Activation of the mTOR Signaling Pathway by L-Leucine in RPS14-Deficient Erythroid Cells

Abstract 171

The 5q- syndrome is the most distinct of all myelodysplastic syndromes with a clear genotype/phenotype relationship. Haploinsufficiency of the ribosomal protein gene RPS14 underlies the erythroid defect found in the 5q- syndrome. The 5q- syndrome is a disorder of aberrant ribosome biogenesis and we have recently demonstrated that erythrocytes obtained from patients with the 5q- syndrome show impaired translation. This defect in translation may represent a potential therapeutic target in the 5q- syndrome and other ribosomopathies, and there are some indications that the use of the translation enhancer L-leucine may have some efficacy. L-leucine is a branched chain amino acid that has been shown to improve haemoglobin levels and transfusion independence in patients with the ribosomopathy Diamond Blackfan Anemia (DBA). Moreover, the treatment of zebrafish models of DBA and the 5q- syndrome with L-leucine has recently been shown by others to result in partial reversal of the anemia. To model the RPS14 haploinsufficiency observed in the 5q- syndrome, we used lentivirally delivered shRNA sequences to reduce the expression of RPS14 in human bone marrow CD34⁺ cells from healthy controls. We have recently shown that treatment of cultured human erythroid cells derived from CD34⁺ cells of healthy controls with RPS14 knockdown and cultured erythroid cells derived from the CD34⁺ cells of patients with the 5q- syndrome with L-leucine results in an increase in cell proliferation, erythroid differentiation and mRNA translation. There is evidence to suggest that L-leucine activates the mTOR (mammalian target of rapamycin) signaling pathway that controls many cellular processes including cell growth and mRNA translation. In order to investigate the mechanism of action of L-leucine, we have studied the phosphorylation levels of S6K1 and 4EBP1, the key downstream targets of mTORC1 (mTOR Complex 1), by sandwich ELISA, Human Phospho-kinase Antibody Array and flow cytometry in RPS14-deficient human erythroblasts. We have shown a significant increase in the levels of phospho-S6K1 and phospho-4EBP1 following L-leucine treatment of cultured erythroid cells derived from CD34⁺ cells of healthy controls with RPS14 knockdown. The effects of L-leucine on phospho-S6K1 and phospho-4EBP1 were abrogated by rapamycin (an mTOR inhibitor), suggesting that L-leucine has a specific action on the mTOR signaling pathway. Consistent to the results observed in the RPS14 knockdown model, treatment with L-leucine also increased the level of phospho-S6K1 in cultured erythroid cells derived from the bone marrow cells of 5q- MDS patients. These data suggest that L-leucine activates the mTOR pathway in RPS14-deficient human erythroblasts. Our studies support the evaluation of L-leucine as a potential therapeutic agent in the treatment of the 5q- syndrome, and provide evidence on its mode of action through activation of the mTOR signaling pathway.