Potential Use of Jak2 Inhibitors to Limit Ineffective Erythropoiesis and Reverse Splenomegaly in β-Thalassemia.
Authors: Melchiori Luca , Guy Ella , Libani Ilaria , Schirò Raffaella , Ramos Pedro , Breda Laura , Chadburn Amy , Liu Yi-Fang , De Sousa Maria , Rachmilewitz Eliezer , Giardina Patricia J , Grady Robert W , Rivella Stefano 
Published: April 2020

Abstract
β-Thalassemia intermedia and major are characterized by ineffective erythropoiesis (IE), requiring sporadic or chronic blood transfusions, respectively. Some of the major consequences of IE are extra-medullary hematopoiesis (EMH), splenomegaly and systemic iron overload mediated by transfusion therapy and down-regulation of hepcidin. Using mouse models of β-thalassemia intermedia (th3/+) and major (th3/th3), and human specimens we investigated IE in this disorder. Th3/+ and th3/th3 erythroid cells were analyzed with respect to rates of apoptosis and degrees of cell proliferation and differentiation. We found that there was both a relative and absolute expansion of the immature erythroid progenitor cell fraction in thalassemic mice compared to cells in the final stages of differentiation. Further investigation of the thalassemic erythroid cells in vivo and in vitro indicated that a larger number of the thalassemic cells are associated with the phosphorylated form of the Jak2 protein kinase than in normal mice. In fact, their proliferation was prevented by TG101209, a Jak2 inhibitor. Similar compounds are currently utilized or being considered for use in the treatment of myeloproliferative diseases such as polycythemia vera. In order to assess the potential of Jak2 inhibitors in limiting IE in β-thalassemia, we administered TG101209 to th3/+ mice. We found that both 10 and 18 days of treatment were sufficient to dramatically reduce the spleen size and the percentage of immature erythroid progenitors therein compared with administration of a placebo. However, these changes were associated with decreasing hemoglobin levels (Blood, 2008, 112:875–85). We speculated that this problem could be overcome by administration of blood transfusions during treatment, an extension of current management in thalassemia as noted above. To test this hypothesis, we administered TG101209 to th3/th3 mice in conjunction with regular blood transfusions. Administration of TG101209 to transfused th3/+ mice is in progress. Our preliminary data on th3/th3 mice indicates that simultaneous administration of TG101209 and transfused blood not only reverses splenomegaly, but also results in higher Hb levels (N ≥ 3). The increased hemoglobin levels observed, compared to those in mice treated with transfusion therapy alone, suggest that the use of Jak2 inhibitors may reduce the amount of blood per transfusion and/or the rate of transfusion required in thalassemia. Since the increased iron absorption in thalassemic mice is a direct consequence of IE, treatment with TG101209 could also be beneficial in ameliorating this process. According to several observations, suppression of erythropoiesis should lead to increased Hamp1 expression in the presence of iron overload. Therefore, under conditions of Jak2 inhibition, Hamp1 transcription is expected to increase. To test this hypothesis we are currently analyzing organ iron levels and the expression of iron-related genes in drug-treated mice. In conclusion, although our study does not exclude a role for apoptosis in the IE of β-thalassemia, we have demonstrated that increased cell proliferation and limited cell differentiation play a significant role in this process. Moreover, we show for the first time that a Jak2 inhibitor is effective in decreasing the spleen size of thalassemic mice. This could represent a completely new approach to the treatment of splenomegaly in β-thalassemia patients, perhaps coupled with blood transfusion. That administration of a Jak2 inhibitor reverses splenomegaly and also ameliorates the degree of iron overload could provide an opportunity to gain new insight into the dynamic processes of iron absorption and erythropoiesis in this pathological condition.