Myelodysplastic Syndrome Cell Line Research Articles

PDE4 Differential Expression Is a Potential Prognostic Factor and Therapeutic Target in Patients With Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia.

Inflammation has an essential role in the pathogenesis of myelodysplastic syndromes (MDS). Its expression is controlled by phosphodiesterase 4 (PDE4). Thus, PDE4 inhibitors might be useful therapeutic targets for MDS. We evaluated the expression of each isoform of PDE4 (A, B, C, and D) using transcriptomic profiling and examined the potential impact on the outcome of patients with MDS in terms of survival and response to hypomethylating agents. Total RNA was extracted from CD34(+) bone marrow hematopoietic cells from healthy individuals (n = 10) and patients with MDS (n = 24) or chronic myelomonocytic leukemia (n = 19). The study cohort had a median follow-up period of 21.2 months (range, 0.2-68 months) and a median overall survival of 17.6 months (95% confidence interval, 9.6-25.6). The main finding of the present study was that PDE4 mean expression was generally higher in patients with MDS than in healthy individuals. Also, upregulated PDE4 expression seemed to have a possible negative effect on survival (P > .05). Moreover, lower, compared with higher, mean PDE4A and PDE4C expression is indicative of a response to a hypomethylating agent (0.09 and 0.03 vs. 0.54 and 0.49, respectively; P > .05). These results should be confirmed in a larger patient cohort. PDE4 expression could be an effective potential prognostic factor and therapeutic target for patients with MDS and chronic myelomonocytic leukemia. The role of PDE4 inhibitors should be explored in vitro against MDS cell lines and in preclinical mouse models of MDS.

Atg3 Overexpression Enhances Bortezomib-Induced Cell Death in SKM-1 Cell.

BackgroundMyelodysplastic syndrome (MDS) is a group of heterogeneous hematopoietic stem cell malignancies with a high risk of transformation into acute myeloid leukemia (AML). Clonal evolutions are significantly associated with transformation to AML. According to a gene expression microarray, atg3 is downregulated in MDS patients progressing to leukemia, but less is known about the function of Atg3 in the survival and death of MSD/AML cells. Moreover, the role of autophagy as a result of bortezomib treatment is controversial. The current study was designed to investigate the function of Atg3 in SKM-1 cells and to study the effect of Atg3 on cell viability and cell death following bortezomib treatment.MethodsFour leukemia cell lines (SKM-1, THP-1, NB4 and K562) and two healthy patients’ bone marrow cells were analyzed for Atg3 expression via qRT-PCR and Western blotting analysis. The role of Atg3 in SKM-1 cell survival and cell death was analyzed by CCK-8 assay, trypan blue exclusion assay, DAPI staining and Annexin V/PI dual staining with or without bortezomib treatment. Western blotting analysis was used to detect proteins in autophagic and caspase signaling pathways. Electron microscopy was used to observe ultrastructural changes after Atg3 overexpression.ResultsDownregulation of Atg3 expression was detected in four leukemia cell lines compared with healthy bone marrow cells. Atg3 mRNA was significantly decreased in MDS patients’ bone marrow cells. Overexpression of Atg3 in SKM-1 cells resulted in AKT-mTOR-dependent autophagy, a significant reduction in cell proliferation and increased cell death, which could be overcome by the autophagy inhibitor 3-MA. SKM-1 cells overexpressing Atg3 were hypersensitive to bortezomib treatment at different concentrations via autophagic cell death and enhanced sensitivity to apoptosis in the SKM-1 cell line. Following treatment with 3-MA, the sensitivity of Atg3-overexpressing cells to bortezomib treatment was reduced. Atg3 knockdown blocked cell growth inhibition and cell death induced by bortezomib.ConclusionOur preliminary study of Atg3 in the high-risk MDS cell line suggests that Atg3 might be possibly a critical regulator of autophagic cell death and a gene target for therapeutic interventions in MDS.

A calcium- and calpain-dependent pathway determines the response to lenalidomide in myelodysplastic syndromes.

Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.

Hu Jie, He Donghua, Xue Xingkui, Gao Liang, Wu Wenjun, Han Xiaoyan, Cai Zhen, Homoharringtonine-induced apoptosis of MDS cell line MUTZ-1 cells is mediated by the endoplasmic reticulum stress pathway, May 2007;48(5):964–977. http://dx.doi.org/10.1080/10428190701216360

Hu Jie, He Donghua, Xue Xingkui, Gao Liang, Wu Wenjun, Han Xiaoyan, Cai Zhen, Homoharringtonine-induced apoptosis of MDS cell line MUTZ-1 cells is mediated by the endoplasmic reticulum stress pathway, May 2007;48(5):964–977. http://dx.doi.org/10.1080/10428190701216360

The Synergistic Effects of Decitabine Combined with Arsenic Trioxide (ATO) in the Human Myelodysplastic Syndrome Cell Line SKM-1.

Despite recent advances in the treatment of myelodysplastic syndrome (MDS), single-agent clinical effects remain unsatisfactory, and decitabine monotherapy is also associated with a relatively low rate of complete remission. To study the combined effects and mechanism of decitabine (DAC) and arsenic trioxide (ATO) on the human myelodysplastic cell line SKM-1,we used the MTS assay and CalcuSyn software to determine the cytotoxicity and potential synergistic effects, respectively. Furthermore, we determined apoptosis and measured the mRNA expression level of two genes that are considered main regulators of the apoptosis process. The results showed that DAC and/or ATO can inhibit the proliferation of SKM-1 cells and demonstrated significant synergy between the two agents (CI<1). Additionally, combination of 2.5μmol/L DAC and 5μmol/L ATO led to a significantly higher apoptosis rate and more significantly decreased the Bcl2/Bax ratio than either compound alone (P<0.001). Based on the observations of this study, we suggest that combined administration of these two drugs might be considered a novel therapeutic regimen for treating MDS.

PDE4 Differential Expression Is a Potential Prognostic Factor and a Therapeutic Target in Myelodysplastic Syndromes

Inflammation and innate immunity have an essential role in the pathogenesis of myelodysplastic syndromes (MDS). One of the key inflammatory cytokines that is upregulated in MDS and acute myeloid leukemia (AML) is IL-8. [Schinke C et al, 2015]. The expression of IL-8 and other inflammatory cytokines is controlled by phosphodiesterase 4 (PDE4), which has yet to be studied in MDS and AML. Furthermore PDE4 inhibitors may be useful therapeutic targets for MDS. Based on this, we evaluated the expression of PDE4 and examined its potential impact on clinical outcomes in MDS. Transcriptomic profiling was obtained from the Illumina Genome Analyzer IIx platform, using the NuGEN Ovation RNA sequencing (RNA-seq) system. Total RNA was extracted from CD34+ bone marrow hematopoietic cells from healthy individuals (n=10) and patients with MDS (n=24) or CMML (n=19). We interrogated the RNA-seq dataset to analyze expression levels of each isoform of the PDE4 (A, B, C and D) using Z-score to normalize the results. Median overall survival (OS) was estimated using Kaplan-Meier methods with the log-rank test. Survival rates for each isoform of the PDE4 were stratified by negative or positive Z-scores (low or high expression groups, respectively). The study cohort had a median follow-up of 21.2 months (range: 0.2-68). Median age at diagnosis was 69 years (range: 43-87) with 67.5 % of diagnosed patients being ≥65 years. Most of patients (72%) were male, and 21% had a prior chemotherapy and/or radiotherapy. The median count (x 109/L) for white blood cells and platelets was 12.2 (range: 0.7-67) and 97.2 (range: 11-331), respectively. IPSS risk was low and int-1 in 51.2% of patients, int-2 in 37.2%, high in 6.9%, and not available in 4.7%. The cytogenetic groups of risk were: very good and good in 53.5% of patients, intermediate in 30.2%, poor in 14% and not available in 2.3%. The overall response rate (ORR) to hypomethylation agents (HMA) was 51.1%, with 37.2% having complete response (CR), 6.9% having bone marrow CR, and 6.9% having hematologic improvement. Median OS for the whole cohort was 17.6 months (95% confidence interval [CI]: 9.6-25.6). Detailed PDE4 isoform-specific analyses are presented in Table1. Importantly, there appears to be an impact on OS depending on PDE4 isoform expression levels, which should be confirmed in a larger patient cohort. PDE4 expression may be useful as both a prognostic factor and a potential therapeutic target for patients with MDS. The effects of PDE4 inhibitors should be investigated in vitro against MDS cell lines and in preclinical mouse models of MDS. One potential application for this study is to exploit the blockade of the PDE4 pathway on the IL8 expression, which may be a good therapeutic strategy against MDS and AML stem cells [Schinke C et al, 2015]. PDE4 inhibitors could also be included in our therapeutic arsenal in the context of the HMA failure in selected patients with MDS.Table 1PDE4 isoform expression stratification by expression levels and by disease stateDISEASE STATE STRATIFICATIONMean expression (range)p valueEXPRESSION LEVEL STRATIFICATIONNumber of patients (%)Mean expression (range)Median OS, months [95% CI]p valuePDE4A0.26PDE4A0.24MDS0.26 (0-5.2)Low27 (62.7)0.26 (0-0.08)15.8 [11.1-37.1]Normal0.108 (0-0.5)High16 (37.3)0.65 (0.1-5.2)29.2 [7.60-36.4]PDE4B0.90PDE4B0.87MDS4.90 (0.06-23.95)Low28 (65.1)2.25 (0.06-4.87)24.1 [11.7-19.8]Normal4.92 (0.06-23.95)High15 (34.9)9.85 (5.21-23.95)22 [26.7-31.8]PDE4C0.03PDE4C0.25MDS0.004 (0-0.045)Low29 (67.4)0.0003 (0-0.002)26.7 [18.6-34.8]Normal0.0009 (0-0.004)High14 (32.6)0.01 (0.004-0.045)12.3 [3.40-21.1]PDE4D0.89PDE4D0.95MDS1.55 (0.007-5.19)Low25 (58.1)0.92 (0.007-1.49)26.7 [4.60-48.80]Normal1.50 (0-3)High18 (41.9)2.42 (1.51-5.10)16.8 [13.3-20.25] DisclosuresNo relevant conflicts of interest to declare.

Clinical Significance and Pathopysiological Function of the Tim-3/Galection-9 Pathway in Myelodysplastic Syndromes

Abstract Background: Galectin-9 (Gal-9), a member of the galectin family, plays a crucial role in inflammation and tumorigenesis involving angiogenesis, cell adhesion, immune escape, and cancer cell survival. In acute myeloid leukemia (AML), Gal-9 is highly expressed in some AML cell lines and binds with T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) on T cells and leukemic stem cells. The Tim-3/Gal-9 pathway may be associated with disease progression by inducing T-cell dysregulation and mediating autocrine proliferation of leukemic cells. However, the role of the pathway in myelodysplastic syndromes (MDS) remains unclear. In the current study, we investigated the prognostic impact of plasma Gal-9 levels and the pathophysiological roles of Gal-9 and Tim-3 in MDS. Methods and results: 1) To investigate the prognostic impact of Gal-9, plasma Gal-9 levels were measured by ELISA in 92 MDS patients: 30 with refractory anemia (RA); 3 with RA with ringed sideroblasts (RARS); 18 with RA with excess blasts (RAEB); 14 with RAEB in transformation (RAEB-t); and 29 with acute leukemia transformed from MDS (AL-MDS). The plasma Gal-9 level increased with disease progression (mean ± SD: control 3.4 ± 0.9; RA, 9.3 ± 5.7; RARS, 10.2 ± 6.8; RAEB, 9.7 ± 6.5; RAEB-t, 17.2 ± 9.6; AML-MDS, 20.1 ± 14.1 ng/ml). Furthermore, patients with AL-MDS in International Prognostic Scoring System (IPSS) high-risk categories (Intermediate-2/High) had significantly higher Gal-9 levels compared with IPSS low-risk (Low/Intermediate-1) patients (Low/Intermediate-1 vs Intermediate-2/High, p = 0.002; Low/Intermediate-1 vs AML-MDS, p = 0.004). The overall survival of MDS patients with plasma Gal-9 higher than the normal level (10 ng/mL) was significantly shorter than that of other patients (log-rank p &lt; 0.001) even in lower-risk (RA, RARS) cases (log-rank p = 0.0369). Moreover, multivariate analysis showed that a high level of Gal-9 was an independent predictor of shorter survival in MDS patients as well as IPSS. 2) Bone marrow mononuclear cells were isolated from some MDS patients, and Tim-3 expression on granulocytes, monocytes, lymphocytes, and blasts was analyzed using flow cytometry (FCM). Tim-3 expression on blasts tended to be higher in AL-MDS patients than in MDS patients. 3) To investigate whether MDS cells can produce Gal-9, we analyzed Gal-9 levels in supernatants of cell cultures of 3 human AL-MDS cell lines, F-36P, SKM-1, and MDS-L, and MDS blasts obtained from an AL-MDS patient by ELISA. Gal-9 was detected in all cell culture supernatants and its concentration was higher when cultured in medium containing 10% fetal bovine serum (FBS) than in 2.5% FBS medium in F-36P and SKM-1 cells. The concentration of Gal-9 produced from MDS blasts was increased time dependently in 2-6 day culture. Furthermore, recombinant human Gal-9 enhanced the cell proliferation of F-36P cells. 4) After analyzing Tim-3 expression in MDS cell lines using real-time quantitative PCR and FCM, all cell lines expressed relatively low levels of Tim-3 mRNA. Extracellular expression of Tim-3 was detected and induced by adding cell culture supernatants of the human stromal cell line HS-5 in F-36P cells. To elucidate the signaling pathway inducing Tim-3 expression, we investigated Tim-3 mRNA expression in F-36P cells treated with HS-5 supernatant with several signal transduction inhibitors (STAT3 inhibitor, U0126; MAPK/ERK; AG490; JAK2, LY294002; PI3K, PDTC; NF-κB inhibitor). Upregulation of Tim-3 gene expression in F-36P cells treated with HS-5 supernatant was inhibited by the MEK inhibitor U0126. These results suggest that the MAPK/ERK pathway is involved in Tim-3 expression induced by HS-5 supernatant. Conclusions: In MDS, the elevation of plasma Gal-9 levels is associated with disease progression, including leukemic transformation, and with shorter survival. Furthermore, its receptor Tim-3 is upregulated on blasts in AL-MDS patients. Our data suggest that the Gal-9/Tim-3 pathway plays a key role in MDS disease progression. Although further study is required to clarify the detailed functions of the interaction between Gal-9 and Tim-3, the current study could lead to the development of a new immunotherapeutic strategy via the blockade of this pathway in MDS. Disclosures No relevant conflicts of interest to declare.

Increased Expression of HERG1 K+ Channels Contribute to MDS Progression and Display Correlation with Prognosis Stratification

Background: Myelodysplastic syndromes (MDS) are defined as a heterogeneous group of clonal hematopoietic stem cell (HSC) malignant disorders which are characterized by bone marrow failure and dysplasia of blood cells. As continually terminated in AML, MDS are treated as the "pre-leukemia" condition. HERG K+ channels, being three subtypes: HERG1, HERG.2, HERG3, are expressed transiently at early stages of cells such as progenitor and stem cells, disappearing at later stages of cells like mature cells. Our previous work and others demonstrated that HERG1, as an oncoprotein, was over expressed in AML cells and played crucial roles in SDF-1induced leukemia cell migration. The expression and functional role of HERG1 K+ channels in MDS development is not reported. We investigated the HERG1 K+ channels expression and explored the functional link between HERG1 K+ channels and MDS progressionMethods: The expression of HERG1 K+ channels in untreated MDS, AML patients and normal control was detected by flow cytometry. The roles of HERG1K+ channels in regulation of SKM-1 cell proliferation, apoptosis and cell cycle by CCK-8 assay and flow cytometry, respectively.Results: We observed that the expression of HERG1 K+ channels on bone marrow (BM) mononuclear cells (MNCs) in MDS patients was significantly higher than that in the controls (42 ± 7.62% vs 19.8 ± 2.79%, p < 0.01) , but was lower than that in AML (42 ± 7.62% vs 52.18 ± 9.72%, p < 0.01). MDS subtypes mainly contained refractory cytopenia unilineage dysplasia (RCUD), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blast I (RAEB-I), refractory anemia with excess blasts II (RAEB-II) and MDS-unclassified (MDS-U). We next analyzed percentage of HERG1 K+ channels in MDS subtypes and found that level of HERG1 K+ channels on the MNCs in each subtypes of MDS was significant higher than that in the control group (Control: 19.8 ± 2.79%, MDS-RCUD: 30.91 ± 1.48%, MDS-RCMD: 39.06 ± 2.47%, MDS-RAEB-I: 44.76 ± 5.54%, MDS-RAEB-II: 49.69 ± 3.28%, p < 0.01), suggesting that HERG1 K+ channels expression might be positively associated with malignancy degree of MDS.HSCs played important roles in the pathophysiology of MDS. Our results also revealed that with the increase of malignancy degree, the percentage of HERG1K+ channels on CD34+CD38- derived cells from MDS subtypes tended to elevate in corresponding MDS subtypes (MDS-RCUD: 61.21 ± 9.46%, MDS-RCMD: 65.66 ± 4.57%, MDS-RAEB-I: 72.35 ± 9.38%, MDS-RAEB-II: 75.71 ± 4.24%, p < 0.05), which supported the notion in other way that MDS are HSC malignant disorders. The over-expression of HERG1 K+ channels on CD34+CD38- cells in MDS patients might be correlated with the oncogenesis of MDS.In addition, the prognosis stratification of MDS patients was performed according to International Prognostic Scoring System (IPSS) scores and the untreated MDS cohort was categorized as four risk groups: Low-R, Int-1-R, Int-2-R and High-R. Our results showed that there was a positively correlation between HERG1 level and IPSS scores of patients (Low-R: 31.93 ± 3.47%, Int-1-R: 39.95 ± 5.76%, Int-2-R: 45.94 ± 6.34%, High-R: 49.05 ± 3.04%, p < 0.01). This showed that expression level of HERG1 K+ channels was helpful for predicting the prognosis of de novo MDS.Furthermore, we analyzed HERG1 K+ channel role on MDS cell proliferation and apoptosis. Incubation with 0, 10, 20 uM E-4031 ( HERG K+ channels inhibitor) with SKM-1 cells (MDS cell line) for 48 h, the results showed that blockage of HERG1 decreased the proliferation of SKM-1 cells but had rarely effects on cell apoptosis and cell cycle distribution. In consistent with other studies, HERG1 K+ channels had already been shown to be necessary for growth of cancer cells through specific activities independent of cell cycle such as interacting with TNFR1 protein which could activate NF-κB to facilitate cell proliferation and favoring transduction of growth signals by MAP kinase/c-fos pathway. These findings showed that physiological activity of HERG1 K+ channels was crucial for MDS cell proliferation and HERG1 K+ channels may be a potential therapeutic target for MDS.Conclusion: Briefly, our study firstly showed that HERG1 K+ channels were aberrantly over-expressed on MDS stem cells, mononuclear cells, and positively associated with malignancy degree of MDS. HERG1 K+ channels functionally contribute to MDS progression and may be a potential therapeutic target for MDS. DisclosuresNo relevant conflicts of interest to declare.

EZH2-Mediated Activation of Serine Biosynthetic Pathway Is Critical for Resistance of Clonal Cells to Stress-Related Apoptosis in Low-Grade Myelodysplastic Syndrome

BackgroundIncreased cell apoptosis mediated by P53 or activated autoimmunity was frequent in low-grade MDS especially in 5q-syndrome and those with trisomy 8. However, MDS clonal cells can gradually acquired apoptosis resistance under different stressed conditions. The mechanism of apoptosis resistance is still not fully understood. The polycomb group protein EZH2 as an anti-apoptosis protein drives tumorigenesis. The association of EZH2 with apoptosis resistance of MDS clonal cells is worth further investigating.ObjectiveThe objective of this study is to investigate the role of EZH2 in the mechanism of apoptosis resistance of MDS.MethodsThe expression of EZH2 protein was analyzed by flow cytometry in 71 MDS patients. EZH2-targeted genes were identified through an integrated analysis of gene expression microarray analysis in MDS patients and cell line. EZH2-targeted genes were confirmed through chromatin immunoprecipitation and luciferase reporter gene assay. Cell functional and metabolic experiments were also performed.ResultsWe observed upregulation of the EZH2 expression in the patients with low-grade MDS especially in the individuals with trisomy 8 although downregulation in the patients with high-grade MDS. Based on the distinct EZH2 expression pattern, we identified an EZH2-targeted gene PSAT1, a serine biosynthetic critical enzyme through an integrated analysis of gene expression microarray analysis in MDS patients and cell line. Functionally, EZH2 can directly activate PSAT1 transcription by binding to its promoter and this effect is independent of its H3K27me3 enzymatic activity in vitro. Expanded sample analysis revealed the overexpression of PSAT1 in MDS patients especially in low-grade MDS. High expression of PSAT1 was associated with severe cytopenias, iron overload and activation of T lymphocytes-mediated autoimmunity. In vitro, overexpression of PSAT1 led to a significant proliferation advantage in the MDS and leukemia cell lines. Overexpression of PSAT1 could also downregulate apoptosis sensitivity to inflammatory cytokines and cytotoxic agents in vitro. However, knockdown of PSAT1 resulted in reduced cell growth capacity and increased the apoptosis sensitivity to cytotoxic agents. Further cancer metabolic experiments showed that overexpression of PSAT1 inhibited the ROS generation and induced an increase in glucose uptake, lactate and ATP production, which may make MDS clonal cells adaptive in the stress-related apoptosis environment.ConclusionEZH2/PSAT1 axis-mediated serine biosynthetic pathway is activated in low-grade MDS, which may confer MDS clonal cells growth advantages and resistance to stress-related apoptosis. DisclosuresNo relevant conflicts of interest to declare.

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.

Effect of Azacytidine on the Hematopoietic Stem and Progenitor Cell Compartments of MDS Mouse Models: Unveiling the Mechanisms of Remission and Relapse

Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic malignancies characterized by the defective production of mature blood cells and the risk of progression to myeloid leukemia. Hypomethylating agents (HMA) are currently the first-line therapy for high-risk MDS and induce hematological improvement in 50% of the patients. However, complete responses occur in less than 20% of these patients, and a majority of MDS patients treated with HMA will eventually relapse, with a subsequently fatal prognosis. Although the presence of mutations in certain genes, such as Tet2, has been reported to predict response to HMA, the causes of resistance and relapse have yet to be described.MDS research has been traditionally a slow-moving field owing to the lack of well-established MDS cell lines and animal models. This landscape recently changed with the development of transgenic mouse models that mirror the phenotype of high-risk MDS patients. In mice from the 4th/5th generations (G4/G5) of our group's telomere-dysfunctional TERTER/ER mouse model, the DNA-damage response triggered by telomere erosion causes profound cell-intrinsic abnormalities in hematopoietic stem and progenitor cells (HSPC) that lead to myeloid-skewed hematopoiesis. These mice exhibit peripheral blood (PB) cytopenias, age-related granulocytosis and myeloid infiltration of the BM and the spleen (Colla et al. Cancer Cell 2015). Similarly, the conditional deletion of Tet2 in mouse hematopoietic cells generates a phenotype of HSC expansion and myeloid skewing of hematopoiesis. These defects, which are also cell-intrinsic, induce progressive PB neutrophilia and granulocytosis, with myeloid infiltration of the bone marrow (BM) and spleen and severe splenomegaly (Moran-Crusio et al. Cancer Cell 2011).Using the TERTER/ER and Tet2-/- mouse models as a platform for cellular and molecular studies, we have investigated the mechanism of action of HMA in MDS, with a focus on the characterization of response by the different HSPC populations and the mechanisms involved in resistance and relapse. Our results show that the administration of the HMA azacytidine (AZA) for 7 days induces in both mouse models a 20-40% drop in white blood cell counts and a 17-24% decrease in hemoglobin levels that mimic patient responses to AZA. After the last day of treatment and during the 3 last weeks of the cycle, cell counts and hemoglobin levels returned to normal values. These changes in PB were explained by an 80-86% decrease in the number of BM common myeloid progenitors (CMP; n = 8, P = 0.0046) and granulo-monocytic progenitors (GMP; n = 8, P <0.0001) after 1 week of AZA treatment, while megakaryocytic-erythroid progenitors remained largely unaffected. However, the number of CMP and GMP in the BM of both G5 and Tet2-/- mice recovered by the end of each cycle, and the characteristic myeloid skewing was not overcome after 2 cycles of treatment, which indicates that AZA cannot reverse the cell-intrinsic defects of these progenitors. Most importantly, BM analysis in both mouse models after one week of treatment and during the recovery period revealed that AZA has no effect on long and short-term HSC populations.To study the effect of AZA in a BM context that mimicked the coexistence of MDS and normal cells in patients, we performed competitive transplantation experiments in both models. Our preliminary results indicate that, in mixed populations, AZA induces a 2.6-fold expansion of the wild type long-term HSC compartment (n = 4, P = 0.0018). The fact that AZA gives a repopulation advantage to normal HSC suggest that, after several cycles, there is a shift in the abundance of normal versus MDS cells in patients BM, and this explains why patients enter morphologic remission. However, in both transgenic mouse models, AZA failed to decrease the size of the MDS-like HSC compartment. This indicates that, although this drug allows a temporary hematologic recovery, it does not eliminate the cell populations that maintain the disease, and therefore it cannot eliminate MDS.Ongoing unbiased transcriptomic and methylomic analyses in the sorted MDS-like HSC that survived AZA treatment will identify the HSC signature of resistance to HMA, and the integration of these results with similar analyses in MDS patients will provide valuable insights into the mechanistic bases of relapse and resistance to HMA and facilitate the design of better combination therapies for high-risk MDS. DisclosuresNo relevant conflicts of interest to declare.

Chidamide, a Novel Histone Deacetylase Inhibitor, Displays Potent Antitumor Activity Against MDS Cells Mainly through JAK2/STAT3 Signaling Inhibition

Background:Many studies have indicated that histone deacetylases (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity are promising new strategies in the treatment of cancers. Chidamide (CS055/HBI-8000), a novel histone deacetylases inhibitor (HDACi) of the benzamide class, is currently under clinical trials. Despite emerging information on the effect of chidamide in multiple cancers, little is yet known about mechanism of action, and there were few reports about this drug's effects on myelodysplastic syndromes (MDS). In this study, we aimed to investigate the antitumor activities of chidamide on MDS cell line SKM-1 and to explore the possible mechanism.Methods:We treated MDS cells with different concentrations of chidamide. The effect of chidamide on HDAC activity of MDS cells was studied by using a HDAC Fluorometric Activity Assay Kit. The induction of histone acetylation was confirmed by detecting acetylated histone H3 and H4 using Western blot analysis. The effect of chidamide on the proliferation of MDS cells was analyzed by Cell Counting Kit (CCK-8) assay. Apoptosis were detected by Annexin V/propidium iodide (PI) double-labeled cytometry. Cell cycle was analyzed by a PI method. The acetylation levels of genes promoterassociated histone H3 and H4 were examined by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR. Quantitativereal-time PCR and Western blot were used to detect the expression of signaling pathway factors (SOCS3, JAK2, STAT3,Bcl-XL,Bcl-2 and Mcl-1).Results:Our results demonstrated chidamide suppressed MDS cells growth in a time- and dose-dependent manner, and induced cell apoptosis and cell cycle arrest at G0/G1phase. Chidamide was able to significantly inhibit the HDAC activity and increase the acetylated histoneH3 and H4 of MDS cells. ChIP analysis further indicated that chidamide induced acetylated histones accumulation in the promoter of SOCS3.Moreover, chidamide upregulated the mRNA and protein expression ofSOCS3, and also significantly downregulated JAK2/STAT3 signaling. Further, we identified reduced expression of STAT3 downstream targets with treatment of chidamide, including Bcl-XL, Bcl-2 and Mcl-1, which may explain the cytotoxic effects of chidamide on MDS cells.Conclusion:These results demonstrate that chidamide possesses significant cytotoxic effects on MDS cells mainly through histone modifications of SOCS3 and consequently JAK2/STAT3 signaling inhibition. Therefore, Our data provide rationale for clinical investigation of chidamide in MDS. DisclosuresNo relevant conflicts of interest to declare.

Synergistic Effect and Molecular Mechanism of Homoharringtonine and Bortezomib on SKM-1 Cell Apoptosis.

BackgroundMyelodysplastic syndromes (MDS) are clonal marrow stem-cell disorders with a high risk of progression to acute myeloid leukemia (AML). Treatment options are limited and targeted therapies are not available for MDS. In the present study, we investigated the cytotoxicity and the molecular mechanism of Homoharringtonine (HHT) and Bortezomib towards high-risk MDS cell line SKM-1 in vitro and the role of miR-3151 was first evaluated in SKM-1 cells.MethodsSKM-1 cells were treated with different concentrations of HHT or Bortezomib, and cell viability was analyzed with CCK-8 assay. The influence on cell proliferation, cell cycle distribution and the percentage of apoptosis cells were analyzed by flow cytometry. Calcusyn software was used to calculate combination index (CI) values. Western blot was used to analysis phosphorylation of Akt and nuclear NF-κB protein expression in SKM-1 cells. Mature miR-3151 level and p53 protein level were detected after HHT or Bortezomib treatment. The cell proliferation and p53 protein level were reassessed in SKM-1 cells infected with lentivirus to overexpress miR-3151.ResultsSimultaneous exposure to HHT and Bortezomib (10.4:1) resulted in a significant reduction of cell proliferation in SKM-1 cells (P < 0.05). Cell cycle arrest at G0/G1 and G2/M phase was observed (P < 0.05). HHT and Bortezomib synergistically induced cell apoptosis by regulating members of caspase 9, caspase 3 and Bcl-2 family (P < 0.01). The mechanisms of the synergy involved Akt and NF-κB signaling pathway inhibition, downregulation of mature miR-3151 and increment of downstream p53 protein level. Overexpression of miR-3151 promoted cell proliferation and inhibited p53 protein expression in SKM-1 (P < 0.01).ConclusionsHHT and Bortezomib synergistically inhibit SKM-1 cell proliferation and induce apoptosis in vitro. Inhibition of Akt and NF-κB pathway signaling contribute to molecular mechanism of HHT and Bortezomib. miR-3151 abundance is implicated in SKM-1 cell viability, cell proliferation and p53 protein expression.

Clinical Significance of ID4 Gene Mehtylation in Demethylation-Treated MDS Cell Line and 2 MDS Patients

To evaluate significance of ID4 gene mehtylation in demethylating myelodysplastic syndrome(MDS) cell Line MUTZ1 and 2 patients with MDS. The methylation-specific PCR (MS-PCR) and reverse transcription-PCR (RT-PCR) were applied to identify the methylation status and gene expression of ID4 gene in MDS cell line MUTZ1, a patient with aplastic anemia(AA) and a donor with normal bone marrow (NBM). RT-PCR was applied to detect the ID4 gene expression status in MUTZ1 cell line treated with decitabine at 3 different concentrations. Then bisulfite sequencing PCR (BSP) was applied to detect ID4 gene methylation status in 2 MDS parients treated with decitabine. The MDS cell line MUTZ-1 displayed a complete methylation of ID4 gene promoter with little mRNA expression. Inversely, bone marrow of an AA patient and NBM showed complete unmethylation of this gene with intensity mRNA expression. With the increase of decitabine concentration, ID4 gene mRNA expression was more and more increased. After decitabine treatment, ID4 gene methylation-positive frequencies of both the 2 MDS patients were much more decreased than that of the first treatment. So, ID4 gene mRNA expression inhibited by promoter hypemethylation could be recovered by using demethylation medicine. ID4 as a new potential anti-oncogene suggests that its methylation may become a marker for selection and assessment of therapeutic schedules in patients with MDS.

Downregulation of HO-1 promoted apoptosis induced by decitabine via increasing p15INK4B promoter demethylation in myelodysplastic syndrome.

Decitabine, which reverses hypermethylation of the p15(INK4B) gene in vitro, has been used to relieve cytopenias and blast excess in over 50% of patients with high-risk myelodysplastic syndrome (MDS). In this study, heme oxygenase-1 (HO-1) was overexpressed in MDS cell line SKM-1, which was closely related to resistance to decitabine-induced apoptosis. We aimed to further investigate the role of HO-1 in apoptosis induced by low-dose decitabine in SKM-1 cells. Upregulation of HO-1 by transfecting it into SKM-1 cells with lentivirus vector promoted cell proliferation and protected them against apoptosis. In contrast, downregulation of HO-1 enhanced decitabine-induced apoptosis but reduced accumulation of the S phase in cell cycle. To explore the mechanism, the expressions of cell cycle-related proteins were detected after the cells were treated by decitabine in each group. p15(INK4B) and CDK4 were overexpressed in SKM-1 cells in which HO-1 was inhibited, and the expression-depending apoptosis was related to the caspase-3 pathway. Even though HO-1 was silenced, the apoptotic rate never increased as the caspase-3 pathway was blocked. It is well known that p15(INK4B) dominantly regulates the S phase of the cell cycle. p15(INK4B) was herein demethylated more evidently in the group of SKM-1 cells in which HO-1 was downregulated, as well as in the mononuclear cells of patients suffering from MDS. In the case of poor prognosis, the mRNA level of HO-1 was raised. In conclusion, overexpression of HO-1 indicated resistance to demethylation of p15(INK4B) induced by decitabine.

Sonic hedgehog produced by bone marrow-derived mesenchymal stromal cells supports cell survival in myelodysplastic syndrome.

The role of marrow microenvironment in the pathogenesis of myelodysplastic syndrome (MDS) remains controversial. Therefore, we studied the influence of bone marrow-derived mesenchymal stromal cells (BMSCs) from patients with different risk types of MDS on the survival of the MDS cell lines SKM-1 and MUTZ-1. We first demonstrated that the expression of Sonic hedgehog (Shh), smoothened (Smo), and glioma-associated oncogene homolog 1 (Gli1) was increased in MDS patients (n = 23); the increase in expression was positively correlated with the presence of high-risk factors. The Shh signaling inhibitor, cyclopamine, inhibited high-risk MDS BMSC-induced survival of SKM-1 and MUTZ-1 cells, suggesting a role for Shh signaling in MDS cell survival. Furthermore, cyclopamine-mediated inhibition of Shh signaling in SKM-1 and MUTZ-1 cells resulted in decreased DNMT1 expression and cell survival; however, exogenous Shh peptide had the opposite effect, suggesting that Shh signaling could regulate the expression of DNMT1, thereby modulating cell survival in MDS. In addition, the apoptosis of SKM-1 and MUTZ-1 cell increased significantly when cultured with cyclopamine and a demethylation agent, 5-Aza-2′-deoxycytidine. These findings suggest that Shh signaling from BMSCs is important in the pathogenesis of MDS and could play a role in disease progression by modulating methylation.

Lenalidomide Promotes Degradation of Casein Kinase 1a (CK1a) through Cereblon: Implications for the Efficacy of Lenalidomide in MDS and AML

Background: Lenalidomide has broad clinical activity in hematologic malignancies, including lymphoid malignancies (multiple myeloma (MM), non-Hodgkin’s lymphomas, B cell chronic lymphocytic leukemia) and myeloid malignancies (myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML)). Lenalidomide’s molecular mechanism involves modulation of the cullin 4 RING E3 ubiquitin ligase complex (CRL4-CRBN), with downstream effects on protein homeostasis. The binding of lenalidomide to CRL4-CRBN promotes ubiquitination and degradation of Aiolos and Ikaros in B cell lineages (MM, lymphoma), and regulation of Ikaros has been shown to be a key effector of lenalidomide’s anti-myeloma tumoricidal and T cell immunomodulatory activities. Multiple mechanisms of clinical efficacy have been hypothesized for lenalidomide activity in del(5q) MDS; however, lenalidomide-regulated substrates of therapeutic relevance to myeloid malignancies have not been defined. We undertook a proteomics approach to identify such substrates.Methods: Changes in global cellular protein levels were measured by tandem-mass-tagged proteomics in a del(5q) MDS cell line (MDS-L) and an AML cell line (HNT-34), following treatment with vehicle or 10 uM lenalidomide for 8, 24 and 72 hours. Western blot analysis was used to subsequently validate proteins that were differentially regulated in these lenalidomide-sensitive cell lines. Sensitivity to lenalidomide treatment in a panel of myeloid cancer cell lines was evaluated by tritiated thymidine and/or BrdU assays.Results: Across a panel of myeloid cancer cell lines evaluated for sensitivity to lenalidomide, HNT-34 and MDS-L cells were the most sensitive to lenalidomide, with EC50’s of 0.6 and 1.5 µM, respectively, while other cell lines were predominantly insensitive (EC50 > 10 µM). Proteomic analysis of lenalidomide-regulated proteins in MDS-L cells revealed Ikaros as the most down-regulated protein at 72 hrs (5.3 fold), and CK1α was the second most down-regulated protein (2.8 fold). The decrease of Ikaros and CK1α proteins by lenalidomide was confirmed by Western blot analysis in MDS-L and HNT-34 cells. Ikaros protein levels were reduced by lenalidomide in cell lines that were sensitive or insensitive to lenalidomide, suggesting that modulation of other proteins may be responsible for lenalidomide sensitivity of myeloid cancer cells. Ikaros levels were also decreased by a CRBN-binding glutarimide analog in HNT-34 cells, which were insensitive to the analog, further suggesting that Ikaros was not a substrate of consequence to sensitivity. In contrast, an initial study of five cell lines showed that lenalidomide promoted the greatest degradation of CK1a in the most sensitive lines (HNT-34, MDS-L), but did not degrade CK1a in the most insensitive line (MOLM-13). Mechanistic studies addressing CK1α regulation in HNT-34 cells revealed that CK1α protein levels were reduced by lenalidomide treatment in a time- and dose-dependent manner, with maximal reduction of 3.3 fold observed at 4 hrs using 10 µM lenalidomide, and CK1α degradation observed with lenalidomide at a dose as low as 0.1 µM. Pre-treating HNT-34 cells with the proteasome inhibitor MG-132 stabilized CK1α protein levels in the presence of lenalidomide, demonstrating proteasome-dependent degradation. A competition experiment performed by pre-treating HNT-34 cells with the glutarimide analog that binds CRBN, but does not result in the degradation of CK1a, resulted in CK1α protein stabilization in the presence of lenalidomide, demonstrating CRBN-dependence of the lenalidomide-induced degradation. Degradation of CK1α was also observed in primary peripheral blood mononuclear cells of AML patients treated with lenalidomide.Conclusions: CK1α is a lenalidomide-induced substrate of CRL4-CRBN, with initial links to myeloid cancer cell line sensitivity to lenalidomide. As the CSNK1A1 gene is located at 5q32, a commonly deleted region in MDS, further reduction of haplo-insufficient expression of CK1α is a potential mechanism of sensitivity to lenalidomide in del(5q) MDS. The therapeutic relevance of CK1α regulation by lenalidomide in AML requires further exploration. CK1α gene silencing and additional correlative studies of lenalidomide-induced degradation of CK1α are ongoing to define mechanistic links to myeloid cancer cell sensitivity to lenalidomide. DisclosuresHollenbach:Celgene: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Gandhi:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. MacBeth:Celgene: Employment, Equity Ownership.

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 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.

Overexpression of miR-125a in Myelodysplastic Syndrome CD34+ Cells Modulates NF-κB Activation and Enhances Erythroid Differentiation Arrest

Myelodysplastic syndromes (MDS) are characterized by impaired proliferation and differentiation of hematopoietic stem cells. The participation of toll-like receptor (TLR)-mediated signaling in MDS is well documented. Increased TLR signaling leads to the constitutive activation of NF-κB, which mediates inflammation, cell proliferation and apoptosis. In addition, the TLR pathway induces the expression of miRNAs which participate in the fine-tuning of the inflammatory response. miRNAs also regulate other biological processes, including hematopoiesis. miR-125a and miR-125b are known modulators of hematopoiesis and are abnormally expressed in several hematologic malignancies. However, little is known about their role in MDS. NF-κB-activating ability has been described for both miRNAs. We studied the role of miR-125a/miR-125b in MDS and their relationship with TLR signaling and hematopoietic differentiation. Our results indicate that miR-125a is significantly overexpressed in MDS patients and correlates negatively with patient survival. Expression of miR-99b, which is clustered with miR-125a, is also directly correlated with prognosis of MDS. Both miR-125a and miR-99b activated NF-κB in vitro; however, we observed a negative correlation between miR-99b expression and the levels of TLR2, TLR7 and two downstream genes, suggesting that NF-κB activation by the miRNA cluster occurs in the absence of TLR signaling. We also show that TLR7 is negatively correlated with patient survival in MDS. In addition, our data suggest that miR-125a may act as an NF-κB inhibitor upon TLR stimulation. These results indicate that miR-125a is involved in the fine-tuning of NF-κB activity and that its effects may depend on the status of the TLR pathway. Furthermore, we observed that miR-125a inhibits erythroid differentiation in leukemia and MDS cell lines. Therefore, this miRNA could serve as a prognostic marker and a potential therapeutic target in MDS.