Alterations In Wnt Signalling In the Megakaryocytic Lineage Leads to Bone Marrow Failure and Myelofibrosis

Introduction: Wnt signalling is fundamental in controlling stem cell self-renewal, cell proliferation and development in multicellular organisms. Stabilization of beta catenin or loss of the scaffold protein adenomatis polyposis coli (APC) causes aberrant activation of wnt signalling and often leads to cancer. Mutations to wnt pathway members in haematopoietic stem cells leads to haematopoietic failure and rapid lethality. In this study, we demonstrate that aberrant wnt signalling in the megakaryocyte lineage underlies myelofibrosis. Methods: We created a series of mice with altered wnt pathway signalling in their megakaryocytic lineage using PF4-Cre (platelet factor 4 cre) as follows: Ctnnb1fx(ex3)/wt_ Expresses stabilized active beta catenin henceforth termed PF4bcat+; APCfx/fx_ Loss of APC stabilises beta catenin termed PF4APC-KO; Ctnnb1fx/fx b-catenin knockout termed PF4bcat-KO. Results: By day 40, PF4bcat+ and PF4APC-KO mice are severely underweight, anaemic (wt 6.8+0.2×106/ml v. PF4bcat+ 4.2+0.3×106/ml, PF4APC-KO 5.5+0.5×106/ml), and have a significant reduction in platelet number (wt 1033+37×106/ml v. PF4bcat+ 717+57×106/ml, PF4APC-KO 747+68×106/ml). Furthermore PF4bcat+ and PF4APC-KO mice develop bone marrow fibrosis and consistently die within 50 days of birth. Both populations of mice have splenic extramedullary haemopoiesis with hyperplasia of splenic megakaryocytes, leading to a dramatic increase in spleen to body size ratio. In addition, both PF4bcat+ and PF4APC-KO mice have increased peripheral blood levels of active TGFb, providing a likely molecular basis of the induction in bone marrow fibrosis. PF4APC-KO and PF4bcat+ mutant mice show a dramatic (>10-fold) increase in platelet b-catenin protein levels over wt samples. By comparison, human myelofibrosis patients (n=16) show a 2.7+0.6-fold increase in platelet b-catenin expression over controls. Moreover, overexpression of b-catenin within human patient samples is correlated with a worsening Primary Myelofibrosis prognostic score (Blood, 2009 vol 113 p. 2895), with those patients with a low score (n=7) having a 1.3+/−0.37-fold increase over control, intermediate score (n=4) 4.52+/−1.23-fold, and high score (n=1) 4.56-fold. In contrast PF4bcat-KO mice show no changes in whole blood counts, weight, or evidence of splenic extramedullary haematopoiesis, indicating that b-catenin removal does not adversely affect megakaryocyte development or function. Conclusions: Stabilisation of b-catenin within mouse megakaryocytes leads to a myelodysplastic disorder and myelofibrosis. This finding demonstrates a defined role for aberrant activation of the wnt signalling pathway and marks the wnt pathway and the megakaryocyte lineage as important potential drug targets for the treatment of myelodysplastic disorders