Ectopic Expression of the Extracellular Domain of Mpl Is Sufficient to Induce a Hematopoietic Population Crisis
Authors: Wicke Daniel C. , Meyer Johann , Büsche Guntram , Kreipe Hans , Li Zhixiong , Welte Karl , Ballmaier Matthias , Baum Christopher , Modlich Ute 
Published: April 2020

Abstract
The thrombopoietin receptor Mpl is required for regeneration of hematopoietic stem cells and governs megakaryocytic differentiation. Patients with inherited MPL deficiency suffer from severe thrombocytopenia which progresses to aplastic anemia, a disorder called congenital amegakaryocytic thrombocytopenia (CAMT). As a first step towards a potential gene therapy for MPL deficiency, we retrovirally expressed the receptor in a murine bone marrow transplantation model. An initial series of vectors used a strong enhancer-promoter derived from the spleen-focus forming virus (SFFV). Mice transplanted with hematopoietic cells modified by these constructs developed a profound yet transient elevation of multi-lineage hematopoiesis due to increased signaling of the Thpo receptor on target and non-target cells. Mice developed increased leukocyte, erythrocyte and platelet counts (2–3 times elevated) compared to GFP control animals. Histopathology revealed an elevated number of mature megakaryocytes with atypical features like numerous giant forms in the bone marrow and the spleen and atypical neoformation of bone in the spleen leading to the diagnosis of chronic myeloproliferative disorder (CMPD). A minority of mice (3/27) developed erythroleukemia with almost 100% CD71 and TER119 double positive cells as detected by flow cytometry. Histopathology presented infiltration of erythroblasts in all hematopoietic tissues like the BM, spleen and liver. Molecular analysis revealed retroviral vector insertions in sfpi1, fli1 and klf3 that seem to be the major driving force for the development of leukemia in these animals. Somewhat unexpectedly, in the majority of mice the CMPD converted into a progressive, potentially lethal pancytopenia. Animals had severely reduced blood cell counts with only 50% of leukocyte, 20% of erythrocyte and 10% of platelet counts compared to GFP control animals. This population crisis affected all major blood lineages and also involved co-existing unmodified hematopoiesis. Histopathology presented a dysmegakaryopoiesis with an increased number of atypical micro-megakaryocytes, histiocytes with erythrocytophagocytosis and atypical mast cell proliferation diagnosed as a myelodysplastic syndrome (MDS)-like disorder. In the bone marrow, pancytopenic mice had reduced cell numbers of the primitive cell fraction (LSK cells). To address the mechanism of pancytopenia, we expressed a dominant negative form of Mpl (dnMpl) consisting of the extracellular and transmembrane domain and lacking the intracellular signal transduction domain. Animals transplanted with dnMpl-modified cells failed to show the initial CMPD but developed the same pancytopenic, MDS-like end stage. A vector expressing Mpl under control of the PGK promoter or a fragment of the Mpl-promoter reduced or completely avoided the side effects (CMPD, MDS-like disorder) observed with vectors using stronger promoters. The induction of a hematopoietic population homeostasis thus depends upon Mpl expression levels, indicating the need for strictly regulated transgene expression in gene therapy for MPL deficiency. As ectopic expression of the extracellular domain is sufficient to cause MDS, sequestration of crucial niche factors like Thpo may contribute to the pathogenesis of this disorder. This study demonstrates that ectopic expression of a hematopoietic growth factor receptor may disturb organ homeostasis through interference with intra- and extracellular mechanisms of cell communication.