Publications by authors named "Florian Grebien"

58 Publications

Impact of Specific -Glycan Modifications on the Use of Plant-Produced SARS-CoV-2 Antigens in Serological Assays.

Front Plant Sci 2021 27;12:747500. Epub 2021 Sep 27.

Department of Applied Genetics and Cell Biology, Institute of Plant Biotechnology and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in the virus-host cell interaction, and viral infection. The RBD is a major target for neutralizing antibodies, whilst recombinant RBD is commonly used as an antigen in serological assays. Such assays are essential tools to gain control over the pandemic and detect the extent and durability of an immune response in infected or vaccinated populations. Transient expression in plants can contribute to the fast production of viral antigens, which are required by industry in high amounts. Whilst plant-produced RBDs are glycosylated, -glycan modifications in plants differ from humans. This can give rise to the formation of carbohydrate epitopes that can be recognized by anti-carbohydrate antibodies present in human sera. For the performance of serological tests using plant-produced recombinant viral antigens, such cross-reactive carbohydrate determinants (CCDs) could result in false positives. Here, we transiently expressed an RBD variant in wild-type and glycoengineered leaves and characterized the impact of different plant-specific -glycans on RBD reactivity in serological assays. While the overall performance of the different RBD glycoforms was comparable to each other and to a human cell line produced RBD, there was a higher tendency toward false positive results with sera containing allergy-related CCD-antibodies when an RBD carrying β1,2-xylose and core α1,3-fucose was used. These rare events could be further minimized by pre-incubating sera from allergic individuals with a CCD-inhibitor. Thereby, false positive signals obtained from anti-CCD antibodies, could be reduced by 90%, on average.
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http://dx.doi.org/10.3389/fpls.2021.747500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8503525PMC
September 2021

Discovery of a Non-Nucleoside SETD2 Methyltransferase Inhibitor against Acute Myeloid Leukemia.

Int J Mol Sci 2021 Sep 17;22(18). Epub 2021 Sep 17.

Medicinal Chemistry Research Group, Research Centre for Natural Sciences, 1117 Budapest, Hungary.

Histone methyltransferases (HMTs) have attracted considerable attention as potential targets for pharmaceutical intervention in various malignant diseases. These enzymes are known for introducing methyl marks at specific locations of histone proteins, creating a complex system that regulates epigenetic control of gene expression and cell differentiation. Here, we describe the identification of first-generation cell-permeable non-nucleoside type inhibitors of SETD2, the only mammalian HMT that is able to tri-methylate the K36 residue of histone H3. By generating the epigenetic mark H3K36me3, SETD2 is involved in the progression of acute myeloid leukemia. We developed a structure-based virtual screening protocol that was first validated in retrospective studies. Next, prospective screening was performed on a large library of commercially available compounds. Experimental validation of 22 virtual hits led to the discovery of three compounds that showed dose-dependent inhibition of the enzymatic activity of SETD2. Compound C13 effectively blocked the proliferation of two acute myeloid leukemia (AML) cell lines with MLL rearrangements and led to decreased H3K36me3 levels, prioritizing this chemotype as a viable chemical starting point for drug discovery projects.
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http://dx.doi.org/10.3390/ijms221810055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8471172PMC
September 2021

Exploring protein hotspots by optimized fragment pharmacophores.

Nat Commun 2021 05 27;12(1):3201. Epub 2021 May 27.

Medicinal Chemistry Research Group, Research Centre for Natural Sciences, Budapest, Hungary.

Fragment-based drug design has introduced a bottom-up process for drug development, with improved sampling of chemical space and increased effectiveness in early drug discovery. Here, we combine the use of pharmacophores, the most general concept of representing drug-target interactions with the theory of protein hotspots, to develop a design protocol for fragment libraries. The SpotXplorer approach compiles small fragment libraries that maximize the coverage of experimentally confirmed binding pharmacophores at the most preferred hotspots. The efficiency of this approach is demonstrated with a pilot library of 96 fragment-sized compounds (SpotXplorer0) that is validated on popular target classes and emerging drug targets. Biochemical screening against a set of GPCRs and proteases retrieves compounds containing an average of 70% of known pharmacophores for these targets. More importantly, SpotXplorer0 screening identifies confirmed hits against recently established challenging targets such as the histone methyltransferase SETD2, the main protease (3CLPro) and the NSP3 macrodomain of SARS-CoV-2.
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http://dx.doi.org/10.1038/s41467-021-23443-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159961PMC
May 2021

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

EBioMedicine 2021 May 25;67:103348. Epub 2021 Apr 25.

Department of Medicine III, Division of Nephrology and Dialysis, Medical University of Vienna, Austria.

Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups.

Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification.

Findings: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus.

Interpretation: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms.

Funding: WWTF, Project No. COV20-016; BOKU, LBI/LBG.
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http://dx.doi.org/10.1016/j.ebiom.2021.103348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099623PMC
May 2021

Regulation of the Mitochondrion-Fatty Acid Axis for the Metabolic Reprogramming of Chlamydia trachomatis during Treatment with β-Lactam Antimicrobials.

mBio 2021 03 30;12(2). Epub 2021 Mar 30.

Department of Infectious Diseases and Microbiology, University of Lübeck, Lübeck, Germany.

Infection with the obligate intracellular bacterium is the most common bacterial sexually transmitted disease worldwide. Since no vaccine is available to date, antimicrobial therapy is the only alternative in infection. However, changes in chlamydial replicative activity and the occurrence of chlamydial persistence caused by diverse stimuli have been proven to impair treatment effectiveness. Here, we report the mechanism for regulating host signaling processes and mitochondrial function, which can be used for chlamydial metabolic reprogramming during treatment with β-lactam antimicrobials. Activation of signal transducer and activator of transcription 3 (STAT3) is a well-known host response in various bacterial and viral infections. In infection, inactivation of STAT3 by host protein tyrosine phosphatases increased mitochondrial respiration in both the absence and presence of β-lactam antimicrobials. However, during treatment with β-lactam antimicrobials, increased the production of citrate as well as the activity of host ATP-citrate lyase involved in fatty acid synthesis. Concomitantly, chlamydial metabolism switched from the tricarboxylic acid cycle to fatty acid synthesis. This metabolic switch was a unique response in treatment with β-lactam antimicrobials and was not observed in gamma interferon (IFN-γ)-induced persistent infection. Inhibition of fatty acid synthesis was able to attenuate β-lactam-induced chlamydial persistence. Our findings highlight the importance of the mitochondrion-fatty acid interplay for the metabolic reprogramming of during treatment with β-lactam antimicrobials. The mitochondrion generates most of the ATP in eukaryotic cells, and its activity is used for controlling the intracellular growth of Furthermore, mitochondrial activity is tightly connected to host fatty acid synthesis that is indispensable for chlamydial membrane biogenesis. Phospholipids, which are composed of fatty acids, are the central components of the bacterial membrane and play a crucial role in the protection against antimicrobials. Chlamydial persistence that is induced by various stimuli is clinically relevant. While one of the well-recognized inducers, β-lactam antimicrobials, has been used to characterize chlamydial persistence, little is known about the role of mitochondria in persistent infection. Here, we demonstrate how undergoes metabolic reprogramming to switch from the tricarboxylic acid cycle to fatty acid synthesis with promoted host mitochondrial activity in response to treatment with β-lactam antimicrobials.
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http://dx.doi.org/10.1128/mBio.00023-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092193PMC
March 2021

Downregulation of MTSS1 in acute myeloid leukemia is associated with a poor prognosis, chemotherapy resistance, and disease aggressiveness.

Leukemia 2021 Oct 29;35(10):2827-2839. Epub 2021 Mar 29.

Division of Oncology, Department of Medicine I, Medical University of Vienna, Vienna, Austria.

Despite recent approval of targeted drugs for acute myeloid leukemia (AML) therapy, chemotherapy with cytosine arabinoside and anthracyclines remains an important pillar of treatment. Both primary and secondary resistance are frequent and associated with poor survival, yet the underlying molecular mechanisms are incompletely understood. In previous work, we identified genes deregulated between diagnosis and relapse of AML, corresponding to therapy naïve and resistant states, respectively. Among them was MTSS1, whose downregulation is known to enhance aggressiveness of solid tumors. Here we show that low MTSS1 expression at diagnosis was associated with a poor prognosis in AML. MTSS1 expression was regulated by promoter methylation, and reduced by cytosine arabinoside and the anthracycline daunorubicin. Experimental downregulation of MTSS1 affected the expression of numerous genes. It induced the DNA damage response kinase WEE1, and rendered human AML cell lines more resistant to cytosine arabinoside, daunorubicin, and other anti-cancer drugs. Mtss1 knockdown in murine MLL-AF9-driven AML substantially decreased disease latency, and increased leukemic burden and ex vivo chemotherapy resistance. In summary, low MTSS1 expression represents a novel factor contributing to disease aggressiveness, therapy resistance, and poor outcome in AML.
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http://dx.doi.org/10.1038/s41375-021-01224-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478650PMC
October 2021

Precision Medicine in Hematology 2021: Definitions, Tools, Perspectives, and Open Questions.

Hemasphere 2021 Mar 17;5(3):e536. Epub 2021 Feb 17.

Department of Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Austria.

During the past few years, our understanding of molecular mechanisms and cellular interactions relevant to malignant blood cell disorders has improved substantially. New insights include a detailed knowledge about disease-initiating exogenous factors, endogenous (genetic, somatic, epigenetic) elicitors or facilitators of disease evolution, and drug actions and interactions that underlie efficacy and adverse event profiles in defined cohorts of patients. As a result, precision medicine and personalized medicine are rapidly growing new disciplines that support the clinician in making the correct diagnosis, in predicting outcomes, and in optimally selecting patients for interventional therapies. In addition, precision medicine tools are greatly facilitating the development of new drugs, therapeutic approaches, and new multiparametric prognostic scoring models. However, although the emerging roles of precision medicine and personalized medicine in hematology and oncology are clearly visible, several questions remain. For example, it remains unknown how precision medicine tools can be implemented in healthcare systems and whether all possible approaches are also affordable. In addition, there is a need to define terminologies and to relate these to specific and context-related tools and strategies in basic and applied science. To discuss these issues, a working conference was organized in September 2019. The outcomes of this conference are summarized herein and include a proposal for definitions, terminologies, and applications of precision and personalized medicine concepts and tools in hematologic neoplasms. We also provide proposals aimed at reducing costs, thereby making these applications affordable in daily practice.
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http://dx.doi.org/10.1097/HS9.0000000000000536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892291PMC
March 2021

Identification of gene targets of mutant C/EBPα reveals a critical role for MSI2 in CEBPA-mutated AML.

Leukemia 2021 09 23;35(9):2526-2538. Epub 2021 Feb 23.

University of Veterinary Medicine, Institute of Medical Biochemistry, Vienna, Austria.

Mutations in the gene encoding the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) occur in 10-15% of acute myeloid leukemia (AML). Frameshifts in the CEBPA N-terminus resulting in exclusive expression of a truncated p30 isoform represent the most prevalent type of CEBPA mutations in AML. C/EBPα p30 interacts with the epigenetic machinery, but it is incompletely understood how p30-induced changes cause leukemogenesis. We hypothesized that critical effector genes in CEBPA-mutated AML are dependent on p30-mediated dysregulation of the epigenome. We mapped p30-associated regulatory elements (REs) by ATAC-seq and ChIP-seq in a myeloid progenitor cell model for p30-driven AML that enables inducible RNAi-mediated knockdown of p30. Concomitant p30-dependent changes in gene expression were measured by RNA-seq. Integrative analysis identified 117 p30-dependent REs associated with 33 strongly down-regulated genes upon p30-knockdown. CRISPR/Cas9-mediated mutational disruption of these genes revealed the RNA-binding protein MSI2 as a critical p30-target. MSI2 knockout in p30-driven murine AML cells and in the CEBPA-mutated human AML cell line KO-52 caused proliferation arrest and terminal myeloid differentiation, and delayed leukemia onset in vivo. In summary, this work presents a comprehensive dataset of p30-dependent effects on epigenetic regulation and gene expression and identifies MSI2 as an effector of the C/EBPα p30 oncoprotein.
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http://dx.doi.org/10.1038/s41375-021-01169-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611617PMC
September 2021

Biomolecular condensation of NUP98 fusion proteins drives leukemogenic gene expression.

Nat Struct Mol Biol 2021 02 21;28(2):190-201. Epub 2021 Jan 21.

Institute for Medical Biochemistry, University of Veterinary Medicine, Vienna, Austria.

NUP98 fusion proteins cause leukemia via unknown molecular mechanisms. All NUP98 fusion proteins share an intrinsically disordered region (IDR) in the NUP98 N terminus, featuring repeats of phenylalanine-glycine (FG), and C-terminal fusion partners often function in gene control. We investigated whether mechanisms of oncogenic transformation by NUP98 fusion proteins are hardwired in their protein interactomes. Affinity purification coupled to mass spectrometry (MS) and confocal imaging of five NUP98 fusion proteins expressed in human leukemia cells revealed that shared interactors were enriched for proteins involved in biomolecular condensation and that they colocalized with NUP98 fusion proteins in nuclear puncta. We developed biotinylated isoxazole-mediated condensome MS (biCon-MS) to show that NUP98 fusion proteins alter the global composition of biomolecular condensates. An artificial FG-repeat-containing fusion protein phenocopied the nuclear localization patterns of NUP98 fusion proteins and their capability to drive oncogenic gene expression programs. Thus, we propose that IDR-containing fusion proteins combine biomolecular condensation with transcriptional control to induce cancer.
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http://dx.doi.org/10.1038/s41594-020-00550-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116736PMC
February 2021

Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in Acute Myeloid Leukemia.

Cancers (Basel) 2020 Sep 26;12(10). Epub 2020 Sep 26.

Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, 30625 Hannover, Germany.

NUP98-NSD1-positive acute myeloid leukemia (AML) is a poor prognostic subgroup that is frequently diagnosed in pediatric cytogenetically normal AML. NUP98-NSD1-positive AML often carries additional mutations in genes including and . The purpose of our study was to characterize the cooperative potential of the fusion and its associated Neuroblastoma rat sarcoma (NRAS) mutation. By constitutively expressing NUP98-NSD1 and NRASG12D in a syngeneic mouse model and using a patient-derived xenograft (PDX) model from a NUP98-NSD1-positive AML patient, we evaluated the functional role of these genes and tested a novel siRNA formulation that inhibits the oncogenic driver NUP98-NSD1. NUP98-NSD1 transformed murine bone marrow (BM) cells in vitro and induced AML in vivo. While NRASG12D expression was insufficient to transform cells alone, co-expression of NUP98-NSD1 and NRASG12D enhanced the leukemogenicity of NUP98-NSD1. We developed a NUP98-NSD1-targeting siRNA/lipid nanoparticle formulation that significantly prolonged the survival of the PDX mice. Our study demonstrates that mutated NRAS cooperates with NUP98-NSD1 and shows that direct targeting of the fusion can be exploited as a novel treatment strategy in NUP98-NSD1-positive AML patients.
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http://dx.doi.org/10.3390/cancers12102766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600396PMC
September 2020

STAT5 is required for lipid breakdown and beta-adrenergic responsiveness of brown adipose tissue.

Mol Metab 2020 10 28;40:101026. Epub 2020 May 28.

Institute of Animal Breeding and Genetics, University of Veterinary Medicine, 1210, Vienna, Austria; Ludwig Boltzmann Institute for Cancer Research, 1090, Vienna, Austria. Electronic address:

Objective: Increasing energy expenditure through activation of brown adipose tissue (BAT) thermogenesis is an attractive approach to counteract obesity. It is therefore essential to understand the molecular mechanisms that control BAT functions. Until now several members of the Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway have been implicated as being relevant for BAT physiology. However, whether the STAT family member STAT5 is important for the thermogenic property of adipose tissues is unknown. Therefore, we have investigated the role of STAT5 in thermogenic fat in this paper.

Methods: We performed metabolic and molecular analyses using mice that harbor an adipocyte-specific deletion of Stat5a/b alleles.

Results: We found that STAT5 is necessary for acute cold-induced temperature maintenance and the induction of lipid mobilization in BAT following β-adrenergic stimulation. Moreover, mitochondrial respiration of primary differentiated brown adipocytes lacking STAT5 was diminished. Increased sensitivity to cold stress upon STAT5 deficiency was associated with reduced expression of thermogenic markers including uncoupling protein 1 (UCP1), while decreased stimulated lipolysis was linked to decreased protein kinase A (PKA) activity. Additionally, brown remodeling of white adipose tissue was diminished following chronic β-adrenergic stimulation, which was accompanied by a decrease in mitochondrial performance.

Conclusion: We conclude that STAT5 is essential for the functionality and the β-adrenergic responsiveness of thermogenic adipose tissue.
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http://dx.doi.org/10.1016/j.molmet.2020.101026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322099PMC
October 2020

CDK6 is an essential direct target of NUP98 fusion proteins in acute myeloid leukemia.

Blood 2020 07;136(4):387-400

Institute for Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria.

Fusion proteins involving Nucleoporin 98 (NUP98) are recurrently found in acute myeloid leukemia (AML) and are associated with poor prognosis. Lack of mechanistic insight into NUP98-fusion-dependent oncogenic transformation has so far precluded the development of rational targeted therapies. We reasoned that different NUP98-fusion proteins deregulate a common set of transcriptional targets that might be exploitable for therapy. To decipher transcriptional programs controlled by diverse NUP98-fusion proteins, we developed mouse models for regulatable expression of NUP98/NSD1, NUP98/JARID1A, and NUP98/DDX10. By integrating chromatin occupancy profiles of NUP98-fusion proteins with transcriptome profiling upon acute fusion protein inactivation in vivo, we defined the core set of direct transcriptional targets of NUP98-fusion proteins. Among those, CDK6 was highly expressed in murine and human AML samples. Loss of CDK6 severely attenuated NUP98-fusion-driven leukemogenesis, and NUP98-fusion AML was sensitive to pharmacologic CDK6 inhibition in vitro and in vivo. These findings identify CDK6 as a conserved, critical direct target of NUP98-fusion proteins, proposing CDK4/CDK6 inhibitors as a new rational treatment option for AML patients with NUP98-fusions.
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http://dx.doi.org/10.1182/blood.2019003267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115844PMC
July 2020

Gain-of-Function Effects of N-Terminal CEBPA Mutations in Acute Myeloid Leukemia.

Bioessays 2020 02 23;42(2):e1900178. Epub 2019 Dec 23.

Institute for Medical Biochemistry, University of Veterinary Medicine, Vienna, 1210, Austria.

Mutations in the CEBPA gene are present in 10-15% of acute myeloid leukemia (AML) patients. The most frequent type of mutations leads to the expression of an N-terminally truncated variant of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), termed p30. While initial reports proposed that p30 represents a dominant-negative version of the wild-type C/EBPα protein, other studies show that p30 retains the capacity to actively regulate gene expression. Recent global transcriptomic and epigenomic analyses have advanced the understanding of the distinct roles of the p30 isoform in leukemogenesis. This review outlines direct and indirect effects of the C/EBPα p30 variant on oncogenic transformation of hematopoietic progenitor cells and discusses how studies of N-terminal CEBPA mutations in AML can be extrapolated to identify novel gain-of-function features in oncoproteins that arise from recurrent truncating mutations in transcription factors.
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http://dx.doi.org/10.1002/bies.201900178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115832PMC
February 2020

HBO1 is required for the maintenance of leukaemia stem cells.

Nature 2020 01 11;577(7789):266-270. Epub 2019 Dec 11.

Cancer Therapeutics CRC, Melbourne, Victoria, Australia.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs). Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
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http://dx.doi.org/10.1038/s41586-019-1835-6DOI Listing
January 2020

All-trans retinoic acid enhances, and a pan-RAR antagonist counteracts, the stem cell promoting activity of EVI1 in acute myeloid leukemia.

Cell Death Dis 2019 12 10;10(12):944. Epub 2019 Dec 10.

Division of Oncology, Clinic of Medicine I, Medical University of Vienna, Vienna, Austria.

Ecotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1 AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1 AML.
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http://dx.doi.org/10.1038/s41419-019-2172-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904467PMC
December 2019

A kinase-independent role for CDK8 in BCR-ABL1 leukemia.

Nat Commun 2019 10 18;10(1):4741. Epub 2019 Oct 18.

Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria.

Cyclin-dependent kinases (CDKs) are frequently deregulated in cancer and represent promising drug targets. We provide evidence that CDK8 has a key role in B-ALL. Loss of CDK8 in leukemia mouse models significantly enhances disease latency and prevents disease maintenance. Loss of CDK8 is associated with pronounced transcriptional changes, whereas inhibiting CDK8 kinase activity has minimal effects. Gene set enrichment analysis suggests that the mTOR signaling pathway is deregulated in CDK8-deficient cells and, accordingly, these cells are highly sensitive to mTOR inhibitors. Analysis of large cohorts of human ALL and AML patients reveals a significant correlation between the level of CDK8 and of mTOR pathway members. We have synthesized a small molecule YKL-06-101 that combines mTOR inhibition and degradation of CDK8, and induces cell death in human leukemic cells. We propose that simultaneous CDK8 degradation and mTOR inhibition might represent a potential therapeutic strategy for the treatment of ALL patients.
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http://dx.doi.org/10.1038/s41467-019-12656-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802219PMC
October 2019

STAT3β is a tumor suppressor in acute myeloid leukemia.

Blood Adv 2019 07;3(13):1989-2002

Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria.

Signal transducer and activator of transcription 3 (STAT3) exists in 2 alternatively spliced isoforms, STAT3α and STAT3β. Although truncated STAT3β was originally postulated to act as a dominant-negative form of STAT3α, it has been shown to have various STAT3α-independent regulatory functions. Recently, STAT3β gained attention as a powerful antitumorigenic molecule in cancer. Deregulated STAT3 signaling is often found in acute myeloid leukemia (AML); however, the role of STAT3β in AML remains elusive. Therefore, we analyzed the β/α messenger RNA (mRNA) expression ratio in AML patients, where we observed that a higher β/α mRNA ratio correlated with a favorable prognosis and increased overall survival. To gain better understanding of the function of STAT3β in AML, we engineered a transgenic mouse allowing for balanced β expression. Transgenic β expression resulted in decelerated disease progression and extended survival in PTEN- and MLL-AF9-dependent AML mouse models. Our findings further suggest that the antitumorigenic function of STAT3β depends on the tumor-intrinsic regulation of a small set of significantly up- and downregulated genes, identified via RNA sequencing. In conclusion, we demonstrate that STAT3β plays an essential tumor-suppressive role in AML.
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http://dx.doi.org/10.1182/bloodadvances.2018026385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616266PMC
July 2019

High activation of STAT5A drives peripheral T-cell lymphoma and leukemia.

Haematologica 2020 31;105(2):435-447. Epub 2020 Jan 31.

Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria.

Recurrent gain-of-function mutations in the transcription factors and much more in were found in hematopoietic malignancies with the highest proportion in mature T- and natural killer-cell neoplasms (peripheral T-cell lymphoma, PTCL). No targeted therapy exists for these heterogeneous and often aggressive diseases. Given the shortage of models for PTCL, we mimicked graded STAT5A or STAT5B activity by expressing hyperactive or variants at low or high levels in the hematopoietic system of transgenic mice. Only mice with high activity levels developed a lethal disease resembling human PTCL. Neoplasia displayed massive expansion of CD8 T cells and destructive organ infiltration. T cells were cytokine-hypersensitive with activated memory CD8 T-lymphocyte characteristics. Histopathology and mRNA expression profiles revealed close correlation with distinct subtypes of PTCL. Pronounced STAT5 expression and activity in samples from patients with different subsets underline the relevance of JAK/STAT as a therapeutic target. JAK inhibitors or a selective STAT5 SH domain inhibitor induced cell death and ruxolitinib blocked T-cell neoplasia We conclude that enhanced STAT5A or STAT5B action both drive PTCL development, defining both STAT5 molecules as targets for therapeutic intervention.
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http://dx.doi.org/10.3324/haematol.2019.216986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012494PMC
April 2021

Dependence on Myb expression is attenuated in myeloid leukaemia with N-terminal CEBPA mutations.

Life Sci Alliance 2019 04 15;2(2). Epub 2019 Mar 15.

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

Mutations at the N- or C-terminus of C/EBPα are frequent in acute myeloid leukaemia (AML) with normal karyotype. Here, we investigate the role of the transcription factor Myb in AMLs driven by different combinations of CEBPA mutations. Using knockdown of Myb in murine cell lines modelling the spectrum of CEBPA mutations, we show that the effect of reduced Myb depends on the mutational status of the two Cebpa alleles. Importantly, Myb knockdown fails to override the block in myeloid differentiation in cells with biallelic N-terminal C/EBPα mutations, demonstrating for the first time that the dependency on Myb is much lower in AML with this mutational profile. By comparing gene expression following Myb knockdown and chromatin immunoprecipitation sequencing data for the binding of C/EBPα isoforms, we provide evidence for a functional cooperation between C/EBPα and Myb in the maintenance of AML. This co-dependency breaks down when both alleles of CEBPA harbour N-terminal mutations, as a subset of C/EBPα-regulated genes only bind the short p30 C/EBPα isoform and, unlike other C/EBPα-regulated genes, do so without a requirement for Myb.
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http://dx.doi.org/10.26508/lsa.201800207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421631PMC
April 2019

Roles of SETD2 in Leukemia-Transcription, DNA-Damage, and Beyond.

Int J Mol Sci 2019 Feb 27;20(5). Epub 2019 Feb 27.

Ludwig Boltzmann Institute for Cancer Research, Waehringer Strasse 13A, 1090 Vienna, Austria.

The non-redundant histone methyltransferase SETD2 (SET domain containing 2; KMT3A) is responsible for tri-methylation of lysine 36 on histone H3 (H3K36me3). Presence of the H3K36me3 histone mark across the genome has been correlated with transcriptional activation and elongation, but also with the regulation of DNA mismatch repair, homologous recombination and alternative splicing. The role of SETD2 and the H3K36me3 histone mark in cancer is controversial. SETD2 is lost or mutated in various cancers, supporting a tumor suppressive role of the protein. Alterations in the SETD2 gene are also present in leukemia patients, where they are associated with aggressive disease and relapse. In line, heterozygous SETD2 loss caused chemotherapy resistance in leukemia cell lines and mouse models. In contrast, other studies indicate that SETD2 is critically required for the proliferation of leukemia cells. Thus, although studies of SETD2-dependent processes in cancer have contributed to a better understanding of the SETD2⁻H3K36me3 axis, many open questions remain regarding its specific role in leukemia. Here, we review the current literature about critical functions of SETD2 in the context of hematopoietic malignancies.
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http://dx.doi.org/10.3390/ijms20051029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429614PMC
February 2019

Altered splicing and cytoplasmic levels of tRNA synthetases in SF3B1-mutant myelodysplastic syndromes as a therapeutic vulnerability.

Sci Rep 2019 02 25;9(1):2678. Epub 2019 Feb 25.

Centre for Cancer Research and Cell Biology (CCRCB), Queen's University Belfast, Belfast, United Kingdom.

Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have uncovered recurrent somatic mutations within RNA splicing factors, including SF3B1, SRSF2, U2AF1 and ZRSR2. The most frequently mutated gene is SF3B1, mutated in 17% of MDS patients. While SF3B1 mutations and their effects on splicing have been well characterised, much remains to be explored about their more far-reaching effects on cellular homeostasis. Given that mRNA splicing and nuclear export are coordinated processes, we hypothesised that SF3B1 mutation might also affect export of certain mRNAs and that this may represent a targetable pathway for the treatment of SF3B1-mutant MDS. We used CRISPR/Cas9-genome editing to create isogenic cellular models. Comprehensive transcriptome and proteome profiling of these cells identified alterations in the splicing and export of components of the translational machinery, primarily tRNA synthetases, in response to the SF3B1 K700E mutation. While steady-state protein synthesis was unaffected, SF3B1 mutant cells were more sensitive to the clinically-relevant purine analogue, 8-azaguanine. In this study, we also demonstrated that 8-azaguanine affects splicing. Our results suggest that the simultaneous targeting of RNA metabolism and splicing by 8-azaguanine represents a therapeutic opportunity for SF3B1-mutant myelodysplastic syndromes.
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http://dx.doi.org/10.1038/s41598-019-39591-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390101PMC
February 2019

CEBPA-mutated leukemia is sensitive to genetic and pharmacological targeting of the MLL1 complex.

Leukemia 2019 07 24;33(7):1608-1619. Epub 2019 Jan 24.

Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria.

The gene encoding the transcription factor C/EBPα is mutated in 10-15% of acute myeloid leukemia (AML) patients. N-terminal CEBPA mutations cause ablation of full-length C/EBPα without affecting the expression of a shorter oncogenic isoform, termed p30. The mechanistic basis of p30-induced leukemogenesis is incompletely understood. Here, we demonstrate that the MLL1 histone-methyltransferase complex represents a critical actionable vulnerability in CEBPA-mutated AML. Oncogenic C/EBPα p30 and MLL1 show global co-localization on chromatin and p30 exhibits robust physical interaction with the MLL1 complex. CRISPR/Cas9-mediated mutagenesis of MLL1 results in proliferation arrest and myeloid differentiation in C/EBPα p30-expressing cells. In line, CEBPA-mutated hematopoietic progenitor cells are hypersensitive to pharmacological targeting of the MLL1 complex. Inhibitor treatment impairs proliferation and restores myeloid differentiation potential in mouse and human AML cells with CEBPA mutations. Finally, we identify the transcription factor GATA2 as a direct critical target of the p30-MLL1 interaction. Altogether, we show that C/EBPα p30 requires the MLL1 complex to regulate oncogenic gene expression and that CEBPA-mutated AML is hypersensitive to perturbation of the MLL1 complex. These findings identify the MLL1 complex as a potential therapeutic target in AML with CEBPA mutations.
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http://dx.doi.org/10.1038/s41375-019-0382-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612510PMC
July 2019

SETD2 in MLL-rearranged leukemia - a complex case.

Mol Cell Oncol 2018 13;5(4):e1503492. Epub 2018 Aug 13.

Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria.

Oncogenic MLL-fusion proteins often hijack essential molecular mechanisms during leukemogenesis. The histone methyltransferase SETD2 was implicated in the regulation of transcription, DNA damage and other cellular processes. Recent studies identified a critical role for SETD2 in MLL-rearranged leukemia. These results may help to unravel important functions of SETD2 in hematopoiesis.
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http://dx.doi.org/10.1080/23723556.2018.1503492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150042PMC
August 2018

Dependency on the TYK2/STAT1/MCL1 axis in anaplastic large cell lymphoma.

Leukemia 2019 03 21;33(3):696-709. Epub 2018 Aug 21.

Cancer Science Institute of Singapore, National University of Singapore, 117599, Singapore, Singapore.

TYK2 is a member of the JAK family of tyrosine kinases that is involved in chromosomal translocation-induced fusion proteins found in anaplastic large cell lymphomas (ALCL) that lack rearrangements activating the anaplastic lymphoma kinase (ALK). Here we demonstrate that TYK2 is highly expressed in all cases of human ALCL, and that in a mouse model of NPM-ALK-induced lymphoma, genetic disruption of Tyk2 delays the onset of tumors and prolongs survival of the mice. Lymphomas in this model lacking Tyk2 have reduced STAT1 and STAT3 phosphorylation and reduced expression of Mcl1, a pro-survival member of the BCL2 family. These findings in mice are mirrored in human ALCL cell lines, in which TYK2 is activated by autocrine production of IL-10 and IL-22 and by interaction with specific receptors expressed by the cells. Activated TYK2 leads to STAT1 and STAT3 phosphorylation, activated expression of MCL1 and aberrant ALCL cell survival. Moreover, TYK2 inhibitors are able to induce apoptosis in ALCL cells, regardless of the presence or absence of an ALK-fusion. Thus, TYK2 is a dependency that is required for ALCL cell survival through activation of MCL1 expression. TYK2 represents an attractive drug target due to its essential enzymatic domain, and TYK2-specific inhibitors show promise as novel targeted inhibitors for ALCL.
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http://dx.doi.org/10.1038/s41375-018-0239-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076043PMC
March 2019

Oxytocin-like signaling in ants influences metabolic gene expression and locomotor activity.

FASEB J 2018 Jun 25:fj201800443. Epub 2018 Jun 25.

Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

Ants are emerging model systems to study cellular signaling because distinct castes possess different physiologic phenotypes within the same colony. Here we studied the functionality of inotocin signaling, an insect ortholog of mammalian oxytocin (OT), which was recently discovered in ants. In Lasius ants, we determined that specialization within the colony, seasonal factors, and physiologic conditions down-regulated the expression of the OT-like signaling system. Given this natural variation, we interrogated its function using RNAi knockdowns. Next-generation RNA sequencing of OT-like precursor knock-down ants highlighted its role in the regulation of genes involved in metabolism. Knock-down ants exhibited higher walking activity and increased self-grooming in the brood chamber. We propose that OT-like signaling in ants is important for regulating metabolic processes and locomotion.-Liutkevičiūtė, Z., Gil-Mansilla, E., Eder, T., Casillas-Pérez, B., Di Giglio, M. G., Muratspahić, E., Grebien, F., Rattei, T., Muttenthaler, M., Cremer, S., Gruber, C. W. Oxytocin-like signaling in ants influences metabolic gene expression and locomotor activity.
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http://dx.doi.org/10.1096/fj.201800443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174076PMC
June 2018

MLL-fusion-driven leukemia requires SETD2 to safeguard genomic integrity.

Nat Commun 2018 05 18;9(1):1983. Epub 2018 May 18.

Ludwig Boltzmann Institute for Cancer Research, Vienna, 1090, Austria.

MLL-fusions represent a large group of leukemia drivers, whose diversity originates from the vast molecular heterogeneity of C-terminal fusion partners of MLL. While studies of selected MLL-fusions have revealed critical molecular pathways, unifying mechanisms across all MLL-fusions remain poorly understood. We present the first comprehensive survey of protein-protein interactions of seven distantly related MLL-fusion proteins. Functional investigation of 128 conserved MLL-fusion-interactors identifies a specific role for the lysine methyltransferase SETD2 in MLL-leukemia. SETD2 loss causes growth arrest and differentiation of AML cells, and leads to increased DNA damage. In addition to its role in H3K36 tri-methylation, SETD2 is required to maintain high H3K79 di-methylation and MLL-AF9-binding to critical target genes, such as Hoxa9. SETD2 loss synergizes with pharmacologic inhibition of the H3K79 methyltransferase DOT1L to induce DNA damage, growth arrest, differentiation, and apoptosis. These results uncover a dependency for SETD2 during MLL-leukemogenesis, revealing a novel actionable vulnerability in this disease.
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http://dx.doi.org/10.1038/s41467-018-04329-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959866PMC
May 2018
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