Apoptosis In Myelodysplastic Syndromes Research Articles

Thiotert Induces Myelodysplastic Syndromes Cells Apoptosis by Activating Oxidative Stress

To explore whether thiotert treatment can inhibit proliferation and induce apoptosis in myelodysplastic syndromes (MDS) cells. CCK-8 assay was used for determining the cytotoxicity of thiotert to MDS cell line SKM-1 and the reversal effect of GSH, NAC, and Z-VAD-FMK on thiotert-induced inhibition of cell viability. EdU assay was deployed to detect the cell proliferation ability. Intracellular reactive oxygen species (ROS) was measured by flow cytometry after DCFH-DA staining. The expression of DNA damage- and apoptosis-related proteins was detected by Western blot. Thiotert treatment significantly suppressed the cell viability and proliferation ability in SKM-1 cells. A large amount of ROS generation and markedly elevated C-PARP, C-Caspase 3, and γ-H2AX were observed after thiotert administration, while BCL-2 was significantly decreased. In addition, GSH, NAC, and Z-VAD-FMK were able to mitigate the cytotoxicity of thiotert on SKM-1 cells. Thiotert can promote MDS cell apoptosis by mediating ROS production and pro-apoptotic proteins expression.

Abstract 74: Osteoblasts efferocytosis in bone marrow microenvironment induces osteoblasts’ senescence and apoptosis

Abstract Increased cellular apoptosis is commonly seen in myelodysplastic syndrome (MDS), but how clearance of apoptotic cells, especially neutrophils, impacts disease progression is unknown. Moreover, while dysfunction of bone marrow (BM) stromal cell populations has been described in patients with MDS, the mechanisms inducing stromal defects are not well understood. Our laboratory found that BM macrophages are defective in a murine model of MDS (Vav-Nup98 HoxD13, aka NHD13), with decreased efferocytotic rates and increased inflammatory mediators. The increased apoptotic burden and defective macrophages would be expected to recruit non-professional phagocytes (osteoblasts) in the bone and bone marrow. Based on these data, we hypothesize that OBs (osteoblasts) participate in the clearance of apoptotic cells in the bone marrow microenvironment (BMME) to maintain BM homeostasis. However, the increased osteoblastic efferocytosis burden in MDS contributes to OBs’ dysfunction as a mechanism of MDS-dependent BMME disruption that may lead to MDS progression. We isolated from wild-type (B6) mice bone-associated cells (BAC), which are enriched with OBs. In vitro co-culture with labeled human end-stage neutrophils at 1:1 and 1:10 (OB: PMN) increased OB apoptosis, while senescence was increased only in high-dose PMN co-culture. We found that these changes were cell-autonomous. In vivo injection of neutrophils into WT mice showed around 5-10% efferocytosis rate in OBs. In efferocytotic OBs, apoptosis rates were significantly increased. Together, these data show that OBs participate in efferocytosis and that efferocytosis disrupts OBs, causing apoptosis and senescence. To mimic the MDS microenvironment, we used a genetic model where one of the Isocitrate dehydrogenase 2 (IDH2) mutations found in MDS (R140Q) is targeted to hematopoietic cells via the Vav promoter (IDH2R140Q mice) as well as NHD13 mice, representing early and late MDS BMME respectively. We observed that 6-to-8 months old NHD13 mice experienced more end-stage neutrophils[CL1] in the BMME (4.6% rate of end-stage neutrophils in WT compared to 9.5% rate in NHD13 mice), where OB apoptosis was also increased in vivo. In IDH2R140Q mice at 3 months of age, we confirmed the presence of mutated IDH2 in bone marrow cells by increased intracellular levels of the oncometabolite 2-hydroxyglutarate. At this early time point, IDH2R140Q mice did not have increased rates of apoptosis in CD45+ cells, and we found no increase in OB apoptosis. Analysis of IDH2R140Q mice at later time points is ongoing. Our data identify novel mechanisms to explain the relationship between defective myeloid cells and OB apoptosis in MDS that may contribute to the inflammatory MDS microenvironment, impact disease progression, and serve as future therapeutic approaches for MDS patients. Citation Format: Chunmo Chen, Emily R. Quarato, Yuko Kawano, Noah A. Salama, Hiroki Kawano, Michael W. Becker, Jeevisha Bajaj, Laura Calvi. Osteoblasts efferocytosis in bone marrow microenvironment induces osteoblasts’ senescence and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 74.

Apoptosis in myelodysplasia: Association with patient age, bone marrow cellularity and karyotypes

Background: Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal hematopoietic stem cell diseases, characterized by dysplasias and apoptosis in bone marrow (BM) and cytopenias in peripheral blood. In this study, we analyzed apoptosis in MDS to verify associations with patient age, bone marrow cellularity and karyotypes and to investigate the role of apoptosis in MDS pathogenesis. Methods: Bone marrow cells were collected from 81 patients with primary MDS, of which 60 were adults and 21 children. BM cells were also collected from 10 healthy donors for bone marrow transplants, 5 adults and 5 children, as controls. The patients and controls came from public onco-hematology institutions in Rio de Janeiro. The percentage of apoptotic BM cells was assessed by flow cytometry using two combinations: annexin V-FITC/CD34PE/CD45PerCP and annexin V-FITC/CD14PE/CD45PerCP in BM cells. Cytogenetic analysis was performed by G-banding. Results: The comparison between adult and pediatric patients showed that these patients show a similar behavior with regard to apoptotic cells percentages in BM samples. Apoptosis occurs independently of BM cellularity, being more prominent in patients with hyper/normocellular BM. Patients with normal karyotypes, del(5q), del(17p) had higher apoptosis rates than patients with del(11q) and complex karyotypes. Cells committed to a differentiation program were associated with high rates of apoptosis, suggesting that apoptosis may be a consequence of inefficient hematopoiesis, such that the hematopoietic system may eliminate dysplastic cells at the beginning of the disease. Conclusions: Our results suggest that apoptosis is an important characteristic of BM cells from adult and pediatric MDS patients and may be a consequence of inefficient hematopoiesis. In addition, we suggest that apoptosis is not the main mechanism associated with hypocellular MDS, and it occurs preferentially in MDS cases of hyper/normocellular BM and is associated with a good prognosis. Keywords: Myelodysplastic Syndrome; Apoptosis; Patient age; Bone marrow cellularity; Karyotypes.

Selective ERBB2 and BCL2 Inhibition Is Synergistic for Mitochondrial-Mediated Apoptosis in MDS and AML Cells.

The ERBB2 proto-oncogene is associated with an aggressive phenotype in breast cancer. Its role in hematologic malignancies is incompletely defined, in part because ERBB2 is not readily detected on the surface of cancer cells. We demonstrate that truncated ERBB2, which lacks the extracellular domain, is overexpressed on primary CD34+ myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells compared with healthy hematopoietic cells. This overexpression of ERBB2 is associated with aberrant, oncogenic signaling with autophosphorylation of multiple tyrosine sites. Like in breast cancers, ERBB2 can exist as truncated isoforms p95ERBB2 and p110ERBB2 in MDS and AML. Neutralization of ERBB2 signaling with ERBB2 tyrosine kinase inhibitors (i.e., lapatinib, afatinib, and neratinib) increases apoptotic cell death and reduces human engraftment of MDS cells in mice at 21 weeks posttransplantation. Inhibition of ERBB2 modulates the expression of multiple pro- and anti-apoptotic mitochondrial proteins, including B-cell lymphoma 2 (BCL2). Dual blockade with ERBB2 and BCL2 inhibitors triggers additional reductions of BCL2 phosphorylation and myeloid cell leukemia-1 (MCL1) expression compared with single drug treatment. Dual therapy was synergistic at all tested doses, with a dose reduction index of up to 29 for lapatinib + venetoclax compared with venetoclax alone. Notably, these agents operated together and shifted cancer cells to a pro-apoptotic phenotype, resulting in increased mitochondrial cytochrome c release and activated caspase-3-mediated cell death. IMPLICATIONS: These findings warrant study of ERBB2 and BCL2 combination therapy in patients with MDS and AML. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/886/F1.large.jpg.

Upregulation of SPAG6 in Myelodysplastic Syndrome: Knockdown Inhibits Cell Proliferation via AKT/FOXO Signaling Pathway.

Recently, sperm-associated antigen 6 (SPAG6), a member of the cancer-testis antigen family, has been shown to be involved in tumorigenesis. An increasing number of studies have shown that SPAG6 expression is associated with the pathogenesis of myelodysplastic syndrome (MDS). However, the mechanism has not been clearly elucidated. Our previous results indicated that SPAG6 affected cell apoptosis in MDS. In this study, we used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to demonstrate that the mRNA expression of SPAG6 in bone marrow cells of patients with MDS or MDS-derived acute myeloid leukemia (MDS-AML) was higher than that of cancer-free patients. Kaplan-Meier survival curve analysis of published AML found that patients with high expression of SPAG6 had poor survival. The results of the cell counting kit-8, FACS, RT-qPCR, and Western blotting assays indicated that SPAG6 knockdown in the SKM-1 cell line inhibited cell proliferation, and affected cell cycle and differentiation. Furthermore, we found that SPAG6 knockdown affected the proliferation of SKM-1 cells by mediating the G1-to-S transition of the cell cycle. Moreover, we demonstrated that the antiproliferative effect of SPAG6 knockdown was associated with the upregulation of the cyclin-dependent kinase inhibitor p27Kip1, and regulation of the AKT/FOXO pathway. These findings indicated that SPAG6 might be a potential therapeutic target.

MicroRNA‑143 increases cell apoptosis in myelodysplastic syndrome through the Fas/FasL pathway both invitro and invivo.

Whilst the role of microRNA‑143 (miR‑143) in myelodysplastic syndrome (MDS) remains unclear, abnormally expressed microRNA‑143 has been detected in many types of cancer tissues. In this study, we describe a cohort study for the verification of miR‑143 expression, as well as the investigation of the molecular mechanisms of miR‑143 in MDS/acute myeloid leukaemia (AML). In a series of experiments, miR‑143 recombinant lentiviral vectors transformed into SKM‑1 cells were either overexpressed or knocked down, and the results illustrated that the overexpression of miR‑143 inhibited SKM‑1 cell growth, arrested the SKM‑1 cells in the G0/G1 phase, interfered with cell proliferation and induced cell apoptosis via the Fas/FasL pathway. Conversely, miR‑143 knockdown induced a decrease in the apoptosis and promoted the proliferation of SKM‑1 cells. Moreover, miR‑143 was shown to suppress MLLT3/AF9 expression by binding to its 3'‑UTR. Taken together, the findings of this study indicate that miR‑143 may be a critical regulator of MDS/AML cell carcinogenesis, acting as a potent antitumour molecular target for the diagnosis or treatment of cancers associated with the abnormal expression of MLLT3/AF9, hence facilitating the development of potential therapeutics against MDS/AML.

Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS.

A novel histone deacetylase inhibitor Chidamide induces G0/G1 arrest and apoptosis in myelodysplastic syndromes.

Chidamide as a newly designed and synthesized histone deacetylase inhibitor induces an antitumor effect in various cancer, and it has been used in several clinical trials such as peripheral T cell lymphoma (PTCL). Here we demonstrate that Chidamide was able to increase the acetylation levels of histone H3 and decrease HDAC activity in MDS cell lines(SKM-1,MUTZ-1)and AML cell line(KG-1). In vitro, at low concentration (<250nM) of Chidamide inhibited cell proliferation and delayed G0/G1 cell cycle progression by down-regulating CDK2 and regulating p-P53 and P21 protein expression. Meanwhile,it also induced cell apoptosis by down-regulating Bcl-2 and up-regulating cleaved Caspase-3 and Bax protein expression.The results of the present study demonstrates the potential utility of Chidamide for the treatment of Myelodysplastic syndromes.

Curcumin reduces the expression of survivin, leading to enhancement of arsenic trioxide-induced apoptosis in myelodysplastic syndrome and leukemia stem-like cells.

Low response, treatment-related complications and relapse due to the low sensitivity of myelodysplastic syndrome (MDS) and leukemia stem cells (LSCs) or pre-LSCs to arsenic trioxide (ATO), represent the main problems following treatment with ATO alone in patients with MDS. To solve these problems, a chemosensitization agent can be applied to increase the susceptibility of these cells to ATO. Curcumin (CUR), which possesses a wide range of anticancer activities, is a commonly used chemosensitization agent for various types of tumors, including hematopoietic malignancies. In the present study, we investigated the cytotoxic effects and potential mechanisms in MDS-SKM-1 and leukemia stem-like KG1a cells treated with CUR and ATO alone or in combination. CUR and ATO exhibited growth inhibition detected by MTT assays and apoptosis analyzed by Annexin V/PI analyses in both SKM-1 and KG1a cells. Apoptosis of SKM-1 and KG1a cells determined by Annexin V/PI was significantly enhanced in the combination groups compared with the groups treated with either agent alone. Further evaluation was performed by western blotting for two hallmark markers of apoptosis, caspase-3 and cleaved-PARP. Co-treatment of the cells with CUR and ATO resulted in significant synergistic effects. In SKM-1 and KG1a cells, 31 and 13 proteins analyzed by protein array assays were modulated, respectively. Notably, survivin protein expression levels were downregulated in both cell lines treated with CUR alone and in combination with ATO, particularly in the latter case. Susceptibility to apoptosis was significantly increased in SKM-1 and KG1a cells treated with siRNA-survivin and ATO. These results suggested that CUR increased the sensitivity of SKM-1 and KG1a cells to ATO by downregulating the expression of survivin.

Apoptosis in human myelodysplastic syndrome CD34+ cells is modulated by the upregulation of TLRs and histone H4 acetylation via a β-arrestin 1 dependent mechanism

Excessive apoptosis of hematopoietic precursors in the bone marrow underlies the ineffective hematopoiesis characteristic of myelodysplastic syndrome (MDS). Toll-like receptor (TLR) signaling is abnormally activated in MDS and may be involved in excessive programmed cell death in the pathogenesis of MDS. TLRs expression and global histone H3/H4 acetylation were analyzed in bone marrow (BM) CD34+ cells from 20 lower-risk and 20 higher-risk MDS patients and 10 healthy controls. Apoptosis of BM CD34+ cells was examined by flow cytometry, and its correlation to histone acetylation and the expression of TLR2 and β-arrestin1 (β-arr1), measured by enzyme-linkedimmunosorbent assay and qRT-PCR, was assessed. TLR1, TLR2 and TLR6 expression and H4 acetylation levels were higher in lower-risk MDS patients than in higher-risk MDS patients or controls, and TLR2 expression and H4 acetylation levels were positively correlated with an increased rate of apoptosis. Lower-risk MDS was associated with increased β-arr1 expression and histone acetyltransferase p300 activity. In in vitro-cultured primary normal and lower-risk MDS CD34+ cells, TLR2 activation-induced apoptosis was mediated by the upregulation of β-arr1 leading to the recruitment of p300 and increased histone H4 acetylation. The nuclear accumulation of βarr1 following TLR2 activation promote H4 acetylation at specific target gene promoters and may thus affect transcription of target genes in BM CD34+ cells. The mechanisms underlying the deregulation of TLR2 and increased apoptosis in MDS may involve the β-arr1 mediated recruitment of p300 leading to increased levels of histone H4 acetylation.

Downregulation of hTERT: An Important As2O3 Induced Mechanism of Apoptosis in Myelodysplastic Syndrome

Two myelodysplastic syndrome (MDS) celllines, MUTZ-1 and SKM-1 cells, were used to study the effect of arsenic trioxide (As2O3) on hematological malignant cells. As2O3 induced this two cell lines apoptosis via activation of caspase-3/8 and cleavage of poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme. As2O3 reduced NF-κB activity, which was important for inducing MUTZ-1 and SKM-1 cells apoptosis. As2O3 also inhibited the activities of hTERT in MUTZ-1 and SKM-1 cells. Moreover, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), had no effect on caspase-8 activation, although PDTC did inhibit MUTZ-1 and SKM-1 cells proliferation. Incubation of MUTZ-1 cells with a caspase-8 inhibitor failed to block As2O3-induced inhibition of NF-κB activity. Our findings suggest that As2O3 may induce apoptosis in MUTZ-1 and SKM-1 cells by two independent pathways: first, by activation of caspase-3/8 and PARP; and second, by inhibition of NF-κB activity, which results in downregulation of hTERT expression. We conclude that hTERT and NF-κB are important molecular targets in As2O3-induced apoptosis.

Increased CD34+CD38 -CD123 + cells in myelodysplastic syndrome displaying malignant features similar to those in AML.

Leukocyte interleukin-3 receptor α (CD123) is regarded as a marker of leukemia stem cells. We previously found that CD123 was also highly expressed on CD34(+)CD38(-) cells in myelodysplastic syndrome (MDS) patients, but it is unclear whether the level and the characteristics of CD34(+)CD38(-)CD123(+) cells in MDS are similar to those in acute myeloid leukemia (AML). Based on previous research by our team, we further enlarged the specimens and found that the mean proportion and the mean MFI of CD34(+)CD38(-)CD123(+) cells in low-grade MDS were lower than that in AML, and those in high-grade MDS were similar to those in AML. CD34(+)CD38(-)CD123(+) cells expressed lower granulocyte stimulating factor receptor, CD11b, and apoptosis molecule (Annexin V), meanwhile, these cells showed upregulation of transcription factors (GATA-1, GATA-2) and transferrin receptor (CD71) in MDS and AML. Furthermore, an increase in CD34(+)CD38(-)CD123(+) cells was closely related to the number of cytopenias involving hematopoietic lineages, anemia, blast count in bone marrow smear, fluorescence in situ hybridization analysis and WHO prognostic scoring system score. Thus, increases in CD34(+)CD38(-)CD123(+) cells may reflect malignant clonal cells with aberrant differentiation, overproliferation, and decreased apoptosis in MDS, which were similar to AML. CD123 may thus be a promising indicator for identifying malignant clonal cells in MDS and a candidate for targeted therapy.

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.

Transcriptional regulation of miR-10a/b by TWIST-1 in myelodysplastic syndromes

The transcription factor TWIST-1 is up-regulated in CD34(+) cells in myelodysplastic syndrome and is involved in resistance to apoptosis. There is evidence that TWIST-1 affects apoptosis via microRNAs (miRs). Expression of miRs was determined in myeloid cell lines and primary CD34(+) marrow cells from patients with myelodysplastic syndrome and healthy donors using NanoString/array and validated by real-time-polymerase chain reaction. Expression levels of miR10a and miR10b were significantly higher in CD34(+) marrow cells from 28 patients with myelodysplastic syndrome than in CD34(+) cells from healthy donors (P=0.05 and P=0.012, respectively). Levels of miR10a/b correlated with TWIST-1 miR levels in CD34(+) myelodysplastic marrow cells (miR10a, R=+0.69, P<0.0001; miR10b, R=+0.56, P=0.0008). Inhibition of miR10a/10b in clonal cells interfered with proliferation and enhanced sensitivity to apoptosis, which involved NF-κB-dependent p53 activation. These data support a role for miR10a/10b in the regulation of apoptosis in myelodysplastic syndrome and suggest the TWIST-1/miR10a/b-axis as a therapeutic target in myelodysplastic syndrome.

Overexpression of MCM2 in myelodysplastic syndromes: Association with bone marrow cell apoptosis and peripheral cytopenia

Myelodysplastic syndromes (MDS) are characterized by proliferation and apoptosis of bone marrow cells. Minichromosome maintenance protein (MCM) 2, which is known to be essential for regulating DNA replication, has proven to have a pro-apoptotic effect in our recent study. Thus, to determine the role of MCM2 in MDS, real-time PCR, immunohistochemistry and in vitro analysis were performed. Our results showed higher MCM2 expression in MDS than in control and AML. Notably, there was no correlation between MCM2 and Ki67-labeling indices (LIs) in MDS, while MCM2 LIs were significantly correlated with cleaved caspase 3 LIs in MDS. In vitro analysis revealed that MCM2 overexpression induced apoptosis in HL60 cells. Furthermore, MDS bone marrow exhibited higher ratio of MCM2 and cleaved caspase 3 double-positive cells and the ratio was correlated with the degree of leukocytopenia. These results suggest that the up-regulated expression of MCM2 is associated with frequent apoptosis in MDS and may have an important role in the pathogenesis of MDS.

Dissecting the Mechanism of Apoptosis and Leukemic Transformation In Myelodysplasia

AbstractAbstract 610A central question in the pathogenesis of the myelodysplastic syndromes (MDS) is the mechanism of apoptosis in early disease, and how this is overcome during disease progression to acute myeloid leukemia (AML). In humans, increased serum and bone marrow levels of TNF and/or FASL in early MDS implicate the cell-extrinsic apoptotic pathway, whereas increased expression of anti-apoptotic BCL2-related proteins (the cell-intrinsic pathway) has been linked to leukemic transformation. Here, we have employed the Vav-NUP98-HOXD13 (NHD13) transgenic mouse model to genetically dissect the molecular mechanisms underpinning apoptosis and leukemic progression in MDS. Three month old NHD13 mice recapitulate ‘low-risk’ human MDS with peripheral blood cytopenias, impaired in vitro progenitor cell growth, dysplasia of myeloid cells and megakaryocytes, and prominent apoptosis. Leukemic transformation in NHD13 mice occurs between 6 and 14 months of age. Using gene targeted mouse lines crossed with NHD13 mice, we show that neither TNF nor FasL are required for apoptosis. In contrast, enforced expression of Bcl2 rescues the early MDS phenotype including the macrocytic anemia, dysplasia of megakaryocytes, apoptosis and impaired erythroid and myeloid progenitor cell growth. Remarkably, enforced expression of Bcl2 completely prevented transformation to AML. Our results challenge many of the concepts regarding apoptosis in MDS. First, apoptosis is the predominant mechanism of peripheral cytopenias including erythroid-megakaryocyte dysplasia. Second, apoptosis is mediated by the BCL2-related cell intrinsic pathway. Third, overcoming apoptosis is unlikely to be an important mechanism of leukemic transformation. Indeed, our results extend recent evidence that apoptosis may be a ‘driver’ of leukemic transformation rather than a protective mechanism. These findings raise the therapeutic potential of BCL2 mimetic drugs for treating cytopenias and preventing leukemic transformation of MDS. Disclosures:No relevant conflicts of interest to declare.

Identification of DJ-1/PARK-7 as a determinant of stroma-dependent and TNF-α–induced apoptosis in MDS using mass spectrometry and phosphopeptide analysis

AbstractIn patients with myelodysplastic syndromes (MDS), apoptosis in hematopoietic cells is up-regulated in low-grade disease, whereas advanced disease is characterized by apoptosis resistance. We have shown that marrow stroma–derived signals convey sensitivity to tumor-necrosis-factor alpha (TNF-α)–mediated apoptosis in otherwise-resistant KG1a myeloid cells and CD34+ cells from MDS marrow. Here, we used a PhosphoScan proteomic liquid chromatography–mass spectrometry method to identify signals relevant for this effect. The transcription factor DJ-1/PARK-7 (DJ-1) was highly phosphorylated in KG1a cells cultured without stroma but dephosphorylated after stroma coculture, whereas expression of p53 increased significantly, suggesting a stroma contact-dependent effect of DJ-1 on p53. In CD34+ marrow cells from advanced MDS, expression of DJ-1 was up-regulated, whereas p53 levels were low, resulting in significantly greater DJ-1/p53 ratios than in patients with low-grade MDS (P = .01). DJ-1 levels were correlated with increasing International Prognostic Scoring System scores (P = .006). Increasing DJ-1/p53 ratios were associated with an increased risk of mortality, although the correlation did not reach statistical significance (P = .18). These data suggest that DJ-1/p53 interactions contribute to apoptosis resistance in clonal myeloid cells and may serve as a prognostic marker in patients with MDS.

Hematologic Disorders and Bone Marrow–Peripheral Blood Dynamics

Hematologic disorders such as the myelodysplastic syndromes (MDS) are discussed. The lingering controversies related to various diseases are highlighted. A simple biomathematical model of bone marrow - peripheral blood dynamics in the normal state is proposed and used to investigate cell behavior in normal hematopoiesis from a mathematical viewpoint. Analysis of the steady state and properties of the model are used to make postulations about the phenomenon of massive apoptosis in MDS. Simulations of the model show situations in which homeostatic equi- librium can be achieved and maintained. Consequently, it is postulated that hematopoietic growth factors may possess the capabilities of preventing oscillatory dynamics and enhancing faster evo- lution towards homeostatic equilibrium.

Amifostine has the potential to induce haematologic responses and decelerate disease progression in individual patients with low- and intermediate-1-risk myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are characterized by hypercellular bone marrow, peripheral cytopenia and an increased rate of intramedullary apoptosis. Oxidative stress is known as an important factor that leads to apoptosis in MDS. Thus, amifostine was investigated in a randomized, multicentre phase II-study ( n = 44 pts.; 22 amifostine, 22 best supportive care). We found an overall haematologic response rate of 18%. One patient developed a complete and persisting haematologic remission. Haematologic progression rate was 46% in the treatment group and 64% in the control group. We conclude that amifostine has the potential to induce haematologic response in individual patients suffering from MDS.

C-FLIP and apoptosis in myelodysplastic syndromes

In recent years,the morbidity of myelodysplastic syndromes(MDS) has been rising gradu-ally,a part of MDS patients may develop to malignant leukemia.It has already been proved that apoptosis plays an important role in the pathogenesy of MDS.c-FLIP is an apoptosis inhibitory protein,which takes part in the onset and development of many neoplasmas.Recently,c-FLIP has been studied generally,for its important role in regulating the process of apoptosis,as well as its various forms and effects. Key words: Apoptosis; Myelodysplastic syndromes; Apoptosis regulatory proteins