Therapeutic Target In Myelodysplastic Syndrome Research Articles

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.

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.

Increased expression of interferon signaling genes in the bone marrow microenvironment of myelodysplastic syndromes.

IntroductionThe bone marrow (BM) microenvironment plays an important role in the pathogenesis of myelodysplastic syndromes (MDS) through a reciprocal interaction with resident BM hematopoietic cells. We investigated the differences between BM mesenchymal stromal cells (MSCs) in MDS and normal individuals and identified genes involved in such differences.Materials and MethodsBM-derived MSCs from 7 MDS patients (3 RCMD, 3 RAEB-1, and 1 RAEB-2) and 7 controls were cultured. Global gene expression was analyzed using a microarray.ResultWe found 314 differentially expressed genes (DEGs) in RCMD vs. control, 68 in RAEB vs. control, and 51 in RAEB vs. RCMD. All comparisons were clearly separated from one another by hierarchical clustering. The overall similarity between differential expression signatures from the RCMD vs. control comparison and the RAEB vs. control comparison was highly significant (p = 0), which indicates a common transcriptomic response in these two MDS subtypes. RCMD and RAEB simultaneously showed an up-regulation of interferon alpha/beta signaling and the ISG15 antiviral mechanism, and a significant fraction of the RAEB vs. control DEGs were also putative targets of transcription factors IRF and ICSBP. Pathways that involved RNA polymerases I and III and mitochondrial transcription were down-regulated in RAEB compared to RCMD.ConclusionGene expression in the MDS BM microenvironment was different from that in normal BM and exhibited altered expression according to disease progression. The present study provides genetic evidence that inflammation and immune dysregulation responses that involve the interferon signaling pathway in the BM microenvironment are associated with MDS pathogenesis, which suggests BM MSCs as a possible therapeutic target in MDS.

Novel Therapeutic Approach to Improve Hematopoiesis By Targeting Myeloid Derived Suppressor Cells with a Humanized Anti-CD33 Antibody

Myelodysplastic syndrome (MDS) is one of the major types of acquired bone marrow failure characterized by impaired peripheral blood cell production (anemia and cytopenias). Although much has been learned regarding the molecular genetic events involved in the pathogenesis of the MDS hematopoietic stem/progenitor cell compartment (HSC/HPC), investigations of the environmental pressures underlying disease initiation have been limited. Prior investigations have shown that inflammation in the bone marrow microenvironment contributes to hematopoietic impairment, with inflammatory molecules providing regulatory cues driving the proliferation and apoptotic death of HSC/HPC. We have reported that, the accumulation of myeloid-derived suppressor cells (MDSCs) in the local inflammatory bone marrow microenvironment plays a major role in the direct pathogenesis of MDS. MDSCs function by producing mediators and inflammatory cytokines capable of suppressing hematopoiesis and are defined by the lack of all lineage markers and only one key surface receptor, CD33 (CD33+HLA-DR−Lin−). We recently uncovered that this receptor is greatly expressed in MDSC isolated from patients with MDS and plays an important role in MDSC-mediated hematopoietic suppressive function. We also found that this ITIM (immune-receptor tyrosine-based inhibitor motif) containing molecule could induce suppressive cytokines after its engagement with its newly identified ligand S100A9. Therefore, we tested the hypothesis that with a fully human IgG1 monoclonal antibody against CD33 (mAb33.1) we could prevent the engagement of this ligand/receptor pair as well as induce ADCC of pathogenic MDSC through the reduction in the accumulation of CD33+ MDSC, the immune suppression and the restoration of hematopoiesis in MDS bone marrow specimens. After testing primary specimens with mAb33.1 we saw a significant increase in ADCC activity via NK as evidenced by significant reduction in the proportion of MDSC in culture as compared to isotype control treated cells. This decrease correlated with an increase in CD107a granule mobilization as well as an increased cytotoxicity in a killing assay without changing the proportion of NK cells in the culture. Functionally, the concentration of secreted IL-10, TGFb and VEGF were decreased, as was the gene expression of these suppressive cytokines after treatment with mAb33.1 but was restored when the antibody was cross-linked with an anti-human IgG antibody demonstrating the blocking ability of the antibody in preventing CD33 downstream signaling. Importantly, all of these observations correlated with the restoration of hematopoiesis, as there was a significant increase in the formation of CFU-GM and BFU-E colonies on a methylcellulose assay (n=9). In addition mAb33.1 also displayed a protective effect on HSC by blocking ROS production and reducing DNA damage, as demonstrated by comet and H2AX assays. This work provides the ground for the development of a novel group of therapies directly aimed at the suppressive MDSC and blockage of their signaling, rather than directly targeting the malignant clone, with the long term goal of improving the local microenvironment. This strategy will provide the background to assess its clinical potential to serve as a therapeutic target in MDS and AML. DisclosuresNo relevant conflicts of interest to declare.

Strategic Role of Nuclear Inositide Signalling in Myelodysplastic Syndromes Therapy

Nuclear inositide signalling is implicated in normal and pathological cell proliferation and differentiation in several distinct models. Among the key molecules of nuclear inositide pathways, phosphoinositide-phospholipase (PI-PLC) C &#946;1 is essential for regulating hematopoiesis, particularly along myeloid and erythroid lineage. Moreover, Akt activation is associated with protein synthesis, via mTOR pathway, and with erythroid induction, through PI-PLCγ1 activation. Myelodysplastic syndromes (MDS) are a series of heterogeneous diseases characterized by ineffective hemopoiesis, with a variable risk of evolution into acute myeloid leukemia (AML). Therapeutic approaches for MDS include demethylating agents, such as azacitidine, aiming at reducing cell proliferation, and erythropoietin, useful for sustaining a normal erythropoiesis. In the last few years, a role for nuclear inositide signalling as a therapeutic target in MDS has been disclosed, in that PI-PLC&#946;1 increase is associated with azacitidine responsiveness, even when this drug is used in combination with other agents, and Akt is specifically activated in MDS at higher risk of AML evolution. On the other hand, recent data demonstrated that inositide signalling can also be involved in erythroid therapy, given the inhibitory effect of erythropoietin on PI-PLC&#946;1 and the activation of Akt/PI-PLCγ1 pathway, following the administration of erythropoietin. Here, we review the strategic role of nuclear inositide signalling in MDS, in pathogenesis and therapy.

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.

NS-018, a selective JAK2 inhibitor, preferentially inhibits CFU-GM colony formation by bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients

JAK2/STAT signaling promotes survival and expansion of myelodysplastic syndrome (MDS) clones, but little is known about the potential of JAK2/STAT as a therapeutic target in MDS. We investigated the effect of NS-018, a novel antagonist for JAK2, on the colony-forming ability of bone marrow mononuclear cells (BMMNCs) from high-risk MDS patients. NS-018 decreased colony-forming unit-granulocyte/macrophage (CFU-GM) colony numbers from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent than against normal BMMNCs. In addition, NS-018 suppressed the phosphorylation of STAT3 in colony-forming cells from MDS patients. Collectively, NS-018 could be a new therapeutic option for high-risk MDS.

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.

C-Terminal-Truncating ASXL1 Mutations Induce MDS Via Inhibition Of PRC2

Recurrent mutations of ASXL1 (Additional sex combs-like1) are found in various hematological malignancies including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia, and acute myeloid leukemia (AML) with myelodysplasia-related changes. Additionally, ASXL1 mutations are linked with adverse survival in a variety of myeloid malignancies. A previous study demonstrated that loss of ASXL1 in mice promotes myeloid transformation by impairing polycomb repressive complex 2 (PRC2)-mediated gene repression at a number of critical loci and leads to myeloid transformation. However, most ASXL1 mutations are heterozygous and located in the 5’ region of the last exon, indicating a dominant-negative or gain-of-function feature of a truncated ASXL1 protein. Therefore, we investigated if the C-terminal truncated form of ASXL1 (ASXL1-MT) contributes to the development of myeloid malignancies. To this end, we examined the effects of ASXL1-MT using in vitro and in vivo experiments. In in vitro experiment, expression of ASXL1-MT inhibited G-CSF-induced myeloid differentiation of 32Dcl3 cells. In a mouse bone marrow transplantation (BMT) model, ASXL1-MT induced multilineage dysplasia, differentiation block, slowly progressive pancytopenia, BM hyperplasia and splenomegaly. The transduced mice died of severe anemia after a long latency (median survival, 400.5 days), and some of the mice progressed to overt leukemia. Thus, the current model displays all of the features of human MDS. In addition, ASXL1-MT collaborated with N-RAS-G12V, which confers a proliferative advantage, in inducing progression of N-RAS-G12V-induced myeloproliferative neoplasm (MPN) to AML, suggesting that ASXL1-MT contributes to leukemic transformation by inhibiting differentiation of MPN cells. To clarify the molecular mechanism for differentiation block and MDS development induced by ASXL1-MT, we performed expression profiles of 32Dcl3 cells transduced with ASXL1-MT and BM cells of the MDS mice. Of note, gene set enrichment analysis (GSEA) of BM cells of the MDS mice indicated that ASXL1-MT induced an expression profile which inversely correlated with known PRC target genes. In fact, ASXL1-MT remarkably derepressed expression of posterior Hoxa genes, including Hoxa5, Hoxa9 and Hoxa10, which are epigenetically silenced by PRC2 in mature cells. In consistent with this, H3K27me3 was globally reduced in ASXL1-MT transduced cells. We also found ASXL1-MT as well as wild type ASXL1 (ASXL1-WT) can bind to EZH2 and, importantly, co-expression of ASXL1-MT with ASXL1-WT efficiently inhibited the binding between ASXL1-WT and EZH2, suggesting a dominant-negative role of ASXL1-MT against the PRC2 function. Using a chromatin immunoprecipitation (ChIP) assay, we confirmed that H3K27me3 and Ezh2-bindig profoundly decreased around the promoter regions of Hoxa5, Hoxa9, and Hoxa10 in the MDS mice, correlating with the upregulation of their mRNA expression. On the other hand, we found that ASXL1-MT reduced the expression of Clec5a, a type 2 transmembrane receptor and that this reduction was associated with differentiation block of the 32Dcl3 cells. Moreover, utilizing an shRNA or a mutant form of Clec5a, we identified that Clec5a plays essential roles in myeloid differentiation of 32Dcl3 cells. Lastly, we searched for microRNAs deregulated by ASXL1-MT since a large subset of microRNAs are found to be transcriptionally regulated by PRC2. Among upregulated microRNAs related to myeloid malignancies, we found that miR-125a targeted 3’UTR of Clec5a gene, repressed Clec5a expression and inhibited granulocytic differentiation in vitro. Intriguingly, H3K27me3 and Ezh2-bindig greatly decreased around the miR-125a gene in the BM cells of the MDS mice, similar to the results of ChIP assays around Hoxa genes. The present results indicate that ASXL1-MT which results in a truncated protein product may (1) inhibit PRC2-function by impairing the interaction of EZH2 with the ASXL1-WT and (2) promote myeloid transformation through impaired PRC2-mediated repression of posterior HOXAs and miR-125a, and subsequent suppression of CLEC5A. HOXA9 and CLEC5A expression were shown to be high and low, respectively, in MDS patients with ASXL1-MT. Our data provide evidence for a novel axis in MDS pathogenesis and implicate both mutant forms of ASXL1 and miR-125a as therapeutic targets in MDS. Disclosures: No relevant conflicts of interest to declare.

Myelodysplastic syndromes are induced by histone methylation–altering ASXL1 mutations

Recurrent mutations in the gene encoding additional sex combs-like 1 (ASXL1) are found in various hematologic malignancies and associated with poor prognosis. In particular, ASXL1 mutations are common in patients with hematologic malignancies associated with myelodysplasia, including myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal–truncating Asxl1 mutations (ASXL1-MTs) inhibited myeloid differentiation and induced MDS-like disease in mice. ASXL1-MT mice displayed features of human-associated MDS, including multi-lineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. ASXL1-MT resulted in derepression of homeobox A9 (Hoxa9) and microRNA-125a (miR-125a) expression through inhibition of polycomb repressive complex 2–mediated (PRC2-mediated) methylation of histone H3K27. miR-125a reduced expression of C-type lectin domain family 5, member a (Clec5a), which is involved in myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1-MT, while CLEC5A expression was generally low. Thus, ASXL1-MT–induced MDS-like disease in mice is associated with derepression of Hoxa9 and miR-125a and with Clec5a dysregulation. Our data provide evidence for an axis of MDS pathogenesis that implicates both ASXL1 mutations and miR-125a as therapeutic targets in MDS.

IRAK1 Is a Therapeutic Target in Myelodysplastic Syndrome

Abstract An IRAK1 inhibitor blocks MDS clone expansion and prolongs survival in an MDS xenograft model.

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

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

Upregulation of Focal Adhesion Kinase, a Potential Therapeutic Target, in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS).

Abstract 2827 IntroductionThe myelodysplastic syndromes (MDS) are a group of leukemia characterized by bone marrow failure and increased risk of transformation to acute myelogenous leukemia (AML). Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase (PTK), plays a key role in the integration of protein-mediated signal transduction. It is a critical mediator of the integrin signaling cascade, which modulates cell proliferation, apoptosis, adhesion, spreading and migration. FAK is upregulated in a wide variety of human cancers. It has been reported that expression of FAK in acute myeloid leukemia (AML) is associated with enhanced blast migration, increased cellularity and poor prognosis. Furthermore, FAK silencing inhibited leukemogenesis in BCR-ABL-transformed hematopoietic cells. FAK has been proposed a potential target in cancer therapy. In this study, we studied the expression patterns of FAK in 98 patients with MDS and performed preclinical studies of FAK inhibition in leukemia cell lines. MethodsCD34+ cells from 98 newly diagnosed patients with MDS and 5 normal donor bone marrow specimens were sorted with CD34 isolation kit from Miltenyi Biotec. Total cellular RNA was isolated using Trizol, cDNA was obtained by using TaqMan reverse transcription reagent (Applied Biosystems). For real-time PCR, FAK assay were purchased from Applied Biosystems and analyzed with an Applied Biosystems Prism 7500 Sequencing detection system. GAPDH was used as internal control. Immunohistochemistry was used to detect FAK protein level in cytospin from MDS CD34+ cells. FAK antibody was obtained from abcam. FAK inhibitor was purchased from Santa Cruz Biotechnology. ResultsFor the 98 MDS patients studied, 78% were older than 60 years; by IPSS score, 17 (17.3%) low risk, 35 (35.7%) INT-1, 24 (24.5%) INT-2, 10 (10.2%) high risk, 12 (12.2%) not available. By cytogenetics, diploid 44 (45%), 20q- 7 (7%), -5/5q- 7 (7%), -7/7q- 7 (7%), -5/5q- and -7/7q- 6 (6%), 8+ 6 (6%), IM 6 (6%), MISC 14 (14%). By real-time PCR, we observed FAK overexpression in 28% of MDS CD34+ cells (fold 2.2–26). We then analyzed FAK protein expression in 10 MDS CD34+ cell cytospin with either higher or lower RNA expression by QPCR using immunohistochemistry. The protein expression patterns were 100% consistent with RNA expression level. This result suggests that FAK expression is upregulated in MDS CD34+ cells. We then performed analysis of clinical associations between FAK expression and clinical characteristics. No association was observed in particular between FAK expression and survival. Because of the potential role of FAK as a therapeutic target, we then studied the effects of FAK inhibition in cell lines. We studied FAK expression level in leukemia cell lines of AML origin and found high protein expression level of FAK in AML leukemia cell line OCI-AML3 and KG1. We treated these cells with FAK inhibitor 14 and detected a dose dependent anti-proliferative effect on both cell lines. Using Annexin V - FITC analysis by flow cytometry, we found the FAK inhibition could induce apoptosis in both cell lines at concentrations of 10uM both at 24 hours and 48 hours. ConclusionsThis study shows that FAK is aberrantly expressed in MDS CD34+ cells, together with the anti-proliferative and apoptosis induction effect of FAK inhibitor, our study demonstrates that FAK may be a potential therapeutic target in MDS and AML patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

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

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

Directed therapy for patients with myelodysplastic syndromes (MDS) by suppression of cyclin D1 with ON 01910.Na

BackgroundWe previously demonstrated upregulation of c-myc, survivin, and cyclin D1 in CD34+ bone marrow mononuclear cells (BMMNCs) of patients with trisomy 8 and monosomy 7 myelodysplastic syndromes (MDS). “Knockdown” of cyclin D1 by RNA interference decreased trisomy 8 cell growth, suggesting that this might be a therapeutic target in MDS. Experimental designWe performed preclinical studies using BMMNCs from patients with MDS and AML to examine the effects of the styryl sulfone ON 01910.Na on cyclin D1 accumulation, aneuploidy, and CD34+ blast percentage. We next treated twelve patients with higher risk MDS and two trisomy 8 AML patients with ON 01910.Na on a phase I clinical protocol (NCT00533416). ResultsON 01910.Na inhibited cyclin D1 expression, and was selectively toxic to trisomy 8 cells in vitro. Flow cytometry studies demonstrated increased mature CD15+ myeloid cells and decreased CD34+ blasts. Three patients treated with ON 01910.Na on a clinical had decreased bone marrow blasts by ≥50%, and three patients had hematologic improvements, one of which was sustained for 33 months. Patients with hematologic responses to ON 01910.Na had decreased cyclin D1 expression in their CD34+ cells. ConclusionsThe preclinical results and responses of patients on a clinical trial warrant further investigation of ON 01910.Na as a potential novel targeted therapy for higher risk MDS patients.

Loss of SHIP-1 protein expression in high-risk myelodysplastic syndromes is associated with miR-210 and miR-155.

The myelodysplastic syndromes (MDSs) comprise a group of disorders characterized by multistage progression from cytopenias to acute myeloid leukemia (AML). They display exaggerated apoptosis in early stages, but lose this behavior during evolution to AML. The molecular basis for loss of apoptosis is unknown. To investigate this critical event, we analyzed phosphatidylinositol (PI) 3'kinase signaling, implicated as a critical pathway of cell survival control in epithelial and hematological malignancies. PI 3'kinase activates Akt through its production of 3' phosphoinositides. In turn, the phosphoinositides are dephosphorylated by two lipid phosphatases, PTEN and SHIP-1, in myeloid cells. We studied primary MDS-enriched bone marrow cells and bone marrow sections by western blotting, immunohistochemistry, immunocytochemistry and quantitative PCR for components of the SHIP/PTEN/PI 3'kinase signaling circuit. We reported constitutively activated Akt, variable levels of PTEN and uniformly decreased SHIP-1 expression in MDS progenitor cells. Overexpression of SHIP-1, but not the phosphatase-deficient form, inhibited myeloid leukemic growth. Levels of microRNA (miR)-210 and miR-155 transcripts, which target SHIP-1, were increased in CD34(+) MDS cells compared with their normal counterparts. Direct binding of miR-210 to the 3' untranslated region of SHIP-1 was confirmed by luciferase reporter assay. Transfection of a myeloid cell line with miR-210 resulted in loss of SHIP-1 protein expression. These data suggest that miR-155 and miR-210/SHIP-1/Akt pathways could serve as clinical biomarkers for disease progression, and that miR-155 and miR-210 might serve as novel therapeutic targets in MDS.

PI3K/AKT Pathway as a Potential Therapeutic Target In Myelodysplastic Syndrome

AbstractAbstract 1871 Introduction:PI3K/AKT pathway is involved in cell growth, proliferation and apoptosis. A key downstream effector is the phosphorylated serine-threonine Akt (p-AKT). Constitutive activation of PI3K/AKT has been observed in solid tumours and leukemic cells. Inhibition of PI3K/AKT activity, results in apoptosis in cell lines (CL) after treatment with different compounds, e.g. deguelin, a natural product from the leguminous Mundulea sericea, with antitumour effects. Aims: To evaluate PI3K/AKT activation in MDS patients and its therapeutic potential in MDS. Methods:PI3K/AKT activation was evaluated by flow cytometry (FC) using an alexa-fluor 488-antibody Ser 473 p-AKT (Cell Signalling Technology). A triple immunostaining procedure using CD45-PerCP and CD34-PE was used for p-AKT expression in CD34+ primary samples. The p-AKT activity was determined using Kolmogorov-Smirnov test (D). CD34+ cells from healthy donors and Jurkat cells were used as negative and positive controls respectively. Apoptosis (determined by Annexin V and PI/7AAD) and cell cycle arrest (using RNAse and PI) were determined following treatments with LY294002 (50uM), and deguelin (100-500nM) in P-39 myeloid leukemia cell line, with constitutive PI3K/AKT activation. Apoptosis was determined in bone marrow mononuclear cells and CD34+ cells from MDS patients with the same treatments. To evaluate in vivo activity of deguelin, we used a xenotransplant model. Briefly, NODSCID mice were injected intrafemurally with P-39 CL and 12 days post transplant a three week-course of treatment, every other day, was started (deguelin 4mg/Kg, n=3 vs vehicle, n=3). Results:P-39 CL showed constitutive PI3K/AKT activation with levels significantly higher than in CD34+cells from controls (median±SD= 0.73. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

NF-κB as a potential therapeutic target in myelodysplastic syndromes and acute myeloid leukemia

Importance of the field: The inactive NF-κB–inhibitor of NF-κB (IκB) complex is activated by stimuli including pro-inflammatory cytokines, mitogens, growth factors and stress-inducing agents. The release of NF-κB facilitates its translocation to the nucleus, where it promotes cell survival by initiating transcription of genes encoding stress-response enzymes, cell-adhesion molecules, pro-inflammatory cytokines and anti-apoptotic proteins. NF-κB and associated regulatory factors (IκB kinase subunits and bcl-3) are implicated in hematological and solid tumour malignancies. NF-κB appears to be involved in cell proliferation control, apoptosis control, angiogenesis promotion and possibly regulation of diffusion of metastases. There are several reports that inhibition of NF-κB as a therapeutic target may have a role in tumour cell death or growth inhibition.Area covered in this review: We review data about inhibition of NF-κB in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). We describe the molecular mechanisms underlying NF-κB deregulation in these haematological malignancies.What the reader will gain: Constitutive activation of NF-κB in the nucleus has been reported in some varieties of MDS/AML. The in vitro and in vivo results of NF-κB inhibition in myeloid malignancies are highlighted.Take home message: NF-κB selective inhibitory drugs may be useful, either as single agents or associated with conventional chemotherapy.

Nuclear inositide signaling in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are defined as clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis in one or more of the lineages of the bone marrow. Although distinct morphologic subgroups exist, the natural history of MDS is progression to acute myeloid leukemia (AML). However, the molecular the mechanisms the underlying MDS evolution to AML are not completely understood. Inositides are key cellular second messengers with well-established roles in signal transduction pathways, and nuclear metabolism elicited by phosphoinositide-specific phospholipase C (PI-PLC) beta1 and Akt plays an important role in the control of the balance between cell cycle progression and apoptosis in both normal and pathologic conditions. Recent findings evidenced the role played by nuclear lipid signaling pathways, which could become promising therapeutic targets in MDS. This review will provide a concise and updated revision of the state of art on this topic.

ID4 methylation predicts high risk of leukemic transformation in patients with myelodysplastic syndrome

Epigenetic gene silencing due to promoter methylation is observed in human cancers like acute myeloid leukemia (AML). Little is known about aberrant methylation in myelodysplastic syndrome (MDS), a heterogeneous clonal stem cell disorder with a ∼30% risk of transformation into secondary AML. Recent evidence demonstrated that ID4, a negative regulator of transcription, may act as a tumor-suppressor gene. To clarify the role of ID4 in MDS, we employed methylation-specific PCR (MSP) to examine the methylation status of ID4 in 144 adult de novo MDS patients. We found that ID4 methylation was present in 35.4% ( n = 51) of these MDS patients and methylaiton was correlated significantly with World Health Organization (WHO) subtypes and International Prognostic Scoring System (IPSS) risk groups. Patients with advanced stages of WHO subtypes (45.8% vs. 21.3%, P = 0.002) and higher risk IPSS subgroups (45.7% vs. 26.0%, P = 0.014) exhibited a significantly higher frequency of ID4 methylation. The median survival of patients with ID4 methylation was shorter than patients without ID4 methylation (12.2 months vs. 26.9 months, P = 0.005). Multivariate analysis indicated that ID4 methylation status was the independent factor that impacted leukemia-free survival (LFS). Disease in patients with ID4 methylation progressed more rapidly than those without ID4 methylation ( P = 0.047, HR = 2.11). Our results suggest that ID4 may be a therapeutic target in MDS.