Devil facial tumour (DFT) disease, a transmissible cancer where the infectious agent is the tumour itself, has caused a dramatic decrease in Tasmanian devil numbers in the wild. The purpose of this ...study was to take a candidate gene/pathway approach to identify potentially perturbed genes or pathways in DFT. A fusion of chromosome 1 and X is posited as the initial event leading to the development of DFT, with the rearranged chromosome 1 material now stably maintained as the tumour spreads through the population. This hypothesis makes chromosome 1 a prime chromosome on which to search for mutations involved in tumourigenesis. As DFT1 has a Schwann cell origin, we selected genes commonly implicated in tumour pathways in human nerve cancers, or cancers more generally, to determine whether they were rearranged in DFT1, and mapped them using molecular cytogenetics. Many cancer-related genes were rearranged, such as the region containing the tumour suppressor NF2 and a copy gain for ERBB3, a member of the epidermal growth factor receptor family of receptor tyrosine kinases implicated in proliferation and invasion of tumours in humans. Our mapping results have provided strong candidates not previously detected by sequencing DFT1 genomes.
After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8–A9, are found in ...high amounts in human breast milk. We studied levels of S100A8–A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system.
We collected stool samples (n = 517) from full-term (n = 72) and preterm infants (n = 49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8–A9 by enzyme-linked immunosorbent assay and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9–/– mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays.
Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wild-type mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wild-type mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wild-type mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8–A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean delivery had lower levels of S100A8–A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria—particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants’ fecal samples associated with development of sepsis and obesity by age 2 years.
S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years.
Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed ...infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air–liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.
Although regulation of stem cell homeostasis by microRNAs (miRNAs) is well studied, it is unclear how individual miRNAs genomically encoded within an organized polycistron can interact to induce an ...integrated phenotype. miR-99a/100, let-7, and miR-125b paralogs are encoded in two tricistrons on human chromosomes 11 and 21. They are highly expressed in hematopoietic stem cells (HSCs) and acute megakaryoblastic leukemia (AMKL), an aggressive form of leukemia with poor prognosis. Here, we show that miR-99a/100∼125b tricistrons are transcribed as a polycistronic message transactivated by the homeobox transcription factor HOXA10. Integrative analysis of global gene expression profiling, miRNA target prediction, and pathway architecture revealed that miR-99a/100, let-7, and miR-125b functionally converge at the combinatorial block of the transforming growth factor β (TGFβ) pathway by targeting four receptor subunits and two SMAD signaling transducers. In addition, down-regulation of tumor suppressor genes adenomatous polyposis coli (APC)/APC2 stabilizes active β-catenin and enhances Wnt signaling. By switching the balance between Wnt and TGFβ signaling, the concerted action of these tricistronic miRNAs promoted sustained expansion of murine and human HSCs in vitro or in vivo while favoring megakaryocytic differentiation. Hence, our study explains the high phylogenetic conservation of the miR-99a/100∼125b tricistrons controlling stem cell homeostasis, the deregulation of which contributes to the development of AMKL.
Pyroptosis is a fulminant form of macrophage cell death, contributing to release of pro‐inflammatory cytokines. In humans, it depends on caspase 1/4‐activation of gasdermin D and is characterized by ...the release of cytoplasmic content. Pathogens apply strategies to avoid or antagonize this host response. We demonstrate here that a small accessory protein (PB1‐F2) of contemporary H5N1 and H3N2 influenza A viruses (IAV) curtails fulminant cell death of infected human macrophages. Infection of macrophages with a PB1‐F2‐deficient mutant of a contemporary IAV resulted in higher levels of caspase‐1 activation, cleavage of gasdermin D, and release of LDH and IL‐1β. Mechanistically, PB1‐F2 limits transition of NLRP3 from its auto‐repressed and closed confirmation into its active state. Consequently, interaction of a recently identified licensing kinase NEK7 with NLRP3 is diminished, which is required to initiate inflammasome assembly.
Synopsis
Contemporary influenza A viruses limit pyroptosis and IL‐1β production in human macrophages by the expression of PB1‐F2. Via its C‐terminus PB1‐F2 binds to the pyrin and LRR domains of NLRP3 and locks it in its closed confirmation.
PB1‐F2 deficient influenza A viruses induce more pyroptosis in human macrophages than isogenic wildtype strains.
PB1‐F2 binds to the pyrin and LRR domains of NLRP3.
The NLRP3‐PB1‐F2 complex does not bind to NEK7.
Contemporary influenza A viruses limit pyroptosis and IL‐1β production in human macrophages by the expression of PB1‐F2. Via its C‐terminus PB1‐F2 binds to the pyrin and LRR domains of NLRP3 and locks it in its closed confirmation.
Summary
Chemotherapy resistance is a major contributor to poor treatment responses and tumour relapse, the development of which has been strongly linked to the action of cancer stem cells (CSCs). ...Mounting evidence suggests that CSCs are reliant on low oxygen conditions and hypoxia‐inducible factors 1α and 2α (HIF1α and HIF2α) to maintain their stem cell features. Research in the last decade has begun to clarify the functional differences between the two HIFα subtypes (HIFαs). Here, we review and discuss these differences in relation to CSC‐associated drug resistance. Both HIFαs contribute to CSC survival but play different roles –HIF1α being more responsible for survival functions and HIF2α for stemness traits such as self‐renewal – and are sensitive to different degrees of hypoxia. Failure to account for physiologically relevant oxygen concentrations in many studies may influence the current understanding of the roles of HIFαs. We also discuss how hypoxia and HIFαs contribute to CSC drug resistance via promotion of ABC drug transporters Breast cancer resistance protein (BCRP), MDR1, and MRP1 and through maintenance of quiescence. Additionally, we explore the PI3K/AKT cell survival pathway that may support refractory cancer by promoting CSCs and activating both HIF1α and HIF2α. Accordingly, HIF1α and HIF2α inhibition, potentially via PI3K/AKT inhibitors, could reduce chemotherapy resistance and prevent cancer relapse.
Abstract 109
Three human miR-99∼125 clusters, each containing one miR-99/100, let-7 and miR-125 family member in identical polycistronic configuration, are located within or next to long non-coding ...RNA (lncRNA) host genes on distinct chromosomal regions. In this study, we investigated the integrative function of these three polycistron-miRNAs. Expression profiling in sorted leukemic blasts indicated an enrichment of mature miR-99∼125 miRNAs in acute megakaryoblastic leukemia (AMKL) and promyelocytic leukemia (APL). We demonstrated that intronic miR-99∼125 miRNAs are produced from one primary transcript with their lncRNA host genes and that the leukemogenic transcription factor and stem cell regulator HOXA10 acts as an upstream transcriptional activator. Therefore, we assessed the consequences of concerted activation of miR-125b, miR-99/100 and let-7 in the megakaryocytic and myelomonocytic system.
During megakaryocytic in vitro differentiation of hematopoietic stem and progenitor cells (HSPCs), lentiviral overexpression of the tricistron (miR-99a/let-7c/miR-125b-2) or miR-125b-2 alone increased proliferation and survival of megakaryocytic cells. Megakaryocytic/erythroid progenitors (MEP) were expanded upon tricistron and miR-125b-2 overexpression, and not by miR-99a and/or let-7c overexpression. In an unbiased triple-transduction RGB-competition assay using distinct color-marked (red/green/blue) monocistronic miRNA constructs, miR-125b-2 alone or in combination with miR-99a or let-7 conferred a selective growth advantage to murine and human HSPCs. In murine transplantation experiments, only combined expression of all three cluster-miRNAs led to the expansion and retention of an immature Gr-1(low)/Mac-1(+)/B220(-) cell population in the bone marrow. Similarly when the miR-99a/let-7c/miR-125b-2 tricistron-transduced HSPCs differentiated along the myelomonocytic lineage (liquid culture and methylcellulose colony-forming assay), we observed a major cell population of monomorphic, non-adherent cells devoid of granulocytic and monocytic markers, which was not present in the miR-99a-, let-7c- or miR-125b-2 single transduced cells.
Global gene expression profiling followed by qRT-PCR-validation of transduced HSPCs identified high confidence targets commonly or individually downregulated by the three miRNAs. Among these targets, we found TGFβ pathway agonists and Wnt antagonists to be enriched. miR-125b and -even stronger- the tricistron decreased the reporter activity of a SMAD response element in 293T cells treated with TGFβ and increased the activity of a TCF response element in synergy with a Wnt-activator (GSK3-inibitor). Western blotting and luciferase reporter assays demonstrated cooperative targeting of TGFBR1, ALK7, BMPR1a, BMPR2, SMAD2, SMAD4 and APC by the tricistron. Strikingly, inhibition of miR-125b by a lentiviral decoy sensitized TGFβ-resistant AMKL-cell lines (CMK and CMY) to TGFβ-mediated apoptosis and proliferation arrest. Conversely, overexpression of miR-125b or the tricistron rendered MV4:11 and NB4 cells resistance to TGFβ. miR-125b or the tricistron-transduced HSPCs displayed a marked increase in cell viability and cells in S-Phase upon TGFβ-treatment during megakaryocytic differentiation. The application of a TGFβ-inhibitor phenocopied the effects of miR-125b or the tricistron during megakaryocytic differentiation. These effects were not further enhanced by combined Wnt activation. In contrast, recapitulating the effects of the polycistron in the myelomonocytic lineage required Wnt-activation.
Collectively we deciphered the functional linkage of miR-99/100, let-7 and miR-125, which are produced from one primary transcript with their lncRNA host genes and are regulated by the stem cell regulator HOXA10. Our study creates a microRNA interaction network, wherein the concerted action of the three miRNAs converges at the TGFβ pathway by a combinatorial block of this pathway. During myelomonocytic commitment only the combination of all polycistron-miRNAs enhanced Wnt signaling to inhibit differentiation. These synthetic phenotypes form an epistatic circuit regulating stemness and developmental fate.
No relevant conflicts of interest to declare.
Abstract 213
Three human miR-99∼125 clusters, each containing one miR-99/100, let-7 and miR-125 family member in identical polycistronic configuration, are located within or next to long non-coding ...RNA (lncRNA) host genes on distinct chromosomal regions. The cluster miRNAs are highly expressed in hematopoietic stem and progenitor cells (HSPCs). The individual importance for stem cell homeostasis and differentiation of let-7 and miR-125 was extensively studied. However, genomic redundancy and high phylogenetic conservation of this ncRNA ensemble suggest a functionally linked role on gene regulatory networks, which remained elusive. Here we describe a synthetic phenotype pattern of the miR-99∼125 clusters on the hematopoietic system.
Expression profiling in CD34+-HSPCs (n=2) and megakaryocytes (n=1) from healthy donors, in sorted leukemic blasts from patients with Down syndrome (DS) acute megakaryoblastic leukemia (AMKL; n=5), DS-transient leukemia (n=4), non-DS-AMKL (n=3), acute myeloid leukemia (AML FAB M5; n=2) and in various cell lines (CMK, CMY, Meg01, M07e all megakaryoblastic leukemia, K562, KG1α, NB4, THP1 and Jurkat) indicated an enrichment of mature miR-99∼125 miRNAs and their lncRNA host genes in acute megakaryoblastic leukemia. Quantification of primary miRNA/mRNA transcripts indicated that intronic miR-99∼125 miRNAs are produced in one primary transcript with their lncRNA host genes C21ORF34 and MIR100HG. 5’RACE revealed the transcription start sites of those transcripts, which allowed mapping of phylogenetically conserved transcription factor binding sites in their promoter regions. Subsequent chromatin immunoprecipitation (ChIP) and luciferase reporter assays confirmed the binding and positive regulation by the leukemogenic transcription factor and stem cell regulator HOXA10.
Retroviral overexpression of miR-99∼125 cluster miRNAs in cord blood (CB)-HSPCs showed a recurrent phenotype. The tricistron (miR-99a/let-7c/miR-125b-2) and miR-125b-2 alone accelerated proliferation during megakaryocytic, neutrophilic and monocytic in vitro differentiation, while let-7c and miR-99a/let-7c blocked proliferation. Likewise, common myeloid (CMP) and megakaryocytic/erythroid progenitors (MEP) were expanded upon tricistron (CMP: 1.5-fold; MEP: 2-fold increase relative to control) and miR-125b-2 (CMP: 1.6-fold; MEP: 2.4-fold increase relative to control) overexpression and reduced upon let-7c (CMP: 2.1-fold; MEP: 2.5-fold reduction relative to control) and miR-99a/let-7c (CMP: 2.2-fold; MEP: 3-fold reduction relative to control) overexpression. Inversely, during induced megakaryocytic differentiation stable inhibition of miR-125b using sponge-technology (SP-miR125) or shRNA-mediated downregulation of the C21ORF34 host gene conferred a selective growth disadvantage (SP-miR125: 1.6-fold; shC21ORF34: 1.7-fold reduction relative to control) and inhibited colony-formation of AMKL cell lines (SP-miR125: 1.4-fold; shC21ORF34: 2-fold reduction relative to control) and CB-HSCs (SP-miR125: 1.7-fold; shC21ORF34: 3.7-fold reduction relative to control). Ectopic expression or stable inhibition of miR-99a alone had no effect in any assay.
Collectively we deciphered the functional linkage of miR-99/100, let-7 and miR-125, which are produced from one primary transcript with their lncRNA host genes. The stem cell regulator HOXA10 acts upstream of this ncRNA ensemble. Our study creates a microRNA interaction network, wherein miR-125b dominates let-7 to induce proliferation and expansion of CMPs and MEPs, favoring megakaryoblastic leukemia. This suggests an epistatic circuit regulating stemness and developmental fate.
No relevant conflicts of interest to declare.
Abstract 3614
Despite well-described anti-cancer effects and promising results in clinical trials, little is known regarding the selectivity of histone deacetylase (HDAC) inhibitors in killing ...malignant and sparing normal cells. Using Down syndrome associated myeloid leukemia (DS-AMKL) as a model for an exceptionally sensitive form of acute myeloid leukemia (AML), we elucidated a role of HDAC inhibitors in blocking autophagy, which exploits a specific vulnerability of several types of cancer.
We could show that HDAC1/2 inhibition efficiently inversed the DS-AMKL gene signature, resulting in p53-independent cell cycle arrest (BrdU incorporation and CFSE assay) and apoptosis (Annexin V/ 7-AAD and Nicoletti staining, caspase 3/7 activity assay) of DS-AMKL cell lines (CMK and CMY) and primary DS-AMKL leukemic blast. In contrast, control cell lines K562 and M07 were resistant to VPA-induced apoptosis. DS-AMKL is characterized by the high activation of the insulin-like growth factor (IGF) signaling cascade, which in turn results in the constitutive activation of mammalian target of rapamycin (mTOR). mTOR has been characterized as a major suppressor of autophagy, a lysosomal degradation process that takes place constitutively at a basal level and protects cells against damaged or unnecessary organelles and protein aggregates. Measuring endogenous LC3-I to LC3-II conversion levels by Western blot, a posttranslational modification during autophagy, we confirmed a lower basal autophagic activity in DS-AMKL cells compared to the control cell lines, K562 and M07 (LC3B-II/actin RU: 1.5 CMK, 1.0 CMY, 6.0 K562 and 3.0 M07).
Studying the effect of HDAC inhibition on autophagy, we detected an initial, dose-dependent increase in LC3-II band formation, punctae formation and decrease in total LC3-GFP cellular abundance (i.e., autophagy activation) 12h after VPA treatment in all cell lines (CMK, CMY, K562 and M07), indicative for induction of autophagy. However, prolonged exposure to VPA for up to 24h led to a block of autophagic flux, as shown by a gradual accumulation of LC3-GFP (fold increase in LC3-GFP MFI compared to untreated control: 1.3 CMK**, 1.1 CMY*, 2.0 K562** and 2.0 M07** at 2mM VPA; **p<0.01; *p<0.05).
Autophagy participates in the removal of damaged mitochondria, which prevents the initiation of intrinsic apoptotic pathway or DNA damage by increased ROS generation. Our results demonstrated a dose-dependent relationship between VPA-associated accumulation of mitochondrial mass and the intrinsic autophagic activity of the respective leukemic cell lines (fold increase in MitoTracker green MFI: 4.7 CMK**, 4.8 CMY**, 1.3 K562 and 1.2 M07 at 10mM VPA; **p<0.01). Likewise, the VPA-mediated mitochondrial mass accumulation correlated with ROS formation (fold increase in CM-H2DCFDA MFI: 3.3 CMK**, 2.7 CMY**, 1.5 K562** and 1.2 M07** at 10mM VPA; **p<0.01) and reflects the sensitivity to VPA induced apoptosis. In addition, we detected DNA-double strand breaks as indicated by an increase of phosphorylated H2AX.
The effects of HDAC inhibitors on DS-AMKL cells could be recapitulated by pharmacologic (autophagosome-lysosome fusion using vinblastine and nocodazole; autophagolysosomal degradation using ammonium chloride, chloroquine and hydroxychloroquine) and genetic (shRNA-mediated knockdown of ATG5 or ATG7) inhibition of autophagy, underlining dependency on their low basal level of autophagy. Inversely, induction of autophagy by starvation reversed the effects of HDAC inhibition on cell lines and primary DS-AMKL cells (i.e. induction of apoptosis, accumulation of mitochondria and ROS-production).
Thus, our study identified the role of HDAC inhibitors in blocking autophagy, exploiting the specific vulnerability of DS-AMKL cells with suppressed basic autophagy due to high mTOR activation. This was unexpected as previous studies proposed HDAC inhibitors as autophagy activators. VPA treatment of DS-AMKL cell lines and primary cells repressed autophagy below a critical threshold leading to accumulation of mitochondria, production of ROS, DNA-damage and apoptosis, which could be reverted upon autophagy activation. Our findings suggest repression of autophagy by HDAC inhibition as a novel treatment strategy in leukemic cells with highly active mTOR pathway. Display omitted
No relevant conflicts of interest to declare.
Abstract 3646
Children with Down syndrome (DS) are at high risk to develop acute megakaryoblastic leukemia (DS-AMKL) and the antecedent transient leukemia (DS-TL). Acquired mutations in the ...hematopoietic transcription factor GATA1, leading to expression of a shorter GATA1 variant (referred to as GATA1s) truncated at its N-terminus, are consistently present in the affected cells of children with DS-AMKL and DS-TL. Mechanistically, we recently found that in fetal megakaryocytic progenitor cells, GATA1 coordinates proliferation and differentiation by repressing E2F target genes through a direct interaction with E2F activators. Failure of this GATA1-E2F interaction in mutated GATA1s likely converges with overactive IGF signaling to promote cellular transformation.
The treatment of DS-AMKL is hampered by their sensitivity against current cytostatic agents, resulting in treatment-related mortality as the main cause of death. To develop novel targeted and less toxic treatment options for DS-AMKL and DS-TL, we conducted a gene expression-based chemical genomic screen. We connected a DS-AMKL gene expression signature (compared to non-DS-AMKL, i.e. GATA1s vs. GATA1) to a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules (Connectivity Map). We discovered the histone deacetylase (HDAC) inhibitor valproic acid (VPA) reverses the DS-AMKL gene expression program.
Cell viability assays, cell cycle analyses, growths curves and colony-forming assays revealed exceptional sensitivity of DS-AMKL cell lines (CMK, CMY; IC50 1mM) and primary DS-AMKL and DS-TL blasts to VPA treatment compared to control cell lines K562 (IC50 4.75mM), M07 (IC50 6.75mM) and CD34+ hematopoietic stem and progenitor cells (IC50 4.75mM). VPA induces apoptosis (26.8% 7-AAD-)Annexin V+ and 38.8% 7-AAD+ CMK cells after 48h at 2mM VPA) and cell cycle arrest (50% reduction of CMK cells in S-phase at 2mM) via activation of the cell cycle inhibitor p21 and the proapoptotic genes BAX and BAK. Gene expression profiles indicated that VPA interferes with the oncogenic effects of GATA1s by globally repressing the deregulated E2F targets. The effects of VPA on leukemic growth in DS-AMKL could be attributed to its HDAC inhibitory function, as the global HDAC inhibitors SAHA and TSA induced a similar response.
Thus, by using a gene expression-based chemical genomic approach, we identified VPA as an efficient and well-tolerated treatment option for DS-AMKL and DS-TL by targeting GATA1s-mediated deregulation of the E2F transcription network.
No relevant conflicts of interest to declare.
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