A gene expression profile associated with relapse of cytogenetically normal acute myeloid leukemia is enriched for leukemia stem cell genes

Some 50 – 80% of patients with acute myeloid leukemia (AML) achieve a complete remission with contemporary chemotherapy protocols, yet the majority of them eventually relapse with resistant disease: some patients no longer respond to chemotherapy at disease recurrence; others accomplish second and even third remissions whose decreasing duration nevertheless indicates that the pool of residual leukemic cells, i.e. of cells that persisted during treatment with cytotoxic drugs, increases with every round of therapy [1]. Either of these clinical courses therefore refl ects an enhanced chemotherapy resistance of leukemic cells at relapse as compared to the cell population at diagnosis. Molecular changes enabling malignant cells to survive exposure to cytotoxic drugs may already have been present in a subset of the leukemic cell population at presentation, or may emerge during treatment [2,3], but in any case are thought to be selected as a consequence of drug therapy, and to play a major role in therapy resistance at relapse. Remarkably, however, even though various types of molecular alterations may be acquired at relapse, neither specifi c cytogenetic alterations nor functionally relevant point mutations as identifi ed by whole genome sequencing were associated with relapse in a recurrent manner [2,3]. Certain copy number variations and known AML associated point mutations were newly present at relapse in small proportions of patients (usually 10%), but the latter were lost in other patients, indicating that they are unlikely to represent drivers of therapy resistance at disease recurrence [4]. Th ese fi ndings could either indicate that chemotherapy resistance at relapse is acquired through a large variety of different mechanisms, or that molecular changes of other types than those mentioned above are of more general relevance in this context. Indeed, an earlier study has suggested that the expression of specifi c genes may change in a consistent manner between diagnosis and relapse of AML [5]. However, only a limited number of genes and mostly unpaired samples were probed in this investigation. Th erefore, in the present study, genes whose expression changed in a relapse-specifi c manner were sought in a set of paired AML samples and on a genome-wide scale. To limit the genetic heterogeneity of the study population, only samples from patients with cytogenetically normal (CN) AML were used.Letter to the Edito

Overexpression of primary microRNA 221/222 in acute myeloid leukemia

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations. METHODS: The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures. RESULTS: 64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease. CONCLUSIONS: Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease

Mg(2+) Deprivation Elicits Rapid Ca(2+) Uptake and Activates Ca(2+)/Calcineurin Signaling in Saccharomyces cerevisiae

To learn about the cellular processes involved in Mg(2+) homeostasis and the mechanisms allowing cells to cope with low Mg(2+) availability, we performed RNA expression-profiling experiments and followed changes in gene activity upon Mg(2+) depletion on a genome-wide scale. A striking portion of genes up-regulated under Mg(2+) depletion are also induced by high Ca(2+) and/or alkalinization. Among the genes significantly up-regulated by Mg(2+) starvation, Ca(2+) stress, and alkalinization are ENA1 (encoding a P-type ATPase sodium pump) and PHO89 (encoding a sodium/phosphate cotransporter). We show that up-regulation of these genes is dependent on the calcineurin/Crz1p (calcineurin-responsive zinc finger protein) signaling pathway. Similarly to Ca(2+) stress, Mg(2+) starvation induces translocation of the transcription factor Crz1p from the cytoplasm into the nucleus. The up-regulation of ENA1 and PHO89 upon Mg(2+) starvation depends on extracellular Ca(2+). Using fluorescence resonance energy transfer microscopy, we demonstrate that removal of Mg(2+) results in an immediate increase in free cytoplasmic Ca(2+). This effect is dependent on external Ca(2+). The results presented indicate that Mg(2+) depletion in yeast cells leads to enhanced cellular Ca(2+) concentrations, which activate the Crz1p/calcineurin pathway. We provide evidence that calcineurin/Crz1p signaling is crucial for yeast cells to cope with Mg(2+) depletion stress

A gene expression profile associated with relapse of cytogenetically normal acute myeloid leukemia is enriched for leukemia stem cell genes

Some 50 – 80% of patients with acute myeloid leukemia (AML) achieve a complete remission with contemporary chemotherapy protocols, yet the majority of them eventually relapse with resistant disease: some patients no longer respond to chemotherapy at disease recurrence; others accomplish second and even third remissions whose decreasing duration nevertheless indicates that the pool of residual leukemic cells, i.e. of cells that persisted during treatment with cytotoxic drugs, increases with every round of therapy [1]. Either of these clinical courses therefore refl ects an enhanced chemotherapy resistance of leukemic cells at relapse as compared to the cell population at diagnosis. Molecular changes enabling malignant cells to survive exposure to cytotoxic drugs may already have been present in a subset of the leukemic cell population at presentation, or may emerge during treatment [2,3], but in any case are thought to be selected as a consequence of drug therapy, and to play a major role in therapy resistance at relapse. Remarkably, however, even though various types of molecular alterations may be acquired at relapse, neither specifi c cytogenetic alterations nor functionally relevant point mutations as identifi ed by whole genome sequencing were associated with relapse in a recurrent manner [2,3]. Certain copy number variations and known AML associated point mutations were newly present at relapse in small proportions of patients (usually 10%), but the latter were lost in other patients, indicating that they are unlikely to represent drivers of therapy resistance at disease recurrence [4]. Th ese fi ndings could either indicate that chemotherapy resistance at relapse is acquired through a large variety of different mechanisms, or that molecular changes of other types than those mentioned above are of more general relevance in this context. Indeed, an earlier study has suggested that the expression of specifi c genes may change in a consistent manner between diagnosis and relapse of AML [5]. However, only a limited number of genes and mostly unpaired samples were probed in this investigation. Th erefore, in the present study, genes whose expression changed in a relapse-specifi c manner were sought in a set of paired AML samples and on a genome-wide scale. To limit the genetic heterogeneity of the study population, only samples from patients with cytogenetically normal (CN) AML were used.Letter to the Edito

EVI1 inhibits apoptosis induced by antileukemic drugs via upregulation of CDKN1A/p21/WAF in human myeloid cells.

Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec). Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs

EVI1 promotes tumor growth via transcriptional repression of MS4A3

Background The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells. Methods U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice. Results Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis. Conclusions Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.(VLID)486732

Effects of <i>EVI1</i> on growth dynamics <i>in vitro</i> and <i>in vivo</i>.

<p>A) Examples of cell cycle profiles of U937_vec and U937_EVI1 cells. B) Cell cycle distribution of U937_vec and U937_EVI1 cells. Shown are the means+standard errors of the mean (SEM) from 3 independent experiments. None of the differences between the two cell lines are statistically significant (Student’s t-test). C) Tumor growth after subcutaneous injection of U937_vec and U937_EVI1 cells into the flanks of CB-17 scid/scid mice. The adjusted area under the curve (aAUC) was calculated for each tumor, and the two groups of tumors were compared by nonparametric bootstrap inference. *, p<0.05. D) Immunohistochemical (IHC) staining showing persistent expression of EVI1 in U937_EVI1 derived tumor xenografts. E) IHC revealing the presence of similar proportions of CD11b positive cells in U937_EVI1 and U937_vec derived tumors.</p

<i>EVI1</i> and etoposide regulate overlapping sets of genes.

<p>cRNAs from two independent cultures of U937_vec and U937_EVI1 cells treated or not treated with 400 nM etoposide for 48 h were hybridized onto Affymetrix ST1.1 arrays. Only genes deregulated at least 2-fold in both experiments were considered as differentially expressed. Gene set enrichment analysis (GSEA) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056308#pone.0056308-Subramanian1" target="_blank">[68]</a> was performed to evaluate concordant differences of A) etoposide regulated genes within the gene expression list ranked by average log2-fold changes between U937_EVI1 and U937_vec cells, and B) <i>EVI1</i> regulated genes within the gene expression list ranked by average log2-fold changes between etoposide treated and untreated U937_vec cells. The normalized enrichment scores were 1.9 in A) and 2.2 in B); the p-values and the q-values of the false discovery rate were 0.0 in both analyses.</p

Primers used for amplification and sequence analysis of the integrated, vector borne <i>EVI1</i> gene from U937_EVI1 cells.

*<p>Primers F1-F and F5-R match the vector, rather than the <i>EVI1</i>, sequence.</p