Poster Presentation 35th Lorne Cancer Conference 2023

Mpl Signalling in Normal Hematopoiesis and Myeloproliferative Neoplasms (#112)

Kira Behrens 1 2 , Maria Kauppi 1 2 , Elizabeth M Viney 2 , Andrew J Kueh 1 2 , Craig D Hyland 2 , Tracy A Willson 2 , Jeff J Babon 1 2 , Marco J Herold 1 2 , Nicos N Nicola 1 2 , Warren S Alexander 1 2
  1. Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia
  2. Walter and Eliza Hall Institute, Parkville, VIC, Australia


Thrombopoietin (Tpo), which binds to its specific receptor, Mpl, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo-binding to Mpl on the surface of target cells induces phosphorylation of Janus kinase 2 (Jak2) and the intracellular Mpl-receptor domain, which in turn serves as docking sites for adaptor proteins inducing activation of several signalling cascades.

Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation: Mpl-Y565, Mpl-Y599 and Mpl-Y604. The precise role of theses Y-residues in normal Tpo-mediated signalling and under pathological conditions remains elusive. 



To define the roles of individual tyrosine residues in the Mpl intracellular domain in steady-state and pathophysiological Tpo/Mpl-signalling.



Using CRISPR/CAS9 genome editing, mutations were introduced in the mouse germline to generate Mpl receptor tyrosine-to-phenylalanine mutants of the three Mpl C-terminal tyrosine residues.



Phenotyping of the hematopoietic stem cell (HSC) and megakaryocytic compartments revealed an increase in megakaryocytes, megakaryocyte progenitors and platelets in MplY565F/Y565F mice, as well as the number of HSC, accompanied by enhanced stem cell activity in transplantation assays. In contrast, Mpl-Y599F resulted in thrombocytopenia, deficient megakaryopoiesis and low HSC numbers with a competitive disadvantage. Lastly the Mpl Y604F mutation slightly increased platelet numbers but had no effect on stem and progenitor numbers and function.

In line with the critical function of Mpl-Y599 in megakaryopoiesis and stem cell homeostasis Mpl-Y599 was critical for activation of Tpo-induced signalling. Strikingly, when crossed to mouse models of MPN, where Mpl is required for pathogenesis, Mpl-Y599F completely abrogated MPN development, while Mpl-Y565F accentuated the disease phenotype.  



We report a consistent physiological requirement for Mpl-Y599 for steady-state megakaryopoiesis and MPN-associated thrombocytosis. In contrast, Mpl-Y565 is a negative regulator of Tpo signalling, required to attenuate steady-state and pathogenic Mpl-signalling.