High-risk Myelodysplastic Syndromes Cells Research Articles

Micrornas Analysis in Patients with Myelodysplastic Syndrome. Possible Implications in Risk Stratification

Background. MicroRNAs (miRNAs) are significant regulators of human hematopoietic stem cells (HSC), and their deregulation contributes to hematological malignancies. Systematic analysis of miRNA expression and function during hematopoiesis has revealed regulatory loops where miRNAs contribute to hematopoietic differentiation and function. Among the identified miRNAs, miR-155 can regulate myelopoiesis and erythropoiesis and its overexpression along with the overexpression of miR-125 and miR-181 has been suggested to have functional effects in the regulation of HSC homeostasis. Myelodysplastic syndromes (MDS) are characterized by dysfunctional HSC, ineffective blood cell production, progressive marrow failure, and exhibit a significant risk of progression to acute myeloid leukemia (AML). Although the pathogenesis of MDS has not been fully understood, various alterations of microRNAs (miRNAs) have been reported in MDS. There is recent evidence that the miRNAs mediated post-transcriptional control of gene expression is key component in the pathogenesis of MDS and AML. Several studies have reported that differentially expressed microRNAs are associated with the transformation of MDS to AML and clinical outcomes. Aim. In this study we examined the potential prognostic impact of six previously described miRNAS (miR-181a-2-3p, miR-124-3p, miR-550a-3p, miR-155-5p, miR-151a-3p, and miR-125b-5p) on the pathogenesis of MDS. Methods. Total RNA was extracted from diagnostic bone marrow samples of 41 MDS patients. Patients were stratified per the revised International Prognostic Scoring System (IPSS-R). After polyadenylation of 1 μg total RNA by poly(A) polymerase and subsequent reverse transcription with an oligo-dT adapter primer, miRNAs transcript levels were determined using a reverse transcription quantitative real-time PCR method, based on SYBR Green chemistry. U6 was used as reference gene. Relative microRNA transcript levels were calculated by the 2-ΔΔct method. The IBM SPSS statistics, version 26 (IBM Corporation, North Castle, NY, USA) was used for the statistical analysis of the results. Results. The basic characteristics of the patients are shown in Table 1. Of the microRNAs analyzed in this study, miR-125b-5p, miR-155-5p, miR-181a-2-3p transcript levels were significantly elevated in higher (intermediate, high, and very high) risk MDS than in lower (very low and low) risk MDS (p=0.012, p=0.010, and p=0.024 respectively). MiR-125b-5p, miR-151a-3p, miR-155-5p, miR-181a-2-3p, and miR-550a-3p presented a positive correlation with the percentage of bone marrow blasts [p(0.001-0.05)], while a negative correlation was observed between the expression levels of mir-151-a-3p, miR181a-2-3p, miR-550a-3p, and the neutrophil count (p=0.05, 0.013, 0.010, and 0.007 respectively). None of the remaining factors that were tested (MDS type per the 2016 WHO classification, gender, age at diagnosis, hemoglobin levels, platelet count, and cytopenias) were correlated with the miRNAs levels. Discussion. In this study we demonstrated the notable difference in the pattern of miRNA expression between lower and higher risk MDS patients. It has been shown that deregulation of miR-155-5p and miR-181a-2-3p may promote aberrant hematopoietic stem cell proliferation, differentiation, and progression to AML. Moreover, mice transplanted with miR-155-5p transfected stem cells developed a myeloproliferative disorder with abnormal granulocyte morphology analogous to MDS, suggesting a role in the higher risk MDS phenotype. Finally, it has been reported that miR-181a-3p was strongly expressed in the cytoplasm of high risk MDS cells, in contrast to low risk samples which displayed reduced levels. The identification of miRNAs that distinguish lower risk from higher risk MDS indicates that microRNA expression profile may be exploited to assist with making treatment decisions. Given that certain miRNAs can discriminate MDS subtypes and risk groups, it is not unlikely to think that an miRNA signature could be a useful tool for MDS risk stratification. Based on our current findings and other previously reported results, we continue the study in a larger cohort of patients. Targeting miRNAs may provide new treatment options for MDS Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Daunorubicin-Loaded CdTe QDs Conjugated with Anti-CD123 mAbs: A Novel Delivery System for Myelodysplastic Syndromes Treatment.

IntroductionThe myelodysplastic syndromes (MDS) are a very heterogeneous group of myeloid disorders characterized by peripheral blood cytopenias and increase risk of transformation to acute myeloid leukemia (AML). Daunorubicin (DNR) is an indispensable drug for the treatment of MDS and AML. However, its side effects including cardiac toxicity and bone marrow suppression severely limit clinical application. Many researches reported high expression of CD123 antigen on high-risk MDS cells, so we constructed a novel drug delivery system comprising daunorubicin-loaded CdTe QDs conjugated with anti-CD123 mAbs (DNR-CdTe-CD123) to develop targeted combination chemotherapy for MDS.MethodsCdTe conjugated antiCD123 through amide bond, co-loaded with DNR with electrostatic bonding. Then, we determined characterization and release rate of DNR-CdTe-CD123. The therapeutic effect and side effect of drug delivery system were evaluated through in vitro and in vivo experiments.ResultsCdTe showed appropriate diameter and good dispersibility and DNR was loaded into CdTes with high encapsulation efficiency and drug loading. The maximum drug loading and encapsulation efficiency were 42.08 ± 0.64% and 74.52 ± 1.81%, respectively, at DNR concentration of 0.2mg/mL and anti-CD123 mAbs volume of 5ul (100ug/mL). Flow cytometry (FCM) showed that CD123 antigen was highly expressed on MUTZ-1 cells, and its expression rate was 72.89 ± 10.67%. In vitro experiments showed that the inhibition rate and apoptosis rate of MUTZ-1 cells treated with DNR-CdTe-CD123 were higher than those in the other groups (P<0.05). Compared with the other groups, the level of apoptosis-related protein (P53, cleaved caspase-9, Bax and cleaved caspase-3) were upregulated in DNR-CdTe-CD123 group (P<0.05). In vivo experiments, DNR-CdTe-CD123 can effectively inhibit the tumor growth of MDS-bearing nude mice and reduce the side effects of DNR on myocardial cells.ConclusionThe system of DNR-CdTe-CD123 enhances the therapeutic effects and reduce the side effects of DNR, thus providing a novel platform for MDS treatment.

R-2HG/KDM2B/RIPK1 Signaling Mediates Necroptosis in Myelodysplastic Syndromes

Introduction: Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders and manifested as ineffective hematopoiesis, refractory cytopenia and a propensity to evolve into acute myeloid leukemia (AML). Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in both MDS and AML. Mutated IDH produces R-2-hydroxyglutarate (R-2HG) which inhibits multiple α-ketoglutarate/α-KG-dependent dioxygenases by competing against α-KG binding. Recent studies have demonstrated that R-2-HG can abrogates leukemic growth and induce leukemia cell death. Several nonapoptotic cell death have been identified, including phosphoribosyl pyrophosphate (PPRP)-1 mediated necrosis, pyroptosis, ferroptosis and necroptosis. By now, the specific type of cell death by which R-2-HG exerts anti-tumor effects is still unknown. Results: (1) (2R)-Octyl-2-HG exhibited anti-tumor effect on SKM-1, THP-1, Molm-13, HL-60 cell lines and bone marrow mononuclear cells from MDS and AML patients in a dose- and time-dependent manner. The overexpression of IDH2 mutation induced by doxycycline significantly decreased the viability of AML cell lines, and the inhibition rate was related to the dose of doxycycline. (2R)-Octyl-2-HG promotes apoptosis and causes G0/G1 phase arrest. (2) R-2-HG leads to increased expression of RIPK1 in high-risk MDS cells. The results of gene enrichment analysis indicated that the apoptotic pathway was enriched in (2R)-Octyl-2-HG groups, and the expression of RIPK1 gene was increased in all three (SKM-1, NOMO-1 and MA9.3ITD) (2R)-Octyl-2-HG groups. After treatment with (2R)-Octyl-2-HG, the mRNA and protein expression levels of RIPK1 gene were increased. (3) R-2-HG triggers RIPK1-dependent necroptosis and occurs earlier than apoptosis. Within 10 hours after treatment with (2R)-Octyl-2-HG, cell membrane permeability was disrupted, interactions between RIPK1 and caspase 8 increased, as well as phosphorylated MLKL level. However, caspase activity did not increase significantly, suggesting that necroptosis occurs earlier than apoptosis. With RIPK1 inhibitor Necrostatin-1 and (2R)-Octyl-2-HG co-treating cells, proliferation inhibition was reduced, cell membrane permeability was more stable and RIPK1-caspase8 complex was difficult to form. The same phenomenon occurs in SKM-1 cells stably transfected with RIPK1 shRNA virus, suggesting that RIPK1-dependent necroptosis is involved in cell death caused by (2R)-Octyl-2-HG. (4) In vivo experiments demonstrated that necroptosis by R-2-HG is dependent on the expression of RIPK. In RIPK1 shRNA MDS mice, the tumor burden showed a decreasing trend, but did not show a significant change in the R-2-HG treatment group. Treatment of scramble shRNA MDS mice with R-2-HG resulted in significantly smaller tumors in the spleen and less engrafment of CD45+ cells in bone marrow. (5) Low RIPK1 expression predicts poor prognosis in MDS patients. Data from the TCGA and GEO public databases indicate that RIPK1 expression is reduced in MDS and AML patients compared to healthy controls. Survival analysis showed that patients with lower RIPK1 expression levels had significantly shorter overall survival (OS) than patients with higher RIPK1 expression levels. MDS patients with lower RIPK1 expression levels progress to leukemia more frequently. (6) Inhibition of KDM2B induces necroptosis independently. Western blot assay shows the knockdown of KDM2B, upregulation of RIPK1 and increased levels of p-MLKL. Analysis of cell numbers showed that proliferation ceased from 4 days after doxycycline treatment onwards in shKDM2B cells. Co-IP assay shows the formation of RIPK1-caspase8 complex. Conclusion: This study confirmed that R-2-HG inhibited the viability of MDS and AML cells. R-2-HG increased the expression of RIPK1 in MDS cells, inducing necroptosis. Necroptosis occurred earlier than apoptosis. Inhibition of RIPK1 can alleviate the inhibitory effect of R-2-HG on MDS and AML cells. Clinical studies have shown that low expression of the RIPK1 gene is associated with poor prognosis in patients with MDS and AML. MDS patients with low expression of RIPK1 was more likely to progress to leukemia. Inhibition of KDM2B can induce necroptosis independently. Disclosures No relevant conflicts of interest to declare.

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.

Artesunate Enhanced Apoptosis of Human High Risk MDS Cells Induced By the DNMT Inhibitor Decitabine

Abstract: The present study evaluated whether the artesunate (ART) could enhance the apoptosis induced by decitabine (DAC) in SKM-1 cells, and to explore its potential mechanisms. Cytotoxicity of the combination of ART and DAC on SKM-1 cells was detected by CCK-8 assay and the apoptosis rate of high-risk myelodysplastic syndrome (MDS) cell line, SKM-1 cells was measured by flow cytometry. Protein expression levels of activated caspase-3, caspase-9, caspase-8, cleaved PARP and AIF in SKM-1 cells were measured by Western blot. Laser confocal microscope analysis showed AIF transfer to the nucleus. The growth inhibition rate and the apoptosis rate of SKM-1cells treated with the combination of ART and DAC was significantly increased compared with that of SKM-1 cells treated with the single agent (P<0.05). Both ART and DAC could induce caspase-dependent apoptosis, while ART, but not DAC, also induced caspase-independent apoptosis via AIF transfer from mitochondria to the nucleus. Additionally, the combination of the two agents induced cell death was not attenuated by caspase3,7 inhibitor Ac-DEVD-CHO. Our result suggested that the ART–DAC combination was more effective than single-agent therapy in vitro. Both compounds not only activated caspase-dependent pathway but also activated a caspase-independent mitochondrial pathway. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Cell Kinase (HCK) Is Highly Expressed in Myelodysplastic Syndrome (MDS) and Regulates Proliferation, Cell Cycle Progression and Hematopoietic Differentiation

AbstractAbstract 3813The pathogenesis of MDS is complex and remains elusive. The molecular characterization of this disease has been a tool to obtain a better understanding. Our group recently identified new possible target genes involved in MDS pathophysiology through the microarray analysis of CD34+ cells from MDS patients. One of the interesting overexpressed genes found was the HCK gene, a Src kinase family member. This family integrates signaling from a variety of cell-surface receptors, such as RTK, EPO receptor, chemokine receptor, MET and CXCR4, to regulate diverse cellular responses including proliferation, differentiation, apoptosis and migration. HCK is restrictedly expressed in hematopoietic cells and overexpressed in some types of solid cancer and in acute lymphoid leukemia; however, functional role of HCK in MDS and myeloid cells is still unknown. Here, using qPCR we investigated the HCK mRNA expression in CD34+ and total bone marrow (BM) cells from 54 MDS patients (FAB 39 low-risk and 15 high-risk), receiving no treatment, and 24 normal donors. We evaluated the role of HCK, studying its effect on proliferation, cell cycle and apoptosis by silencing this gene in U937 myeloid cell line. To inhibit HCK, specific shRNA expressing lentiviral vectors targeting the HCK gene or no specific sequence were used. Cell growth was measured by MTT colorimetric assay. Annexin-PI was used to evaluate apoptosis and cell cycle was analyzed by flow cytometry. These assays were carried out in lentiviral transduced cells treated, or not, with rapamycin(10 or 100nM) and LY294002(50nM). We further analyzed the HCK expression during granulocytic and erythroid differentiation of established leukemia cell lines models. Granulocytic differentiation was induced in NB4 or HL60 cells by treatment with ATRA(10−6 M) for 4 days. Erythroid differentiation was induced in KU812 cells by treatment with HE(50μM) and HU(100μM) for 4 days. We observed a significant increase in HCK mRNA expression of MDS CD34+ cells (P=0.002) and BM cells (P=0.03) compared to normal cells. HCK mRNA expression in low-risk MDS cells was higher compared to high-risk MDS cells. In U937 cells, HCK inhibition resulted in a significant decrease in proliferation compared to control cells (P<0.0001). Interestingly, the combination of HCK inhibition/rapamycin or LY treatment, inhibitors of PI3K/mTOR pathway, showed no reduction in cell proliferation compared to HCK inhibition alone. HCK inhibition induced cell cycle delay in G0/G1 phase and a consequent decrease in cell percentages in the S phase. The combination of HCK inhibition/rapamycin or LY treatment showed a significant decrease in cell number in G0/G1 phase (P=0.007) and increase in S phase (P=0.01) compared to HCK inhibited alone. Apoptosis was unaffected by HCK knockdown. HCK expression was significantly increased in NB4 or HL60 cells after granulocytic differentiation and in KU812 cells after erythroid differentiation (P<0.01). Furthermore, HCK mRNA was significantly up-regulated during erythroid differentiation of MDS low-risk CD34+ cells but not in normal CD34+ cells. Considering that some transmembrane receptor (CXCR4, MET, chemokine) expressions are altered in MDS patients and their downstream pathway is mediated by Scr family/PI3K, our results suggest that HCK overexpression in MDS may be related to alteration in the expression of some of these cell-surface receptors. Loss of HCK expression may contribute to disease progression by skewing the balance between proliferation and differentiation, perhaps giving malignant cells a proliferative advantage. Further, HCK inhibition in U937 cells potently decreases proliferation through PI3K/mTOR pathway since the combination of HCK inhibition/rapamycin or LY treatment showed no alteration in the proliferation. In addition, HCK protein might have role in cell cycle control by modulating cell cycle gene expression in G0/G1 phase check point. Moreover, granulocyte and erythroid differentiation results in an HCK overexpression probably because this gene is an adaptor protein that is essential for intermediating downstream signaling of activated cell-surface receptors. Our findings also indicate that HCK plays a role in myeloid cell abnormalities, through different cell activities, including proliferation, cell cycle and differentiation, suggesting that HCK represents a possible therapeutic target in MDS. Support: FAPESP, CNPq. Disclosures:No relevant conflicts of interest to declare.

Antitumor activity and drug interactions of proteasome inhibitor Bortezomib in human high-risk myelodysplastic syndrome cells

The purpose of this study was to investigate the antitumor effects and drug interactions of the proteasome inhibitor Bortezomib against high-risk myelodysplastic syndrome (MDS) cells in vitro and in vivo. The high-risk MDS-derived MUTZ-1 cell line and bone marrow mononuclear cells from primary high-risk MDS patients were used to examine antitumor activity and drug interactions for Bortezomib. Apoptotic proteins, including caspase and Bcl-2 family members, as well as the protein FLIP, were studied. Phosphoinositide 3-kinase (PI3K)/Akt and MAPK signaling pathways were also examined. The PI3K inhibitor LY294002 was used to examine the involvement of the PI3K/Akt signaling pathway in the induction of apoptosis. Cytarabine (AraC) and daunorubicin (DNR) were used to test for synergistic effects between Bortezomib and chemotherapeutic agents. SCID mice xenografted with MUTZ-1 cells were used for in vivo study. We found that Bortezomib could induce growth arrest and apoptosis in high-risk MDS cells in vitro and in vivo. The mechanisms were related to decreased activation of the PI3K/Akt survival signaling pathway, but not the MAPK pathway, and involved inhibition of the NF-κB activity and downregulation of the Bcl-2/Bax and FLIPL/FLIPS ratios, triggering the activation of caspase cascades. This phenomenon was inhibited by the PI3K inhibitor LY294002. Bortezomib also acted synergistically with the chemotherapeutic agents AraC and DNR, which are associated with the inhibition of NF-κB activity. Our results demonstrate that Bortezomib can induce growth arrest and apoptosis of high-risk MDS cells and had a synergistic effect with two chemotherapeutic agents. Our findings provide new insights for the treatment of high-risk MDS, using either Bortezomib alone, or in combination with conventional antineoplastic agents.

Identification of defects in the transcriptional program during lineage-specific in vitro differentiation of CD34 + cells selected from patients with both low- and high-risk myelodysplastic syndrome

Development of myelodysplastic syndrome (MDS) is suggested to follow a multistep pathogenesis and is characterized by accumulation of molecular defects of the hematopoietic stem/progenitor cells, resulting in aberrant differentiation and proliferation. To detect alterations within the transcriptional program in MDS-derived CD34(+) cells during lineage-specific differentiation, we performed serial gene expression analysis of in vitro differentiated erythro-, granulo-, and megakaryopoietic cells using oligonucleotide microarrays (HG-U133A, Affymetrix, Santa Clara, CA, USA). For selected genes, expression data were confirmed using real-time polymerase chain reaction. We identified genes with altered expression during lineage-specific differentiation in either low- or high-risk MDS cells compared to the expression patterns of continuously up- or downregulated genes from the normal transcriptional program of hematopoiesis. In cluster analyses, we could show that MDS samples have a distinct expression pattern of a set of selected genes compared to normal cells, which allows prediction of the affiliation of a sample to one group. Furthermore, this study gives an overview of genes that are differentially expressed in MDS cells compared to normal hematopoiesis. Our data provide the first comprehensive transcriptional analysis of differentiating human CD34(+) cells derived from MDS patients compared to normal individuals. It gives new insights into the alteration of differentiation and proliferation of MDS stem cells.

Antitumor Activity of Cda-2, An Extract from Unine, in a High-Risk Myelodysplastic Syndrome Cells in Vitro

DNA methyltransferase inhibitors (DNMTI), including 5-azacytidine and 5-aza-2′- deoxycytidine, are a new class of epigenetic drug, which exhibit higher response rates in myelodysplastic syndrome (MDS) patients. Cell differentiation agent (CDA-2) is a kind of urine extracts, which contains several DNMTIs. A phase IV clinical trials for MDS showed total response rate is 69.22%. In the present study, we investigated the mechanism of CDA-2 on MDS using high-risk MDS cell line namely MuTz-1. MTT assay results showed that CDA-2 significantly inhibit the cell growth at a dose and time-dependent manner. Flow cytometer anlyasis showed that this growth inhibition was remarkblely associated with cycle arrest in G1-phase, but not associated with apoptosis. In addition, CDA-2 increased the expression of CD11b/CD14, a pair markers representing cell differentiation. we found the spectrum of hypermethylated tumor suppressor genes (TIMP3, CDKN2B, CHFR, CD44, RASSF1, TP73, IGSF4, CDH13 and DAPK) in MuTz-1 cells by Methylation-Specific Multiplex ligation-dependent Probe amplification (MS-MLPA), but the hypermethylation of these genes were remarkable decreased, as well as the expressions of DNA methyltransferase genes DNMT1 and DNMT3B at mRNA and protein level were downregulated in the treatment for 3 days with CDA-2. Also, we used CDA-2 for treatment of three MDS patients, whose several tumor suppressor genes are hypermethylated. After tour weeks of treatment, all the hypermethylation genes were undetected, part of this result was verified by methylation specific PCR (MSP) and bisulphite sequencing. In conclusion, our results demonstrated that CDA-2 may be an effective agent targeting hepermethylated tumor suppressor genes on MDS.

The Akt/Mammalian Target of Rapamycin Signal Transduction Pathway Is Activated in High-Risk Myelodysplastic Syndromes and Influences Cell Survival and Proliferation

The Akt/mammalian target of rapamycin (mTOR) signaling pathway is important for both cell growth and survival. In particular, an impaired regulation of the Akt/mTOR axis has been strongly implicated in mechanisms related to neoplastic transformation, through enhancement of cell proliferation and survival. Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders characterized by ineffective hematopoiesis and by a high risk of evolution into acute myelogenous leukemia (AML). The pathogenesis of the MDS evolution into AML is still unclear, although some recent studies indicate that aberrant activation of survival signaling pathways could be involved. In this investigation, done by means of immunofluorescent staining, we report an activation of the Akt/mTOR pathway in high-risk MDS patients. Interestingly, not only mTOR was activated but also its downstream targets, 4E-binding protein 1 and p70 ribosomal S6 kinase. Treatment with the selective mTOR inhibitor, rapamycin, significantly increased apoptotic cell death of CD33(+) (but not CD33(-)) cells from high-risk MDS patients. Rapamycin was ineffective in cells from healthy donors or low-risk MDS. Moreover, incubation of high-risk MDS patient CD34(+) cells with rapamycin decreased the in vitro clonogenic capability of these cells. In contrast, the phosphoinositide 3-kinase inhibitor, LY294002, did not significantly affect the clonogenic activity of high-risk MDS cells. Taken together, our results indicate that the Akt/mTOR pathway is critical for cell survival and proliferation in high-risk MDS patients. Therefore, this signaling network could become an interesting therapeutic target for treating more advanced MDS cases.