, with and without the presence of α5 integrin inhibitory antibody Hmα5-1. Culture on FN had a positive effect on the number of CD41+ megakaryocytes after four days of culture, especially in JAK2^V617F+ BM. Importantly, inhibition of α5 integrin decreased the number of CD41+ megakaryocytes in JAK2^V617F+ BM to nearly wild-type levels.

To determine the effect of in vivo inhibition of α5 integrin on megakaryocyte numbers, the antibody clone 5H10-27, or MFR-5, previously reported to be effective in vivo, was used. Effective targeting in vivo of BM megakaryocytes by the 5H10-27 antibody was confirmed using a fluorochrome-labelled secondary antibody specific to the isotype of the injected clone (Rat IgG_2a, κ). The same method was used to determine minimum required dose and interval of administration. JAK2^V617F+ mice were treated with three doses of 5H10-27 or isotype control antibody (1mg/Kg IV every 48 hours). Analysis of treated animals on day 5 detected a decrease in megakaryocyte α5 integrin expression in animals treated with 5H10-27 antibody relative to control isotype antibody-treated animals. Platelet counts in peripheral blood and percentage of BM CD41+ megakaryocytes were consistently, albeit non-significantly, lower in 5H10-27 antibody-treated than in isotype antibody-treated animals.

Corroborating our findings in mice, analysis of megakaryocytes from patients carrying the JAK2^V617F mutation revealed elevated cell surface expression of α5 integrin subunit and increased adhesion to FN, which was dampened by an anti-α5 integrin antibody (Sam-1).

Our results uncovered a global de-regulation of FN-binding integrin expression in megakaryocytes carrying the JAK2^V617F mutation and the central role of FN-α5 integrin in development of megakaryocytosis in PMF. These results challenge the current paradigm by bringing ECM and myelofibrosis from a bystander position to center stage in the pathology of PMF.