Early Stage Myelodysplastic Syndromes Research Articles

Effects of Black Raspberry Supplementation on Methylation Pathways in Vav-creAsxl1fl/flTet2fl/fl Double Knockout Mice with Early-stage Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDS) are a subset of myeloid malignancies defined by clonality of immature hematopoietic stem cells that leads to faulty blood cell development. These syndromes can lead to an increased risk of infection and may transform into acute myeloid leukemia, making it critical to determine effective treatments for the condition. While hypomethylating agents such as azacitidine and decitabine, as well as stem cell transplants, have been delineated as favored treatments for MDS, not all patients are physiologically receptive to these treatments. However, black raspberries (BRBs) have been shown to exert hypomethylating effects in various malignancies, with minimal adverse effects and thus a broader range of potential candidacies. This study aimed to investigate the potential of BRBs to exert such effects on MDS using Addition of Sex Combs Like/Tet Methylcytosine Dioxygenase 2 (Asxl1/Tet2) double knockout mice (Vav-cre Asxl1fl/fl Tet2fl/fl), which typically manifest symptoms around 25 weeks of age, mirroring genetic mutations found in humans with MDS. Following a 12-week dietary supplementation of Vav-cre Asxl1fl/fl Tet2fl/fl mice with 5% BRBs, we observed both hyper- and hypomethylation at multiple transcription start sites and intragenic locations linked to critical pathways, including hematopoiesis. This methylation profile may have implications for delaying the onset of MDS, prompting a need for in-depth investigation. Our results emphasize the importance of exploring whether an extended BRB intervention can effectively alter MDS risk and elucidate the relationship between BRB-induced methylation changes, thus further unlocking the potential benefits of BRBs for MDS patients.

Clinical and Biological Effects of Canakinumab in Lower-Risk Myelodysplastic Syndromes (MDS): Results from a Phase 2 Clinical Trial

Interleukin 1 beta (IL-1B) induced inflammasome activation is a mechanism of cancer initiation and progression (Schroder K, Cell, 2010). Our previous studies (Ganan-Gomez I, Blood 2021) showed that IL-1B was one of the most overexpressed pro-inflammatory cytokines in the hematopoietic stem and progenitor cells (HSPCs) of patients with clonal cytopenia of undetermined significance (CCUS) carrying DNMT3a and TET2 mutations. Given that IL-1B upregulation induces aberrant myeloid differentiation of HSPCs (Pietras EM, Nat Cell Biol., 2016), we hypothesize that IL-1B plays a critical role in MDS pathogenesis, and that targeting IL-1B signaling overcomes aberrant MDS HSPCs' myeloid skewing. To test this hypothesis, we designed a clinical trial to evaluate the IL-1B inhibitor canakinumab's (NCT04239157) safety profile and its clinical and biological effects in patients with lower-risk MDS. Patients and Methods: Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS, normal renal and hepatic functions, and performance status, who had previously received at least 1 line of other therapy were included in the study (Table1). Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. Bone marrow (BM) samples were obtained before therapy and after 1, 2, and 4 cycles of therapy during the trial and after every 3 or 4 cycles of therapy thereafter. Single-cell RNA sequencing (scRNA-seq; 10X Genomics) analyses were performed in lineage negative (Lin-)CD34+ HSPCs and mononuclear cells (MNCs) isolated at different therapy timepoints from the BM of 1 patient who achieved hematological improvement after canakinumab treatment, 2 patients who achieved stable disease, and 2 patients who did not respond to this therapy. The clinical characteristics of the 5 patients whose samples were analyzed by scRNA-seq analyses are included in Table 1. Results: Twenty-six patients have been enrolled in the clinical trial. Patients' clinical characteristics are included in Table 1. Twenty-three patients were evaluable for response. Among the 22 out of the 23 patients who were transfusion dependent at baseline, one achieved transfusion independence, and another had platelet count improvement. Three out of the 23 had progressive disease, and 18 had stable disease. Treatment was well tolerated with no severe adverse events. One patient died of sepsis unrelated to therapy. Overall, we did not observe any improvement in the blast counts and cytogenetic and/or mutational burden. To evaluate the biological effects of canakinumab, we performed scRNA-seq of Lin-CD34+ HSPCs (Figure 1) and MNCs at therapy baseline and after 2, 4, 5, 8, and 11 cycles of treatment. Consistent with our initial hypotheses, these analyses revealed that canakinumab decreased the frequencies of hematopoietic stem cells (HSCs) and multipotent myeloid progenitors (MPPs). Differential analysis between the samples showed that HSCs and MPPs significantly downregulated the expression of genes involved in cytokine signaling (e.g., IL-1B, CXCL2, and CXCL8), TNFα-induced activation of NF-κB, and hypoxia pathways, which demonstrated efficient target engagement of canakinumab on MDS cells. Similar results were also observed in downstream monocytes and B cells. In contrast, we did not observe any significant change in the HSPC architecture in the BM of patients with stable disease or whose disease did not respond to canakinumab. Consistent with our previous results in early stage MDS, the patient who achieved hematological improvement after canakinumab therapy was the only one among the cohort who carried a single mutation affecting DNMT3A (Table1). Conclusion. This study demonstrates that canakinumab is safe in MDS and efficiently targets IL-1B signaling. However, the biological effect of canakinumab has not yet translated in clinical responses in the subset of patients enrolled in this clinical trial. The study will accrue transfusion independent patients and patients with early stage MDS as CCUS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Modeling ASXL1 Mutation Revealed Myelodysplasia Caused By Derepression of p16Ink4a through Aberrant PRC1-Mediated Histone Modification

Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant condition in patients with blood cells that possess somatic mutations of genes known to be recurrently mutated in hematologic malignancies. Among them is additional sex combs like 1 (ASXL1), whose mutation is frequent in various myeloid malignancies and associated with poor prognosis, and ASXL1 is known to act as an epigenetic modulator. Despite the clinical impact of ASXL1 mutations in myeloid malignancies, lack of animal models expressing mutated Asxl1 at endogenous levels mimicking human ASXL1-mutated diseases hinders from dissecting precise molecular mechanisms of mutated Asxl1. Here in this study, with newly generated knock-in mice carrying a frameshift mutation at the exon 13 of the Asxl1 gene (Asxl1G643WfsX12), which is identical to the most frequent human ASXL1 G646WfsX12 mutation, we extensively investigated the biological effects of the mutant.Asxl1G643fs/+ mice showed decreased body size and weight, resembling hypomorphic features in patients with Bohring-Opitz syndrome carrying de novo constitutive ASXL1 mutation.Asxl1G643fs/+ mice developed slowly progressive peripheral leukopenia, and Wright-Giemsa stain of bone marrow (BM) cells from Asxl1G643fs/+ mice showed dysplastic myeloid cells represented by hypersegmented neutrophils, apoptotic neutrophils and hyposegmented neutrophils with fine nuclear bridging consistent with pseudo-Pelger-Huët anomaly. Flow cytometric analyses of Asxl1G643fs/+ BM cells confirmed aberrant myeloid differentiation with significant increase of granulocytic and monocytic cell fraction, significant decrease of hematopoietic stem cell (HSC) pool (c-kit+ Sca-1+ Lin- (LSK) cells and CD34- CD135- LSK cells), increased apoptosis in hematopoietic stem/progenitor cells (HSPC) and significant accumulation of G1-phase HSC in the cell cycle analysis, all of which were reminiscent of human myelodysplastic syndromes (MDS). Interestingly, Asxl1G643fs/+ mice became moribund about 600 days after birth when compared to Asxl1+/+ littermates with anemia, leukocytosis, neutrophilia/monocytosis and hepatosplenomegaly, suggesting the development of MDS/myeloproliferative neoplasm (MPN)-like disease, most relevant to human cases with chronic myelomonocytic leukemia.RNA sequencing and gene set enrichment analysis in Asxl1G643fs/+ mice identified derepression of sets of genes that are normally repressed by BMI1, a critical regulator of histone modification in polycomb repressive comprex 1 (PRC1). Among these genes were p16Ink4a, p21, and p15Ink4b all of which were significantly upregulated in Asxl1G643fs/+ LSK cells. We performed chromatin immunoprecipitation (ChIP) assay with an anti-H2AK119ub1 antibody using LSK cells from Asxl1G643fs/+ mice and found that H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in Asxl1G643fs/+ LSK cells, suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutated Asxl1. Co-immunoprecipitation assay and in vitro pull-down assay revealed that the mutant form of ASXL1 had no ability to interact with BMI1 as opposed to wild type ASXL1 protein. In addition, we performed ChIP assay with an anti-BMI1 and anti-FLAG antibody using a cell line stably expressed FLAG-tagged wild-type (WT) ASXL1 or mutant form (MT), and found that BMI1 level around the promoter region of p16Ink4a was significantly decreased in the ASXL1 MT-expressed cell line, whereas no significant change in the FLAG level between WT and MT. Furthermore, we intercrossed Asxl1G643fs/+ mice with p16Ink4a heterozygous knock out mice to elucidate the effect of p16Ink4a up-regulation in Asxl1G643fs/+ mice, and found restoration of HSC pool and amelioration of increased apoptosis in HSPC obtained from Asxl1G643fs/+p16Ink4a+/- mice. These results indicated that loss of protein interaction between Asxl1 mutant and Bmi1 affected the activity of PRC1, and subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in MDS-like phenotypes represented by decreased HSC pool and aberrant myeloid differentiation in Asxl1G643fs/+ mice.Taken together, our Asxl1 G643fs mutation knock-in mouse line is useful as a model of CHIP or early stage MDS, and our detailed analysis highlights the functional role of ASXL1 mutation on MDS and MDS /MPN development. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Kurokawa:Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Celgene Corporation: Speakers Bureau; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau.

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.

PS1327 PRO‐NECROPTOTIC RIPK3 AS A STAGE‐DEPENDENT MARKER IN THE BONE MARROW OF PATIENTS WITH MDS AND CMML

Background:Chronic inflammation plays a central role in the development of hematological neoplasms. In Myelodysplastic syndromes (MDS) pro‐inflammatory microenvironment is suspected to drive disease development. Several studies have shown increased rates of programmed cell death in marrow cells of patients with early stage MDS. In these patients improvement of cytopenia is a major therapeutic approach. Overcoming resistance to programmed cell death is a central therapeutic aim in patients with high‐risk MDS/sAML. RIPK3 is a crucial player of regulated necrosis (necroptosis). Necroptosis has a strong inflammatory capacity and pro‐inflammatory processes trigger necroptosis.Aims:The aim of this project is to identify pro‐necroptotic proteins in the bone marrow of patients with MDS, CMML and sAML and to correlate the necroptotic capacity with the stage of the disease. By pharmacological modulation of necroptosis we would like to evaluate the effects of necroptotic signaling on inflammation, cell death and cell differentiation.Methods:We therefore analyzed the impact of pro‐necroptotic signaling on bone marrow bulk and the subset of CD34+stem/progenitor cells of patients with MDS and chronic myelomonocytic leukemia (CMML) in vitro. Bone marrow mononuclear cells (BMMNCs) were isolated from BM aspirates of 5 patients with early MDS (MDS‐MLD, MDS‐RS‐SLD and MDS‐RS‐MLD), 5 patients with late MDS (MDS‐EB1 and MDS‐EB2), 5 patients with CMML‐1, 7 patients with CMML‐2 and 4 patients with sAML after a history of MDS and 4 patients with sAML after a history of CMML. Bulk of BMMNCs and purified CD34+stem/progenitor cells were stained intracellular for RIPK3 and analyzed by flow cytometry. The patient samples were compared to 12 age‐matched BM samples obtained from hip replacement surgery from otherwise healthy individuals. Furthermore, immunohistochemistry was performed in an enlarged cohort of patients. Therefore paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression. Additionally BMMNCs of patients with MDS were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitors for 72 h. Short term viability analysis by flow cytometry were performed and long‐term analysis using growth factor enriched methylcellulose.Results:Expression of RIPK3 was significantly increased in sAML after a history of MDS compared to late MDS and compared to healthy controls. Furthermore patients with sAML after a history of CMML showed a significantly higher expression of RIPK3 than patients with CMML‐1, CMML‐2 and healthy controls. These findings were confirmed by immunohistochemistry in an enlarged cohort of patients. Here paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression.Additionally BMMNCs of patients with MDS or CMML were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitorsfor 72 h. Short term viability analysis by flow cytometry showed no impact of inhibitor treatment. However after culture in growth factor enriched methylcellulose for 10–14 days colony forming capacity was significantly increased in low‐risk MDS patients after inhibition of pro‐necroptotic signaling. Here increase in colony numbers was not restricted to a specific lineage.Summary/Conclusion:We conclude that RIPK3 plays a critical stage‐dependent role in patients with MDS and CMML. Disease progression to sAML is associated with an increased RIPK3‐Expression. We further hypothesize that pro‐inflammatory processes like necroptosis have an impact on aberrant myeloid homeostasis.

SKI controls MDS-associated chronic TGF-β signaling, aberrant splicing, and stem cell fitness

AbstractThe transforming growth factor beta (TGF-β) signaling pathway controls hematopoietic stem cell (HSC) behavior in the marrow niche; however, TGF-β signaling becomes chronic in early-stage myelodysplastic syndrome (MDS). Although TGF-β signaling normally induces negative feedback, in early-stage MDS, high levels of microRNA-21 (miR-21) contribute to chronic TGF-β signaling. We found that a TGF-β signal–correlated gene signature is sufficient to identify an MDS patient population with abnormal RNA splicing (eg, CSF3R) independent of splicing factor mutations and coincident with low HNRNPK activity. Levels of SKI messenger RNA (mRNA) encoding a TGF-β antagonist are sufficient to identify these patients. However, MDS patients with high SKI mRNA and chronic TGF-β signaling lack SKI protein because of miR-21 activity. To determine the impact of SKI loss, we examined murine Ski−/− HSC function. First, competitive HSC transplants revealed a profound defect in stem cell fitness (competitive disadvantage) but not specification, homing, or multilineage production. Aged recipients of Ski−/− HSCs exhibited mild phenotypes similar to phenotypes in those with macrocytic anemia. Second, blastocyst complementation revealed a dramatic block in Ski−/− hematopoiesis in the absence of transplantation. Similar to SKI-high MDS patient samples, Ski−/− HSCs strikingly upregulated TGF-β signaling and deregulated expression of spliceosome genes (including Hnrnpk). Moreover, novel single-cell splicing analyses demonstrated that Ski−/− HSCs and high levels of SKI expression in MDS patient samples share abnormal alternative splicing of common genes (including those that encode splicing factors). We conclude that miR-21–mediated loss of SKI activates TGF-β signaling and alternative splicing to impair the competitive advantage of normal HSCs (fitness), which could contribute to selection of early-stage MDS-genic clones.

3122 - Understanding Early Stage Myelodysplastic Syndrome Pathobiology

3122 - Understanding Early Stage Myelodysplastic Syndrome Pathobiology

Ex vivo T-cell–depleted allogeneic stem cell transplantation for hematologic malignancies: The search for an optimum transplant T-cell dose and T-cell add-back strategy

BackgroundT-cell depletion (TCD) of allogeneic stem cell transplants (SCT) can reduce graft-versus-host disease but may negatively affect transplant outcome by delaying immune recovery. To optimize TCD in HLA-matched siblings with hematologic malignancies, we explored varying the transplant CD3+ T-cell dose between 2 and 50 × 104/kg (corresponding to 3–4 log depletion) and studied the impact of 0–6 × 107/kg CD3+ donor lymphocyte infusion (DLI) “add-back” on immune recovery post-SCT. MethodsTwo hundred seventeen consecutive patients (age range, 10–75 years) with hematologic malignancy (excluding chronic leukemias) underwent ex vivo TCD SCT from HLA-identical sibling donors from 1994–2015. Ninety-four patients had standard-risk disease (first remission acute leukemia [AL] and early stage myelodysplastic syndromes [MDS]) and 123 had high-risk disease (AL beyond first complete remission, advanced MDS or refractory B-cell malignancy). ResultsMedian follow-up was 8.5 years. At 20 years post-SCT, overall survival (OS) was 40%, nonrelapse mortality (NRM) was 27% and relapse incidence was 39%. Factors affecting outcome in multivariate analysis were transplantation era, with OS increasing from 38% in the period 1994–2000 to 58% in 2011–2015, disease risk (hazard ratio [HR], 1.68 for high risk) and increasing age (HR, 1.19 per decade). Neither the T-cell dose or the add back of T cells in the first 100 days had any effect on OS, NRM and relapse. ConclusionsOutcomes for TCD SCT have greatly improved. However, our data do not support the need to precisely manipulate transplant CD3+ T-cell dose provided at least 3-log depletion is achieved or the use of T-cell add-back. Future improvements for TCD SCT await better strategies to prevent relapse, especially in high-risk recipients.

Detection of erythroblast antibodies in mitogen-stimulated bone marrow cultures from patients with myelodysplastic syndromes.

Low-risk myelodysplastic syndromes (MDS) show several immunologic abnormalities, including increased frequency of autoimmune manifestations and/or overt autoimmune diseases, whose prognostic significance still remains controversial. We studied the presence of erythroblast antibodies in mitogen-stimulated bone marrow (BM) cultures of 70 patients with early-stage MDS (refractory anemia and refractory anemia with ringed sideroblasts). Sixty-six percent of patients showed positive erythroblast antibodies, along with BM erythroid hyperplasia and a hemolytic picture in the peripheral blood. Supernatants from positive cultures induced an increase of overall cellularity, the appearance of erythroblastic clustering, and dyserythropoietic signs in normal BM. We identified CD45(dim) Gly-A(dim) CD71(bright) cells (red blood cell precursors at different maturation stage) as the target of the antibodies. Erythropoietin (EPO) levels were reduced and EPO receptors (EPO-R) increased in BM culture supernatants from positive patients. However, flow cytometric analysis showed that neither EPO nor EPO-R was involved in an abnormal stimulation driven by these autoantibodies. Values of the proapoptotic protein Bax were increased in positive patients and Bcl-2 levels were decreased, although not significantly. MDS patients with anti-erythroblast autoimmunity showed increased BM apoptosis, suggesting that the autoimmune reaction may contribute to an unfavorable BM microenvironment for optimal erythropoiesis.

Abstract 1499: Expression and prognostic significance of microRNAs in Korean patients with myelodysplastic syndrome

Abstract Introduction: MicroRNAs (miRNAs) are short non-coding RNAs that are involved in post-transcriptional regulation of gene expression, which may result in translational inhibition or destabilization of the target mRNA. Various alterations of miRNA expression have been reported in myelodysplastic syndrome (MDS). We aimed to investigate the unique patterns and prognostic significance of miRNA expression in Korean patients with MDS. Materials and Methods: Bone marrow mononuclear cells were collected from 10 healthy controls and 26 MDS patients, and miRNAs were isolated using a miRNeasy isolation kit (Qiagen) according to the manufacturer's instructions. Quantitative real-time PCR for selected miRNAs including miR-21, miR-124a, miR-126, miR-146b, miR-155, miR-200c, miR-342-5p, miR-708, and Let-7a was performed using a miScript SYBR green PCR kit (Qiagen). RNU6 was used to normalize the miRNA values of all RNA samples. Calculations were made using the comparative Ct (2-ΔΔCt ) method. Results: Eleven patients with early stage MDS (2 RA, 1 RARS, and 8 RCMD), 10 patients with advanced MDS (3 RAEB-1 and 7 RAEB-2), 4 patients with unknown subtype of MDS, and 1 patient with chronic myelomonocytic leukemia were included in this study. Median age was 58.5 years (range, 13-76). MiR-124a, miR-155, miR-200c, miR-342-5p, and let-7a were significantly under-expressed in MDS patients compared to healthy controls. Levels of miR-124, miR-146b, and miR-155 transcripts were significantly lower in patients with early stage MDS than in those with advanced MDS. Secondary MDS patients (n=6) showed significantly under-expressed MiR-146b and miR-342-5p transcript levels than de novo MDS patients (n=19) (p=0.009 and p=0.043, respectively). We failed to find statistically significant survival differences according to the expression levels of miRNAs, although high expression of miR-124, miR-126, miR-155 and low expression of miR-342-5p tended to be associated with poor overall survival and leukemia free survival. Conclusion: We first report under-expression of miR-200c in MDS patients. Expression of miRNAs appears to be different according to the stage of MDS. Further investigations are needed to evaluate genetic functions and prognostic significances of miRNAs in MDS. Citation Format: Yunsuk Choi, Eun-Hye Hur, Ju Hyun Moon, Bon-Kwan Goo, Dae Ro Choi, Je-Hwan Lee. Expression and prognostic significance of microRNAs in Korean patients with myelodysplastic syndrome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1499. doi:10.1158/1538-7445.AM2014-1499

The difficulty to define progression patterns in patients with early stage myelodysplastic syndromes and deletion 5q—New prognostic markers are needed

The difficulty to define progression patterns in patients with early stage myelodysplastic syndromes and deletion 5q—New prognostic markers are needed

The New CD95 Ligand Inhibitor APG101 Leads To Decreased Apoptosis and Improved Erythroid Differentiation In Primary CD34+ Cells From Patients With Low Risk Myelodysplastic Syndrome (MDS)

IntroductionMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid malignancies characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. Particularly early stage MDS are at least in part characterized by an increased apoptosis of myeloid and erythroid progenitors that causes peripheral cytopenia. APG101 is a glycosylated fusion protein consisting of the extracellular domain of human CD95 (Fas receptor) and the Fc domain of human IgG1. APG101 effectively binds to the CD95 ligand (CD95L) expressed on effector cells as well as to functionally active ligand in solution, by that blocking the interaction between CD95 and its ligand. The aim of our study was to evaluate whether APG101 treatment of primary CD34+ reduces the apoptotic rate and improves the differentiation capacity of these cells. MethodsBone marrow cells were obtained during routine bone marrow aspiration after all patients gave their written informed consent. Isolated primary CD34+ cells from 11 MDS patients were cultured in complete supplemented IMDM medium for 6 days with increasing concentrations of APG101 (1 µg/mL, 3 µg/mL, 10 µg/mL, 30 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL). After incubation time, cells were multicolor- stained with the following dye combination: Annexin-FITC + CD235a-PE + CD34-PECy7 + CD71-APC + 7-AAD and analyzed immediately on a flow cytometer (FACSCanto, BD Bioscience, Heidelberg, Germany). Analysis of raw FACS data was done with the FACSDiva software. To analyze the differentiation capacity of CD34+ progenitors, methylcellulose assays were performed in parallel to the aforementioned experiments. However, due to limited cell numbers, colony assays were performed on 9 MDS patients only. Cells were cultured in triplicates with increasing concentrations of APG101 for 14 days. Colonies were counted and the mean number of colonies was determined. ResultsTreatment of differentiating CD34+ cells with APG101 led to a decreased apoptosis in both CD34+ cells and CD71+ cells, respectively, indicated by decreased Annexin-FITC fluorescence. Interestingly, this effect was particularly seen at low APG101 concentrations (maximum of 10 µg/ml), while the effect was abrogated at higher APG101 concentrations. The anti-apoptotic effect was more pronounced in low risk MDS patients compared to high risk MDS patients. No effect was seen when the CD235a+ fraction of cells was analyzed. With regard to colony formation, an improvement of erythroid differentiation, indicated by an increase in CFU-E, was found in 3 out of 4 low risk patients (less than 5% blasts in the bone marrow). No effect was seen on erythroid differentiation in high risk patients (more than 5% blasts in the bone marrow). ConclusionAPG101 shows promising in vitro activity in viable CD34+ cells with regard to inhibition of apoptosis and promotion of differentiation. The observation that the anti-apoptotic effect was more pronounced in low risk MDS patients as compared to high risk MDS patients supports the concept of increased apoptosis particularly in early stage MDS progenitors. Although the numbers in the differentiation experiments are small, we found a promising effect of APG101 on CFU-E formation at lower doses in patients with less than 5% bone marrow blasts. Moreover, the same dose-dependent effect was observed in the apoptosis assays. Since the activation of the CD95 pathway seems not only to be involved in apoptosis induction, but is also required for terminal erythroid differentiation in normal hematopoiesis, this dose-dependent effect might particularly reflect these ambivalent roles of CD95 and its ligand in both MDS and healthy hematopoiesis, repsectively. Disclosures:Kunz:APOGENIX GmbH: Employment. Fricke:APOGENIX GmbH: Employment.

Puma Is The Critical BH3-Only Protein Mediating Apoptosis In The Nup98-HoxD13 (NHD13) Mouse Model Of Human MDS

One of the key hallmarks of myelodysplastic syndromes (MDS) is that of ineffective haematopoiesis. The main factor contributing to this is increased apoptosis of bone marrow progenitors – a prominent feature particularly of early-stage MDS. Although apoptosis has been attributed to cell extrinsic triggers such as increased levels of Fas and TNF-α, there is increasing evidence implicating the cell intrinsic pathway – in which Bcl-2 and its related proteins play an important role and which is activated by p53. Consistent with this, we have recently shown that apoptosis in the Nup98-HoxD13 (NHD13) transgenic mouse model of MDS can be effectively blocked by enforced expression of Bcl-2. To further define the molecular mechanism of apoptosis upstream of Bcl-2, we mated NHD13 mice with mice deficient for p53. Consistent with a lineage specific role for p53, we observed p53-dependent apoptosis of the erythroid, but not the myeloid lineage. In NHD13 progenitors, gene expression analysis for members of the intrinsic pathway, showed elevated expression of Noxa but not Puma – both of which are BH3-only proteins and transcriptional targets of p53. We proceeded to mate NHD13 mice with mice deficient in Noxa and Puma. Unexpectedly, the expression levels were in fact misleading as genetic ablation studies showed that apoptosis of both erythroid and myeloid lineages was Puma dependent, with no role for Noxa. These studies have demonstrated that gene expression of Bcl-2 proteins cannot be used to predict functional relevance and in this case, Puma is the critical BH3-only protein mediating apoptosis in MDS. Disclosures:Guirguis:Leukaemia Foundation - Australia: Research Funding.

Relationship between clinical characteristics and myelodysplastic syndrome patients with isocitrate dehydrogenase gene mutations

To assess the prevalence and clinical characteristics of isocitrate dehydrogenase 1 and 2 (IDH 1 and IDH2) gene mutations in myelodysplastic syndrome (MDS) patients. Pretreatment bone marrow specimens were enriched for mononuclear cells in 108 adult patients with de novo MDS from January 2006 to August 2012. Genomic DNA was extracted from mononuclear cells. And PCR and direct sequencing were performed to sequence exon 4 of IDH gene. IDH mutations were discovered in 11 MDS patients (10.19%, 11/108) and all were heterozygous. The frequencies of IDH1 and IDH2 mutations were 5.56% (6/108) and 4.63% (5/108) respectively. Only one type of IDH1 mutation (c.394C→, p.R132C) was identified in our cohort. All IDH2 mutations caused the changes of R140 (c.419G→A, p.R140Q). However IDH2 R172 mutation was not detected. The combined mutations of IDH1 and IDH2 were not simultaneously observed in the same patient. The prevalence of IDH mutation was higher in advanced-stage MDS than those early-stage MDS patients. Mutated and wild-type groups had significantly difference in bone marrow blast percentage (median 12.5% vs 6.0%, P = 0.013) at diagnosis, but not in white blood cell count, hemoglobin level and platelet count, etc. In the normal karyotype group, the frequencies of IDH mutations were as similar as those in the abnormal karyotype group (10.61% (7/66) vs 10.00% (4/40), P > 0.05). The median follow-up time was 472 d, our data indicated that IDH mutations were correlated with poor overall survival (median time 512 vs 740 d, P = 0.017). IDH mutations were also an inferiorly predictive factor in the intermediate-1 group patients of International Prognostic Scoring System (IPSS) (median survival time 512 d vs not reached, P = 0.038). There was also better efficacies than other treatments in IDH mutation positive patients (median survival time 623 vs 165 d, P = 0.049). IDH mutation is a vital biomarker for better risk stratification of MDS patients with and improving IPSS. Hypomethylation agents may be effective for treating IDH mutation positive patients.

Stress-induced hematopoietic failure in the absence of immediate early response gene X-1 (IEX-1, IER3)

Expression of the immediate early response gene X-1 (IEX-1, IER3) is diminished significantly in hematopoietic stem cells in a subgroup of patients with early stage myelodysplastic syndromes, but it is not clear whether the deregulation contributes to the disease. The current study demonstrates increased apoptosis and a concomitant decrease in the number of hematopoietic stem cells lacking this early response gene. Null mutation of the gene also impeded platelet differentiation and shortened a lifespan of red blood cells. When bone marrow cells deficient in the gene were transplanted into wild-type mice, the deficient stem cells produced significantly fewer circulating platelets and red blood cells, despite their enhanced repopulation capability. Moreover, after exposure to a non-myeloablative dose of radiation, absence of the gene predisposed to thrombocytopenia, a significant decline in red blood cells, and dysplastic bone marrow morphology, typical characteristics of myelodysplastic syndromes. These findings highlight a previously unappreciated role for this early response gene in multiple differentiation steps within hematopoiesis, including thrombopoiesis, erythropoiesis and in the regulation of hematopoietic stem cell quiescence. The deficient mice offer a novel model for studying the initiation and progression of myelodysplastic syndromes as well as strategies to prevent this disorder.

Fbw7-dependent cyclin E regulation ensures terminal maturation of bone marrow erythroid cells by restraining oxidative metabolism

The mechanisms that coordinate the final mitotic divisions of terminally differentiated bone marrow erythroid cells with components of their structural and functional maturation program remain largely undefined. We previously identified phenotypes resembling those found in early-stage myelodysplastic syndromes, including ineffective erythropoiesis, morphologic dysplasia, and hyper-cellular bone marrow, in a knock-in mouse model in which cyclin E mutations were introduced at its two Cdc4 phosphodegrons (CPDs) to ablate Fbw7-dependent ubiquitination and degradation. Here we have examined the physiologic consequences of cyclin E dysregulation in bone marrow erythroid cells during terminal maturation in vivo. We found cyclin E protein levels in bone marrow erythroid cells are dynamically regulated in a CPD-dependent manner and that disruption of Fbw7-dependent cyclin E regulation impairs terminal erythroid cell maturation at a discrete stage prior to enucleation. At this stage of erythroid cell maturation, CPD phosphorylation of cyclin E regulates both cell cycle arrest and survival. We also found normal regulation of cyclin E restrains mitochondrial reactive oxygen species accumulation and expression of genes that promote mitochondrial biogenesis and oxidative metabolism during terminal erythroid maturation. In the setting of dysregulated cyclin E expression, p53 is activated in bone marrow erythroid cells as part of a DNA damage response-type pathway, which mitigates ineffective erythropoiesis, in contrast to the role of p53 induction in other models of dyserythropoiesis. Finally, cyclin E dysregulation and ROS accumulation induce histone H3 lysine 9 hyper-methylation and disrupt components of the normal terminal erythroid maturation gene expression program. Thus, ubiquitin-proteasome pathway control of G1-to-S-phase progression is intrinsically linked to regulation of metabolism and gene expression in terminally differentiating bone marrow erythroid cells.

Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome

BackgroundImmunological mechanisms are increasingly recognized in the progression of myelodysplastic syndrome (MDS). Early-stage MDS (E-MDS) is characterized by autoimmune-mediated myelosuppression whereas late-stage MDS (L-MDS) involves immune evasion, giving dysplastic cells growth potential to progress into acute myeloid leukemia. T-helper (Th) 22 is involved in the pathogenesis of inflammatory autoimmunity and tumorigenesis. The roles of Th22 cells in the pathophysiology of E-MDS and L-MDS remain unsettled.Design and MethodsWe studied 37 MDS patients (E-MDS, n = 17; L-MDS, n = 20) and 20 healthy controls to characterize their peripheral blood (PB), as well as 25 MDS patients and 10 healthy controls to characterize their bone marrow(BM). The expression of Interleukin-22 (IL-22), IL-17 or interferon gamma (IFN-γ) was examined in E-MDS, L-MDS patients and controls by flow cytometry. The mRNA expression levels of RAR-related orphan receptor C (RORC), IL-6, tumor necrosis factor alpha (TNF-α) and IL-23 in peripheral blood mononuclear cells (PBMCs) were determined by real-time quantitative polymerase chain reaction. The levels of IL-22 and IL-17 both in PB and BM plasma were examined by enzyme-linked immunosorbent assay.ResultsIn E-MDS, peripheral Th17 cells were significantly elevated and correlated with peripheral Th22 cells compared with healthy controls and L-MDS. Significantly higher levels of peripheral Th22 expansion, mRNA expression of IL-6, TNF-α and lower level of RORC mRNA expression were observed in L-MDS compared with E-MDS. No statistical difference was found in IL-23 mRNA expression or plasma IL-22, IL-17 levels among E-MDS, L-MDS and controls.ConclusionsOur data demonstrated that L-MDS cohort had increased frequencies of peripheral Th22 cells and higher mRNA expression levels of IL-6 and TNF-α, indicating that Th22 cells along with Th17 cells or not are involved in the dynamic immune responses of MDS.

197 Patients with early stage myelodysplastic syndromes show increased frequency of CD4+CD25high+CD127low regulatory T-cells

197 Patients with early stage myelodysplastic syndromes show increased frequency of CD4+CD25high+CD127low regulatory T-cells

Alterations In the Signaling Profile of Leukemic Progenitors Can Predict the Response of Myelodysplastic Syndrome (MDS) Patients to Azacytidine

AbstractAbstract 2921STAT3 and STAT5 regulate fundamental cellular processes and comprise the most studied signaling molecules of both normal and malignant hematopoiesis. Deregulation of STAT signaling contributes to leukemogenesis and may serve as a treatment target. In leukemic progenitors (LPs), the clustering of STAT3 and STAT5 phosphorylation patterns, both basal and after growth factor stimulation, can be achieved by flow cytometry, leading to the identification of distinct signaling profiles (SPs). In acute myeloid leukemia patients, SPs reflect the biological behavior of the LPs and can distinguish patient subgroups with worse prognosis and/or resistance to treatment. As epigenetic defects of genes involved in cell signaling are frequently observed in cancer cells we investigated the alterations in the SPs of MDS progenitors during azacytidine treatment and their correlation with response, cytogenetics and transfusion requirements.Bone marrow samples of 24 high risk MDS patients were obtained before and 15 days after the initiation of azacytidine in order to assess potential changes in SP before the disappearance of the LPs. According to the IWG response criteria patients were divided into group A (CR, PR and HI, n=10) and group B (stable disease and failure, n=14). Immunomagnetically purified LPs were either left untreated or stimulated with G-CSF and GM-CSF for 15' and then stained intracellularly with monoclonal antibodies against STAT3 and STAT5.The comparisons of basal and potentiated responses before and 15 days after azacytidine initiation were made with Mann-Whitney U-test. Clustering of SPs was performed with hierarchical cluster analysis and was correlated with treatment response, cytogenetics and transfusion dependence by using Chi square or Fisher Exact tests as appropriate. All analyses were performed using SPSS 14.0 software (SPSS Science, Chicago, IL).By clustering the SPs before and 15 days after the initiation of azacytidine we distinguished two subgroups of patients based on both the basal levels and potentiated response to growth factors. Patients with generally weak expression of STAT3 and STAT5 had significantly better response to azacytidine compared to those with strong expression of the same molecules (p=0.035), whereas there were no correlation of SPs with the karyotype (p=0.45) and transfusion rate (p=0.39). In line with the above, we further identified a STAT3+STAT5+ double positive population of MDS progenitors whose pretreatment levels after G-CSF and GM-CSF stimulation were inversely associated with treatment response (figure 1). Additionally, SP kinetics were following the disease course and response to therapy. In two late-stage MDS patients who achieved complete remission the SP was restored to early-stage MDS levels in day 15 after azacytidine initiation (figure 2A). In contrast, the SPs in the majority of non-responding patients remained unaltered (figure 2B), whereas the SP of a relapsing patient reverted to pretreatment levels after an initial restoration to early-stage MDS levels (figure 3). [Display omitted] [Display omitted] [Display omitted] In conclusion, we demonstrate that SP alterations of MDS progenitors during azacytidine treatment can predict clinical response. Moreover, it appears that azacytidine can restore the leukemic signaling in MDS by modifying both the basal and potentiated expression of STAT3 and STAT5. Our findings advocate the differentiating activity of hypomethylating agents, potentially via epigenetic reprogramming of pivotal signaling networks of leukemic progenitors. Disclosures:No relevant conflicts of interest to declare.

MicroRNA Diagnostic Signature of Myelodysplastic Syndrome.

Abstract 1763Poster Board I-789 IntroductionThe myelodysplastic syndromes (MDS) are a group of aging-associated hematopoietic disorders characterized by ineffective maturation that, in 6-33% of cases, progress to acute leukemia (AML), a disease of blocked differentiation. Since microRNAs (miRNAs) regulate cell differentiation/maturation as well as cell identity, miRNAs may play critical roles in both the development of MDS as well as transformation to AML. With the advent of new therapies and treatment modalities for MDS and AML, the availability of biomarkers capable of detecting early MDS and predicting progression to AML would have tremendous impact on the management of these patients. MethodsThe study utilizes twenty samples of bone marrow mononuclear cells, ten from MDS patients and ten from normal controls, isolated and stored at the University of Pennsylvania Stem Cell Core Facility between 2003 and 2007. The MDS specimens included seven (7) from patients in whom it was known that no transformation to acute leukemia occurred within five (5) years of the available specimen and three (3) from patients in whom acute leukemia was diagnosed within two (2) years of the available specimen. The patient specimens were selected to represent early stage MDS and therefore not at high risk for transformation based on morphologic analysis (circulating blasts '5%) and the 2001 WHO classification system. Total RNA was obtained from each sample and arrayed on a custom Agilent miRNA microarray at the University of Pennsylvania Microarray Facility in a dual channel experiment in which each sample was arrayed against a pool of all twenty samples. Statistical analysis was performed using Genespring v 7.31 and Partek Genomics Suite v6.3. Subsequently, thirteen miRNAs of interest and one amplification control were examined by RT-PCR using ABI commercial primers for those targets. Total RNA extracted from formalin-fixed paraffin-embedded tissue was also examined for the relative expression of these fourteen miRNAs. ResultsClass discovery ANCOVA algorithms were applied to the data to identify 13 miRNAs which differentiate the MDS samples from the normal controls. Real time PCR was performed upon the sample RNAs to verify the microarray results. Of the thirteen candidate miRNAs, miR-150, miR-342, and miR-103, miRNAs documented in other studies to be important in hematopoiesis, demonstrate particular promise as classifiers for MDS based upon the high degree of correlation between the microarray results and the RT-PCR studies as well as their ability to independently differentiate MDS patients from normal controls. When the three miRNAs are taken together, the microarray and RT-PCR results correlate in 83% of cases, with only 2 major discrepancies. Furthermore, comparison between the total RNA isolated from the bone marrow mononuclear cells suspensions of two patients with total RNA isolated from the corresponding formalin-fixed paraffin-embedded clot sections, across fourteen different miRNAs demonstrate extremely high levels of correlation (R2 = 0.94 and 0.85), proving the feasibility of future studies utilizing the readily available paraffin-embedded clot sections as a source of patient samples. Microarray probes to predicted miRNAs were also identified which distinguished the three patients who progressed to acute myeloid leukemia within 24 months from the seven that did not. ConclusionsA miRNA signature was identified which distinguishes early cases of MDS from normal controls. These miRNAs are predicted to regulate many of those mRNAs identified by traditional transcriptional profiling. Ultimately, it is hoped that assessment of miRNAs or their downstream targets will provide practicing pathologists with new markers for the identification of MDS and provide clinicians with additional prognostic information to guide the use of therapeutic interventions. DisclosuresCarroll:Sanofi Aventis Corp: Research Funding; Cephalon Oncoloy: Consultancy.