β-thalassemia (β-thal) is one of the most common monogenic disorders worldwide, characterized by ineffective erythropoiesis leading to a chronic, debilitating anemia associated with high morbidity and mortality. Erythroid maturation is a dynamic process tightly regulated by complex signaling mechanisms, only partially described either in normal and diseased erythropoiesis. To investigate this issue, we carried out a high throughput kinome analysis by taking advantage of Kinexus array technology (http://www.kinexus.ca), in sorted erythroid precursors from a mouse model of β-thalassemia (Hbb3th/+) compared to wildtype animals. In β-thal mice, we found differential modulation of many protein kinases. Network computational analysis unveiled common as well as erythroid precursor-specific signaling mechanisms of altered erythrocyte differentiation in beta thalassemia, suggesting a selective perturbation in protein kinase/phosphatase balance in β-thal erythropoiesis.
We reasoned that balancing kinome anomalies, by increasing phosphatome activity, could normalize kinome signaling pathways, thus ameliorating erythropoiesis. We explored the expression and function of different protein phosphatase and we found reduced expression and function of protein Tyr-phosphatase receptor type gamma (PTPRG). To investigate PTPRG role in erythropoiesis, we exploited a novel Trojan-fusion protein (TAT-ICD), we recently patented, that delivers intracellularly the catalytic domain of PTPRG and up-modulates its signaling cascade, as both a research tool to map dysfunctional pathways and as a potential therapeutic agent. In β-thal mice, TAT-ICD acted on multiple abnormally activated targets identified by computational analysis. TAT-ICD significantly reduced the activation of (i) Jak2- STAT5 pathway; (ii) Bruton tyrosine kinase (BTK), that has been reported to be part of the erythropoietin cascade; (iii) Akt, that is involved in TGF-β -SMAD signaling pathway. This was associated with down-regulation of Erfe and Gdf11 gene expression in sorted erythroblasts from TAT-ICD treated β-thal mice. Collectively, TAT-ICD treatment resulted in amelioration of β-thal ineffective erythropoiesis, evaluated by multiple approaches, including the profile of erythroid maturation and the amount of Annexin-V+ erythroid cells, reticulocyte count, circulating erythroblasts and hemolytic indices (U.S. Patent #62/109,555). The improvement of anemia was also associated with reduction in alpha aggregates and membrane bound hemichromes in circulating erythrocytes. We also found a reduction of liver and spleen iron accumulation in agreement with the beneficial effects on the hematologic phenotype. It is of note that TAT-ICD treatment did not affect either peripheral leukocyte counts or spleen lymphocyte pattern.
Our data unveil abnormalities in signal transduction pathways as new mechanism involved in β-thal erythropoiesis, and validate a novel, breakthrough, therapeutic approach to reset back to homeostatic equilibrium altered kinome in diseased erythropoiesis.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.