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  • 1
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    Circular RNAs of the Nucleophosmin 1 (NPM1) Gene in Acute Myeloid Leukemia Patients
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 2695-2695
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 2695-2695
    Abstract: Background: The nucleophosmin 1 (NPM1) gene is not only commonly mutated in acute myeloid leukemia (AML), but also encodes several linear splice isoforms, one of which was recently shown to be of prognostic importance. Furthermore, circular RNAs (circRNAs) are transcribed from the NPM1 gene which demands further investigation with regard to function in normal hematopoiesis and impact on leukemogenesis. Aims: We aimed to investigate circRNAs derived from NPM1 and gain insights into their regulation and function. Additionally, we wanted to determine changes in the circular RNAome in the course of hematopoietic differentiation and leukemic transformation. Methods: Circular NPM1 transcripts were detected by PCR and sequenced in leukemic cell lines (n=7) and healthy control samples (n=3, peripheral blood-derived mononuclear cells). Expression of hsa_circ_0075001 and total NPM1 was measured in a cohort of 23 NPM1 wildtype (NPM1wt) and 23 NPM1 mutated (NPM1mut) AML patients via quantitative real-time PCR (qPCR), and Affymetrix U133plus2 microarray data was set in relation to the expression levels. Principal component analysis (PCA) was conducted to identify groups with similarities in gene expression patterns and differentially expressed genes were subjected to pathway analysis. Next, ribosomal RNA-depleted RNA-seq was performed for 5 NPM1mut and 5 NPM1wt AML cases, as well as 10 healthy control samples derived from 4 FACS-sorted myeloid differentiation stages (myeloblasts, promyelocytes, metamyelocytes and neutrophils). PCA and unsupervised hierarchical clustering were performed based on circRNA expression. Results: We detected and sequenced multiple circular NPM1 transcripts (n=23) in leukemic as well as in healthy control cells. As hsa_circ_0075001 showed differential expression between different AML cell lines in a semi-quantitative PCR analysis, quantification in 46 AML patients via qPCR was performed. This analysis revealed that total NPM1 and hsa_circ_0075001 expression were independent of the NPM1 mutational status. Furthermore, the hsa_circ_0075001 expression status defined distinct leukemia subgroups characterized by similarities in gene expression as determined by PCA. For example, differentially expressed genes between high versus low hsa_circ_0075001 expression groups (dichotomized at the median) were significantly enriched in components of the Toll-like receptor (TLR) signaling pathway, which was downregulated in patients with high hsa_circ_0075001 expression. Expression of hsa_circ_0075001 correlated positively with total NPM1 expression, and RNA-seq analysis further revealed a global correlation of circRNA and parental gene expression. In total, in our cohort circRNAs were found for 19 % of all expressed genes. PCA based on circRNA expression illustrated that immature and mature hematopoietic cells, as well as NPM1wt and NPM1mut AML samples, exhibit distinct circRNA signatures (Figure 1). Thus, circRNA expression seems to play a role during differentiation of normal hematopoietic cells, but also seems to be severely deregulated in AML. Figure 1: Altered circular RNA expression in AML patients compared to healthy control samples. Principal component analysis (PCA) of circRNA expression data of 5 NPM1mut patients (red), 5 NPM1wt patients (green), and 10 healthy control samples, of which 4 were derived from immature (blue) and 6 from more mature myeloid differentiation stages (purple). Data was generated via RNA-Seq and reads derived from circRNAs were aligned and quantified using STAR, and normalized and transformed using DESeq2. PCA was performed based on 500 genes with the highest variance of circRNA expression across all samples. Conclusions: circRNAs transcribed from the NPM1 gene showed differential expression in AML cell lines and healthy cells, and higher hsa_circ_0075001 expression defined an AML subgroup characterized by downregulation of the TLR signaling pathway. These findings provide evidence for the relevance of circular NPM1 transcripts and add another level of complexity to the multifaceted gene NPM1. In general, circRNA expression seems to be involved in the regulation of hematopoietic differentiation, which is in line with previous observations, but, based on distinct circRNA expression profiles in AML, they might also play a significant pathogenic role in leukemic transformation. Figure 1 Figure 1. Disclosures Paschka: Celgene: Honoraria; Pfizer Pharma GmbH: Honoraria; Bristol-Myers Squibb: Honoraria; Medupdate GmbH: Honoraria; Novartis: Consultancy; ASTEX Pharmaceuticals: Consultancy.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.2695.2695
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
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  • 2
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    Circular RNAs of the nucleophosmin (NPM1) gene in acute myeloid leukemia
    Ferrata Storti Foundation (Haematologica) ; 2017
    In:  Haematologica Vol. 102, No. 12 ( 2017-12), p. 2039-2047
    Details
    In: Haematologica, Ferrata Storti Foundation (Haematologica), Vol. 102, No. 12 ( 2017-12), p. 2039-2047
    Type of Medium: Online Resource
    ISSN: 0390-6078 , 1592-8721
    URL: Article
    DOI: 10.3324/haematol.2017.172866
    Language: English
    Publisher: Ferrata Storti Foundation (Haematologica)
    Publication Date: 2017
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    detail.hit.zdb_id: 2030158-3
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  • 3
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    Comprehensive micro RNA expression profiling in cerebrospinal fluid distinguishes between neurological disease classes
    Wiley ; 2019
    In:  Neuropathology and Applied Neurobiology Vol. 45, No. 3 ( 2019-04), p. 318-323
    Details
    In: Neuropathology and Applied Neurobiology, Wiley, Vol. 45, No. 3 ( 2019-04), p. 318-323
    Type of Medium: Online Resource
    ISSN: 0305-1846 , 1365-2990
    URL: Issue
    URL: Article
    DOI: 10.1111/nan.2019.45.issue-3
    DOI: 10.1111/nan.12491
    Language: English
    Publisher: Wiley
    Publication Date: 2019
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  • 4
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    Members of the microRNA-106a-363 Cluster Associate with Unfavorable Outcome in Adult Acute Myeloid Leukemia Patients and Promote Leukemogenesis invivo through Increased Metabolic Activity
    American Society of Hematology ; 2018
    In:  Blood Vol. 132, No. Supplement 1 ( 2018-11-29), p. 3924-3924
    Details
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 3924-3924
    Abstract: The miR-106a-363 cluster, encoding six miRNAs (miR-106a, miR-18b, miR-20b, miR-19b, miR-92a and miR-363), is a paralogue of the oncogenic miR-17-92a polycistron and its role in leukemia is at present largely unknown. We aimed to investigate the putative oncogenic role of the miR-106a-363 cluster in adult acute myeloid leukemia (AML) and to dissect the contributions of its individual members to disease formation and progression. First, we analyzed the expression of each miRNA in AML patient samples as well as their clinical relevance. To determine the association of the miR-106a-363 cluster in AML with active disease, we quantified all six miRNAs individually in AML patient samples at initial diagnosis (n=33) and in AML patients in complete remission after chemotherapy (n=6). Hereof, miR-106a-5p, miR-19b-3p and miR-92a-3p levels were significantly lower in remission samples (p=0.0015, p=0.0013 and p=0.0004, respectively), confirming that these miRNAs are upregulated in AML. Stratifying AML patients within the LAML miRNA-Seq dataset of The Cancer Genome Atlas (TCGA) Research Network (n=187) (Ley et al., NEJM, 2013) according to their cytogenetic risk group demonstrated that all members of the cluster, except for miR-18b-5p, significantly associated with adverse cytogenetics. In addition, with the exception of miR-18b-5p, all members associated with an inferior overall survival (OS) in AML patients within the TCGA-LAML dataset, further supporting a pro-leukemogenic role for the cluster. Of note, miR-106a-5p was the most abundantly expressed unique miRNA of the polycistron, both in the TCGA patient cohort and in 11 myeloid leukemia cell lines quantified by quantitative real-time PCR (qRT-PCR). Since the miR-106a-363 cluster is associated with high risk AML, we hypothesized that increased levels of the entire cluster as well as individual members would significantly shorten the survival time in a murine transplantation model mimicking aggressive AML. Therefore, we engineered transplantable, primary murine AML cell lines based on retroviral overexpression of Hoxa9 and Meis1 exhibiting a median disease latency of 39 days (n=14) after syngeneic transplantation in mice. Enforced lentiviral expression of miR-106a-363 (n=13, p 〈 0.0001), miR-106a (n=15, p=0.0003), miR-18b (n=8, p 〈 0.0001), miR-20b (n=13, p 〈 0.0001) and miR-363 (n=13, p 〈 0.0001) in Hoxa9/Meis1 cells significantly accelerated leukemogenesis compared to the control arm. The most pronounced anemia (p=0.03) and the most immature phenotype, based on a significantly higher proportion of c-kit+ (p=0.0147) and a concurrent lower percentage of Mac-1+ and Gr-1+ (p=0.0051) cells, were observed in mice transplanted with Hoxa9/Meis1/miR-106a cells. Based on these results, we focused on the mechanism by which miR-106a contributed to the pathogenesis of AML and performed a proteomics screen comparing Hoxa9/Meis1/miR-106a and Hoxa9/Meis1/control cells. In particular, mitochondrial respiration processes, such as oxidative phosphorylation and electron transport chain components were induced by miR-106a as shown by Gene Set Enrichment Analysis. Preliminary results using high-resolution respirometry further indicated an increased number of mitochondria in Hoxa9/Meis1/miR-106a cells, supporting these findings. In conclusion, we highlight the previously unrecognized oncogenic contribution of the miR-106a-363 polycistron in adult AML. Functional dissection of this cluster, in particular miR-106a, revealed a new therapeutic angle for high risk AML. Disclosures Döhner: Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Pfizer: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; AROG Pharmaceuticals: Research Funding; Agios: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2018-99-116035
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
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  • 5
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    Towards Stratification of Lymphomas with Involvement of the Central Nervous System Using miRNA-Seq from Small Volumes of Cerebrospinal Fluid
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 1751-1751
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 1751-1751
    Abstract: Early diagnosis and therapy monitoring have improved overall survival of lymphoma patients. Biomarkers from body fluids are an active field of research for diagnosing and monitoring malignant diseases. MicroRNAs (miRNAs) are present in all body fluids and have already been described as biomarkers in lymphoma patients. However, only few studies addressed the impact of miRNAs as diagnostic tools for central nervous system (CNS) involvement of systemic lymphomas (CNS positive lymphomas). In addition, due to technical limitations such as low cerebrospinal fluid (CSF) starting amounts, translation into clinical application has been very limited. In order to facilitate development of an applicable diagnostic assay of circulating miRNAs from low CSF starting volumes (400ml), we compared quantitative realtime PCR (qRT-PCR) arrays with next generation sequencing (miRNA-Seq) and established a protocol to robustly quantify a miRNA signature for CNS positive lymphomas from diagnostic volumes of CSF. First, we could show that circulating miRNAs are highly stable in CSF and that miRNA levels can be assessed robustly with qRT-PCR and miRNA-Seq. For this purpose, miRNAs were extracted from 400µL of CSF using an optimized miRNA extraction protocol. MiRNAs were quantified by qRT-PCR with the Exiqon Human miRNome Panel v2 which assays 752 miRNAs or by miRNA-Seq of libraries prepared with the Seqmatic TailorMix v2 miRNA Sample Preparation Kit using the Illumina HiSeq. In total, eight patients with diffuse large B-cell lymphoma (DLBCL) that showed involvement of the central nervous system (CNS) were analyzed. Of each patient, two paired CSF samples were available both from initial diagnosis and complete remission after treatment. The presence of CNS manifestation was established via magnetic resonance imaging (MRI) and flow cytometry at the Erasmus MC Daniel den Hoed Cancer Center in Rotterdam. CSF from ten patients with cephalalgia was used as a control. After inter-plate calibration and normalization, 260 miRNAs were detected on average with the Exiqon Human miRNome Panel v2. In order to assess the probability of deriving false positive signals from e.g. primer dimers, two miRNome Panels (four 384-well plates) were processed without template. Surprisingly, after 40 cycles of qRT-PCR, 10% of assays showed amplicons in duplicates. Furthermore, false positive amplicons were not reproducible using this method, underlining that qRT-PCR had a low overall specificity that could not be adjusted by exclusion of erroneous amplicons. In contrast, miRNA seq produced a median of ~40,000 mappable reads per sample (range 2x104 - 1x106) with a Phred score of 〉 20. Prediction analysis using all detectable miRNAs segregated CSF samples from CSF positive lymphoma patients before treatment and after treatment based on a 14-miRNA signature with a misclassification error of 〈 0.1. Unsupervised clustering of the 14-miRNA signature correctly stratified 7/8 samples after treatment i.e. CSF negative lymphomas, while 4/5 samples from patients with CSF positive lymphomas clustered separately. Thus, using the expression levels of only 14 miRNAs determined with miRNA-Seq, the two disease stages could be differentiated with each group containing one misclassification. The robust quantification of miRNAs from small volumes of CSF with next generation sequencing has potential to monitor minimal residual disease diagnosis or even to stratify other pathologic entities such as dementias or inflammatory diseases where only unreliable markers or no biomarkers at all are currently available. Disclosures Mulaw: NuGEN: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.1751.1751
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
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  • 6
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    The non-coding RNA landscape of human hematopoiesis and leukemia
    Springer Science and Business Media LLC ; 2017
    In:  Nature Communications Vol. 8, No. 1 ( 2017-08-09)
    Details
    In: Nature Communications, Springer Science and Business Media LLC, Vol. 8, No. 1 ( 2017-08-09)
    Abstract: Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs—such as LINC00173 in granulocytes — and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy.
    Type of Medium: Online Resource
    ISSN: 2041-1723
    URL: Article
    DOI: 10.1038/s41467-017-00212-4
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2017
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  • 7
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    Identification of Novel Lncrnas That Predict Survival in AML Patients and Modulate Leukemic Cells
    American Society of Hematology ; 2018
    In:  Blood Vol. 132, No. Supplement 1 ( 2018-11-29), p. 3909-3909
    Details
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 3909-3909
    Abstract: Long noncoding RNAs (lncRNAs) have complex, mainly chromatin-associated functions and their expression is highly coordinated and cell-type specific. Based on their tight regulation in normal differentiation, we set out to investigate whether lncRNAs are dysregulated in diseases where differentiation is impaired, such as in acute myeloid leukemia (AML). To identify lncRNAs that are essential for both normal hematopoiesis as well as AML maintenance, we sequenced the long polyA- and non-polyA-tagged transcriptome from successive stages of human myelopoiesis (myeloblasts, promyelocytes, metamyelocytes, and neutrophils) isolated from bone marrow of healthy donors (n=3). Applying a high-dimensional data portraying approach (OposSOM, Löffler-Wirth et al., BMC Bioinformatics, 2015), we identified functional expression modules of lncRNAs that are either positively or negatively associated with myeloid lineage commitment in our dataset. Seven out of the top15 differentiation-associated lncRNAs exhibit significant prognostic relevance in overall and event-free survival analyses of independent AML patient datasets and improve the predictive power of the current prognosis standards (cytogenetic risk/age/TP53-status). In particular, a combination of 3 transcripts, PROMYS (Promoter of Myelopoiesis, annotated as uncharacterized ncRNA LOC107985167), ANTAMY (Antagonist of Myelopoiesis, uncharacterized ncRNA LOC101927745) and LINC00677, outperformed the recently reported prognostic benefit of the LSC17high score (Ng et al, Nature, 2016) by a factor of Ø 22.7 based on concordance index score increase (Ø 4.8% vs. 0.21%). All three lncRNAs are highly conserved, expressed in 10 tested human AML cell lines as well as significantly differentially expressed in distinct cytogenetic patient subgroups of The Cancer Genome Atlas (TCGA) LAML cohort (n=171). PROMYS is downregulated in t(15;17) and t(8;21) cases, supporting its strong association with worse OS in the TCGA-LAML dataset (p=0.0001). In contrast, ANTAMY shows high expression in AML with t(8;21), and LINC00677 in NPM1+/FLT3- mutated AML patient samples with normal karyotype (CN-AML) and in core Binding factor (CBF) AMLs. Accordingly, high expression levels of both lncRNAs associate with a significantly better OS in the TCGA LAML dataset (p=0.01 and 0.02, respectively). To investigate their function in vitro, we knocked out each lncRNA individually in the human OCI/AML-5 AML cell line using CRISPR/Cas9. Loss of ANTAMY impaired proliferation (p=0.04) and increased both monocytic differentiation upon treatment with 2-0-tetradecanoylphorbol-13-acetate (TPA) (p=0.0001) and granulocytic differentiation with all-trans retinoic acid (ATRA) (p=0.0002) compared to the empty vector control. Loss of LINC00677 in OCI/AML-5 cells specifically increased granulocytic differentiation through ATRA (p=0.0002). In contrast, inactivation of PROMYS led to reduced differentiation induced by ATRA (p=0.00004) and TPA (p=0.002). Furthermore, we found that PROMYS is involved in the regulation of the Macrophage colony-stimulating factor 1 (CSF1), which is deregulated in ATRA- and TPA-induced differentiation in PROMYS knockout but not in control cells (p 〈 0.002 and 〈 0.00002, respectively), explaining its negative impact on differentiation. Through screening of human myelopoiesis, we identified three unexplored lncRNAs: LINC00677, PROMYS, and ANTAMY, which play a role in myeloid differentiation and have an impact on patient prognosis. Our in vitro findings confirm that ANTAMY, LINC00677, and PROMYS are active modulators of leukemic cells, which influence their proliferation, morphology, myeloid marker expression as well as apoptosis rate. These transcripts and their interaction partners add an additional layer of regulation to the understanding of differentiation and might represent previously unknown vulnerabilities of AML cells, which warrants their further investigation in vivo. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2018-99-118331
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
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  • 8
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    Prospective identification of resistance mechanisms to HSP90 inhibition in KRAS mutant cancer cells
    Impact Journals, LLC ; 2017
    In:  Oncotarget Vol. 8, No. 5 ( 2017-01-31), p. 7678-7690
    Details
    In: Oncotarget, Impact Journals, LLC, Vol. 8, No. 5 ( 2017-01-31), p. 7678-7690
    Type of Medium: Online Resource
    ISSN: 1949-2553
    URL: Issue
    URL: Article
    DOI: 10.18632/oncotarget.v8i5
    DOI: 10.18632/oncotarget.13841
    Language: English
    Publisher: Impact Journals, LLC
    Publication Date: 2017
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  • 9
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    Abstracts from the 3rd Conference on Aneuploidy and Cancer: Clinical and Experimental Aspects : Berkeley, CA, USA. 26 - 29 January 2017
    Springer Science and Business Media LLC ; 2017
    In:  Molecular Cytogenetics Vol. 10, No. S2 ( 2017-7)
    Details
    In: Molecular Cytogenetics, Springer Science and Business Media LLC, Vol. 10, No. S2 ( 2017-7)
    Type of Medium: Online Resource
    ISSN: 1755-8166
    URL: Article
    DOI: 10.1186/s13039-017-0320-x
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2017
    detail.hit.zdb_id: 2420849-8
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