ALBERT

Description: Microarray analysis with 40 000 cDNA gene chip arrays determined differential gene expression profiles (GEPs) in CD34+ marrow cells from myelodysplastic syndrome (MDS) patients compared with healthy persons. Using focused bioinformatics analyses, we found 1175 genes significantly differentially expressed by MDS versus normal, requiring a minimum of 39 genes to separately classify these patients. Major GEP differences were demonstrated between healthy and MDS patients and between several MDS subgroups: (1) those whose disease remained stable and those who subsequently transformed (tMDS) to acute myeloid leukemia; (2) between del(5q) and other MDS patients. A 6-gene “poor risk” signature was defined, which was associated with acute myeloid leukemia transformation and provided additive prognostic information for International Prognostic Scoring System Intermediate-1 patients. Overexpression of genes generating ribosomal proteins and for other signaling pathways was demonstrated in the tMDS patients. Comparison of del(5q) with the remaining MDS patients showed 1924 differentially expressed genes, with underexpression of 1014 genes, 11 of which were within the 5q31-32 commonly deleted region. These data demonstrated (1) GEPs distinguishing MDS patients from healthy and between those with differing clinical outcomes (tMDS vs those whose disease remained stable) and cytogenetics [eg, del(5q)]; and (2) molecular criteria refining prognostic categorization and associated biologic processes in MDS.

Description: Gene expression profiles (GEPs) were obtained from marrow hematopoietic precursor cells (HPC)(CD34+ cells) from 30 myelodysplastic syndrome (MDS) patients: RARS 2, RA 15, RAEB 9, RAEBT 4; IPSS Low 11, Int-1 10, Int-2 5, High 4, and 6 Normal individuals. Fluorescently labeled cDNA was prepared from CD34+ cells (〉90% purity), isolated by immunomagnetic column separation, after reverse transcription of high fidelity PCR-amplified poly(A) RNA (aRNA). The Cy-conjugated nucleotides for aRNA were hybridized to 40,000 gene chip microarrays obtained from the Stanford Functional Genomics Microarray Facility. aRNA from pooled normal CD34+ marrow cells was used as a Reference standard. High resolution scans were obtained to compile a dataset for each microarray, through files submitted to the Stanford Microarray Database. Dendrograms generated by unsupervised hierarchical gene clustering indicated major differences of GEP between Normal and MDS patients. Significance Analysis for Microarray (SAM) yielded 2327 genes significantly differentially expressed by MDS vs Normal: 2269 genes overexpressed, 58 underexpressed, with a false positive rate of ~10%. Prediction Analysis of Microarray (PAM) distinctly separated the MDS and Normal patients, requiring a minimum of 31 genes (which were also SAM significant). Class analysis by PAM correctly predicted 29 of the 30 to be MDS and 5 of the 6 to be Normal. Four disparate differential GEP regions in the dendrograms, comprising predominantly genes of differing functional categories provided signatures associated with differing MDS clinical subgroups. Nine of 10 patients with poor clinical outcomes were associated with a differing GEP signature than that which occurred in 14 of 20 patients with relatively good outcomes. Compared to the remainder of MDS patients, those with 5q- syndrome (n=5) had a differing GEP signature, with under-expression of 1018 genes, 11 of which were within the 5q31–32 CDS. Two of these genes (antioxidant protein1 and interferon regulatory factor1) have previously been proffered as candidate genes for this syndrome. Analysis of FACS-sorted highly purified marrow HPC subsets: CD34+38+ (late) and CD34+38- (early HPCs), indicated these ratios to be 4.3±2.1 (n=2) for MDS and 3.2±1.2 (n=12) for Normals. These findings suggest that the differing GEPs between the MDS and Normal CD34+ cells were not due to major differences in their proportions of CD38 cell subsets. SAM and PAM significant differential GEPs were noted between these cell subsets (also differing between MDS and Normal), indicating alteration of gene expression during differentiation. Wnt1 and β-catenin1 (genes involved in cell self-renewal) were over-expressed in both MDS CD38- and CD38+ cells compared to Normal. These data demonstrate: (1) molecular differences between MDS and Normal HPCs and within HPC subsets; (2) GEP signatures characterizing MDS patients with differing cytogenetic abnormalities (eg, 5q-) and clinical outcomes; (3) molecular criteria refining the prognostic categorization of MDS; and (4) gene expression data aiding characterization of the heterogeneous nature of this spectrum of diseases.

Description: Recombinant human erythropoietin (EPO) has been evaluated in MDS using a spectrum of doses and schedules, usually daily or thrice weekly, with an average erythroid response rate of 20% in published trials. Darbepoetin alfa (DA) is a hyperglycosylated analog of EPO with a longer serum half-life and greater in vivo potency. We evaluated the efficacy and safety profile of weekly subcutaneous DA dosing in MDS patients (pts) who had not received prior EPO treatment, with a hemoglobin (Hb) ≤ 10g/dL and/or red blood cell transfusion-dependence. During the induction phase, DA was begun at 4.5 mcg/kg/week (wk). For pts who did not achieve a major erythroid response by 6 wks (International Working Group response criteria), the DA dose was doubled to 9 mcg/kg/wk for 6 wks. In patients without a major erythroid response, G-CSF 2.5 mcg/kg twice weekly was added to DA 9 mcg/kg/wk for an additional 6 wks. Patients achieving an erythroid response underwent a ≥ 3-month maintenance phase. Dose reduction occurred for ≥ grade 3 non-hematologic toxicity or excess Hb response (Hb ≥ 13 g/dL). To date, 12 pts have been enrolled. FAB diagnoses were: RA (n=3), RARS (n=5), CMML (n=1), RAEB (n=3); divided into IPSS Low (n=6), Intermediate-1 (n=4) and Intermediate-2 (n=2). Of the 10 pts evaluable for erythroid responses (2 were too early), 7 responded: 5 major (3 RA, 2 RARS), 2 minor (RARS, RAEB), with 3 non-responders (2 RAEB, 1 RARS). The median durability of the major erythroid responses was 12.5 months. Doses for the 5 major erythroid responders were: DA 4.5 mcg/kg/wk (1), DA 9 mcg/kg/wk (1), and DA 9 mcg/kg/wk + G-CSF (3). DA was dose-reduced in 2 patients for excess Hb response. DA was generally well tolerated with no unexpected non-hematologic toxicity. One pt with baseline diabetic chronic renal insufficiency developed grade 2 proteinuria and increased creatinine levels, and grade 3 hypertension. Of 3 patients hospitalized for pneumonia, 1 (RAEB) expired. Progressive thrombocytopenia (grade 2 to 3 or worsening grade 3) occurred in 3 pts (2 RAEB, 1 RARS). Evaluation of the stimulatory effects of DA and EPO on in vitro marrow erythroid colony formation indicated that for those MDS pts with BFU-E growth (4 of 7 pts), similar BFU-E efficacy occurred compared with normal marrow (n=7), with DA being less stimulatory than EPO. These findings are consistent with known differences between in vitro responses (a function of receptor binding affinities) and in vivo efficacy (related to the extended serum half life of DA compared with EPO). Plateau (peak)/2 stimulatory concentrations were: DA 1nM (37ng/ml), EPO 0.2nM (0.78U/ml). In vivo/in vitro correlations are being assessed. These initial data indicate good tolerability and encouraging erythroid responses with once weekly DA dosing with or without G-CSF in MDS pts. Updated results of ongoing accrual will be presented.

Description: Abstract 4010 Patients with IPSS intermediate or high risk myelodysplastic syndrome (MDS) have few treatment options once their disease fails to respond to hypomethylating agent therapy. The styryl sulfone mitotic inhibitor ON 01910.Na, inhibits Polo-1 kinase, PI3-kinase and AKT pathways, and the drug has shown promising results in patients (pts) with advanced solid tumors (Jimeno et al, J Clin Onc 26:5504, 2008) and in Phase I/II studies of MDS pts, including those with trisomy 8 (Sloand at al, Proc ASH 2007, #822, Proc ASH 2008, #1651). In an ongoing Phase II clinical trial, we have treated 10 MDS pts unresponsive to at least 4 cycles (range 4–12 cycles) of hypomethylating agent therapy (5 post-azacytidine, 4 post-decitabine, and 1 pt treated with both agents) with ON 01910.Na. The pts had IPSS Intermediate-1 (n=3), Intermediate-2 (n=4) and High (n=3) risk MDS. The study cohort comprised pts with RAEB-1 (4 pts), RAEB-2 (3 pts) and RAEB-T (3 pts), with a median age of 80 years (range 65–86) and 2.3 year median (range 0.4–5.4) prior duration of MDS. Their cytogenetic profile included 5 pts with Good, 4 with Intermediate [t(8,10), t(14,18), +8, and (8+, 19+)], 1 with Poor risk cytogenetics (+8, 5q-, 1p-). At baseline, all pts were red blood cell transfusion-dependent. After the initial 2 pts were treated with 800mg/m2/day × 2day continuous IV infusion (CIVI)/week × 3weeks/month, the subsequent 8 patients received 1800mg/day × 3day CIVI q2weeks/month × 2 months, then monthly. Patients underwent bone marrow sampling and evaluation after every other cycle of treatment. To date, 7 pts have completed at least 2 cycles of therapy, with 2 pts having completed the full treatment course of 7 monthly cycles (median 4.7 for all pts). Responses according to IWG 2006 criteria were: Marrow CR (mCR) (2), Partial response (1), stable disease (SD) with hematologic improvement (HI) (2; 1 HI-E, N, 1 HI-P, N) = 5/10 overall responses (50%). Four pts had SD without HI, 1 pt progressed to AML. mCRs occurred in those with

Description: Introduction: Flow cytometry is commonly used to characterize bone marrow (BM) cells of patients with myelodysplastic syndrome (MDS). However the diagnostic utility of this technique has been limited. To address this, we utilized 31-parameter single cell mass cytometry (MCM) to comprehensively analyze primary MDS BM samples. Methods: Expression levels of 31 surface markers, including most previously reported aberrant markers in MDS, were measured on 30 whole BM samples from 10 patients with higher-risk MDS (HR-MDS; IPSS = Int2/High/RAEB-T), 10 with lower-risk MDS (LR-MDS; IPSS = Low/Int1), and 3 patients with non-clonal cytopenias. In addition, 5 BM samples from normal donors were simultaneously analyzed as internal reference comparisons. All samples were barcoded, such that 20 samples (MDS and healthy) could be combined into a single tube for simultaneous antibody staining and analysis. Aberrant marker expression was defined as a median expression level falling outside 4 times the absolute variance of the normal samples in each gated population. Further analysis compared manual gating with unsupervised clustering (spanning tree progression analysis of density-normalized events [SPADE]). Results: MCM analysis generated 31-parameter single-cell data that defined 28 major immunophenotypic populations for each sample. This enabled detection of an aberrant expression of 25/31 markers in at least one population, encompassing essentially every previously reported surface marker aberrancy in MDS. Additionally, 3 previously unrecognized aberrant expression patterns were identified by both manual gating and SPADE: increased CD321 (64% of samples) and CD99 (36% of samples); and decreased CD47 (14% of samples). We focused further analyses on the stem and progenitor cell compartment (HSC, MPP, CMP), in which 20 of the 22 MDS samples exhibited at least one aberrancy (average 2.7) in one of these 3 populations (RAEB-T samples exhibited an average of 4). By contrast, no aberrancies were detected within these populations in the 3 samples from patients with non-clonal cytopenias. In addition to the identification of aberrant expression patterns within the subdivided stem and progenitor cell populations (HSPC) of individual samples, analysis of the HSPC population (CD34+CD38low) as a whole, revealed significant increases (~2-fold) in median expression of CD117 (p=0.003) and HLA-DR (p=0.028) for MDS samples compared to normal. Differences in CD117 and HLA-DR could also be appreciated as aberrant expression patterns (outside 4-fold the variance of normal) in 12/22 and 13/22 samples, respectively. Comparison of marker expression within the HSPCs between patients with HR-MDS and LR-MDS also revealed significant differences. HR-MDS HSPCs were characterized by a ~2-fold increase in CD99 compared to LR-MDS (p=0.0018) and a ~3-fold decrease in CD45 compared to LR-MDS (p=8.8x10-5). Differences in CD99 and CD45 could also be appreciated as aberrant expression patterns in 7/12 and 6/12 of the HR-MDS samples, respectively. Finally, the distribution of cell frequencies across the immunophenotypic populations (by SPADE analysis or manual gating) was used to perform a hierarchical clustering of all samples. This clustered patients into groups with different clinical risk. The most significant single distinguishing feature between clinical risk groups was the increased frequency (〉40-fold) of HSPCs in HR-MDS compared to LR-MDS (p=9x10-7) or normal (p=6.3x10-6). Furthermore, this high-parameter analysis detected a 〉12-fold increase in the HSPC frequency in 2 patients with IPSS Int-2 disease with blast frequencies of

Description: Background: Prior studies using microarray platforms have shown alterations of gene expression profiles (GEPs) in MDS CD34+ marrow cells related to clinical outcomes (Sridhar et al, Blood 2009, Pellagatti et al, JCO 2013). Given the increased sensitivity and accuracy of high-throughput RNA sequencing (RNA-Seq) (Mortazavi et al, Nat Meth 2008, Soon et al, Mol Syst Bio 2012) for detecting and quantifying mRNA transcripts, we applied this methodology for evaluating differential gene expression between MDS and normal CD34+ marrow cells. Methods:RNA was isolated from magnetic bead affinity-enriched CD34+ (〉90%) marrow aspirate cells (Miltenyi Biotec, Auburn, CA) and amplified using the Smarter Kit (Clontech, Mt View, CA). The amplified product (ds DNA) was fragmented to a size distribution of ~200-300bp using the E220 Focused Ultrasonicator (Covaris Inc, Woburn, MA). End repair, adapter ligation and PCR amplification were performed using the NEBNext Ultra RNA library prep kit for Illumina (New England Biolabs, Ipswich, MA). The indexed cDNA libraries were sequenced (paired end, 100bp) on an Illumina HiSeq2000 platform with median read counts of 69 million. The sequences were aligned to Human Reference sequence hg19 using DNAnexus mapper with gene detection focused on known annotated genes. The differential expression was analyzed using edgeR. DAVID and Ingenuity IPA programs were used for pathway analyses. Gene Set Enrichment Analysis (GSEA) was used to identify biologic processes in our dataset present across phenotypes. Results: Correlations of RNA-Seq data from unamplified to amplified transcripts demonstrated high fidelity of transcripts obtained (Pearson and Spearman R2 = 0.80). After filtering samples for adequate read counts, 12,323 genes were evaluated. Differential expression analysis yielded 719 differentially expressed genes (DEGs) in MDS (n=30) vs normal (n=21) with FDR 50% of the patients. Hierarchical cluster analysis using these DEGs confirmed clear separation of MDS patients from normals, with 2 differential expression clusters—one region overexpressed and one underexpressed. A distinctive trend toward clustering of the patients was seen which related to their IPSS categories and marrow blast %. In functional pathway analysis of the 2 distinctive gene clusters which distinguished MDS from normal, the underexpressed MDS DEGs demonstrated enrichment of inflammatory cytokines, oxidative stress and interleukin signaling pathways, plus mitochondrial calcium transport; whereas the MDS overexpressed DEG cluster showed enrichment of adherens junction/cytokeletal remodeling, cell cycle control of chromosome replication and DNA damage response pathways. Using GSEA analysis, significantly increased numbers of genes in MDS vs normal, common to those in gene sets present within curated public databases, were involved with TP53 targets and mTOR signaling pathways. Conclusions: Our study demonstrated that RNA-Seq methodology, a high-throughput and more comprehensive technique than most gene expression microarrays, was capable of showing significant and distinctive differences in gene expression between MDS and normal marrow CD34+ cells. Specific clustering of the DEGs was demonstrated to distinguish patient subsets associated with their major clinical features. Further, the stringently identified DEGs shown to be engaged in functional pathways and biologic processes highly relevant for MDS were extant within the patients’ CD34+ cells. These transcriptomic data provide information complementary to exomic mutational findings contributing to improved understanding of biologic mechanisms underlying MDS. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 738 Myelodysplastic syndromes (MDS) are characterized by defective hematopoietic stem/progenitor cell maturation, resulting in ineffective hematopoiesis. This group of disorders is characterized by cytogenetic abnormalities and approximately 25% of cases progress to acute myeloid leukemia (AML). NPM1 is frequently mutated in AML and translocations involving NPM1 occur in a number of hematopoietic malignancies including MDS. NPM1 heterozygous mice (NPM1 +/−) have been shown to have a MDS-like phenotype. Taken together, these data suggest an important role for NPM1 in the function of hematopoietic stem cells (HSC) and/or committed progenitors. In order to evaluate NPM1 function in early hematopoiesis, we have evaluated NPM1 expression in both the mouse and human hematopoietic systems. Using quantitative RT-PCR, we show that NPM1 expression levels are 2-3-fold higher in normal CD34+ bone marrow progenitor cells compared to total bone marrow in humans. Furthermore, NPM1 expression levels are decreased by ∼50% in 9/37 MDS CD34+ cells when compared to normal controls. Of interest, NPM1 expression is reduced primarily in patients with poor or intermediate prognosis. Consistent with a functional role for NPM1 in HSC, NPM1 +/− mice (developed by gene trapping and obtained from the MMRRC at UC-Davis) contained significantly increased numbers of HSC (Lin-cKit+Sca+CD34-CD150+) within the Lin-cKit+Sca+ population compared to those from the littermate controls (52 ± 2.6% vs,74 ± 12%, p 〈 0.01). Consistent with prior reports, NPM +/− mice contained significantly fewer mature erythrocytes (Ter119+CD71lo) in the bone marrow compared to WT controls (6.5 ± 1.8% vs 10 ± 0.5% p 〈 0.01). In order to study NPM +/− HSC function, we tested the ability of these HSCs to form colonies in methylcellulose. NPM1 +/− HSCs formed increased numbers of both CFU-GM and CFU-GEMM colonies and decreased numbers of CFU-E colonies compared to WT HSC. Flow cytometric analysis of pooled day 14 colonies from individual mice revealed a 〉2 fold increase in cKit+ progenitor cells from NPM1 +/− colonies (2.0 ± 1.0% vs. 0.2 ± 0.2%, p = 0.02), suggesting that the differentiation potential of NPM+/− HSCs is impaired. To characterize HSC function in vivo, equal numbers of double-sorted HSCs from WT and NPM1 +/− mice were transplanted in triplicate into lethally irradiated C57B6 (CD45.2) recipients. Analysis of peripheral blood donor chimerism (CD45.1+CD45.2+) 21 days post-transplantation showed that NPM1 +/− HSC-transplanted recipients exhibited markedly lower granulocyte chimerism than WT HSC recipients (5.5 fold reduction, 2 ± 2% vs. 11 ± 5%, p 〈 0.01). This finding suggests that although NPM1 +/− mice have increased numbers of HSC, these HSC exhibit either altered myeloid fate decisions or decreased bone marrow homing capacity. We are currently investigating long-term engraftment potential to further elucidate the function of HSC in NPM1 +/− mice in vivo. In aggregate, these data demonstrate a functional role for NPM1 in early myeloid differentiation and strongly suggest that NPM effects may be exerted as early as at the level of the HSC. Disclosures: Weissman: Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: Equity Ownership, Founder; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Description: Abstract 1887 Myelodysplastic syndromes (MDS) are clonal bone marrow failure disorders with variably progressive bone marrow failure and risk of leukemic transformation. Risk stratification based on the International Prognostic Scoring System (IPSS) is a powerful tool, but much clinical variability remains within risk categories. Gene expression profiling (GEP) of disaggregated bone marrow mononuclear cells has yielded many potential prognostic biomarkers; such studies are limited by (1) the need for fresh bone marrow and labor-intensive analysis of individual specimens, leading to a bottleneck between identification of biomarkers and clinical implementation, and (2) by the loss of cell-specific and architectural information. We have demonstrated the feasibility of quantitative cell-type-specific evaluation of biomarkers in intact archival MDS bone marrow (BM). Routinely processed archival core biopsy specimens were sampled to create tissue microarrays (TMAs). We chose to test the protein products of 4 genes overexpressed in a “poor risk” gene signature for early leukemic transformation in MDS (Sridhar K et al, Blood 114:4847, 2009). These include ribosomal subunit components RPS4 and RPL23 and proteases TPP2 and KLK3. A TMA was constructed from 1 mm cores of 5 normal, 4 MDS, and 5 acute myeloid leukemia (AML) BM core biopsies. Immunohistochemistry showed reproducible cytoplasmic staining of immature mononuclear cells by antibodies against RPL23, RPS4Y, TPP2, and KLK3; TPP2 also stained megakaryocytes. Reactivity with a correctly-sized band was confirmed by Western blotting of frozen BM from normal and MDS subjects. Double immunofluorescence (IF) staining was then performed to allow simultaneous identification of cell types of interest [CD34+ progenitors, Glycophorin C(GPC)+ erythroid precursors] in combination with quantitative analysis of the marker of interest. Representative image Figure 1: ribosomal marker RPL23 (red) and erythroid marker GPC (green); DAPI nuclei (blue): After scanning of the TMA on the Ariol platform, CellProfiler image analysis software (Broad Institute) was used to identify primary objects (nuclei) in the DAPI stained-image, and associated secondary objects (cells) in the green channel; fluorescence intensity was then quantified in the red channel. An example of object identification in CellProfiler is shown in Figure 2: When the mean per cell intensities from pooled MDS specimens were compared with the pooled normal specimens, all four putative “poor prognosis” biomarkers were significantly more highly expressed in the CD34+ population of MDS as compared to normal BM; even higher expression was seen in AML (see table below; Kruskal Wallis test with Bonferroni adjustment for multiple comparisons, p

Description: Abstract 3808 The ability to quantify changes in protein activity in a clinical setting is important for the development of therapeutics that target cancer signaling pathways. We have developed a highly sensitive microfluidic nano-immunoassay (NIA) to quantify unphosphorylated, single- and multi- phosphorylated isoforms of proteins in patient specimens. Using as little as 2 nanoliters of cellular lysate, we can measure over 40 proteomic parameters, and assess changes in proteins that mediate signal transduction, cell cycle progression and apoptosis. The styryl sulfone mitotic inhibitor, rigosertib, inhibits multiple kinases in vitro, including the PI3 Kinase, MAP Kinase and PLK-1 signaling pathways. In order to develop potential biomarkers of response to this agent, we first used NIA to demonstrate that rigosertib decreases specific phosphorylated MEK isoforms in the MAPK pathway and AKT isoforms in the PI3K pathway in the TF1 erythroleukemia cell line in vitro. Next, to determine if these changes occur in MDS patients treated with rigosertib, we used NIA to quantify MEK and AKT isoforms in bone marrow CD34+ cells sampled before and at sequential time points after initiating rigosertib treatment in patients enrolled in our prospective single institution Phase II study “A Phase 2, Single-Arm Study To Assess The Efficacy and Safety Of 72-Hour Continuous Intravenous Dosing Of ON 01910.Na (Rigosertib) Administered Every Other Week in Myelodysplastic Syndrome Patients with Trisomy 8 or Classified as Intermediate-1, 2 or High Risk”. To date, we have analyzed 14 specimens from 5 patients. Three patients who responded to treatment (marrow complete response or stable disease) exhibited a 20% mean decrease in MEK1 phosphorylation. In contrast, two patients whose disease progressed on treatment exhibited a 15% mean increase in MEK1 phosphorylation. In the PI3 Kinase pathway, we found that AKT2 phosphorylation decreased by an average of 15% in responders, whereas the two patients whose disease progressed on treatment exhibited a 2% decrease and an 18% increase in AKT2 phosphorylation, respectively. NIA is able to measure changes in different phosphorylated isoforms of AKT2; thus, we were able to further resolve changes in AKT2 phosphorylation into 4 distinct phosphorylated isoforms. Interestingly, NIA revealed that the most abundant of these isoforms, isoform 3, was preferentially suppressed upon successful rigosertib treatment: isoform 3 decreased by a mean of 28% in the three patients who responded to the drug. In contrast, the two patients whose disease progressed on treatment exhibited a 10% decrease and a 22% increase in isoform 3, respectively. Our results suggest that a possible mechanism of action of rigosertib in MDS patients might be through the inhibition of both the PI3K and the MAPK pathways, and raise the hypothesis that the drug may preferentially target specific phosphorylated isoforms within each pathway. We have shown that NIA can be used to measure isoforms of phospho-MEK and phospho-AKT as potential biomarkers of rigosertib activity in MDS. Disclosures: Fan: Onconova: Research Funding. Wilhelm:Onconova Therapeutics: Employment, Equity Ownership. Greenberg:onconova: Research Funding. Felsher:Onconova: Consultancy, Research Funding.

Description: Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis. The DNA-hypomethylating agents 5-azacytidine and 5-aza-2′-deoxycytidine are effective treatments for patients with MDS, increasing the time to progression to acute myelogenous leukemia and improving overall response rates. Although genome-wide increases in DNA methylation have been documented in BM cells from MDS patients, the methylation signatures of specific gene promoters have not been correlated with the clinical response to these therapies. Recently, attention has been drawn to the potential etiologic role of decreased expression of specific ribosomal proteins in MDS and in other BM failure states. Therefore, we investigated whether rRNA expression is dysregulated in MDS. We found significantly decreased rRNA expression and increased rDNA promoter methylation in CD34+ hematopoietic progenitor cells from the majority of MDS patients compared with normal controls. Treatment of myeloid cell lines with 5-aza-2′-deoxycytidine resulted in a significant decrease in the methylation of the rDNA promoter and an increase in rRNA levels. These observations suggest that an increase in rDNA promoter methylation can result in decreased rRNA synthesis that may contribute to defective hematopoiesis and BM failure in some patients with MDS.