Myelodysplastic Syndromes Progenitor Cells Research Articles

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Deep immunophenotypic analysis of the bone marrow progenitor cells in myelodysplastic syndromes

BackgroundDiagnosis of myelodysplastic syndromes (MDS) is often challenging and requires integration of clinical, morphologic, cytogenetics and molecular information. Flow cytometry immunophenotyping (FCIP) can support the diagnosis by demonstration of numerical and immunophenotypic abnormalities of progenitor and maturing myelomonocytic and erythroid populations. We have previously shown that comprehensive immunophenotypic analysis of the progenitor population is valuable in the diagnosis of MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). This study was designed to improve the analysis method and confirm its value in a larger cohort of patients. MethodsFCIP of bone marrow samples from 105 patients with cytopenia(s) (with or without leukocytosis) and clinical concern for MDS or MDS/MPN was performed using a single-tube/10-color/13-marker assay. A modified analysis approach was used to obtain 11 progenitor parameters and 2 myelomonocytic parameters. ResultsSignificantly higher number of abnormalities were identified in MDS and MDS/MPN cases when compared to cytopenic patients not meeting the diagnostic criteria for MDS (Non-MDS). A FCIP score that combined the 13 parameters showed a sensitivity of 89.8% and specificity of 93.5% for the diagnosis of MDS and MDS/MPN. The sensitivity was 100% for both MDS/MPN and higher-risk MDS, and 81.3% for lower-risk MDS. ConclusionThis study confirms that detailed immunophenotypic analysis of the progenitor population is powerful in the diagnosis of MDS and MDS/MPN. The combination of markers used in the panel allowed for evaluation of two relatively new parameters, namely myeloid progenitor heterogeneity and stem cell aberrancy, which improved the sensitivity of the assay for lower-risk MDS.

Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

PLAGL2 Independently Drives Aberrant Erythropoiesis and Initiation of Preleukemic State

BackgroundThe identification and understanding of early drivers in malignancy is crucial to prevent or revert preleukemic events. Del(20q) is one of the most common primary cytogenetic abnormalities found in preleukemic malignancies from myeloproliferative neoplasms to myelodysplastic syndrome (MDS). Previous studies have identified a “common retained region” within 20q11.21 that is often amplified in a subset of MDS patients. PLAGL2 is one of the 4 genes identified to be within the minimally conserved amplified region. (MacKinnon et al. 2010) Indeed, in previously published datasets of MDS hematopoietic stem and progenitor cells (HSPCs) transcriptome, PLAGL2 is significantly elevated in del(20q) patients compared to healthy controls. However, we have found that its level is also higher in HSPCs of cytogenetically normal MDS patients with low blasts. Given these findings, we sought to define the role of PLAGL2 as a potential early driver of myeloid malignancies.ResultsIn healthy cord blood (CB) HSPCs, PLAGL2 overexpression enhanced proliferation ex vivo, better maintained stemness and decreased apoptosis. Colony formation assays also identified increased output of the erythroid lineage. Xenotransplanted CB CD34+ HSPCs overexpressing PLAGL2 exhibited increased engraftment competitiveness and led to splenomegaly with signs of hypercellularity after 20 weeks, features consistent with clinical observations of hematological malignancy. Grafts derived from PLAGL2 overexpressing cells reproducibly maintained a significantly larger CD34+ HSPC compartment. Intriguingly we also identified that ~50% of PLAGL2-overexpressing grafts exhibited a significant erythroid (CD71+/CD235a+) component where none was observed in the control group. This unique finding of aberrant erythropoiesis is reminiscent of clinical observations in patients with 20q11.21 amplification, where a high proportion of erythroblasts in the marrow and in some cases progression to erythroleukemia was noted. To evaluate the progression of PLAGL2-overexpressing grafts, further secondary transplantations were carried out and showed the persistence of only immature erythroid progenitors (CD71+/CD235a-) coupled with a near complete absence of lymphopoiesis in the same grafts. Together, our data strongly suggests ectopic levels of PLAGL2 can independently drive the expansion of human HSPCs and enforce features of myeloid malignancy.To uncover the molecular mechanism underlying PLAGL2 function, we performed RNA-seq and Cut&Run in human CB CD34+ HSPCs overexpressing PLAGL2. Geneset enrichment analysis of the transcriptome and over-representation analysis of bound genes both identified signatures consistent with LSCs. We compared these findings with identically-derived omics profiles of HSPCs overexpressing PLAG1, a closely related family member that our lab has identified as a potent expander of HSCs ex vivo but not capable of promoting malignant features. We found a strong common feature in the downregulation of ribosomal components and translation machinery, then functionally validated reduced protein synthesis in PLAGL2 overexpressing HSPCs through OP-Puro assays. We have shown dampened mRNA translation to be one of the mechanisms by which PLAG1 enhances stemness and survival of HSCs, one that potentially extends to PLAGL2 as well. However, we also identified discordant signatures, notably PLAGL2's unique capacity to reduce mitochondrial translation, a pathway associated with ineffective erythropoiesis and MDS and one that we are currently exploring as a means by which PLAGL2 can enforce malignant phenotypes.Finally, to investigate the potential of PLAGL2 as a therapeutic target in MDS, we performed shRNA knockdown in MDSL, a human MDS cell line. In vitro competitive assays with mixed wildtype cells showed steady dropout of PLAGL2 depleted cells. Currently, we are continuing to purse the dependence of primary MDS cells on PLAGL2 through in vivo xenograft models.ConclusionWe have identified PLAGL2's potential as an early independent driver of myeloid malignancy and aberrant erythroid differentiation. An understanding of PLAGL2 and its downstream mechanisms will not only further our understanding on the development of early myeloid malignancies but also potentially provide another avenue to treat or prevent leukemia before it manifests. DisclosuresDick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

PI3-Kinase Deletion Dysregulates Autophagy in HSCs and Promotes Myelodysplasia

▪Myelodysplastic Syndrome (MDS) is a heterogeneous clonal malignancy arising in hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis, cytopenias, and the potential to progress to acute myeloid leukemia (AML). However, the perturbations in HSCs that lead to MDS initiation are poorly understood. It has been reported that HSCs are particularly dependent on autophagy for the maintenance of differentiation and self-renewal. We observed that, compared to healthy donor bone marrow hematopoietic stem and progenitor cells (HSPCs), MDS patient stem and progenitor cells (Lin-CD33-CD34+CD38-) have abnormal levels of autophagic degradation, as demonstrated by abnormal intracellular LC3II and P62 staining (Figure 1A). Autophagy is known to be regulated by the (PI3K)/AKT pathway, which transduces hematopoietic growth factor and cytokine signals in HSCs. PI3K/AKT is frequently activated in AML, but its role in MDS is less clear. Surprisingly, we found that CD34+ cells from a subset of MDS patients have upregulated expression of PTEN, the major negative regulator of the PI3K/AKT pathway, suggesting that PI3K/AKT may be downregulated in MDS stem cells. Therefore, we hypothesized that the Class IA PI3K isoforms (P110α, β, and δ) are required to maintain HSC differentiation and self-renewal.To understand the consequences of PI3K downregulation in HSCs, we generated a triple knockout (TKO) mouse model with conditional deletion of P110α and P110β in hematopoietic cells, and germline deletion of P110δ. Surprisingly, we found that PI3K deletion causes transplantable pancytopenia and decreased survival, despite the abnormal expansion of donor TKO HSCs (Figure 1 B,C). Consistent with this inefficient hematopoiesis, TKO bone marrow cells exhibited dysplastic features in multiple blood lineages and multiple chromosomal abnormalities (Figure 1 E,F), suggesting that PI3K inactivation in HSCs can promote MDS initiation. To determine whether impaired HSC differentiation in TKO mice could be due to dysregulated autophagy, we assessed autophagy in TKO HSCs by flow cytometry and immunofluorescence with the autophagosomal marker, LC3II. Our results showed that, compared to the WT controls, TKO HSCs have inefficient autophagic flux and decreased degradation of the cargo protein P62. We also discovered that TKO HSCs have significantly enlarged autophagic vesicles (Figure 1 G), and impaired fusion of autophagosomes with lysosomes, consistent with a marked defect in autophagic degradation. Treatment of TKO mice with two pharmacologic inducers of autophagy, rapamycin or metformin, improved HSC differentiation with an increase in Flk2+ MPPs (Figure 1 H), reduced dysplasia, and decreased the size of the TKO mutant clone in chimeric mice. Thus, our results uncover an important role for PI3K in regulating autophagy in HSCs to maintain the proper balance between self-renewal and differentiation. Our new mouse model of MDS will be a useful tool to study the mechanisms of MDs initiation. In addition, our findings open exciting avenues for future investigations of autophagy-inducing agents in MDS. [Display omitted] DisclosuresVerma: Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company; Acceleron: Consultancy; Novartis: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Curis: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; BMS: Research Funding; GSK: Research Funding. Gritsman: iOnctura: Research Funding.

Unique Metabolic Vulnerabilities of Myelodysplastic Syndrome Stem Cells

Background: The mechanisms that cause the progression of myelodysplastic syndrome (MDS) are poorly understood. Little is known about major signaling networks and energy metabolism in MDS cells as patients progress from low risk (LR) to high risk (HR) disease and from high risk to secondary acute myelogenous leukemia (sAML). As many as 30% of HR MDS patients progress to sAML and a portion of LR MDS patients progress to HR. The goal of this project is preventing progression by identifying MDS-specific targets for therapy. A deeper understanding of the metabolic properties of leukemia stem cells (LSCs) in AML has shown these cells are uniquely vulnerable to venetoclax and azacitidine (Ven/Aza) (Pollyea et al, Nat. Medicine, 2018) and metabolic changes cause resistance to Ven/Aza (Stevens et al, Nat. Cancer, 2021). Little however is known about the contribution of metabolism to the pathogenesis of MDS. The contributing factors to progression including metabolic properties, transcriptional programs, and immunophenotype are examined in this study.Methods: Bone marrow specimens from MDS patients at various disease stages, including serial samples during progression, were obtained. Single cell techniques including mass cytometry, antibody based single cell RNA sequencing (CITE-Seq) and transcriptional profiling with RNA sequencing were used to elucidate novel mechanisms of progression. Selective targeting of primitive MDS cells was tested using several agents.Results: Our previous work characterizing MDS stem cells (MDSC) showed significant similarities between MDSCs and AML LSCs (Stevens et al, Nat. Communications, 2018). However, little is known about lower risk disease. In order to understand transcriptional changes and their relationship to metabolism across pathogenesis, the transcriptome of blasts from patients with LR, intermediate (INT), or HR IPSS scores was investigated. The first major transcriptional difference identified was enrichment of glycolysis pathway at LR and INT stage. In contrast, HR MDS demonstrated enrichment of oxidative phosphorylation. Furthermore, comparison of intermediate to HR MDS showed increased RNA polymerase and Ribosome pathways at the HR stage. These changes demonstrate the progressive alteration of metabolic properties during MDS pathogenesis with cells first relying on mechanisms associated with normal stem cells (i.e. glycolysis) and later transitioning to a state associated with AML stem cells (i.e. reliance on oxidative phosphorylation).Using serial specimens of patients of who progressed from LR to HR MDS we performed CITE-Seq and mass cytometry. CITE-seq in serial specimens showed up-regulation of protein translation and oxidative phosphorylation in a subset of MDS stem and progenitor cells (CD34+ at transcript and antibody level) present at LR stage and conserved at HR stage (Fig 1A-C). MDSCs also acquired surface antigens including CD99 and CD52 upon progression from LR to HR. Analysis of the mass cytometry data showed significant overlap with CITE-Seq data including increased CD123+ and MCL1 expression in MDS stem cells upon progression.In order to understand therapeutic vulnerabilities as they relate to progression, we investigated ex vivo drug response in LR and HR specimens. MDS samples were challenged with two regimens, Ven/Aza, a regimen known to inhibit OXPHOS; and omacetaxine and azacitidine (Oma/Aza), which inhibits translation. CITE-seq showed that MDSC were selectively sensitive to these agents (Fig 1D). Importantly, addition of either drug regimen caused ablation of MDSC at LR and HR stages and these changes were most profound in cells with LSC properties.Based on preclinical findings, we are investigating MDS patients treated with Ven/Aza or Oma/Aza via CITE-seq and metabolomics for correlation of clinical response with properties of MDSC. Preliminary studies show that patients that respond to Oma/Aza present with a population of MDSC with transcriptional signatures of protein translation and LSCs (Fig. 1E). Studies are underway to investigate overlapping properties of ven/aza resistance in AML to resistance in MDS specifically investigating fatty acid metabolism in MDSC.Conclusions: Analysis of MDS patient bone marrow reveals acquisition of aberrant metabolic properties at both low and high risk stages of disease. These distinct aspects of MDSC biology create unique and targetable features. [Display omitted] DisclosuresPollyea: Genentech: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Syndax: Consultancy; Takeda: Consultancy; Daiichi Sankyo: Consultancy; Celgene/BMS: Consultancy; Amgen: Consultancy; AbbVie: Consultancy, Research Funding; Agios: Consultancy; Glycomimetics: Other.

Adaptive response to inflammation contributes to sustained myelopoiesis and confers a competitive advantage in myelodysplastic syndrome HSCs.

Despite evidence of chronic inflammation in myelodysplastic syndrome (MDS) and cell-intrinsic dysregulation of Toll-like receptor (TLR) signaling in MDS hematopoietic stem and progenitor cells (HSPCs), the mechanisms responsible for the competitive advantage of MDS HSPCs in an inflammatory milieu over normal HSPCs remain poorly defined. Here, we found that chronic inflammation was a determinant for the competitive advantage of MDS HSPCs and for disease progression. The cell-intrinsic response of MDS HSPCs, which involves signaling through the noncanonical NF-κB pathway, protected these cells from chronic inflammation as compared to normal HSPCs. In response to inflammation, MDS HSPCs switched from canonical to noncanonical NF-κB signaling, a process that was dependent on TLR-TRAF6-mediated activation of A20. The competitive advantage of TLR-TRAF6-primed HSPCs could be restored by deletion of A20 or inhibition of the noncanonical NF-κB pathway. These findings uncover the mechanistic basis for the clonal dominance of MDS HSPCs and indicate that interfering with noncanonical NF-κB signaling could prevent MDS progression.

Oxidized Mitochondrial DNA Engages TLR9 to Activate the NLRP3 Inflammasome in Myelodysplastic Syndromes

Myelodysplastic Syndromes (MDS) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, Nlrp3 inflammasome (IFM) driven pyroptotic cell death and the induction of interferon-stimulated genes (ISG). Toll-like receptor 9 (TLR9) is an endosomal, DNA sensing pattern recognition receptor that primes and activates the IFM and ISG response through myddosome signaling upon engagement by hypomethylated, CpG-rich DNA. Oxidized newly synthesized mitochondrial DNA (ox-mtDNA) is released into the cytosol upon TLR/IL-1R activation to trigger Nlrp3 IFM activation. Upon lytic pyroptotic cell death, however, ox-mtDNA is released into the extracellular space. We previously reported that concentrations of ox-mtDNA, a native TLR9 ligand, are profoundly increased in MDS patient plasma compared to age-matched controls and other hematologic malignancies (Ward G, et. al. ASH 2018). The aim of this investigation was to determine if ox-mtDNA acts as a danger associated molecular pattern (DAMP) to propagate the inflammatory response and IFM activation in neighboring cells through TLR9. We have shown that MDS hematopoietic stem and progenitor cells (HSPC) redistribute TLR9 to display cell surface TLR9 expression. We hypothesized that this increased surface expression is induced in response to ox-mtDNA in the BM plasma. To test this, SKM1 and U937 cells were incubated for 2 hours with 50ng/mL ox-mtDNA (ND1 gene, unmethylated, amplified with oxidized guanosine) and TLR9 expression was assessed by flow cytometry (FC). Following incubation, both cell lines significantly increased TLR9 surface expression (n=3, p<0.03).We next confirmed that TLR9 interacts with ox-mtDNA by immunofluorescence (IF) microscopy in MDS samples and murine somatic gene mutation (SGM) models (Tet2-/- and Srsf2P95H). We further investigated the relationship between TLR9 and ox-mtDNA during IFM activation. Treatment of SKM1 and U937 cells with the TLR4 ligand LPS and priming agents ATP/nigericin initiates a robust increase in ox-mtDNA that co-localized intracellularly with TLR9 by IF microscopy. Furthermore, upon incubation with ox-mtDNA, ox-mtDNA bound TLR9 is internalized. Additionally, interferon regulatory factor 7 (IRF7), an ISG transcription factor induced by TLR9, translocates to the nucleus following treatment, confirming TLR9 activation in response to ox-mtDNA. We next assessed whether IFM activation by ox-mtDNA is TLR9-dependent. 50ng/mL ox-mtDNA was incubated with 3 leukemic cell lines: SKM1, U937, and THP1 that display varying levels of endogenous TLR9 expression, as well as corresponding TLR9-KO cells generated by CRISPR-Cas9 editing. We found that TLR9 was necessary for IFM initiated caspase-1 cleavage in response to ox-mtDNA, thereby demonstrating the specificity of ox-mtDNA for the TLR9 sensor. Notably, receptor density determined response tempo. SKM1 cells, which have the highest TLR9 receptor density, responded within 1 hour, U937 cells which have an intermediate receptor density respond in 2 hours, while THP1 cells, which do not express endogenous TLR9 never responded to ox-mtDNA treatment. TLR9 knockout abrogated IFM activation in response to ox-mtDNA as demonstrated by caspase-1 glo ® assay (n=5, p<0.03). By western blot, TLR9 knockout cells no longer demonstrate phosphorylated NFk-B, cleaved caspase-1, and cleaved IL-1β in response to ox-mtDNA treatment. We conclude that MDS HSPC display functionally competent TLR9 on the plasma membrane which primes them for response to ox-mtDNA released by neighboring pyroptotic cells. Blocking TLR9 activation may prove to be a novel therapeutic strategy for MDS and suppression of inflammatory BM failure. Disclosures Hou: Celgene: Research Funding; Abbvie, Astellas, BMS, Celgene, Chugai, Daiichi Sankyo, IQVIA, Johnson & Johnson, Kirin, Merck Sharp & Dohme, Novartis, Pfizer, PharmaEssential, Roche, Takeda: Honoraria. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The NLRP3 Inflammasome as a Driver of the MDS Phenotype

Myelodysplastic syndromes (MDS) share features of cytological dysplasia and ineffective hematopoiesis while demonstrating profound molecular and genetic heterogeneity. Our investigations show that many of the hallmark features of MDS arise from activation of the NLRP3 inflammasome that directs clonal expansion and pyroptotic cell death, a caspase-1–dependent pro-inflammatory, lytic form of cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPC) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct the activation of caspase-1, IL-1β and IL-18 generation, and pyroptosis. NLRP3 is a redox-sensitive, cytosolic sensors of danger signals, which upon stimulation, undergoes a conformational change fostering homotypic pyrin domain interaction with the adaptor protein ASC [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD)] and ASC polymerization to create large, cytoplasmic filaments. The ASC speck formed by ASC filament clusters creates abundant caspase-1 activation sites, thereby acting as an inflammasome signal amplification complex. Mechanistically, pyroptosis is triggered by the alarmin S100A9 found in excess in MDS HSPC, myeloid-derived suppressor cells (MDSC) and bone marrow plasma, which like founder gene mutations, induces reactive oxygen species (ROS) to initiate cation influx, cell swelling and β-catenin activation. Accordingly, knockdown of caspase-1, neutralization of S100A9 in BM plasma, and pharmacologic inhibition of the NLRP3 inflammasome or NADPH oxidase suppresses pyroptosis, ROS generation and nuclear β-catenin in MDS HSPC and restores effective hematopoiesis. NLRP3 inflammasome activation appears specific to MDS compared to other bone marrow malignancies, with ASC specks detected and quantified in the peripheral blood providing an index of ineffective hematopoiesis and pyroptotic cell death in MDS patients These data indicate that DAMP signals and oncogenic mutations in MDS HSPC license a common redox-sensitive inflammasome platform, and that targeted disruption of the inflammasome signal can restore effective hematopoiesis.

The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype

AbstractDespite genetic heterogeneity, myelodysplastic syndromes (MDSs) share features of cytological dysplasia and ineffective hematopoiesis. We report that a hallmark of MDSs is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct activation of caspase-1, generation of interleukin-1β (IL-1β) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX), increasing levels of reactive oxygen species (ROS) that initiate cation influx, cell swelling, and β-catenin activation. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear β-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention.

TNF-Alpha Promotes Clonal Expansion of Tet2 Deficient Bone Marrow Progenitors in MDS

Background: Recent studies suggest that pro-inflammatory cytokines such as TNF-α are increased in myelodysplastic syndromes (MDS), but their contribution to disease pathogenesis is unknown. Here we uncover a mechanistic role of TNF-α in promoting clonal dominance of Tet2 murine mutant haematopoietic stem and progenitor cells (HSPCs). We hypothesized that Tet2 mutation induces TNFα expression and confers TNFα resistance to MDS progenitor cells by mechanisms involving the suppression of apoptotic cues. Methods: We isolated lineage negative cells (Lin-), enriched for HSPCs, from the bone marrow(BM) of 10-14 weeks old Tet2wild(+/+) type and mutant (-/-) C57BL/6 mice strain, (EasySep; StemCell Technologies) and cultured these +/- TNFα (0.1, 1, or 10 ng/ml) and or anti-TNF antibody (1 or 10 ug/ml) in a colony formation assay (MethoCult; StemCell) and then examined their colony growth, immunophenotypic characteristics and apoptosis over a period of 12 days. Where indicated serial re-plating was performed. Expression of apoptotic regulators was assessed by qRT-PCR. Results: In our initial triplicate experiments, starting with equal amount of Tet2 +/+ and -/- Lin- cells (104 cells/MethoCult well), cultured in appropriate growth factors and graded concentrations of TNFα , we observed that Tet2 -/- Lin- cells displayed superior colony forming ability over +/+ in serial re-plating assays under stress of increasing TNFα. To further confirm these initial results, we cultured the cells in-vitro Blocking Assays. We observed that in-vitro blockade of TNF-α with anti-TNF antibody, significantly limits the colony forming superiority of re-plated Tet2 -/- progenitors over +/+ in a dose dependent manner. (P<0.05). Immunophenotypic analysis of the colonies after replating showed significantly increased proportion of Mac1+Gr1+ and Sca1+Kit1+ populations in Tet2-/-, as compared to +/+, in the presence of TNF-α (P<0.05). These findings suggest that deregulation of innate and inflammatory cytokine signaling may provide a permissive environment for clonal dominance of Tet2 mutant HSPCs with an associated myeloid bias. To address the mechanisms behind these observations, we examined the susceptibility of both wild type and -/- cells to apoptosis using an annexin V- propidium iodide flow assay and then measured their apoptotic index. Our results showed that Tet2 -/- HSPCs have a lower apoptotic index compared to +/+ under stress of increasing TNF-α, suggesting that Tet2-/- BM cells may be resistant to TNFα induced apoptosis. Next, we searched for components of TNFα signaling pathway that might have been altered in resistant Tet2 -/- clones. Specifically, we measured the fold change in mRNA expression levels (using SYBR green qRT-PCR method) of some pro-apoptotic (TNFR1 and II, Fas-R, caspase 3 and 8) and anti-apoptotic genes (BCL-2, BCL-XL, IAP1, IAP2,) in both +/+ and -/- progenitor cells that have been cultured +/-TNF-α. Our results showed that mRNA levels of TNFR1, Fas receptors and caspase 8 were significantly lower in Tet2 -/- cells, compared to +/+ under inflammatory stress of TNF-α (P<0.05). In contrast, the mRNA levels of BCL2, IAP1 and IAP2 were significantly elevated in Tet2 -/- compared to +/+ (P<0.05). We also measured TNFα mRNA expression levels. Our preliminary data showed increased expression of TNF α mRNA in Tet2 -/-progenitors, compared to +/+ in absence of TNF-α (P<0.05). We also examined colony growths of TET2 mutant and non-mutant human MDS under varying TNF-α concentrations, using frozen BM mononuclear cells (BMMNCs) from MDS patients and normal healthy controls. Colony formation at 0.0 ng/ml TNFα was normalised to 100%. Our preliminary data showed that BFU-E colonies in TET2 mutant MDS show relatively increased survival at high TNFα concentrations, compared to wild type and controls, while CFU-GM colony formation in Tet2 mutant MDS was enhanced by low TNFα concentrations (P< 0.05). We are currently examining gene signatures in these groups of patients. Conclusion: Our data introduce a model where Tet2 mutation promotes clonal dominance by conferring TNFα resistance to TNFα sensitive progenitors, while also generating a TNFα rich environment. Mutations that promote resistance to environmental stem cell stressors are a known mechanism of clonal selection in aplastic anaemia and JAK2-mutant MPN and our findings suggest that this mechanism may be critical to clonal selection and dominance in MDS. Disclosures No relevant conflicts of interest to declare.

Myelodysplastic syndromes: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

P. Fenaux1, D. Haase2, G. F. Sanz3, V. Santini4 & C. Buske5 on behalf of the ESMO Guidelines Working Group* Service d’Hematologie Clinique, Groupe Francophone des Myelodysplasies (GFM), Hopital St Louis (Assistance Publique, Hopitaux de Paris) and Paris 7 University, Paris, France; Clinics of Hematology and Medical Oncology, University Medicine, Goettingen, Germany; Department of Haematology, Hospital Universitario y Politecnico La Fe, Valencia, Spain; Functional Unit of Haematology, AOU Careggi, University of Florence, Firenze, Italy; Comprehensive Cancer Center Ulm, Institute of Experimental Cancer Research, University Hospital, Ulm, Germany

Signal transduction inhibitors in treatment of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of hematologic disorders characterized by ineffective hematopoiesis that results in reduced blood counts. Although MDS can transform into leukemia, most of the morbidity experienced by these patients is due to chronically low blood counts. Conventional cytotoxic agents used to treat MDS have yielded some encouraging results but are characterized by many adverse effects in the predominantly elderly patient population. Targeted interventions aimed at reversing the bone marrow failure and increasing the peripheral blood counts would be advantageous in this cohort of patients. Studies have demonstrated over-activated signaling of myelo-suppressive cytokines such as TGF-β, TNF-α and Interferons in MDS hematopoietic stem cells. Targeting these signaling cascades could be potentially therapeutic in MDS. The p38 MAP kinase pathway, which is constitutively activated in MDS, is an example of cytokine stimulated kinase that promotes aberrant apoptosis of stem and progenitor cells in MDS. ARRY-614 and SCIO-469 are p38 MAPK inhibitors that have been used in clinical trials and have shown activity in a subset of MDS patients. TGF-β signaling has been therapeutically targeted by small molecule inhibitor of the TGF-β receptor kinase, LY-2157299, with encouraging preclinical results. Apart from TGF-β receptor kinase inhibition, members of TGF-β super family and BMP ligands have also been targeted by ligand trap compounds like Sotatercept (ACE-011) and ACE-536. The multikinase inhibitor, ON-01910.Na (Rigosertib) has demonstrated early signs of efficacy in reducing the percentage of leukemic blasts and is in advanced stages of clinical testing. Temsirolimus, Deforolimus and other mTOR inhibitors are being tested in clinical trials and have shown preclinical efficacy in CMML. EGF receptor inhibitors, Erlotinib and Gefitinib have shown efficacy in small trials that may be related to off target effects. Cell cycle regulator inhibitors such as Farnesyl transferase inhibitors (Tipifarnib, Lonafarnib) and MEK inhibitor (GSK1120212) have shown acceptable toxicity profiles in small studies and efforts are underway to select mutational subgroups of MDS and AML that may benefit from these inhibitors. Altogether, these studies show that targeting various signal transduction pathways that regulate hematopoiesis offers promising therapeutic potential in this disease. Future studies in combination with high resolution correlative studies will clarify the subgroup specific efficacies of these agents.

Durable erythroid response after discontinuation of epoetin-alpha: an unexpected outcome in a patient with myelodysplastic syndrome.

Dear Sir, Human recombinant erythropoietin (Hu-Epo) has enabled significant advances in the management of anaemic patients with myelodysplastic syndrome (MDS)1. Hu-Epo provides erythroid precursors protection from apoptosis and stimulates the residual normal haematopoiesis2,3, although possible effects on abnormal clones have also been reported4,5. An excessive erythroid response may be observed in some patients who are usually managed by an appropriate dose reduction of the Hu-Epo and/or by an extended interval of administration. However, we recently observed an erythroid response that was maintained for up to several months despite therapy discontinuation. This extremely rare phenomenon occurred in a 76-year old man who was admitted to hospital in July 2011 because of severe malaise and fatigue. On admission, besides the severe macrocytic anaemia (haemoglobin [Hb] =5.2 g/dL) with decreased reticulocyte count requiring the transfusion of six units of red blood cells; the neutrophil count (950/μL) and platelet count (123,000/μL) were also slightly reduced. The bone marrow was hypercellular with erythrodysplasia and 2% of blasts but no dysgranulopoietic and/or dysmegakaryocytic features were observed. All other causes of anaemia were carefully ruled out; the serum level of endogenous erythropoietin was in the normal range. Therefore, a diagnosis of MDS, subtype refractory anaemia, was made. Conventional cytogenetic and fluorescence in situ hybridization analyses identified a 20q deletion in 80% of metaphases. According to the International Prognostic Scoring System and the World Health Organization organisation-based Prognostic Scoring System, the MDS risk was classified as Intermediate-1 and very low risk, respectively. The patient received epoetin-α 40,000 IU twice a week and was followed up regularly. After 2 weeks, a major erythroid response was achieved so the dose of epoetin-α was reduced to 40,000 IU once a week. After 4 weeks, a complete haematological recovery was recorded: in particular, the patient’s Hb level was 12.5 g/dL and he had normal platelet and neutrophil counts, so the interval of administration of epoetin-α was extended to 2 weeks. After 8 weeks the haemoglobin level was 15.4 g/dL and the epoetin-α was withdrawn. Thereafter, he maintained near-normal Hb levels, ranging from 12.4 and 13.5 g/dL, without any therapeutic intervention. At present, 11 months after discontinuation of epoetin-α, the patient’s Hb level is 13.2 g/dL, although he continues to show the same bone marrow findings detected at disease onset, such as erythrodysplasia and the original clonal cytogenetic alteration in 50% of metaphases. In daily clinical practice it is very rare that patients responding to Hu-Epo have a prolonged normalisation of peripheral blood counts, without any further therapy, once treatment has been discontinued because of an excessive therapeutic response. To the best of our knowledge, only one case of MDS with a similar outcome after treatment with Hu-Epo and human granulocyte-macrophage colony-stimulating factor has been reported so far5. A possible interpretation of our findings is that the Hu-Epo may have enhanced erythropoieis both through maturation of MDS progenitor cells as well as through the reduction of karyotypically abnormal clones5 and the expansion of a non-clonal erythroid matrix2,3. The cytogenetic mosaicism in this case allows us to postulate that the therapy with Hu-Epo expanded the polyclonal erythropoiesis at the expense of the very low malignant clonal erythropoiesis. In conclusion, in our low-risk MDS patient a short course of Hu-Epo at high doses appears to have exerted, through unknown mechanisms, some durable effects on the processes of haematopoiesis and/or apoptosis restoring normal erythropoiesis in spite of the discontinuation of epoetin-α.

Cytogenetic Characteristic of Mesenchymal and Hematopoetic Progenitor Cells in Myelodysplastic Syndromes and Acute Myeloid Leukemias with Myelodysplasia-Related Changes

Abstract 4899 Background:Abnormal karyotype in bone marrow (BM) cells is detected in 30–70% of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias with myelodysplasia-related changes (AML). The same cytogenetic abnormalities are found in isolated CD34+ hematopoetic progenitor cells (HPCs) of these patients. According to recent studies cytogenetic abnormalities are described in 10–70% MDS and AML patients in BM derived mesenchymal stromal cells (MSCs) [Flores-Figueroa et al. 2008, Blau et al. 2011]. These abnormalities can be clonal and nonclonal (spontaneous). The goal of the study was to characterize and compare the cytogenetic changes in BM derived MSCs and in CD34+ HPCs isolated from BM and peripheral blood (PB) in MDS and AML patients. Patients and methods:The data from 35 patients is presented: 6 pts with AML (1 pt with therapy-related AML), 29 pts with MDS (refractory anemia − 4 pts, refractory cytopenia with multilineage dysplasia − 13 pts, refractory anemia with ringed sideroblasts − 2 pts, refractory anemia with excess blasts − 7 pts, 5q-syndrome – 3 pts). Median age was 60 years (range 19 to 77). Patients’ assignment to different groups was made according to the 2008 World Health Organization (WHO) classification. Cytogenetic analysis was performed using G-banding and FISH. MSCs were derived from bone marrow mononuclear cells then plated in 75 cm3flacks and harvested after 2–5 passages. CD34+ HPCs were collected from BM and PB by magnetic separation with anti-CD34 MicroBead Kit human (Miltenyi Biotec). Results:BM karyotype was normal in 17 pts, cytogenetic abnormalities were found in 18 pts − 4 AML and 14 MDS: in 9 pts (2 AML/7 MDS) – isolated del(5q), monosomy 7, i(14), inv(3) or trisomy 8; in 8 pts (2 AMl/6 MDS) – two or more abnormalities; and 1 pt displayed only constitutional abnormality (inv(9)(p13q21)). It is worth to note that routine cytogenetic analysis didn’t present any mitosis in 3 MDS pts however FISH analysis revealed del(5) and del(7) simultaneously in one of these pts, trisomy 8 in one pt and normal karyotype in another one pt. Cytogenetic analysis of MSCs was carried out in 23 pts – 4 AML and 19 MDS pts. BM karyotype was normal in 10 pts and abnormal in 13 pts. Karyotype in MSCs was normal in all AML pts and in 16 MDS pts. There were cytogenetic changes in 3 MDS pts: in 1 pt - constitutional inversion (inv9(p13q21)), in 1 pt - nonclonal translocation and constitutional inversion (46XY,t(2;22)(p10;q11),inv(9)(p13q21)[1]/46XY,inv(9)(p13q21)[19]) and in 1 pt - clonal abnormalities (46XY,add(2q)[7]/46XY[13]). Patient with clonal cytogenetic abnormalities in MSCs had complex karyotype in BM (45–46XY,−5,−13,der(19),add(q13?or p13?),−20,+marx2,+mar del(13q21)?[15]/45–46XY idem, +dmin[2]/46XY[3]), but these changes were different. Constitutional inversion was confirmed in both cases by cytogenetic analysis in PB lymphocytes. FISH analysis was performed in CD 34+ HPCs magnetically isolated from BM and PB in 24 pts. Fourteen of these pts had normal BM karyotype and we confirmed that using FISH with LSI (5q33-q34), LSI (7q31)/CEP 7, CEP 8 (Vysis). We used the same DNA probes for evaluation of CD34+ HPCs from BM and PB in pts with normal BM karyotype. We used FISH in 10 pts with ascertained cytogenetic abnormalities in BM to confirm these findings with DNA probes LSI (5q33-q34), LSI (7q31)/CEP 7, CEP 8, LSI AML1/ETO, CEP X/CEP Y, LSI (20q12) (Vysis). FISH analysis didn’t reveal any aberration in BM and PB CD34+ HPCs in patients with normal BM karyotype. In pts with distinguished abnormalities in BM the same pattern was demonstrated by FISH in CD34+ HPCs. We counted the percent of abnormal nuclei per 200. The means of percentages by FISH of BM, BM CD 34+ HPCs and PB CD34+ HPCs didn’t differ and constituted 65.8%, 73.1% and 74.8% respectively. Conclusion:Cytogenetic analysis in MSCs revealed aberrations only in MDS patients (3 from 16). No cytogenetic abnormalities in MSCs in AML pts was found. Karyotype in MSCs didn't coincide with BM karyotype in the same patients. Hematopoetic progenitor cells from BM and PB of MDS and AML patients displayed the same cytogenetic abnormalities as the full population of bone marrow cells. The data shows that hematopoetic and mesenchymal precursors in MDS and AML are cytogenetically distinct. Disclosures:No relevant conflicts of interest to declare.

Severe Hypoxia Promotes Selection of Repopulating Progenitor CELLS in Primary MDS BONE Marrow CELL Culture

AbstractAbstract 4747 Introduction:Myelodysplastic Syndromes (MDS) are clonal neoplasms. Whether the transforming event occurs in a myeloid committed cell or in earlier hematopoietic progenitor/stem cell it is still not ascertained. In this study, we evaluated the repopulating ability of hypoxia selected cells obtained from primary MDS bone marrow cultures. We then characterized the “stemness” of MDS maintaining cells in different subtypes of this disease. Methods:We studied 31 samples obtained at diagnosis from patients with different WHO subtypes of MDS (6 RA, 12 RCMD, 7 RAEB, 6 AL/post MDS). Mononuclear bone marrow cells were isolated after density gradient centrifugation and cultured in RPMI 1640 medium supplemented with 20 % FBS and a cocktail of cytokines (TPO, FLT3-L, SCF, IL-3). Cells were incubated and selected in Ruskin Concept 400 anaerobic incubator, in severe hypoxia conditions by flushing with a performed gas mixture (0,3 % O2,5%CO2,95% N2), for 10–13 days (LC1) and daily counted (by Trypan blue dye). The stem and progenitor cell potential of these cultures at different times of incubation was explored by transferring cells to growth-permissive secondary cultures in normoxia (LC2), in the presence of SCF, G-CSF, IL-6, IL-3, according to the Culture-Repopulating Ability assay methodology (Cipolleschi et al, Leukemia 2000). At the same time, we evaluated clonal potential of selected cells by semisolid culture in methylcellulose, in the presence of the same cocktail of cytokines. The phenotype of hypoxia selected cells was evaluated by determining the expression of CD34, CD38, CD117, CD133, as well as beta-catenin expression and localization at fluoresence microscopy. When present, we analyzed by FISH the persistence of chromosomal aberrations in MDS cells, before and after hypoxic selection. Results:The hypoxic culture system allowed selection of a minute cell population: in 31/31 cases viable cell number decreased of one log after 10–13 days of culture in extreme hypoxic conditions. Only 11/31 MDS cases showed a significant repopulating ability at day 17 of LC2 and in the same cases we observed parallel formation of colonies ( CFU-GM and CFU-GEMM) in semisolid cultures. Ten of these eleven cases that showed repopulating ability belonged to the Low/INT1 IPSS risk category. In the other 20, all IPSS high and INT-2 risk cases, repopulating ability was absent. In particular, MDS cases presenting > 10% blasts in the bone marrow did not show repopulating cells after selective hypoxic conditions. In 12 cases, after hypoxia selection we observed a reduction in the number of CD34+ cells. Within this shrinked CD34+ population, we could isolate highly repopulating progenitors CD34+ /CD38−, that confirmed the selection of earlier hematopoietic progenitor cells. We analyzed by interphase FISH MDS cases presenting chromosomal aberration ( +8, −Y, complex karyotype and del7q). In all cases, the marker chromosomal aberrations were maintained after hypoxic culture. In particular, in one of these cases with marker chromosomal aberration, the percentage of cells carrying –Y at interphase FISH was 66% at baseline, and 82% after hypoxic selection, while 66% of the repopulating cells, after 8 days of normoxic LC2, still had −Y. Conclusions:We demonstrate here that it is possible to select by severe hypoxic conditions primary MDS progenitor cells with repopulating ability. MDS cases in earlier phases of the disease still show near-normal repopulating abilityafter hypoxic selection, whereas more progressive dysplastic disease is accompanied by a loss of repopulating ability, as measured by CRA assay and colony formation. Functional characterization of the neoplastic isolated repopulating cells is warranted. Disclosures:Santini:Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria.

Stem and progenitor cells in myelodysplastic syndromes show aberrant stage-specific expansion and harbor genetic and epigenetic alterations

AbstractEven though hematopoietic stem cell (HSC) dysfunction is presumed in myelodysplastic syndrome (MDS), the exact nature of quantitative and qualitative alterations is unknown. We conducted a study of phenotypic and molecular alterations in highly fractionated stem and progenitor populations in a variety of MDS subtypes. We observed an expansion of the phenotypically primitive long-term HSCs (lineage−/CD34+/CD38−/CD90+) in MDS, which was most pronounced in higher-risk cases. These MDS HSCs demonstrated dysplastic clonogenic activity. Examination of progenitors revealed that lower-risk MDS is characterized by expansion of phenotypic common myeloid progenitors, whereas higher-risk cases revealed expansion of granulocyte-monocyte progenitors. Genome-wide analysis of sorted MDS HSCs revealed widespread methylomic and transcriptomic alterations. STAT3 was an aberrantly hypomethylated and overexpressed target that was validated in an independent cohort and found to be functionally relevant in MDS HSCs. FISH analysis demonstrated that a very high percentage of MDS HSC (92% ± 4%) carry cytogenetic abnormalities. Longitudinal analysis in a patient treated with 5-azacytidine revealed that karyotypically abnormal HSCs persist even during complete morphologic remission and that expansion of clonotypic HSCs precedes clinical relapse. This study demonstrates that stem and progenitor cells in MDS are characterized by stage-specific expansions and contain epigenetic and genetic alterations.

Loss of SHIP-1 protein expression in high-risk myelodysplastic syndromes is associated with miR-210 and miR-155.

The myelodysplastic syndromes (MDSs) comprise a group of disorders characterized by multistage progression from cytopenias to acute myeloid leukemia (AML). They display exaggerated apoptosis in early stages, but lose this behavior during evolution to AML. The molecular basis for loss of apoptosis is unknown. To investigate this critical event, we analyzed phosphatidylinositol (PI) 3'kinase signaling, implicated as a critical pathway of cell survival control in epithelial and hematological malignancies. PI 3'kinase activates Akt through its production of 3' phosphoinositides. In turn, the phosphoinositides are dephosphorylated by two lipid phosphatases, PTEN and SHIP-1, in myeloid cells. We studied primary MDS-enriched bone marrow cells and bone marrow sections by western blotting, immunohistochemistry, immunocytochemistry and quantitative PCR for components of the SHIP/PTEN/PI 3'kinase signaling circuit. We reported constitutively activated Akt, variable levels of PTEN and uniformly decreased SHIP-1 expression in MDS progenitor cells. Overexpression of SHIP-1, but not the phosphatase-deficient form, inhibited myeloid leukemic growth. Levels of microRNA (miR)-210 and miR-155 transcripts, which target SHIP-1, were increased in CD34(+) MDS cells compared with their normal counterparts. Direct binding of miR-210 to the 3' untranslated region of SHIP-1 was confirmed by luciferase reporter assay. Transfection of a myeloid cell line with miR-210 resulted in loss of SHIP-1 protein expression. These data suggest that miR-155 and miR-210/SHIP-1/Akt pathways could serve as clinical biomarkers for disease progression, and that miR-155 and miR-210 might serve as novel therapeutic targets in MDS.

Expression of the CAMPATH-1 Antigen (CD52) on CD34+/CD38- Progenitor Cells in Patients with 5q- MDS and in a Subset of AML

Abstract 1728The target antigen CAMPATH-1 (CD52) is widely expressed in various hematopoietic lineages inlcuding lymphocytes, basophils, and blood monocytes. The anti-CD52 antibody Alemtuzumab is used successfully to treat patients with chemotherapy-refractory chronic lymphocytic leukemia. Based on its strong immunosuppressive effects, Alemtuzumab has also been considered for patients with aplastic anemia and hypoplastic myelodysplastic syndromes (MDS). Indeed, more recently, Alemtuzumab was found to induce major hematologic responses in a group of patients with MDS. Although the immunosuppressive effect was considered to play a role, the exact mechanisms underlying this drug effect remained speculative. In the current study, we asked whether CD34+ bone marrow (BM) progenitor cells in MDS and acute myeloid leukemia (AML) express the CAMPATH-1 antigen. Twelve patients with MDS (5 females, 7 males; median age: 70 years), 25 patients with AML (16 females, 9 males; median age: 62 years), and 34 control cases (normal reactive BM, n=12; idiopathic cytopenia of unknown significance, n=11; chronic myeloid leukemia, CML, n=4; chronic myelomonocytic leukemia, CMML, n=3; JAK2 V617F+ myeloproliferative neoplasms, MPN, n=4) were examined. Surface expression of CD52 on CD34+/CD38+ and CD34+/CD38- BM progenitor cells was analyzed by monoclonal antibodies and multicolor flow cytometry. In the group of MDS, CD52 was detectable on CD34+/CD38- stem cells in 3/4 patients with isolated 5q-. In most of the other MDS patients, CD52 was weakly expressed or not detectable on CD34+/CD38- cells. In AML, CD34+/CD38- cells displayed CD52 in 12/25 patients, namely 3 with complex karyotype including 5q-, 2 with inv(3), one with t(8;21), one with inv(16), one with del13q, one with trisomy 8, one with monosomy 7, and 2 with normal karyotype. Expression of CD52 mRNA in CD34+/CD38- AML stem cells was confirmed by qPCR in all patients tested (n=14). In addition, a good correlation was found between surface CD52 expression and CD52 mRNA expression in AML progenitor fractions. In patients with normal hematopoiesis (n=12) or idiopathic cytopenia (n=11), CD34+/CD38- cells stained weakly positive or negative for CD52. Almost in all cases tested, blood monocytes and blood basophils stained positive for CD52. Together, our data suggest that the target antigen CAMPATH-1 (CD52) is expressed on primitive CD34+/CD38- progenitor cells in MDS, preferentially in 5q- patients, and in a subset of patients with AML. These observations may have clinical implications and explain recently described effects of Alemtuzumab in patients with MDS. Our data also suggest that Alemtuzumab may be an interesting targeted drug in patients with refractory or relapsed AML in whom neoplastic stem cells express the target antigen CD52. Disclosures:No relevant conflicts of interest to declare.

5-Azacytidine and Decitabine Induce Demethylation and Re-Expression of FAS (CD95) and Promote Apoptosis in Neoplastic Cells in Acute Myeloid Leukemia (AML),

Abstract 3463Epigenetic and apoptosis-regulating mechanisms have been implicated as critical factors contributing to the progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML). However, the exact molecular mechanisms and genes involved in disease evolution have not been identified yet. We screened for epigenetically regulated pro-apoptotic effector molecules in neoplastic cells in patients with MDS (n=50) and AML (n=30). Among a series of potential regulators, we identified FAS (CD95) as an epigenetically regulated critical death regulator in neoplastic cells. As assessed by qPCR, bone marrow cells obtained from patients with low risk MDS were found to display high levels of FAS, whereas FAS mRNA levels were lower or undetectable in patients with advanced MDS (with excess of blasts) or secondary AML. Moreover, we were able to show by multicolor flow cytometry that CD34+/CD38+ progenitor cells and CD34+/CD38- stem cells in MDS and AML display measurable FAS (CD95) on their surface, with slightly higher levels detectable in progenitor cells in low risk MDS compared to high risk MDS and secondary AML. Methylation-specific PCR and qPCR revealed that the FAS-promoter is hypermethylated in primary AML cells as well as in various AML cell lines including KG1 and HL60 thus repressing mRNA-synthesis. In addition, we found that exposure to 5-Azacytidine or Decitabine leads to demethylation of CpG-rich regions closest to the transcription starting sites, and thus to re-expression of FAS in AML cells. In vitro-targeting of AML cells by demethylating drugs was also found to revert epigenetic inactivation of other tumor suppressor genes such as CDKN2B (P15), CDKN2A (P16), ESR1 (estrogen-receptor alpha), with subsequent normalization of mRNA expression levels. Next, we asked whether CD95 acts as a critical death regulator involved in drug-induced apoptosis in neoplastic cells. In these experiments, both demethylating agents, 5-Azacytidine or Decitabine, were found to induce dose-dependent apoptosis and growth inhibition in primary AML cells, primary MDS cells, and in all AML cell lines examined. Drug-induced apoptosis in AML cells was accompanied by activation of caspase 8 and caspase 3 as well as increased expression of proapoptotic FAS/CD95 as determined by qPCR, Western blotting, and flow cytometry. Moreover, both drugs were found to induce expression of the FAS-ligand and DAPK1 in neoplastic cells. We then applied a siRNA against FAS. The siRNA-induced knock-down of FAS was found to block drug-induced FAS expression and FAS-induced apoptosis in KG1 cells and HL60 cells. In conclusion, our data show that FAS is hypermethylated in neoplastic cells in patients with advanced MDS and AML, that demethylating agents lead to re-expression of FAS, and that drug-induced FAS expression mediates apoptosis in leukemic cells. As FAS is also expressed on neoplastic stem cells, these observations may have clinical implications and may explain beneficial effects seen with 5-Azacytidine or Decitabine in patients with advanced MDS. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.