B. Fogliardi

COSTANTINI, BENEDETTA

LEONI, Pietro

M. Fanelli

MANCINI, Stefania

MARIANI, MARIANNA

MATTIUCCI, DOMENICO

MAURIZI, GIULIA

OLIVIERI, Attilio

POLONI, Antonella

S. Amatori

IRIS UniversitÃ  Politecnica delle Marche

LETTER TO THE EDITOROverexpression of CDKN2B (p15INK4B) and altered globalDNA methylation status in mesenchymal stem cells of high-riskmyelodysplastic syndromesLeukemia advance online publication, 29 July 2014; doi:10.1038/leu.2014.197Myelodysplastic syndromes (MDSs) are a heterogeneous group ofclonal hematopoietic stem cell (HSC) malignancies that arecharacterized by ineffective bone marrow hematopoiesis, periph-eral blood cytopenias and a substantial risk of progression toacute myeloid leukemia.1–3 There is evidence that alterations ofthe direct interaction between HSCs and stroma contribute toabnormal HSC growth and maturation.4Mesenchymal stem cells (MSCs) have been defined as non-hematopoietic cells that primarily reside in bone marrow (BM) andare capable of producing different cell lineages. They areimportant components of the hematopoietic microenvironmentand are important in supporting and regulating the proliferationand differentiation of hematopoietic stem cells, as well as inimmunoregulatory functions.5We characterized the molecular and functional properties ofBM-derived MSCs isolated from 24 high-risk IPSS MDS patientsand compared them with 10 older healthy donors (5 ml, medianage 79 years, range 60–82 years) and 10 younger healthy donors(5 ml, median age 17 years, range 16–45 years).Clinical data and BM samples were obtained from the local MDSregistry with informed consent from each patient. This protocolwas approved by the Ethics Committee and followed theDeclaration of Helsinki guidelines. Healthy BM was harvestedfrom BM donors or from patients undergoing hip surgery afterobtaining written informed consent from all participants or fromparents or guardians of those under 18 years of age. Their clinicaland biological characteristics are shown in Table 1.After cellular adhesion in in vitro culture, MDS-MSCs displayedfibroblast-like shapes under a phase–contrast microscope; how-ever, since the beginning of the culture they exhibited an alteredmorphology and appeared larger, flattened and granular.Using flow cytometry we compared the expression of moleculesthat have important roles in cell adhesion, such as CD90, CD44and CD29, and typical mesenchymal markers, such as CD105 andCD73. The MDS-MSCs and the healthy donor MSCs exhibited thesame phenotype and were positively stained for the antigenslisted above. They were negative for hematopoietic and endo-thelial markers CD34, CD45, CD14 and CD31.MDS-MSCs showed reduced proliferative capacity comparedwith controls (expansion potential of MDS-MSCs vs old donors:0.88±1.1 logs, 2.5±3.5 population doublings (PDs) vs 2.93±0.64logs, 9.5±2.3 population doublings) (Po0.05). Moreover, 40% ofthe samples could not be expanded in vitro (Figure 1A). This resultTable 1. Clinical and laboratory data of patients studiedNo. Age/gender FAB WHO % Blasts Karyotype IPSS r-IPSS1 69/M RAEB RAEB-1 10 del7 (15%) 1.5 INT2 5 INT2 68/M RAEB RAEB-2 15 Normal 2 INT2 6.5 VH3 77/M RAEB RAEB-2 15 Not evaluable4 73/M RAEB-T AML 25 Normal 2.5 HIGH 7 VH5 56/M RAEB RAEB-2 12 tris1q, mon7q (25%) 2.5 HIGH 6 VH6 82/F RAEB RAEB-2 15 del5, tris1, tris11, adm14 3 HIGH 10 VH7 72/M RAEB-T AML 28 Normal 2.5 HIGH 6.5 VH8 79/M RAEB-T AML 30 Not evaluable9 84/M RAEB RAEB-2 20 Normal 2 INT2 6.5 VH10 77/F RAEB-T AML 28 Normal 2.5 HIGH 5 INT11 74/F RAEB RAEB-2 20 Normal 2 INT2 8 VH12 71/M RAEB-T AML 30 Not evaluable13 69/M RAEB RAEB-2 15 ct5, del5q, del7q, t(12;13)(12p;13q) (85%) 2.5 HIGH 9 VH14 80/F RAEB RAEB-2 11 Normal 2 INT2 8 VH15 62/F RAEB RAEB-2 13 mon7 2.5 HIGH 7 VH16 61/M RAEB RAEB-2 15 del5q, hyperdiploide 2 INT2 6 HIGH17 75/M RAEB RAEB-2 15 Not evaluable18 66/M RAEB RAEB-2 12 tris11 (7%) 2 INT2 6 HIGH19 64/F RAEB RAEB-2 15 tris1q (50%) 2.5 HIGH 7.5 VH20 82/F RAEB RAEB-2 15 del5q (35%) 1.5 INT2 5.5 HIGH21 60/M RAEB RAEB-2 20 Normal 2 INT2 8 VH22 77/M RAEB-T RAEB-2 30 del5q, del12p, del 3.5 HIGH 9 VH23 79/M RAEB RAEB-1 10 Not evaluable24 75/M RAEB RAEB-2 15 Normal 2 INT2 6.5 VHAbbreviations: adm, additional materials; AML, acute myeloid leukemia; ct, complex translocation; F, female; FAB, French–American–British classification; HIGH,high risk; INT2, intermediate 2; IPSS, International Prognostic Scoring System; M, male; RAEB, refractory anemia with excess blasts; RAEB-T, refractory anemiawith excess blasts in transformation; r-IPSS, revised International Prognostic Scoring System; VH, very high; WHO, World Health Organization.Accepted article preview online 19 June 2014Leukemia (2014), 1–4& 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14www.nature.com/leuFigure 1. (A) Expansion capacity of the BM-derived MDS-MSCs calculated at every passage according to the equation: log10 (number of harvestedcells/number of seeded cells). Finite logs were determined by the cumulative addition of the total numbers generated from each passage until thecells stopped dividing. The results are shown as the average values±s.d. Each point on the growth curve represents a single passage of culture.(B) Molecular analysis for CDKN2B expression in MDS-MSC samples, in old and young MSC donors. The samples were run in duplicate andthe values are the average±s.d. *Po0.05. (C) Southern blot analysis of telomere length in the MDS-MSCs, in older and younger MSC donors. Thevalues are the average±s.d. *Po0.05. (D) Single cell gel electrophoresis assay. (a) No comet tails were seen in MDS-MSCs, suggesting that therewas no damaged DNA (containing cleavage and strand breaks), magnification  10. (b) DNA damage of treated cell line A549 was separated fromintact DNA, yielding a comet tail shape, magnification  10. (E) Hoechst 33342 staining in MDS-MSC samples for apoptotic cells, magnification 10. (F) Southern blot analysis of satellite 2 methylation levels. (Upper panel) Csp45I-digested genomic DNA separated by 1% agarose gelelectrophoresis, before blotting; (lower panel) satellite 2 hybridization of digested genomic DNA by using a PCR probe (sat2-384U/781L). GenomicDNA, digested with Csp45I, from T98G glioblastoma cell line has been used as control of satellite 2 demethylation. (G) Methylated DNAquantification assay of the MDS-MSCs, in older and younger MSC donors. The values are the average±s.d. *Po0.05.Letter to the Editor2Leukemia (2014) 1 – 4 & 2014 Macmillan Publishers Limitedwas in line with the clonogenic potential of MDS-MSCs performedwith colony-forming unit fibroblast (CFU-F) assay, plating 5 106mononuclear cells. CFU-F count of MDS samples was significantlyreduced in comparison with healthy donors (clonogenic potentialof MDS-MSCs vs old donors: 9±6 vs 53±26 CFU-F).To study the ability to provide long-term hematopoieticsupport, peripheral blood progenitor CD133þ cells isolated fromleukapheresis of patients undergoing autologous transplantationwere co-cultured onto feeder-layers of irradiated MDS-MSCs.6These cells were able to support the proliferation of both myeloidand erythroid progenitor cells but not to the level of MSCs fromolder donors (38±15 vs 62±20 colony-forming units,respectively) (Po0.05).The significantly reduced capacity of MDS-MSCs for proliferationtogether with the morphological changes suggested possiblealteration in cell cycle progression and, as reported by otherauthors, a possible senescent-like phenotype.7 Therefore, westudied the gene expression profiles for 37 genes (directly orindirectly involved in the cell cycle regulation) in MDS-MSCs atsecond culture passage.8 The only significant difference found wasrepresented by CDKN2B upregulation in the MDS-MSCs with anexpression level of 8–11 times higher than that in the MSCs fromolder and younger donors (Po0.05) (Figure 1B). These data maybe associated with a negative regulation of the cell cycle. Theymay be at least partially responsible for the reduced proliferationpotential and there could be a premature cellular senescence-likephenotype induction. Upregulation of CDKN2B has been indicatedas being at least part of the molecular mechanisms responsible forthe induction of senescence in MSCs. Recent data revealed thatlong-term culture induces continuous changes in gene expression;in particular an upregulation of CDKN2B in late-culture passageshas been demonstrated, indicating an association betweenreplicative senescence in long-term culture and this gene.Interestingly, in MDS-MSCs CDKN2B appeared overexpressed inearly-culture passage demonstrating an impaired regulation of cellcycle progression independently from culture conditions.9However, we cannot detect any other elaboration of the geneexpression that often relates to a senescence phenotype, such asthe upregulation of CDKN1A and CDKN2A. In an attempt toelucidate the biological status of MDS-MSCs, we studied thepresence of senescence-associated markers such as expression ofbeta-galactosidase, telomere shortening and genomic instability.Quantitative data of beta-galactosidase expression showed thatthere was no statistical difference between MDS-MSCs andhealthy donors. We then evaluated the telomere length of MDS-MSC samples and controls using the telomere restriction fragmentlength assay.10 The MDS-MSCs had shorter telomeres than thecontrols, but this difference was not statistically significant(7.8±0.6 vs 8.6±2.6 kb, respectively), although both MDS-MSCsand MSCs from older donors exhibited significantly shortertelomeres than MSCs from younger donors (7.8±0.6 vs10.1±1.4 kb, respectively) (Po0.05) (Figure 1C), as expected. Inaddition, MSC-MDS did not show signs of DNA damage onceanalyzed by comet assay (Figure 1D). Moreover, MDS-MSCs werestained with Hoechst 33342 to evaluate chromatin condensationand nuclear fragmentation, showing a low frequency of apoptoticcells (range 5–10%), similar to the MSCs of healthy donors(Figure 1E).Globally reduced CpG methylation levels are often reported notonly in cancer cells but also correlated with a premature senescentstatus. It is well known that replicative senescence of normalhuman fibroblast is accompanied by decreased methylation levelsof heterochromatic sequences such as satellite 2 and 3.11Moreover, fibroblast-replicative senescence can be anticipated(premature senescence) by treatment with demethylating agents.In function of its correlation with both replicative- andpremature-senescence, and also as part of the phenomenon ofglobal DNA hypomethylation in cancer, pericentromeric satellite 2(Sat2) methylation was investigated in MDS-MSCs. Unexpectedly,the methylation-sensitive Sat2 digestion assay showed equal highlevels of methylation between MSCs from donors and MDS-MSCs(Figure 1F). Thus, the global methylation levels of MDS-MSCs werefurther investigated comparing it with MSCs of older and youngerhealthy donors using 5-methyl-cytosine enzyme-linked immuno-sorbent assay quantification assay. In a recent work,12 wedemonstrated a global hypermethylated DNA status ofhematopoietic cells in 134 MDS biopsies at diagnosis and thisresult correlated with the prognosis of these patients. Also, theMDS-MSCs exhibited globally hypermethylated DNA that was 2.2times higher than that of the MSCs from older and youngerhealthy donors (0.36±0.16% vs 0.16±0.02% and 0.02±0.01%,respectively) (Po0.05) (Figure 1G).The degree of aberrant methylation correlated with reducedmRNA and decreased protein expression and appeared to beassociated with higher expression of DNMT3a (DNA methyltrans-ferase 3a).13 However, gene expression data obtained by real-timequantitative PCR approaches11 from the MDS-MSCs showed nodifferences in the levels of DNMT1, DNMT3a and DNMT3b withrespect to the donor MSCs.Recently, Geyh et al.7 conducted a detailed analysis of themolecular and functional properties of MDS-MSCs. Theydemonstrated a hypermethylation pattern of MDS-MSCscorrelating it with impaired growth capacities and osteogenicdifferentiation. In particular, they found a hypermethylation status inthe TBX15 and HOXB1 promoter region that resulted in reducedmRNA expression. They also showed the hypermethylation of aPTX2 gene body that resulted in a significant overexpression of PTX2mRNA in MDS-MSCs. In line with these data, the reducedproliferative potential in the MDS-MSCs was directly linked withfunctional alterations. Neither increased apoptosis nor cell cycledefects were observed in MDS-MSCs, so their impaired proliferativecapacity and altered morphology may be due to increasedsenescence. However, the absence of SA-b-Gal expression in MDS-MSCs in our data excluded some senescence mechanisms ofteninvolved in MDS-MSC-impaired growth, which is in line with arecently published observation.14 The data demonstrated thatpremature cell senescence seems unlikely to account for theimpaired proliferative potential of MDS-MSCs, which express fewersenescence characteristics such as the downregulation of CDKN1A,CDKN2A, CDKN2B and PARG1 compared with their normalcounterparts. However, the low proliferation rate of MDS-MSCsmight be associated with the upregulation of WNT signaling.Moreover, other authors15 analyzed MDS-MSC transcriptomesand demonstrated that these cells have an intrinsically alteredpattern of gene expression, including a number of processesinvolved in intercellular crosstalk, that may contribute to theircapacity to support MDS hematopoietic cells.Our data showed that MDS-MSCs were structurally, epigeneti-cally and functionally altered. The alterations indicated impairedstromal support and the possible involvement of MSCs in theineffective MDS hematopoiesis. Moreover, the mechanismsunderlying the impaired proliferative potential and cellularmethylation status of MSCs are interesting avenues for furtherinvestigation. In conclusion, we suggest that MSCs could have acritical role in MDS pathogenesis and may be a possible target fornew therapeutic strategies.CONFLICT OF INTERESTThe authors declare no conflict of interest.ACKNOWLEDGEMENTSThis work was supported by grants from Associazione Italiana contro leleucemie, linfomi e mieloma, sezione di Ancona-ONLUS. The English was editedby Timothy Wills.Letter to the Editor3& 2014 Macmillan Publishers Limited Leukemia (2014) 1 – 4A Poloni1,3, G Maurizi1,3, D Mattiucci1, S Amatori2, B Fogliardi2,B Costantini1, M Mariani1, S Mancini1, A Olivieri1,M Fanelli2 and P Leoni11Clinica di Ematologia, Dipartimento di Scienze Cliniche e Molecolari,Università Politecnica delle Marche, Ancona, Italy and2Laboratorio di Patologia Molecolare ‘PaoLa’, Dipartimento diScienze Biomolecolari, Università di Urbino ‘Carlo Bo’, Fano, ItalyE-mail: a.poloni@univpm.it or mirco.fanelli@uniurb.it3These authors contributed equally to this work.REFERENCES1 Mufti GJ. Pathobiology, classification, and diagnosis of myelodysplastic syn-dromes. Best Pract Res Clin Haematol 2004; 17: 543–557.2 Nimer SD. Myelodysplastic syndromes: molecular pathogenesis and genomicchanges. Ann Hematol 2008; 87: 777–795.3 Kerbauy DB, Deeg HJ. Apoptosis and antiapoptotic mechanisms in the progres-sion of myelodysplastic syndromes. Exp Hematol 2007; 35: 1739–1746.4 Tauro S, Hepburn MD, Peddie CM, Bowen DT, Pippard MJ. Functional disturbanceof marrow stromal microenvironment in the myelodysplastic syndromes.Leukemia 2002; 16: 785–790.5 Dazzi F, Ramasamy R, Glennie S, Jones SP, Roberts I. The role of mesenchymalstem cells in hematopoiesis. Blood Rev 2006; 8: 315–317.6 Poloni A, Leoni P, Buscemi L, Balducci F, Pasquini R, Masia MC et al. Engraftmentcapacity of mesenchymal cells following hematopoietic stem cell transplantationin patients receiving reduced-intensity conditioning regimen. Leukemia 2006; 20:329–335.7 Geyh S, Oz S, Cadeddu RP, Fröbel J, Brückner B, Kündgen A et al. Insufficientstromal support in MDS results from molecular and functional deficits ofmesenchymal stromal cells. Leukemia 2013; 27: 1841–1851.8 Guerzoni C, Amatori S, Giorgi L, Manara MC, Landuzzi L et al. An aza-macrocyclecontaining maltolic side-arms (maltonis) as potential drug against humanpediatric sarcomas. BMC Cancer 2014; 14: 137.9 Wagner W, Bork S, Lepperdinger G, Joussen S, Ma N, Strunk D et al. How to trackcellular aging of mesenchymal stromal cells? Aging (Albany NY) 2010; 2: 224–230.10 Poloni A, Maurizi G, Babini L, Serrani F, Berardinelli E, Mancini S et al. Humanmesenchymal stem cells from chorionic villi and amniotic fluid are not susceptibleto transformation after extensive in vitro expansion. Cell Transplant 2011; 20: 643–654.11 Fanelli M, Caprodossi S, Ricci-Vitiani L, Porcellini A, Tomassoni-Ardori F, Amatori Set al. Loss of pericentromeric DNA methylation pattern in human glioblastoma isassociated with altered DNA methyltransferases expression and involves the stemcell compartment. Oncogene 2008; 27: 358–365.12 Poloni A, Goteri G, Zizzi A, Serrani F, Trappolini S, Costantini B et al. Prognostic roleof immunohistochemical analysis of 5 mc in myelodysplastic syndromes. Eur JHaematol 2013; 91: 219–227.13 Romermann D, Hasemeier B, Metzing K, Schlegelberger B, Länger F, Kreipe H et al.Methylation status of LINE-1 sequences in patients with MDS or secondary AML.Verh Dtsch Ges Pathol 2007; 91: 338–342.14 Pavlaki K, Pontikoglou CG, Demetriadou A, Batsali AK, Damianaki A, Simantirakis Eet al. Impaired proliferative potential of bone marrow mesenchymal stem cells inpatients with myelodysplastic syndromes is associated with abnormal WNTsignaling pathway. Stem Cells Dev 2014; 23: 1568–1581.15 Medyouf H, Mossner M, Jann JC, Nolte F, Raffel S, Herrmann C et al. Myelodys-plastic cells in patients reprogram mesenchymal stromal cells to establish atransplantable stem cell niche disease unit. Cell Stem Cell 2014; 14: 824–837.Letter to the Editor4Leukemia (2014) 1 – 4 & 2014 Macmillan Publishers Limited

English

Overexpression of CDKN2B (p15INK4B) and altered global
DNA methylation status in mesenchymal stem cells of high-risk
myelodysplastic syndromes

https://iris.univpm.it/bitstream/11566/206335/1/leu2014197a.pdf

Overexpression of CDKN2B (p15INK4B) and altered global DNA methylation status in mesenchymal stem cells of high-risk myelodysplastic syndromes

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