Jacqueline Boultwood

Jacqueline Boultwood

Andrea Pellagatti

Andrea Pellagatti
 

 

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.

References

1. Van den Berghe H, Cassiman JJ, David G, Fryns JP, Michaux JL, Sokal G. Distinct haematological disorder with deletion of long arm of no. 5 chromosome. Nature 1974; 251: 437-438.
2. Boultwood J, Pellagatti A, McKenzie AN, Wainscoat JS. Advances in the 5q- syndrome. Blood 2010; 116: 5803-5811.
3. Boultwood J, Fidler C, Lewis S, Kelly S, Sheridan H, Littlewood TJ, Buckle VJ, Wainscoat JS. Molecular mapping of uncharacteristically small 5q deletions in two patients with the 5q- syndrome: delineation of the critical region on 5q and identification of a 5q- breakpoint. Genomics 1994; 19: 425-432.
4. Boultwood J, Fidler C, Strickson AJ, Watkins F, Gama S, Kearney L, Tosi S, Kasprzyk A, Cheng JF, Jaju RJ, Wainscoat JS. Narrowing and genomic annotation of the commonly deleted region of the 5q- syndrome. Blood 2002; 99: 4638-4641.
5. Boultwood J, Pellagatti A, Cattan H, Lawrie CH, Giagounidis A, Malcovati L, Della Porta MG, Jadersten M, Killick S, Fidler C, Cazzola M, Hellstrom-Lindberg E, Wainscoat JS. Gene expression profiling of CD34+ cells in patients with the 5q- syndrome. Br J Haematol 2007; 139: 578-589.
6. Largaespada DA. Haploinsufficiency for tumor suppression: the hazards of being single and living a long time. J Exp Med 2001; 193: F15-18.
7. Ebert BL, Pretz J, Bosco J, Chang CY, Tamayo P, Galili N, Raza A, Root DE, Attar E, Ellis SR, Golub TR. Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature 2008; 451: 335-339.
8. Pellagatti A, Hellstrom-Lindberg E, Giagounidis A, Perry J, Malcovati L, Della Porta MG, Jadersten M, Killick S, Fidler C, Cazzola M, Wainscoat JS, Boultwood J. Haploinsufficiency of RPS14 in 5q- syndrome is associated with deregulation of ribosomal- and translation-related genes. Br J Haematol 2008; 142: 57-64.
9. Draptchinskaia N, Gustavsson P, Andersson B, Pettersson M, Willig TN, Dianzani I, Ball S, Tchernia G, Klar J, Matsson H, Tentler D, Mohandas N, Carlsson B, Dahl N. The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia. Nat Genet 1999; 21: 169-175.
10. McGowan KA, Li JZ, Park CY, Beaudry V, Tabor HK, Sabnis AJ, Zhang W, Fuchs H, de Angelis MH, Myers RM, Attardi LD, Barsh GS. Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects. Nat Genet 2008; 40: 963-970.
11. Barlow JL, Drynan LF, Hewett DR, Holmes LR, Lorenzo-Abalde S, Lane AL, Jolin HE, Pannell R, Middleton AJ, Wong SH, Warren AJ, Wainscoat JS, Boultwood J, McKenzie AN. A p53-dependent mechanism underlies macrocytic anemia in a mouse model of human 5q- syndrome. Nat Med 2010; 16: 59-66.
12. Pellagatti A, Marafioti T, Paterson JC, Barlow JL, Drynan LF, Giagounidis A, Pileri SA, Cazzola M, McKenzie AN, Wainscoat JS, Boultwood J. Induction of p53 and up-regulation of the p53 pathway in the human 5q- syndrome. Blood 2010; 115: 2721-2723.
13. Dutt S, Narla A, Lin K, Mullally A, Abayasekara N, Megerdichian C, Wilson FH, Currie T, Khanna-Gupta A, Berliner N, Kutok JL, Ebert BL. Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells. Blood 2011; 117: 2567-2576.
14. Fernandez-Mercado M, Burns A, Pellagatti A, Giagounidis A, Germing U, Agirre X, Prosper F, Aul C, Killick S, Wainscoat JS, Schuh A, Boultwood J. Targeted resequencing analysis of 25 genes commonly mutated in myeloid disorders in del(5q) myelodysplastic syndromes. Haematologica 2013; [Epub ahead of print].
15. Fidler C, Watkins F, Bowen DT, Littlewood TJ, Wainscoat JS, Boultwood J. NRAS, FLT3 and TP53 mutations in patients with myelodysplastic syndrome and a del(5q). Haematologica 2004; 89: 865-866.
16. Jadersten M, Saft L, Smith A, Kulasekararaj A, Pomplun S, Gohring G, Hedlund A, Hast R, Schlegelberger B, Porwit A, Hellstrom-Lindberg E, Mufti GJ. TP53 mutations in low-risk myelodysplastic syndromes with del(5q) predict disease progression. J Clin Oncol 2011; 29: 1971-1979.
17. List A, Dewald G, Bennett J, Giagounidis A, Raza A, Feldman E, Powell B, Greenberg P, Thomas D, Stone R, Reeder C, Wride K, Patin J, Schmidt M, Zeldis J, Knight R, Myelodysplastic Syndrome-003 Study I. Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N Engl J Med 2006; 355: 1456-1465.
18. Sokol L, List AF. Immunomodulatory therapy for myelodysplastic syndromes. Int J Hematol 2007; 86: 301-305.
19. Caceres G, McGraw K, Yip BH, Pellagatti A, Johnson J, Zhang L, Liu K, Zhang LM, Fulp WJ, Lee JH, Al Ali NH, Basiorka A, Smith LJ, Daugherty FJ, Littleton N, Wells RA, Sokol L, Wei S, Komrokji RS, Boultwood J, List AF. TP53 suppression promotes erythropoiesis in del(5q) MDS, suggesting a targeted therapeutic strategy in lenalidomide-resistant patients. Proc Natl Acad Sci U S A 2013; [Epub ahead of print].
20. Payne EM, Virgilio M, Narla A, Sun H, Levine M, Paw BH, Berliner N, Look AT, Ebert BL, Khanna-Gupta A. L-Leucine improves the anemia and developmental defects associated with Diamond-Blackfan anemia and del(5q) MDS by activating the mTOR pathway. Blood 2012; 120: 2214-2224.
21. Yip BH, Pellagatti A, Vuppusetty C, Giagounidis A, Germing U, Lamikanra AA, Roberts DJ, Fernandez-Mercado M, McDonald EJ, Killick S, Wainscoat JS, Boultwood J. Effects of L-leucine in 5q- syndrome and other RPS14-deficient erythroblasts. Leukemia 2012; 26: 2154-2158.
22. Starczynowski DT, Kuchenbauer F, Argiropoulos B, Sung S, Morin R, Muranyi A, Hirst M, Hogge D, Marra M, Wells RA, Buckstein R, Lam W, Humphries RK, Karsan A. Identification of miR-145 and miR-146a as mediators of the 5q- syndrome phenotype. Nat Med 2010; 16: 49-58.
23. Kumar MS, Narla A, Nonami A, Mullally A, Dimitrova N, Ball B, McAuley JR, Poveromo L, Kutok JL, Galili N, Raza A, Attar E, Gilliland DG, Jacks T, Ebert BL. Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q- syndrome. Blood 2011; 118: 4666-4673.



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