L-Leucine Improves the Anemia of the 5q- Syndrome Via Activation of the mTOR (mammalian target of rapamycin) Pathway

Abstract 970

The “5q- syndrome” is a subtype of myelodysplastic syndrome (MDS) characterized by a profound macrocytic anemia that is thought to arise from the heterozygous loss of the RPS14 gene on chromosome 5q. The 5q- syndrome shares many characteristics with another ribosomopathy, Diamond Blackfan anemia (DBA), wherein half the patients have a heterozygous loss of a ribosomal protein gene, with RPS19 being the most frequently mutated. Both RPS19 and RPS14 are components of the 40S small ribosomal subunit. Heterozygous loss of both genes is thought to contribute to the pathophysiology of these ribosomopathies by impairing ribosome function and thereby decreasing mRNA translational efficiency. L-Leucine, a branched-chain amino acid has been shown to be a potent stimulator of mRNA translation. In a case report, administration of L-Leucine to a DBA patient resulted in complete recovery of the anemia and improvement in appetite and growth parameters. We therefore hypothesized that L-Leucine could improve the anemia associated with heterozygous loss of RPS14. To test this hypotheisis, we used zebrafish as an in vivo model for del(5q) MDS. Using antisense morpholinos, we knocked down Rps14 expression to haploinsufficient levels and observed anemia in the resulting morphants. Treatment of these morphants with L-Leucine, but not D-Leuicne, resulted in a dramatic increase in hemoglobinization as well as an increase in total erythroid cells. This observation was further validated in vitro using human CD34⁺ hematopoietic progenitor cells infected with RPS14 shRNA. Flow cytometric analysis demonstrated that treatment with L-Leucine increased the total number of erythroid cells (glycophorin-A/CD71 expressing cells) compared to untreated cells. In previous studies, L-leucine has been shown to upregulate mRNA translation by activating the mTOR (mammalian target of rapamycin) pathway and its downstream targets S6Kinase (S6K1) and 4E-Binding proteins (4E-BPs). We demonstrated increased levels of phospho-S6K1 as a result of L-Leucine treatment in the rps14 zebrafish morphant embryos. This increased phosphorylation of S6K1 was inhibited by rapamycin, suggesting specificity of mTOR activation in bringing about the L-Leucine effect. In summary, we have successfully demonstrated, in both a zebrafish model and in human primary hematopoietic stem cells, that L-Leucine alleviates the anemia associated with del(5q) MDS. This effect is likely mediated by activation of the mTOR pathway resulting in increased mRNA translation and an improvement in the anemia associated with loss of RPS14. Our studies support the further evaluation of L-Leucine as a potential therapeutic agent in the treatment of the 5q- syndrome.