| 1. |
- Alzrigat, Mohammad
(författare)
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Targeted Inhibition of Polycomb Repressive Complexes in Multiple Myeloma : Implications for Biology and Therapy
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Multiple myeloma (MM) is a hematological malignancy of antibody producing plasmablasts/plasma cells. MM is characterized by extensive genetic and clonal heterogeneity, which have hampered the attempts to identify a common underlying mechanism for disease establishment and development of appropriate treatment regimes. This thesis is focused on understanding the role of epigenetic regulation of gene expression mediated by the polycomb repressive complexes 1 and 2 (PRC1 and 2) in MM and their impact on disease biology and therapy.In paper I the genome-wide distribution of two histone methylation marks; H3K27me3 and H3K4me3 were studied in plasma cells isolated from newly diagnosed MM patients or age-matched normal donors. We were able to define targets of H3K27me3, H3K4me3 and bivalent (carry both marks) which are, when compared to normal individuals, unique to MM patients. The presence of H3K27me3 correlated with silencing of MM unique H3K27me3 targets in MM patients at advanced stages of the disease. Notably, the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also showed that inhibition of the PRC2 enzymatic subunit EZH2 using highly selective inhibitors (GSK343 and UNC1999) demonstrated anti-myeloma activity using relevant in vitro models of MM. These data suggest an important role for gene repression mediated by PRC2 in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM.In paper II we further explored the therapeutic potential of UNC1999, a highly selective inhibitor of EZH2 in MM. We showed that EZH2 inhibition by UNC1999 downregulated important MM oncogenes; IRF-4, XBP-1, BLIMP-1and c-MYC. These oncogenes have been previously shown to be crucial for disease establishment, growth and progression. We found that EZH2 inhibition reactivated the expression of microRNAs genes previously found to be underexpressed in MM and which possess potential tumor suppressor functions. Among the reactivated microRNAs we identified miR-125a-3p and miR-320c as predicted negative regulators of the MM-associated oncogenes. Notably, we defined miR-125a-3p and miR-320c as targets of EZH2 and H3K27me3 in MM cell lines and patients samples. These findings described for the first time PRC2/EZH2/H3K27me3 as regulators of microRNA with tumor suppressor functions in MM. This further strengthens the oncogenic features of EZH2 and its potential as a therapeutic target in MM.In paper III we evaluated the therapeutic potential of targeting PRC1 in MM using the recently developed chemical PTC-209; an inhibitor targeting the BMI-1 subunit of PRC1. Using MM cell lines and primary cells isolated from newly diagnosed or relapsed MM patients, we found that PTC-209 has a potent anti-MM activity. We showed, for the first time in MM, that PTC-209 anti-MM effects were mediated by on-target effects i.e. downregulation of BMI-1 protein and the associated repressive histone mark H2AK119ub, but that other subunits of the PRC1 complex were not affected. We showed that PTC-209 reduced MM cell viability via significant induction of apoptosis. More importantly, we demonstrated that PTC-209 shows synergistic anti-MM activity with other epigenetic inhibitors targeting EZH2 (UNC1999) and BET-bromodomains (JQ1). This work highlights the potential use of BMI-1 and PRC1 as potential therapeutic targets in MM alone or in combination with other anti-MM agents including epigenetic inhibitors.
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| 2. |
- Kalushkova, Antonia
(författare)
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Epigenetic gene regulation in multiple myeloma and mood disorders
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Epigenetics continues to be redefined and new discoveries are likely to revolutionise the field still further. This thesis explores different aspects of how epigenetic regulation of gene expression contributes to human disease.Paper I explores the function of the IKKα kinase in regulating gene expression through the nuclear retinoic acid receptor (RAR). We define a set of genes requiring IKKα for their expression and found recruitment of IKKα to the RAR dependent on structural motifs in its protein sequence. This interplay between the NFκB pathway and nuclear receptor regulated transcription is important to consider when designing therapeutic strategies.Papers II and III focus on the plasma cell malignancy multiple myeloma (MM) and define a gene regulatory circuit defining an underexpressed gene profile in MM dependent on the Polycomb proteins. We provide proof-of-principle that the use of small chemical inhibitors may be operational in reactivating genes silenced by H3K27me3 and that this leads to decreased tumour load and increased survival in the 5T33 in vivo model of MM. We explored the genome-wide distribution of H3K27me3 and H3K4me3, and defined their association with gene expression in freshly-isolated malignant plasma cells from MM patients. Importantly, H3K27me3-marked genes in MM associated with more aggressive stages of the disease and less favourable survival. We present evidence that gene targeting by H3K27me3 is likely to not only involve a small population of tumour cells, but rather represent a common MM profile and further provide a rationale for evaluating epigenetic therapeutics in MM.Paper IV shows that pro-inflammatory gene expression in monocytes of psychiatric patients can be induced in vitro by sodium pump inhibitors, as the steroid hormone ouabain. We suggest that the ouabain-induced gene expression is regulated by an intricate network involving microRNAs, Polycomb and the H3K27me3 demethylase JMJD3. Our data indicates that epigenetic regulators play a role in transmitting cues between intrinsic and/extrinsic stimuli and gene expression in psychiatric illness.This thesis provides novel insights on how seemingly unrelated pathways may converge on transcriptional regulation and evidence that epigenetic modifiers contribute to the pathogenesis of human complex diseases such as multiple myeloma and mood disorders.
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| 3. |
- Kårehed, Karin, 1972-
(författare)
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Signal Transduction in Malignant Cells – Transformation, Activation and Differentiation
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation.We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K.The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway.ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation.Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.
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| 4. |
- Larsson, Oscar, 1978-
(författare)
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The Governmentality of Meta-governance : Identifying Theoretical and Empirical Challenges of Network Governance in the Political Field of Security and Beyond
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Meta-governance recently emerged in the field of governance as a new approach which claims that its use enables modern states to overcome problems associated with network governance. This thesis shares the view that networks are an important feature of contemporary politics which must be taken seriously, but it also maintains that networks pose substantial analytical and political challenges. It proceeds to investigate the potential possibilities and problems associated with meta-governance on both theoretical and empirical levels.The theoretical discussion examines meta-governance in relation to governmentality, and it puts forward the claim that meta-governance may be understood as a specific type of neo-liberal governmentality. The meta-governance perspective regards networks as a complementary structure to traditional administration that can be utilized in the implementation and realization of public policy, but which also preserves the self-regulating and flexible character of networks. This generates a contradiction between the goals of public management and the character of networks that requires further investigation.The combination of the specific dynamics of the political field of security, the diminishing role of sovereign powers, the emergence of security networks, and the meta-governance stance adopted by the Swedish state constitutes a situation that should have been favorable for the successful employment of meta-governance. The empirical investigation of meta-governance is divided into two parts. The first part reviews the historical process involved and shows how the Swedish government and public authorities have adopted a meta-governance stance. The second analyzes the specific instruments and strategies that have been deployed in the governance of security communications and in the management of Sweden’s new security communications system which is an important aspect of security networks. The historical study together with the analysis of the meta-governance tools deployed reveals that the meta-governors neither reached the goals specified, nor fulfilled the overall purpose of successful security communications.I argue on the basis of the theoretical and empirical findings obtained in the present study that it is very difficult to successfully employ meta-governance in respect to security and crisis management, and that we have sound reasons to suspect that meta-governance will run into similar difficulties in other political fields as well. I conclude that meta-governance is a far more difficult practice than has been anticipated by existing theories and policy recommendations. Turning to meta-governance as a way to govern and control organizations may in fact lead to further fragmentation and distortion of public politics.
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| 5. |
- Mark, Charlotta, 1970-
(författare)
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Three Subfamilies of KRAB Zinc Finger Proteins : A Structural, Functional and Evolutionary Analysis
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Krüppel-related zinc finger proteins constitute the largest single class of transcription factors within the human genome. Members of this protein family have the ability to either activate or repress transcription depending on the presence of specific activator or repressor domains within the protein. Approximately one third of the Krüppel-related zinc finger proteins contain an evolutionarily well-conserved repressor domain termed the KRAB domain. This domain acts as a potent repressor of transcription by interacting with the co-repressor protein, TIF1β. TIF1β then, in turn, recruits HP1 proteins, HDACs and probably other proteins involved in gene silencing. In order to identify novel KRAB-containing zinc finger proteins, one mouse monocytic cDNA library and two testis cDNA libraries were screened for novel members of this multigene family. Six novel KRAB-ZNF cDNAs, four mouse and two human, were isolated. The corresponding proteins were all shown to contain N-terminally located KRAB domains as well as varying numbers of C-terminally located zinc finger motifs. An extensive comparative sequence analysis of the KRAB domains of these proteins together with KRAB domains from a large number of previously identified KRAB-ZNF proteins resulted in a clear subdivision into three different subfamilies, A+B, A+b and A. Later, we also isolated a fourth KRAB box, which is present downstream of the KRAB A box in a few proteins of the KRAB A family. This module was named KRAB C. Potential functional differences between these different subfamilies were investigated. In line with previous observations, the KRAB A box was shown to repress transcription, an activity which was enhanced by the presence of the KRAB B box. However, addition of neither the KRAB b box nor the KRAB C box had any effect on repression. Moreover, all KRAB A motifs had the ability to bind TIF1β, and this binding was increased both by the presence of the KRAB B box and by the KRAB C box. The KRAB b box, however, did not seem to contribute to TIF1β-binding. One of the novel human cDNAs, HKr19, was found to be a member of the large ZNF91 family of KRAB zinc finger genes. Interestingly, the expression of HKr19 and a number of other closely related genes were restricted to lymphoid cells, indicating that these genes may be involved in regulating lineage commitment. The effect of HKr19 on cell viability was investigated by transfection into human embryonic kidney cells (HEK 293). The results indicated that HKr19, or its zinc finger domain in isolation, were toxic to these cells when expressed at high levels. The MZF6D protein, on the other hand, showed a testis-specific expression. In situ hybridization analysis located this expression to meiotic germ cells, suggesting a role for this protein in spermatogenesis. Further, the evolutionary perspectives of this large gene family were addressed, and its enormous expansion throughout evolution probably includes numerous duplication events. The results from two extensive sequence analyses give clues to how the repetitive nature of the ZNF motif has given rise to both internal duplications of single motifs as well as duplications of entire genes resulting in gene clusters.
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| 6. |
- Nylund, Patrick
(författare)
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Targeting molecular mechanisms for epigenetic silencing in multiple myeloma : Implications for biology and precision medicine
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Multiple myeloma (MM) is a heterogeneous haematological cancer where malignant plasma cells clonally expand within the bone marrow. The transcriptional repressor PRC2 and its catalytic subunit EZH2 play a major role in MM, as PRC2 re-targeting results in a MM-specific gene silencing profile. In paper I, we explored the metabolic response to EZH2 inhibition (EZH2i). A global loss of H3K27me3 was found in all EZH2i-treated MM cell lines. EZH2i-sensitive cell lines acquired a unique metabolic signature, following the upregulation of a cluster of miRNAs which target methionine cycling-associated genes and are silenced by H3K27me3. These miRNAs were not upregulated in resistant cell lines, due to additional DNA methylation-mediated silencing.Therefore, in paper II we sought to evaluate the combinatorial effect of DNA demethylation agents and EZH2 inhibitors. Here, we provided a comprehensive map of the reconfiguration of the epigenome in primary MM samples. Furthermore, we demonstrated a direct protein-protein interaction between DNMT1 and EZH2 and showed that co-inhibition of these enzymes has an enhanced effect in synergistically activating genes regulating apoptosis and cell cycling. PRC2 lacks sequence specificity but contains a lncRNA binding pocket. In paper III, we hypothesized that PRC2 targeting to specific genomic regions could be mediated by lncRNAs in the context of MM. Coupling RIP- and RNA-seq, we identified a physical interaction between the lncRNA PVT1 and EZH2, as well as 270 genes potentially targeted by the EZH2-PVT1 axis. In addition, we found that independent inhibition of EZH2 and PVT1 resulted in the upregulation of the tumour suppressor genes ZBTB7C, RNF144A and CCDC136, suggesting a functional interdependency between these two epigenetic regulators. In paper IV we investigated the effects of dual G9a/DNMT inhibition in MM cells, resulting in suppressed expression of MM-associated oncogenes and increased tumour cell death. By coupling ChIP-seq, DNA methylation arrays and RNA-seq, we identified a group of genes silenced by G9a and/or DNMTs that when activated, blocked MM proliferative potential by activating genes with tumour suppressor function. In summary, this thesis highlights the strong interconnection between the dysregulation of epigenetic/metabolic regulatory mechanisms and MM pathogenesis, providing insights into how these mechanisms can be targeted to promote anti-MM effects.
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| 7. |
- Párraga, Alba Atienza, 1988-
(författare)
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The Epigenome of Multiple Myeloma : From genome-wide analysis to pharmacological manipulation
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nowadays epigenetic dysregulation is known to play a crucial role in virtually all cancers. In multiple myeloma (MM), an extensively heterogeneous malignancy, the key common feature among patients is the gene silencing imposed by the PRC2 complex through the addition of H3K27me3. This thesis focuses on the exploration of the MM epigenomic landscape, with an emphasis on both the interplay between H3K27me3 and other epigenetic tags, and on the effects of a series of inhibitors altering this profile.In paper I we provide the genome-wide H3K27me3 distribution unique to MM and demonstrate that the silencing of genes in the profile correlates with an advanced and poor-outcome disease. Reduction of H3K27me3 using the EZH2 inhibitor UNC1999 reactivates genes with anti-tumor activity and induces apoptosis in vitro.EZH2 inhibition also leads to downregulation of the MM oncogenes IRF-4, BLIMP-1, XBP-1 and c-MYC. Paper II identifies miR-125a-3p and miR-320c, predicted to target these oncogenes, as part of the PRC2 targets induced upon treatment.In addition, H3K27me3 can be recognized and bound by the PRC1 complex. In paper III we show that inhibition of PRC1 using PTC-209 induces apoptosis and this is further enhanced when PTC-209 is combined with UNC1999. Moreover, PTC-209 has been previously shown to reduce the expression of c-MYC. Combined treatment using PTC-209 and JQ1, demonstrated to downregulate c-MYC, results in additive and synergistic effects in reducing MM cell viability.In paper IV we present the first catalogue of genomic regulatory regions in normal plasma cells, as predicted by their combinations of histone marks. Using this, we demonstrate that in MM a subset of TSSs and enhancers become targeted by H3K27me3 and display high DNA methylation, pointing towards a possible silencing. Conversely, poised TSSs lose H3K27me3 and seemingly become de novo activated. Furthermore, we show that EZH2 physically interacts with the DNA methyltransferase DNMT1 and that combined inhibition using UNC1999 and the DNA hypomethylating agent AZA blocks the G2/M arrest triggered by AZA and induces apoptosis.In summary, this thesis highlights the complex interconnectivity of epigenetic mechanisms in MM and provides proof-of-principle of the anti-MM effects derived from inhibiting epigenetic components in single or combinatorial regimens.
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| 8. |
- Tshuikina Wiklander, Marina, 1978-
(författare)
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Epigenetic Regulation of Gene Transcription in Hematopoietic Tumors
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Epigenetic modifications were shown to play an essential role in tumorigenesis. Epigenetic mechanisms can alter transcription in several ways, through DNA methylation and/or through histone modification. DNA methylation at the TSS (transcriptional start site) has been implicated in tumor development and gene silencing. However, several examples of atypical methylation were shown. In Paper I we present the ICSBP/IRF8 gene that belongs to the IRF family and has characteristics of a tumor suppressor gene. The ICSBP/IRF8 is fully methylated in the promoter and TSS regions in U-937 and despite high expression of the gene. Presence of positive histone marks suggests that methylated DNA can be overridden by histone modification.In Paper II a panel of 13 MM (multiple myeloma) cell lines and 9 primary patient tumors were analysed for methylation status of the ICSBP/IRF8 gene. In most cell lines (8/13) the gene was partially or fully methylated and partial methylation was also observed in 1/9 primary tumors. In vitro methylation analysis and treatment with 5-aza-2’deoxycytidine (DAC) proved that the ICSBP/IRF8 gene is silenced by methylation and may be associated with the malignant phenotype.In Paper III and IV the NFκB signalling pathway was analysed and the role of ATRA and TNFα induction. In Paper III the data shows that activation of the NFκB pathway is essential in ATRA-induced terminal differentiation in the U-937 cell line and IκBα (S32A/S36A) inhibits ATRA-induced differentiation and G1 cell cycle arrest. This was accompanied by delayed down-regulation of several cyclins (A and E) and up-regulation of p21WAF1/CIP1 (CDKN1A) and p27KIP1 (CDKN1B).TNFα alone did not induce expression of RA-induced genes analysed in Paper IV. However, ATRA in combination with TNFα showed enhanced activation of RA-induced genes. TNFα triggers demethylation of H3K9me3/H3K9me2 and H3K4me3 at RAR/RXR target genes, which were not accompanied by changes in the level of H3K9-ac. This decrease in H3 methylation by TNFα may pave way for the later ATRA-induced gene transcription.
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| 9. |
- Öberg, Fredrik, 1982
(författare)
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AQUAPORINS: Production Optimization and Characterization
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism – including humans. They are vital molecules and their malfunction can lead to several severe disorders. An increased understanding of their structure, function and regulation is of utmost importance for developing current and future drugs. The first problem to overcome is to acquire the proteins in sufficient amounts to enable characterization. To achieve this, proteins are often produced in a host organism. One of the most successful hosts for recombinant overproduction is the yeast Pichia pastoris. Using this yeast we could obtain exceptional yield of aquaporin 1, whereas some others were below the threshold needed for successful subsequent characterization. In this process, we have established methods allowing fast and accurate determination of the initial production yield. Furthermore, we optimized the yield for low producing targets, enabling studies of proteins previously out of reach, exemplified with human aquaporin 4. Characterization has been performed on aquaporins obtained in sufficient quantities, and the functionality of aquaporin 1, 5 and 10 has been assessed. Furthermore, a glycosylation was found to stabilize the aquaporin 10 tetramer although only a minority of the monomers where modified. Moreover, we used protein crystallography to determine the three dimensional structure of a hAQP5 mutant, providing insight into regulation of the protein by trafficking. Taken together, these results provide insight into factors directing high production of eukaryotic membrane proteins. The subsequent characterization, including functional and structural determination, reveals new knowledge about aquaporin activity and regulation.
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| 10. |
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