Proinflammatory cytokines suppress nonsense-mediated RNA decay to impair regulated transcript isoform processing in pancreatic β cells.

Introduction: Proinflammatory cytokines are implicated in pancreatic ß cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of nonsense-mediated RNA decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß cells.

Methods: A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3, or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity.

Proinflammatory Cytokines Suppress Nonsense-Mediated RNA Decay to Impair Regulated Transcript Isoform Processing in Pancreatic β-Cells.

Proinflammatory cytokines are implicated in pancreatic β-cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of Nonsense-Mediated RNA Decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in β-cells. A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-βH3 or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity.

BloodSpot 3.0: a database of gene and protein expression data in normal and malignant haematopoiesis.

BloodSpot is a specialised database integrating gene expression data from acute myeloid leukaemia (AML) patients related to blood cell development and maturation. The database and interface has helped numerous researchers and clinicians to quickly get an overview of gene expression patterns in healthy and malignant haematopoiesis. Here, we present an update to our framework that includes protein expression data of sorted single cells.

TET2 lesions enhance the aggressiveness of CEBPA-mutant acute myeloid leukemia by rebalancing GATA2 expression.

The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPA) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal mutations (CEBPA), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations.

Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition.

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential.

The impact of SWI/SNF and NuRD inactivation on gene expression is tightly coupled with levels of RNA polymerase II occupancy at promoters.

SWI/SNF and NuRD are protein complexes that antagonistically regulate DNA accessibility. However, repression of their activities often leads to unanticipated changes in target gene expression (paradoxical), highlighting our incomplete understanding of their activities. Here we show that SWI/SNF and NuRD are in a tug-of-war to regulate PRC2 occupancy at lowly expressed and bivalent genes in mouse embryonic stem cells (mESCs).

ERG Functionally Overlaps with Other Ets Proteins in Promoting TH9 Cell Expression of Il9 during Allergic Lung Inflammation.

CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.

The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes.

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML.

Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis.

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment.

Transcription factor-driven coordination of cell cycle exit and lineage-specification in vivo during granulocytic differentiation : In memoriam Professor Niels Borregaard.

Differentiation of multipotent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues, and requires the coordinated induction of lineage-specific transcriptional programs and cell cycle withdrawal. To understand the underlying regulatory mechanisms of this fundamental process, we investigated how the tissue-specific transcription factors, CEBPA and CEBPE, coordinate cell cycle exit and lineage-specification in vivo during granulocytic differentiation. We demonstrate that CEBPA promotes lineage-specification by launching an enhancer-primed differentiation program and direct activation of CEBPE expression.

PTBP1 promotes hematopoietic stem cell maintenance and red blood cell development by ensuring sufficient availability of ribosomal constituents.

Ribosomopathies constitute a range of disorders associated with defective protein synthesis mainly affecting hematopoietic stem cells (HSCs) and erythroid development. Here, we demonstrate that deletion of poly-pyrimidine-tract-binding protein 1 (PTBP1) in the hematopoietic compartment leads to the development of a ribosomopathy-like condition. Specifically, loss of PTBP1 is associated with decreases in HSC self-renewal, erythroid differentiation, and protein synthesis.

H3K9 dimethylation safeguards cancer cells against activation of the interferon pathway.

Activation of interferon genes constitutes an important anticancer pathway able to restrict proliferation of cancer cells. Here, we demonstrate that the H3K9me3 histone methyltransferase (HMT) suppressor of variegation 3-9 homolog 1 (SUV39H1) is required for the proliferation of acute myeloid leukemia (AML) and find that its loss leads to activation of the interferon pathway. Mechanistically, we show that this occurs via destabilization of a complex composed of SUV39H1 and the two H3K9me2 HMTs, G9A and GLP.

Real-Time Search-Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics.

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed scMS requires high ion injection times and high-resolution spectra to quantify the single-cell signal; however, the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time-intensive quantification scan.

Quantitative single-cell proteomics as a tool to characterize cellular hierarchies.

Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy.

Basement membrane stiffness determines metastases formation.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores.

Targeted inhibition of cooperative mutation- and therapy-induced AKT activation in AML effectively enhances response to chemotherapy.

Most AML patients exhibit mutational activation of the PI3K/AKT signaling pathway, which promotes downstream effects including growth, survival, DNA repair, and resistance to chemotherapy. Herein we demonstrate that the inv(16)/KIT AML mouse model exhibits constitutive activation of PI3K/AKT signaling, which was enhanced by chemotherapy-induced DNA damage through DNA-PK-dependent AKT phosphorylation. Strikingly, inhibitors of either PI3K or DNA-PK markedly reduced chemotherapy-induced AKT phosphorylation and signaling leading to increased DNA damage and apoptosis of inv(16)/KIT AML cells in response to chemotherapy.

The ASXL1-G643W variant accelerates the development of CEBPA mutant acute myeloid leukemia.

ASXL1 is one of the most commonly mutated genes in myeloid malignancies, including Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). In order to further our understanding of the role of ASXL1 lesions in malignant hematopoiesis, we generated a novel knock-in mouse model carrying the most frequent ASXL1 mutation identified in MDS patients, p.G643WfsX12.

CCAAT enhancer binding protein alpha (CEBPA) biallelic acute myeloid leukaemia: cooperating lesions, molecular mechanisms and clinical relevance.

Recent advances in sequencing technologies have allowed for the identification of recurrent mutations in acute myeloid leukaemia (AML). The transcription factor CCAAT enhancer binding protein alpha (CEBPA) is frequently mutated in AML, and biallelic CEBPA-mutant AML was recognised as a separate disease entity in the recent World Health Organization classification. However, CEBPA mutations are co-occurring with other aberrations in AML, and together these lesions form the clonal hierarchy that comprises the leukaemia in the patient.

Inhibition of Upf2-Dependent Nonsense-Mediated Decay Leads to Behavioral and Neurophysiological Abnormalities by Activating the Immune Response.

In humans, disruption of nonsense-mediated decay (NMD) has been associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorder and intellectual disability. However, the mechanism by which deficient NMD leads to neurodevelopmental dysfunction remains unknown, preventing development of targeted therapies. Here we identified novel protein-coding UPF2 (UP-Frameshift 2) variants in humans with NDD, including speech and language deficits.

Mutant CEBPA directly drives the expression of the targetable tumor-promoting factor CD73 in AML.

The key myeloid transcription factor (TF), CEBPA, is frequently mutated in acute myeloid leukemia (AML), but the direct molecular effects of this leukemic driver mutation remain elusive. To investigate mutant AML, we performed microscale, in vivo chromatin immunoprecipitation sequencing and identified a set of aberrantly activated enhancers, exclusively occupied by the leukemia-associated CEBPA-p30 isoform. Comparing gene expression changes in human mutant AML and the corresponding mouse model, we identified , encoding CD73, as a cross-species AML gene with an upstream leukemic enhancer physically and functionally linked to the gene.

Heterozygous loss of Srp72 in mice is not associated with major hematological phenotypes.

Objectives: Familial cases of hematological malignancies are associated with germline mutations. In particular, heterozygous mutations of SRP72 correlate with the development of myelodysplasia and bone marrow aplasia in two families. The signal recognition particle 72 kDa protein (SRP72) is part of the SRP complex, responsible for targeting of proteins to the endoplasmic reticulum.

The splicing factor RBM25 controls MYC activity in acute myeloid leukemia.

Cancer sequencing studies have implicated regulators of pre-mRNA splicing as important disease determinants in acute myeloid leukemia (AML), but the underlying mechanisms have remained elusive. We hypothesized that "non-mutated" splicing regulators may also play a role in AML biology and therefore conducted an in vivo shRNA screen in a mouse model of CEBPA mutant AML. This has led to the identification of the splicing regulator RBM25 as a novel tumor suppressor.