Use of Jak2 Inhibitors to Limit Ineffective Erythropoiesis and Iron Absorption in Mice Affected by β-Thalassemia and Other Disorders of Red Cell Production.
Authors: Melchiori Luca , Gardenghi Sara , Guy Ella C. , Rachmilewitz Eliezer , Giardina Patricia J , Grady Robert W. , Narla Mohandas , An Xiuli , Rivella Stefano 
Published: October 2019

Abstract
Abstract 2020
Poster Board I-1042
β-thalassemia intermedia (TI) and major (TM) are characterized by Ineffective Erythropoiesis (IE). We hypothesized that the kinase Jak2 plays a major role in IE and splenomegaly. To test this hypothesis we administered a Jak2 inhibitor (TG101209) to mice affected by TI, showing that this treatment was associated with a marked decrease in IE, and a moderate decrease in hemoglobin (Hb) levels (∼1 g/dL). This last observation indicates that the use of a Jak2 inhibitor might exacerbate anemia in thalassemia. However, we hypothesized that using standard transfusion to treat TM mice would also be adequate to prevent any further anemia caused by Jak2 inhibition while still allowing for decreased splenomegaly. Therefore, we analyzed the erythropoiesis and iron metabolism in TM animals treated with a Jak2 inhibitor and transfused. Use of TG101209 in TM mice not only reduced the spleen size dramatically (0.42±0.15 g and 0.19±0.10 g respectively in transfused+placebo (N=4) vs transfused+TG101209 (N=8), P= 0.007), but also allowed the mice to maintain higher Hb levels (respectively 7.3±1.1 g/dl vs 9.3±1.2 g/dl, P=0.019). This was likely due to reduced spleen size and limited red cell sequestration. Contrary to TM mice treated with transfusion+placebo, no foci of extra-medullary hematopoiesis were detectable in the parenchema of mice treated with TG101209. Hamp1 expression inversely correlated with the spleen weight, possibly indicating that suppression of IE (due both to blood transfusion and TG101209 administration) had a positive effect on Hamp1 expression. Hb levels also directly correlated with Hamp1 expression in the same animals. In this case, however, only transfusion played a role in increasing Hamp1 expression, although TG101209 undoubtedly had a positive effect by reducing the spleen size and thereby indirectly increasing the Hb levels.
The suppression of erythropoiesis by blood transfusion limits the extent of our interpretations as it may mask the effect of the Jak2 inhibitor. Therefore we hypothesized that the administration of a tailored and reduced dose of the drug could be effective in reducing the splenomegaly in non-transfused TI mice, without affecting the Hb levels. We also hypothesized that the suppression of erythropoiesis would also lead to increased Hamp1 expression in the presence of iron overload. Compared to mice treated with placebo (N=5), analysis of TI mice treated with a tailored dose of 100mg/kg/day of the drug (N=11) showed a significant decrease in spleen size (0.18±0.05 g and 0.27±0.05 g, P=0.006 for drug treated mice and placebo treated mice respectively). Of note no significant difference of Hb levels was detectable between the 2 groups. In the drug treated mice we observed a significant decrease of the immature erythroid cell population (P=0.012) and amelioration of the architecture of the spleen, with the reappearance of white pulp foci and a significant restoration of the splenic lymphocitic populations. Drug treated mice showed increased levels of Hamp1 mRNA that inversely correlated with the spleen weight, suggesting a direct feedback between erythropoietic rate and expression of Hamp1.
To determine if the use of Jak2 inhibitors could be beneficial in a mouse model mimicking a human form of hereditary ellyptocytosis, we treated mice KO for the 4.1R protein isoforms with TG101209, in presence or absence of blood transfusions. These mice exhibit moderate splenomegaly and anemia and the drug treatment was effective in reducing the spleen weight and the associated IE. We also plan to analyze Sickle Cell mice that we are treating with a Jak2 inhibitor. In conclusion our data show that the administration of Jak2 inhibitors is efficient in decreasing the spleen size and ameliorating the pathologic iron metabolism in thalassemia, both in the presence or absence of blood transfusions. Moreover we show that Jak2 inhibitors could transform the therapeutic approach for other forms of anemias.

Disclosures:
No relevant conflicts of interest to declare.