Deregulated Gene Expression Pathways in Myelodysplastic Syndrome Hematopoietic Stem Cells

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Deregulated gene expression pathways in myelodysplastic syndrome hematopoietic stem cells

A Pellagatti1, M Cazzola2, A Giagounidis3, J Perry1, L Malcovati2, MG Della Porta2,MJa¨dersten4, S Killick5, A Verma6, CJ Norbury7, E Hellstro¨m-Lindberg4, JS Wainscoat1 and J Boultwood1

1LRF Molecular Haematology Unit, NDCLS, John Radcliffe Hospital, Oxford, UK; 2Department of Hematology Oncology, University of Pavia Medical School, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 3Medizinische Klinik II, St Johannes Hospital, Duisburg, Germany; 4Division of Hematology, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; 5Department of Haematology, Royal Bournemouth Hospital, Bournemouth, UK; 6Albert Einstein College of Medicine, Bronx, NY, USA and 7Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

To gain insight into the molecular pathogenesis of the the World Health Organization.6,7 Patients with refractory myelodysplastic syndromes (MDS), we performed global gene anemia (RA) with or without ringed sideroblasts, according to expression profiling and pathway analysis on the hematopoietic stem cells (HSC) of 183 MDS patients as compared with the the French–American–British classification, were subdivided HSC of 17 healthy controls. The most significantly deregulated based on the presence or absence of multilineage dysplasia. In pathways in MDS include interferon signaling, thrombopoietin addition, patients with RA with excess blasts (RAEB) were signaling and the Wnt pathways. Among the most signifi- subdivided into two categories, RAEB1 and RAEB2, based on the cantly deregulated gene pathways in early MDS are immuno- percentage of bone marrow blasts. Patients with 420% blasts deficiency, apoptosis and chemokine signaling, whereas advanced were classified as AML. MDS patients with o5% bone marrow MDS is characterized by deregulation of DNA damage response and checkpoint pathways. We have identified distinct gene blasts and an isolated deletion of the long arm of chromosome expression profiles and deregulated gene pathways in patients 5 [del(5q)] were categorized as 5qÀ syndrome, and a new with del(5q), trisomy 8 or À7/del(7q). Patients with trisomy 8 category termed as MDS-unclassifiable was created incorporat- are characterized by deregulation of pathways involved in ing patients not fulfilling the other criteria. The main prognostic the immune response, patients with À7/del(7q) by pathways factors of MDS, for progression to AML and survival, include involved in cell survival, whereas patients with del(5q) show the number of cytopenias, percentage of marrow blasts and deregulation of integrin signaling and cell cycle regulation pathways. This is the first study to determine deregulated gene cytogenetic abnormalities. These factors are combined in an pathways and ontology groups in the HSC of a large group of International Prognostic Scoring System that distinguishes four MDS patients. The deregulated pathways identified are likely to subgroups with distinct probabilities of progression to AML and be critical to the MDS HSC phenotype and give new insights survival.8,9 into the molecular pathogenesis of this disorder, thereby Early MDS (RA) are characterized by an increased apoptosis providing new targets for therapeutic intervention. in the bone marrow, which may in part underlie the ineffective Leukemia (2010) 24, 756–764; doi:10.1038/leu.2010.31; published online 11 March 2010 hematopoiesis and peripheral blood cytopenia found in these Keywords: gene expression pathways; myelodysplastic subgroups. In contrast, in advanced MDS (high-risk MDS), a syndromes; hematopoietic stem cells; gene expression profiling progressive increase in myeloblasts, coupled with a decrease in apoptosis, and increased genomic instability is observed.1,2 Karyotypic abnormalities are reported in about 50% of patients with primary MDS and 80% of patients with secondary MDS.10 The characteristic MDS abnormalities involve loss of Introduction genetic material, whereas balanced translocations are rare. The most common cytogenetic abnormalities in MDS include The myelodysplastic syndromes (MDS) are a heterogeneous the del(5q), trisomy 8 and À7/del(7q).10 Patients with the group of clonal hematopoietic stem cell (HSC) malignancies that 5qÀ syndrome, in which the del(5q) is the sole karyotypic are characterized by ineffective hematopoiesis resulting in abnormality, are characterized by a macrocytosis, anemia, peripheral cytopenias (thrombopenia, leucopenia and anemia) 1,2 normal or high platelet count and hypolobulated megakaryo- and typically a hypercellular bone marrow. The MDS are cytes in the bone marrow and a good prognosis.11–13 The pre-leukemic conditions showing frequent progression to acute presence of À7/del(7q) is associated with a frequent progression myeloid leukemia (AML). Approximately 40% of MDS patients 1,2 to AML, and chromosome 7 abnormalities are an indicator of a develop AML. MDS is one of the commonest myeloid 8 3,4 poor prognosis in the International Prognostic Scoring System. malignancies and is at least as common as AML. In comparison, MDS with trisomy 8 typically has an inter- The classification of MDS is based on morphologic anomalies mediate prognosis;8 peripheral blood cytopenias are often according to the French–American–British Cooperative Study 14 5 responsive to immunosuppressive treatment, and have a more Group. More recently, this classification has been modified by favorable prognosis. A number of gene abnormalities have been implicated in the development or progression of MDS, includ- Correspondence: Dr J Boultwood, Nuffield Department of Clinical ing, for example, mutations in the Ras, p53, RUNX1 and TET2 Laboratory Sciences, University of Oxford, LRF Molecular Haemato- genes.15,16 However, the molecular pathogenesis of MDS logy Unit, John Radcliffe Hospital, Oxford OX3 9DU, UK. E-mail: [email protected] remains incompletely understood. Received 29 October 2009; revised 15 December 2009; accepted 14 The disease course and prognosis are highly variable in MDS. January 2010; published online 11 March 2010 The majority of MDS patients will succumb to the disease over Deregulated pathways in myelodysplastic syndromes A Pellagatti et al 757 time, because of infection as a result of neutropenia and/or Microarray data analysis neutrophilic dysfunction or the development of AML.1,2 With Cell intensity calculation and scaling was performed using the exceptions of lenalidomide, which is effective in MDS with GeneChip Operating Software. Data analysis was performed the del(5q)17 and hypomethylating agents such as azacitidine, using GeneSpring 7.3.1 (Agilent Technologies). The GeneChip which is used to treat advanced MDS and may improve overall Operating software was used to perform quality control after survival by several months,18 there are few effective drug scaling the signal intensities of all arrays to a target of 100. The treatments for this disorder.19 MDS-associated leukemia does values obtained for scale factors, background levels, percentage not respond well to chemotherapy and patients have a poor of present calls, 30/50 GAPDH ratio and intensities of spike prognosis.1,2 Allogeneic stem cell transplantation remains the hybridization controls were within the acceptable range for all only option for curative treatment in MDS, yet is only applicable samples. Affymetrix CEL files were pre-processed using robust in 15–20% of all patients.20 The identification of new targets in multichip analysis.23 Data from 17 healthy controls were used to MDS for drug treatment is, therefore, of major importance. obtain patient/control expression ratios. Hierarchical clustering Recent studies have identified several core signaling pathways was performed with GeneSpring software using standard altered in the development of certain solid tumors, including correlation. Pathway analysis was performed using Ingenuity glioblastoma21 and pancreatic cancer,22 that greatly further our Pathway Analysis 7.5 software (Ingenuity Systems, Redwood understanding of the molecular pathogenesis of these malig- City, CA, USA) based on the Ingenuity Pathways Knowledge nancies and importantly may represent new targets for Base, which is the most comprehensive database of biological therapeutic intervention. findings. Gene ontology was performed using the Database for We have chosen to determine deregulated gene expression Annotation, Visualization and Integrated Discovery (DAVID). pathways and ontology (functional) groups in the CD34 þ cells The data discussed in this publication have been deposited of a large group of patients with MDS to advance our in NCBI’s Gene Expression Omnibus24 and are accessible understanding of these disorders. through GEO Series accession number GSE19429 (http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc ¼ GSE19429).

Materials and methods Real-time quantitative PCR The expression data for selected genes were validated in a Sample collection and cell separation subset of MDS patients (n ¼ 12) and controls (n ¼ 11) using real- A total of 183 patients with MDS and 17 healthy controls were time quantitative PCR. The b2-microglobulin gene was used included in the study. Classification of MDS patients was to normalize for differences in input cDNA. Pre-developed according to the French–American–British criteria,5 with RAEB TaqMan Assays were used (Assays-on-Demand, Applied patients further subdivided into RAEB1 and RAEB2. At the time Biosystems, Foster City, CA, USA) and reactions were run on a of sample, 55 patients had RA, 48 RARS, 37 RAEB1 and 43 LightCycler 480 Real-Time PCR System (Roche Diagnostics, RAEB2. The MDS patient samples were collected from several Lewes, UK). Each sample was run in triplicate and the centers: Oxford and Bournemouth (UK), Duisburg (Germany), expression ratios were calculated using the DDC method. Stockholm (Sweden) and Pavia (Italy). The study was approved T by the ethics committees (Oxford C00.196, Bournemouth 9991/ 03/E, Duisburg 2283/03, Stockholm 410/03, Pavia 26264/2002) Results and informed consent was obtained. Bone marrow samples were obtained and CD34 þ cells isolated from MDS patients The gene expression profiles of CD34 þ cells obtained from the and healthy controls. Mononuclear cells were separated using bone marrow aspirates of 183 MDS patients were compared Histopaque (Sigma-Aldrich, Gillingham, UK) density gradient with those of CD34 þ cells from the bone marrow aspirates of centrifugation, labeled with CD34 MicroBeads, and then 17 healthy individuals. We identified several genes that were CD34 þ cells were isolated using MACS magnetic cell up- or down-regulated in the CD34 þ cells of the majority of the separation columns (Miltenyi Biotec, Bergisch Gladbach, MDS patients. Thirty-five genes were up-regulated with a ratio Germany) according to the manufacturer’s recommendations. 42.0 in at least 92 (half) MDS patients (Supplementary Table The purity of CD34 þ cell preparations was evaluated with S1). IMP3 was the most commonly up-regulated gene (130 of FACS and was X90%. 183 patients). Several of the genes commonly up-regulated in the MDS patients included in our study were interferon- stimulated genes. IFIT1, IFITM1, IFI44L and IFIT3 were up- Affymetrix experiments regulated by more than twofold in at least 60% of the patients Total RNA was extracted using TRIZOL (Invitrogen, Paisley, UK) (Supplementary Table S1). A total of 139 genes were down- following the manufacturer’s protocol. The quality of the RNA regulated with a ratio o0.5 in at least 92 MDS patients, samples was evaluated using Agilent Bioanalyzer 2100 (Agilent including ARPP-21, Gravin/AKAP12, CD24 and LEF1 (Supple- Technologies, Santa Clara, CA, USA). For each sample, 50 ng of mentary Table S1). The expression levels of five commonly total RNA were amplified and labeled with the Two-Cycle deregulated genes (IFITM1, DLK1, PTHR2, ARPP-21 and cDNA Synthesis and the Two-Cycle Target Labelling and Gravin/AKAP12) were validated using real-time quantitative Control Reagent packages (Affymetrix, Santa Clara, CA, USA). PCR (Supplementary Figure S1). Real-time quantitative PCR 10 mg of biotin-labeled fragmented cRNA was hybridized to experiments confirmed the up-regulation of IFITM1, DLK1 and GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix), PTHR2, and the down-regulation of ARPP-21 and Gravin/ covering over 47 000 transcripts representing 39 000 human AKAP12 (Supplementary Figure S1). genes. Hybridization was performed at 45 1C for 16 h in Pathway analysis was performed on those genes commonly Hybridization Oven 640 (Affymetrix). Chips were washed and up- or down-regulated by twofold or more in at least one third stained in a Fluidics Station 450 (Affymetrix) and scanned using of MDS patients (Table 1; Figure 1; Supplementary Figure S2), a GeneChip Scanner 3000 (Affymetrix). using Ingenuity Pathway Analysis software. The most significant

Leukemia Deregulated pathways in myelodysplastic syndromes A Pellagatti et al 758 Table 1 Top ﬁve deregulated pathways for the list of up-regulated and down-regulated genes in MDS

Ingenuity canonical pathway P-value Up-regulated molecules

Interferon signaling 2.57E-06 IFIT3, IFIT1, IFITM1, MX1, IRF9, STAT1 Thrombopoietin signaling 2.09E-05 PIK3R3, RRAS2, MPL (includes EG:4352), SOS1, STAT1, PRKCZ, PRKCA IL-3 signaling 0.00010 PIK3R3, PTPN6, RRAS2, SOS1, STAT1, PRKCZ, PRKCA Natural killer cell signaling 0.00019 PIK3R3, FCGR3B, PTPN6, RRAS2, TYROBP, SOS1, PRKCZ, PRKCA Function of pattern recognition receptors 0.00021 PIK3R3, CLEC7A, DDX58, TLR8 (includes EG:51311), C1QC, C1QA, CCL5 in recognition of bacteria and viruses

Ingenuity canonical pathway P-value Down-regulated molecules

Primary immunodeﬁciency signaling 2.57E-05 BLNK, RAG2, IGHM, NR3C1, CD79A, IGLL1 B-cell receptor signaling 0.00016 BLNK, CD19, CD79B, GAB1, PAG1, PIK3AP1, MAP3K8, CD79A Cell cycle regulation by BTG family proteins 0.0068 BTG1, PPP2R2C, E2F2 Wnt/b-catenin signaling 0.0074 TCF4, CD44, TLE4, TLE1, PPP2R2C, LEF1 FcgRIIB signaling in B lymphocytes 0.0085 BLNK, CD79B, CD79A Abbreviation: MDS, myelodysplastic syndromes.

Figure 1 Interferon signaling pathway. Up-regulated genes in MDS mapping to the pathway are colored red.

deregulated pathway for commonly up-regulated genes was patients with RAEB2 and healthy controls (4670 genes, P-value the ‘interferon signaling’ pathway (Table 1; Figure 1). The most o0.05) were obtained and pathway analysis was performed signiﬁcant deregulated pathways for commonly down- using Ingenuity software (Tables 2 and 3; Figure 2; Supplemen- regulated genes include the ‘Wnt/b-catenin signaling’ pathway tary Figure S3). Gene ontology analysis was also performed on (Table 1). these gene lists using DAVID, and the signiﬁcant main ontology To identify deregulated gene pathways and ontology groups themes for the comparison RA vs healthy controls include in early MDS (RA) and in advanced MDS (RAEB2), differentially ‘immune response’ and ‘apoptosis’ (Supplementary Table S2), expressed genes between the 55 patients with RA and healthy and for the comparison RAEB2 vs healthy controls include ‘cell controls (534 genes, P-value o0.05), and between the 43 cycle’ and ‘DNA repair’ (Supplementary Table S2).

Leukemia Deregulated pathways in myelodysplastic syndromes A Pellagatti et al 759 Table 2 Top 10 deregulated pathways in RA compared with healthy controls

Ingenuity canonical pathway P-value Molecules

Primary immunodeﬁciency signaling 8.13E-05 PTPRC, RAG2, RAG1, IGHG1, NR3C1, CD79A, IGLL1 Bile acid biosynthesis 0.0016 CEL, DHRS9, SRD5A1, ALDH3B1, HSD3B7 FcgRIIB signaling in B lymphocytes 0.0066 CD79B, RRAS, DOK1, CD79A B-cell receptor signaling 0.0068 PTPRC, VAV2, CD19, CD79B, DAPP1, RRAS, PAG1, CD79A Apoptosis signaling 0.029 RRAS, BAX, CASP7, FAS, PRKCA Glycolysis/gluconeogenesis 0.029 HK1, DHRS9, LRRC16A, ALDH3B1, GALM (includes EG:130589) IL-4 signaling 0.035 IRF4, RRAS, TYK2, NR3C1 Prolactin signaling 0.052 RRAS, SOCS6, NR3C1, PRKCA Chemokine signaling 0.052 PLCB2, RRAS, CXCR4, PRKCA fMLP signaling in neutrophils 0.055 GNAS, PLCB2, ARPC1B, RRAS, PRKCA Abbreviation: RA, refractory anemia. Genes in regular font are expressed at lower levels in RA compared with healthy controls, whereas genes in bold font are expressed at higher levels in RA compared with healthy controls.

Table 3 Top 10 deregulated pathways in RAEB2 compared with healthy controls

Ingenuity canonical pathway P-value Molecules

Mitotic roles of 5.89E-10 KIF23, CDC20, PTTG1, PPP2R5B, CCNB2, CDC23 (includes EG:8697), SMC1A, CDC25B, polo-like kinase PLK4, TGFB1, PPP2R2C, CDC16, CDC25A, CDC25C, ESPL1, CCNB1, PPP2R5C, WEE1, PRC1, ANAPC13, CDC2, PPP2CB, PPP2R4, PKMYT1, PPP2R1B, CDC27, STAG2, KIF11 ATM signaling 1.86E-05 CDC25C, CCNB1, CCNB2, MDM2, MRE11A, CREB3L4, MAPK12, MAPK11, CDC2, SMC1A, CHEK1, RAD51, MDM4, JUN, SMC2, FANCD2, TLK1, H2AFX, TP53BP1, CDC25A Role of CHK proteins in 0.00012 CDC25C, MRE11A, RFC5, CDC2, CHEK1, E2F6, PCNA, TLK1, RFC4, E2F5, RFC2, E2F2, cell-cycle checkpoint control RFC3, CDC25A Role of BRCA1 in DNA 0.00013 RBBP8 (includes EG:5932), MRE11A, RBL1, RFC5, MLH1, CHEK1, RAD51, E2F6, RB1, damage response FANCD2, RFC4, MSH6, RFC2, E2F5, BRCA2, FANCA, E2F2, RFC3 B-cell receptor signaling 0.00021 BLNK, RAC2, RELA (includes EG:5970), MAP3K11, POU2F2, INPPL1, MAPK11, CD79A, PTEN, PTPRC, IKBKB, IKBKG, CAMK2D, JUN, NFAT5, PPP3CB, CD22, VAV2, ETS1, PTPN6, CD19, PIK3C2A, CD79B, RRAS, EGR1, CSK, CREB3L4, MALT1, MAPK12, MAPK14, GAB1, DAPP1, PAG1, LYN, VAV1, PIK3AP1, CAMK2G Cell cycle regulation by BTG 0.00098 CCNE2, PPP2R5C, PPP2R5B, BTG1, PPP2CB, RB1, E2F6, CCNE1, PPP2R4, E2F5, family proteins PPP2R2C, PPP2R1B, E2F2 Cell cycle: G2/M DNA damage 0.0018 CDC25C, CCNB1, WEE1, CCNB2, MDM2, CDC2, SKP2, CHEK1, CDC25B, MYT1, TOP2B, checkpoint regulation TOP2A, BTRC Cell cycle: G1/S checkpoint 0.010 CCNE2, TFDP1, RBL1, SKP2, HDAC5, RB1, E2F6, CCNE1, MAX, TGFB1, HDAC11, E2F5, regulation BTRC, E2F2, CDC25A CDK5 signaling 0.011 ITGB1, PPP1CC, LAMA5, PPP2R5C, RRAS, EGR1, PPP2R5B, ITGA6, PPP1R14A, PPP1R11, MAPK12, CACNA1A, LAMC1, PPP2CB, PPP1R3D, PRKAR2B, PPP2R4, CAPN1, PRKAG2, PPP2R2C, ADCY7, PPP2R1B Pyrimidine metabolism 0.016 POLR2D, POLA1, DKC1, POLQ, POLR2J, POLI, DNTT, DTYMK, POLD3, MAD2L2, PUS3, TXN, POLR1D, TYMS, APOBEC3B, DUT (includes EG:1854), DCK, POLE2, PRIM2, RRM2, TK2, DCTD, RFC5, APOBEC3G, CTPS, RRM1 (includes EG:6240), CRMP1, POLA2, TK1, CANT1, RFC3, POLE3 Abbreviation: RAEB, refractory anemia with excess blasts. Genes in regular font are expressed at lower levels in RAEB2 compared with healthy controls, whereas genes in bold font are expressed at higher levels in RAEB2 compared with healthy controls.

We determined whether a distinct gene expression profile was pathway analyses were performed (Tables 4–6). Gene ontology associated with specific cytogenetic groups. We investigated analysis was also performed on these gene lists using DAVID differences in gene expression between the 29 MDS patients (Supplementary Table S2) and the significant main ontology with a del(5q), five MDS patients with À7/del(7q) and five MDS themes for the comparison of del(5q) vs healthy controls include patients with þ 8. We applied ANOVA using 0.05 P-value ‘chromatin assembly’ and ‘translation,’ for the comparison of cutoff and Benjamini–Hochberg multiple testing correction, and À7/del(7q) vs healthy controls include ‘activation of JNK 290 significantly different probe sets were identified. Hierarch- activity’ and ‘signal transduction’ and for the comparison of ical clustering performed using these 290 probe sets could þ 8 vs healthy controls include ‘immune response’ and separate the MDS patients into different clusters according to ‘leukocyte activation.’ their cytogenetic group (Supplementary Figure S4). Leave-one- out cross-validation classified all samples correctly. Differentially expressed genes between patients with del(5q) Discussion and healthy controls (507 genes, P-value o0.001), between patients with À7/del(7q) and healthy controls (353 genes, The identification of genes and gene pathways deregulated in P-value o0.05) and between patients with þ 8 and healthy cancer can lead to a better understanding of the molecular controls (430 genes, P-value o0.01) were obtained and pathogenesis of the disease and identify new targets for

Leukemia Deregulated pathways in myelodysplastic syndromes A Pellagatti et al 760

Figure 2 ATM signaling pathway. Signiﬁcantly differentially expressed genes between RAEB2 and normals were mapped to the pathway and colored red if the expression levels were higher or green if lower in RAEB2 cases compared with normals.

Table 4 Top 10 deregulated pathways in del(5q) compared with healthy controls

Ingenuity canonical pathway P-value Molecules

Primary immunodeﬁciency signaling 2.95E-06 BLNK, RAG2, RAG1, STAT3, IGHG1, NR3C1, CD79A, IGLL1 FcgRIIB signaling in B lymphocytes 0.0039 BLNK, CD79B, LYN, CD79A Wnt/b-catenin signaling 0.0050 TCF4, PPP2CA, CSNK1G3, CSNK1A1, TLE1, PPP2R2C, LEF1, FZD7 Role of NFAT in regulation of the immune response 0.0056 BLNK, GNG11, CD79B, CSNK1G3, MS4A2, CSNK1A1, LYN, CD79A B-cell receptor signaling 0.010 BLNK, CD19, CD79B, PAG1, LYN, PIK3AP1, CD79A Integrin signaling 0.015 RAP2B, PARVB, WIPF1, ARHGAP26, ARPC1B, ARPC2, CAPN2, CAPN3 Mitotic roles of polo-like kinase 0.015 CDC25C, PPP2CA, PPP2R2C, CDC23 (includes EG:8697) Cell cycle regulation by BTG family proteins 0.021 PPP2CA, PPP2R2C, E2F2 Hypoxia signaling in the cardiovascular system 0.022 UBE2B, UBE2D2, UBE2E3, UBE2F Huntington’s disease signaling 0.023 HSPA4, GNG11, HDAC3, HSPA9, TCERG1, CAPN2, BAX, CAPN3 Genes in regular font are expressed at lower levels in del(5q) compared with healthy controls, whereas genes in bold font are expressed at higher levels in del(5q) compared with healthy controls.

treatment. We have used the Affymetrix GeneChip U133 Plus Multiple deregulated pathways have been identified in MDS. 2.0 platform, on which most human genes are represented, to We have shown that the ‘interferon signaling’ pathway is the determine the transcriptome of the CD34 þ cells of a large most significantly up-regulated pathway in MDS, with multiple group of MDS patients as compared with CD34 þ cells of genes in this pathway, including STAT1, IRF9, IFIT1 and IFITM1, healthy controls. Combining global gene expression analysis showing up-regulation in MDS. IFN-g, which is overexpressed with detailed annotated pathway analyses and gene ontology in the bone marrow of MDS patients,25 has been shown to analyses has facilitated the identification of key pathways and exhibit a strong inhibitory effect on hematopoietic progenitor functional groups perturbed in MDS HSC compared with and stem cells.26,27 We have earlier reported the up-regulation normal HSC. of several interferon-stimulated genes in MDS28 and this present

Leukemia Deregulated pathways in myelodysplastic syndromes A Pellagatti et al 761 Table 5 Top 10 deregulated pathways in À7/del7q compared with healthy controls

Ingenuity canonical pathway P-value Molecules

B-cell receptor signaling 2.57E-06 MAP3K9, CD19, RRAS2, CD79B, GAB1, PAG1, PIK3C3, CD22, MALT1, MAPK11, CD79A Primary immunodeﬁciency signaling 0.00068 RAG2, IGL@, RAG1, IGHG1, CD79A FcgRIIB signaling in B lymphocytes 0.0016 RRAS2, CD79B, PIK3C3, CD79A Nicotinate and nicotinamide metabolism 0.0019 NAPRT1, MAP3K9, NADSYN1, PIM1, DFFB, NAMPT SAPK/JNK signaling 0.0057 MAP3K9, RRAS2, GAB1, PIK3C3, ZAK T-cell receptor signaling 0.0079 SHB, RRAS2, PAG1, PIK3C3, MALT1 NF-kB signaling 0.0081 CSNK2A2, TRAF3, IL1F9, RRAS2, PIK3C3, MALT1 Docosahexaenoic acid (DHA) signaling 0.011 FOXO1, PIK3C3, BAX LXR/RXR activation 0.012 IL1F9, SREBF1, NR1H3, NCOR1 PI3K/AKT signaling 0.017 RRAS2, FOXO1, GAB1, PPP2R2C, RHEB Genes in regular font are expressed at lower levels in À7/del7q compared with healthy controls, whereas genes in bold font are expressed at higher levels in À7/del7q compared with healthy controls.

Table 6 Top 10 deregulated pathways in +8 compared with healthy controls

Ingenuity canonical pathway P-value Molecules

Primary immunodeﬁciency signaling 2.19E-05 LCK, RAG2, ZAP70, RAG1, IGHG1, MAPK12, CD79A FcgRIIB signaling in B lymphocytes 0.00035 CD79B, PIK3R1, DOK1, MAPK12, CD79A B-cell receptor signaling 0.00044 VAV2, MAP3K9, CD19, CD79B, POU2F2, PAG1, PIK3R1, MAPK12, CD79A Antigen presentation pathway 0.0010 B2M, HLA-DMB, HLA-G, HLA-F Allograft rejection signaling 0.0011 HLA-DMB, IGHG1, HLA-G, HLA-F Autoimmune thyroid disease signaling 0.0015 HLA-DMB, IGHG1, HLA-G, HLA-F CTLA4 signaling in cytotoxic T lymphocytes 0.0016 PPP2CB, LCK, PIK3R1, ZAP70, PPP2R2C, HLA-DMB Dendritic cell maturation 0.0017 STAT4, B2M, PIK3R1, FSCN1, HLA-DMB, IGHG1, MAPK12, DDR1 IL-4 signaling 0.0032 B2M, IRF4, PIK3R1, TYK2, HLA-DMB T-cell receptor signaling 0.0040 VAV2, LCK, PAG1, PIK3R1, ZAP70, MAPK12 Genes in regular font are expressed at lower levels in +8 compared with healthy controls, whereas genes in bold font are expressed at higher levels in +8 compared with healthy controls.

study confirms and extends these earlier observations. We of the Wnt pathway in MDS may lead to defective self-renewal suggest that the up-regulation of interferon-stimulated genes and of HSC in this disorder. of the interferon signaling pathway is a major feature of MDS To better understand disease progression in MDS, we sought and may be responsible for some of the hematological charac- to identify genes and deregulated gene pathways in early and teristics of MDS, including cytopenias. advanced MDS. There is mounting evidence, such as the Megakaryocytic differentiation is frequently defective in MDS; presence of autoreactive T-cell clones and increased levels of thrombocytopenia occurs in about 50% of patients and is a preapoptotic cytokines, suggesting that immune deregulation, major cause of morbidity and mortality.1,29 Thrombopoietin and especially in early MDS, results in an autoimmune process its receptor (TPOR) have an important function in maintaining leading to ineffective hematopoiesis and bone marrow failure, production of controlled numbers of megakaryocytes and whereas advanced MDS is characterized by the immune platelets.30 Moreover, it has recently been shown that thrombo- evasion and selective growth advantage of the malignant poietin signaling has a function in HSC self-renewal.31 Intrigu- clone.35,36 In accord with this hypothesis, we have found that ingly, ‘thrombopoietin signaling’ was the second most among the most significantly deregulated gene pathways in RA up-regulated pathway in MDS, with several genes in this are ‘immunodeficiency signaling,’ ‘B-cell receptor signaling,’ pathway, including TPOR, STAT1 and SOS1 showing ‘IL4 signaling’ and ‘chemokine signaling.’ The ‘primary up-regulation. Up-regulation of the thrombopoietin pathway in immunodeficiency signaling’ pathway was the most signifi- MDS represents a possible mechanism for the observed cantly deregulated pathway in RA. This pathway was also megakaryocytic abnormalities in MDS. The up-regulation of deregulated in RAEB2, but with a lower significance. Deregula- the interferon and thrombopoietin signaling pathways may tion of the ‘B-cell receptor signaling’ pathway in RA is in accord relate to a bone marrow compensatory reaction to a with earlier observations from our group and others that reduced fundamental defect in these pathways. expression of genes involved in B-lymphocyte development is One of the most significantly down-regulated pathways in a feature of MDS.28,37 It has recently been shown that MDS was the Wnt canonical pathway. Deregulation of this T-Regulatory cells in early MDS, in contrast to advanced MDS, pathway has been implicated in the pathogenesis of several have a reduced ability to suppress immune responses and, different types of human cancer, including leukemia. In contrast through reduced expression of CXCR4, impaired bone marrow to our findings in MDS, the Wnt pathway has been shown to be trafficking.35 In agreement with these findings, we found that up-regulated in chronic myeloid leukemia and AML.32,33 The chemokine signaling through CXCR4 was deregulated in RA, with Wnt pathway has a critical function in the regulation of the CXCR4 being down-regulated in RA by approximately 50% self-renewal of HSC,34 and we speculate that down-regulation compared with healthy controls in our study.

Leukemia Deregulated pathways in myelodysplastic syndromes A Pellagatti et al 762 Early MDS is characterized by a high level of apoptosis and in CDC2/CDK1 and cyclin B) and topoisomerase II, which is accord with this, we found that the apoptosis signaling pathway normally expressed only in the S and G2 phases. However, the was one of the most significantly deregulated pathways (up- interpretation of these data is complex as we also observe regulated) in RA, but not in RAEB2. It is well recognized that up-regulation of the proto-oncogenes c-jun49 and CREB50 and there is a higher bone marrow apoptotic index in the bone down-regulation of RB1, a negative regulator of the cell cycle,51 marrow of low-risk MDS patients compared with high-risk MDS in these pathways in RAEB2. The proliferative index of advanced and MDS-AML.38–40 MDS is uncertain, with some studies showing a decreased Advanced MDS is characterized by high levels of genomic proliferative rate and others an increased proliferative rate, instability with a concomitant increase in cytogenetic abnorm- compared with early MDS.52–54 Our data support a defect in alities in comparison with early MDS. DNA damage facilitates regulation of the cell cycle in advanced MDS. The down- activation of the DNA damage response (DDR) and DNA regulation of several pathways involving the cell cycle in AML, damage checkpoint pathways, which can then lead to cell cycle including cell cycle regulation by BRCA1 and CDC25, has also arrest, apoptosis, repair or cell senescence. The study of recently been shown.32 advanced solid tumors has shown that once DNA damage has The del(5q) is the most commonly reported deletion in de occurred in genes having a crucial function in the DDR, the novo MDS and is found in 10–15% of all patients.12 Trisomy 8 DNA damage checkpoint pathway is compromised or inacti- and À7/del(7q) are also among the most frequently reported vated and the cells show increased genomic instability and karyotypic abnormalities in MDS.55 The association of karyo- acquire a growth advantage leading to malignant transforma- typic abnormalities with some particular clinical features of tion.41 Intriguingly, the most significantly deregulated gene MDS suggests specific functions in the pathogenesis of MDS.8,56 pathways in RAEB2 in our study concern the DDR and Indeed, the clinical behavior of patients with del(5q), þ 8or checkpoints, including, for example, ‘CHK proteins in cell À7/del(7q) is different.8,56,57 In our study, hierarchical clustering cycle checkpoint control,’ ‘ATM signaling,’ ‘BRCA1 in DDR,’ grouped patients with a del(5q), trisomy 8 or À7/del(7q) into ‘G2/M DNA damage checkpoint regulation’ and the ‘mitotic distinct clusters. The demonstration that each of these major roles of polo-like kinase (PLK)’ pathways, with most of the karyotypic groups has a particular gene expression profile significantly deregulated genes mapping to these pathways suggests a common underlying pathophysiologic basis to each showing down-regulation in RAEB2 compared with healthy disease group. Moreover, our study identified deregulated gene controls. For example, PLK4 mapping to the ‘mitotic roles of pathways for MDS defined by del(5q), trisomy 8 or À7/del(7q). PLK’ pathway is down-regulated in RAEB2 by approximately Strikingly, the 10 most significantly deregulated gene path- twofold. Plk4 haploinsufficiency has been shown to cause ways in trisomy 8 MDS in our study are associated with the mitotic infidelity and carcinogenesis.42 CHK1 is also a immune response, including ‘immunodeficiency signaling,’ haploinsufficient tumor suppressor,43 is an important compo- ‘B-cell receptor signaling,’ ‘antigen presentation,’ ‘CTLA4 nent of DDR pathways44 and is down-regulated in RAEB2 by signaling in cytotoxic T lymphocytes’ and ‘T-cell receptor- approximately twofold. Interestingly, MDS-derived cell lines signaling’ pathways. Moreover, ‘immune response’ was among (MDS-related AML) have been shown to exhibit defects in their the most significant gene ontology/functional groups in trisomy capacity to mount a checkpoint response.20 A recent study 8 MDS identified by our study. These data are in accord with an concerning the identification of deregulated gene pathways in earlier microarray study by Chen et al.58 showing that multiple AML has similarly shown the down-regulation of several DDR immune and inflammatory response genes are dysregulated in and checkpoint signaling pathways in AML.32 RAEB2 is clearly trisomy 8 CD34 þ cells; for example TGFBR3, MAF and IGF1 closely related to AML and both disorders seem to share a were found to be up-regulated in both studies. Trisomy 8 MDS similar deregulation of the DDR and checkpoint signaling. has been earlier associated with oligoclonal T-cell expansion, The efficient repair of damaged DNA is critical for the suggestive of an antigen-driven T-cell pathophysiology, and maintenance of genome stability and cell survival.45 Several of some patients’ T-cell clones are specifically cytotoxic for trisomy the genes mapping to the DDR pathways that we have shown to 8 cells compared with normal cells from the same indivi- be down-regulated in RAEB2, for example RAD51 and BRCA2, dual.59,60 These results are consistent with an immune system have an important function in this process. We have shown function in the development of pancytopenia in trisomy 8 that RAD51, which mediates homologous recombination repair MDS.58 Thus, an important role for the immune system in of DNA double-strand breaks, is down-regulated in RAEB2 by trisomy 8 MDS has been suggested by several groups and our 4twofold compared with healthy controls. Repression of gene expression and deregulated pathway data strongly support RAD51 gene expression has been described in some tumors and extend this proposal. and is associated with genomic instability.46,47 Intriguingly, The most significantly deregulated gene pathways in del(5q) several genes, which when mutated give rise to Fanconi anemia, MDS in our study include ‘Wnt/b-catenin signaling,’ ‘integrin a chromosomal instability disorder with an increased incidence signaling’ and ‘cell cycle regulation.’ We have earlier shown of MDS,48 are down-regulated in RAEB2, including FANCA and that patients with the 5q- syndrome show deregulation of similar FANCD2. The repression of DNA repair and checkpoint gene pathways.61 Moreover, ‘translation’ and ‘chromatin components in RAEB2 would suggest that the clonal MDS cells assembly’ were among the most significantly deregulated would accumulate and/or mis-repair DNA strand breaks. We ontology groups in del(5q) MDS. Interestingly, marked dereg- suggest that the increased genomic instability that characterizes ulation of ribosomal- and translation-related genes in patients RAEB2 may be associated with these abnormalities. with the 5q- syndrome has been shown by our group.62 The ‘cell cycle’ was the most significantly deregulated Multiple pathways that have an important function in cell ontology/functional group in RAEB2 and several of the most survival, differentiation, apoptosis and growth63–66 were sig- significantly deregulated pathways concern the cell cycle. The nificantly deregulated in À7/del(7q) MDS, including SAPK/JNK, overall expression pattern suggests that the RAEB2 patients’ NF-kB, PI3K/AKT and ceramide signaling pathways. Interest- CD34 þ cells are proliferating less actively than the controls. ingly, CK2, an enzyme with a master regulatory function in Wnt The down-regulated genes include known E2F targets such as and NF-kB signaling, is up-regulated in À7/del(7q) MDS by cyclin E, as well as other markers of proliferative index (such as 4twofold. Overexpression of CK2 has been shown to promote

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The World Health Organization classification of function in tumorigenesis and cell growth regulation.69 The neoplastic diseases of the hematopoietic and lymphoid tissues. R-Ras2 oncogenic signals are mediated through the PI3K/Akt Report of the Clinical Advisory Committee meeting, Airlie House, and NF-kB signaling pathways, both of which are significantly Virginia, November, 1997. Ann Oncol 1999; 10: 1419–1432. 8 Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G et al. À deregulated in MDS patients with 7/del(7q) in our study. International scoring system for evaluating prognosis in myelodys- Intriguingly, four pathways involving the retinoid X receptor plastic syndromes. Blood 1997; 89: 2079–2088. (RXR) and LXR system, ‘LXR/RXR activation,’ ‘FXR/RXR activa- 9 Sanz GF, Sanz MA, Greenberg PL. Prognostic factors and scoring tion,’ ‘TR/RXR activation’ and ‘PPAR signaling’ were also systems in myelodysplastic syndromes. Haematologica 1998; 83: deregulated in À7/del(7q) MDS. 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