Myelodysplastic Syndrome Marrow Research Articles

Bone marrow fibrosis impact on response to azacitidine in myelodysplastic syndromes

Bone marrow fibrosis in myelodysplastic syndromes (MDS) has been associated with poor outcome. However, these studies were conducted prior to the widespread use of azacitidine in the management of MDS. Our study aimed to assess whether treatment with azacitidine ameliorates the inferior outcome in MDS with fibrosis. A retrospective study of all patients diagnosed with MDS and treated with azacitidine over 3 years in two institutions was performed. A total of 21 patients were included in this study. Approximately half of these had moderate to severe bone marrow fibrosis at the start of treatment with azacitidine. The median overall survival was 34 months in patients with non-fibrotic bone marrow compared to 14 months in patients with fibrotic marrow (p=0.0007). Median event-free survival was 26 months versus 12 months (p=0.0027) in patients with non-fibrotic and fibrotic marrow, respectively. In multivariate analysis, bone marrow fibrosis was an independent factor in overall survival. Transfusion requirement was not different between the two groups. Despite the small sample size, we observed a worse outcome in azacitidine treated patients with MDS and fibrotic marrow. We suggest a prospective larger study to confirm the above finding.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Novel In Vivo Models Uncover Non-Cell-Autonomous Effects of Myelodysplastic Syndrome (MDS) on Marrow Osteolineage Cells and Hematopoietic Dysfunction

Myelodysplastic syndromes (MDS) remain a therapeutic challenge, leading to significant morbidity and mortality due to blood cytopenias and leukemic transformation. We and others have demonstrated that abnormalities of the bone marrow microenvironment (BMME) contribute to disease pathogenesis and progression. However, current models fail to assess in vivo the impact of the malignant cells on the BMME and on residual non-clonal hematopoiesis. We previously identified BMME abnormalities in Vav1-NUP98/HOXD13 (NHD13) mice, a well-established transgenic murine model that recapitulates pathologic hallmarks of human MDS including blood cytopenias, dysmyelopoiesis, and transformation. NHD13 mice had increased osteoblastic-lineage cells (OBC, lineage-/CD45-/CD31-/CD51+/Sca1-), as well as hematopoietic dysfunction and hematopoietic stem cell (HSC) loss.To determine if MDS can initiate BMME alterations, we transplanted MDS marrow of NHD13 mice or wild-type (WT) littermate marrow (CD45.2+/CD45.1+) into irradiated WT recipients (CD45.2+/CD45.1-). Using flow cytometry to distinguish donor- vs. host-derived hematopoietic cells, we found that NHD13 and WT donor-derived cells contributed to approximately 60% of recipient marrow, generating an in vivo model of MDS cells among genetically-WT hematopoietic cells. This transplant model recapitulates human MDS, where MDS clones emerge and expand among normal hematopoietic cells in a BMME that is initially genetically normal. At 6 weeks post-transplant, OBCs were increased in recipients of MDS marrow (p<0.05), indicating that interaction with MDS cells induced osteolineage cells to take on features of an MDS-BMME. Concurrent with BMME remodeling,HSCs were decreased in recipients of MDS marrowcompared to recipients of WT marrow (p<0.05). Notably, HSCs in recipients of MDS marrow were also decreased compared to surviving irradiated but non-transplanted mice (p<0.05), consistent with disruption of normal HSC function by MDS cells. In line with acquired hematopoietic dysfunction, recipients of MDS marrow exhibited leukopenia and anemia compared to recipients of WT marrow. To assess the hematopoietic function of genetically-WT recipient cells in the presence of MDS cells, we quantified donor- vs. recipient-derived peripheral white blood cells (WBC). The number of recipient-derived WBCs in mice transplanted with MDS marrow was lower compared to irradiated, non-transplanted mice. This suggests that cytopenias in recipients of MDS marrow were not only due to defective cell-autonomous hematopoiesis by MDS cells, but also due to the impaired hematopoietic function of residual WT recipient cells. Together, these data indicate that MDS cells may decrease hematopoietic support, leading to dysfunctional hematopoiesis.To assess hematopoietic function of non-irradiated WT hematopoietic cells in the presence and absence of MDS, we transplanted NHD13/GFP- marrow mixed 1:1 with WT/GFP+ marrow (NHD13/GFP-:WT/GFP+) or WT/GFP-:WT/GFP+ marrow into irradiated WT recipients. Although NHD13 donor-derived cells contributed to only 4% of reconstituted recipient marrow in this model, HSC depletion was unexpectedly observed in recipients of NHD13/GFP-:WT/GFP+ marrow compared to recipients of WT/GFP-:WT/GFP+ marrow, showing that, even when recipient marrow is comprised of over 90% normal hematopoietic cells, the HSC pool is reduced in the presence of MDS cells. These data demonstrate a strong non-cell-autonomous effect of MDS on hematopoietic function.Because human MDS is associated with diverse genetic abnormalities, we sought to determine if MDS-dependent BMME remodeling transcends the genetic makeup of MDS by assessing the BMME in mice with inducible EVI1 overexpression (EVI1++), an oncogenic transcription factor important in myeloid progenitor function and commonly implicated in human MDS. Marrow from EVI++ mice (mixed 1:1 with WT marrow) was transplanted into WT mice. By 14 weeks post-transplant, recipients of EVI1++ cells exhibited BMME alterations, demonstrated by increased OBCs (p<0.05) compared to recipients of WT cells. Thus, exposure to both NHD13 and EVI1++ marrow induces OBC remodeling in our transplant models.Together, these data uncover non-cell-autonomous effects of MDS causing hematopoietic dysfunction in vivo, and provide novel tools to discern the MDS signals and BMME changes impairing normal hematopoiesis. DisclosuresLiesveld:Seattle Genetics: Honoraria; Onconova: Honoraria.

Evidence of Targetable Immune Dysfunction in the Bone Marrow of Patients with Intermediate/High-Risk Myelodysplastic Syndrome Refractory to Hypomethylating Agents

Background Growing evidence indicates that a dysfunctional adaptive immune response contributes to the pathogenesis of myelodysplastic syndrome (MDS). Intermediate/high (I/H)-risk MDS is associated with immunosuppression, and median overall survival in hypomethylating agent (HMA)-refractory patients (pts) remains poor at < 6 months. Thus, new immune checkpoint-targeting therapies may be important for restoring effective anti-tumor T-cell mechanisms in these pts. Here we aim to evaluate the relationship between immunomodulatory and regulatory targets in lymphocytes and CD34+ blasts, cytogenetics and prior treatment regimens to inform future novel immunotherapy combinations.Methods Bone marrow aspirates from HMA-refractory, I/H-risk pts (n = 18) and normal donors (NDs; n = 20) were assessed by flow cytometry (FC), immunohistochemistry and RNA sequencing (RNAseq). An independent cohort of treatment-naive, I/H-risk pts (n = 9) was assessed for regulatory T-cell (Treg) counts by FC. T-cell subsets (Treg, T-naive, T-effector memory, T-central memory, T-late effector memory) were identified by FC based on established markers (CD3, CD4, CD8, CD197, CD45RO, CD127, CD25, FOXP3). Activation/proliferation status of T-cell subsets was measured by HLA-DR and Ki-67. MDS blasts were identified by CD45, CD34 and CD117. Immune checkpoint markers programmed death-1 (PD-1) and programmed death-ligands 1 and 2 (PD-L1/2) were measured on T cells and CD34+ blasts. T-cell immunoglobulin and ITIM domain (TIGIT) was also measured on CD4 and CD8 T cells. T cells (CD4, CD8) and PD-L1 were identified in decalcified bone marrow biopsies by immunohistochemistry. Whole transcriptome gene expression analysis was performed using the Illumina TruSeq RNA Access Library Prep Kit on bulk bone marrow aspirates.Results T cells were detected in bone marrow aspirates from HMA-refractory, I/H-risk pts at levels comparable to those in NDs. Treg counts (cells/µL) were increased in HMA-refractory vs treatment-naive pts (P= 0.017), with the highest Treg count associated with complex cytogenetic karyotypes (≥ 3 abnormalities). We observed increased T-cell activation and proliferation in these pts compared with NDs (MDS vs ND medians [% positive cells]; HLA-DR−/Ki-67+: 1.23 vs 0.75; HLA-DR+/Ki-67+: 1.71 vs 0.33; HLA-DR+/Ki-67−: 14.14 vs 4.80). No trends were seen in CD4-naive and memory subsets, whereas CD8-naive cells were lower and late effector memory cells were higher than in NDs (P= 0.035 each). We observed higher expression of immune checkpoint and regulatory molecules (PD-L1, PD-1, TIGIT) in MDS except for PD-L2, which was minimally detected (< 5%). Frequency (%) of PD-L1 on CD34+ blasts was higher in pts with MDS than in NDs (P= 0.02) and was associated with complex karyotype (P= 0.026) and Treg frequency (P= 0.021). PD-1+ T cells were higher in pts with MDS than in NDs (CD4, P= 0.004; CD8, P= 0.012), with a trend toward increased PD-1 expression associated with number of prior therapies. TIGIT expression on CD4 T cells correlated with PD-1 (P= 0.008) and Treg frequency (P= 0.022). Lymphocyte-activation gene 3 expression by RNAseq was also higher in MDS vs ND marrow (P < 0.001). Some signals, such as PD-L1 and Tregs, correlated by FC and RNAseq.Conclusions Bone marrow in pts with I/H-risk MDS demonstrate evidence of prolonged T-cell activation, an increase in suppressive immune cell populations (i.e., Tregs) and expression of immune checkpoint molecules (PD-L1, TIGIT) compared with normal bone marrow. Patients with complex karyotype had subtle differences, potentially relevant for identifying subsets responsive to checkpoint inhibitors. These data provide new insight into the immune landscape of HMA-refractory I/H-risk MDS. DisclosuresGreen:Genentech, Inc.: Employment. Yan:F. Hoffmann-La Roche Ltd: Employment. Nalle:Genentech, Inc.: Employment; Roche: Equity Ownership. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Zhong:F. Hoffmann-La Roche Ltd.: Employment. Krishnan:Genentech, Inc.: Employment. Phuong:Genentech, Inc.: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment, Equity Ownership. Adamkewicz:Genentech Inc.: Employment. Venstrom:Genentech, Inc.: Employment. Dail:Genentech, Inc.: Employment.

Understanding Early Stage Myelodysplastic Syndrome Pathobiology

Understanding myelodysplastic-syndrome (MDS) pathobiology is critical to derive and evaluate new therapeutic interventions. Transforming growth factor-beta (TGFβ) signaling is abnormally active in early-stage MDS patient hematopoietic stem and progenitors, and the level of TGFβ-signal-induced transcriptional changes may have prognostic value. However, the mechanism of TGFβ signaling resulting in ineffective hematopoiesis in MDS is not known. TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7) which block promiscuous activity. Upon TGFβ receptor signaling, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by downstream signaling and subsequently terminate the TGFβ signal (a negative feedback loop). The Verma lab demonstrated that human MDS marrow cells have elevated levels of microRNA-21 (miR-21) that target SMAD7. Their work identifies miR-21 as one factor in MDS that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal. We used transient transcription assays to reveal that miR-21 targets the SKI corepressor, a TGFβ-signaling antagonist that binds to R-SMADS to block downstream SMAD-target gene activation. Immunofluorescence staining of primary bone-marrow samples from early-stage MDS patients (with elevated miR-21 and chronic TGFβ) shows significant reduction of not only SMAD7 but also SKI protein. TGFβ signaling induces ubiquitin mediated degradation of SKI, so low SKI expression in MDS is the sum of miR-21 and chronic TGFβ effects. To determine the functional impact of low SKI we examined Ski-/- mice. We show that SKI loss of function is itself sufficient to impair hematopoietic stem cell (HSC) fitness. While flow cytometric evaluation of Ski-/- hematopoiesis indicates little phenotypic change in HSC fractions, and Ski-/- HSC engraft lethally irradiated recipients, competitive HSC transplantation reveals profound Ski-/- HSC fitness defects. Ski-like (Sno/Skil) encodes a SKI-related compressor that is not a miR-21 target. Ski-/- defects are not phenocopied in Skil-/- HSC; underscoring the specificity of the HSC defects to the hypothesized miR-21-SKI axis. In agreement with this, retroviral or lentiviral vector forced expression of miR-21 in HSC is counter-selected within 60 days after transplantation. The Growth factor independent-1 (GFI1) transcriptional repressor is required for HSC fitness and is an independent prognostic indicator in AML/MDS. We previously demonstrated GFI1 transcriptional repression of miR21 . Subsequently, SKI protein levels are significantly decreased in Gfi1-/- bone marrow cells (which express elevated miR-21). SKI expression is rescued in Gfi1-/-miR-21-/- knockout mice but Gfi1-/- HSC fitness is not restored. Thus, it is unlikely that loss of SKI downstream of Gfi1 loss of function explains Gfi1-/- HSC fitness defects. Instead, we show that the SKI corepressor binds the GFI1 transcriptional repressor. Native GFI1 and SKI proteins are in the same complex by immunoprecipitation, and synthetic-protein pull-down assays validated direct interaction and mapped the interacting domains. ChIP Seq reveals that Ski is coincident with Gfi1 on a subset of common peaks. We conclude that: 1) in normal cells GFI1 repression of miR-21 protects expression of SKI, an HSC-relevant GFI1 corepressor, and 2) chronic TGFβ signaling in early stage MDS directly affects HSC fitness by targeting SKI. Disclosures No relevant conflicts of interest to declare.

Pediatric MDS and bone marrow failure-associated germline mutations in SAMD9 and SAMD9L impair multiple pathways in primary hematopoietic cells

Pediatric myelodysplastic syndromes (MDS) are a heterogeneous disease group associated with impaired hematopoiesis, bone marrow hypocellularity, and frequently have deletions involving chromosome 7 (monosomy 7). We and others recently identified heterozygous germline mutations in SAMD9 and SAMD9L in children with monosomy 7 and MDS. We previously demonstrated an antiproliferative effect of these gene products in non-hematopoietic cells, which was exacerbated by their patient-associated mutations. Here, we used a lentiviral overexpression approach to assess the functional impact and underlying cellular processes of wild-type and mutant SAMD9 or SAMD9L in primary mouse or human hematopoietic stem and progenitor cells (HSPC). Using a combination of protein interactome analyses, transcriptional profiling, and functional validation, we show that SAMD9 and SAMD9L are multifunctional proteins that cause profound alterations in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, our molecular and functional studies also demonstrated that expression of these genes and their mutations leads to a cellular environment that promotes DNA damage repair defects and ultimately apoptosis in hematopoietic cells. This study provides novel functional insights into SAMD9 and SAMD9L and how their mutations can potentially alter hematopoietic function and lead to bone marrow hypocellularity, a hallmark of pediatric MDS.

In search for a haemoglobin threshold in myelodysplastic syndromes.

In search for a haemoglobin threshold in myelodysplastic syndromes.

Aberrant Patterns of Alternative Splicing Are Frequent Events and Harbor Prognostic Significance in Patients with Myelodysplastic Syndrome

▪BackgroundAberrant patterns of alternative splicing (AS) are reported in acute myeloid leukemia and other malignances, but have not yet been investigated in myelodysplastic syndrome (MDS). In addition, mutations in spliceosome genes are especially common in MDS. Therefore, it would be highly interesting to analyze the clinical and biological significance of aberrant AS in MDS.Materials and MethodsA total of 243 newly diagnosed MDS patients, including 78 with refractory anemia (RA), 22 with RA with ring sideroblasts(RARS), 76 with RA with excess blasts (RAEB), 24 with RAEB in transformation (RAEBT), and 43 with chronic myelomonocyticleukemia (CMMoL), who had complete clinical and genetic information were enrolled. We also included 20 healthy donors of hematopoietic stem cell transplantation for comparison. We extracted RNA from mononuclear cells (MNC) isolated from the bone marrow (BM), followed by hybridization on the microarrays of AffymetrixHuman Transcriptome Array 2.0, which contained > 6 million distinct probes covering coding, non-coding transcripts, and the exon-exon junctions among 67539 genes (44710 coding genesand 22829 noncoding genes). This comprehensive coverage of exons and junctions facilitated investigation of the splicing patterns of the genes. The aberrant AS was analyzed with the splicing index (SI) method using the default setting of AffymetrixTranscriptome Analysis Console version 3.0 software. The SI of a probe in a gene was derived from the ratio between MDS patients and normal controls in terms of the probe intensity divided by the gene expression level, ie. [intensityof probe A in gene A /gene A expression level]MDS/[intensity of probe A in gene A /gene A expression level]normal control. SI had to be less than -2 or more than 2 to be defined as an aberrant AS event.ResultsTotally 52730 of 67539 genes (78.1%) on the array were expressed in BM from both MDS patients and normal donor controls. Approximately 17565 of the 52730 expressed genes (33.3%) were differentially spliced between the MDS and normal marrow MNC (P < 0.05). As a whole, 88.9% of these aberrant spliced events were mapped to coding genome and 11.1% to noncoding regions. These aberrant AS exons fell into five common patterns, including exon skipping (57.8%), alternative donor sites (18.1%), alternative acceptor sites (17.8%), intron retention (5.6%) and mutually exclusive exons (0.7%). In average, when compared with the normal marrow MNC, there were 6.4 (4.8-12.0) more aberrant AS events in each AS gene (ASG) in the marrow MNC of 243 MDS patients.More aberrant AS events per ASG implied a more complex aberrant AS pattern.The numbers of aberrant AS events per ASG were not significantly associated with MDS subtypes and frequencies of spliceosome gene mutations. However, with a median follow up of 48.4 months, the patients with more aberrant AS events per ASG had a trend of shorter overall survival (OS) (median 20.1 vs 29.6 months, P=0.074) and a trend of shorter time to acute leukemic transformation (P=0.212) than the others (Fig.1). Further subgroup analysis showed a significantly shorter OS in the patients with more aberrant AS events per ASG than those with less events among RAEB patients (median OS: 10.8 months vs 16.9 months, P=0.007, Fig.2A). In addition, RA patients with more aberrant AS events per ASG had shorter time to acute leukemic transformation than RA patients with fewer aberrant AS events per ASG (P=0.006, Fig.2B). Among the patients without detectable mutations in spliceosome genes or genes related to epigenetic modification, those with more aberrant AS events per ASG also had a shorter OS than the others (median OS: 27.9 months vs not reached, P=0.013, Fig.3). Multivariate analysis in our cohort of 243 MDS patients revealed that higher aberrant AS events per ASG was an unfavorable prognostic factor for OS (P=0.016) independent of age, IPSS-R risk score, and mutations of SF3B1, ASXL1 and TP53.ConclusionTo our knowledge, we are the first to present the prognostic impact of aberrant AS pattern in MDS patients. Large prospective cohorts are needed to confirm our observations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Beyond the Niche: Myelodysplastic Syndrome Topobiology in the Laboratory and in the Clinic.

We review the murine and human microenvironment and hematopoietic stem cell niche in the context of intact bone marrow architecture in man and mouse, both in normal and in myelodysplastic syndrome marrow. We propose that the complexity of the hematopoietic stem cell niche can usefully be approached in the context of its topobiology, and we provide a model that incorporates in vitro and in vivo models as well as in situ findings from intact human marrow to explain the changes seen in myelodysplastic syndrome patients. We highlight the clinical application of the study of the bone marrow microenvironment and its topobiology in myelodysplastic syndromes.

Recurrent Abnormal Clones in Myelodysplastic Syndrome Marrow Originate from Cells at a Pluripotent Stem Level and Maintain Their Early Differentiation Potency

Objective: The present study aimed to investigate the origins and differentiation potencies of 4 common malignant clonal cell types (+8, 5q−/−5, 20q−/−20, 7q−/−7) in myelodysplastic syndrome (MDS) and to investigate whether the trisomy of chromosome 8 occurs subsequently to other chromosomal abnormalities. Methods: The present study analyzed a total of 46 cases of chromosomal abnormalities in MDS patients. The magnetic activated cell sorting technique (MACS) was used to sort the CD34+CD38− (pluripotent hematopoietic stem cells) and CD34+CD38+ cells (committed progenitor cells) from the bone marrow mononuclear cells (BMNCs) of these patients; the sorted cells were then individually smeared. Meanwhile, cytospins were prepared from the remaining CD34− BMNCs after cell sorting. The clonal cell proportions in these three types of smears were detected by fluorescence in situ hybridization (FISH). Cases in which +8 was associated with another abnormality (2 cases each in combination with abnormalities in chromosomes 7, 5, and 20) were dually hybridized with the cep8 probe and another corresponding probe. Results: (1) for abnormalities of +8, 5q−/−5, 20q−/−20 or chromosome 7 involvements, clonal cells above the baseline level were detected in the pluripotent stem cell level. (2) The average clonal cell proportion in the committed progenitor cells of the 46 cases increased to 75.3% from 57.3% at the level of stem cell (p < 0.001). The groups with +8 and chromosome 5 abnormalities showed a statistically significant increase in clonal cells at the progenitor cell stage. At the individual level, 33 of 46 cases showed significant increases in clonal cells at the progenitor cell stage relative to the stem cell stage, whereas the clonal cell proportion in the CD34− BMNCs generally did not increase relative to the committed progenitor cell population. (3) The dual hybridization analysis showed that if +8 and another abnormality were present in the same abnormal clone according to G-banding, +8 always coexisted with the other chromosomal abnormality at the single cell level; there were no situations in which +8 occurred later than the other chromosomal abnormality. Conclusion: It seems that the all malignant MDS clones originated at the pluripotent hematopoietic stem cell stage and that the proliferation and differentiation potencies were retained partly in these clonal cells. The present study failed to confirm that the trisomy 8 occurred subsequently to the other abnormalities, but some in vitro or transplant experiments maybe prove the succession of clonal origination.

Transcriptomic Evaluation of CD34+ Marrow Cells from Myelodysplastic Syndrome (MDS) Patients

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 <.05. Among the DEGs, 548 and 171 were over- and under-expressed ≥2 fold in MDS vs Normal, respectively: 20% of the overexpressed genes were present in >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. DisclosuresNo relevant conflicts of interest to declare.

A Calcium-Dependent Pathway Determines Response to Lenalidomide in Del(5q) Myelodysplastic Syndromes

Deletions of the long arm of chromosome 5 (del(5q)) are common cytogenetic alterations in myelodysplastic syndrome (MDS), a disease characterized by refractory anemia, megakaryocyte dysplasia, and thrombocytosis. The thalidomide analogue lenalidomide (LEN) produces durable erythroid responses in ~60% of del(5q) MDS patients, including a majority of cytogenetic responses in which the del(5q) clone becomes undetectable in the bone marrow. Despite high response rates, clinical and cytogenetic relapse occur within 2-3 years. Mechanisms of clinical response, resistance and relapse with LEN therapy remain to be elucidated. The target of LEN has recently been identified as the cereblon (CRBN) component of the cullin 4 RING E3 ubiquitin ligase complex (CRL4-CRBN). Upon LEN binding, the substrate-specificity of the CRL4-CRBN complex is altered, and LEN-regulated substrates are beginning to be identified.An RNA interference screen was performed to identify genes/pathways that mediate LEN sensitivity and resistance in del(5q) MDS. The LEN-sensitive del(5q) MDS patient-derived cell line MDSL was screened with a genome-wide shRNA library (SBI GeneNet Human 50K Library) in the presence and absence of LEN treatment (0 and 10 μM) for 7 days. Three independent shRNAs targeting the proton-sensing G protein-coupled receptor 1 (GPR68 or OGR1) were among the most enriched shRNAs in LEN-treated cells, suggesting that loss of GPR68 expression conferred resistance to LEN. This finding was validated in MDSL cells, using an independent set of shRNAs. Conversely, a GPR68 agonist (N-cyclopropoyl-5-[thiophen-2-yl]-isoxazole-3-carboxamid) enhanced LEN-induced cytotoxicity to MDSL cells. GPR68 is a proton-sensing G-protein coupled receptor that stimulates inositol phosphate production and/or intracellular calcium (Ca2+) mobilization. Curiously, CRBN was originally identified as a binding protein of calcium-activated potassium channels. These data led us to hypothesize that Ca2+ signaling may be responsible for LEN-mediated cytotoxic effect in MDS cells. Reducing intracellular Ca2+ level with chelators reversed LEN’s cytotoxic effects, while increasing intracellular Ca2+ level with ionomycin enhanced LEN’s cytotoxic effect, indicating that intracellular Ca2+ levels determine cellular responsiveness to LEN. Although LEN did not induce an instant burst of Ca2+ influx, a gradual increase of basal intracellular free Ca2+ was observed following LEN treatment in LEN-sensitive cell lines and primary MDS marrow cells, but not in LEN-resistant cells, suggesting that LEN cytotoxicity was dependent on the cell’s ability to release Ca2+ from intracellular stores. GPR68 and CRBN were both necessary for the LEN-induced increase in Ca2+, as knockdown of GPR68 or CRBN in LEN-sensitive cells prevented the Ca2+increase.To identify the Ca2+-dependent signaling pathway responsible for mediating the cytotoxic effect of LEN, a panel of seven inhibitors that blocked mitochondrial/caspase-, calpain-, autophagy-, or lysosomal-dependent cell death pathways was tested in combination with LEN on MDSL cells. Only the inhibitor of calpains (PD150606) prevented LEN-induced cytotoxic effects in MDSL cells, indicating that calpain activation was necessary for mediating cell death in LEN-treated cells. Calpains are Ca2+-dependent cysteine proteases that can induce apoptotic and necrotic cell death by proteolytic cleavage of protein substrates. Calpastatin, the only endogenous calpain inhibitor, is localized to 5q15 and its expression is haploinsufficient in del(5q) MDS as compared to normal karyotype MDS. Taken together, our results show that LEN increased intracellular Ca2+ levels by a CRBN- and GPR68-dependent mechanism, leading to calpain-mediated cytotoxicity in del(5q) MDS cells. We propose a model in which haploinsufficient expression of calpastatin in del(5q) MDS sensitizes cells to cytotoxic effects of LEN. Further studies are required to identify the direct LEN-modulated substrates of CRBN that mediate this effect. DisclosuresOliva:Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria. MacBeth:Celgene: Employment, Equity Ownership. Starczynowski:Celgene: Research Funding.

Myelodysplastic Syndrome Marrow Stroma Shows Widespread Aberrant Hypermethylation That Is Abrogated By Treatment with Dnmt Inhibitors

The marrow microenvironment contributes to the pathogenesis of ineffective hematopoiesis in Myelodysplastic Syndromes (MDS). Since mutations and cytogenetic alterations seen in the hematopoietic compartment are generally not present in marrow stromal cells, we hypothesized that epigenetic alterations may be responsible for altered stroma function in MDS. To elucidate the epigenome of MDS microenvironment, we used the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay to study cytosine methylation patterns of 50,000 CpGs loci covering 14,000 promoters in primary stromal cells. Global DNA methylation of 6 MDS marrow-derived stroma was analyzed by HELP assay and compared to 3 healthy controls. MDS stroma showed aberrant hypermethylation compared with controls (3626 hypermethylated vs 306 hypomethylated loci in untreated MDS stromal cells) that preferentially occurred outside of CpG islands (Test of Proportions, P value <0.01) and involved important signaling pathways and binding sites for transcription factors (Sp1, NFY, MYB,). Aberrant genomic methylation was seen to be significantly enriched at chromosomal regions chr12q15, chr5q32, chr4q21, chr7q31, chr3q13, chr2p12 and chr8q24 when compared with the genomic distribution of all HpaII fragments from the HELP array (P value <0.05; MDSig Program). Comparison with stroma derived from 5-Azacytidine (5-Aza) treated MDS patients revealed abrogation of aberrant methylation in treated samples. Integrative expression analysis revealed that the WNT pathway was epigenetically dysregulated in MDS stroma, and the WNT antagonists FRZB and SFRP1 were aberrantly hypermethylated and underexpressed. These epigenetic changes were validated in a co-culture model of stroma (HS27) and leukemic cells (Kg1a) and analyzed at a single base pair resolution using next gen sequencing based HELP-Tagging assay. Epigenetic changes in MDS stroma involving the WNT antagonists FRZB/SFRP1 were also validated at the protein level by IHC in an independent set of MDS stroma samples .After demonstrating aberrant hypermethylation in MDS stroma, we next examined the functional role of these alterations by treatment with DNMT inhibitors. 5-Aza treatment of MDS stroma enhanced hematopoietic activity and erythroid differentiation from co-cultured healthy CD34+ cells. Significantly increased erythroid differentiation was seen in CD34+ cells cocultured with aza pretreated MDS stroma as shown by their glycophorin A positivity.Conclusions: These results reveal widespread aberrant epigenetic changes in the MDS marrow microenvironmentthat affect many important pathways. A recent report had shown that activation of beta-catenin in the bone marrow niche can trigger MDS/AML in vivo. Our results demonstrate that epigenetic silencing of WNT antagonists can potentially lead to activation of the WNT pathway in MDS stroma. Finally, we demonstrate that DNA methyl transferase inhibitors alter the epigenomic profiles of stromal cells and it raises the possibility that epigenetic modulation of the stroma by these drugs contributing to their therapeutic efficacy. DisclosuresNo relevant conflicts of interest to declare.

The KDM2B- let-7b -EZH2 axis in myelodysplastic syndromes as a target for combined epigenetic therapy.

Both DNA and histone methylation are dysregulated in the myelodysplastic syndromes (MDS). Based on preliminary data we hypothesized that dysregulated interactions of KDM2B, let-7b and EZH2 signals lead to an aberrant epigenetic landscape. Gene expression in CD34+ cells from MDS marrows was analyzed by NanoString miR array and validated by real-time polymerase chain reaction (PCR). The functions of KDM2B, let-7b and EZH2 were characterized in myeloid cell lines and in primary MDS cells. Let-7b levels were significantly higher, and KDM2B and EZH2 expression was lower in primary CD34+ MDS marrow cells (n = 44) than in healthy controls (n = 21; p<0.013, and p<0.0001, respectively). Overexpression of let-7b reduced EZH2 and KDM2B protein levels, and decreased cells in S-phase while increasing G0/G1 cells (p = 0.0005), accompanied by decreased H3K27me3 and cyclin D1. Silencing of KDM2B increased let-7b expression. Treatment with the cyclopentanyl analog of 3-deazaadenosine, DZNep, combined with the DNA hypomethylating agent 5-azacitidine, decreased levels of EZH2, suppressed methylation of di- and tri-methylated H3K27, and increased p16 expression, associated with cell proliferation. Thus, KDM2B, via let-7b/EZH2, promotes transcriptional repression. DZNep bypassed the inhibitory KDM2B/let-7b/EZH2 axis by preventing H3K27 methylation and reducing cell proliferation. DZNep might be able to enhance the therapeutic effects of DNA hypomethylating agents such as 5-azacitidine, currently considered standard therapy for patients with MDS.

Paraneoplastic inflammation in myelodysplastic syndrome or bone marrow failure: case series with focus on 5‐azacytidine and literature review

Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal disorders of haematopoietic stem cells, characterised by dysplastic haematopoiesis and dysregulated apoptosis resulting in various degrees of cytopenia, whereas canonical cytologic, cytogenetic and histopathologic findings guiding the diagnosis MDS are widely accepted, the MDS-phenotype can be masked by coexisting/paraneoplastic immunologic disease. Autoimmune disorders have an estimated incidence of 10% among patients suffering from MDS and are causally related to increased morbidity and mortality, younger age at diagnosis and more complex genetics. Conversely, systemic inflammatory disorders may be an early manifestation of MDS, show good response to immunosuppressive therapy and frequently disappear during the course of specific haematologic therapy. Monocentric report on clinical phenotypes found in MDS or bone marrow failure with paraneoplastic inflammatory disease. Clinical case reports and systematic review about MDS pathophysiology and treatment. We report eight patients diagnosed with MDS or bone marrow failure, who presented with paraneoplastic autoimmune diseases. Six of eight patients were treated with the hypomethylating agent 5-azacytidine, three of which achieved meaningful response with regard to inflammation control and haematologic recovery. As paraneoplastic syndromes are often mistakenly diagnosed as idiopathic autoimmune disorders, we propose that coexistence of an underlying myelodysplastic syndrome should be considered early in the diagnostic work up. 5-Azacytidine is effective in controlling paraneoplastic inflammation.

Deletion Of Tifab, a Novel Candidate Gene On Chromosome 5q, Results In Hematopoietic Defects By Changing The Dynamic Range Of Innate Immune Pathway Activation

Myelodysplastic syndromes (MDS) are defined by blood cytopenias due to ineffective hematopoiesis, and predisposition to acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic alteration in MDS is deletion of chromosome 5q (del(5q)). A search of annotated genes within or near the commonly-deleted regions (CDR) in del(5q) revealed a novel uncharacterized gene, TRAF-interacting protein with forkhead-associated domain B (TIFAB). TIFAB resides within the proximal CDR on band 5q31.1, and consistent with haploinsufficiency, expression of TIFAB mRNA is at least 50% lower in del(5q) MDS as compared to diploid 5q MDS and age-matched control marrow cells. Restoring TIFAB in human AML cell lines with low endogenous levels of TIFAB results in increased apoptosis, diminished proliferation, and impaired leukemic progenitor function, suggesting that it functions as a tumor suppressor. To investigate whether loss of TIFAB is important to the pathophysiology of del(5q) MDS/AML, we characterized a novel germline TIFAB knockout (KO) mouse for hematopoietic stem/progenitor cell (HSPC) function. Whole-body TIFAB KO mice do not exhibit changes in steady-state hematopoiesis even beyond 1 year of age. However, the consequences of 5-fluorouracil (5-FU) treatment on TIFAB KO mice were more severe as compared to wild type (WT)-treated mice, suggesting that HSPC function of TIFAB KO mice is affected following hematopoietic stress. To further investigate the consequences of TIFAB loss on HSPC function, TIFAB KO marrow cells were transplanted into WT syngeneic recipient mice. For 3 months post-BM transplant, WT and TIFAB KO-transplanted mice displayed similar blood and BM proportions. However, beyond 6 months post-BM transplantation, TIFAB KO mice displayed progressive hematopoietic defects, including skewed HSPC proportions, altered myeloid differentiation, and pancytopenia. Importantly, approximately 40% of mice transplanted with TIFAB KO BM cells developed a BMF-like disease associated with BM dysplasia and pancytopenia. Cell-intrinsic HSC defects in TIFAB KO mice were confirmed by performing competitive BM transplantations. In support of a HSC defect, TIFAB KO HSC were out-competed by co-transplanted WT HSC. To uncover the molecular consequences that explain the HSPC defects in TIFAB KO mice, we isolated Lin-cKit+Sca1+ (LSK) and performed a microarray analysis. TIFAB KO LSK exhibited an increase in HSC-, IFN-, and p53-related gene signatures and downregulation of LPS-stimulated gene signatures as compared to WT LSK. Despite a repression of an LPS-stimulated gene signature, TIFAB KO BM cells are hyper-sensitive to LPS stimulation, suggesting that loss of TIFAB alters the innate immune pathway. As a potential explanation for altered LPS sensitivity, TIFAB loss induces an increase in TRAF6 protein, a key modulator of the innate immune pathway. Taken together, our results provide evidence that TIFAB exhibits tumor suppressor-like functions and that deletion of TIFAB contributes to an MDS-like phenotype in mice by changing the dynamic range of the innate immune pathway reactivity in HSPC cells. Disclosures:No relevant conflicts of interest to declare.

Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow

Mesenchymal stromal cells (MSCs) support hematopoiesis and are cytogenetically and functionally abnormal in myelodysplastic syndrome (MDS), implying a possible pathophysiologic role in MDS and potential utility as a diagnostic or risk-stratifying tool. We have analyzed putative MSC markers and their relationship to CD34+ hematopoietic stem/progenitor cells (HSPCs) within intact human bone marrow in paraffin-embedded bone marrow core biopsies of benign, MDS and leukemic (AML) marrows using tissue microarrays to facilitate scanning, image analysis and quantitation. We found that CD271+, ALP+ MSCs formed an extensive branching perivascular, periosteal and parenchymal network. Nestin was brightly positive in capillary/arteriolar endothelium and occasional subendothelial cells, whereas CD146 was most brightly expressed in SMA+ vascular smooth muscle/pericytes. CD271+ MSCs were distinct by double immunofluorescence from CD163+ macrophages and were in close contact with but distinct from brightly nestin+ and from brightly CD146+ vascular elements. Double immunofluorescence revealed an intimate spatial relationship between CD34+ HSPCs and CD271+ MSCs; remarkably, 86% of CD34+ HSPCs were in direct contact with CD271+ MSCs across benign, MDS and AML marrows, predominantly in a perivascular distribution. Expression of the intercrine chemokine CXCL12 was strong in the vasculature in both benign and neoplastic marrow, but was also present in extravascular parenchymal cells, particularly in MDS specimens. We identified these parenchymal cells as MSCs by ALP/CXCL12 and CD271/CXCL12 double immunofluorescence. The area covered by CXCL12+ ALP+ MSCs was significantly greater in MDS compared with benign and AML marrow (P=0.021, Kruskal–Wallis test). The preservation of direct CD271+ MSC/CD34+ HSPC contact across benign and neoplastic marrow suggests a physiologically important role for the CD271+ MSC/CD34+ HSPC relationship and possible abnormal exposure of CD34+ HSPCs to increased MSC CXCL12 expression in MDS.

Highlights of This Issue

Lee and colleagues examined serial measurements of cholesterol, triglycerides, and glucose from patients randomized to interferon or temsirolimus in the Global Advanced Renal Cell Carcinoma Phase III Trial. Temsirolimus treatment was associated with larger mean increases in cholesterol (1.02 mmol/L; P &lt; 0.0001), triglycerides (0.32 mmol/L; P = 0.0008), and glucose (1.28 mmol/L; P &lt; 0.0001) compared with interferon, and improved survival (OS: HR 0.76, P = 0.02; PFS: HR = 0.70; P = 0.001). Cholesterol increase is a potential predictor for temsirolimus efficacy; these data have important implications for the design of other mTOR inhibitor studies.Erythropoiesis-stimulating agents (ESA) are widely used in treatment of myelodysplastic syndromes (MDS). Their efficacy is variable, and clinical parameters may only partially predict response. To refine prediction of response, Spinelli and colleagues set up a cytofluorimetric assay to evaluate in vitro activation of the erythropoietin signaling pathway in primary MDS marrow cell subpopulations. Erythropoietin in vitro was able to phosphorylate STAT5 in erythroid progenitors of MDS. This activation was significantly weaker than in progenitors from normal marrows, but correlated with clinical response. This test successfully identifies, prior to therapy, MDS patients who will respond to ESAs, thus avoiding noneffective treatments.GDC-0973 is a potent and selective MEK1 inhibitor currently in clinical trials. Wong and colleagues designed a series of preclinical studies to characterize GDC-0973 tumor disposition and pharmacodynamics in preclinical melanoma tumor models. PK–PD modeling was used to integrate preclinical and limited early clinical data in order to identify a minimum target plasma concentration in patients. Prospective predictions of active doses compared well with clinical responses. This work illustrates how PK–PD modeling can be used to improve the predictive value of preclinical data in the phase I setting.Demetri and colleagues present the complete longitudinal analyses, including final overall survival (OS), from the randomized, placebo-controlled, phase III study that led to regulatory approval of sunitinib for the treatment of patients with advanced imatinib-resistant/intolerant gastrointestinal stromal tumor. The confounding effect of the study's crossover design (allowing placebotreated patients to receive sunitinib upon disease progression) on OS was demonstrated: Conventional analyses resulted in biased comparisons favoring the control arm, despite a statistically significant difference in time to tumor progression. However, rank-preserving structural failure-time analysis modeling the absence of crossover estimated a substantial OS benefit for sunitinib versus placebo.

History of autoimmunity to predict clinical response to DNA methyltransferase inhibitors (DNMTI) in myelodysplastic syndromes (MDS), and MDS-derived acute myeloid leukemias (AML).

6629 Background: MDS is comprised of a heterogeneous group of clonal myeloid disorders. Chronic immune stimulation has been reported as a trigger for the development of a subset of MDS, with increased autoreactive cytotoxic T cells present in the bone marrow. Autoimmunity has been associated with MDS and other marrow failures. DNMTIs, 5-azacytidine and decitabine, are approved for the treatment of MDS. In addition to epigenetic impacts, these agents may have immunomodulatory effects, including augmentation of MAGE-related anti-tumor response. These reports and clinical observations led us to hypothesize that MDS patients with a history of autoimmunity may be more responsive to DNMTIs. Methods: To identify patients with MDS, a retrospective database review (2007-2011) was performed at Johns Hopkins Hospital (JHH) and Massachusetts General Hospital (MGH). The MGH data also included those with AML whose disease had progressed from MDS. Past medical history of autoimmune disorders, diagnosis, blood counts, flow cytometry and cytogenetics were reviewed. Patients with aplastic anemia, paroxysmal nocturnal hemoglobinuria or non-malignant etiologies of cytopenia were excluded. Patients with MDS were further studied if they were treated with DNMTI. Results: Of 137 patients with MDS or MDS/AML, 23 had a documented history of autoimmunity in the medical record. Of these, 15 (65.2%) experienced a response to therapy as defined by the International Working Group. Of 114 patients without a documented history of autoimmunity, 34 (29.8%) achieved a response during therapy, a significantly lower percentage as compared to those with autoimmune conditions (p-value 0.002, t-test). The majority of responding patients with a history of autoimmunity displayed a normal karyotype (9 of 15 patients). Conclusions: A history or co-presence of an autoimmune disorder may predict a high likelihood of achieving a clinical response to DNMTIs. Correlative studies in this unique population of patients with MDS and MDS/AML and prospective clinical studies are needed to improve our understanding of the possible mechanism of action of DNMTIs in these patients.

A systematic modeling study on the pathogenic role of p38 MAPK activation in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell diseases. In addition to intrinsic genetic alterations, the effects of the extrinsic microenvironment also play a pathological role in MDS development. The presence of increased inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), in marrow and abnormal activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in hematopoietic cells are associated with the ineffective hematopoiesis in MDS. However, the molecular mechanism of p38 MAPK activation triggered by microenvironment cytokines remains poorly understood. To address this question, we combined computational modeling analysis and molecular biology studies to perform a systematic investigation of signaling events regulated by microenvironment cytokines in hematopoietic cells from MDS patients. We examined dynamic changes of key signaling events, including the p38 MAPK and the c-Jun N-terminal kinase (JNK) pathway in bone marrow mononuclear cells from MDS patients or normal donors in response to TNF-α stimulation using reverse phase protein array technology. The results were analyzed by a novel computational model and preliminarily validated by immunohistochemistry analysis of the bone marrow tissues from twelve MDS patients and normal donors. Our systematic model revealed that the dynamic response patterns of p38 MAPK and JNK to TNF-α stimulation in MDS were different from that observed in normal marrow cells. Particularly, B-cell lymphoma-X (BCL-XL) protein degradation was regulated by the JNK pathway in normal cells, but by p38 MAPK in MDS cells. By immunohistochemistry, BCL-XL was highly expressed in hematopoietic cells from normal marrow, but was minimally expressed in MDS marrow. Additionally, immunostaining for phosphorylated p38 MAPKα showed much higher p38 MAPK activation in MDS marrows, supporting over-activation of p38 MAPK-enhanced degradation of BCL-XL in MDS. The degradation of BCL-XL triggered by p38 MAPK over-activation may contribute to the increasing apoptosis of marrow cells, a phenomenon commonly observed in MDS, and lead to ineffective hematopoiesis. Our study suggests that the combination of molecular biological studies and systematic modeling is a powerful tool for comprehensive investigation of the complex cellular mechanisms involved in MDS pathogenesis.