MDS-L Cells Research Articles

Withaferin A suppresses the growth of myelodysplasia and leukemia cell lines by inhibiting cell cycle progression.

Treatment outcomes for acute myeloid leukemia and myelodysplastic syndromes (MDS) remain unsatisfactory despite progress in various types of chemotherapy and hematopoietic stem cell transplantation. Therefore, there is a need for the development of new treatment options. We investigated the growth‐suppressive effects of withaferin A (WA), a natural plant steroidal lactone, on myelodysplasia and leukemia cell lines. WA exhibited growth‐suppressive effects on the cell lines, MDS‐L, HL‐60, THP‐1, Jurkat and Ramos, and induction of cell cycle arrest at G2/M phase at relatively low doses. Evaluation by annexin V/PI also confirmed the induction of partial apoptosis. Gene expression profiling and subsequent gene set enrichment analysis revealed increased expression of heme oxygenase‐1 (HMOX1). HMOX1 is known to induce autophagy during anticancer chemotherapy and is considered to be involved in the treatment resistance. Our study indicated increased HMOX1 protein levels and simultaneous increases in the autophagy‐related protein LC3A/B in MDS‐L cells treated with WA, suggesting increased autophagy. Combined use of WA with chloroquine, an autophagy inhibitor, enhanced early apoptosis and growth suppression. Together with the knowledge that WA had no apparent suppressive effect on the growth of human normal bone marrow CD34‐positive cells in the short‐term culture, this drug may have a potential for a novel therapeutic approach to the treatment of leukemia or MDS.

Quantitative proteomic analysis of histone modifications in decitabine sensitive and resistant leukemia cell lines

BackgroundThe refractory nature of many cancers remains the main health challenge over the past century. The epigenetic drug, decitabine (DAC), represents one of the most promising therapeutic agents in cancers particularly in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). However, its ambiguous anti-tumor mechanism and the unpredictable drug-resistant nature in some population compromise its application in cancer therapy. In crosstalk with DNA methylation, histone post-translational modifications (PTMs) are the key players in modulating the downstream epigenetic status of tumor suppressor genes. This study targets the role of decitabine in epigenetic regulation in leukemia therapy and searches responsive predictors and therapeutic targets for pretreatment evaluation and drug development.ResultsA simple, fast, and robust proteomic strategy identified 15 novel PTMs and 60 PTM combinations in two leukemia cell lines (MDS-L and TF-1). Histone modification profiles have been generated and compared between DAC sensitive and resistant groups (n = 3) in response to DAC treatment. Among these histone PTMs, five of which were found differentially upon DAC treatment in drug sensitive and resistant cells: H3.3K36me3, H4K8acK12acK16ac in MDS-L cells; and H3.1K27me1, H3.1K36me1, H3.1K27me1K36me1 in TF-1 cells. They may serve as biomarkers in predicting leukemia and drug responsiveness. In addition, we also explored PTM differences in two cell lines which were developed from early and advanced stages of AML. Three PTMs (H3.1K27me3, H3.1K27me2K36me2 and H3.3K27me2K36me2) are highly abundant in TF-1 cells (advanced AML cell line), suggesting their relevance to leukemogenesis. Our method allowed deep analysis of histone proteins and elucidation of a large number of histone PTMs with high precision and sensitivity.ConclusionDAC-induced DNA hypomethylation has wide impact on chromatin modifications. This study represents first effort to investigate the undefined epigenetic mechanism of decitabine in leukemia therapy. The identification of 15 novel PTMs and the discovery of several marks have relevance to epigenetic directed therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-016-9115-z) contains supplementary material, which is available to authorized users.

Growth inhibitory effects of Withaferin A on MDS-L cells – A human 5q Myelodysplastic Syndrome (MDS) cell line

Abstract Myelodysplastic syndrome (MDS) affects bone marrow production of white blood cells and erythrocytes. At least 40% of MDS patients who go into remission after treatment eventually progress to acute myelogenous leukemia (AML) which is very difficult to treat. Hence, there is a compelling need for new and better therapies. Withaferin A (WFA), a withanolide isolated from Winter cherry (Withania somnifera), has been shown to have potent anti-cancer properties in several animal models of cancer. We found that WFA inhibited the growth of MDS-L cells, a human cell line derived from a 5q MDS patient. Treatment with WFA induced apoptosis of MDS-L cells, indicated by the cleavage of caspase-3 as early as 6hrs after treatment and a dose dependent increase of annexin positive cells. Cell cycle analysis revealed that WFA caused MDS-L cells to arrest at S and G2/M and accordingly there was a reduction in cyclin A/CDK2 and cyclin B/CDK1 respectively. WFA also inhibited the activity of the heat shock protein (Hsp 90) chaperone as shown by the degradation of its client proteins such as Akt, an important pro-survival kinase and increased expression of Hsp70 which partially compensates for decrease in Hsp90 function. Interestingly, the mechanism by which WFA acts on MDS-L cells is independent of its well-recognized action on Nf-κB. Microarray analysis of gene expression in MDS-L cells showed a dramatic increase in several genes that may have a role in Nf-κB independent growth inhibitory effects of WFA. Studies with a xenograft model of MDS-L cells in NSGS mice suggest WFA might also be effective in vivo. The potent anti-proliferative and anti-survival effects of WFA on MDS-L cells, suggest that it could be a novel therapeutic agent for the treatment of 5q MDS.

Targeting the Hedgehog Signaling Pathway By PF-04449913 Limits the Self-Renewal of MDS-Derived Induced Potent Stem Cells (iPSC): Molecular Mechanisms

Background:Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by no efficient hematopoiesis and frequent progression to acute myeloid leukemia (AML). Even in low risk MDS, clonal hematopoiesis already dominates at diagnosis, and clones found in secondary AML originate from the MDS stage of disease, highlighting the need to specifically target the MDS-initiating clone. PF-0449913 is a potent and selective hedgehog pathway inhibitor that act by binding Smoothened (SMO) and blocking signal transduction. In xenograft models of human coloirectal and pancreatic cancer, treatment with PF-04449913 in combination with other anticancer agents reduced the tumor growth. Furthermore, PF-04449913 demonstrated preliminary antitumor activity in a phase I trial, when given as monotherapy in patients with several hematopoietic malignancy. In the present study, we investigated the molecular mechanisms by which PF-04449913 regulate the self-renewal of MDS-derived iPS cells (iPSCs) in vivo.Methods:We generated iPSCs from bone marrow mononuclear cells of two MDS patients (RAEB1 and RAEB2 by WHO clssification) with chromosome 5 deletion and complex karyotypic abnormalities, respectivly. Karyotyping analysis revealed that MDS-derived iPSCs have identical abnormalities to primary MDS cells. We also generated iPSCs from bone marrow mononuclear cells of normal volunteer as control. To investigate the effects of PF-04449913 on self-renewal and the relevance as a therapeutic target in MDS initiating cells, we examined the activity of PF-04449913 against MDS-derived iPSCs transferred NOD/SCID mice in vivo. NOD/SCID mice were injected sucutaneously with MDS-derived iPSCs or normal iPSCs then treated with PF-04449913 (100 mg/kg; p.o.) from day 10 for 28 days. We also used MDS-L, a myelodysplastic cell line establised from MDS patient with del(5q) and complex karyotypic abnormalities for in vitro studies.Results:Both MDS-derived iPSCs transferred NOD/SCID mice and normal iPSCs transferred NOD/SCID mice demonstrated the engraftment of CD34+CD38- positive cells by flow cytometry. However, the treatment with PF-04449913 reduced the population of CD34+CD38-positive cells in MDS-derived iPSCs transferred NOD/SCID mice. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of CD45+ cells into secondary recipients. Following 50 days, all mice treated with vehicle engrafted with CD34+CD38- positive cells. In contrast, CD34+CD38-positive cells engraftment was not detected in recipient mice (n=3) from PF-04449913-treated donors. These results demonstrate the persistent effects of PF-0449913 on long term self-renewing MDS-initiating cells. We further examined the effects of Nanog pathway modulation on in vitro clonogenic growth. CD34+CD38- cells from MDS-derived iPSCs transferred NOD/SCID mice and MDS-L cells were treated with 2 mM of PF-04449913 for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. PF-04449913 had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by PF-04449913 (p<0.001). To identify the mechanisms that limit the self-renewal of MDS-initiating ells by PF-04449913, NOD/SCID mice engrafted with CD34+CD38- fractions from MDS-derived iPSCs were treated with PF-04449913 (100 mg/kg; p.o.) for 14 days. PF-04449913 induced the expressions of p21Cip1, cleaved PARP and reduced the expression of BMI-1, c-Myc, Nanog, and Bcl-XL.Conclusion:Our preclinical results indicate that PF-04449913 have potential as an important option for controlling the drug-resistant MDS-initiating cells. It is expected that the PF-04449913 may become extremely useful therapeutic interventions in a number of hematological neoplasms, including MDS, where the persistence of cancer stem cells. DisclosuresOhyashiki:Sumitomo Dainippon: Membership on an entity's Board of Directors or advisory committees; Chugai Pharna KK: Research Funding; Bristol Meyer Squib KK: Research Funding; Jansen Pharma KK: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis Pharma KK: Honoraria, Research Funding, Speakers Bureau; Kyowa Kirin KK: Honoraria; MSD KK: Honoraria; Nippo Shinyaku KK: Speakers Bureau; Toyama Kagaku KK: Speakers Bureau; Shinbaio Pharma KK: Honoraria; Asteras: Research Funding; Alexion Pharma KK: Research Funding; Teijin Pharma KK: Research Funding; Asahikasei: Research Funding; Taiho Yakuhin KK: Research Funding.

Pharmacological Inhibition of Histone Demethylase JMJD3 Reduces Leukemia Cell Survival and Represses Production of the Cytokine CCL2 in MDS/AML

Deregulation of pro-inflammation signals plays an important role in MDS and AML. We have previously demonstrated that the histone H3 demethylase JMJD3 (KDM6B), a key regulator of inflammatory genes, is significantly overexpressed in the bone marrow progenitor cell population of patients with MDS (Wei et al, 2013). GSK-J4, a novel selective inhibitor against JMJD3, has been shown to modulate pro-inflammatory signals and affect cell growth in T-ALL and glioma. We therefore sought to evaluate the effect of GSK-J4 in MDS and AML.First, we observed that GSK-J4 significantly reduces the survival of MDS and AML cell lines (MDS-L and TF1) by 70% at concentrations 1-3 uM. These doses have no obvious cytotoxic effect on primary normal peripheral blood cells. The repression of survival was associated with induction of apoptosis, cell cycle blockade, and inhibition of p38MAPK activation. Second, we evaluated the impact of GSK-J4 on histone H3 methylation and found that the compound did not affect overall histone H3K4 and K27 methylation, which is consistent with previous findings (Ntziachristos et al, 2014).To pinpoint functionally relevant targets of JMJD3 inhibition in AML/MDS, we sought to identify disease-relevant inflammatory cytokines because JMJD3 binds and modulates promoters of many pro-inflammatory genes, particularly cytokines. We analyzed 38 inflammatory cytokines in bone marrow plasma specimens of patients with MDS (N=37) using a Luminex-based cytokine array, which revealed that levels of the cytokines CCL2 (also called MCP-1) and CCL11 (also called eotaxin-1) are significantly elevated in patients compared to healthy controls by 700 and 330 fold respectively (p=0.002 and 0.04, respectively). Both CCL2 and 11 are c-c motif chemokines whose encoding genes are located in the human chromosome 17q cytokine gene cluster. Furthermore, compared to pre-treatment samples, CCL2 was significantly elevated in paired plasma samples that were collected after hypomethylating agent (HMA) treatment (2.5 fold increase, N=11 pairs, p=0.04). Luminex results were confirmed by cytokine-specific ELISA. Previous studies have shown that JMJD3 binds to CCL2 gene promoter in mouse macrophage cells (De Santa et al, 2009). Together these results implicate that CCL2 may be a relevant pro-inflammatory signal modulated by JMJD3 in MDS/ AML.We therefore sought to evaluate impact of JMJD3 inhibition on CCL2 production by MDS/AML cells. In MDS-L and TF1 cells, GSK-J4 significantly repressed RNA expression and cytokine release of CCL2. Furthermore, in HMA-resistant MDS-L and TF1 derivative cell lines, productions of CCL2 are elevated by 1.8 and 2.5 fold respectively compared to their parental lines. Treatment of HMA-resistant lines with GSK-J4 improved the anti-leukemia effect of decitabine and had a synergistic effect with HMA.In summary, we have demonstrated that pharmacological inhibition of JMJD3 by GSK-J4 has anti-leukemia effects in MDS/AML cells and has the potential to improve hypomethylation-based therapy in this disease. A possible mechanism for this effect is through the modulation of the inflammatory cytokine CCL2. To further evaluate this hypothesis, we will characterize the effect of JMJD3 and its inhibition on histone methylation of the CCL2 promoter in future work. DisclosuresNo relevant conflicts of interest to declare.

Five-aza-2'-deoxycytidine-induced hypomethylation of cholesterol 25-hydroxylase gene is responsible for cell death of myelodysplasia/leukemia cells.

DNA methyltransferase inhibitors (DNMT inhibitors) are administered for high-risk MDS, but their action mechanisms are not fully understood. Hence, we performed a genome-wide DNA methylation assay and focused on cholesterol 25-hydroxylase (CH25H) among the genes whose expression was up-regulated and whose promoter region was hypomethylated after decitabine (DAC) treatment in vitro. CH25H catalyzes hydroxylation of cholesterol and produces 25-hydroxycholesterol (25-OHC). Although CH25H mRNA expression level was originally low in MDS/leukemia cell lines, exposure to DNMT inhibitors enhanced CH25H mRNA expression. The promoter region of CH25H was originally hypermethylated in HL-60 and MDS-L cells, but DAC treatment induced their hypomethylation together with increased CH25H mRNA expression, activation of CH25H-oxysterol pathway, 25-OHC production and apoptotic cell death. We further confirmed that normal CD34-positive cells revealed hypomethylated status of the promoter region of CH25H gene. CH25H-knockdown by transfection of shRNA lentiviral vector into the cell lines partially protected the cells from DAC-induced cell death. Exogenous addition of 25-OHC suppressed leukemic cell growth. The present study raises a possibility that DNMT inhibitors activate CH25H-oxysterol pathway by their hypomethylating mechanism and induce leukemic cell death. Further investigations of the promoter analysis of CH25H gene and therapeutic effects of DNMT inhibitors on MDS/leukemia will be warranted.

Rigosertib induces cell death of a myelodysplastic syndrome-derived cell line by DNA damage-induced G2/M arrest.

A multi-kinase inhibitor, rigosertib (ON 01910.Na) has recently been highlighted as a novel type of anti-cancer agent for the treatment of the myelodysplastic syndromes (MDS), but its action mechanisms remain to be clarified. We investigated the in vitro effects of rigosertib on an MDS-derived cell line MDS-L and a myeloid leukemia cell line HL-60. Rigosertib suppressed the proliferation of both HL-60 and MDS-L cells and induced apoptosis by inhibition of the PI3 kinase/Akt pathway. As the effects on cell cycle, rigosertib treatment promoted the phosphorylation of histone H2AX and led to the DNA damage-induced G2/M arrest. In addition, an immunofluorescence staining study demonstrated the abnormal localization of aurora A kinase, suggesting that rigosertib causes perturbation of spindle assembly and deregulated mitotic patterns towards cell cycle arrest and apoptosis. We also found that rigosertib exerted growth inhibitory effects on two lymphoid cell lines, Jurkat and Ramos. We further examined the molecular pathways influenced by rigosertib from the gene expression profiling data of MDS-L cells and found a possible involvement of rigosertib treatment in the upregulation of the genes related to microtubule kinetics and the downregulation of the mRNA degradation system. The gene set enrichment analysis showed the suppression of “nonsense-mediated mRNA decay (NMD)” as the most significantly affected gene set. These data provide a new aspect and a potential utility of rigosertib for the treatment of refractory hematopoietic malignancies.

Decitabine Induced the Cell Differentiation, Decreased Malignant Potential, and Increased Susceptibility to Apoptosis Via a Death Receptor Pathway with Hypomethylation of MS4A3, PAR-4, APITD1, LOX in Myelodysplastic Syndrome Cell Line, MDS-L

Introduction: Myelodysplastic syndrome (MDS) is a group of clonal myeloid disorders characterized by ineffective hematopoiesis and cytopenias. Treatment strategies are based on the balance between patients’ predicted lifespan and the anticipated risk-benefit of intervention. Current guidelines recommend to classify patients into low-risk and high-risk groups. Hypomethylating agents azacitidine and decitabine are recommended for patients with high-risk MDS, who are not eligible for intensive therapy including stem cell transplantation. Azacitidine showed a survival benefit, however, treatment with this agent has not been standardized. To further optimize the use of hypomethylating agents, the target molecules of the agents should be identified to select patients, who would best benefit from the hypomethylating treatment. In the present study, the cellular drug sensitivity, the cell differentiation, the induction of apoptosis, and DNA methylation status after the treatment with decitabine were extensively investigated using cultured MDS cell line MDS-L that had been previously established.Methods: MDS-L cells, which had been established in the previous study (Br J Haematol, 87,235-242,1994) were used. This cell line is a blastic subline derived from the bone marrow blasts of a 50-year Japanese male patients with MDS. Trypan dye exclusion assay was used for determining growth inhibition effects. The cellular morphology was observed by staining the cells with May- Grunwald-Giemsa dye. Cell surface markers were detected by using flow cytometry. The transcript of Wilms’ tumor 1 was measured by real time RT-PCR. The induction of apoptosis was determined either by the sub-G1 population in the cell cycle analysis or the caspase cleavage. The enzymatic activity of DNA methyltransferase 1 (DNMT1) was determined by using a DNMT1 activity assay kit (Active Motif, Carlsbad, CA). DNA methylation array was performed using Infinium Human Methylation 450 BeadChip (Illumina, San Diego, CA).Results: The 50%-growth inhibitory concentration of decitabine was 64 nM in MDS-L cells. The cells were incubated with a non-toxic concentration (5 nM) of decitabine for 6 months (named as MDS-L-DAC cell line). MDS-L-DAC cells proliferated more slowly than untreated MDS-L cells (doubling time:51 h). Morphologically, MDS-L-DAC cells were maturated with a lobulated nuclei and cytoplasmic granulation. Differentiation induction was also indicated by an increased CD15 positivity (9.3→30.5%) and a decreased CD34 positivity (98.8→6.4%) in MDS-L-DAC cells. Wilms’ tumor 1 transcript level was also reduced (1,400,000→420,000 copies/μgRNA) in MDS-L-DAC cells, suggesting a decreased malignant potential by the effect of decitabine. Decitabine induced greater amount apoptosis (sub-G1 :12.3%) was with caspase 3 and caspase 8 cleavage in MDS-L-DAC cells, but not in MDS-L cells (sub-G1: 5.6%). The enzymatic activity of DNMT1 was successfully inhibited by decitabine in MDS-L-DAC cells (11.2→4.5OD/h/mg). The epigenome-wide association study using DNA methylation array compared the methylation status at CpG islands between MDS-L cells and MDS-L-DAC cells. Demethylation occurred in MS4A3 (Membrane-spanning 4-domains subfamily A member 3), PAR-4 (Prostate apoptosis response 4), CD40LG (CD40 ligand), APITD1 (Apoptosis-inducing, TAF 9-like domain 1), LOX (Lysyl Oxidase), and AMPH (Amphipysin) in MDS-L-DAC cells. Western blotting confirmed that the corresponding protein levels were increased.Discussion and Conclusions: MS4A3 is associated with myeloid differentiation. Caspase-8-mediated PAR-4 cleavage is required for tumor necrosis factor α-induced apoptosis. Low expression of APITD1 interferes with the ability for apoptosis through the p53 pathway. LOX is one of the commonly methylated genes in gastric cancer. Thus, the long-term incubation with decitabine at a low concentration demethylated these gene promoters and regained their protein expression. Decitabine induced the cell differentiation, decreased malignant potential, and increased susceptibility to apoptosis via a death receptor pathway, provably due to the functions of these proteins. DisclosuresNo relevant conflicts of interest to declare.

A Calcium-Dependent Pathway Determines Response to Lenalidomide in Del(5q) Myelodysplastic Syndromes

Deletions of the long arm of chromosome 5 (del(5q)) are common cytogenetic alterations in myelodysplastic syndrome (MDS), a disease characterized by refractory anemia, megakaryocyte dysplasia, and thrombocytosis. The thalidomide analogue lenalidomide (LEN) produces durable erythroid responses in ~60% of del(5q) MDS patients, including a majority of cytogenetic responses in which the del(5q) clone becomes undetectable in the bone marrow. Despite high response rates, clinical and cytogenetic relapse occur within 2-3 years. Mechanisms of clinical response, resistance and relapse with LEN therapy remain to be elucidated. The target of LEN has recently been identified as the cereblon (CRBN) component of the cullin 4 RING E3 ubiquitin ligase complex (CRL4-CRBN). Upon LEN binding, the substrate-specificity of the CRL4-CRBN complex is altered, and LEN-regulated substrates are beginning to be identified.An RNA interference screen was performed to identify genes/pathways that mediate LEN sensitivity and resistance in del(5q) MDS. The LEN-sensitive del(5q) MDS patient-derived cell line MDSL was screened with a genome-wide shRNA library (SBI GeneNet Human 50K Library) in the presence and absence of LEN treatment (0 and 10 μM) for 7 days. Three independent shRNAs targeting the proton-sensing G protein-coupled receptor 1 (GPR68 or OGR1) were among the most enriched shRNAs in LEN-treated cells, suggesting that loss of GPR68 expression conferred resistance to LEN. This finding was validated in MDSL cells, using an independent set of shRNAs. Conversely, a GPR68 agonist (N-cyclopropoyl-5-[thiophen-2-yl]-isoxazole-3-carboxamid) enhanced LEN-induced cytotoxicity to MDSL cells. GPR68 is a proton-sensing G-protein coupled receptor that stimulates inositol phosphate production and/or intracellular calcium (Ca2+) mobilization. Curiously, CRBN was originally identified as a binding protein of calcium-activated potassium channels. These data led us to hypothesize that Ca2+ signaling may be responsible for LEN-mediated cytotoxic effect in MDS cells. Reducing intracellular Ca2+ level with chelators reversed LEN’s cytotoxic effects, while increasing intracellular Ca2+ level with ionomycin enhanced LEN’s cytotoxic effect, indicating that intracellular Ca2+ levels determine cellular responsiveness to LEN. Although LEN did not induce an instant burst of Ca2+ influx, a gradual increase of basal intracellular free Ca2+ was observed following LEN treatment in LEN-sensitive cell lines and primary MDS marrow cells, but not in LEN-resistant cells, suggesting that LEN cytotoxicity was dependent on the cell’s ability to release Ca2+ from intracellular stores. GPR68 and CRBN were both necessary for the LEN-induced increase in Ca2+, as knockdown of GPR68 or CRBN in LEN-sensitive cells prevented the Ca2+increase.To identify the Ca2+-dependent signaling pathway responsible for mediating the cytotoxic effect of LEN, a panel of seven inhibitors that blocked mitochondrial/caspase-, calpain-, autophagy-, or lysosomal-dependent cell death pathways was tested in combination with LEN on MDSL cells. Only the inhibitor of calpains (PD150606) prevented LEN-induced cytotoxic effects in MDSL cells, indicating that calpain activation was necessary for mediating cell death in LEN-treated cells. Calpains are Ca2+-dependent cysteine proteases that can induce apoptotic and necrotic cell death by proteolytic cleavage of protein substrates. Calpastatin, the only endogenous calpain inhibitor, is localized to 5q15 and its expression is haploinsufficient in del(5q) MDS as compared to normal karyotype MDS. Taken together, our results show that LEN increased intracellular Ca2+ levels by a CRBN- and GPR68-dependent mechanism, leading to calpain-mediated cytotoxicity in del(5q) MDS cells. We propose a model in which haploinsufficient expression of calpastatin in del(5q) MDS sensitizes cells to cytotoxic effects of LEN. Further studies are required to identify the direct LEN-modulated substrates of CRBN that mediate this effect. DisclosuresOliva:Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria. MacBeth:Celgene: Employment, Equity Ownership. Starczynowski:Celgene: Research Funding.

A Novel Simulation Method for Mapping Dysregulated Pathways and Predicting Effective Therapeutics in the Myelodysplastic Syndromes

Background: The myelodysplastic syndromes (MDS) are a heterogeneous group of malignancies characterized by multiple genomic abnormalities. New technologies are needed that bridge the complex molecular biology of MDS and available therapeutics.Methods: Therefore, we developed a predictive simulation software program to create MDS patient avatars based on disease-specific genomic profile. The avatar technology has three layers: the first layer is a proprietary mathematics solver engine architected to handle millions of differential equations representing biological cross-talk reactions needed for modeling MDS physiology; the middle layer is the comprehensive functional proteomics representation of disease physiology network; and the topmost layer is a semiconductor engineering-based automaton engine which allows high-throughput simulation of multi-million interventions through assembly code language. The MDS avatars were intended to map the complex interplay of dysregulated pathways and predict potential therapeutics. The human MDS-L cell line is one of few bona fide MDS cell lines and was analyzed by conventional G-banding karyotyping, FISH, array CGH and Sanger sequencing of MDS-relevant genes. Using this genomic information, a simulation patient avatar was created. Next, a library of over 80 FDA approved agents was simulated against the dysregulated pathways. A list of predicted drugs where then prospectively validated using in vitro culture of MDS-L cells.Results: The MDS-L cells harbored an activating NRAS mutation (c.35G>C, p.G12A) and complex chromosome abnormalities. The simulation MDS-L patient avatar predicted activation of the RAF-ERK pathway due to NRAS mutation and various copy number variations (CNVs) including high CN of IGFR, AURKA, PAR5 that predicted high AKT activation, high CN of mTOR, high CN of IL6 and JAK3 with predicted activation of STAT3 and STAT5, high CN of MDM4 with predicted lower levels of TP53, high CN of RCE1, MAPK1 and MAP2K1, and low CN of RASA1 and DUSP1 (Figure 1). Given the strong dominance of AKT and ERK loops in this profile (Figure 1), we simulated potential drugs individually and in combinations to predict impact on these pathways. Iterative simulations identified four FDA-approved drugs: (A) nelfinavir and celecoxib acting on AKT, (B) sorafenib and trametinib that inhibit RAF-ERK targets, and (C) statins that inhibit the RAS-RAF-ERK pathway through inhibition of prenylation. In vitro validation experiments showed statistically significant reductions in MDS-L cell viability when treating with nelfinavir alone, celecoxib alone, and enhanced additive reduction in viability with the combination of nelfinavir and celecoxib (Table 1).Conclusions: This study shows how a novel simulation method can be employed to use patient-specific MDS genomic profiling to map dysregulated pathways and predict potentially therapeutic agents. The MDS case presented led to the discovery of a novel combination of nelfinavir and celecoxib. Results from this study serve as the basis for MDS clinical trials that assign treatment based on genetic mutations. [Display omitted] Table 1MDS-L Cell In Vitro Validation Experiments of Drug Treatments.NelfinavirDrug Dose0μM5μM10μMCelecoxib0 μM100.00%81.45% ± 7.8%53.49% ± 6.9%30 μM71.34% ± 4.89%60.58% ± 3.25%35.78% ± 3.59%MDS-L cells were treated with vehicle control, nelfinavir alone (incremental dose increases), celecoxib alone (incremental dose increases), and combination nelfinavir and celecoxib. MDS-L cell viability was quantified and compared among treatment groups. Both nelfinavir alone and celecoxib alone reduce MDS-L cell viability in a dose-dependent manner. The combination of nelfinavir and celecoxib showed enhanced additive reduction of MDS-L cell viability, again in a dose-dependent manner. DisclosuresCogle:PHAR: Consultancy. Vali:CellWorks: Employment. Kumar:CellWorks: Employment. Singh:CellWorks: Employment. Tyagi:CellWorks: Employment. Abbasi:CellWorks: Employment, Equity Ownership.

The Pharmacological Inhibition of KDM1A Displays Preclinical Efficacy in AML and MDS By Inducing Myelomonocytic Differentiation

Background: Histone methylation is one of the major systems of epigenetics and reversibly regulated by lysine (K) specific methyltransferases (KMTs) and demethylases (KDMs). Dysregulation of KMTs such as EZH2 and MLL play a key role in the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) while the precise function of KDMs remains unclear.KDM1A is the first reported histone demethylase, which mainly catalyzes demethylation of mono- and di-methylated lysine 4 of histone 3 (H3K4me1 and me2 respectively). According to recent reports, the inhibition of KDM1A either alone or in combination of all-trans retinoic acid is effective for AML expressing MLL-AF9 or for several types of AML respectively, suggesting KDM1A could be a therapeutic target of AML. However, other reports argue that KDM1A is essential in hematopoiesis, raising concern that KDM1A-targeted therapy could lead to severe hematological toxicity. Here, we try to clarify what types of leukemia can be ameliorated by the pharmacological inhibition of KDM1A with a possible therapeutic window and what are functional and molecular mechanisms utilizing highly selective KDM1A inhibitors we have newly designed.Results: First we validated the effect of our novel inhibitors on murine leukemia cells harboring MLL-AF9. In accordance with a previous report, our novel KDM1A inhibitors suppressed cell proliferation, diminished clonogenic capacity and induced G1-S cell cycle arrest and myelomonocytic differentiation but not apoptosis in quite low concentration that clonogenicity of normal murine bone marrow cells was spared. Next we examined the effect of these drugs on diverse types of human myeloid leukemia cells and found that our drugs were particularly effective in erythroid leukemia cells (HEL), megakaryocytic leukemia cells (CMK11-5), and a blastic subline from a MDS patient with complex karyotype (MDS-L). MDS-L cells were changed phenotypically and morphologically towards myelomonocytic differentiation such as the increase of CD11b expression level and the induction of neutrophil-like cells. HEL and CMK cells which are negative for myelomonocytic markers also gained CD11b expression and decreased erythroid markers such as CD235a and CD71. These data suggest that the inhibition of KDM1A induces myeloid differentiation across various types of leukemia.To investigate an underlying molecular basis for the cell fate conversion in HEL and myeloid differentiation in MDS-L by the inhibition of KDM1A, we performed gene expression profiling and analyzed a change of gene signatures. The expression pattern of transcriptional factors was changed from the erythroid signature (e.g. GATA1 and TAL1) to the myeloid signature (e.g. SPI1 and CEBPA) in HEL. Gene Set Enrichment Analysis (GSEA) showed that the myeloid differentiation-associated gene signature was positively enriched and the leukemia stem cell-associated gene signature was negatively enriched in both HEL and MDS-L.Finally, we investigated the effect of our KDM1A inhibitors on primary human samples such as AML with MLL-AF9 and MDS in the phase of overt leukemia (MDS/AML) with complex karyotype. The colony formation capacity was clearly impaired in relatively low concentration that normal colonies were spared. We transplanted primary MDS/AML cells with complex karyotype to immunodeficient mice and treated with a KDM1A inhibitor or vehicles after confirming the engraftment. In one MDS/AML case, all mice treated with vehicles (n=4) died of anemia and the increase of human leukemia cells within four months while 3 of 4 mice treated with a KDM1A inhibitor have survived for more than six months. Also in another case, both of two vehicle-treated mice died while all drug-treated mice had survived for more than six months. At day 200 after transplantation, we sacrificed all survived mice treated with a KDM1A inhibitor and found that human blasts were displaced from the bone marrow of treated mice. Those data suggest that our KDM1A inhibitor is effective in vivo and have a possibility for the clinical application.Conclusion: Our study suggests that KDM1A involves with myeloid differentiation and the leukemia stem cell signature and that the pharmacological inhibition of KDM1A by highly selective inhibitors is a promising way to ameliorate AML with poor prognosis such as erythroleukemia and MDS/AML with complex karyotype, without impairing normal hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

A Novel Method of Using Molecular Profiling in Myelodysplastic Syndromes to Predict Patient-Specific Potential Therapeutics

Background: The myelodysplastic syndromes (MDS) are a heterogeneous group of malignancies characterized by multiple genomic abnormalities. Precise characterization of MDS molecular abnormalities in individual patients and treatment with drugs that target the specific constellation of molecular abnormalities may provide a practical approach in this complex disease.Methods: Therefore, bone marrow cells from 90 MDS patients were molecularly profiled by testing for mutations in the following 14 MDS-specific genes: ASXL1, ETV6, EZH2, IDH1, IDH2, NRAS, CBL, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. We used these profiles in a novel predictive simulation software program to create MDS patient avatars. The avatar technology has three layers: the first layer is a proprietary mathematics solver engine architected to handle millions of differential equations representing biological cross-talk reactions needed for modeling MDS physiology; the middle layer is the comprehensive functional proteomics representation of disease physiology network; and the topmost layer is a semiconductor engineering-based automaton engine which allows high-throughput simulation of multi-million interventions through assembly code language. Bone marrow conventional cytogenetics, FISH and molecular profiling data from 90 patients were used in the simulation. A library of over 80 FDA-approved agents was simulated against the predicted dysregulated pathways. A list of predicted drugs where then prospectively validated using in vitro culture of MDS-L cells.Results: Of the 90 MDS patients, the IPSS-R distribution was 25% Very Low, 58% Low, 9% Intermediate, 7% High, and 1% Very High. Median BM blast was 2% (range 0-16%). All 90 patients had detectable genomic abnormalities. 86/90 (96%) of patients had detectable genetic mutations. The 4 patients with undetectable genetic mutations all had structural chromosome abnormalities (e.g., del(11q), +8, del(20q)). Among the 90 patients, recurrent constellations of genomic mutations were identified, which permitted clustering into 63 unique MDS genomic profiles. Using these unique MDS genomic profiles, simulation avatars of dysregulated pathways were created for each MDS patient (Figure 1 as example). Next, we used the MDS avatars to simulate FDA-approved drugs individually and in combinations to predict impact on these dysregulated pathways. Iterative simulations identified 12 new drug regimens depending on the MDS patient’s constellation of genomic abnormalities: (1) nelfinavir, (2) bortezomib, (3) metformin + simvastatin, (4) metformin + seliciclib, (5) nelfinavir + bortezomib, (6) nelfinavir + ruxolitinib, (7) nelfinavir + simvastatin, (8) nelfinavir + sorafenib, (9) nelfinavir + trametinib, (10) ponatinib + bortezomib, (11) nelfinavir + ponatinib, and (12) trametinib + simvastatin. In vitro validation experiments of drug regimens with MDS-L cells showed significant, dose-dependent reductions of MDS-L cell viability.Conclusions: This study shows how molecular profiling using 14 genes and a novel simulation method can be used to map dysregulated pathways and predict potentially therapeutic agents for patients with MDS. Results from this study serve as the basis for an MDS clinical study that assigns treatment based on genomic abnormalities. [Display omitted] DisclosuresCogle:Leukemia & Lymphoma Society: Research Funding. Off Label Use: Treating MDS with one of these regimens: (1) nelfinavir, (2) bortezomib, (3) metformin + simvastatin, (4) metformin + seliciclib, (5) nelfinavir + bortezomib, (6) nelfinavir + ruxolitinib, (7) nelfinavir + simvastatin, (8) nelfinavir + sorafenib, (9) nelfinavir + trametinib, (10) ponatinib + bortezomib, (11) nelfinavir + ponatinib, and (12) trametinib + simvastatin.. Vali:Cellworks: Employment. Kumar:Cellworks: Employment. Singh:Cellworks: Employment. Nair:Cellworks: Employment. Lala:Cellworks: Employment. Basu:Cellworks: Employment. Abbasi:Cellworks: Employment. Albitar:Neogenomics: Employment.

Arsenic disulfide-triggered apoptosis and erythroid differentiation in myelodysplastic syndrome and acute myeloid leukemia cell lines

ObjectivesEffects of arsenic disulfide (As2S2) were investigated by focusing on growth inhibition, apoptosis induction, and erythroid differentiation in MDS-L, F-36p and HL-60 cells, derived from myelodysplastic syndrome (MDS), MDS/acute myeloid leukemia (AML), and de novo AML, respectively.MethodsCell viability was determined by MTT assay. Apoptosis induction was analyzed using Annexin V/propidium iodide staining. Erythroid differentiation was assessed by the expression level of CD235a, a marker for detection of the erythroid cell lineage. The activation of p38 MAPK and the expression profile of apoptosis-related proteins Bcl-2 and Bid were analyzed using western blot.ResultsAs2S2 inhibited cell growth of these cell lines. Of note, the IC50 value of As2S2 in MDS-L cells was comparable to that in F-36p cells, and was half of that in HL-60 cells. A dose-dependent decrease in cell viability and concomitant increase in the percentage of apoptotic cells were observed in F-36p cells treated with 8 and 16 µM As2S2 for 72 hours. However, similar phenomena were only observed in HL-60 cells when treated with as high as 16 µM As2S2. Furthermore, As2S2 exerted more potent erythroid differentiation-inducing activity on F-36p cells than HL-60 cells. Interestingly, negative correlation between p38 MAPK signaling pathway and As2S2-induced erythroid differentiation was observed in HL-60 cells. Treatment with relatively high concentration of As2S2 resulted in the downregulation of Bcl-2 and Bid proteins in HL-60 cells.DiscussionThese results suggest that compared to AML cell line, MDS and MDS/AML cell lines are more sensitive to not only the erythroid differentiation-inducing activity of As2S2, but also its cytotoxicity associated with apoptosis induction. These findings further provide novel insight into As2S2 action toward its use for clinical application in patients with hematological disorders.

Overexpression Of Mir-125a In Bone Marrow CD34+ cells Of Patients With Myelodysplastic Syndrome Is Correlated To a Poor Prognosis and May Contribute To The Pathogenesis Of The Disease Through The Modulation Of NF-Kb Activation and Enhancement Of Differentiation Arrest

Myelodysplastic syndromes (MDS) are a group of clonal malignancies characterized by impaired proliferation and differentiation of hematopoietic stem cells and precursors. The involvement of toll-like receptor (TLR)-mediated signalling in the modulation of myeloid differentiation and its participation in the pathogenesis of MDS are well documented (Wei et al 2013). Increased signaling through this pathway leads to the constitutive activation of NF-kB, which regulates the production of cytokines and mediates cell proliferation and apoptosis (Starczynowski 2010). In addition to the expression of proteins involved in inflammation, the TRL pathway also induces the expression of microRNAs (miRNAs) which participate in the fine-tuning of the inflammatory response (Kawai and Akira 2010). miR-125a and miR-125b are known modulators of hematopoiesis (Gerrits et al. 2012) and have been reported to be involved in several lymphoid and myeloid diseases. Little is known about their role in the pathogenesis of MDS. Interestingly, NF-kB-activating ability has been described for both miR-125a/b (Kim et al. 2012), and miR-125b appears to be upregulated by NF-kB within a positive feedback loop (Zhou et al. 2009; Tan et al. 2012). The aim of this work was to analyze the expression of miR-125a/b in MDS CD34+ cells and to study their relationship with the TLR pathway and differentiation.For this purpose, we analyzed the expression of miR-125a/b by qPCR in bone marrow CD34+ cells of 48 MDS patients, compared it with expression in healthy donors and studied the correlation with overall survival. In our study, we included miR-99b, which is clustered with miR-125a in the genome. Levels of TLR pathway components were detected by qPCR and correlated to those of the miRNAs. Activation of NF-kB was determined in Meg-01 and KG1 cells by the luciferase reporter gene assay, using a vector containing NF-kB responsive elements. Differentiation was studied in K562 and MDS-L cells through colony formation assays combined with analyses of the expression of specific markers by qPCR. For these experiments we used miRNA analogs and a miR-125a anti-sense oligonucleotide.Our results showed that miR-125a, but not miR-125b, is strongly overexpressed in MDS patients (∼15-fold of controls; P<0.01) and that miR-125a levels are significantly and negatively correlated to overall survival of MDS patients (P<0.05). Moreover, expression of miR-99b is also directly connected to the progression of the disease (P<0.05). Both miR-125a and miR-99b cooperate in vitro in the activation of NF-kB (P<0.001); however, we observed a negative correlation between miR-99b/miR-125a expression and levels of TLR2, TLR7 and their downstream proteins MyD88 and JMJD3 (P<0.05), suggesting that NF-kB activation by the miRNA cluster occurs in the absence of TLR signaling. Furthermore, we observed a ∼4-fold increase in NF-kB activity after miR-125a inhibition in the presence of a TLR2 agonist (P<0.001), indicating that miR-125a acts as an NF-kB inhibitor upon TLR stimulation. These results show that miR-125a is involved in the fine-tuning of NF-kB activity and that its effects may depend on the status of the TLR pathway. We then investigated the role of miR-125a in hematopoiesis and found that this miRNA contributes to the blockade of differentiation in the cell lines studied. Therefore, miR-125a could be involved in the pathogenesis or progression of MDS through the modulation of NF-kB activity and differentiation arrest. Thus, this miRNA could be a good prognostic marker and is a potential therapeutic target in MDS. Disclosures:No relevant conflicts of interest to declare.

The Jumanjii Histone Demethylase KDM2B controls EZH2 Expression In Myelodysplastic Syndromes (MDS) Via Mir Let-7b (let7b), a Pathway That Is Bypassed By The Histone Methylation Inhibitor DZNep

Background and HypothesisDNA and histone methylation as well as microRNA (miR) expression are dysregulated in MDS. The histone demethylase KDM2B suppresses let7b, which post-transcriptionally controls EZH2, a histone-lysine N-methyl transferase that methylates lysine 27 on histone 3. We hypothesized that epigenetic dysregulation in MDS involves the KDM2B/ let7b/EZH2 axis (Figure). DZNep (cyclopentanyl analog of 3-deazaadenosine) is a novel compound, which results in loss of lysine27 methylation, re-expression of epigenetically silenced genes, and decreased cell proliferation.We propose that DZNep will bypass cellular dysregulation via the KDM2B/let7b/EZH2 axis and thereby enhance therapeutic efficacy when combined with azanucleosides. MethodsCD34+ MDS marrow cells were obtained by density separation and enriched by magnetic-activated cell sorting (MACS; Milteny Biotec, Auburn, CA); purity 95% to 98% by flow cytometry. For NanoString nCounter miR array total cell derived RNA or synthetic miR pools (IDT; 30 pmol per oligonucleotide) were used as input. Nano-string data were validated by real-time polymerase chain reaction (PCR). RNA was extracted using the RNeasy Mini kit (QIAGEN, Valencia, CA). Primers for let7b, U6, KDM2b and EZH2 were synthesized by Applied Biosystems. Total RNA was reverse transcribed with Taqman MiR Reverse Transcriptase kit (Applied Biosystems, Carlsbad, CA). Potential functional roles of KDM2B, let7b and EZH2 were determined in myeloid cell lines MDS-L, PL-21, KG1a,OCI-AML3 and ML1. Conditional knock-down of KDM2B was achieved with 2 short hairpins (open-biosystems). Expression of KDM2B was forced using pCMV-Myc-KDM2B (gift of Dr. Tzatsos) in cell lines with low/absent constitutive levels of KDM2B and high levels of let7b. miRZip plasmids (miRZip anti-sense) were used to knock-down let7b; pre-BAB-let7b (origene) served to overexpress let7b. Gene expression, protein levels and cell proliferation were analyzed by RQ-PCR, western blot and BrdU uptake. To determine the impact of modified let7b and KDM2B expression on treatment responses, MDS-L cells (high constitutive levels of EZH2) were treated with DZNep and methyltransferase inhibitor, 5-azacytidine, and subjected to the same analyses. ResultsLet7b levels were significantly higher in primary CD34+ MDS marrow cells (n=44) than in healthy controls (p= 0.0304), while KDM2B and EZH2 expression was reduced (n=21, p<0.0126, p<0.0001, respectively). Overexpression of let-7b in cells with low constitutive levels (PL21, ML1 and KG1a) reduced EZH2 and KDM2B protein levels, and decreased S-phase while increasing G0/G1 cells (p=0.0005); this was associated with a decrease of H3K27me3 and cyclin D1. Silencing of KDM2B in cells with high (MDS-L) and low (KG1a) constitutive levels of let-7b resulted in increased levels of let7b. This increase in let-7b was associated with reduced levels of EZH2 protein and H3K27me3. These findings were accompanied by reduced proliferation (fewer cells in S-phase, more cells in G0/G1). Addition of DZNep to the myeloid cell lines under study resulted in a decrease of let-7b expression, increased levels of EZH2, but reduced cell proliferation. When DZNep (1 uM/ml) was combined with 5-azacytidine (AZA, 5 uM/ml), levels of EZH2 increased in parallel with reduction of H3K27me3 and cell proliferation. KG1a cells overexpressing let-7b (to mimic conditions in primary MDS cells) showed further increase of EZH2 in response to DZNep and AZA. Summary and ConclusionThe KDM2B/ let7b/EZH2 axis is involved in epigenetic regulation in MDS. KDM2B, via let-7b/EZH2, promotes transcriptional repression in myeloid cell lines and primary MDS cells; KDM2B controls epigenetic modifications and cell proliferation. DZNep bypasses the “inhibitory” KDM2B/let7b/EZH2 axis by preventing H3K27 methylation and reducing cell proliferation, independent of EZH2 expression. Thus, the use of DZNep would add histone demethylation to the effect of azanucleosides on DNA methylation, thereby enhancing the therapeutic success in MDS. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Development and Characterization of a Novel Human Xenograft Model Using an MDS Patient-Derived Cell Line

Abstract 3814Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders, defined by ineffective hematopoiesis, blood cytopenias, myeloid dysplasia, and an increased risk of acute myeloid leukemia (AML). Immunodeficient mice engineered to support human tumor xenografts represent a pivotal development in the study of human cancers. The xenograft model has been an attractive approach to interrogate human AML; in contrast, MDS patient samples exhibit negligible or poor engraftment, with no evidence of disease in immunocompromised recipient mice. To circumvent this limitation we developed a novel xenograft mouse model using an MDS cell line, MDSL. The MDSL cell line was derived from the non-leukemic phase of an MDS patient with refractory anemia-ringer sideroblasts, and has maintained growth-factor dependence and sensitivity to Lenalidomide. Two immunocomprismised mouse stains were used for xenotransplantation of MDSL cell: NOD/LtSz-scid IL2RG (NSG) and NOD/LtSz-scid IL2RG-SGM3 (NSGS). NSGS mice are identical to NSG except that they have been engineered to express three human cytokines, SCF, GM-CSF and IL-3, to improve engraftment of human hematopoietic cells. Following transplantation of MDSL cells by either intrafemoral or intravenous injection, NSG and NSGS animals exhibited human cell grafts in the bone marrow, peripheral blood, and spleen. Subsequently, mice developed a fatal disease exhibiting features of human MDS, including anemia and thrombocytopenia, which coincides with clonal expansion and increased human cell graft in all hematopoietic organs. The median survival for NSGS and NSG mice was 21 and 55 days post-injection, respectively. The shortened latency in NSGS mice suggests that the expression of human cytokines accelerates the growth and enhances survival of the MDSL cells in vivo. MDSL cells isolated from the bone marrow of primary recipient mice were capable of initiating an MDS-like disease in secondary recipients. Given that few preclinical options for MDS are available, we also evaluated whether this model is suitable for therapeutic studies. Since MDSL cells are sensitive to Lenalidomide and IRAK1 Inhibitor (Rhyasen et al, ASH 2011), we evaluated the effects of these drugs in the xenograft model. After xenotransplantation of MDSL cells, NSGS mice were administered either Lenalidomide or IRAK1 Inhibitor. Our preliminary findings indicate that mice xenografted with MDSL cells are amenable to drug delivery and show a response to treatment. In summary this work describes a novel xenograft model of human MDS, the first to develop clinical features resembling human disease. In addition, we predict that this model will also be useful for preclinical studies to examine novel and existing MDS therapies. Disclosures:No relevant conflicts of interest to declare.

TC11, a Novel Phthalimide Derivative, Directly Binds to NPM1 and Induced Apoptosis of High-Risk Myeloma Cells Via Centrosomal Disruption

Abstract 1841 PURPOSE:Despite recent advances in the treatment of multiple myeloma (MM), patients with high-risk chromosomal changes such as del13q, t(4;14) or del17p revealed significantly shorter survival. In addition, bone disease markedly reduces quality of life of the patients with MM. To overcome these problems, we have designed and screened synthetic phthalimides which significantly inhibited the growth of MM cell lines with high-risk chromosomal abnormalities. The purposes of this study are to explore novel drugs which possess anti-tumor activity against high-risk MM cells and to examine the anti-osteoclastogenic activity and to isolate directly binding molecules. METHODS AND RESULTS:Thirty synthetic phthalimides were screened for anti-proliferative effect on KMS34 cells with t(4;14) and deletion of chromosome 17. A phthalimide derivative, 2-(2,6-diisopropylphenyl)- 5-amino- 1H-isoindole- 1,3- dione (TC11) significantly inhibited growth of KMS34 cells as well as other MM cells lines with high-risk chromosomal abnormalities (IC50 to KMS34 cells= 4μM). TC11 increased annexin V fraction and induced apoptosis in a caspase-dependent manner. In vivo anti-myeloma activity was evaluated using KMS34-bearing lcr/SCID mice by intraperitoneal injection of TC11. Twenty mg/kg of TC11 significantly inhibited growth of TC11-derived tumor cells, and apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not suppressed, suggesting low hematopoietic toxicity. In pharmacokinetic study using lcr/SCID mice, the plasma concentrations of TC11 was examined; Cmax=18.1μM, Tmax=1.5hr, T1/2 =4.5hr when 100mg/kg of TC11 was injected, and Cmax=2.1μM Tmax=1.0hr, T1/2 =4.5hr when 20mg/kg was injected. In order to examine efficacy to bone disease, anti-osteoclastogenic activity was examined by adding TC11 to M-CSF/RANK ligand-induced osteoclastogenic culture of mouse bone marrow mononuclear cells. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in the presence of 1μM of TC11. It was also found that TC11 inhibited bone resorption by pit assay. We also tried to isolate directly binding proteins to TC11 by our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. We identified nucleophosmin 1 (NPM1) as a TC11-binding molecule. Knockdown assay introducing siRNA for NPM1 into HeLa cells induced emergence of the cells with multipolar spindles, suggesting centrosomal disruption during cell division. Since NPM1 gene localizes at chromosome 5q, anti-MDS effect of TC11 was also examined. TC11 also inhibited growth of MDS-L cells (IC50=7μM). CONCLUSION:A novel phthalimide derivative, TC11, has anti-MM activity in vivo and is a potentially effective drug for high-risk MM with bone lesions. TC11 directly binds to NPM1 and induces apoptosis of MM and MDS cells with low toxicity to normal hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Effects of DNA methyltransferase inhibitors (DNMTIs) on MDS-derived cell lines

DNA methyltransferase inhibitors (DNMTIs), including decitabine (DAC) and azacitidine (AZA), have recently been highlighted for the treatment of high-risk myelodysplastic syndrome (MDS); however, their action mechanisms have not been clearly defined. Therefore, we investigated the effects of DNMTIs on MDS-derived cell lines invitro. An MDS-derived cell line MDS92 and its blastic subline MDS-L and HL-60 were used. All three cell lines were sensitive to DNMTIs, but MDS-L was the most susceptible. DAC-induced cell death in MDS-L was preceded by DNA damage-induced G2 arrest via a p53-independent pathway. AZA did not influence the pattern of cell cycle, although it induced DNA damage response. The IC(50) of DAC or AZA on MDS-L cells was associated with the dose inducing the maximal hypomethylation in long interspersed nuclear elements-1 (LINE-1) methylation assay. AZA suppressed the level of methylation in a time-dependent manner (days 4, 7, and 10), whereas DAC maintained the level of methylation from day 4 to 11. The protein expression of DNMT1 and DNMT3a decreased with the suppression of growth and methylation. We conclude that this study provides invitro models for understanding the effects of DNMTIs on cell growth and gene regulation, including differences in the possible action mechanism of DAC and AZA.

Approach to New Therapeutics: Investigation by the Use of MDS-derived Cell Lines

Myelodysplastic syndromes (MDS) are a group of aquired hematopoietic disorders characterized by ineffective hematopoiesis, and increased risk of progression of acute myeloid leukemia. For a long period of time, the standard therapy for MDS was hematopoietic stem cell transplantation, however DNA methyltransferase inhibitors (DNMT inhibitors) including decitabine (DAC) and azacitidine (AZA), and lenalidomide, a derivative of thalidomide have been highlighted as new chemotherapeutic agents for MDS. However, the underlying mechanisms of action of these drugs have not been fully defined yet. Therefore, we investigated the in vitro effects of DNMT inhibitors and lenalidomide on an MDS-derived cell line, MDS92 and its blastic subline MDS-L, both of which carry del(5q). MDS-L cells were found to be quite sensitive to DAC, which induced to cell death through DNA damage-mediated G2 arrest via p53- independent pathways. Gene expression profiling suggested that DAC affects biogroups representing hematological systems, cellular development, cell death and apoptosis. Next, we examined the effects of lenalidomide on MDS-L. Cell growth was inhibited and multinucleated cells were frequently formed prior to cell death by lenalidomide treatment. Time-lapse microscopic observation and DNA ploidy analysis revealed that lenalidomide does not affect DNA synthesis itself but inhibits cytokinesis of MDS-L cells. The gene expression profiling showed decreased expression of M phase-related genes. These data contribute to the understanding of action mechanisms of lenalidomide on MDS with del(5q).

Overexpression of BMI1 confers clonal cells resistance to apoptosis and contributes to adverse prognosis in myelodysplastic syndrome

The polycomb group (PcG) protein BMI1 plays a critical role in regulating self renewal capacity of both normal and leukemic stem cells. BMI1 is frequently overexpressed in several types of cancer, which is associated with poor prognosis. However, there are few researches on BMI1 in myelodysplastic syndromes (MDS). In this study, we reported that overexpression of BMI1 protein was detected in MDS patients, and inversely correlated with the apoptosis of CD34+ cells. In vitro overexpression of BMI1 facilitated proliferation and inhibited apoptosis of MDS-L cells. The overexpression of BMI1 could downregulate apoptosis sensitivity to cytotoxic agents in MDS-L cells; on the contrary, MDS-L cells could be rendered apoptosis-sensitive by BMI1 knockdown. Overexpression of BMI1 antagonised apoptosis by downregulating several apoptosis-related proteins, such as p16INK4a, phospho-p53 (Ser46) and caspase 3/9. In addition, overexpression of BMI1 was correlated with an elevated IPSS score and a shorter survival. Collectively, overexpression of BMI1 induces resistance to apoptosis and contributes to adverse prognosis in MDS. BMI1 could serve as a therapeutic target for patients with MDS.