Publications by authors named "Johannes Zuber"

127 Publications

A functional LSD1 coregulator screen reveals a novel transcriptional regulatory cascade connecting R-loop homeostasis with epigenetic regulation.

Nucleic Acids Res 2021 Apr 6. Epub 2021 Apr 6.

Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, 70569 Stuttgart, Germany.

The lysine specific demethylase 1 (LSD1) plays a pivotal role in cellular differentiation by regulating the expression of key developmental genes in concert with different coregulatory proteins. This process is impaired in different cancer types and incompletely understood. To comprehensively identify functional coregulators of LSD1, we established a novel tractable fluorescent reporter system to monitor LSD1 activity in living cells. Combining this reporter system with a state-of-the-art multiplexed RNAi screen, we identify the DEAD-box helicase 19A (DDX19A) as a novel coregulator and demonstrate that suppression of Ddx19a results in an increase of R-loops and reduced LSD1-mediated gene silencing. We further show that DDX19A binds to tri-methylated lysine 27 of histone 3 (H3K27me3) and it regulates gene expression through the removal of transcription promoting R-loops. Our results uncover a novel transcriptional regulatory cascade where the downregulation of genes is dependent on the LSD1 mediated demethylation of histone H3 lysine 4 (H3K4). This allows the polycomb repressive complex 2 (PRC2) to methylate H3K27, which serves as a binding site for DDX19A. Finally, the binding of DDX19A leads to the efficient removal of R-loops at active promoters, which further de-represses LSD1 and PRC2, establishing a positive feedback loop leading to a robust repression of the target gene.
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http://dx.doi.org/10.1093/nar/gkab180DOI Listing
April 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

XPO7 is a tumor suppressor regulating p21-dependent senescence.

Genes Dev 2021 Mar 18;35(5-6):379-391. Epub 2021 Feb 18.

MRC London Institute of Medical Sciences (LMS), London W12 0NN, United Kingdom.

Senescence is a key barrier to neoplastic transformation. To identify senescence regulators relevant to cancer, we screened a genome-wide shRNA library. Here, we describe exportin 7 (XPO7) as a novel regulator of senescence and validate its function in telomere-induced, replicative, and oncogene-induced senescence (OIS). XPO7 is a bidirectional transporter that regulates the nuclear-cytoplasmic shuttling of a broad range of substrates. Depletion of XPO7 results in reduced levels of TCF3 and an impaired induction of the cyclin-dependent kinase inhibitor p21 during OIS. Deletion of correlates with poorer overall survival in several cancer types. Moreover, depletion of XPO7 alleviated OIS and increased tumor formation in a mouse model of liver cancer. Our results suggest that XPO7 is a novel tumor suppressor that regulates p21 expression to control senescence and tumorigenesis.
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http://dx.doi.org/10.1101/gad.343269.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919420PMC
March 2021

Ubiquitylation of MYC couples transcription elongation with double-strand break repair at active promoters.

Mol Cell 2021 02 15;81(4):830-844.e13. Epub 2021 Jan 15.

Theodor Boveri Institute, Department of Biochemistry and Molecular Biology, Biocenter, University of Würzburg, 97074 Würzburg, Germany. Electronic address:

The MYC oncoprotein globally affects the function of RNA polymerase II (RNAPII). The ability of MYC to promote transcription elongation depends on its ubiquitylation. Here, we show that MYC and PAF1c (polymerase II-associated factor 1 complex) interact directly and mutually enhance each other's association with active promoters. PAF1c is rapidly transferred from MYC onto RNAPII. This transfer is driven by the HUWE1 ubiquitin ligase and is required for MYC-dependent transcription elongation. MYC and HUWE1 promote histone H2B ubiquitylation, which alters chromatin structure both for transcription elongation and double-strand break repair. Consistently, MYC suppresses double-strand break accumulation in active genes in a strictly PAF1c-dependent manner. Depletion of PAF1c causes transcription-dependent accumulation of double-strand breaks, despite widespread repair-associated DNA synthesis. Our data show that the transfer of PAF1c from MYC onto RNAPII efficiently couples transcription elongation with double-strand break repair to maintain the genomic integrity of MYC-driven tumor cells.
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http://dx.doi.org/10.1016/j.molcel.2020.12.035DOI Listing
February 2021

Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors.

Cell Host Microbe 2021 02 16;29(2):267-280.e5. Epub 2020 Dec 16.

Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA. Electronic address:

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks.
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http://dx.doi.org/10.1016/j.chom.2020.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833927PMC
February 2021

Correction to: Combining the differentiating effect of panobinostat with the apoptotic effect of arsenic trioxide leads to significant survival benefit in a model of t(8;21) acute myeloid leukemia.

Clin Epigenetics 2020 Nov 18;12(1):178. Epub 2020 Nov 18.

Cancer Therapeutics Program, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, VIC, 3002, Australia.

An amendment to this paper has been published and can be accessed via the original article.
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http://dx.doi.org/10.1186/s13148-020-00964-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677833PMC
November 2020

A human tissue screen identifies a regulator of ER secretion as a brain-size determinant.

Science 2020 11 29;370(6519):935-941. Epub 2020 Oct 29.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria.

Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional cell cultures, which hinders the testing of gene functions requiring tissue context. Here, we present CRISPR-lineage tracing at cellular resolution in heterogeneous tissue (CRISPR-LICHT), which enables parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes, revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized , which regulates the endoplasmic reticulum (ER) function and extracellular matrix protein secretion crucial for tissue integrity, the dysregulation of which results in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain-size control.
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http://dx.doi.org/10.1126/science.abb5390DOI Listing
November 2020

SFPQ Depletion Is Synthetically Lethal with BRAF in Colorectal Cancer Cells.

Cell Rep 2020 09;32(12):108184

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health. Laboratory of Molecular Tumor Pathology and Systems Biology, Institute of Pathology, 10117 Berlin, Germany; German Cancer Consortium (DKTK), Partner Site Berlin and German Cancer Research Center (DKFZ), Heidelberg, Germany. Electronic address:

Oncoproteins such as the BRAF kinase endow cancer cells with malignant properties, but they also create unique vulnerabilities. Targeting of BRAF-driven cytoplasmic signaling networks has proved ineffective, as patients regularly relapse with reactivation of the targeted pathways. We identify the nuclear protein SFPQ to be synthetically lethal with BRAF in a loss-of-function shRNA screen. SFPQ depletion decreases proliferation and specifically induces S-phase arrest and apoptosis in BRAF-driven colorectal and melanoma cells. Mechanistically, SFPQ loss in BRAF-mutant cancer cells triggers the Chk1-dependent replication checkpoint, results in decreased numbers and reduced activities of replication factories, and increases collision between replication and transcription. We find that BRAF-mutant cancer cells and organoids are sensitive to combinations of Chk1 inhibitors and chemically induced replication stress, pointing toward future therapeutic approaches exploiting nuclear vulnerabilities induced by BRAF.
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http://dx.doi.org/10.1016/j.celrep.2020.108184DOI Listing
September 2020

Rational discovery of molecular glue degraders via scalable chemical profiling.

Nat Chem Biol 2020 11 3;16(11):1199-1207. Epub 2020 Aug 3.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Targeted protein degradation is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are molecular glues that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, we describe a scalable strategy toward glue degrader discovery that is based on chemical screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led us to identify compounds that induce ubiquitination and degradation of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, our data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.
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http://dx.doi.org/10.1038/s41589-020-0594-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116640PMC
November 2020

Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters.

Nat Biotechnol 2021 02 27;39(2):174-178. Epub 2020 Jul 27.

Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria.

We developed a functional lineage tracing tool termed CaTCH (CRISPRa tracing of clones in heterogeneous cell populations). CaTCH combines precise clonal tracing of millions of cells with the ability to retrospectively isolate founding clones alive before and during selection, allowing functional experiments. Using CaTCH, we captured rare clones representing as little as 0.001% of a population and investigated the emergence of resistance to targeted melanoma therapy in vivo.
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http://dx.doi.org/10.1038/s41587-020-0614-0DOI Listing
February 2021

Cohesin-Dependent and -Independent Mechanisms Mediate Chromosomal Contacts between Promoters and Enhancers.

Cell Rep 2020 07;32(3):107929

Nuclear Dynamics Programme, Babraham Institute, Cambridge CB22 3AT, UK; MRC London Institute of Medical Sciences, London W12 0NN, UK; Institute of Clinical Sciences, Faculty of Medicine, Imperial College, London W12 0NN, UK. Electronic address:

It is currently assumed that 3D chromosomal organization plays a central role in transcriptional control. However, depletion of cohesin and CTCF affects the steady-state levels of only a minority of transcripts. Here, we use high-resolution Capture Hi-C to interrogate the dynamics of chromosomal contacts of all annotated human gene promoters upon degradation of cohesin and CTCF. We show that a majority of promoter-anchored contacts are lost in these conditions, but many contacts with distinct properties are maintained, and some new ones are gained. The rewiring of contacts between promoters and active enhancers upon cohesin degradation associates with rapid changes in target gene transcription as detected by SLAM sequencing (SLAM-seq). These results provide a mechanistic explanation for the limited, but consistent, effects of cohesin and CTCF depletion on steady-state transcription and suggest the existence of both cohesin-dependent and -independent mechanisms of enhancer-promoter pairing.
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http://dx.doi.org/10.1016/j.celrep.2020.107929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383238PMC
July 2020

Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe.

Cell Death Dis 2020 07 1;11(7):499. Epub 2020 Jul 1.

Institute of Pathology, Charité Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.

To unravel vulnerabilities of KRAS-mutant CRC cells, a shRNA-based screen specifically inhibiting MAPK pathway components and targets was performed in CaCo2 cells harboring conditional oncogenic KRAS. The custom-designed shRNA library comprised 121 selected genes, which were previously identified to be strongly regulated in response to MEK inhibition. The screen showed that CaCo2 cells expressing KRAS were sensitive to the suppression of the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whereas KRAS CaCo2 cells were largely resistant to MCM7 suppression. Similar results were obtained in an isogenic DLD-1 cell culture model. Knockdown of MCM7 in a KRAS-mutant background led to replication stress as indicated by increased nuclear RPA focalization. Further investigation showed a significant increase in mitotic cells after simultaneous MCM7 knockdown and KRAS expression. The increased percentage of mitotic cells coincided with strongly increased DNA damage in mitosis. Taken together, the accumulation of DNA damage in mitotic cells is due to replication stress that remained unresolved, which results in mitotic catastrophe and cell death. In summary, the data show a vulnerability of KRAS-mutant cells towards suppression of MCM7 and suggest that inhibiting DNA replication licensing might be a viable strategy to target KRAS-mutant cancers.
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http://dx.doi.org/10.1038/s41419-020-2704-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330027PMC
July 2020

Leukemia Cell of Origin Influences Apoptotic Priming and Sensitivity to LSD1 Inhibition.

Cancer Discov 2020 Oct 30;10(10):1500-1513. Epub 2020 Jun 30.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts.

The cell of origin of oncogenic transformation is a determinant of therapeutic sensitivity, but the mechanisms governing cell-of-origin-driven differences in therapeutic response have not been delineated. Leukemias initiating in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and highly express the transcription factor (EVI1) compared with leukemias derived from myeloid progenitors. Here, we compared leukemias initiated in either HSCs or myeloid progenitors to reveal a novel function for EVI1 in modulating p53 protein abundance and activity. HSC-derived leukemias exhibit decreased apoptotic priming, attenuated p53 transcriptional output, and resistance to lysine-specific demethylase 1 (LSD1) inhibitors in addition to classical genotoxic stresses. p53 loss of function in progenitor-derived leukemias induces resistance to LSD1 inhibition, and EVI1 leukemias are sensitized to LSD1 inhibition by venetoclax. Our findings demonstrate a role for in p53 wild-type cancers in reducing p53 function and provide a strategy to circumvent drug resistance in chemoresistant acute myeloid leukemia. SIGNIFICANCE: We demonstrate that the cell of origin of leukemia initiation influences p53 activity and dictates therapeutic sensitivity to pharmacologic LSD1 inhibitors via the transcription factor EVI1. We show that drug resistance could be overcome in HSC-derived leukemias by combining LSD1 inhibition with venetoclax...
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http://dx.doi.org/10.1158/2159-8290.CD-19-1469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584353PMC
October 2020

Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles.

Nat Methods 2020 07 8;17(7):708-716. Epub 2020 Jun 8.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna BioCenter (VBC), Vienna, Austria.

CRISPR-Cas9 screens have emerged as a transformative approach to systematically probe gene functions. The quality and success of these screens depends on the frequencies of loss-of-function alleles, particularly in negative-selection screens widely applied for probing essential genes. Using optimized screening workflows, we performed essentialome screens in cancer cell lines and embryonic stem cells and achieved dropout efficiencies that could not be explained by common frameshift frequencies. We find that these superior effect sizes are mainly determined by the impact of in-frame mutations on protein function, which can be predicted based on amino acid composition and conservation. We integrate protein features into a 'Bioscore' and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR-Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles.
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http://dx.doi.org/10.1038/s41592-020-0850-8DOI Listing
July 2020

STAG1 vulnerabilities for exploiting cohesin synthetic lethality in STAG2-deficient cancers.

Life Sci Alliance 2020 07 28;3(7). Epub 2020 May 28.

Boehringer Ingelheim Regional Center Vienna (RCV) GmbH & Co KG, Vienna, Austria

The cohesin subunit has emerged as a recurrently inactivated tumor suppressor in human cancers. Using candidate approaches, recent studies have revealed a synthetic lethal interaction between and its paralog To systematically probe genetic vulnerabilities in the absence of STAG2, we have performed genome-wide CRISPR screens in isogenic cell lines and identified STAG1 as the most prominent and selective dependency of STAG2-deficient cells. Using an inducible degron system, we show that chemical genetic degradation of STAG1 protein results in the loss of sister chromatid cohesion and rapid cell death in STAG2-deficient cells, while sparing -wild-type cells. Biochemical assays and X-ray crystallography identify STAG1 regions that interact with the RAD21 subunit of the cohesin complex. STAG1 mutations that abrogate this interaction selectively compromise the viability of -deficient cells. Our work highlights the degradation of STAG1 and inhibition of its interaction with RAD21 as promising therapeutic strategies. These findings lay the groundwork for the development of STAG1-directed small molecules to exploit synthetic lethality in -mutated tumors.
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http://dx.doi.org/10.26508/lsa.202000725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266993PMC
July 2020

The EMT modulator SNAI1 contributes to AML pathogenesis via its interaction with LSD1.

Blood 2020 08;136(8):957-973

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.
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http://dx.doi.org/10.1182/blood.2019002548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441169PMC
August 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

Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis.

Oncotarget 2020 Mar 3;11(9):875-890. Epub 2020 Mar 3.

Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and . We show that BCL6 knock-out in SU-DHL-4 cells results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction.
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http://dx.doi.org/10.18632/oncotarget.27506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061739PMC
March 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

Interconversion between Tumorigenic and Differentiated States in Acute Myeloid Leukemia.

Cell Stem Cell 2019 08;25(2):258-272.e9

Australian Centre for Blood Diseases, Monash University, Commercial Road, Melbourne, VIC 3004, Australia. Electronic address:

Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within this hierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that specifically eradicate CSCs or induce their differentiation. The clinical success of these approaches depends on CSC differentiation being unidirectional rather than reversible, yet this question remains unresolved even in prototypically hierarchical malignancies, such as acute myeloid leukemia (AML). Here, we show in murine and human models of AML that, upon perturbation of endogenous expression of the lineage-determining transcription factor PU.1 or withdrawal of established differentiation therapies, some mature leukemia cells can de-differentiate and reacquire clonogenic and leukemogenic properties. Our results reveal plasticity of CSC maturation in AML, highlighting the need to therapeutically eradicate cancer cells across a range of differentiation states.
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http://dx.doi.org/10.1016/j.stem.2019.07.001DOI Listing
August 2019

Publisher Correction: Sequencing cell-type-specific transcriptomes with SLAM-ITseq.

Nat Protoc 2019 Aug;14(8):2597

Department of Genetics, University of Cambridge, Cambridge, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41596-019-0224-9DOI Listing
August 2019

Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy.

EMBO Mol Med 2019 08 24;11(8):e9266. Epub 2019 Jun 24.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria.

Angiogenesis is a hallmark of cancer, promoting growth and metastasis. Anti-angiogenic treatment has limited efficacy due to therapy-induced blood vessel alterations, often followed by local hypoxia, tumor adaptation, progression, and metastasis. It is therefore paramount to overcome therapy-induced resistance. We show that Apelin inhibition potently remodels the tumor microenvironment, reducing angiogenesis, and effectively blunting tumor growth. Functionally, targeting Apelin improves vessel function and reduces polymorphonuclear myeloid-derived suppressor cell infiltration. Importantly, in mammary and lung cancer, Apelin prevents resistance to anti-angiogenic receptor tyrosine kinase (RTK) inhibitor therapy, reducing growth and angiogenesis in lung and breast cancer models without increased hypoxia in the tumor microenvironment. Apelin blockage also prevents RTK inhibitor-induced metastases, and high Apelin levels correlate with poor prognosis of anti-angiogenic therapy patients. These data identify a druggable anti-angiogenic drug target that reduces tumor blood vessel densities and normalizes the tumor vasculature to decrease metastases.
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http://dx.doi.org/10.15252/emmm.201809266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685079PMC
August 2019

Sequencing cell-type-specific transcriptomes with SLAM-ITseq.

Nat Protoc 2019 08 26;14(8):2261-2278. Epub 2019 Jun 26.

Department of Genetics, University of Cambridge, Cambridge, UK.

Analysis of cell-type-specific transcriptomes is vital for understanding the biology of tissues and organs in the context of multicellular organisms. In this Protocol Extension, we combine a previously developed cell-type-specific metabolic RNA labeling method (thiouracil (TU) tagging) and a pipeline to detect the labeled transcripts by a novel RNA sequencing (RNA-seq) method, SLAMseq (thiol (SH)-linked alkylation for the metabolic sequencing of RNA). By injecting a uracil analog, 4-thiouracil, into transgenic mice that express cell-type-specific uracil phosphoribosyltransferase (UPRT), an enzyme required for 4-thiouracil incorporation into newly synthesized RNA, only cells expressing UPRT synthesize thiol-containing RNA. Total RNA isolated from a tissue of interest is then sequenced with SLAMseq, which introduces thymine to cytosine (T>C) conversions at the sites of the incorporated 4-thiouracil. The resulting sequencing reads are then mapped with the T>C-aware alignment software, SLAM-DUNK, which allows mapping of reads containing T>C mismatches. The number of T>C conversions per transcript is further analyzed to identify which transcripts are synthesized in the UPRT-expressing cells. Thus, our method, SLAM-ITseq (SLAMseq in tissue), enables cell-specific transcriptomics without laborious FACS-based cell sorting or biochemical isolation of the labeled transcripts used in TU tagging. In the murine tissues we assessed previously, this method identified ~5,000 genes that are expressed in a cell type of interest from the total RNA pool from the tissue. Any laboratory with access to a high-throughput sequencer and high-power computing can adapt this protocol with ease, and the entire pipeline can be completed in <5 d.
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http://dx.doi.org/10.1038/s41596-019-0179-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997029PMC
August 2019

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.

Nat Genet 2019 06 27;51(6):990-998. Epub 2019 May 27.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.
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http://dx.doi.org/10.1038/s41588-019-0413-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952269PMC
June 2019

Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets.

BMC Bioinformatics 2019 May 20;20(1):258. Epub 2019 May 20.

Center for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories, University of Vienna, Medical University of Vienna, Dr. Bohrgasse 9, VBC, 1030, Vienna, Austria.

Background: Methods to read out naturally occurring or experimentally introduced nucleic acid modifications are emerging as powerful tools to study dynamic cellular processes. The recovery, quantification and interpretation of such events in high-throughput sequencing datasets demands specialized bioinformatics approaches.

Results: Here, we present Digital Unmasking of Nucleotide conversions in K-mers (DUNK), a data analysis pipeline enabling the quantification of nucleotide conversions in high-throughput sequencing datasets. We demonstrate using experimentally generated and simulated datasets that DUNK allows constant mapping rates irrespective of nucleotide-conversion rates, promotes the recovery of multimapping reads and employs Single Nucleotide Polymorphism (SNP) masking to uncouple true SNPs from nucleotide conversions to facilitate a robust and sensitive quantification of nucleotide-conversions. As a first application, we implement this strategy as SLAM-DUNK for the analysis of SLAMseq profiles, in which 4-thiouridine-labeled transcripts are detected based on T > C conversions. SLAM-DUNK provides both raw counts of nucleotide-conversion containing reads as well as a base-content and read coverage normalized approach for estimating the fractions of labeled transcripts as readout.

Conclusion: Beyond providing a readily accessible tool for analyzing SLAMseq and related time-resolved RNA sequencing methods (TimeLapse-seq, TUC-seq), DUNK establishes a broadly applicable strategy for quantifying nucleotide conversions.
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http://dx.doi.org/10.1186/s12859-019-2849-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6528199PMC
May 2019

The histone chaperone CAF-1 cooperates with the DNA methyltransferases to maintain silencing in cytotoxic T cells.

Genes Dev 2019 06 11;33(11-12):669-683. Epub 2019 Apr 11.

The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, New York 10016, USA.

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the gene is stably repressed in CD8 T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in silencing. When CAF-1 was lacking, derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the locus. Instead, Dnmt3a deficiency sensitized CD8 T cells to derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the gene in CD8 T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.
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http://dx.doi.org/10.1101/gad.322024.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546056PMC
June 2019

Functional-genetic approaches to understanding drug response and resistance.

Curr Opin Genet Dev 2019 02 2;54:41-47. Epub 2019 Apr 2.

Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), 1030 Vienna, Austria; Medical University of Vienna, Vienna BioCenter (VBC), 1030 Vienna, Austria. Electronic address:

Drug development remains a slow and expensive process, while the effective use of established therapeutics is widely hampered by our limited understanding of response and resistance mechanisms. Functional-genetic tools such as CRISPR/Cas9, advanced RNAi methods, and targeted protein degradation, together with other emerging technologies such as time-resolved and single-cell transcriptomics, fundamentally change the way we can search for candidate therapeutic targets and evaluate them before drug development. In addition, for already available therapeutics these tools open vast opportunities for probing response mechanisms and predictive biomarkers, and thereby guide the development of personalized therapies. Here, we review promising applications and remaining limitations of recently established functional-genetic tools for high-throughput screening and the in-depth analysis of candidate targets and established drugs.
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http://dx.doi.org/10.1016/j.gde.2019.03.003DOI Listing
February 2019

Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute.

Nat Protoc 2019 03 1;14(3):756-780. Epub 2019 Feb 1.

Shanghai Key Laboratory of Tuberculosis, Clinical Translational Research Center, Shanghai Pulmonary Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.

Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by its comprehensive pipeline, which contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control (QC), normalization, batch effect removal, copy-number bias correction, gene hit identification and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires ~3 h on a desktop computer running Linux or Mac OS with R support.
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http://dx.doi.org/10.1038/s41596-018-0113-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6862721PMC
March 2019