LLR Molecular Haematology Unit, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford
The 5q- syndrome was first described by Van den Berghe et al in 19741 and is the most distinct of all the myelodysplastic syndromes (MDS) with a clear genotype-phenotype relationship. Patients with the 5q- syndrome have macrocytic anaemia, normal or high platelet count, hypolobulated megakaryocytes, a medullary blast count of less than 5%, and deletion of the long arm of chromosome 5 [del(5q)] as the sole karyotypic abnormality. Approximately 10% of patients transform to acute myeloid leukaemia (AML).2
The commonly deleted region (CDR) of the 5q- syndrome was identified by our group in Oxford as the 1.5Mb interval at 5q32-33 flanked by the DNA marker DS5413 and the GLRA1 gene. Genomic annotation of the CDR of the 5q- syndrome was performed and several promising candidate genes mapping within the CDR were noted, including the tumour suppressor gene SPARC, RPS14 (a component of the 40S ribosomal subunit) and several microRNA genes. All the genes within the CDR were sequenced and no mutations were found,5 suggesting that haploinsufficiency (a dosage effect resulting from the loss of a single allele of a gene)6 might be the basis of the 5q- syndrome. The transcriptome of the CD34+ cells of patients with the 5q- syndrome has been determined using gene expression profiling.5 Genes identified as showing haploinsufficiency in the haematopoietic stem cells of patients with the 5q- syndrome include the ribosomal protein gene RPS14.5
A pivotal report from Ebert et al in 2008 identified RPS14 as a 5q- syndrome gene by an RNA interference screen of each gene within the CDR.7 Knockdown of RPS14 caused a block in differentiation with relative preservation of megakaryocytic differentiation. Forced expression of an RPS14 cDNA in primary bone marrow cells from patients with the 5q- syndrome rescued the phenotype.7 RPS14 haploinsufficiency caused a block in the processing of pre-ribosomal RNA and in the formation of the 40S ribosomal subunit.7 We subsequently showed that CD34+ cells from patients with the 5q- syndrome have a defect in the expression of many genes involved in ribosome biogenesis and in the control of translation.8 These data suggest that the 5q- syndrome represents a disorder of aberrant ribosome biogenesis. The 5q- syndrome is now considered to be a ribosomopathy, with strong analogy to Diamond-Blackfan anaemia (DBA),5 a disorder similarly caused by haploinsufficiency of ribosomal protein genes.9
The study of animal models of DBA show that ribosomal stress leads to activation of the p53 pathway in this disorder and that this mechanism underlies the anaemia observed.10 Barlow et al generated a mouse model of the 5q- syndrome using large-scale chromosomal engineering.11 Mice with haploinsufficiency of Rps14 show key features of the human disease, including a macrocytic anaemia. The ‘5q- mouse’ has defective bone marrow progenitor development and the bone marrow cells show an accumulation of p53 protein with increased apoptosis. Intercrossing the ‘5q- mouse’ with p53 deficient mice completely rescued the progenitor cell defect, suggesting, for the first time, that a p53-dependent mechanism underlies the pathophysiology of the 5q- syndrome.11 Importantly, induction of p53 and up-regulation of the p53 pathway was subsequently shown to occur in the human 5q- syndrome.12 Immunohistochemical analysis of p53 protein expression in bone marrow trephine sections from patients with 5q- syndrome showed moderate to strong p53 expression in erythroid cells and gene expression profiling demonstrated that the p53 pathway is significantly deregulated in the haematopoietic stem cells (HSC) of patients with the 5q- syndrome.12 P53 activation has been shown to occur selectively in human erythroid progenitor cells; expression of shRNAs targeting RPS14 in human HSC resulted in erythroid-specific accumulation of p21, cell cycle arrest and apoptosis, consistent with the haematopoietic phenotype of the 5q- syndrome.13 The subsequent inhibition of p53 by the p53 inhibitor PFT-alpha in culture rescued the erythroid defect, suggesting that p53 activation may represent a therapeutic target in MDS with the del(5q).13 However, this will be a therapeutic option in humans only if this intervention does not abrogate the critical tumour suppressor function of p53.
Recent data suggests that mutation of p53, resulting in the inactivation of the p53 protein, may be one of the molecular events necessary for clonal progression of the 5q- syndrome to AML.14-16 Using deep-sequencing technology, Jädersten et al have demonstrated that small subclones of haematopoietic cells with p53 mutation may occur at an early disease stage in 18% of patients with MDS with the del(5q). The mutations were present years before disease progression and were associated with an increased risk of leukaemic evolution.16 It is well recognised that mutated p53 may lead to genetic instability and disease progression in cancer and leukaemia. Thus p53 may play a pivotal role in both the development and progression of the 5q- syndrome; with p53 (wildtype) activation leading to increased apoptosis and consequent defective erythropoiesis in the early stage of the disease, followed, in some patients, by an expansion of a small subclone harbouring mutant (inactivated) p53, and leading to leukaemic transformation as the disease progresses.
The drug lenalidomide has been shown to have dramatic therapeutic effects in MDS patients with the 5q- syndrome and other patients with MDS and a del(5q). A large phase II study by List et al, on 148 MDS patients with a chromosome 5q31 deletion showed transfusion independency in 67% of the patients, as well as a complete cytogenetic remission in 45% of the patients treated with lenalidomide.17 This and other studies have clearly demonstrated that lenalidomide is effective in lower-risk, transfusion-dependent patients with MDS and the del(5q). However, not all patients respond to lenalidomide and approximately 50% of del(5q) MDS patients acquire resistance to lenalidomide within two years.17 Interestingly, there is evidence to suggest that the presence of p53 mutations negatively influences response to lenalidomide in del(5q) MDS.16 There is thus a clinical need for novel treatments for del(5q) MDS. Potential new therapeutic agents for del(5q) MDS, include the p53 inhibitor Cenersen19 and the translation enhancer L-leucine. Recently, Cenersen, a clinically active 20-mer antisense oligonucleotide complementary to p53 exon10, has been shown to suppress p53 expression and restore erythropoiesis in del(5q) MDS patient cells in culture.19
It is now widely accepted that p53 activation secondary to ribosomal haploinsufficiency is the mechanism that underlies the anaemia in the 5q- syndrome. There is evidence to suggest that haploinsufficiency of the miRNA genes miR-145 and miR-146a, mapping within and adjacent to the CDR of the 5q- syndrome,5 may be the cause of some of the other key features of the 5q- syndrome, namely hypolobulated megakaryocytes and peripheral thrombocytosis. Starczynowski et al have shown that miR-145 and miR-146a are down-regulated in the CD34+ cells of 5q- syndrome patients and that knockdown of miR-145 and miR-146a in mouse HSCs resulted in thrombocytosis, mild neutropenia and megakaryocytic dysplasia.22 The FLI1 gene, encoding a transcription factor involved in megakaryopoiesis, has been identified as a critical target of miR-145.23 Inhibition of miR-145 or overexpression of Fli-1 increases the production of megakaryocytic cells relative to erythroid cells.23 Therefore, the thrombocytosis observed in some patients with the 5q- syndrome may be the result of deficiency of miR-145 and miR-146a.
Great progress has been made over the past decade in the elucidation of the molecular basis of the 5q- syndrome. New insights into disease mechanisms are leading to the development of novel treatments for this disorder. The determination of the molecular abnormality that confers a clonal growth advantage in the 5q- syndrome remains a key unanswered question in this disease.
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