| 1. |
- Deneberg, Stefan, et al.
(författare)
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microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia
- 2014
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Ingår i: Epigenetics. - : Informa UK Limited. - 1559-2294 .- 1559-2308. ; 9:6, s. 910-917
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Tidskriftsartikel (refereegranskat)abstract
- A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22-23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.
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| 2. |
- Deneberg, Stefan, et al.
(författare)
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Prognostic DNA methylation patterns in cytogenetically normal acute myeloid leukemia are predefined by stem cell chromatin marks
- 2011
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 118:20, s. 5573-5582
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Tidskriftsartikel (refereegranskat)abstract
- Cytogenetically normal acute myeloid leukemia (CN-AML) comprise between forty and fifty percent of all adult acute myeloid leukemia (AML) cases. In this clinically diverse group molecular aberrations such as FLT3ITD, NPM1 and CEBPA mutations recently have added to the prognostic accuracy. Aberrant DNA methylation is a hallmark of cancer including AML. We investigated in total 118 CN-AML samples in a test and a validation cohort for genome-wide promoter DNA methylation with Illumina Methylation Bead arrays and compared them to normal myeloid precursors and global gene expression. IDH and NPM1 mutations were associated with different methylation patterns (p=0.0004 and 0.04, respectively). Genome-wide methylation levels were elevated in IDH mutated samples (p=0.006). We observed a negative impact of DNA methylation on transcription. Genes targeted by Polycomb group (PcG) proteins and genes associated with bivalent histone marks in stem cells showed increased aberrant methylation in AML (p<0.0001). Furthermore, high methylation levels of PcG target genes were independently associated with better progression free (OR 0.47, p=0.01) and overall survival (OR 0.36, p=0.001). In summary, genome wide methylation patterns show preferential methylation of PcG targets with prognostic impact in CN-AML.
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| 3. |
- Mareschal, Sylvain, et al.
(författare)
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Challenging conventional karyotyping by next-generation karyotyping in 281 intensively treated patients with AML
- 2021
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Ingår i: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 5:4, s. 1003-1016
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Tidskriftsartikel (refereegranskat)abstract
- Although copy number alterations (CNAs) and translocations constitute the backbone of the diagnosis and prognostication of acute myeloid leukemia (AML), techniques used for their assessment in routine diagnostics have not been reconsidered for decades. We used a combination of 2 next-generation sequencing-based techniques to challenge the currently recommended conventional cytogenetic analysis (CCA), comparing the approaches in a series of 281 intensively treated patients with AML. Shallow whole-genome sequencing (sWGS) outperformed CCA in detecting European Leukemia Net (ELN)-defining CNAs and showed that CCA overestimated monosomies and suboptimally reported karyotype complexity. Still, the concordance between CCA and sWGS for all ELN CNA-related criteria was 94%. Moreover, using in silico dilution, we showed that 1 million reads per patient would be enough to accurately assess ELN-defining CNAs. Total genomic loss, defined as a total loss 200 Mb by sWGS, was found to be a better marker for genetic complexity and poor prognosis compared with the CCA-based definition of complex karyotype. For fusion detection, the concordance between CCA and whole-transcriptome sequencing (WTS) was 99%. WTS had better sensitivity in identifying inv(16) and KMT2A rearrangements while showing limitations in detecting lowly expressed PML-RARA fusions. Ligation-dependent reverse transcription polymerase chain reaction was used for validation and was shown to be a fast and reliable method for fusion detection. We conclude that a next-generation sequencing-based approach can replace conventional CCA for karyotyping, provided that efforts are made to cover lowly expressed fusion transcripts.
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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. |
- Neddermeyer, Anne, et al.
(författare)
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A new mutant NPM1/IDH2R140- and PML-RARA-associated lncRNA MALNC plays a role in AML biology, prognosis and drug response
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by poor prognosis that requires better understanding of its disease biology and new tools for suitable risk stratification and effective treatments. Long non-coding RNAs (lncRNAs) are involved in numerous molecular mechanisms, are implicated in tumor biology and can serve as clinical biomarkers, yet their role remains mostly unclear in AML. In this study, the aim was to discover and characterize lncRNAs implicated in AML and to describe their role in AML biology. Further aims were to explore their use as prognostic or predictive biomarkers. Using whole-transcriptome analysis, a novel lncRNA, here named MALNC, was identified. MALNC had elevated expression in two large AML cohorts compared to normal CD34+ cells. Clinical correlation analyses indicated that MALNC was almost uniquely expressed in patients with PML-RARA fusion gene and with co-mutated isocitrate dehydrogenase-2 R140 and nucleophosmin-1 (IDH2R140/NPM1). MALNC expression was specifically high at the promyelocytic stage and decreased with maturation in leukemic and normal cells. High MALNC expression associated independently with better overall survival. CRISPR-Cas9-knockout in promyelocytic cell lines impaired proliferation, colony formation and all-trans retinoic acid (ATRA)-induced differentiation. Also, MALNC-knockout dramatically sensitized cells to arsenic trioxide (ATO), ATO-ATRA combinatorial and Bcl-2-inhibitor venetoclax treatment as well as associated with cyclin-dependent kinase (Cdk)-inhibitor resistance. In conclusion, MALNC is overexpressed in certain subgroups of AML and could play a role during normal and leukemic hematopoietic maturation. Furthermore, it correlates with response to anti-leukemic drugs, which suggests a role as a predictive marker to drug response and survival in AML.
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| 6. |
- Paul, Esbjörn, et al.
(författare)
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Low p14ARF expression in de novo acute myeloid leukemia with normal karyotype is associated with poor survival
- 2009
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Ingår i: Leukemia and Lymphoma. - : Informa UK Limited. - 1042-8194 .- 1029-2403. ; 50:9, s. 1512-1518
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Tidskriftsartikel (refereegranskat)abstract
- The p14ARF protein activates the p53 tumor suppressor by binding to and inhibiting its negative regulator HDM-2. We have studied the prognostic impact of p14ARF in acute myeloid leukemia (AML). Leukemic cells from 57 adult patients with normal karyotype de novo AML were analyzed for p14ARF mRNA expression level using real-time polymerase chain reaction (RT-PCR). We also tested the effect of conventional anti-leukemic drugs and the mutant p53-targeting small molecule PRIMA-1 in vitro. Patients whose cells expressed more p14ARF mRNA than the 75th percentile (0.26) had significantly better survival compared with those expressing lower levels, 61 vs. 30% 3-year survival (p = 0.046). The difference remained significant also when NPM1/FLT3 status was considered. The mean effects of all the tested conventional anti-leukemic drugs were greater in leukemic cell samples expressing p14ARF mRNA >or= 0.26, but the differences were not statistically significant. In contrast, PRIMA-1 had a significantly greater effect on leukemic cell samples with low levels of p14ARF mRNA. We conclude that low levels of p14ARF mRNA in leukemic cells from patients with normal karyotype AML is associated with poor prognosis. Treatment with drugs targeting p53 may be a future possibility to improve outcome for these patients.
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| 7. |
- Qu, Ying, et al.
(författare)
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Differential methylation in CN-AML preferentially targets non-CGI regions and is dictated by DNMT3A mutational status and associated with predominant hypomethylation of HOX genes
- 2014
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Ingår i: Epigenetics. - : Informa UK Limited. - 1559-2294 .- 1559-2308. ; 9:8, s. 1108-1119
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Tidskriftsartikel (refereegranskat)abstract
- The extent and role of aberrant DNA methylation in promoter CpG islands (CGIs) have been extensively studied in leukemia and other malignancies. Still, CGIs represent only a small fraction of the methylome. We aimed to characterize genome-wide differential methylation of cytogenetically normal AML (CN-AML) cells compared with normal CD34(+) bone marrow cells using the Illumina (R) 450K methylation array. Differential methylation in CN-AML was most prominent in genomic areas far from CGIs, in so called open sea regions. Furthermore, differential methylation was specifically found in genes encoding transcription factors (TFs), with WT1 being the most differentially methylated TF. Among genetic mutations in AML, DNMT3A mutations showed the most prominent association with the DNA methylation pattern, characterized by hypomethylation of CGIs (as compared with DNMT3A wild type cases). The differential methylation in DNMT3A mutant cells vs. wild type cells was predominantly found in HOX genes, which were hypomethylated. These results were confirmed and validated in an independent CN-AML cohort. In conclusion, we show that, in CN-AML, the most pronounced changes in DNA methylation occur in non-CGI regions and that DNMT3A mutations confer a pattern of global hypomethylation that specifically targets HOX genes.
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| 8. |
- Struyf, Nona, et al.
(författare)
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Delineating functional and molecular impact of ex vivo sample handling in precision medicine
- 2024
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Ingår i: npj Precision Oncology. - : Springer Nature. - 2397-768X. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Consistent handling of samples is crucial for achieving reproducible molecular and functional testing results in translational research. Here, we used 229 acute myeloid leukemia (AML) patient samples to assess the impact of sample handling on high-throughput functional drug testing, mass spectrometry-based proteomics, and flow cytometry. Our data revealed novel and previously described changes in cell phenotype and drug response dependent on sample biobanking. Specifically, myeloid cells with a CD117 (c-KIT) positive phenotype decreased after biobanking, potentially distorting cell population representations and affecting drugs targeting these cells. Additionally, highly granular AML cell numbers decreased after freezing. Secondly, protein expression levels, as well as sensitivity to drugs targeting cell proliferation, metabolism, tyrosine kinases (e.g., JAK, KIT, FLT3), and BH3 mimetics were notably affected by biobanking. Moreover, drug response profiles of paired fresh and frozen samples showed that freezing samples can lead to systematic errors in drug sensitivity scores. While a high correlation between fresh and frozen for the entire drug library was observed, freezing cells had a considerable impact at an individual level, which could influence outcomes in translational studies. Our study highlights conditions where standardization is needed to improve reproducibility, and where validation of data generated from biobanked cohorts may be particularly important.
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| 9. |
- Österroos, Albin, et al.
(författare)
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Integrated multi-omic profiling of azacitidine-venetoclax in AML reveals additional targetable pathways to improve the treatment
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The combination of venetoclax and azacitidine constitutes the first-line option for patients with acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy. Azacitidine-venetoclax has shown pronounced clinical and preclinical ecacy but the molecular mechanisms causing this synergy remain to be clarified. We applied an integrative multi-omic approach with focus in epigenetics on two AML cell lines to characterize the effects of azacitidine-venetoclax in vitro on chromatin accessibility, DNA methylation and gene expression patterns.We report distinct epigenetic and transcriptomic eects when combining azacitidine and venetoclax as compared to either substance alone. With the application of ATAC-seq, we delineate combination-unique gained pathways including the activation of mRNA splicing and NOTCH-HSF1 signaling but also more widespread heterochromatin compared to azacitidine or venetoclax alone. When assessing methylation status, we observed combination-unique hypermethylation of Rac1- and Rho-associated signaling. We integrate the epigenetic alterations of azacitidine-venetoclax with RNA-seq data and report activation of genes involved in the serine synthesis pathway and NTRK signaling that represent potentially upregulated survival pathways upon azacitidine-venetoclax exposure.We also propose potential synergistic triplet therapies based on in silico drug predictions using condition-wise weighted co-expression networks including inhibitors of proteaseomes, MCL-1 and histone deacetylase. Future studies are needed to investigate whether the inhibition of the mentioned pro-survival pathways or proposed triplets may enhance the effects of azacitidine-venetoclax in AML.
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