| 1. |
- Neddermeyer, Anne H.
(författare)
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Identification and Functional Significance of Aberrant Long Non-coding RNAs in Acute Myeloid Leukemia : Biological and Prognostic Implications
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Acute myeloid leukemia (AML) is the most frequently diagnosed type of acute leukemia in adults. It commonly affects people aged 60 or older, as incidence increases with age, and it is characterized by the accumulation of immature hematopoietic progenitor cells in the bone marrow. Despite recent treatment advances and improvements for certain subtypes, as acute promyelocyte leukemia (APL), AML remains difficult to cure. While many patients reach remission after induction treatment, relapses are common and 5-year overall survival remains dismal. Long non-coding RNAs (lncRNAs) are involved in various regulatory cellular functions and, like coding genes, they are frequently dysregulated in cancer. In this thesis, the aim was to elucidate the functional implications of lncRNAs in the biology and treatment response of AML and normal hematopoiesis in order to improve understanding of AML pathology. In Paper I, whole-transcriptome sequencing identified the novel lncRNA MALNC. Clinical correlation analyses and CRISPR-knockout cell models were used to functionally explore its implications in AML. It was identified that enhanced MALNC expression is specifically associated with the AML-subtypes APL and AML with co-mutant NPM1/IDH2R140. Further, it was shown that MALNC is implicated in key factors of leukemogenesis, like differentiation and proliferation, and that MALNC expression associates with better overall survival in AML patients. Moreover, knockout of the MALNC gene sensitized AML cells to arsenic trioxide (ATO), all-trans retinoic acid (ATRA)-ATO combination and venetoclax treatment. In Paper II, three high-throughput functional CRISPR interference screens were performed to identify lncRNAs implicated in proliferation, differentiation or venetoclax response. Several novel lncRNAs were identified to potentially play a positive or negative role in these processes and furthermore were found to implicate AML prognosis. In Paper III, the lncRNA NEAT1 was studied in respect to its role in normal hematopoiesis and AML using CAGE- and RADICL-sequencing. It could be illustrated that NEAT1 expression positively correlates with cell maturity during normal hematopoiesis, in particular monocytes, and associates with core-binding factor AML inv(16) and t(8;21). Further, RADICL-sequencing identified that lncRNA NEAT1 binds to the genomic loci of key hematopoietic transcription factor RUNX2. In contrast to solid cancers, it was demonstrated, that higher NEAT1 expression correlated with better outcome in AML, independent of known risk factors. In summary, these studies have outlined the scope of functional implications of lncRNAs in normal and dysregulated hematopoiesis and have highlighted their potential roles as biomarkers for prognosis and drug sensitivity. These findings support the efforts to understand how lncRNAs could serve as novel biomarkers for personalized treatment.
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| 2. |
- Björnsson, Jon Mar, et al.
(författare)
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Reduced proliferative capacity of hematopoietic stem cells deficient in hoxb3 and hoxb4
- 2003
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Ingår i: Blood. - 0006-4971 .- 1528-0020. ; 23:11, s. 3872-3883
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Tidskriftsartikel (refereegranskat)abstract
- Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin(-) Scal(+) c-kit(+) hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response.
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| 3. |
- Schmiderer, Ludwig, et al.
(författare)
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Efficient and nontoxic biomolecule delivery to primary human hematopoietic stem cells using nanostraws
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 117:35, s. 21267-21273
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Tidskriftsartikel (refereegranskat)abstract
- Introduction of exogenous genetic material into primary stem cells is essential for studying biological function and for clinical applications. Traditional delivery methods for nucleic acids, such as electroporation, have advanced the field, but have negative effects on stem cell function and viability. We introduce nanostraw-assisted transfection as an alternative method for RNA delivery to human hematopoietic stem and progenitor cells (HSPCs). Nanostraws are hollow alumina nanotubes that can be used to deliver biomolecules to living cells. We use nanostraws to target human primary HSPCs and show efficient delivery of mRNA, short interfering RNAs (siRNAs), DNA oligonucleotides, and dextrans of sizes ranging from 6 kDa to 2,000 kDa. Nanostraw-treated cells were fully functional and viable, with no impairment in their proliferative or colony-forming capacity, and showed similar long-term engraftment potential in vivo as untreated cells. Additionally, we found that gene expression of the cells was not perturbed by nanostraw treatment, while conventional electroporation changed the expression of more than 2,000 genes. Our results show that nanostraw-mediated transfection is a gentle alternative to established gene delivery methods, and uniquely suited for nonperturbative treatment of sensitive primary stem cells.
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| 4. |
- Wong, Wan Man, et al.
(författare)
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Expression of Integrin alpha 2 Receptor in Human Cord Blood CD34+CD38-CD90+Stem Cells Engrafting Long-Term in NOD/SCID-IL2R gamma(c)null Mice
- 2013
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Ingår i: Stem Cells. - : AlphaMed Press. - 1066-5099 .- 1549-4918. ; 31:2, s. 360-371
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Tidskriftsartikel (refereegranskat)abstract
- Human hematopoietic stem cells reside in the CD34+CD38-CD90+ population in cord blood and bone marrow. However, this cell fraction is heterogeneous, and the phenotype of the rare primitive stem cells remains poorly defined. We here report that primitive cord blood CD34+CD38-CD90+ stem cells, with the ability to reconstitute NOD/SCID-IL2R gamma(c)null (NSG) mice long-term, at 24 weeks after transplantation, can be prospectively isolated at an increased purity by using integrin alpha 2 receptor as an additional stem cell marker. Using a limiting dilution transplantation assay, we found a highly significant enrichment of multilineage reconstituting stem cells in the CD34+CD38-CD90+ cell fraction expressing the integrin alpha 2 receptor, with a frequency of 1/29 cells, as compared to a frequency of 1/157 in the corresponding integrin alpha 2- cells. In line with this, long-term reconstituting stem cells within the cord blood CD34+CD38- cell population were significantly enriched in the integrin alpha 2+ fraction, while stem cells and progenitors reconstituting short-term, at 8-12 weeks, were heterogeneous in integrin alpha 2 expression. Global gene expression profiling revealed that the lineage-marker negative (Lin-) CD34+CD38-CD90+CD45RA- integrin alpha 2+ cell population was molecularly distinct from the integrin alpha 2- cell population and the more mature Lin-CD34+CD38-CD90-CD45RA- cell population. Our findings identify integrin alpha 2 as a novel stem cell marker, which improves prospective isolation of the primitive human hematopoietic stem cells within the CD34+CD38-CD90+ cell population for experimental and therapeutic stem cell applications. STEM CELLS 2013;31:360-371
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| 5. |
- Magnusson, Mattias, et al.
(författare)
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HOXA10 is a critical regulator for hematopoietic stem cells and erythroid/megakaryocyte development
- 2007
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 109:9, s. 3687-3696
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Tidskriftsartikel (refereegranskat)abstract
- The Homeobox (Hox) transcription factors are important regulators of normal and malignant hematopoiesis because they control proliferation, differentiation, and self-renewal of hematopoietic cells at different levels of the hematopoietic hierarchy. In transgenic mice we show that the expression of HOXA10 is tightly regulated by doxycycline. Intermediate concentrations of HOXA10 induced a 15-fold increase in the repopulating capacity of hematopoietic stem cells (HSCs) after 13 days of in vitro culture. Notably, the proliferation induction of HSC by HOXA10 was dependent on the HOXA10 concentration, because high levels of HOXA10 had no effect on HSC proliferation. Furthermore, high levels of HOXA10 blocked erythroid and megakaryocyte development, demonstrating that tight regulation of HOXA10 is critical for normal development of the erythroid and megakaryocytic lineages. The HOXA10-mediated effects on hematopoietic cells were associated with altered expression of genes that govern stem-cell self-renewal and lineage commitment (eg, hepatic leukemia factor [HlF], Dickkopf-1 [Dkk-1], growth factor independent-1 [Gfi-1], and Gata-1). Interestingly, binding sites for HOXA10 were found in HLF, Dkk-1, and Gata-1, and Dkk-1 and Gfi-1 were transcriptionally activated by HOXA10. These findings reveal novel molecular pathways that act downstream of HOXA10 and identify HOXA10 as a master regulator of postnatal hematopoietic development.
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| 6. |
- Karlsson, Göran, et al.
(författare)
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Gene expression profiling demonstrates that TGF-{beta}1 signals exclusively through receptor complexes involving Alk5 and identifies targets of TGF-{beta} signaling.
- 2005
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Ingår i: Physiological Genomics. - : American Physiological Society. - 1094-8341 .- 1531-2267. ; 21:3, s. 396-403
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-β 1 (TGF-β) regulates cellular functions like proliferation, differentiation, and apoptosis. On the cell surface, TGF-β binds to receptor complexes consisting of TGF-β receptor type II (Tβ RII) and activin-like kinase receptor-5 (Alk5), and the downstream signaling is transduced by Smad and MAPK proteins. Recent data have shown that alternative receptor combinations aside from the classical pairing of Tβ RII/Alk5 can be relevant for TGF-β signaling. We have screened for alternative receptors for TGF-β and also for gene targets of TGF-β signaling, by performing functional assays and microarray analysis in murine embryonic fibroblast (MEF) cell lines lacking Alk5. Data from TGF-β-stimulated Alk5(-/-) cells show them to be completely unaffected by TGF-β. Additionally, 465 downstream targets of Alk5 signaling were identified when comparing Alk5(-/-) or TGF-β-stimulated Alk5(+/+) MEFs with unstimulated Alk5(+/+) cells. Our results demonstrate that, in MEFs, TGF-β signals exclusively through complexes involving Alk5, and give insight to its downstream effector genes.
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| 7. |
- Lopez de Lapuente Portilla, Aitzkoa, et al.
(författare)
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Genome-wide association study on 13,167 individuals identifies regulators of hematopoietic stem and progenitor cell levels in human blood
- 2021
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Understanding how hematopoietic stem and progenitor cells (HSPCs) are regulated is of central importance for the development of new therapies for blood disorders and stem cell transplantation. To date, HSPC regulation has been extensively studied in vitro and in animal models, but less is known about the mechanisms in vivo in humans. Here, in a genome-wide association study on 13,167 individuals, we identify 9 significant and 2 suggestive DNA sequence variants that influence HSPC (CD34+) levels in human blood. The identified loci associate with blood disorders, harbor known and novel HSPC genes, and affect gene expression in HSPCs. Interestingly, our strongest association maps to the PPM1H gene, encoding an evolutionarily conserved serine/threonine phosphatase never previously implicated in stem cell biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD34+ cell levels downregulates PPM1H. By functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates a MYB transcription factor binding site in PPM1H intron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, rs772557-A selectively increases HSPC subpopulations in which the MYB site is active, and PPM1H shRNA- knockdown increased CD34+ and CD34+90+ cell proportions in umbilical cord blood cultures. Our findings represent the first large-scale association study on a stem cell trait, illuminating HSPC regulation in vivo in humans, and identifying PPM1H as a novel inhibition target that can potentially be utilized clinically to facilitate stem cell harvesting for transplantation.
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| 8. |
- Chapellier, Marion, et al.
(författare)
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Arrayed molecular barcoding identifies TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells
- 2019
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 104:10, s. 2006-2016
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Tidskriftsartikel (refereegranskat)abstract
- Dysregulation of cytokines in the bone marrow microenvironment promotes acute myeloid leukemia cell growth. Due to the complexity and low throughput of in vivo stem-cell based assays, studying the role of cytokines in the bone marrow niche in a screening setting is challenging. Herein, we developed an ex vivo cytokine screen using 11 arrayed molecular barcodes, allowing for a competitive in vivo readout of leukemia-initiating capacity. With this approach, we assessed the effect of 114 murine cytokines on MLL-AF9 acute myeloid leukemia mouse cells and identified the tumor necrosis factor ligand superfamily member 13 (TNFSF13) as a positive regulator of leukemia-initiating cells. By using Tnfsf13-/- recipient mice, we confirmed that TNFSF13 supports leukemia-initiation also under physiological conditions. TNFSF13 was secreted by normal myeloid cells but not by leukemia mouse cells, suggesting that mature myeloid bone marrow cells support leukemia cells by secreting TNFSF13. TNFSF13 supported leukemia cell proliferation in an NF-κB-dependent manner by binding TNFRSF17 and suppressed apoptosis. Moreover, TNFSF13 supported the growth and survival of several human myeloid leukemia cell lines, demonstrating that our findings translate to human disease. Taken together, using arrayed molecular barcoding, we identified a previously unrecognized role of TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells. The arrayed barcoded screening methodology is not limited to cytokines and leukemia, but can be extended to other types of ex vivo screens, where a multiplexed in vivo read-out of stem cell functionality is needed.
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| 9. |
- Galeev, Roman, et al.
(författare)
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Cohesin in haematopoiesis and leukaemia
- 2018
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Ingår i: Current Opinion in Hematology. - 1065-6251. ; 25:4, s. 259-265
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Forskningsöversikt (refereegranskat)abstract
- Purpose of review Disturbance of the delicate balance between self-renewal and differentiation in haematopoietic stem cells (HSCs) can lead to both leukaemia and bone marrow failure. The regulation of this balance in HSC biology has been intensely investigated in several model systems, and lately the importance of epigenetic modifications as well as the organization and architecture of chromatin has become increasingly recognized. In this review, we will focus on the role of the chromatin organizing protein complex cohesin in regulation of normal and malignant haematopoiesis. Recent findings Several functional studies in both mouse and human systems have implicated cohesin as a critical regulator of self-renewal and differentiation in HSCs. Together with the discovery of recurrent mutations of cohesin genes in myeloid malignancies, this points towards a direct role of perturbed cohesin function in leukemogenesis. Summary The work reviewed here provides new insights about the role of the cohesin complex and chromatin architecture in normal and malignant HSCs, and indicates how cohesin may be specifically targeted for therapeutic benefit in the future.
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| 10. |
- Komorowska, Karolina, et al.
(författare)
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Hepatic Leukemia Factor Maintains Quiescence of Hematopoietic Stem Cells and Protects the Stem Cell Pool during Regeneration
- 2017
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 21:12, s. 3514-3523
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Tidskriftsartikel (refereegranskat)abstract
- The transcription factor hepatic leukemia factor (HLF) is strongly expressed in hematopoietic stem cells (HSCs) and is thought to influence both HSC self-renewal and leukemogenesis. However, the physiological role of HLF in hematopoiesis and HSC function is unclear. Here, we report that mice lacking Hlf are viable with essentially normal hematopoietic parameters, including an intact HSC pool during steady-state hematopoiesis. In contrast, when challenged through transplantation, Hlf-deficient HSCs showed an impaired ability to reconstitute hematopoiesis and became gradually exhausted upon serial transplantation. Transcriptional profiling of Hlf-deficient HSCs revealed changes associated with enhanced cellular activation, and cell-cycle analysis demonstrated a significant reduction of quiescent HSCs. Accordingly, toxic insults targeting dividing cells completely eradicated the HSC pool in Hlf-deficient mice. In summary, our findings point to HLF as a critical regulator of HSC quiescence and as an essential factor for maintaining the HSC pool during regeneration. Komorowska et al. report that the transcription factor HLF is required to maintain hematopoietic stem cell (HSC) function during regeneration. Moreover, Hlf-deficient HSCs are less quiescent. In accordance with this, toxic insults targeting dividing cells completely eradicate the HSC pool in Hlf-deficient mice.
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