[Show abstract][Hide abstract] ABSTRACT: Artemisinins, endoperoxide-containing molecules, best known as antimalarials, have potent antineoplastic activity. The established antimalarial, artesunate (AS), and the novel artemisinin-derived trioxane diphenylphosphate dimer 838 (ART-838) inhibited growth of all 23 tested acute leukemia cell lines, reduced cell proliferation and clonogenicity, induced apoptosis, and increased intracellular levels of reactive oxygen species (ROS). ART-838 was 88-fold more potent that AS in vitro, inhibiting all leukemia cell lines at submicromolar concentrations. Both ART-838 and AS cooperated with several established antileukemic drugs and newer kinase inhibitors to inhibit leukemia cell growth. ART-838 had a longer plasma half-life than AS in immunodeficient NOD-SCID-IL2Rgnull (NSG) mice, remaining at effective antileukemic concentrations for >8h. Intermittent cycles of ART-838 inhibited growth of acute leukemia xenografts and primagrafts in NSG mice, at higher potency than AS. Based on these preclinical data, we propose that AS, with its established low toxicity and low cost, and ART-838, with its higher potency and longer persistence in vivo, should be further developed toward integration into antileukemic regimens.
[Show abstract][Hide abstract] ABSTRACT: Complex phenotypic differences among different acute leukemias cannot be
fully captured by analyzing the expression levels of one single molecule, such
as a miR, at a time, but requires systematic analysis of large sets of miRs.
While a popular approach for analysis of such datasets is principal component
analysis (PCA), this method is not designed to optimally discriminate different
phenotypes. Moreover, PCA and other low-dimensional representation methods
yield linear or non-linear combinations of all measured miRs. Global human miR
expression was measured in AML, B-ALL, and T-ALL cell lines and patient RNA
samples. By systematically applying support vector machines to all measured
miRs taken in dyad and triad groups, we built miR networks using cell line data
and validated our findings with primary patient samples. All the coordinately
transcribed members of the miR-23a cluster (which includes also miR-24 and
miR-27a), known to function as tumor suppressors of acute leukemias, appeared
in the AML, B-ALL and T-ALL centric networks. Subsequent qRT-PCR analysis
showed that the most connected miR in the B-ALL-centric network, miR-708, is
highly and specifically expressed in B-ALLs, suggesting that miR-708 might
serve as a biomarker for B-ALL. This approach is systematic, quantitative,
scalable, and unbiased. Rather than a single signature, our approach yields a
network of signatures reflecting the redundant nature of biological signaling
pathways. The network representation allows for visual analysis of all
signatures by an expert and for future integration of additional information.
Furthermore, each signature involves only small sets of miRs, such as dyads and
triads, which are well suited for in depth validation through laboratory
experiments such as loss- and gain-of-function assays designed to drive changes
in leukemia cell survival, proliferation and differentiation.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRs) regulate essentially all cellular processes, but few miRs are known to inhibit growth of precursor-B acute lymphoblastic leukemias (B-ALLs). We identified miR-509 via a human genome-wide gain-of-function screen for miRs that inhibit growth of the NALM6 human B-ALL cell line. MiR-509-mediated inhibition of NALM6 growth was confirmed by 3 independent assays. Enforced miR-509 expression inhibited 2 of 2 additional B-ALL cell lines tested, but not 3 non-B-ALL leukemia cell lines. MiR-509-transduced NALM6 cells had reduced numbers of actively proliferating cells and increased numbers of cells undergoing apoptosis. Using miR target prediction algorithms and a filtering strategy, RAB5C was predicted as a potentially relevant target of miR-509. Enforced miR-509 expression in NALM6 cells reduced RAB5C mRNA and protein levels, and RAB5C was demonstrated to be a direct target of miR-509. Knockdown of RAB5C in NALM6 cells recapitulated the growth inhibitory effects of miR-509. Co-expression of the RAB5C open reading frame without its 3' untranslated region (3'UTR) blocked the growth-inhibitory effect mediated by miR-509. These findings establish RAB5C as a target of miR-509 and an important regulator of B-ALL cell growth with potential as a therapeutic target.
[Show abstract][Hide abstract] ABSTRACT: Expression levels of MIR144 and MIR451 increase during erythropoiesis, a pattern that is conserved from zebrafish to humans. As these two miRs are expressed from the same polycistronic transcript, we manipulated MIR144 and MIR451 in human erythroid cells individually and together to investigate their effects on human erythropoiesis. Inhibition of endogenous human MIR451 resulted in decreased numbers of erythroid (CD71hiCD235ahiCD34−) cells, consistent with prior studies in zebrafish and mice. In addition, inhibition of MIR144 impaired human erythroid differentiation, unlike in zebrafish and mouse studies where the functional effect of MIR144 on erythropoiesis was minimal. In this study, we found RAB14 is a direct target of both MIR144 and MIR451. As MIR144 and MIR451 expression increased during human erythropoiesis, RAB14 protein expression decreased. Enforced RAB14 expression phenocopied the effect of MIR144 and/or MIR451 depletion, whereas shRNA-mediated RAB14 knockdown protected cells from MIR144 and/or MIR451 depletion-mediated erythropoietic inhibition. RAB14 knockdown increased the frequency and number of erythroid cells, increased β-haemoglobin expression, and decreased CBFA2T3 expression during human erythropoiesis. In summary, we utilized MIR144 and MIR451 to identify RAB14 as a novel physiological inhibitor of human erythropoiesis.
No preview · Article · Oct 2014 · British Journal of Haematology
[Show abstract][Hide abstract] ABSTRACT: Increased hepcidin antimicrobial peptide correlates with hypoferremia and anemia in various disease states, but its requirement for anemia of inflammation has not been adequately demonstrated. Anemia of inflammation is usually described as normocytic and normochromic, while diseases associated with over expression of hepcidin, alone, are often microcytic and hypochromic. These differences in erythrocyte parameters suggest anemia in many inflammatory states may not be fully explained by hepcidin-mediated iron sequestration. We used turpentine-induced sterile abscesses to model chronic inflammation in mice with targeted disruption of Hepcidin 1 [Hepc1 (-/-)] or its positive regulator, Interleukin-6 [IL-6 (-/-)], to determine whether these genes are required for features characteristic of anemia of inflammation. Although hemoglobin levels did not decline in Hepc1 (-/-) mice with sterile abscesses, erythrocyte numbers were significantly reduced compared to untreated Hepc1 (-/-) mice. In contrast, both hemoglobin concentration and erythrocyte number declined significantly in wild type and IL-6 (-/-) mice with sterile abscesses. Both Hepc1 (-/-) and IL-6 (-/-) mice had increased erythrocyte mean cell volume and mean cell hemoglobin following sterile abscesses, while wild types had no change. Thus, IL-6 (-/-) mice with sterile abscesses exhibit an intermediate phenotype between wild type and Hepc1 (-/-). Our results demonstrate the requirement of Hepc1 for the development of anemia in this rodent model. Simultaneously, our results demonstrate hepcidin-independent effects of inflammation on the suppression of erythropoiesis. Our results suggest chronic anemia associated with inflammation may benefit from interventions protecting erythrocyte number in addition to anti-hepcidin interventions aimed at enhancing iron availability.
No preview · Article · May 2014 · American Journal of Hematology
[Show abstract][Hide abstract] ABSTRACT: Several individual miRNAs (miRs) have been implicated as potent regulators of important processes during normal and malignant hematopoiesis. In addition, many miRs have been shown to fine-tune intricate molecular networks, in concert with other regulatory elements. In order to study hematopoietic networks as a whole, we first created a map of global miR expression during early murine hematopoiesis. Next, we determined the copy number per cell for each miR in each of the examined stem and progenitor cell types. As data is emerging indicating that miRs function robustly mainly when they are expressed above a certain threshold (∼100 copies per cell), our database provides a resource for determining which miRs are expressed at a potentially functional level in each cell type. Finally, we combine our miR expression map with matched mRNA expression data and external prediction algorithms, using a Bayesian modeling approach to create a global landscape of predicted miR-mRNA interactions within each of these hematopoietic stem and progenitor cell subsets. This approach implicates several interaction networks comprising a "stemness" signature in the most primitive hematopoietic stem cell (HSC) populations, as well as "myeloid" patterns associated with two branches of myeloid development.
[Show abstract][Hide abstract] ABSTRACT: Complex phenotypic differences such as those among different acute leukemias
cannot be fully captured by analyzing the expression levels of one single
microRNA at a time. We introduce a data-driven approach that identifies which
and how many microRNAs are needed to differentially characterize acute myeloid
(AML) and lymphoblastic (B-ALL and T-ALL) leukemias. First, global human
microRNA expression was measured on AML, B-ALL, and T-ALL cell lines and
patient samples. Then, by systematically applying support vector machines to
all microRNAs taken pairwise, we built an AML-centric microRNA-dyad network
based only on the cell line data. These dyads were able to classify patient
samples very reliably (accuracy >94%). In order to differentially characterize
B-ALL and T-ALL, however, at least 3 microRNAs were needed. We thus built
microRNA-triad network ensembles using a scalable framework that preselects
top-scoring microRNAs using a GenePattern class prediction method. We validated
our findings by analyzing some well-characterized microRNAs. For instance, all
the members of the miR-23a cluster (which includes also miR-24 and miR-27a),
known to function as tumor suppressors of acute leukemias, appeared in the AML,
B-ALL and T-ALL centric networks playing similar roles from a
network-topological perspective. The most connected microRNA in the
B-ALL-centric network was miR-708 (70% of all triads). Microarray and qRT-PCR
analyses across cell lines and patient samples showed that miR-708 is highly
and specifically expressed in B-ALL, suggesting that miR-708 might serve as a
biomarker for B-ALL. However, by experimentally manipulating the expression
levels of miR-708 in B-ALL cell lines, no functional effect was revealed on
cell survival or proliferation, which underscores the cooperative nature of
cellular function involving multiple microRNAs.
[Show abstract][Hide abstract] ABSTRACT: Anemia of inflammation or chronic disease is a highly prevalent form of anemia. The inflammatory cytokine interleukin-6 (IL-6) negatively correlates with hemoglobin concentration in many disease states. The IL-6-hepcidin antimicrobial peptide axis promotes iron-restricted anemia; however the full role of IL-6 in anemia of inflammation is not well-defined. We previously reported that chronic inflammation had a negative impact on maturation of erythroid progenitors in a mouse model. We hypothesized that IL-6 may be responsible for impaired erythropoiesis, independent of iron restriction. To test the hypothesis we utilized the human erythroleukemia TF-1 cell line to model erythroid maturation and exposed them to varying doses of IL-6 over six days. At 10ng/ml, IL-6 significantly repressed erythropoietin-dependent TF-1 erythroid maturation. While IL-6 did not decrease the expression of genes associated with hemoglobin synthesis, we observed impaired hemoglobin synthesis as demonstrated by decreased benzidine staining. We also observed that IL-6 down regulated expression of the gene SLC4a1 which is expressed late in erythropoiesis. Those findings suggested that IL-6-dependent inhibition of hemoglobin synthesis might occur. We investigated the impact of IL-6 on mitochondria. IL-6 decreased the mitochondrial membrane potential at all treatment doses, and significantly decreased mitochondrial mass at the highest dose. Our studies indicate that IL-6 may impair mitochondrial function in maturing erythroid cells resulting in impaired hemoglobin production and erythroid maturation. Our findings may indicate a novel pathway of action for IL-6 in the anemia of inflammation, and draw attention to the potential for new therapeutic targets that affect late erythroid development.
Full-text · Article · Oct 2013 · Blood Cells Molecules and Diseases
[Show abstract][Hide abstract] ABSTRACT: Anemia is common in older adults and associated with adverse health outcomes in epidemiologic studies. A thorough understanding of the complex pathophysiological mechanisms driving anemia in the elderly is lacking; but inflammation, iron restriction, and impaired erythroid maturation are thought to influence the phenotype. We hypothesized that interleukin-6 contributes to this anemia, given its pro-inflammatory activities, its ability to induce hepcidin antimicrobial peptide, and its negative impact on several tissues in older adults. We tested this hypothesis by comparing changes in indices of inflammation, iron metabolism and erythropoiesis in aged C57BL/6 mice to aged mice with targeted deletions of interleukin-6 or hepcidin antimicrobial peptide. Circulating neutrophil and monocyte numbers and inflammatory cytokines increased with age. Declines in hemoglobin concentration and red blood cell number indicated that C57BL/6, interleukin-6 knockout mice, and hepcidin antimicrobial peptide knockout mice all demonstrated impaired erythropoiesis by 24 months. However, the interleukin-6 knock out genotype and the hepcidin antimicrobial peptide knock out genotype resulted in improved erythropoiesis in aged mice. Increased erythropoietic activity in the spleen suggested that the erythroid compartment was stressed in aged C57BL/6 mice compared to aged interleukin-6 knockout mice. Our data suggest C57BL/6 mice are an appropriate mammalian model for the study of anemia with age. Furthermore, although interleukin-6 and hepcidin antimicrobial peptide are not required, they can participate in the development of anemia in aging mice, and could be targeted, pre-clinically, with existing interventions to determine the feasibility of such agents for the treatment of anemia in older adults.
[Show abstract][Hide abstract] ABSTRACT: Homologous recombination repair (HRR) protects cells from lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51 and RAD52-RAD51 serves as back-up. To target HRR in tumor cells a phenomenon called "synthetic lethality" was applied, which relies on addiction of cancer cells to one DNA repair pathway whereas normal cells operate two or more mechanisms. Using mutagenesis and peptide aptamer approach we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-F79) as valid target to induce synthetic lethality in BRCA1 and/or BRCA2 -deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts RAD52 - DNA interaction resulting in accumulation of toxic DNA double stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52 - DNA interaction enhanced anti-leukemia effect of the already approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, and mutations in BRCA1 and/or BRCA2 genes, and also by gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe that this work may initiate personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA-deficiency.
[Show abstract][Hide abstract] ABSTRACT: We recently reported the anti-cancer and anti-cytomegalovirus (CMV) activity of artemisinin-derived trioxane diphenylphosphate dimer 838. To probe the relationship between chemical structure and anti-CMV and anti-cancer activities, we now report synthesis and evaluation of a series of eight new dimer phosphate ester analogs of 838. This series of novel molecules was screened against human foreskin fibroblasts (HFFs) infected with CMV and against the human Jurkat T cell acute lymphoblastic leukemia cell line. This SAR study confirms the very high anti-CMV and anti-cancer potencies of dimer diphenyl phosphate ester 838 without its being toxic to normal cells.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and protein synthesis. To characterize functions of miRNAs and to assess their potential applications, we carried out an integrated multi-omics analysis to study miR-145, a miRNA that has been shown to suppress tumor growth. We employed gene expression profiling, miRNA profiling and quantitative proteomic analysis of a pancreatic cancer cell line. In our transcriptomic analysis, overexpression of miR-145 was found to suppress the expression of genes that are implicated in development of cancer such as ITGA11 and MAGEA4 in addition to previously described targets such as FSCN1, YES1 and PODXL. Based on miRNA profiling, overexpression of miR-145 also upregulated other miRNAs including miR-124, miR-133b and miR-125a-3p, all of which are implicated in suppression of tumors and are generally co-regulated with miR-145 in other cancers. Using the SILAC system, we identified miR-145-induced downregulation of several oncoproteins/cancer biomarkers including SET, RPA1, MCM2, ABCC1, SPTBN1 and SPTLC1. Luciferase assay validation carried out on a subset of downregulated candidate targets confirmed them to be novel direct targets of miR-145. Overall, this multi-omics approach provided insights into miR-145-mediated tumor suppression and could be used as a general strategy to study the targets of individual miRNAs.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRs) regulate cellular processes by modulating gene expression. Although transcriptomic studies have identified numerous miRs differentially expressed in diseased versus normal cells, expression analysis alone cannot distinguish miRs driving a disease phenotype from those merely associated with the disease. To address this limitation, we developed miR-HTS, a method for unbiased high-throughput screening of the miRNome to identify functionally relevant miRs. Herein, we applied miR-HTS to simultaneously analyze the effects of 578 lentivirally transduced human miRs or miR clusters on growth of the IMR90 human lung fibroblast cell line. Growth-regulatory miRs were identified by quantitating the representation (i.e., relative abundance) of cells overexpressing each miR over a one-month culture of IMR90, using a panel of custom-designed quantitative real-time PCR (qPCR) assays specific for each transduced miR expression cassette. The miR-HTS identified 4 miRs previously reported to inhibit the growth of human lung-derived cell lines and 55 novel growth-inhibitory miR candidates. Nine of 12 (75%) selected candidate miRs were validated and shown to inhibit IMR90 cell growth. Thus, this novel lentiviral library- and qPCR-based miR-HTS technology provides a sensitive platform for functional screening that is straightforward and relatively inexpensive.