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

Background:

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. 

 

Aims:

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

 

Methods:

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.

 

Results:

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.  

 

Summary

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.