60 results on '"Heckl D"'
Search Results
2. MPN development caused by activating mutations in Mpl depends on Ras/MapK and PI3K/Akt signalling in addition to Jak/Stat signalling: V694
- Author
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Heckl, D., Meyer, J., Wicke, D., Büsche, G., Wolf, S., Wintterle, S., Kreipe, H., Baum, C., and Modlich, U.
- Published
- 2011
3. FRI-255 - Impact of Genetic Polymorphisms in the SCARB1 Gene on Hepatitis C Virus
- Author
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Westhaus, S., Deest, M., Stanke, F., Heckl, D., Schambach, A., Manns, M.P., Berg, T., Sarrazin, C., Ciesek, S., and von Hahn, T.
- Published
- 2016
- Full Text
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4. Correction to: Efficient generation of gene-modified human natural killer cells via alpharetroviral vectors.
- Author
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Suerth JD, Morgan MA, Kloess S, Heckl D, Neudörfl C, Falk CS, Koehl U, and Schambach A
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- 2024
- Full Text
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5. Myeloid leukemia vulnerabilities embedded in long noncoding RNA locus MYNRL15 .
- Author
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Ng M, Verboon L, Issa H, Bhayadia R, Vermunt MW, Winkler R, Schüler L, Alejo O, Schuschel K, Regenyi E, Borchert D, Heuser M, Reinhardt D, Yaspo ML, Heckl D, and Klusmann JH
- Abstract
The noncoding genome presents a largely untapped source of new biological insights, including thousands of long noncoding RNA (lncRNA) loci. While lncRNA dysregulation has been reported in myeloid malignancies, their functional relevance remains to be systematically interrogated. We performed CRISPRi screens of lncRNA signatures from normal and malignant hematopoietic cells and identified MYNRL15 as a myeloid leukemia dependency. Functional dissection suggests an RNA-independent mechanism mediated by two regulatory elements embedded in the locus. Genetic perturbation of these elements triggered a long-range chromatin interaction and downregulation of leukemia dependency genes near the gained interaction sites, as well as overall suppression of cancer dependency pathways. Thus, this study describes a new noncoding myeloid leukemia vulnerability and mechanistic concept for myeloid leukemia. Importantly, MYNRL15 perturbation caused strong and selective impairment of leukemia cells of various genetic backgrounds over normal hematopoietic stem and progenitor cells in vitro , and depletion of patient-derived xenografts in vivo ., Competing Interests: J.H.K. has advisory roles for Bluebird Bio, Boehringer Ingelheim, Novartis, Roche and Jazz Pharmaceuticals. D.R. has advisory roles for Celgene Corporation, Novartis, Bluebird Bio, Janssen, and receives research funding from CLS Behring and Roche. M.H. receives research funding from Abbvie, Agios, Astellas, BergenBio, Bristol-Myers Squibb, Glycostem, Jazz Pharmaceuticals, Karyopharm, Loxo Oncology, and PinotBio, has received honoraria from Novartis, Pfizer, Jazz Pharmaceuticals, Janssen, Certara, and Sobi, and has advisory roles for Abbvie, BMS, Glycostem, Servier, PinotBio, Amgen, Pfizer, and LabDelbert., (© 2023 The Author(s).)
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- 2023
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6. Preclinical testing of miRNA-193b-3p mimic in acute myeloid leukemias.
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Issa H, Bhayadia R, Winkler R, Swart LE, Heckl D, and Klusmann JH
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- Humans, MicroRNAs genetics, Leukemia, Myeloid, Acute genetics
- Published
- 2023
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7. Healthcare resource utilization and cost of pneumococcal disease in children in Germany, 2014-2019: a retrospective cohort study.
- Author
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Hu T, Podmore B, Barnett R, Beier D, Galetzka W, Qizilbash N, Heckl D, Boellinger T, and Weaver J
- Abstract
Background: Since the introduction of higher valency pneumococcal conjugate vaccines in 2009, recent estimates on the economic burden of pediatric pneumococcal disease (PD) in Germany have been lacking. This study estimates healthcare resource utilization (HCRU) and medical cost associated with PDs in children < 16 years old in Germany from 2014-2019., Methods: A nationally representative sample from the Institute for Applied Health Research (InGef) German claims database was used, covering approximately 5% of the total German population. Episodes of pneumococcal pneumonia (PP), all-cause pneumonia (ACP), invasive pneumococcal disease (IPD), and acute otitis media (AOM) in children aged < 16 years were identified using ICD-10-GM codes. HCRU was estimated from annual rates of outpatient visits, outpatient antibiotic prescriptions and inpatient admissions, divided by person-years (PY) at-risk. Average direct medical costs per episode were estimated as the total cost of all HCRU, divided by the total number of episodes. The Mann-Kendall test was used to assess monotonic time trends from 2014-2019., Results: During 2014-2019, 916,805 children aged < 16 years were followed up for a total of 3,608,716 PY. The average costs per episode for out-versus inpatient care associated with PP and ACP were €67 (95% CI 58-76) versus €2,606 (95% CI 1,338-3,873), and €63 (95% CI 62-63) versus €620 (95% CI 598-641), respectively. For IPD, the average medical cost per episode for out-versus inpatients were €30 (95% CI 19-42) versus €6,051 (95% CI 3,323-8,779), respectively. There were no significant trends in HCRU or costs for IPD or pneumonia over the study period, except for a significant reduction in ACP outpatient visits. A significant decrease in rate of outpatient visits and antibiotic prescribing for recurrent AOM was observed, in addition to an increase in rates of hospital admissions for simple AOM. This was paralleled by a significant increase in inpatient costs per episode for treating AOM overall, and simple AOM, over the study period., Conclusions: The HCRU and cost per episode of pneumonia and IPD did not vary significantly from 2014-2019, but increased for AOM. The economic burden of pneumonia, IPD, and AOM remains substantial in Germany., (© 2023. Merck & Co., Inc., Rahway, NJ, USA and its affiliates.)
- Published
- 2023
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8. RUNX1 isoform disequilibrium promotes the development of trisomy 21-associated myeloid leukemia.
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Gialesaki S, Bräuer-Hartmann D, Issa H, Bhayadia R, Alejo-Valle O, Verboon L, Schmell AL, Laszig S, Regényi E, Schuschel K, Labuhn M, Ng M, Winkler R, Ihling C, Sinz A, Glaß M, Hüttelmaier S, Matzk S, Schmid L, Strüwe FJ, Kadel SK, Reinhardt D, Yaspo ML, Heckl D, and Klusmann JH
- Subjects
- Animals, Child, Humans, Mice, Aneuploidy, Protein Isoforms genetics, Trisomy genetics, Core Binding Factor Alpha 2 Subunit genetics, Down Syndrome complications, Down Syndrome genetics, Leukemia, Myeloid genetics
- Abstract
Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (DS) (ie, trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to DS-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR-CRISPR-associated protein 9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in patients with ML-DS, and mechanistic studies using murine ML-DS models and patient-derived xenografts revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in patient-derived xenografts in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong antileukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
- Published
- 2023
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9. Combining LSD1 and JAK-STAT inhibition targets Down syndrome-associated myeloid leukemia at its core.
- Author
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Grimm J, Bhayadia R, Gack L, Heckl D, and Klusmann JH
- Subjects
- Histone Demethylases metabolism, Humans, Janus Kinases metabolism, STAT Transcription Factors metabolism, Down Syndrome complications, Leukemia, Myeloid
- Published
- 2022
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10. Long noncoding RNAs as regulators of pediatric acute myeloid leukemia.
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Neyazi S, Ng M, Heckl D, and Klusmann JH
- Abstract
Long noncoding RNAs (lncRNAs) are increasingly emerging as regulators across human development and disease, and many have been described in the context of hematopoiesis and leukemogenesis. These studies have yielded new molecular insights into the contribution of lncRNAs to AML development and revealed connections between lncRNA expression and clinical parameters in AML patients. In this mini review, we illustrate the versatile functions of lncRNAs in AML, with a focus on pediatric AML, and present examples that may serve as future therapeutic targets or predictive factors., (© 2022. The Author(s).)
- Published
- 2022
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11. Genetic barcoding systematically compares genes in del(5q) MDS and reveals a central role for CSNK1A1 in clonal expansion.
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Stalmann USA, Ticconi F, Snoeren IAM, Li R, Gleitz HFE, Cowley GS, McConkey ME, Wong AB, Schmitz S, Fuchs SNR, Sood S, Leimkühler NB, Martinez-Høyer S, Banjanin B, Root D, Brümmendorf TH, Pearce JE, Schuppert A, Bindels EMJ, Essers MA, Heckl D, Stiehl T, Costa IG, Ebert BL, and Schneider RK
- Subjects
- Haploinsufficiency, Hematopoietic Stem Cells pathology, Humans, Chromosome Deletion, Myelodysplastic Syndromes pathology
- Abstract
How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1-/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1-/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
12. The megakaryocytic transcription factor ARID3A suppresses leukemia pathogenesis.
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Alejo-Valle O, Weigert K, Bhayadia R, Ng M, Issa H, Beyer C, Emmrich S, Schuschel K, Ihling C, Sinz A, Zimmermann M, Wickenhauser C, Flasinski M, Regenyi E, Labuhn M, Reinhardt D, Yaspo ML, Heckl D, and Klusmann JH
- Subjects
- Animals, Child, GATA1 Transcription Factor genetics, Gene Expression Regulation, Leukemic, Genes, Tumor Suppressor, Humans, Leukemia, Megakaryoblastic, Acute pathology, Mice, Inbred C57BL, MicroRNAs genetics, Mutation, Mice, DNA-Binding Proteins genetics, Leukemia, Megakaryoblastic, Acute genetics, Transcription Factors genetics
- Abstract
Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21-encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease., (© 2022 by The American Society of Hematology.)
- Published
- 2022
- Full Text
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13. Correction: Effective drug treatment identified by in vivo screening in a transplantable patient-derived xenograft model of chronic myelomonocytic leukemia.
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Kloos A, Mintzas K, Winckler L, Gabdoulline R, Alwie Y, Jyotsana N, Kattre N, Schottmann R, Scherr M, Gupta C, Adams FF, Schwarzer A, Heckl D, Schambach A, Imren S, Humphries RK, Ganser A, Thol F, and Heuser M
- Published
- 2021
- Full Text
- View/download PDF
14. Comprehensive CRISPR-Cas9 screens identify genetic determinants of drug responsiveness in multiple myeloma.
- Author
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Bohl SR, Schmalbrock LK, Bauhuf I, Meyer T, Dolnik A, Szyska M, Blätte TJ, Knödler S, Röhner L, Miller D, Kull M, Langer C, Döhner H, Letai A, Damm F, Heckl D, Bullinger L, and Krönke J
- Subjects
- CRISPR-Cas Systems, Humans, Lenalidomide, Neoplasm Recurrence, Local, Multiple Myeloma drug therapy, Multiple Myeloma genetics, Pharmaceutical Preparations
- Abstract
The introduction of new drugs in the past years has substantially improved outcome in multiple myeloma (MM). However, the majority of patients eventually relapse and become resistant to one or multiple drugs. While the genetic landscape of relapsed/ resistant multiple myeloma has been elucidated, the causal relationship between relapse-specific gene mutations and the sensitivity to a given drug in MM has not systematically been evaluated. To determine the functional impact of gene mutations, we performed combined whole-exome sequencing (WES) of longitudinal patient samples with CRISPR-Cas9 drug resistance screens for lenalidomide, bortezomib, dexamethasone, and melphalan. WES of longitudinal samples from 16 MM patients identified a large number of mutations in each patient that were newly acquired or evolved from a small subclone (median 9, range 1-55), including recurrent mutations in TP53, DNAH5, and WSCD2. Focused CRISPR-Cas9 resistance screens against 170 relapse-specific mutations functionally linked 15 of them to drug resistance. These included cereblon E3 ligase complex members for lenalidomide, structural genes PCDHA5 and ANKMY2 for dexamethasone, RB1 and CDK2NC for bortezomib, and TP53 for melphalan. In contrast, inactivation of genes involved in the DNA damage repair pathway, including ATM, FANCA, RAD54B, and BRCC3, enhanced susceptibility to cytotoxic chemotherapy. Resistance patterns were highly drug specific with low overlap and highly correlated with the treatment-dependent clonal evolution in patients. The functional association of specific genetic alterations with drug sensitivity will help to personalize treatment of MM in the future., (© 2021 by The American Society of Hematology.)
- Published
- 2021
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15. Molecular Mechanisms of the Genetic Predisposition to Acute Megakaryoblastic Leukemia in Infants With Down Syndrome.
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Grimm J, Heckl D, and Klusmann JH
- Abstract
Individuals with Down syndrome are genetically predisposed to developing acute megakaryoblastic leukemia. This myeloid leukemia associated with Down syndrome (ML-DS) demonstrates a model of step-wise leukemogenesis with perturbed hematopoiesis already presenting in utero , facilitating the acquisition of additional driver mutations such as truncating GATA1 variants, which are pathognomonic to the disease. Consequently, the affected individuals suffer from a transient abnormal myelopoiesis (TAM)-a pre-leukemic state preceding the progression to ML-DS. In our review, we focus on the molecular mechanisms of the different steps of clonal evolution in Down syndrome leukemogenesis, and aim to provide a comprehensive view on the complex interplay between gene dosage imbalances, GATA1 mutations and somatic mutations affecting JAK-STAT signaling, the cohesin complex and epigenetic regulators., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Grimm, Heckl and Klusmann.)
- Published
- 2021
- Full Text
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16. Effective drug treatment identified by in vivo screening in a transplantable patient-derived xenograft model of chronic myelomonocytic leukemia.
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Kloos A, Mintzas K, Winckler L, Gabdoulline R, Alwie Y, Jyotsana N, Kattre N, Schottmann R, Scherr M, Gupta C, Adams FF, Schwarzer A, Heckl D, Schambach A, Imren S, Humphries RK, Ganser A, Thol F, and Heuser M
- Subjects
- Animals, Antineoplastic Agents administration & dosage, Antineoplastic Agents adverse effects, Antineoplastic Combined Chemotherapy Protocols adverse effects, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Azacitidine pharmacology, Clonal Evolution, Disease Models, Animal, Drug Synergism, Female, GTP Phosphohydrolases genetics, Humans, Leukemia, Myelomonocytic, Chronic genetics, Leukemia, Myelomonocytic, Chronic mortality, Leukemia, Myelomonocytic, Chronic pathology, Membrane Proteins genetics, Mice, Mutation, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Pyridones pharmacology, Pyridones therapeutic use, Pyrimidinones pharmacology, Pyrimidinones therapeutic use, RNA, Small Interfering genetics, Receptor, Notch1 genetics, Antineoplastic Agents pharmacology, Drug Evaluation, Preclinical methods, Drug Evaluation, Preclinical standards, Leukemia, Myelomonocytic, Chronic drug therapy, Xenograft Model Antitumor Assays methods
- Abstract
To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.
- Published
- 2020
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17. Chromosome 21 gain is dispensable for transient myeloproliferative disorder driven by a novel GATA1 mutation.
- Author
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Lukes J Jr, Danek P, Alejo-Valle O, Potuckova E, Gahura O, Heckl D, Starkova J, Stary J, Mejstrikova E, Alberich-Jorda M, Zuna J, Trka J, Klusmann JH, and Zaliova M
- Subjects
- GATA1 Transcription Factor, Humans, Janus Kinase 1 genetics, Male, Chromosomes, Human, Pair 12, Down Syndrome genetics, Leukemoid Reaction genetics, Mutation
- Published
- 2020
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18. LncRNA-SLC16A1-AS1 induces metabolic reprogramming during Bladder Cancer progression as target and co-activator of E2F1.
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Logotheti S, Marquardt S, Gupta SK, Richter C, Edelhäuser BAH, Engelmann D, Brenmoehl J, Söhnchen C, Murr N, Alpers M, Singh KP, Wolkenhauer O, Heckl D, Spitschak A, and Pützer BM
- Subjects
- Adenosine Triphosphate genetics, Cell Line, Tumor, Disease Progression, Gene Expression Regulation, Neoplastic genetics, Glycolysis genetics, Humans, Mitochondria genetics, Oxidation-Reduction, Promoter Regions, Genetic genetics, Transcriptional Activation genetics, Urinary Bladder Neoplasms pathology, Cellular Reprogramming physiology, E2F1 Transcription Factor genetics, Monocarboxylic Acid Transporters genetics, RNA, Long Noncoding genetics, Symporters genetics, Urinary Bladder Neoplasms genetics
- Abstract
Long non-coding RNAs (lncRNAs) have emerged as integral components of E2F1-regulated gene regulatory networks (GRNs), but their implication in advanced or treatment-refractory malignancy is unknown. Methods: We combined high-throughput transcriptomic approaches with bioinformatics and structure modeling to search for lncRNAs that participate in E2F1-activated prometastatic GRNs and their phenotypic targets in the highly-relevant case of E2F1-driven aggressive bladder cancer (BC). RNA immunoprecipitation was performed to verify RNA-protein interactions. Functional analyses including qRT-PCR, immunoblotting, luciferase assays and measurement of extracellular fluxes were conducted to validate expression and target gene regulation. Results: We identified E2F1-responsive lncRNA-SLC16A1-AS1 and its associated neighboring protein-coding gene, SLC16A1/MCT1, which both promote cancer invasiveness. Mechanistically, upon E2F1-mediated co-transactivation of the gene pair, SLC16A1-AS1 associates with E2F1 in a structure-dependent manner and forms an RNA-protein complex that enhances SLC16A1/MCT1 expression through binding to a composite SLC16A1-AS1:E2F1-responsive promoter element. Moreover, SLC16A1-AS1 increases aerobic glycolysis and mitochondrial respiration and fuels ATP production by fatty acid β-oxidation. These metabolic changes are accompanied by alterations in the expression of the SLC16A1-AS1:E2F1-responsive gene PPARA, a key mediator of fatty acid β-oxidation. Conclusions: Our results unveil a new gene regulatory program by which E2F1-induced lncRNA-SLC16A1-AS1 forms a complex with its transcription factor that promotes cancer metabolic reprogramming towards the acquisition of a hybrid oxidative phosphorylation/glycolysis cell phenotype favoring BC invasiveness., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)
- Published
- 2020
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19. RNA-Binding Proteins in Acute Leukemias.
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Schuschel K, Helwig M, Hüttelmaier S, Heckl D, Klusmann JH, and Hoell JI
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- Acute Disease, Animals, Humans, Leukemia metabolism, Nucleic Acid Conformation, Protein Binding, Protein Domains, RNA, Messenger chemistry, RNA, Messenger metabolism, RNA-Binding Proteins chemistry, RNA-Binding Proteins metabolism, Gene Expression Regulation, Leukemic, Leukemia genetics, RNA, Messenger genetics, RNA-Binding Proteins genetics
- Abstract
Acute leukemias are genetic diseases caused by translocations or mutations, which dysregulate hematopoiesis towards malignant transformation. However, the molecular mode of action is highly versatile and ranges from direct transcriptional to post-transcriptional control, which includes RNA-binding proteins (RBPs) as crucial regulators of cell fate. RBPs coordinate RNA dynamics, including subcellular localization, translational efficiency and metabolism, by binding to their target messenger RNAs (mRNAs), thereby controlling the expression of the encoded proteins. In view of the growing interest in these regulators, this review summarizes recent research regarding the most influential RBPs relevant in acute leukemias in particular. The reported RBPs, either dysregulated or as components of fusion proteins, are described with respect to their functional domains, the pathways they affect, and clinical aspects associated with their dysregulation or altered functions.
- Published
- 2020
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20. Meningioma 1 is indispensable for mixed lineage leukemia-rearranged acute myeloid leukemia.
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Sharma A, Jyotsana N, Gabdoulline R, Heckl D, Kuchenbauer F, Slany RK, Ganser A, and Heuser M
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- Animals, Hematopoietic Stem Cells, Humans, Mice, Leukemia, Myeloid, Acute genetics, Trans-Activators genetics, Tumor Suppressor Proteins genetics
- Abstract
Mixed lineage leukemia ( MLL/KMT2A ) rearrangements (MLL-r) are one of the most frequent chromosomal aberrations in acute myeloid leukemia. We evaluated the function of Meningioma 1 (MN1), a co-factor of HOXA9 and MEIS1, in human and murine MLL-rearranged leukemia by CRISPR-Cas9 mediated deletion of MN1. MN1 was required for in vivo leukemogenicity of MLL positive murine and human leukemia cells. Loss of MN1 inhibited cell cycle and proliferation, promoted apoptosis and induced differentiation of MLL-rearranged cells. Expression analysis and chromatin immunoprecipitation with sequencing from previously reported data sets demonstrated that MN1 primarily maintains active transcription of HOXA9 and HOXA10, which are critical downstream genes of MLL, and their target genes like BCL2, MCL1 and Survivin. Treatment of MLL-rearranged primary leukemia cells with anti-MN1 siRNA significantly reduced their clonogenic potential in contrast to normal CD34
+ hematopoietic progenitor cells, suggesting a therapeutic window for MN1 targeting. In summary, our findings demonstrate that MN1 plays an essential role in MLL fusion leukemias and serve as a therapeutic target in MLL-rearranged acute myeloid leukemia., (Copyright© 2020 Ferrata Storti Foundation.)- Published
- 2020
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21. Functional characterization of BRCC3 mutations in acute myeloid leukemia with t(8;21)(q22;q22.1).
- Author
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Meyer T, Jahn N, Lindner S, Röhner L, Dolnik A, Weber D, Scheffold A, Köpff S, Paschka P, Gaidzik VI, Heckl D, Wiese S, Ebert BL, Döhner H, Bullinger L, Döhner K, and Krönke J
- Subjects
- Animals, CRISPR-Cas Systems genetics, Cell Line, Cell Proliferation drug effects, Cell Proliferation genetics, Cytokines genetics, DNA Damage drug effects, DNA Damage genetics, Doxorubicin pharmacology, Granulocyte Colony-Stimulating Factor genetics, HEK293 Cells, Humans, Inflammasomes genetics, Leukemia, Myeloid, Acute drug therapy, Mice, Deubiquitinating Enzymes genetics, Leukemia, Myeloid, Acute genetics, Mutation genetics
- Abstract
BRCA1/BRCA2-containing complex 3 (BRCC3) is a Lysine 63-specific deubiquitinating enzyme (DUB) involved in inflammasome activity, interferon signaling, and DNA damage repair. Recurrent mutations in BRCC3 have been reported in myelodysplastic syndromes (MDS) but not in de novo AML. In one of our recent studies, we found BRCC3 mutations selectively in 9/191 (4.7%) cases with t(8;21)(q22;q22.1) AML but not in 160 cases of inv(16)(p13.1q22) AML. Clinically, AML patients with BRCC3 mutations had an excellent outcome with an event-free survival of 100%. Inactivation of BRCC3 by CRISPR/Cas9 resulted in improved proliferation in t(8;21)(q22;q22.1) positive AML cell lines and together with expression of AML1-ETO induced unlimited self-renewal in mouse hematopoietic progenitor cells in vitro. Mutations in BRCC3 abrogated its deubiquitinating activity on IFNAR1 resulting in an impaired interferon response and led to diminished inflammasome activity. In addition, BRCC3 inactivation increased release of several cytokines including G-CSF which enhanced proliferation of AML cell lines with t(8;21)(q22;q22.1). Cell lines and primary mouse cells with inactivation of BRCC3 had a higher sensitivity to doxorubicin due to an impaired DNA damage response providing a possible explanation for the favorable outcome of BRCC3 mutated AML patients.
- Published
- 2020
- Full Text
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22. The stem cell-specific long noncoding RNA HOXA10-AS in the pathogenesis of KMT2A-rearranged leukemia.
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Al-Kershi S, Bhayadia R, Ng M, Verboon L, Emmrich S, Gack L, Schwarzer A, Strowig T, Heckl D, and Klusmann JH
- Subjects
- Animals, Disease Models, Animal, Flow Cytometry, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Humans, Leukemia diagnosis, Leukemia mortality, Leukemia therapy, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute metabolism, Leukemia, Myeloid, Acute pathology, Mice, NF-kappa B metabolism, Neoplastic Stem Cells pathology, Nucleophosmin, Prognosis, Transcriptome, Gene Rearrangement, Histone-Lysine N-Methyltransferase genetics, Homeobox A10 Proteins genetics, Leukemia genetics, Myeloid-Lymphoid Leukemia Protein genetics, Neoplastic Stem Cells metabolism, RNA, Antisense, RNA, Long Noncoding
- Abstract
HOX genes are highly conserved, and their precisely controlled expression is crucial for normal hematopoiesis. Accordingly, deregulation of HOX genes can cause leukemia. However, despite of intensive research on the coding HOX genes, the role of the numerous long noncoding RNAs (lncRNAs) within the HOX clusters during hematopoiesis and their contribution to leukemogenesis are incompletely understood. Here, we show that the lncRNA HOXA10-AS, located antisense to HOXA10 and mir-196b in the HOXA cluster, is highly expressed in hematopoietic stem cells (HSCs) as well as in KMT2A-rearranged and NPM1 mutated acute myeloid leukemias (AMLs). Using short hairpin RNA- and locked nucleic acid-conjugated chimeric antisense oligonucleotide (LNA-GapmeR)-mediated HOXA10-AS-knockdown and CRISPR/Cas9-mediated excision in vitro, we demonstrate that HOXA10-AS acts as an oncogene in KMT2A-rearranged AML. Moreover, HOXA10-AS knockdown severely impairs the leukemic growth of KMT2A-rearranged patient-derived xenografts in vivo, while high HOXA10-AS expression can serve as a marker of poor prognosis in AML patients. Lentiviral expression of HOXA10-AS blocks normal monocytic differentiation of human CD34+ hematopoietic stem and progenitor cells. Mechanistically, we show that HOXA10-AS localizes in the cytoplasm and acts in trans to induce NF-κB target genes. In total, our data imply that the normally HSC-specific HOXA10-AS is an oncogenic lncRNA in KMT2A-r AML. Thus, it may also represent a potential therapeutic target in KMT2A-rearranged AML., (© 2019 by The American Society of Hematology.)
- Published
- 2019
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23. Mechanisms of Progression of Myeloid Preleukemia to Transformed Myeloid Leukemia in Children with Down Syndrome.
- Author
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Labuhn M, Perkins K, Matzk S, Varghese L, Garnett C, Papaemmanuil E, Metzner M, Kennedy A, Amstislavskiy V, Risch T, Bhayadia R, Samulowski D, Hernandez DC, Stoilova B, Iotchkova V, Oppermann U, Scheer C, Yoshida K, Schwarzer A, Taub JW, Crispino JD, Weiss MJ, Hayashi Y, Taga T, Ito E, Ogawa S, Reinhardt D, Yaspo ML, Campbell PJ, Roberts I, Constantinescu SN, Vyas P, Heckl D, and Klusmann JH
- Published
- 2019
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- View/download PDF
24. The Regulatory Roles of Long Noncoding RNAs in Acute Myeloid Leukemia.
- Author
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Ng M, Heckl D, and Klusmann JH
- Abstract
In this post-genomic era, long noncoding RNAs (lncRNAs) are rapidly gaining recognition for their crucial roles across diverse biological processes and contexts. The human blood system is no exception, where dozens of lncRNAs have been established as regulators of normal and/or malignant hematopoiesis, and where ongoing works continue to uncover novel lncRNA functions. Our review focuses on lncRNAs that are involved in the pathogenesis of acute myeloid leukemia (AML) and the mechanisms through which they control gene expression in this disease context. We also comment on genome-wide sequencing or profiling studies that have implicated large sets of lncRNAs in AML pathophysiology.
- Published
- 2019
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25. Jak2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation.
- Author
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Jacquelin S, Straube J, Cooper L, Vu T, Song A, Bywater M, Baxter E, Heidecker M, Wackrow B, Porter A, Ling V, Green J, Austin R, Kazakoff S, Waddell N, Hesson LB, Pimanda JE, Stegelmann F, Bullinger L, Döhner K, Rampal RK, Heckl D, Hill GR, and Lane SW
- Subjects
- Animals, DNA Methyltransferase 3A, Humans, Janus Kinase 2 genetics, Janus Kinase 2 metabolism, Mice, Mice, Mutant Strains, Amino Acid Substitution, DNA (Cytosine-5-)-Methyltransferases genetics, DNA (Cytosine-5-)-Methyltransferases metabolism, Hematologic Neoplasms enzymology, Hematologic Neoplasms genetics, Hematologic Neoplasms pathology, Hematopoietic Stem Cells enzymology, Hematopoietic Stem Cells pathology, Mutation, Missense, Primary Myelofibrosis enzymology, Primary Myelofibrosis genetics, Primary Myelofibrosis pathology, Signal Transduction genetics
- Abstract
Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a
-/- Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer-driven inflammatory signaling., (© 2018 by The American Society of Hematology.)- Published
- 2018
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26. Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout.
- Author
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Knopp Y, Geis FK, Heckl D, Horn S, Neumann T, Kuehle J, Meyer J, Fehse B, Baum C, Morgan M, Meyer J, Schambach A, and Galla M
- Abstract
The recently discovered CRISPR/Cas9 system is widely used in basic research and is a useful tool for disease modeling and gene editing therapies. However, long-term expression of DNA-modifying enzymes can be associated with cytotoxicity and is particularly unwanted in clinical gene editing strategies. Because current transient expression methods may still suffer from cytotoxicity and/or low efficiency, we developed non-integrating retrovirus-based CRISPR/Cas9 all-in-one particles for targeted gene knockout. By redirecting the gammaretroviral packaging machinery, we transiently delivered Streptococcus pyogenes Cas9 (SpCas9) mRNA and single-guide RNA transcripts into various (including primary) cell types. Spatiotemporal co-delivery of CRISPR/Cas9 components resulted in efficient disruption of a surrogate reporter gene, as well as functional knockout of endogenous human genes CXCR4 and TP53. Although acting in a hit-and-run fashion, knockout efficiencies of our transient particles corresponded to 52%-80% of those obtained from constitutively active integrating vectors. Stable SpCas9 overexpression at high doses in murine NIH3T3 cells caused a substantial G0/G1 arrest accompanied by reduced cell growth and metabolic activity, which was prevented by transient SpCas9 transfer. In summary, the non-integrating retrovirus-based vector particles introduced here allow efficient and dose-controlled delivery of CRISPR/Cas9 components into target cells., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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27. Gli1 + Mesenchymal Stromal Cells Are a Key Driver of Bone Marrow Fibrosis and an Important Cellular Therapeutic Target.
- Author
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Schneider RK, Mullally A, Dugourd A, Peisker F, Hoogenboezem R, Van Strien PMH, Bindels EM, Heckl D, Büsche G, Fleck D, Müller-Newen G, Wongboonsin J, Ventura Ferreira M, Puelles VG, Saez-Rodriguez J, Ebert BL, Humphreys BD, and Kramann R
- Published
- 2018
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- View/download PDF
28. GATA1 s exerts developmental stage-specific effects in human hematopoiesis.
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Gialesaki S, Mahnken AK, Schmid L, Labuhn M, Bhayadia R, Heckl D, and Klusmann JH
- Subjects
- Adult, Cell Differentiation, Female, GATA1 Transcription Factor genetics, Humans, Infant, Newborn, Male, Mutation, Pregnancy, Stem Cells, GATA1 Transcription Factor physiology, Hematopoiesis
- Published
- 2018
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- View/download PDF
29. Endogenous Tumor Suppressor microRNA-193b: Therapeutic and Prognostic Value in Acute Myeloid Leukemia.
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Bhayadia R, Krowiorz K, Haetscher N, Jammal R, Emmrich S, Obulkasim A, Fiedler J, Schwarzer A, Rouhi A, Heuser M, Wingert S, Bothur S, Döhner K, Mätzig T, Ng M, Reinhardt D, Döhner H, Zwaan CM, van den Heuvel Eibrink M, Heckl D, Fornerod M, Thum T, Humphries RK, Rieger MA, Kuchenbauer F, and Klusmann JH
- Subjects
- Animals, Cell Growth Processes genetics, Down-Regulation, Genes, Tumor Suppressor, Heterografts, Homeodomain Proteins genetics, Humans, Leukemia, Myeloid, Acute pathology, Leukemia, Myeloid, Acute therapy, MAP Kinase Signaling System, Mice, Mice, Inbred C57BL, Mice, Inbred NOD, MicroRNAs genetics, Myeloid Ecotropic Viral Integration Site 1 Protein genetics, Prognosis, Leukemia, Myeloid, Acute genetics, MicroRNAs biosynthesis
- Abstract
Purpose Dysregulated microRNAs are implicated in the pathogenesis and aggressiveness of acute myeloid leukemia (AML). We describe the effect of the hematopoietic stem-cell self-renewal regulating miR-193b on progression and prognosis of AML. Methods We profiled miR-193b-5p/3p expression in cytogenetically and clinically characterized de novo pediatric AML (n = 161) via quantitative real-time polymerase chain reaction and validated our findings in an independent cohort of 187 adult patients. We investigated the tumor suppressive function of miR-193b in human AML blasts, patient-derived xenografts, and miR-193b knockout mice in vitro and in vivo. Results miR-193b exerted important, endogenous, tumor-suppressive functions on the hematopoietic system. miR-193b-3p was downregulated in several cytogenetically defined subgroups of pediatric and adult AML, and low expression served as an independent indicator for poor prognosis in pediatric AML (risk ratio ± standard error, -0.56 ± 0.23; P = .016). miR-193b-3p expression improved the prognostic value of the European LeukemiaNet risk-group stratification or a 17-gene leukemic stemness score. In knockout mice, loss of miR-193b cooperated with Hoxa9/Meis1 during leukemogenesis, whereas restoring miR-193b expression impaired leukemic engraftment. Similarly, expression of miR-193b in AML blasts from patients diminished leukemic growth in vitro and in mouse xenografts. Mechanistically, miR-193b induced apoptosis and a G1/S-phase block in various human AML subgroups by targeting multiple factors of the KIT-RAS-RAF-MEK-ERK (MAPK) signaling cascade and the downstream cell cycle regulator CCND1. Conclusion The tumor-suppressive function is independent of patient age or genetics; therefore, restoring miR-193b would assure high antileukemic efficacy by blocking the entire MAPK signaling cascade while preventing the emergence of resistance mechanisms.
- Published
- 2018
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30. Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications.
- Author
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Labuhn M, Adams FF, Ng M, Knoess S, Schambach A, Charpentier EM, Schwarzer A, Mateo JL, Klusmann JH, and Heckl D
- Subjects
- Base Composition, Base Sequence, CRISPR-Associated Protein 9 metabolism, Cell Line, Tumor, Clustered Regularly Interspaced Short Palindromic Repeats, HEK293 Cells, Humans, Leukocytes cytology, Leukocytes metabolism, Nucleic Acid Conformation, RNA, Guide, CRISPR-Cas Systems chemistry, RNA, Guide, CRISPR-Cas Systems metabolism, Algorithms, CRISPR-Associated Protein 9 genetics, CRISPR-Cas Systems, Gene Editing methods, Gene Targeting methods, RNA, Guide, CRISPR-Cas Systems genetics
- Abstract
Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.
- Published
- 2018
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31. Pooled Generation of Lentiviral Tetracycline-Regulated microRNA Embedded Short Hairpin RNA Libraries.
- Author
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Adams FF, Hoffmann T, Zuber J, Heckl D, Schambach A, and Schwarzer A
- Subjects
- Cell Line, Humans, Gene Library, Genetic Vectors genetics, Lentivirus genetics, MicroRNAs genetics, RNA, Small Interfering genetics, Tetracycline
- Abstract
Short hairpin RNA (shRNA) screens are powerful tools to probe genetic dependencies in loss-of-function studies, such as the identification of therapeutic targets in cancer research. Lentivirally delivered shRNAs embedded in endogenous microRNA contexts (shRNAmiRs) mediate efficient long-term suppression of target genes suitable for numerous experimental contexts and clinical applications. Here, an easy-to-use laboratory protocol is described, covering the design and pooled assembly of focused shRNAmiR libraries into an optimized, Tet-inducible all-in-one lentiviral vector, packaging of viral particles, followed by retrieval and quantification of hairpin sequences after cellular DNA-recovery. Starting from a gene list to the identification of hits, the protocol enables shRNA screens within 6 weeks.
- Published
- 2018
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32. An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries.
- Author
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Adams FF, Heckl D, Hoffmann T, Talbot SR, Kloos A, Thol F, Heuser M, Zuber J, Schambach A, and Schwarzer A
- Subjects
- Cell Line, Doxycycline pharmacology, Gene Knockdown Techniques, Humans, Promoter Regions, Genetic, Cloning, Molecular, Genetic Vectors genetics, Lentivirus genetics, MicroRNAs genetics, RNA Interference, RNA, Small Interfering genetics
- Abstract
RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
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33. CRISPR-Cas9-induced t(11;19)/MLL-ENL translocations initiate leukemia in human hematopoietic progenitor cells in vivo .
- Author
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Reimer J, Knöß S, Labuhn M, Charpentier EM, Göhring G, Schlegelberger B, Klusmann JH, and Heckl D
- Subjects
- Cells, Cultured, Chromosomes, Human, Pair 11 metabolism, Chromosomes, Human, Pair 19 metabolism, Hematopoietic Stem Cells pathology, Humans, CRISPR-Cas Systems, Cell Transformation, Neoplastic genetics, Cell Transformation, Neoplastic metabolism, Cell Transformation, Neoplastic pathology, Chromosomes, Human, Pair 11 genetics, Chromosomes, Human, Pair 19 genetics, Hematopoietic Stem Cells metabolism, Leukemia genetics, Leukemia metabolism, Leukemia pathology, Translocation, Genetic
- Abstract
Chromosomal translocations that generate oncogenic fusion proteins are causative for most pediatric leukemias and frequently affect the MLL/KMT2A gene. In vivo modeling of bona fide chromosomal translocations in human hematopoietic stem and progenitor cells is challenging but essential to determine their actual leukemogenic potential. We therefore developed an advanced lentiviral CRISPR-Cas9 vector that efficiently transduced human CD34
+ hematopoietic stem and progenitor cells and induced the t(11;19)/MLL-ENL translocation. Leveraging this system, we could demonstrate that hematopoietic stem and progenitor cells harboring the translocation showed only a transient clonal growth advantage in vitro In contrast, t(11;19)/MLL-ENL-harboring CD34+ hematopoietic stem and progenitor cells not only showed long-term engraftment in primary immunodeficient recipients, but t(11;19)/MLL-ENL also served as a first hit to initiate a monocytic leukemia-like disease. Interestingly, secondary recipients developed acute lymphoblastic leukemia with incomplete penetrance. These findings indicate that environmental cues not only contribute to the disease phenotype, but also to t(11;19)/MLL-ENL-mediated oncogenic transformation itself. Thus, by investigating the true chromosomal t(11;19) rearrangement in its natural genomic context, our study emphasizes the importance of environmental cues for the pathogenesis of pediatric leukemias, opening an avenue for novel treatment options., (Copyright© 2017 Ferrata Storti Foundation.)- Published
- 2017
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34. The non-coding RNA landscape of human hematopoiesis and leukemia.
- Author
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Schwarzer A, Emmrich S, Schmidt F, Beck D, Ng M, Reimer C, Adams FF, Grasedieck S, Witte D, Käbler S, Wong JWH, Shah A, Huang Y, Jammal R, Maroz A, Jongen-Lavrencic M, Schambach A, Kuchenbauer F, Pimanda JE, Reinhardt D, Heckl D, and Klusmann JH
- Subjects
- Cell Lineage, Gene Expression Profiling, HEK293 Cells, Humans, RNA, Long Noncoding physiology, Hematopoiesis, Leukemia metabolism, RNA, Untranslated metabolism
- 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.While micro-RNAs are known regulators of haematopoiesis and leukemogenesis, the role of long non-coding RNAs is less clear. Here the authors provide a non-coding RNA expression landscape of the human hematopoietic system, highlighting their role in the formation and maintenance of the human blood hierarchy.
- Published
- 2017
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35. Scavenger receptor class B member 1 (SCARB1) variants modulate hepatitis C virus replication cycle and viral load.
- Author
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Westhaus S, Deest M, Nguyen ATX, Stanke F, Heckl D, Costa R, Schambach A, Manns MP, Berg T, Vondran FWR, Sarrazin C, Ciesek S, and von Hahn T
- Subjects
- Cell Line, Genetic Variation, Humans, Polymorphism, Single Nucleotide, Viral Envelope Proteins physiology, Viral Load, Virus Internalization, Virus Replication, Hepacivirus physiology, Hepatitis C genetics, Hepatitis C virology, Host-Pathogen Interactions genetics, Host-Pathogen Interactions physiology, Scavenger Receptors, Class B genetics, Scavenger Receptors, Class B physiology
- Abstract
Background & Aims: There are numerous coding and non-coding variants in the SCARB1 gene that encodes scavenger receptor class B member 1 (SR-BI), a key receptor for both high density lipoproteins and hepatitis C virus (HCV). Many have been linked to clinical phenotypes, yet their impact on the HCV replication cycle is incompletely understood. The aim of this study was to analyze the impact of these variants on the molecular biology and clinical course of HCV., Methods: We analyzed key coding non-synonymous as well as non-coding SCARB1 variants using virological in vitro and human genetics approaches., Results: Non-synonymous variants: S112F and T175A have greatly reduced HCV receptor function. When present on the cell surface, these variants are impaired in their ability to interact with HCV E2. Non-coding variants: The G allele in rs3782287 is associated with decreased viral load. Haplotype analysis confirmed these findings and identified haplotype rs3782287 A/rs5888 C as a risk allele associated with increased viral load. We also detected a trend towards lower hepatic SR-BI expression in individuals with the rs3782287 GG genotype associated with low viral load suggesting a potential underlying mechanism., Conclusion: Coding and non-coding genetic SCARB1 variants modulate the HCV replication cycle and possibly clinical features of hepatitis C. These findings underscore the relevance of SR-BI as an HCV receptor and contribute to our understanding of inter-individual variation in HCV infection., Lay Summary: The cell surface receptor SR-BI (scavenger receptor class B member 1), is essential for hepatitis C virus (HCV) entry into hepatocytes. Variations in the gene coding this receptor influence infectivity and viral load. We analyzed these variations to gain a better understanding of inter-individual differences over the course of HCV infection., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)
- Published
- 2017
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36. Gene correction of HAX1 reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells.
- Author
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Pittermann E, Lachmann N, MacLean G, Emmrich S, Ackermann M, Göhring G, Schlegelberger B, Welte K, Schambach A, Heckl D, Orkin SH, Cantz T, and Klusmann JH
- Abstract
Severe congenital neutropenia (SCN, Kostmann disease) is a heritable disorder characterized by a granulocytic maturation arrest. Biallelic mutations in HCLS1 associated protein X-1 ( HAX1 ) are frequently detected in affected individuals, including those of the original pedigree described by Kostmann in 1956. To date, no faithful animal model has been established to study SCN mediated by HAX1 deficiency. Here we demonstrate defective neutrophilic differentiation and compensatory monocyte overproduction from patient-derived induced pluripotent stem cells (iPSCs) carrying the homozygous HAX1
W44X nonsense mutation. Targeted correction of the HAX1 mutation using the CRISPR-Cas9 system and homologous recombination rescued neutrophil differentiation and reestablished an HAX1 and HCLS1 -centered transcription network in immature myeloid progenitors, which is involved in the regulation of apoptosis, apoptotic mitochondrial changes, and myeloid differentiation. These findings made in isogenic iPSC-derived myeloid cells highlight the complex transcriptional changes underlying Kostmann disease. Thus, we show that patient-derived HAX1W44X -iPSCs recapitulate the Kostmann disease phenotype in vitro and confirm HAX1 mutations as the disease-causing monogenic lesion. Finally, our study paves the way for nonvirus-based gene therapy approaches in SCN., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.- Published
- 2017
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37. Gli1 + Mesenchymal Stromal Cells Are a Key Driver of Bone Marrow Fibrosis and an Important Cellular Therapeutic Target.
- Author
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Schneider RK, Mullally A, Dugourd A, Peisker F, Hoogenboezem R, Van Strien PMH, Bindels EM, Heckl D, Büsche G, Fleck D, Müller-Newen G, Wongboonsin J, Ventura Ferreira M, Puelles VG, Saez-Rodriguez J, Ebert BL, Humphreys BD, and Kramann R
- Subjects
- Animals, Cell Differentiation genetics, Humans, Mesenchymal Stem Cells pathology, Mice, Mice, Transgenic, Myofibroblasts pathology, Primary Myelofibrosis genetics, Primary Myelofibrosis metabolism, Primary Myelofibrosis pathology, Zinc Finger Protein GLI1 genetics, Zinc Finger Protein GLI1 metabolism, Cell Differentiation drug effects, Cell Proliferation drug effects, Mesenchymal Stem Cells metabolism, Myofibroblasts metabolism, Primary Myelofibrosis drug therapy, Pyridines pharmacology, Pyrimidines pharmacology, Zinc Finger Protein GLI1 antagonists & inhibitors
- Abstract
Bone marrow fibrosis (BMF) develops in various hematological and non-hematological conditions and is a central pathological feature of myelofibrosis. Effective cell-targeted therapeutics are needed, but the cellular origin of BMF remains elusive. Here, we show using genetic fate tracing in two murine models of BMF that Gli1
+ mesenchymal stromal cells (MSCs) are recruited from the endosteal and perivascular niche to become fibrosis-driving myofibroblasts in the bone marrow. Genetic ablation of Gli1+ cells abolished BMF and rescued bone marrow failure. Pharmacological targeting of Gli proteins with GANT61 inhibited Gli1+ cell expansion and myofibroblast differentiation and attenuated fibrosis severity. The same pathway is also active in human BMF, and Gli1 expression in BMF significantly correlates with the severity of the disease. In addition, GANT61 treatment reduced the myofibroblastic phenotype of human MSCs isolated from patients with BMF, suggesting that targeting of Gli proteins could be a relevant therapeutic strategy., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
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38. Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability.
- Author
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Paolella BR, Gibson WJ, Urbanski LM, Alberta JA, Zack TI, Bandopadhayay P, Nichols CA, Agarwalla PK, Brown MS, Lamothe R, Yu Y, Choi PS, Obeng EA, Heckl D, Wei G, Wang B, Tsherniak A, Vazquez F, Weir BA, Root DE, Cowley GS, Buhrlage SJ, Stiles CD, Ebert BL, Hahn WC, Reed R, and Beroukhim R
- Subjects
- Cell Line, Tumor, Humans, Gene Dosage, Neoplasms genetics, Neoplasms pathology, Phosphoproteins genetics, RNA Splicing Factors genetics
- Abstract
Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent. One of these, the pre-mRNA splicing factor SF3B1 , is also frequently mutated in cancer. We validated SF3B1 as a CYCLOPS gene and found that human cancer cells harboring partial SF3B1 copy-loss lack a reservoir of SF3b complex that protects cells with normal SF3B1 copy number from cell death upon partial SF3B1 suppression. These data provide a catalog of copy-number associated gene dependencies and identify partial copy-loss of wild-type SF3B1 as a novel, non-driver cancer gene dependency.
- Published
- 2017
- Full Text
- View/download PDF
39. Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.
- Author
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Labenski V, Suerth JD, Barczak E, Heckl D, Levy C, Bernadin O, Charpentier E, Williams DA, Fehse B, Verhoeyen E, and Schambach A
- Subjects
- Base Sequence, CRISPR-Cas Systems genetics, Clone Cells, Genes, Reporter, HEK293 Cells, Humans, Jurkat Cells, Reproducibility of Results, T-Lymphocytes immunology, Transduction, Genetic, Transgenes, Alpharetrovirus metabolism, Genetic Techniques, Genetic Vectors metabolism, T-Lymphocytes metabolism, Virus Assembly
- Abstract
Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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40. Multiple genetically engineered humanized microenvironments in a single mouse.
- Author
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Lee J, Heckl D, and Parekkadan B
- Abstract
Background: Immunodeficient mouse models that accept human cell and tissue grafts can contribute greater knowledge to human stem cell research. In this technical report, we used biomaterial implants seeded with genetically engineered stromal cells to create several unique microenvironments in a single mouse. The scope of study was focused on human CD34 hematopoietic stem/progenitor cell (HSPC) engraftment and differentiation within the engineered microenvironment., Results: A mouse model system was created using subdermal implant sites that overexpressed a specific human cytokines (Vascular Endothelial Growth Factor A (hVEGFa), Stromal Derived Factor 1 Alpha (hSDF1a), or Tumor Necrosis Factor Alpha (hTNFa)) by stromal cells in a three-dimensional biomaterial matrix. The systemic exposure of locally overexpressed cytokines was minimized by controlling the growth of stromal cells, which led to autonomous local, concentrated sites in a single mouse for study. This biomaterial implant approach allowed for the local analysis of each cytokine on hematopoietic stem cell recruitment, engraftment and differentiation in four different tissue microenvironments in the same host. The engineered factors were validated to have bioactive effects on human CD34+ hematopoietic progenitor cell differentiation., Conclusions: This model system can serve as a new platform for the study of multiple human proteins and their local effects on hematopoietic cell biology for in vivo validation studies.
- Published
- 2016
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41. Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML.
- Author
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Puram RV, Kowalczyk MS, de Boer CG, Schneider RK, Miller PG, McConkey M, Tothova Z, Tejero H, Heckl D, Järås M, Chen MC, Li H, Tamayo A, Cowley GS, Rozenblatt-Rosen O, Al-Shahrour F, Regev A, and Ebert BL
- Subjects
- Animals, Circadian Rhythm, Disease Models, Animal, Gene Knockout Techniques, Hematopoiesis, Humans, Leukemia, Myeloid, Acute metabolism, Mice, Mice, Inbred C57BL, Neoplastic Stem Cells metabolism, RNA Interference, RNA, Small Interfering metabolism, ARNTL Transcription Factors genetics, CLOCK Proteins genetics, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute pathology, Neoplastic Stem Cells pathology
- Abstract
Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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42. Efficient generation of gene-modified human natural killer cells via alpharetroviral vectors.
- Author
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Suerth JD, Morgan MA, Kloess S, Heckl D, Neudörfl C, Falk CS, Koehl U, and Schambach A
- Subjects
- Cell Line, Tumor, Cytotoxicity, Immunologic immunology, Genetic Therapy methods, Green Fluorescent Proteins genetics, Humans, Immunotherapy, Adoptive methods, Killer Cells, Natural cytology, Leukemia immunology, Receptors, Antigen biosynthesis, Receptors, Antigen immunology, Transduction, Genetic methods, Tumor Escape immunology, Alpharetrovirus genetics, Antigens, CD19 genetics, Cytotoxicity, Immunologic genetics, Genetic Vectors genetics, Killer Cells, Natural immunology, Leukemia therapy, Receptors, Antigen genetics
- Abstract
Unlabelled: Natural killer (NK) cells play an important role in tumor immunotherapy with their unique capability of killing transformed cells without the need for prior sensitization and without major histocompatibility complex (MHC)/peptide restriction. However, tumor cells can escape NK cell cytotoxicity by various tumor immune escape mechanisms. To overcome these escape mechanisms, NK cells can be modified to express chimeric antigen receptors (CARs), enhancing their tumor-specific cytotoxicity. To determine the most efficacious method to modify human NK cells, we compared different retroviral vector systems, retroviral pseudotypes, and transduction protocols. Using optimized transduction conditions, the highest transduction levels (up to 60%) were achieved with alpharetroviral vectors. Alpharetroviral-modified primary human NK cells exhibited no alteration in receptor expression and had similar degranulation activity as untransduced NK cells, thus demonstrating that alpharetroviral modification did not negatively affect NK cell cytotoxicity. Transduction of NK cells with an alpharetroviral vector containing a CD19 CAR expression cassette selectively enhanced NK cell cytotoxicity towards CD19-expressing leukemia cells, achieving nearly complete elimination of leukemia cells after 48 h. Taken together, alpharetroviral vectors are promising tools for NK cell-mediated cancer immunotherapy applications., Key Messages: Efficient modification of human NK cells using alpharetroviral vectors. Anti-CD19-CAR-NK cells exhibited improved cytotoxicity towards CD19(+) leukemia cells. Alpharetroviral vectors are promising tools for immunotherapy applications using NK cells.
- Published
- 2016
- Full Text
- View/download PDF
43. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells.
- Author
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Kowalczyk MS, Tirosh I, Heckl D, Rao TN, Dixit A, Haas BJ, Schneider RK, Wagers AJ, Ebert BL, and Regev A
- Subjects
- Age Factors, Animals, Biomarkers, Cluster Analysis, Computational Biology methods, Female, Gene Expression Profiling, High-Throughput Nucleotide Sequencing, Mice, Models, Biological, Multipotent Stem Cells cytology, Multipotent Stem Cells metabolism, Organ Specificity genetics, Phenotype, Sequence Analysis, RNA, Single-Cell Analysis, Transcription, Genetic, Transcriptome, Cell Cycle genetics, Cell Differentiation genetics, Cellular Senescence genetics, Gene Expression Regulation, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism
- Abstract
Both intrinsic cell state changes and variations in the composition of stem cell populations have been implicated as contributors to aging. We used single-cell RNA-seq to dissect variability in hematopoietic stem cell (HSC) and hematopoietic progenitor cell populations from young and old mice from two strains. We found that cell cycle dominates the variability within each population and that there is a lower frequency of cells in the G1 phase among old compared with young long-term HSCs, suggesting that they traverse through G1 faster. Moreover, transcriptional changes in HSCs during aging are inversely related to those upon HSC differentiation, such that old short-term (ST) HSCs resemble young long-term (LT-HSCs), suggesting that they exist in a less differentiated state. Our results indicate both compositional changes and intrinsic, population-wide changes with age and are consistent with a model where a relationship between cell cycle progression and self-renewal versus differentiation of HSCs is affected by aging and may contribute to the functional decline of old HSCs., (© 2015 Kowalczyk et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2015
- Full Text
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44. Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis.
- Author
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Kramann R, Fleig SV, Schneider RK, Fabian SL, DiRocco DP, Maarouf O, Wongboonsin J, Ikeda Y, Heckl D, Chang SL, Rennke HG, Waikar SS, and Humphreys BD
- Subjects
- Animals, Cell Cycle Checkpoints genetics, Cell Line, Fibrosis, Glutathione pharmacology, Humans, Kidney pathology, Kidney Diseases genetics, Kidney Diseases metabolism, Kidney Diseases pathology, Kruppel-Like Transcription Factors genetics, Kruppel-Like Transcription Factors metabolism, Mice, Mice, Knockout, Myofibroblasts pathology, Zinc Finger Protein GLI1, Zinc Finger Protein Gli2, Arsenicals pharmacology, Cell Cycle Checkpoints drug effects, Glutathione analogs & derivatives, Kidney metabolism, Kidney Diseases drug therapy, Kruppel-Like Transcription Factors antagonists & inhibitors, Myofibroblasts metabolism, Pyridines pharmacology, Pyrimidines pharmacology
- Abstract
Chronic kidney disease is characterized by interstitial fibrosis and proliferation of scar-secreting myofibroblasts, ultimately leading to end-stage renal disease. The hedgehog (Hh) pathway transcriptional effectors GLI1 and GLI2 are expressed in myofibroblast progenitors; however, the role of these effectors during fibrogenesis is poorly understood. Here, we demonstrated that GLI2, but not GLI1, drives myofibroblast cell-cycle progression in cultured mesenchymal stem cell-like progenitors. In animals exposed to unilateral ureteral obstruction, Hh pathway suppression by expression of the GLI3 repressor in GLI1+ myofibroblast progenitors limited kidney fibrosis. Myofibroblast-specific deletion of Gli2, but not Gli1, also limited kidney fibrosis, and induction of myofibroblast-specific cell-cycle arrest mediated this inhibition. Pharmacologic targeting of this pathway with darinaparsin, an arsenical in clinical trials, reduced fibrosis through reduction of GLI2 protein levels and subsequent cell-cycle arrest in myofibroblasts. GLI2 overexpression rescued the cell-cycle effect of darinaparsin in vitro. While darinaparsin ameliorated fibrosis in WT and Gli1-KO mice, it was not effective in conditional Gli2-KO mice, supporting GLI2 as a direct darinaparsin target. The GLI inhibitor GANT61 also reduced fibrosis in mice. Finally, GLI1 and GLI2 were upregulated in the kidneys of patients with high-grade fibrosis. Together, these data indicate that GLI inhibition has potential as a therapeutic strategy to limit myofibroblast proliferation in kidney fibrosis.
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- 2015
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45. Toward Whole-Transcriptome Editing with CRISPR-Cas9.
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Heckl D and Charpentier E
- Subjects
- Humans, CRISPR-Cas Systems genetics, Gene Expression, Genetic Engineering methods, Genetic Techniques, Genome, Human genetics, Melanoma genetics, Transcription, Genetic, Transcriptional Activation genetics
- Abstract
Targeted regulation of gene expression holds huge promise for biomedical research. In a series of recent publications (Gilbert et al., 2014; Konermann et al., 2015; Zalatan et al., 2015), sophisticated, multiplex-compatible transcriptional activator systems based on the CRISPR-Cas9 technology and genome-scale libraries advance the field toward whole-transcriptome control., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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46. Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS.
- Author
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Schneider RK, Ademà V, Heckl D, Järås M, Mallo M, Lord AM, Chu LP, McConkey ME, Kramann R, Mullally A, Bejar R, Solé F, and Ebert BL
- Subjects
- Aged, Animals, Base Sequence, Casein Kinase I genetics, DNA Primers, Female, Flow Cytometry, Haploinsufficiency, Humans, Male, Mice, Mutation, Polymerase Chain Reaction, Young Adult, Casein Kinase I metabolism, Chromosome Deletion, Chromosomes, Human, Pair 5, Myelodysplastic Syndromes genetics
- Abstract
The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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47. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing.
- Author
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Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, Thielke A, Aster JC, Regev A, and Ebert BL
- Subjects
- Animals, Disease Models, Animal, Mice, Bone Marrow Neoplasms genetics, Clustered Regularly Interspaced Short Palindromic Repeats genetics
- Abstract
Genome sequencing studies have shown that human malignancies often bear mutations in four or more driver genes, but it is difficult to recapitulate this degree of genetic complexity in mouse models using conventional breeding. Here we use the CRISPR-Cas9 system of genome editing to overcome this limitation. By delivering combinations of small guide RNAs (sgRNAs) and Cas9 with a lentiviral vector, we modified up to five genes in a single mouse hematopoietic stem cell (HSC), leading to clonal outgrowth and myeloid malignancy. We thereby generated models of acute myeloid leukemia (AML) with cooperating mutations in genes encoding epigenetic modifiers, transcription factors and mediators of cytokine signaling, recapitulating the combinations of mutations observed in patients. Our results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a broad array of in vivo cancer models that better reflect the complexity of human disease.
- Published
- 2014
- Full Text
- View/download PDF
48. Ectopic expression of HOXC6 blocks myeloid differentiation and predisposes to malignant transformation.
- Author
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Wurm M, Kowalski J, Heckl D, Zhang XB, Nelson V, Beard BC, and Kiem HP
- Subjects
- Animals, Hematopoietic Stem Cell Transplantation, Mice, Oligonucleotide Array Sequence Analysis, Bone Marrow pathology, Cell Differentiation genetics, Cell Transformation, Neoplastic, Homeodomain Proteins genetics
- Abstract
Insertional mutagenesis resulting from the integration of retroviral vectors has led to the discovery of many oncogenes associated with leukemia. We investigated the role of HOXC6, identified by proximal provirus integration in a large animal hematopoietic stem cell gene therapy study, for a potential involvement in hematopoietic stem cell activity and hematopoietic cell fate decision. HOXC6 was overexpressed in the murine bone marrow transplantation model and tested in a competitive repopulation assay in comparison to the known hematopoietic stem cell expansion factor, HOXB4. We have identified HOXC6 as a factor that enhances competitive repopulation capacity in vivo and colony formation in vitro. Ectopic HOXC6 expression also induced strong myeloid differentiation and expansion of granulocyte-macrophage progenitors/common myeloid progenitors (GMPs/CMPs) in vivo, resulting in myeloid malignancies with low penetrance (3 of 17 mice), likely in collaboration with Meis1 because of a provirus integration mapped to the 3' region in the malignant clone. We characterized the molecular basis of HOXC6-induced myeloid differentiation and malignant cell transformation with complementary DNA microarray analysis. Overexpression of HOXC6 induced a gene expression signature similar to several acute myeloid leukemia subtypes when compared with normal GMPs/CMPs. These results demonstrate that HOXC6 acts as a regulator in hematopoiesis and is involved in malignant transformation., (Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
49. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells.
- Author
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Krönke J, Udeshi ND, Narla A, Grauman P, Hurst SN, McConkey M, Svinkina T, Heckl D, Comer E, Li X, Ciarlo C, Hartman E, Munshi N, Schenone M, Schreiber SL, Carr SA, and Ebert BL
- Subjects
- Cell Line, Tumor, HEK293 Cells, Humans, Ikaros Transcription Factor genetics, Interleukin-2 biosynthesis, Lenalidomide, Proteolysis, T-Lymphocytes drug effects, T-Lymphocytes metabolism, Thalidomide pharmacology, Ubiquitination, Antineoplastic Agents pharmacology, Ikaros Transcription Factor metabolism, Multiple Myeloma metabolism, Thalidomide analogs & derivatives
- Abstract
Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.
- Published
- 2014
- Full Text
- View/download PDF
50. Genome-scale CRISPR-Cas9 knockout screening in human cells.
- Author
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Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, and Zhang F
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Cullin Proteins genetics, Gene Knockout Techniques, Gene Library, Genes, Neurofibromatosis 1, Genes, Neurofibromatosis 2, Genetic Loci, Genome-Wide Association Study, Humans, Indoles therapeutic use, Lentivirus, Mediator Complex genetics, Melanoma drug therapy, Protein Kinase Inhibitors therapeutic use, Selection, Genetic, Sulfonamides therapeutic use, Transcription Factors genetics, Vemurafenib, raf Kinases antagonists & inhibitors, Cell Survival genetics, Clustered Regularly Interspaced Short Palindromic Repeats, Drug Resistance, Neoplasm genetics, Genetic Testing methods, Melanoma genetics, Pluripotent Stem Cells metabolism
- Abstract
The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
- Published
- 2014
- Full Text
- View/download PDF
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