Shucheng Gu

Senescence of bone marrow mesenchymal stromal cells is accompanied by activation of p53/p21 pathway in myelodysplastic syndromes

The contribution of bone marrow mesenchymal stromal cells (BMMSCs) to the pathogenesis of myelodysplastic syndrome (MDS) has created controversies. In this study, we confirmed that BMMSCs from MDS patients showed prominent features of senescence, which were characterized by increased cell size, decreased proliferation and colony‐forming potential, alteration of cytoskeleton, and increased senescence‐associated β‐galactosidase (SA‐β‐Gal) activity. Interestingly, the apoptosis assay results showed that the percentage of apoptosis cells was very low and the difference was not significant between MDS patients and normal controls. Moreover, the osteogenic differentiation potential of BMMSCs from lower risk but not higher risk MDS was impaired, indicated by cytochemical stainings and reduced expressions of RUNX2. In addition, BMMSCs from MDS patients had impaired hematopoietic supporting function. Furthermore, the expression of p53 and p21 which played an important role in regulating the senescence progress of BMMSCs was significantly increased, whereas levels of p16 and pRb expression were not changed in the BMMSCs from MDS patients. Taken together, our comprehensive analysis shows that BMMSCs from MDS patients exhibited senescent behavior and activation of p53/p21 pathway probably played an important role in the senescence process.

Impaired osteogenic differentiation of mesenchymal stem cells derived from bone marrow of patients with lower-risk myelodysplastic syndromes

The pathogenesis of myelodysplastic syndromes (MDS) has not been completely understood, and insufficiency of the hematopoietic microenvironment can be an important factor. Mesenchymal stem cells (MSCs) and osteoblasts are key components of the hematopoietic microenvironment. Here, we measured the expression of multiple osteogenic genes in 58 MSCs from MDS patients with different disease stages and subtypes by real-time PCR and compared the osteogenic differentiation of MSCs from 20 MDS patients with those of MSCs from eight normal controls quantitatively and dynamically. The mRNA level of Osterix and RUNX2, two key factors involved in the early differentiation process toward osteoblasts, was significantly reduced in undifferentiated MSCs from lower-risk MDS. After osteogenic induction, lower-risk MDS showed lower alkaline phosphatase activity, less intense alizarin red S staining, and lower gene expression of osteogenic differentiation markers; however, higher-risk MDS was normal. Finally, in bone marrow biopsy, the number of osteoblasts was significantly decreased in lower-risk MDS. These results indicate that MSCs from lower-risk MDS have impaired osteogenic differentiation functions, suggesting their insufficient stromal support in MDS.

Interleukin-17 enhances the production of interferon-γ and tumour necrosis factor-α by bone marrow T lymphocytes from patients with lower risk myelodysplastic syndromes

Lower risk myelodysplastic syndromes (MDSs) are characterised by increased apoptosis of haematopoietic cells in the bone marrow (BM). The mechanism driving this excessive apoptosis involves multiple immune molecules, including inflammatory cytokines such as interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and interleukins (ILs). Interleukin‐17 (IL‐17) is the hallmark cytokine produced by CD4+ Th17 cells, and IL‐17 mediates activation of the adaptive T‐cell response inducing an inflammatory cytokine environment. However, little is known about the role of IL‐17 in MDS‐associated immune dysfunction.

Whole-exome and targeted sequencing identify ROBO1 and ROBO2 mutations as progression-related drivers in myelodysplastic syndromes

The progressive mechanism underlying myelodysplastic syndrome remains unknown. Here we identify ROBO1 and ROBO2 as novel progression-related somatic mutations using whole-exome and targeted sequencing in 6 of 16 (37.5%) paired MDS patients with disease progression. Further deep sequencing detects 20 (10.4%) patients with ROBO mutations in a cohort of 193 MDS patients. In addition, copy number loss and loss of heterogeneity (LOH) of ROBO1 and ROBO2 are frequently observed in patients with progression or carrying ROBO mutations. In in vitro experiments, overexpression of ROBO1 or ROBO2 produces anti-proliferative and pro-apoptotic effects in leukaemia cells. However, this effect was lost in ROBO mutants and ROBO-SLIT2 signalling is impaired. Multivariate analysis shows that ROBO mutations are independent factors for predicting poor survival. These findings demonstrate a novel contribution of ROBO mutations to the pathogenesis of MDS and highlight a key role for ROBO-SLIT2 signalling in MDS disease progression.

Bone Marrow Mesenchymal Stem Cells in Myelodysplastic Syndromes: Cytogenetic Characterization

Aim: This study compared genetic aberrations in hematopoietic cells (HCs) and mesenchymal stem cells of myelodysplastic syndrome (MDS-MSCs) patients. Methods: We obtained chromosomes with aberrations from 22 patients with MDS and chromosomes from 7 healthy individuals. Chromosomal aberrations in both HCs and MSCs were identified using G-banding. We then performed DNA content analysis of the HCs and MSCs. Results: Cytogenetic aberrations were detected in HCs from 13 of the 22 MDS patients (59%). Chromosomal aberrations in MSCs were detected in 15 of the 22 MDS patients (68%). No chromosomal abnormalities were identified in MSCs of the 7 healthy volunteers. We demonstrate herein that MSCs have distinct genetic abnormalities compared to HCs from the same individual. We observed a random loss of chromosomal material in significant proportions of MSCs. A high proportion of random loss may be a marker of chromosomal instability of MDS-MSCs. However, two case results showed that HCs and MSCs have different altered structural changes. Conclusion: Our results suggest enhanced genetic susceptibility of these cells in MDS patients. Our data indicates that the genetic alterations in MSCs may constitute a particular biological mechanism of MDS pathogenesis.

High expression of APAF-1 elevates erythroid apoptosis in iron overload myelodysplastic syndrome

Apoptotic protease-activating factor 1 (APAF-1) is a central component of the intrinsic pathway of apoptosis. Our study aims at searching the role of APAF-1 in iron overload myelodysplastic syndrome (MDS). Erythroid apoptosis rate, mRNA expression levels of APAF-1, and caspase-9 activity were determined by flow cytometry, quantitative real-time PCR, and colorimetric assay in MDS patients, respectively. In addition, K562 and MDS-L cell lines were incubated with different concentrations of ferric ammonium citrate (FAC) or ferric ammonium citrate + desferrioxamine (FAC + DFO) in vitro to observe the alteration in erythrocyte apoptosis rate, APAF-1 mRNA, and protein expression levels. Moreover, as control, erythroid apoptosis rate and APAF-1 mRNA expression were detected after silencing APAF-1 expression by endoribonuclease-prepared small interfering RNAs (esiRNAs) in K562 and MDS-L cell lines. Both erythroid apoptosis rate and APAF-1 mRNA expression of the iron overload (IO) group were significantly higher than those of the non-IO group (P < 0.001 and P < 0.001). There is a significant difference of caspase-9 activity between the IO group and the non-IO group (P < 0.001). Erythroid apoptosis rate and APAF-1 mRNA expression of K562 and MDS-L cell lines significantly elevated after FAC incubation in different concentrations (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L), while erythroid apoptosis rate and APAF-1 mRNA expression in the FAC + DFO group declined (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L). After silencing of APAF-1 expression with specific esiRNAs, erythroid apoptosis rate and APAF-1 mRNA expression of K562 and MDS-L cell lines markedly decreased (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L). APAF-1 plays an important role in iron-induced erythroid apoptosis increase in MDS.

Genomic loss of EZH2 leads to epigenetic modifications and overexpression of the HOX gene clusters in myelodysplastic syndrome

The role of EZH2 in cancer is complex and may vary depending on cancer type or stage. We examined the effect of altered EZH2 levels on H3K27 methylation, HOX gene expression, and malignant phenotype in myelodysplastic syndrome (MDS) cell lines and an in vivo xenograft model. We also studied links between EZH2 expression and prognosis in MDS patients. Patients with high-grade MDS exhibited lower levels of EZH2 expression than those with low-grade MDS. Low EZH2 expression was associated with high percentages of blasts, shorter survival, and increased transformation of MDS into acute myeloid leukemia (AML). MDS patients frequently had reductions in EZH2 copy number. EZH2 knockdown increased tumor growth capacity and reduced H3K27me3 levels in both MDS-derived leukemia cells and in a xenograft model. H3K27me3 levels were reduced and HOX gene cluster expression was increased in MDS patients. EZH2 knockdown also increased HOX gene cluster expression by reducing H3K27me3, and H3K27 demethylating agents increased HOX gene cluster expression in MDS-derived cell lines. These findings suggest genomic loss of EZH2 contributes to overexpression of the HOX gene clusters in MDS through epigenetic modifications.

Rigosertib as a selective anti-tumor agent can ameliorate multiple dysregulated signaling transduction pathways in high-grade myelodysplastic syndrome

Rigosertib has demonstrated therapeutic activity for patients with high-risk myelodysplastic syndrome (MDS) in clinical trials. However, the role of rigosertib in MDS has not been thoroughly characterized. In this study, we found out that rigosertib induced apoptosis, blocked the cell cycle at the G2/M phase and subsequently inhibited the proliferation of CD34+ cells from MDS, while it minimally affected the normal CD34+ cells. Further studies showed that rigosertib acted via the activation of the P53 signaling pathway. Bioinformatics analysis based on gene expression profile and flow cytometry analysis revealed the abnormal activation of the Akt-PI3K, Jak-STAT and Wnt pathways in high-grade MDS, while the p38 MAPK, SAPK/JNK and P53 pathways were abnormally activated in low-grade MDS. Rigosertib could markedly inhibit the activation of the Akt-PI3K and Wnt pathways, whereas it activated the SAPK/JNK and P53 pathways in high-grade MDS. A receptor tyrosine kinase phosphorylation array demonstrated that rigosertib could increase the activation of RET and PDGFR-β while reducing the activation of Tie2 and VEGFR2 in MDS cells. Taken together, these data indicate that rigosertib is a selective and promising anti-tumor agent that could ameliorate multiple dysregulated signaling transduction pathways in high-grade MDS.

Downregulation of p21 in Myelodysplastic Syndrome Is Associated With p73 Promoter Hypermethylation and Indicates Poor Prognosis

OBJECTIVES p21 Can both promote and inhibit tumorigenic processes. We explored the role of p21 in myelodysplastic syndrome (MDS). METHODS In this study, we analyzed p21 expression and p73 methylation in 88 patients with de novo MDS. RESULTS We found decreased expression of the p21 gene in higher-risk MDS compared with lower-risk groups or healthy controls (P < .05). Patients with p73 methylation had lower p21 than those in the unmethylated group (P < .001). Moreover, there was a significantly positive correlation between p73 and p21 expression in MDS (r = 0.436, P < .001). In vitro assays further confirm the role of p73 methylation in p21 expression. Compared with patients with normal expression levels of p21, patients with lower p21 expression levels experienced much higher rates of transformation to acute myeloid leukemia and lower overall survival both in univariate as well as multivariate analyses. CONCLUSIONS Our results suggest p21 expression may serve as a new biomarker to predict clinical outcome in patients with MDS.

The prognostic impact of multiparameter flow cytometry immunophenotyping and cytogenetic aberrancies in patients with multiple myeloma

Objectives: The aim of this study was to evaluate the prognostic impact of immunophenotyping in patients with multiple myeloma (MM), as well as other markers of disease, such as serum hyaluronan and cytogenetic aberrancies. Methods: We have prospectively analyzed the prognostic impact of antigenic markers, assessed by multiparametric flow cytometry (MFC), in a series of newly diagnosed MM patients (n = 79). Results and discussion: Our results show that the expression of CD44, CD45, and CD28 and the absence of CD117 were associated with a significantly shorter progression free-survival (PFS). Clinical characteristics were collected; Cytogenetic aberrancies were assessed in 40 patients. Multivariate survival analyses identified that the CD117−, CD28+, CD45+, and the percentage of bone marrow plasma cells by MFC are survival predictor, along with the International Staging System stage. Interestingly, the CD117− patients were associated with chromosomal aberrancies, including del (17p), +1q21, and IgH translocations. Conclusion: The incorporation of multiparameter flow cytometry immunophenotyping into the routine diagnostic evaluation of MM patients can help to identify patients at a high risk of progression.