Myelodysplastic Syndromes Cells Research Articles

Ezh1 Targets Bivalent Genes to Maintain Self-Renewing Stem Cells in Ezh2-Insufficient Myelodysplastic Syndrome

SummaryPolycomb repressive complex (PRC) 2 represses transcription through histone H3K27 trimethylation (H3K27me3). We previously reported that the hematopoietic-cell-specific deletion of Ezh2, encoding a PRC2 enzyme, induced myelodysplastic syndrome (MDS) in mice, whereas the concurrent Ezh1 deletion depleted hematopoietic stem and progenitor cells (HSPCs). We herein demonstrated that mice with only one Ezh1 allele (Ezh1+/-Ezh2Δ/Δ) maintained HSPCs. A chromatin immunopreciptation sequence analysis revealed that residual PRC2 preferentially targeted genes with high levels of H3K27me3 and H2AK119 monoubiquitination (H2AK119ub1) in HSPCs (designated as Ezh1 core target genes), which were mostly developmental regulators, and maintained H3K27me3 levels in Ezh1+/-Ezh2Δ/Δ HSPCs. Even upon the complete depletion of Ezh1 and Ezh2, H2AK119ub1 levels were largely retained, and only a minimal number of Ezh1 core targets were de-repressed. These results indicate that genes marked with high levels of H3K27me3 and H2AK119ub1 are the core targets of polycomb complexes in HSPCs as well as MDS stem cells.

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

The high NRF2 expression confers chemotherapy resistance partly through up-regulated DUSP1 in myelodysplastic syndromes.

Although cytarabine has been widely considered as one of the chemotherapy drugs for high-risk myelodysplastic syndromes (MDS), the overall response rate is only approximately 20-30%. Nuclear factor erythroid 2-related factor 2 (NRF2, also called NFE2L2) has been shown to play a pivotal role in preventing cancer cells from being affected by chemotherapy. However, it is not yet known whether NRF2 can be used as a prognostic biomarker in MDS, or whether elevated NRF2 levels are associated with cytarabine resistance. Here, we found that NRF2 expression levels in bone marrow from high-risk patients exceeded that of low-risk MDS patients. Importantly, high NRF2 levels are correlated with inferior outcomes in MDS patients (n=137). Downregulation of NRF2 by the inhibitor Luteolin, or lentiviral shRNA knockdown, enhanced the chemotherapeutic efficacy of cytarabine, while MDS cells treated by NRF2 agonist Sulforaphane showed increased resistance to cytarabine. More importantly, pharmacological inhibition of NRF2 could sensitize primary high-risk MDS cells to cytarabine treatment. Mechanistically, downregulation of dual specificity protein phosphatase 1, an NRF2 direct target gene, could abrogate cytarabine resistance in NRF2 elevated MDS cells. Silencing NRF2 or dual specificity protein phosphatase 1 also significantly sensitized cytarabine treatment and inhibited tumors in MDS cells transplanted mouse models in vivo. Our study suggests that targeting NRF2 in combination with conventional chemotherapy could pave the way for future therapy for high-risk MDS.

Associations between the peripheral blood Wilms tumor gene 1 level and both bone marrow blast cells and the prognosis in patients with myelodysplastic syndrome

Wilms tumor gene 1 (WT1) is highly expressed in myelodysplastic syndrome (MDS) cells and is known to reflect the tumor burden in MDS. We evaluated the usefulness of WT1 mRNA levels for predicting the prognosis of MDS. At diagnosis, WT1 levels were strongly correlated with the percentage of blasts calculated based on non-erythroid cells, but not with that based on all nucleated cells (r = 0.57, p < .05 vs r = 0.42, p = .13). Among the allogeneic transplant recipients, the presence of two consecutive WT1 levels ≥100 copies/μg RNA with a median interval of one month was associated with a 77.8% relapse rate at nine months from the first detection of a high WT1 level, and the median time to relapse was only 114 [36–257] days. WT1 levels at diagnosis were correlated with known prognostic factors. In addition, the presence of two consecutive high WT1 levels after allogeneic transplantation may predict early relapse of MDS.

Clinical significance of hyaluronan levels and its pro-osteogenic effect on mesenchymal stromal cells in myelodysplastic syndromes

BackgroundHyaluronan (HA), a major component of the extracellular matrix, has been proven to play a crucial role in tumor progression. However, it remains unknown whether HA exerts any effects in myelodysplastic syndromes (MDS).MethodsA total of 82 patients with MDS and 28 healthy donors were investigated in this study. We firstly examined the bone marrow (BM) serum levels of HA in MDS by radioimmunoassay. Then we determined HA production and hyaluronan synthase (HAS) gene expression in BM mesenchymal stromal cells (MSC) and mononuclear cells derived from MDS patients. Finally, we investigated the effects of HA on osteogenic differentiation of MSC.ResultsThe BM serum levels of HA was increased in higher-risk MDS patients compared to normal controls. Meanwhile, patients with high BM serum HA levels had significantly shorter median survival than those with low HA levels. Moreover, the HA levels secreted by MSC was elevated in MDS, especially in higher-risk MDS. In addition, HAS-2 mRNA expression was also up-regulated in higher-risk MDS-MSC. Furthermore, we found that MSC derived from MDS patients with high BM serum HA levels had better osteogenic differentiation potential. Moreover, MSC cultured in HA-coated surface presented enhanced osteogenic differentiation ability.ConclusionsOur results show that elevated levels of BM serum HA are related to adverse clinical outcome in MDS. Better osteogenic differentiation of MSC induced by HA may be implicated in the pathogenesis of MDS.

PO-434 AT1413 antibody derived from a cured AML patient recognisesa unique sialylated CD43 epitope shared by AML, MDS and melanoma cells

IntroductionAT1413 is an antibody derived from B cells of an AML patient who was cured following allogeneic hematopoietic stem cell transplantation. It recognises a sialylated epitope on CD43 (CD43s), which is expressed on myeloid cells but not on B and T cells and is over-expressed on acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) cells. AT1413 kills AML cells in vitro and in vivo via antibody dependent cell-mediated cytotoxicity (ADCC) suggesting that AT1413 played a role in the graft versus leukaemia response of this patient. Because CD43 is broadly expressed in non-hematopoietic cells we explored whether CD43s is present on non-hematopoietic tumours.Material and methodsAT1413 binding on a panel of tumour cell lines was analysed by flow cytometry. AT1413 was assembled into a bispecific T-cell engaging format (AT1413 bTCE) by linking the full-length AT1413 IgG to two single chain variable fragments against CD3e with a combination of site-specific enzymatic and chemical coupling. Two point mutations in the IgG heavy chain were introduced to prevent interactions between AT1413 bTCE and Fc gamma receptors. The cytotoxicity-inducing activities of naked AT1413 and its bTCE format were tested with PBMCs as effector and tumour cells as target cells using standard cytotoxic assays.Results and discussionsAT1413 bound to melanoma cell lines but not to pancreas carcinoma, colon carcinoma, or liver carcinoma. Expression on melanoma cells was confirmed by immunoprecipitation and western blot using a mouse anti-human CD43 antibody. AT1413 bound to 14 out of 21 patient-derived primary melanoma samples with varying intensities. AT1413 induced ADCC of melanoma cell lines and patient-derived melanoma cells. The level of ADCC correlated with CD43s expression levels. To increase the cytotoxicity inducing potential of AT1413 we generated a bTCE format and demonstrated that it induced strong cytotoxic T cell activities against melanoma cells in vitro. The efficacy of AT1413 and AT1413-bTCE on human melanoma cells in vivo in a xenograft mouse model is currently tested.ConclusionThe AT1413 antibody recognises a sialylated epitope on CD43 shared by melanoma, AML and MDS cells. Both the non-modified IgG- and the bispecific TCE form of AT1413 induce strong anti-tumour cytotoxic activities in vitro and in vivo. Because of its broad tumour reactivity and functional activities AT1413 has promising therapeutic potential.

Abstract 3004: Single-cell mutational profiling of clonal evolution in myelodysplastic syndromes (MDS) during therapy and disease progression

Abstract Myelodysplastic syndromes (MDS) are a collection of clonal diseases with dysfunctional hematopoietic stem cells, characterized by ineffective hematopoiesis, cytopenias, and dysplasia. MDS is increasingly being recognized as a molecularly heterogeneous disease with variability in clinical phenotype, prognosis, and response to treatment. The clonal evolution in response to therapy remains a challenge in the management of MDS. However, resolution of complex clonal architectures is difficult with current bulk sequencing approaches. High-throughput single-cell genomic profiling enables the resolution of tumor heterogeneity, and may improve clinical diagnosis and treatment monitoring by allowing for characterization and early identification of resistant subclonal populations. To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cells confined to droplets. The barcodes were then used to reassemble the genetic profiles of cells from next-generation sequencing data. We applied this approach to sequential clinical MDS samples, genotyping the most clinically relevant loci across more than 15,000 individual cells. Additionally, to study effects of subclonal mutations on drug sensitivity, ex vivo functional testing was performed on red blood cell-lysed peripheral blood and/or bone marrow aspirate patient samples. Targeted single-cell sequencing was able to recapitulate bulk sequencing data from both peripheral blood and bone marrow aspirate samples. Furthermore, the single-cell nature of our approach enabled definitive determination of mutational co-occurrence within the same cell. For examples in one sample, bulk sequencing identified mutations in JAK2 and NRAS both at 3% variant allele frequency (VAF), where single-cell analysis suggested that these mutations were mutually exclusive, each defining a distinct subclone. Single-cell sequencing allowed for serial monitoring of clonal evolution, with analysis of sequential samples from this same patient showing increase of NRAS clone from 2.5% to 24.7% at time of disease progression after hypomethylating agent therapy. Furthermore, single-cell analysis was able to identify a distinct subclone characterized by a KRAS mutation (0.4% at initiation of therapy and 6.7% at relapse), missed by serial bulk sequencing. Taken together, our results suggest a greater degree of heterogeneity in MDS samples than suggested with bulk sequencing methods alone, and demonstrate utility of single-cell sequencing for sequential monitoring and identification of resistant clones prior to therapy initiation. We show here that this approach is a feasible, scalable, and effective way to identify and track heterogeneous populations of cells in MDS. Citation Format: Alexey Aleshin, Peter L. Greenberg, Bruno C. Medeiros, Diane Heiser, Marianne Santaguida, Sacha Prashad, Robert Durruthy- Durruthy, Dennis J. Eastburn. Single-cell mutational profiling of clonal evolution in myelodysplastic syndromes (MDS) during therapy and disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3004.

Abstract 5548: The therapeutic potential of AMV564, a novel bispecific bivalent (2x2) T-cell engager, for the treatment of CD33-expressing hematologic malignancies

Abstract AMV564 is a novel bispecific bivalent (2x2) immunotherapy that initiates an immunological synapse between T cells and CD33-expressing cells by binding both CD33 and CD3 with strong avidity. CD33 (Siglec-3) is a clinically-validated antigen target that is broadly expressed on the surface of malignant cells in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and other hematologic malignancies, as well as on cells that potently suppress immune function, such as myeloid-derived suppressor cells (MDSC). AMV564 has been shown to eliminate MDSCs and restore hematopoiesis, induce potent cytolysis of leukemic cells in patient blood and bone marrow samples, and produce robust antitumor activity in a disseminated model of AML. In vitro studies, including cytotoxicity assays with AML patient samples, human T-cell activation assays, and cytokine-release assays, as well as animal safety studies were performed to characterize the pharmacokinetics, pharmacodynamics, and potential toxicities of AMV564. AMV564 demonstrated potent in vitro antitumor activity with EC50 values of approximately 0.7 - 3 pM. In vitro studies also demonstrated that leukemic cells are more sensitive to T-cell engagement using AMV564 than peripheral blood mononuclear cells by approximately 10-fold. In animal safety studies, AMV564 was tolerated at exposures that exceed those expected to have clinical antitumor activity by &amp;gt; 10-fold. Toxicological findings noted in these studies were anticipated based on the pharmacological activity of AMV564; observed findings included events related to CD3-mediated T-cell activation, cytokine release, and decreases in neutrophil and monocyte counts with rapid recovery. Results from a pharmacokinetic/pharmacodynamic model integrating the preclinical data suggest the potential for a wide therapeutic index for patients with AML. Based on the in vitro and in vivo preclinical data, AMV564 is expected to have a wide therapeutic index, with robust antitumor activity in AML and MDS at doses well below those predicted to be associated with dose-limiting toxicities, and a predictable and monitorable adverse event profile. Citation Format: Tae H. Han, Jeanmarie Guenot, William S. Denney, Eric J. Feldman. The therapeutic potential of AMV564, a novel bispecific bivalent (2x2) T-cell engager, for the treatment of CD33-expressing hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5548.

Luteolin induces myelodysplastic syndrome‑derived cell apoptosis via the p53‑dependent mitochondrial signaling pathway mediated by reactive oxygen species.

Luteolin, a common dietary flavonoid, induces the apoptosis of cells in several types of cancer. However, its role in myelodysplastic syndrome (MDS) and the potential underlying mechanisms remain to be elucidated. To evaluate the potential benefit and underlying mechanisms of luteolin in MDS cells, the viability of SKM‑1 cells and primary bone marrow (PBM) mononuclear cells from patients with intermediate‑ or high‑risk MDS were assessed using a Cell Counting Kit‑8 assay. The apoptotic features of cell morphology were assessed using Wright‑Giemsa staining, DNA fragmentation was analyzed by agarose gel electrophoresis, and the extent of apoptosis was quantified by flow cytometry (FCM). Reactive oxygen species (ROS) were measured by FCM with 2,7‑dichlorodihydrofluorescein diacetate staining and mitochondrial membrane potential (ΔΨm) was determined using 5,5',6,6'‑tetrachloro‑1,1',3,3'‑tetraethylbenzimidazolylcarbocyanine iodide staining. Caspase activity was detected using a fluorometric protease assay. Furthermore, the effects of luteolin on the expression of apoptosis‑related proteins were analyzed using western blot analysis. The resulting data revealed that luteolin significantly inhibited the proliferation of SKM‑1 cells invitro, and its half maximal inhibitory concentration was 139.41µM at 24h and 23.95µM at 72h. Luteolin also markedly inhibited the proliferation of mononuclear cells from patients with intermediate‑ or high‑risk MDS. Luteolin suppressed cell proliferation, mainly as a result of the induction of apoptosis, as demonstrated by typical apoptotic morphological features, the ladder pattern of genomic DNA fragmentation, and the results of FCM using Annexin V‑FITC/PI double staining. It was also found that short‑term exposure of SKM‑1 cells to luteolin led to a marked increase in the accumulation of ROS. The increased intracellular level of ROS appeared to induce the activation of p53 and elevate the B‑cell lymphoma 2 (Bcl‑2)‑associated X protein/Bcl‑2 ratio, which modulates ΔΨm and triggers the release of cytochrome c, and may increase the activities of apoptotic protease activating factor 1, caspase‑3, ‑8 and ‑9 to further trigger the destruction of structural and specific proteins and thereby cell apoptosis. Notably, the inhibition of ROS generation by the antioxidant N‑acetyl‑L‑cysteine significantly attenuated the luteolin‑induced loss of ΔΨm and activities of caspase‑3, ‑8 and ‑9. These data suggested that luteolin exerts its pro‑apoptotic action partly through the p53‑dependent mitochondrial signaling pathway mediated by intracellular ROS, which provides a promising therapeutic candidate for patients with MDS.

Transforming growth factor β1-mediated functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia.

Mesenchymal stromal cells are involved in the pathogenesis of myelodysplastic syndromes and acute myeloid leukemia, but the underlying mechanisms are incompletely understood. To further characterize the pathological phenotype we performed RNA sequencing of mesenchymal stromal cells from patients with myelodysplastic syndromes and acute myeloid leukemia and found a specific molecular signature of genes commonly deregulated in these disorders. Pathway analysis showed a strong enrichment of genes related to osteogenesis, senescence, inflammation and inhibitory cytokines, thereby reflecting the structural and functional deficits of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia on a molecular level. Further analysis identified transforming growth factor β1 as the most probable extrinsic trigger factor for this altered gene expression. Following exposure to transforming growth factor β1, healthy mesenchymal stromal cells developed functional deficits and adopted a phenotype reminiscent of that observed in patient-derived stromal cells. These suppressive effects of transforming growth factor β1 on stromal cell functionality were abrogated by SD-208, an established inhibitor of transforming growth factor β receptor signaling. Blockade of transforming growth factor β signaling by SD-208 also restored the osteogenic differentiation capacity of patient-derived stromal cells, thus confirming the role of transforming growth factor β1 in the bone marrow microenvironment of patients with myelodysplastic syndromes and acute myeloid leukemia. Our findings establish transforming growth factor β1 as a relevant trigger causing functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia and identify SD-208 as a candidate to revert these effects.

The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34+CD123+ cells from acute myeloid leukemia/myelodysplastic syndrome patients and healthy donors.

Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123− lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a “bridge-to-transplant” before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.

Changes of signal transductivity and robustness of gene regulatory network in the carcinogenesis of leukemic subtypes via microarray sample data.

Mutation accumulation and epigenetic alterations in genes are important for carcinogenesis. Because leukemogenesis-related signal pathways have been investigated and microarray sample data have been produced in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and normal cells, systems analysis in coupling pathways becomes possible.Based on system modeling and identification, we could construct the coupling pathways and their associated gene regulatory networks using microarray sample data. By applying system theory to the estimated system model in coupling pathways, we can then obtain transductivity sensitivity, basal sensitivity and error sensitivity of each protein to identify the potential impact of genetic mutations, epigenetic alterations and the coupling of other pathways from the perspective of energy, respectively. By comparing the results in AML, MDS and normal cells, we investigated the potential critical genetic mutations and epigenetic alterations that activate or repress specific cellular functions to promote MDS or AML leukemogenesis. We suggested that epigenetic modification of β-catenin and signal integration of CSLs, AP-2α, STATs, c-Jun and β-catenin could contribute to cell proliferation at AML and MDS. Epigenetic regulation of ERK and genetic mutation of p53 could lead to the repressed apoptosis, cell cycle arrest and DNA repair in leukemic cells. Genetic mutation of JAK, epigenetic regulation of ERK, and signal integration of C/EBPα could result in the promotion of MDS cell differentiation. According to the results, we proposed three drugs, decitabine, genistein, and monorden for preventing AML leukemogenesis, while three drugs, decitabine, thalidomide, and geldanamycin, for preventing MDS leukemogenesis.

SPAG6 silencing induces apoptosis in the myelodysplastic syndrome cell line SKM‑1 via the PTEN/PI3K/AKT signaling pathway in vitro and in vivo.

Apoptosis is a multi-step mechanism of cell self‑destruction for maintaining cellular homeostatic balance. Accumulating evidence indicates that abnormal apoptosis promotes the evolution and progression of myelodysplastic syndromes (MDS). As a novel cancer-testis antigen, sperm‑associated antigen6 (SPAG6) has been reported to regulate apoptosis through the tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in the MDS cell line SKM‑1. However, the mechanism of the intrinsic cell death pathway for apoptosis induction by SPAG6 silencing is unclear. In the present study, the invitro effects of SPAG6 silencing were investigated in SKM‑1 cells through extensive biochemical and molecular approaches. Western blotting and reverse transcription-quantitative polymerase chain reaction were used to detect the expression of SPAG6 and activation of PTEN/PI3K/AKT signal pathway. Additionally, SKM‑1 cells transduced with SPAG6 short hairpin RNA (shRNA) lentivirus were treated with the phosphatidylionositol 3-kinase (PI3K) inhibitor LY294002 or pan caspase inhibitor z‑VAD‑fmk and the apoptosis rates were measured by flow cytometry, and the expressions of associated proteins were examined by western blot analysis. A mouse xenograft model was also used to further evaluate the effects of SPAG6 knockdown on inducing tumor apoptosis invivo. Lentivirus-mediated knockdown of SPAG6 in SKM‑1 cells increased phosphatase and tensin homolog (PTEN) expression and reduced protein kinaseB (AKT) phosphorylation, which in turn resulted in cell apoptosis as evidenced by induced myeloid leukaemia cell differentiation protein Mcl‑1 downregulation, cytochromec release and increased caspase‑9 expression. Consistently, the PI3K inhibitor LY294002 synergistically enhanced apoptosis of SKM‑1 cells when co-administered with SPAG6 shRNA lentivirus. Furthermore, treatment with the pan caspase inhibitor z‑VAD‑fmk failed to prevent PTEN activation upon SPAG6 knockdown, suggesting that SPAG6-regulated PTEN expression was caspase activation-independent. In addition, SPAG6 knockdown was associated with DNMT1 downregulation, implying that SPAG6 may indirectly control PTEN expression via DNA methylation. Furthermore, tumor tissues from nonobese diabetic/severe combined immunodeficient mice inoculated with SPAG6-shRNA lentivirus pre-infected SKM‑1 cells exhibited significantly elevated apoptosis in the extrinsic and intrinsic pathways. These results demonstrate that SPAG6 silencing induces PTEN expression to regulate apoptosis though the PI3K/AKT pathway, indicating that SPAG6 may be a potential therapeutic target for MDS.

Cytokines increase engraftment of human acute myeloid leukemia cells in immunocompromised mice but not engraftment of human myelodysplastic syndrome cells.

Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not. We compared the engraftment of acute myeloid leukemia cells and myelodysplastic syndrome cells in NSG mice to that in NSG-S mice, which have transgene expression of human cytokines. We observed that only 50% of all primary acute myeloid leukemia samples (n=77) transplanted in NSG mice provided useful levels of engraftment (>0.5% human blasts in bone marrow). In contrast, 82% of primary acute myeloid leukemia samples engrafted in NSG-S mice with higher leukemic burden and shortened survival. Additionally, all of 5 injected samples from patients with myelodysplastic syndrome showed persistent engraftment on week 6; however, engraftment was mostly low (<2%), did not increase over time, and was only transiently affected by the use of NSG-S mice. Co-injection of mesenchymal stem cells did not enhance human myelodysplastic syndrome cell engraftment. Overall, we conclude that engraftment of acute myeloid leukemia samples is more robust compared to that of myelodysplastic syndrome samples and unlike those, acute myeloid leukemia cells respond positively to human cytokines, whereas myelodysplastic syndrome cells demonstrate a general unresponsiveness to them.

Severe hypoxia selects hematopoietic progenitors with stem cell potential from primary Myelodysplastic syndrome bone marrow cell cultures.

Myelodysplastic Syndromes (MDS) are clonal neoplasms where stem/progenitor cells endowed with self-renewal and capable of perpetuating the disease have been demonstrated. It is known that oxygen tension plays a key role in driving normal hematopoiesis and that hematopoietic stem cells are maintained in hypoxic areas of the bone marrow (BM). Hypoxia could also regulate leukemic/dysplastic hematopoiesis. We evaluated the stem cell potential of MDS cells derived from the BM of 39 MDS patients and selected under severe hypoxia. MDS cells rescued from hypoxia-incubated cultures were subjected to stem and progenitor cell assays in vitro, as well as to hematopoietic reconstitution assay in NOD-SCID mice. Incubation in severe hypoxia of cells explanted from MDS patients selected a cell subset endowed with stem cell potential, as determined in vitro. This occurred only from the BM of patients classified as IPSS low/INT-1 risk. Transplantation into NOD-SCID mice confirmed using an in vivo model that severe hypoxia selects a cell subset endowed with stem cell potential from bone marrow mononuclear cells (BMMC). derived from patients belonging to the IPSS low/int-1 risk group. Data here reported show that cells endowed with stem cell potential and capable of adapting to hypoxia and escaping hypoxia-induced apoptosis exist within MDS cell populations.

Clonal hematopoiesis in aplastic anemia

Aplastic anemia (AA) is an autoimmune-mediated bone marrow failure syndrome. While AA is not a malignant disease, clonal hematopoiesis is commonly detected via next-generation sequencing and single nucleotide polymorphism (SNP) array. Clonal hematopoiesis in AA has been confirmed by the detection of classic X chromosome skewing, PNH clones, UPD6p, and various mutations. The most frequent genetic events in AA are UPD6p and somatic mutations in BCOR/BCORL1, PIGA, DNMT3A, and ASXL1. While some mutations are common between patients with AA and healthy elderly donors, UPD6p and PIGA mutations are specific to clonal cells in AA, which need to manage their highly autoimmune extrinsic environment. During the evolution of AA into myelodysplastic syndrome (MDS), additional genetic events are frequently acquired that provide MDS cells with intrinsic survival benefits. Hematopoietic cells in AA appear to achieve clonal expansion by their escape from recognition and cytotoxicity by CD8 T-cells, accounting for the distinct landscape of genetic events observed in AA.

Abnormal CD25 expression on hematopoietic cells in myelodysplastic syndromes

ObjectiveTo investigate the frequencies and biological characteristics of CD25 positive hematopoietic stem cells (HSC) in myelodysplastic syndromes. MethodsThe expression of CD25 on HSC in bone marrow derived from patients with untreated MDS patients, untreated AML patients and normal controls were accessed by flow cytometry (FCM). The correlation analysis was done between CD25+ HSC and clinical parameters in MDS patients. ResultsThe expression of CD25 on HSC (CD34+CD38− cells) in MDS patients (28.81%) was significantly higher than that in normal controls (9.41%, P = 0.020), which similar to that in AML patients (32.54%, P = 0.410). The CD25 expression on HSC was positively correlated with the CD123 expression on HSC (r = 0.602, P = 0.008). The expression of CD25 on HSC in high-risk MDS group (53.27%) based on IPSS score was significantly higher than that in low-risk MDS group (18.66%, P = 0.003). In MDS patients, CD25+ HSC were negatively correlated with the counts of neutrophils (r = −0.684, P = 0.002) and platelets (r = −0.561, P = 0.015), while positively correlated with the percentage of blasts in bone marrow (r = 0.596, P = 0.009). The CD25 expression on erythroblasts had a significant positive correlation with red blood cell counts in MDS patients (r = 0.536, P = 0.012). ConclusionsCD25 was over-expressed on HSC in MDS patients, especially in high-risk MDS patients. Increased CD25+ HSC was correlated with progression of MDS. Low-expression of CD25 on erythroblasts might correlate with anemia in MDS patients. CD25 could be a specific marker of LSC in MDS, and could involve in the mechanisms of development and progression of MDS.

Effect of Mir-16 Targeting VEGF on Angiogenesis of Myelodysplastic Syndromes

Abstract Background: As key factors in gene post-transcriptional regulation, microRNAs (miRNAs) have been identified to play important roles in carcinogenesis in various tumors. Myelodysplastic syndrome(MDS) is a group of clonal myeloid disorders characterized by refractory quantitative and qualitative abnormalities of hemocytes and its pathogenesis is poorly understood. Some studies have showed that abnormal expressions of some miRNAs have close relationship with the pathogenesis of MDS. Recently, high VEGF expression is found common in all kinds of myelodysplastic syndromes including patients but its mechanism remains unclear. Methods The expression levels of miR-16 in RNA of normal controls, myelodysplastic syndrome (MDS) patients and cell lines including HL-60 cell, THP-1 cell, SKM-1 cell and K562 cell line were determined by qRT-PCR. The VEGF-secreted level in the serum of normal controls, MDS patients and those cell lines were detected by ELISA. The level of VEGF protein in cell lines were detected by the method of immunofluorescence. Then, constracting the lentivirus-based has-miR-16 to overexpress or silence miR-16 in the SKM-1 cell line. The levels of VEGF, bFGF, MMP2 and CTGF were detected by ELISA using the culture supernatant of SKM-1 cell line. The supernatant of SKM-1 cell line overexpressed or silenced miR-16 and HUVEC cell line were co-cultured. The effects of miR-16 on the migration ability and ability of HUVEC cells were measured by transwell chamber penetration test and tube formation test respectively. Results: The expression level of miR-16 in low-risk group was higher than that in normal control group, while it was lower in low-risk group than that in normal control group. The level of VEGF secretion in the low-risk group and the high-risk group were higher than that in the normal control group, and it was higher in the high-risk group than that in the low-risk group.The level of VEGF secretion and VEGF protein expression in the four cell lines were listed: K562>SKM-1>THP-1>HL-60; The expression level of miR-16 were illustarated: THP-1>SKM-1>K562>HL-60. Overexpression/silencing miR-16, the transfection rate was more than 80%, the expression level of miR-16 and VEGF showed: overexpression miR-16, the expression of miR-16 group was higher than that of the control group, while the expression level of VEGF was decreased; silencing miR-16, the expression of miR-16 group was lower than that of the control group, while the expression level of VEGF was increased. The supernatant of overexpression/silencing miR-16 and HUVEC cell were co-cultured: Overexpression miR-16, the migration and tube formation ability of HUVEC cell were reduced in miR-16 group than that of the control group; silencing miR-16, the migration and tube formation ability of HUVEC cell were increased in miR-16 group than that of the control group. Conclusions: Compared with the normal control group, the expression of miR-16 was decreased in high-risk MDS patients and negatively correlated with the expression of VEGF, and the decrease level of miR-16 may be a sign of poor prognosis; Overexpression/silencing miR-16, it was further confirmed that miR-16 was negatively correlated with VEGF expression; meanwhile, miR-16 could significantly affected the tube formation and migration ability of HUVEC cell by negative regulation of VEGF. Download : Download high-res image (96KB) Download : Download full-size image Figure . Disclosures No relevant conflicts of interest to declare.

Overexpression of Arginase 1 is linked to DNMT3A and TET2 mutations in lower-grade myelodysplastic syndromes and chronic myelomonocytic leukemia

Immune dysregulation is a common feature of myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML), particularly in early stages. However, the genetic basis remains poorly understood. We recently reported that macrophages from mice deficient in tet methylcytosine dioxygenase 2 (Tet2), a model of MDS/CMML, are hyperinflammatory and have increased expression of arginase 1 (Arg1). In macrophages and myeloid derived suppressor cells (MDSCs) expression of Arg1 contributes to T-cell suppression and immune evasion by L-arginine depletion, in the setting of chronic inflammation and cancer. Since human MDS and CMML are driven by TET2 mutations and associated with chronic inflammation, we hypothesized that arginase enzymatic activity and ARG1 expression would be increased in human MDS/CMML bone marrow. Elevated arginase activity was observed in bone marrow mononuclear cells of MDS and CMML patients with lower-grade features. Immunohistochemical studies confirmed that myelomonocytic cells overexpress ARG1. Additionally, mutations in the epigenetic regulators TET2 and DNMT3A corresponded to high ARG1 expression and activity. These findings suggest ARG1 is a biomarker of immune dysregulation in early MDS and CMML. Recent murine findings have implicated Tet2 and Dnmt3a in regulation of innate immunity. Our study suggests similar changes may be driven by human TET2 and DNMT3A mutations.

Safety and persistence of WT1-specific T-cell receptor gene−transduced lymphocytes in patients with AML and MDS

AbstractWilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR–gene transduced T-cell (TCR–T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519.