L-leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway

Abstract

Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34 ؉ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS. (Blood. 2012; 120(11):2214-2224) IntroductionDiamond-Blackfan anemia (DBA; MIM# 105650) is a congenital bone marrow failure syndrome of childhood manifested as normochromic macrocytic anemia with absence or insufficient erythroid precursors in the bone marrow. 1,2 Twenty-five percent of DBA patients have mutations in the RPS19 gene, which encodes a component of the 40S ribosomal subunit. 3,4 A further 25% of DBA patients have been shown to have mutations in other ribosomal protein genes, 5 supporting the hypothesis that DBA is a disease of altered ribosome assembly or function. DBA shares a number of its clinical features with several other congenital syndromes that also carry heterozygous mutations affecting ribosome biogenesis, such as Shwachman-Diamond syndrome (SDS), cartilage-hair hypoplasia syndrome, and dyskeratosis congenita (DC) suggesting that all of these conditions share at least some common pathogenic mechanisms; they have thus been termed "ribosomopathies." 6 In addition, evidence suggests that the anemia associated with the 5q minus (5qϪ) syndrome (or myelodysplastic syndrome with loss of all or part of chromosome 5q [del(5q) MDS]), a distinct subtype of myelodysplastic syndrome results from somatic heterozygous loss of the ribosomal protein gene RPS14 in hematopoietic stem cells. 7,8 Efforts to understand why ribosomal protein haploinsufficiencies have such a specific and profound effect on erythroid development at the molecular level have focused on the activation and stabilization of p53 in response to ribosomal stress. 9 However, the precise mechanisms governing how p53 stabilization occurs in response to ribosomal protein haploinsufficiency have not been clearly defined. Furthermore, not all bone marrow samples from patients with del(5q) MDS or DBA sh...

Translation factors and ribosomal proteins control tumor onset and progression: how?

“…154 A possible involvement of the mTORC1 signaling in the response to RP depletion has been recently shown in zebrafish. Payne et al 155 showed that leucine-mediated mTORC1 activation could rescue the phenotype caused by RPS19 or RPS14 depletion. Therefore, the model is that a quantitative inhibition of ribosome synthesis or function could induce a cellular response that, by altering the ratio between initiation and elongation, could affect the translation pattern favoring the synthesis of oncogenic products (model 2, Figure 4).…”

Marrow failure: a window into ribosome biology

“…42 Leucine treatment improved erythroid differentiation in zebrafish rps19 and rps14 morpholino knockdown models 43 and in human CD34 1 cells wherein RPS19 or RPS14 had been knocked down. 43 These data suggest that the beneficial effect of L-leucine on DBA cells may be ascribed to its regulation of protein metabolism through activation of a key kinase known as mammalian target of rapacmycin (mTOR), a master regulator of cell growth and protein synthesis [43][44][45] and the resulting stimulation of translation. 46 The potential effect of leucine on protein translation of specific mRNAs in DBA has yet to be determined and clinical studies are ongoing.…”

Activation of the mTOR signaling pathway by L-leucine in 5q- syndrome and other RPS14-deficient erythroblasts

“…11 Recently, DBA and del(5q) MDS have been modeled in zebrafish using antisense morpholinos to knockdown rps19 and rps14, respectively. 12 The treatment of the zebrafish models of DBA and del(5q) MDS with L-leucine resulted in an improvement in the hemoglobinization, red cell numbers and developmental defects. 12 Similarly, the treatment of Rps19-deficient mice with L-leucine has been shown to result in an improvement in the anemia, with an increase in hemoglobin concentration and the number of erythrocytes.…”

“…12 The treatment of the zebrafish models of DBA and del(5q) MDS with L-leucine resulted in an improvement in the hemoglobinization, red cell numbers and developmental defects. 12 Similarly, the treatment of Rps19-deficient mice with L-leucine has been shown to result in an improvement in the anemia, with an increase in hemoglobin concentration and the number of erythrocytes. 13 In a recent study, we have shown that L-leucine treatment of cultured human erythroblasts derived from CD34 þ bone marrow cells with RPS14 knockdown and from MDS patients with del(5q) results in an increase in the cell proliferation, erythroid differentiation and mRNA translation.…”