| 1. |
- Ali, Dina, et al.
(författare)
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Anti-leukaemic effects induced by APR-246 are dependent on induction of oxidative stress and the NFE2L2/HMOX1 axis that can be targeted by PI3K and mTOR inhibitors in acute myeloid leukaemia cells
- 2016
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Ingår i: British Journal of Haematology. - : Wiley. - 0007-1048 .- 1365-2141. ; 174:1, s. 117-126
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Tidskriftsartikel (refereegranskat)abstract
- The small molecule APR-246 (PRIMA-1(MET)) is a novel drug that restores the activity of mutated and unfolded TP53 protein. However, the mechanisms of action and potential off-target effects are not fully understood. Gene expression profiling in TP53 mutant KMB3 acute myeloid leukaemia (AML) cells showed that genes which protected cells from oxidative stress to be the most up-regulated. APR-246 exposure also induced reactive oxygen species (ROS) formation and depleted glutathione in AML cells. The genes most up-regulated by APR-246, confirmed by quantitative real time polymerase chain reaction, were heme oxygenase-1 (HMOX1, also termed HO-1), SLC7A11 and RIT1. Up-regulation of HMOX1, a key regulator of cellular response to ROS, was independent of TP53 mutational status. NFE2L2 (also termed Nrf2), a master regulator of HMOX1 expression, showed transcriptional up-regulation and nuclear translocation by APR-246. Down-regulation of NFE2L2 by siRNA in AML cells significantly increased the antitumoural effects of APR-246. The PI3K inhibitor wortmannin and the mTOR inhibitor rapamycin inhibited APR-246-induced nuclear translocation of NFE2L2 and counteracted the protective cellular responses to APR-246, resulting in synergistic cell killing together with APR-246. In conclusion, ROS induction is important for antileukaemic activities of APR-246 and inhibiting the protective response of the Nrf-2/HMOX1 axis using PI3K inhibitors, enhances the antileukaemic effects.
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| 2. |
- Kling, Peter, 1968, et al.
(författare)
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Differential regulation of the rainbow trout (Oncorhynchus mykiss) MT-A gene by nuclear factor interleukin-6 and activator protein-1
- 2013
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Ingår i: Bmc Molecular Biology. - : Springer Science and Business Media LLC. - 1471-2199. ; 14:28
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Tidskriftsartikel (refereegranskat)abstract
- Background: Previously we have identified a distal region of the rainbow trout (Oncorhynchus mykiss) metallothionein-A (rtMT-A) enhancer region, being essential for free radical activation of the rtMT-A gene. The distal promoter region included four activator protein 1 (AP1) cis-acting elements and a single nuclear factor interleukin-6 (NF-IL6) element. In the present study we used the rainbow trout hepatoma (RTH-149) cell line to further examine the involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion. Results: Using enhancer deletion studies we observed strong paraquat (PQ)-induced rtMT-A activation via NF-IL6 while the AP1 cis-elements showed a weak but significant activation. In contrast to mammals the metal responsive elements were not activated by oxidative stress. Electrophoretic mobility shift assay (EMSA) mutation analysis revealed that the two most proximal AP1 elements, AP1(1,2), exhibited strong binding to the AP1 consensus sequence, while the more distal AP1 elements, AP1(3,4) were ineffective. Phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, resulted in a robust induction of rtMT-A via the AP1 elements alone. To determine the conservation of regulatory functions we transfected human Hep G2 cells with the rtMT-A enhancer constructs and were able to demonstrate that the cis-elements were functionally conserved. The importance of NF-IL6 in regulation of teleost MT is supported by the conservation of these elements in MT genes from different teleosts. In addition, PMA and PQ injection of rainbow trout resulted in increased hepatic rtMT-A mRNA levels. Conclusions: These studies suggest that AP1 primarily is involved in PMA regulation of the rtMT-A gene while NF-IL6 is involved in free radical regulation. Taken together this study demonstrates the functionality of the NF-IL6 and AP-1 elements and suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.
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| 3. |
- Miliara, Sophia, et al.
(författare)
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The biological and prognostic role of long non-coding RNA NEAT1 in acute myeloid leukemia
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA localized in the cell nucleus that has been associated to promote several malignant solid tumors. Its role in acute myeloid leukemia (AML) remains largely elusive. Therefore, the aim of this study was to define the role of NEAT1 in AML compared to normal hematopoiesis. During normal hematopoiesis, it was identified that NEAT1 expression was low in early progenitors but increased in more differentiated cells, especially in monocytes. NEAT1 expression was increased in AML as a whole compared to normal bone marrow (NBM). It was specifically high in AML with inv(16) and t(8;21), while it was lower in patients with t(15;17). Further, NEAT1 expression correlated positively with ASXL1, KRAS and NRAS mutations and negatively with TP53 mutant AML. Higher NEAT expression was associated to better overall survival in AML, independent of other known risk factors. Antisense oligo-mediated knockdown of NEAT1 in AML cells significantly increased expression of the monocytic marker CD14 while granulocytic markers did not change. Genes affected by NEAT1-knockdown using CAGE-sequencing were significantly enriched for genes involved in glucose metabolism. By investigating genome-wide RNA and DNA interactions using RADICL-sequencing, it was revealed that NEAT1 binds to the loci of key hematopoietic regulator RUNX2 as well as the chromatin regulators KMT2A, KMT5B and CHD7. The results suggest that lncRNA NEAT1 has a potential role in hematopoietic and AML cell differentiation and could be a potential new biomarker in AML.
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| 4. |
- Mujahed, Huthayfa, et al.
(författare)
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AML displays increased CTCF occupancy associated with aberrant gene expression and transcription factor binding
- 2020
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 136:3, s. 339-352
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Tidskriftsartikel (refereegranskat)abstract
- CCTC-binding factor (CTCF) is a key regulator of gene expression through organization of the chromatin structure. Still, it is unclear how CTCF binding is perturbed in leukemia or in cancer in general. We studied CTCF binding by chromatin immunoprecipitation sequencing in cells from patients with acute myeloid leukemia (AML) and in normal bone marrow (NBM) in the context of gene expression, DNA methylation, and azacitidine exposure. CTCF binding was increased in AML compared with NBM. Aberrant CTCF binding was enriched for motifs for key myeloid transcription factors such as CEBPA, PU.1, and RUNX1. AML with TET2 mutations was characterized by a particularly strong gain of CTCF binding, highly enriched for gain in promoter regions, while AML in general was enriched for changes at enhancers. There was a strong anticorrelation between CTCF binding and DNA methylation. Gain of CTCF occupancy was associated with increased gene expression; however, the genomic location (promoter vs distal regions) and enrichment of motifs (for repressing vs activating cofactors) were decisive for the gene expression pattern. Knockdown of CTCF in K562 cells caused loss of CTCF binding and transcriptional repression of genes with changed CTCF binding in AML, as well as loss of RUNX1 binding at RUNX1/CTCF-binding sites. In addition, CTCF knockdown caused increased differentiation. Azacitidine exposure caused major changes in CTCF occupancy in AML patient cells, partly by restoring a CTCF-binding pattern similar to NBM. We conclude that AML displays an aberrant increase in CTCF occupancy that targets key genes for AML development and impacts gene expression.
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| 5. |
- Mujahed, Huthayfa
(författare)
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Genetic and epigenetic studies of acute myeloid leukemia and therapeutic possibilities
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Acute myeloid leukaemia (AML) is malignant tumour that forms in the bone marrow and arises from immature myeloid progenitors. Consequently, this leads to excessive accumulation of dysfunctional blast cells and lack of normal blood cells. The molecular and genetic heterogeneity of the disease is substantial which makes the disease challenging to classify and treat. Although the AML classification is updated continuously and more data and research on AML pathophysiology emerges, first line treatment for the vast majority of AML patients remains a combination of cytarabine and an anthracycline. While most patients attain a complete remission, the majority of AML patients relapse and develop drug resistance. Recently, new drugs have been approved for the treatment of specific AML subtypes. However, there is need for better understanding of disease pathogenesis including better genetic and epigenetic risk factors in order to develop more effective treatment regimens to improve the outcome of the disease. In Study I, we studied off-target effects of APR-246, a drug that originally was developed to restore the activity of mutated TP53 protein. Oxidative stress related genes heme oxygenase-1 (HMOX1, also termed HO-1), SLC7A11 and RIT1 were significantly upregulated. Also, Nrf2 that induces the expression of HO-1 was upregulated and depletion of Nrf2 mRNA resulted in increased cytotoxicity of APR-246. Moreover, blocking Nrf2 from translocating into the nucleus by using PI3K and mTOR inhibitors led to enhanced cell killing. This suggests that a combination of APR-246 with PI3K and mTOR inhibitors improves sensitivity to APR-246 by interfering with the cellular response to ROS activation to achieve better anti-leukemic effects of APR-246. In Study II, we aimed to define the potential of using stroma cells in diagnostic AML samples as a source of germline DNA. To obtain germline DNA, together with DNA from leukemic cells, it is essential to reliably define somatic mutations in AML cells. Consequently, we cultivated and expanded bone marrow stroma cells from vitally frozen mononuclear cells from AML patients with monosomy 7 as well as defined somatic mutations. In vitro expanded bone marrow stroma cells were stable after 6 weeks of culture and were able to differentiate into adipocytes or osteocytes. We could also show that cultivated stroma cells do not harbour the somatic mutations found in the malignant cells. Thus, we could conclude that bone marrow stroma cells from diagnostic bone marrow samples could be used as a source of germline DNA in AML patients. In Study III, we studied the binding occupancy of the chromatin organizer CTCF in AML patient cells and compared it to binding in normal CD34+ cells. We found that AML cells display an aberrant increase of CTCF binding. Motif analysis revealed that gained CTCF sites are enriched for transcription factors such as PU.1, RUNX1 and CEBPA, which is found to be important for normal myeloid development. TET2 mutated AML patients exhibit a greater gain of CTCF occupancy that is mainly annotated to promoters. Generally, gained CTCF sites were found to be hypomethylated and associated with genes that were upregulated in AML. Knockdown of CTCF in K562 cells resulted in loss of CTCF and decreased gene expression of targeted genes as well as loss of RUNX1 binding at common CTCF and RUNX1 binding sites. Knockdown of CTCF also resulted in increased differentiation of K562 cells. In vitro exposure of AML patient cells with azacytidine resulted in major changes in CTCF occupancy where most gained sites restored the binding pattern found in normal CD34+ cells. In conclusion, our results suggest that an aberrant CTCF occupancy in AML can have a role in driving leukemogenic gene expression patterns in AML.
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