Effects of L-leucine in 5q- syndrome and other RPS14-deficient erythroblasts

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The 5q- syndrome is the most distinct of all the myelodysplastic syndromes (MDS) with a clear genotype/phenotype relationship. The RPS14 gene, encoding a ribosomal protein and mapping to the commonly deleted region (CDR) of the 5q- syndrome,1 shows haploinsufficiency in the CD34+ cells of patients with this disorder.2, 3 The genes in the 5q- syndrome CDR were studied by an RNA-mediated interference-based approach and it was shown that haploinsufficiency of RPS14 in normal CD34+ cells resulted in a block in erythroid differentiation with relative preservation of megakaryocyte differentiation, suggesting that RPS14 haploinsufficiency is the probable cause of the erythroid defect in this disorder.3 Several lines of converging evidence suggest that p53 activation secondary to ribosomal haploinsufficiency is the mechanism that underlies the anemia in the 5q- syndrome.4, 5

The bone marrow cells of patients with the 5q- syndrome show a block in the processing of pre-ribosomal RNA3 and the CD34+ cells of these patients show deregulation of multiple ribosomal protein genes and genes involved in the control of translation.6 These data suggest that the 5q- syndrome represents a disorder of aberrant ribosome biogenesis.3, 6 Erythroid progenitor cells have a very high rate of proliferation and a requirement for massive globin synthesis, thus necessitating a very high level of ribosome biogenesis and ribosomal activity. This may explain the particular sensitivity of the erythroid lineage to reduced expression levels of ribosomal proteins.5

In this study, we have first investigated the effects of RPS14 haploinsufficiency on human erythropoiesis using an RPS14 short hairpin RNA (shRNA) culture system. Second, we have investigated the effects of the translation enhancer L-leucine on cell proliferation, erythroid differentiation and mRNA translation in RPS14-deficient erythroblasts, both obtained from patients with MDS and the del(5q) and also generated using shRNA technology.

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. Cells were cultured according to a method developed to study the generation of erythroblasts7 and evaluated at day 14 of culture. RPS14 expression levels were significantly decreased by approximately 50% in cells expressing RPS14 shRNAs (Figure 1a). Decreased RPS14 expression was confirmed at the protein level (Figure 1a). Recently, a p53-dependent mechanism was found to underlie the pathogenesis of the 5q- syndrome.4, 5 Consistent with these reports, a significant activation of p53 expression (>1.5-fold increase in total p53 protein expression measured by intracellular staining and flow cytometry) was observed in the cells with reduced RPS14 expression when compared with the scramble control (Figure 1b). A significant increase in apoptosis (>twofold increase in dead-cell percentage) and cell cycle arrest at G1 phase were observed in the cells with reduced RPS14 expression when compared with the scramble control (Figures 1c and d). Moreover, gene expression profiling data showed deregulation of multiple p53 target genes in cells expressing RPS14 shRNAs, including upregulation of p21 by four-fold. A significant reduction in the cell populations expressing the erythroid differentiation markers CD36 (early erythroid cells), CD71 (proliferating cells and early erythroid cells) and CD235a (late erythroid cells) was observed, suggesting that reduced RPS14 expression leads to an impaired erythroid differentiation (Figure 1e). These data are consistent with earlier reports showing that haploinsufficiency of RPS14 causes an erythroid maturation block and increased apoptosis in vitro.3

Figure 1
figure1

Analysis of RPS14-deficient cells from healthy controls. Cells were cultured according to a method developed to study the generation of erythroblasts7 and evaluated at day 14 of culture. CD34+ cells were cultured for 14 days as described7 and on day 7, erythropoietin (Roche, Basel, Switzerland) at 2 U/ml was added to the medium. shRNA sequences targeting RPS14 and cloned into a pLKO.1 vector were obtained from Sigma Aldrich (Gillingham, UK) (TRCN0000008641 and TRCN0000008644, henceforth termed sh41 and sh44, respectively). An shRNA sequence that does not target human genes (‘scramble’) was used as experimental control. Lentivirus was produced in 293T cells. CD34+ cells were infected with lentivirus for 24 h, and transduced cells were selected with puromycin. (a) Expression levels of RPS14 determined using real-time quantitative PCR. RPS14 protein level measured by western blotting in NB4 cells. (b) Total p53 protein level measured by intracellular staining. The expression level is presented as mean fluorescence intensity obtained by flow cytometry. (c) Apoptosis assay performed using Annexin V and propidium iodide staining and measured by flow cytometry. Live, apoptotic and dead cells correspond to Annexin VPI, Annexin V+PI and Annexin V+PI+, respectively. (d) Cell cycle analysis performed using propidium iodide staining and measured by flow cytometry. (e) Erythroid differentiation measured by staining of erythroid cell surface markers CD36, CD71 and CD235a. The values in cells expressing RPS14 shRNAs are relative to the scramble control. (f) Evaluation of mRNA translation measured by incorporation of [3H]-L-leucine into newly synthesized proteins. Results in each bar graph were obtained from 5, 6, 4, 6, 5 and 6 independent experiments in panel A, B, C, D, E and F, respectively. Bar graphs show mean+s.e.m. (*P<0.05, **P<0.01 and ***P<0.001, paired t-test). Full methods are available as Supplementary Information.

The deregulation (primarily downregulation) of multiple genes involved in ribosome biogenesis and protein synthesis has been described in the cells of patients with the ribosomopathies Diamond–Blackfan anemia (DBA) and Schwachman–Diamond syndrome.8 Similarly, we have previously reported the downregulation of multiple ribosomal genes and translation initiation and elongation factors in the CD34+ cells of patients with the 5q- syndrome.6 It is considered that this phenomenon is likely a consequence of haploinsufficiency of RPS14 in the 5q- syndrome. However, experimental evidence for this hypothesis is lacking. In this current study, using gene expression profiling we showed that 75% of 579 probe sets for multiple ribosomal and translation-related genes were downregulated in cells expressing RPS14 shRNAs compared with the scramble control, thus demonstrating that reduced RPS14 expression in cultured erythroid cells results in co-downregulation of multiple genes involved in ribosome biogenesis.

Defective ribosome biogenesis can result in a reduction in the efficiency of mRNA translation.9 The lymphocytes of patients with DBA, for example, show reduced translation compared with lymphocytes obtained from healthy controls.9 We investigated whether reduced RPS14 expression in cultured erythroid cells results in reduced mRNA translation. We found that total protein production in cells expressing RPS14 shRNAs was significantly decreased compared with cells expressing the scramble control (Figure 1f). This defect in translation represents a potential therapeutic target in the ribosomopathies and there are some indications that the use of the amino acid L-leucine may have some efficacy. A patient with DBA was reported to become transfusion-independent following L-leucine therapy.10 The addition of L-leucine to cultured lymphocytes obtained from some patients with DBA, resulted in an increase in translational efficiency.9 More recently, it has been reported that the treatment of RPS14-deficient or RPS19-deficient zebrafish embryos and RPS19-deficient mice with L-leucine resulted in a partial reversal of the anemia.11, 12 Although the mechanism by which L-leucine enhances translation is not fully understood, there is evidence to suggest that it acts as a nutrient signal that stimulates the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway controls cell growth and proliferation by enhancing the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex, and thus by modulating mRNA translation.13

We investigated whether the treatment with L-leucine ameliorates the phenotype associated with RPS14 deficiency in human-cultured erythroblasts. Cells were evaluated 4 days after addition of L-leucine. The treatment with L-leucine did not show a significant effect on p53 levels in cells expressing RPS14 shRNAs (Supplementary Figure 1a). Using the MTS assay, we have shown that the proliferation of cells expressing RPS14 shRNAs is reduced compared with the scramble control (Figure 2a). We found that cells expressing RPS14 shRNAs treated with L-leucine showed a significant increase in proliferation compared with untreated cells (Figure 2a). This increase in proliferation remained significant 7 days after addition of L-leucine (P<0.05). We then studied the effect of L-leucine on erythroid differentiation by staining for the erythroid cell surface markers CD36, CD71 and CD235a. No significant increase in intermediate (CD36+CD235a+ and CD71+CD235a+) and late (CD36CD235a+ and CD71CD235a+) erythroid cell populations was observed between L-leucine-treated and untreated cells (data not shown). However, a significant increase in early erythroid progenitor cells (CD36+CD235a and CD71+CD235a) was observed in the L-leucine-treated CD34+ cells expressing RPS14 shRNAs compared with untreated cells (Figure 2b).

Figure 2
figure2

Effects of L-leucine in cells expressing RPS14 shRNAs and in cells from MDS patient with del(5q). Cells were cultured under the same conditions described in the Figure 1 legend. L-leucine (Sigma Aldrich) at a concentration of 600 μg/ml was added from day 7 and cells were evaluated 4 days after treatment with L-leucine and compared with untreated cells. (a) Cell proliferation in cells expressing RPS14 shRNAs or the scramble control. Cells were evaluated by MTS assays and cell proliferation is shown as percentage compared with the proliferation in untransduced control cells from the same donors. (b) Erythroid differentiation measured by staining of erythroid cell surface markers CD36, CD71 and CD235a in cells expressing RPS14 shRNAs. (c) Evaluation of mRNA translation in cells expressing RPS14 shRNAs or the scramble control measured by incorporation of [3H]-L-leucine into newly synthesized proteins. (d) Cell counts (trypan blue exclusion) on cultured cells from two MDS patients with del(5q). (e) Cell proliferation in cells from two MDS patients with del(5q) and healthy controls (n=3) as measured by MTS assays. Cell proliferation in L-leucine-treated cells is reported as percentage compared with untreated cells. (f) Erythroid differentiation in cells from two MDS patients with del(5q) and from healthy controls (n=3) measured by staining for the erythroid cell surface markers CD36, CD71 and CD235a. (g) Percentage of CD71+CD235a+ cells in cultured cells from healthy controls (n=3) and from two MDS patients with del(5q). (h) Evaluation of mRNA translation in healthy control and del(5q) patient cells measured by incorporation of [3H]-L-leucine into newly synthesized proteins as previously described.9 Results in each bar graph were obtained from 3, 6 and 6 independent experiments in panel A, B and C, respectively. Bar graphs show mean+s.e.m. (*P<0.05 and ***P<0.001, paired t-test). Full methods are available as Supplementary Information.

We observed decreased mRNA translation in cells expressing RPS14 shRNAs (Figure 1f), and we then investigated whether L-leucine enhanced mRNA translation in these cells. Treatment with L-leucine significantly increased protein production by 2.4–2.9-fold in cells expressing RPS14 shRNAs compared with a 1.2-fold increase in cells expressing the scramble control (Figure 2c).

We next evaluated the effects of L-leucine in bone marrow CD34+ cells from two MDS patients with the del(5q) as the sole karyotypic abnormality. The patient cells were cultured using the same erythroid culture system used for the normal CD34+ bone marrow cells. Consistent with our results obtained in cultured erythroblasts with decreased RPS14 expression, cells from the two patients with del(5q) treated with L-leucine showed an increase in cell number and proliferation compared with untreated cells (Figures 2d and e). Treatment with L-leucine also stimulated erythroid differentiation, with an increase in the proportion of cells stained for the erythroid markers CD36, CD71 and CD235a (Figure 2f) and an increase in the proportion of CD71+CD235a+ cells compared with untreated cells in the two MDS patients with del(5q) (Figure 2g). Colony-forming cell assay was performed on cultured cells from one MDS patient with del(5q) and treatment with L-leucine increased the number of BFU-E colonies (Supplementary Figure 1b). Interestingly, Sen et al.14 have recently shown that treatment with L-leucine resulted in improved BFU-E colony growth in cultured cells from two of the three patients with the ribosomopathy Schwachman–Diamond syndrome.

We studied whether mRNA translation was enhanced after L-leucine treatment in cultured cells from the two patients with del(5q). Treatment with L-leucine increased protein production by 2.7–2.8-fold in cells from the two patients with del(5q) compared with a 1.3-fold increase in healthy control cells (Figure 2h), similar to the increase in translation we observed in cells expressing RPS14 shRNAs.

L-leucine treatment did not show a significant increase on cell proliferation, erythroid differentiation and mRNA translation in two patients with low-risk MDS without del(5q) (Supplementary Figures 1c–g).

Gene expression profiling data showed that L-leucine had a limited effect on the transcriptome of cultured cells from the two MDS patients with del(5q), with seven genes increased and nine genes decreased by >1.5-fold by treatment with L-leucine compared with untreated cells (Supplementary Table 1). These data suggest that the effects of L-leucine occur predominantly at the translation level rather than mRNA level.

In this study, we have confirmed that RPS14 is critical for normal human erythropoiesis; its deficiency resulted in p53 activation, impaired erythroid differentiation and increased cellular apoptosis. Our findings also indicate that some ribosomal genes are closely co-regulated in humans and that RPS14 haploinsufficiency results in downregulation of additional ribosomal genes in erythroblasts. Here we report, for the first time, that RPS14-deficient erythroblasts and cultured erythroblasts from MDS patients with the del(5q), show reduced levels of mRNA translation, and that the translation enhancer L-leucine increased the erythroid differentiation, cell proliferation and translation efficiency of these cells. It is probable that translation insufficiency contributes to the erythroid defect observed in the 5q- syndrome and these data support the development of an exciting new strategy for the treatment of MDS with the del(5q) based on improving translation efficiency by L-leucine or other agents.

Lenalidomide has become an established therapy for MDS patients with the del(5q).15 Although the modes of action of lenalidomide and L-leucine are yet to be fully elucidated, the combined use of L-leucine and lenalidomide might be considered for therapy in MDS, as there is evidence to suggest that these two drugs act through different mechanisms and their effects may be complementary.

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Acknowledgements

This work was supported by Leukaemia and Lymphoma Research of the United Kingdom and in part by the National Institute for Health Research of the United Kingdom (Grant NIHR-RP-PG-0310-1004-AN).

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Correspondence to J Boultwood.

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Supplementary Information accompanies the paper on the Leukemia website

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