Endocytosis of the Thrombopoietin Receptor Mpl Regulates Both Megakaryocyte and Erythroid Progenitors and Is Critical for Myelofibrosis Development

Somatic mutations in the tyrosine kinase JAK2, the thrombopoietin (TPO) receptor Mpl and the chaperone calreticulin cause myelofibrosis due to constitutive TPO/Mpl signaling in abnormal hematopoietic stem cells (HSCs). Impaired Mpl-mediated endocytosis has been reported in myelofibrosis patients carrying the most frequent JAK2-V617F mutation. Mpl-mediated endocytosis is also impaired in Dnm2^fl/fl Pf4-Cre (Dnm2^Plt-/-) mice specifically lacking the highly conserved endocytic GTPase dynamin 2 (DNM2) in the megakaryocyte (MK) lineage. Consequently, Dnm2^Plt-/-mice develop hallmarks of myelofibrosis such as elevated circulating TPO levels, constitutive JAK2 phosphorylation, marked expansion of HSCs, massive MK hyperplasia, bone marrow fibrosis, extramedullary hematopoiesis and splenomegaly.

To determine whether the phenotype is due to unrestrained TPO/Mpl signaling in HSCs, Dnm2^Plt-/- mice were crossed with Mpl^-/-mice.

Mpl^-/- Dnm2^Plt-/- mice were obtained with a normal Mendelian distribution at weaning, and bone marrow HSC and MK numbers were significantly reduced in Mpl^-/- Dnm2^Plt-/- mice, similar to those of Mpl^-/- mice. Surprisingly, Mpl^-/- Dnm2^Plt-/- mice died at a median age of 26 days postnatal and presented a severe splenomegaly, similar to that of Dnm2^Plt-/- mice. Complete blood counts were analyzed from birth to 3 weeks postnatal. Blood platelet counts increased over time in control mice, reaching maximal values at 3 weeks postnatal, and remained low in Mpl^-/-, Dnm2^Plt-/- and Mpl^-/- Dnm2^Plt-/- mice. Blood erythrocyte counts increased over time in control mice, were significantly slower in single Mpl^-/- and Dnm2^Plt-/- mice, and failed to increase in Mpl^-/- Dnm2^Plt-/-mice, resulting in severe anemia.

Erythroid maturation in Mpl^-/- Dnm2^Plt-/- spleens was investigated by flow cytometry analysis using CD71 and Ter119 as erythroid markers, where immature erythroblasts are defined as CD71^high/Ter119^low and mature erythroblasts as CD71^low/Ter119^high. Approximately 75% of erythroid cells in control spleens were mature erythroblasts, and the distribution significantly decreased to 40% and 20% in single Mpl^-/- and Dnm2^Plt-/- spleens, respectively. In Mpl^-/- Dnm2^Plt-/- spleens, only 5% of erythroid cells were mature erythroblasts, consistent with severely impaired erythroid maturation. Flow cytometry phenotypic analysis of bone marrow MK and eryrthroid progenitors revealed a significant increase of the distribution of progenitors with both MK and erythroid potential (Pre-Meg-E) in Mpl^-/- Dnm2^Plt-/-mice.

The data shows that TPO/Mpl signaling and endocytosis orchestrate erythropoiesis and thrombopoiesis to control the bone marrow environment. Mpl regulates both MK and erythroid progenitors, highlighting clinically relevant interactions between these two blood cell compartments in myelofibrosis development.