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Progression of cystic kidney disease has been linked to activation of the mTORC1 signaling pathway. Yet the utility of mTORC1 inhibitors to treat patients with polycystic kidney disease remains controversial despite promising preclinical data. To define the cell intrinsic role of mTORC1 for cyst development, the mTORC1 subunit gene Raptorwas selectively inactivated in kidney tubular cells lacking cilia due to simultaneous deletion of the kinesin family member gene Kif3A. In contrast to a rapid onset of cyst formation and kidney failure in mice with defective ciliogenesis, both kidney function, cyst formation discerned by magnetic resonance imaging and overall survival were strikingly improved in mice additionally lacking Raptor. However, these mice eventually succumbed to cystic kidney disease despite mTORC1 inactivation. In-depth transcriptome analysis revealed the rapid activation of other growth-promoting signaling pathways, overriding the effects of mTORC1 deletion and identified cyclin-dependent kinase (CDK) 4 as an alternate driver of cyst growth. Additional inhibition of CDK4-dependent signaling by the CDK4/6 inhibitor Palbociclib markedly slowed disease progression in mice and human organoid models of polycystic kidney disease and potentiated the effects of mTORC1 deletion/inhibition. Our findings indicate that cystic kidneys rapidly adopt bypass mechanisms typically observed in drug resistant cancers. Thus, future clinical trials need to consider combinatorial or sequential therapies to improve therapeutic efficacy in patients with cystic kidney disease.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) constitutes a rare and aggressive malignancy originating from plasmacytoid dendritic cells (pDCs) with a primarily cutaneous tropism followed by dissemination to the bone marrow and other organs. We conducted a genome-wide analysis of the tumor methylome in an extended cohort of 45 BPDCN patients supplemented by WES and RNA-seq as well as ATAC-seq on selected cases. We determined the BPDCN DNA methylation profile and observed a dramatic loss of DNA methylation during malignant transformation from early and mature DCs towards BPDCN. DNA methylation profiles further differentiate between BPDCN, AML, CMML, and T-ALL exhibiting the most striking global demethylation, mitotic stress, and merely localized DNA hypermethylation in BPDCN resulting in pronounced inactivation of tumor suppressor genes by comparison. DNA methylation-based analysis of the tumor microenvironment by MethylCIBERSORT yielded two, prognostically relevant clusters (IC1 and IC2) with specific cellular composition and mutational spectra. Further, the transcriptional subgroups of BPDCN (C1 and C2) differ by DNA methylation signatures in interleukin/inflammatory signaling genes but also by higher transcription factor activity of JAK-STAT and NFkB signaling in C2 in contrast to an EZH2 dependence in C1-BPDCN. Our integrative characterization of BPDCN offers novel molecular insights and potential diagnostic applications.

BackgroundDuplications at the Xp21.2 locus have previously been linked to 46,XY gonadal dysgenesis (GD), which is thought to result from gene dosage effects of NR0B1(DAX1), but the exact disease mechanism remains unknown.MethodsPatients with 46,XY GD were analysed by whole genome sequencing. Identified structural variants were confirmed by array CGH and analysed by high-throughput chromosome conformation capture (Hi-C).ResultsWe identified two unrelated patients: one showing a complex rearrangement upstream of NR0B1and a second harbouring a 1.2 Mb triplication, including NR0B1. Whole genome sequencing and Hi-C analysis revealed the rewiring of a topological-associated domain (TAD) boundary close to NR0B1associated with neo-TAD formation and may cause enhancer hijacking and ectopic NR0B1expression. Modelling of previous Xp21.2 structural variations associated with isolated GD support our hypothesis and predict similar neo-TAD formation as well as TAD fusion.ConclusionHere we present a general mechanism how deletions, duplications or inversions at the NR0B1locus can lead to partial or complete GD by disrupting the cognate TAD in the vicinity of NR0B1. This model not only allows better diagnosis of GD with copy number variations (CNVs) at Xp21.2, but also gives deeper insight on how spatiotemporal activation of developmental genes can be disrupted by reorganised TADs causing impairment of gonadal development.

Mutated and unmutated IgE and IgG play different and partly opposing roles in allergy development, but the mechanisms controlling their relative production are incompletely understood. Here, we analyzed the IgE-response in murine food allergy. Deep sequencing of the complementary-determining region (CDR) repertoires indicated that an ongoing unmutated extrafollicular IgE response coexists with a germinal center response, even after long-lasting allergen challenges. Despite overall IgG1-dominance, a significant proportion of clonotypes contained several-fold more IgE than IgG1. Clonotypes with differential bias to either IgE or IgG1 showed distinct hypermutation and clonal expansion. Hypermutation rates were associated with different physiochemical binding properties of individual B-cell receptors (BCR). Increasing BCR signaling strength inhibited class switching from IgG1 to IgE in vitro, preferentially constraining IgE formation. These data indicate that antigen-binding properties of individual BCRs determine differential IgE hypermutation and IgE versus IgG1 production on the level of single B-cell clones.

Mutated and unmutated IgE and IgG play different and partly opposing roles in allergy development, but the mechanisms controlling their relative production are incompletely understood. Here, we analyzed the IgE-response in murine food allergy. Deep sequencing of the complementary-determining region (CDR) repertoires indicated that an ongoing unmutated extrafollicular IgE response coexists with a germinal center response, even after long-lasting allergen challenges. Despite overall IgG1-dominance, a significant proportion of clonotypes contained several-fold more IgE than IgG1. Clonotypes with differential bias to either IgE or IgG1 showed distinct hypermutation and clonal expansion. Hypermutation rates were associated with different physiochemical binding properties of individual B-cell receptors (BCR). Increasing BCR signaling strength inhibited class switching from IgG1 to IgE in vitro, preferentially constraining IgE formation. These data indicate that antigen-binding properties of individual BCRs determine differential IgE hypermutation and IgE versus IgG1 production on the level of single B-cell clones.

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240Kand RPA1V227Aproteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270Ahas binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240Kmutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1variants in individuals with TBD/STS.

Plasmablastic lymphoma (PBL) represents a clinically heterogeneous subtype of aggressive B-cell non-Hodgkin lymphoma. Targeted-sequencing studies and a single-center whole-exome sequencing (WES) study in HIV-positive patients recently revealed several genes associated with PBL pathogenesis; however, the global mutational landscape and transcriptional profile of PBL remain elusive. To inform on disease-associated mutational drivers, mutational patterns, and perturbed pathways in HIV-positive and HIV-negative PBL, we performed WES and transcriptome sequencing (RNA-sequencing) of 33 PBL tumors. Integrative analysis of somatic mutations and gene expression profiles was performed to acquire insights into the divergent genotype–phenotype correlation in Epstein-Barr virus–positive (EBV+) and EBV– PBL. We describe a significant accumulation of mutations in the JAK signal transducer and transcription activator (OSMR, STAT3, PIM1, and SOCS1), as well as receptor tyrosine-kinase RAS (ERBB3, NRAS, PDGFRB, and NTRK) pathways. We provide further evidence of frequent perturbances of NF-κB signaling (NFKB2 and BTK). Induced pathways, identified by RNA-sequencing, closely resemble the mutational profile regarding alterations accentuated in interleukin-6/JAK/STAT signaling, NF-κB activity, and MYC signaling. Moreover, class I major histocompatibility complex–mediated antigen processing and cell cycle regulation were significantly affected by EBV status. An almost exclusive upregulation of phosphatidylinositol 3-kinase/AKT/mTOR signaling in EBV+ PBL and a significantly induced expression of NTRK3 in concert with recurrent oncogenic mutations in EBV– PBL hint at a specific therapeutically targetable mechanism in PBL subgroups. Our characterization of a mutational and transcriptomic landscape in PBL, distinct from that of diffuse large B-cell lymphoma and multiple myeloma, substantiates the pathobiological independence of PBL in the spectrum of B-cell malignancies and thereby refines the taxonomy for aggressive lymphomas.

Plasmablastic lymphoma (PBL) represents a clinically heterogeneous subtype of aggressive B-cell non-Hodgkin lymphoma. Targeted-sequencing studies and a single-center whole-exome sequencing (WES) study in HIV-positive patients recently revealed several genes associated with PBL pathogenesis; however, the global mutational landscape and transcriptional profile of PBL remain elusive. To inform on disease-associated mutational drivers, mutational patterns, and perturbed pathways in HIV-positive and HIV-negative PBL, we performed WES and transcriptome sequencing (RNA-sequencing) of 33 PBL tumors. Integrative analysis of somatic mutations and gene expression profiles was performed to acquire insights into the divergent genotype–phenotype correlation in Epstein-Barr virus–positive (EBV+) and EBV–PBL. We describe a significant accumulation of mutations in the JAK signal transducer and transcription activator (OSMR, STAT3, PIM1,and SOCS1), as well as receptor tyrosine-kinase RAS (ERBB3, NRAS, PDGFRB,and NTRK) pathways. We provide further evidence of frequent perturbances of NF-κB signaling (NFKB2and BTK). Induced pathways, identified by RNA-sequencing, closely resemble the mutational profile regarding alterations accentuated in interleukin-6/JAK/STAT signaling, NF-κB activity, and MYC signaling. Moreover, class I major histocompatibility complex–mediated antigen processing and cell cycle regulation were significantly affected by EBV status. An almost exclusive upregulation of phosphatidylinositol 3-kinase/AKT/mTOR signaling in EBV+PBL and a significantly induced expression of NTRK3in concert with recurrent oncogenic mutations in EBV–PBL hint at a specific therapeutically targetable mechanism in PBL subgroups. Our characterization of a mutational and transcriptomic landscape in PBL, distinct from that of diffuse large B-cell lymphoma and multiple myeloma, substantiates the pathobiological independence of PBL in the spectrum of B-cell malignancies and thereby refines the taxonomy for aggressive lymphomas.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mproinduces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood–brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

Deficiency of the transcription factor GATA2 is a highly penetrant genetic disorder predisposing to myelodysplastic syndromes (MDS) and immunodeficiency. It has been recognized as the most common cause underlying primary MDS in children. Triggered by the discovery of a recurrent synonymous GATA2variant, we systematically investigated 911 patients with phenotype of pediatric MDS or cellular deficiencies for the presence of synonymous alterations in GATA2. In total, we identified nine individuals with five heterozygous synonymous mutations: c.351C>G, p.T117T (N= 4); c.649C>T, p.L217L; c.981G>A, p.G327G; c.1023C>T, p.A341A; and c.1416G>A, p.P472P (N= 2). They accounted for 8.2% (9/110) of cases with GATA2 deficiency in our cohort and resulted in selective loss of mutant RNA. While for the hotspot mutation (c.351C>G) a splicing error leading to RNA and protein reduction was identified, severe, likely late stage RNA loss without splicing disruption was found for other mutations. Finally, the synonymous mutations did not alter protein function or stability. In summary, synonymous GATA2substitutions are a new common cause of GATA2 deficiency. These findings have broad implications for genetic counseling and pathogenic variant discovery in Mendelian disorders.

Images of differentiating PC12 cells were used to construct models of cell shape, nuclear shape, and mitochondrial distribution. Likely trajectories that a single cell would have followed if it had been observed at each time point were found, and synthetic movies made that show the extensive cell–cell variation in rate and extent of differentiation.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental non-malignant cells for survival. We compared the transcriptomes of primary CLL cells cocultured or not with protective bone marrow stromal cells (BMSCs) and found that oxidative phosphorylation, mitochondrial function, and hypoxic signaling undergo most significant dysregulation in non-protected CLL cells, with the changes peaking at 6–8 h, directly before induction of apoptosis. A subset of CLL patients displayed a gene expression signature resembling that of cocultured CLL cells and had significantly worse progression-free and overall survival. To identify drugs blocking BMSC-mediated support, we compared the relevant transcriptomic changes to the Connectivity Map database. Correlation was found with the transcriptomic signatures of the cardiac glycoside ouabain and of the ipecac alkaloids emetine and cephaeline. These compounds were highly active against protected primary CLL cells (relative IC50's 287, 190, and 35 nM, respectively) and acted by repressing HIF-1α and disturbing intracellular redox homeostasis. We tested emetine in a murine model of CLL and observed decreased CLL cells in peripheral blood, spleen, and bone marrow, recovery of hematological parameters and doubling of median survival (31.5 vs. 15 days, P= 0.0001). Pathways regulating redox homeostasis are thus therapeutically targetable mediators of microenvironmental support in CLL cells.

Intravenous Ig (IVIg) is used to treat mucous membrane pemphigoid, although its therapeutic effectivity is not sufficiently supported by randomized controlled clinical trials, and its mode of action is only insufficiently understood. We have examined the effect of IVIg in a mouse model of anti–laminin 332 mucous membrane pemphigoid and found that IVIg ameliorates both cutaneous and mucosal inflammatory lesions. Our investigation into the modes of action of IVIg in mucous membrane pemphigoid indicated effective anti-inflammatory mechanisms beyond the enhanced degradation of IgG mediated through inhibition of the FcRn. Our results suggest that IVIg curbs the activation of neutrophils at several levels. This includes a direct, immediate inhibitory effect on neutrophil activation by immune complexes but not C5a, which blunts the release of ROS and leukotriene B4from neutrophils. IVIg also suppresses the formation of neutrophil extracellular traps in response to calcium ion ionophore. In vivo treatment with IVIg altered the transcriptome of blood leukocytes and bone marrow neutrophils toward less proinflammatory phenotypes. Collectively, our results support the effectivity of IVIg in the treatment of mucous membrane pemphigoid and indicate that effects on neutrophils at multiple levels may significantly contribute to its therapeutic effects.

Damage to and loss of glomerular podocytes has been identified as the culprit lesion in progressive kidney diseases. Here, we combine mass spectrometry-based proteomics with mRNA sequencing, bioinformatics, and hypothesis-driven studies to provide a comprehensive and quantitative map of mammalian podocytes that identifies unanticipated signaling pathways. Comparison of the in vivodatasets with proteomics data from podocyte cell cultures showed a limited value of available cell culture models. Moreover, in vivostable isotope labeling by amino acids uncovered surprisingly rapid synthesis of mitochondrial proteins under steady-state conditions that was perturbed under autophagy-deficient, disease-susceptible conditions. Integration of acquired omics dimensions suggested FARP1 as a candidate essential for podocyte function, which could be substantiated by genetic analysis in humans and knockdown experiments in zebrafish. This work exemplifies how the integration of multi-omics datasets can identify a framework of cell-type-specific features relevant for organ health and disease.

ABSTRACTDNA methylation is recognized as one of several epigenetic regulators of gene expression and as potential driver of carcinogenesis through gene-silencing of tumor suppressors and activation of oncogenes. However, abnormal methylation, even of promoter regions, does not necessarily alter gene expression levels, especially if the gene is already silenced, leaving the exact mechanisms of methylation unanswered. Using a large cohort of matching DNA methylation and gene expression samples of colorectal cancer (CRC; n = 77) and normal adjacent mucosa tissues (n = 108), we investigated the regulatory role of methylation on gene expression. We show that on a subset of genes enriched in common cancer pathways, methylation is significantly associated with gene regulation through gene-specific mechanisms. We built two classification models to infer gene regulation in CRC from methylation differences of tumor and normal tissues, taking into account both gene-silencing and gene-activation effects through hyper- and hypo-methylation of CpGs. The classification models result in high prediction performances in both training and independent CRC testing cohorts (0.92

BRAF mutations occur in ~10% of colorectal cancer (CRC) and are associated with poor prognosis. Inhibitors selective for the BRAFV600Eoncoprotein, the most common BRAF mutant, elicit only poor response rates in BRAF-mutant CRC as single agents. This unresponsiveness was mechanistically attributed to the loss of negative feedbacks on the epidermal growth factor receptor (EGFR) and initiated clinical trials that combine BRAF (and MEK) inhibitors, either singly or in combination, with the anti-EGFR antibodies cetuximab or panitumumab. First results of these combinatorial studies demonstrated improved efficacy, however, the response rates still were heterogeneous. Here, we show that BRAF inhibition leads to the upregulation of a variety of receptor tyrosine kinases (RTKs) in CRC cell lines, including not only the EGFR, but also human epidermal growth factor receptor (HER) 2 and HER3. Importantly, combination of the BRAF inhibitors (BRAFi) vemurafenib (PLX4032), dabrafenib, or encorafenib with inhibitors dually targeting the EGFR and HER2 (such as lapatinib, canertinib, and afatinib) significantly reduced the metabolic activity and proliferative potential of CRC cells. This re-sensitization was also observed after genetic depletion of HER2 or HER3. Interestingly, BRAF inhibitors did not only upregulate RTKs, but also increased the abundance of the GRB2-associated binders (Gab) 1 and Gab2, two important amplifiers of RTK signaling. An allele-specific shRNA-mediated knockdown of BRAFV600Erevealed that Gab2 upregulation was directly dependent on the loss of the oncoprotein and was not caused by an “off-target” effect of these kinase inhibitors. Furthermore, Gab2 and Gab2-mediated Shp2 signaling were shown to be functionally important in BRAFi resistance. These findings highlight potential new escape mechanisms to these targeted therapies and indicate that a broad suppression of RTK signaling might be beneficial and should be taken into account in future research addressing targeted therapy in BRAF-mutant CRC.

Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD+leukemia cells. This synergized with the allogeneic CD8+T cell response, leading to long-term survival in six mouse models of FLT3-ITD+AML. Sorafenib-related IL-15 production caused an increase in CD8+CD107a+IFN-γ+T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD+AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8+T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7–IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.

Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously.

-Presence of GFI1-36N impedes homologous recombination and MGMT-mediated DNA repair selectively in AML cells.-Use of temozolomide and olaparib allows selective targeting of GFI1-36Nleukemic cells.

Supplemental Digital Content is available in the text.

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

Cancer cell differentiation is an important field of discussion in the light of cancer stem cells. In a recent study by Herr et al. (2015) “B-RAF inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells” we described how inhibition of mutant BRAF in colorectal cancer cell lines induces cell re-differentiation that is correlated with the loss of tumor growth in vitroand in vivo. We used Illumina HumanHT-12 v4 Expression BeadChip to characterize the gain of differentiation of PLX4720-treated 3D cultures of HT29 and Colo-205 cells. Here, we describe the experimental design and statistical analysis that were performed on the data set leading to the above hypothesis. The data are publicly available at the Gene Expression Omnibus (GEO) database under the accession number GSE50791.

Esophageal cancers are highly aggressive tumors with poor prognosis despite some recent advances in surgical and radiochemotherapy treatment options. This study addressed the feasibility of drugs targeting epigenetic modifiers in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells. We tested inhibition of histone deacetylases (HDACs) by SAHA, MS-275, and FK228, inhibition of DNA methyltransferases by Azacytidine (AZA) and Decitabine (DAC), and the effect of combination treatment using both types of drugs. The drug targets, HDAC1/2/3 and DNMT1, were expressed in normal esophageal epithelium and tumor cells of ESCC or EAC tissue specimens, as well as in non-neoplastic esophageal epithelial (Het-1A), ESCC (OE21, Kyse-270, Kyse-410), and EAC (OE33, SK-GT-4) cell lines. In vitro, HDAC activity, histone acetylation, and p21 expression were similarly affected in non-neoplastic, ESCC, and EAC cell lines post inhibitor treatment. Combined MS-275/AZA treatment, however, selectively targeted esophageal cancer cell lines by inducing DNA damage, cell viability loss, and apoptosis, and by decreasing cell migration. Non-neoplastic Het-1A cells were protected against HDACi (MS-275)/AZA treatment. RNA transcriptome analyses post MS-275 and/or AZA treatment identified novel regulated candidate genes (up: BCL6, Hes2; down: FAIM, MLKL), which were specifically associated with the treatment responses of esophageal cancer cells. In summary, combined HDACi/AZA treatment is efficient and selective for the targeting of esophageal cancer cells, despite similar target expression of normal and esophageal cancer epithelium, in vitroand in human esophageal carcinomas. The precise mechanisms of action of treatment responses involve novel candidate genes regulated by HDACi/AZA in esophageal cancer cells. Together, targeting of epigenetic modifiers in esophageal cancers may represent a potential future therapeutic approach.

Keratinocytes account for 95% of all cells of the epidermis, the stratified squamous epithelium forming the outer layer of the skin, in which a significant number of skin diseases takes root. Immortalized keratinocyte cell lines are often used as research model systems providing standardized, reproducible, and homogenous biological material. Apart from that, primary human keratinocytes are frequently used for medical studies because the skin provides an important route for drug administration and is readily accessible for biopsies. However, comparability of these cell systems is not known. Cell lines may undergo phenotypic shifts and may differ from the in vivosituation in important aspects. Primary cells, on the other hand, may vary in biological functions depending on gender and age of the donor and localization of the biopsy specimen. Here we employed metabolic labeling in combination with quantitative mass spectrometry-based proteomics to assess A431 and HaCaT cell lines for their suitability as model systems. Compared with cell lines, comprehensive profiling of the primary human keratinocyte proteome with respect to gender, age, and skin localization identified an unexpected high proteomic consistency. The data were analyzed by an improved ontology enrichment analysis workflow designed for the study of global proteomics experiments. It enables a quick, comprehensive and unbiased overview of altered biological phenomena and links experimental data to literature. We guide through our workflow, point out its advantages compared with other methods and apply it to visualize differences of cell lines compared with primary human keratinocytes.

Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb−/−, and Ctsl−/−mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl−/−skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl−/−skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl−/−or Ctsb−/−samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.

Beneficial microorganisms on the skin contribute to the first line of defense against attacking pathogens. However, instability of the skin microbiota is associated with skin diseases. Hence, temporal analyses are crucial because they serve as a baseline to understand the development of dysbiosis in disease. In this study, we aim to improve the understanding of the fungal skin microbiota, the mycobiota, in healthy subjects. Skin swabs were taken monthly for a year from four different skin sites, that is, antecubital crease, dorsal neck, glabella, and vertex, and analyzed by DNA sequencing of the internal transcribed spacer 1 region. The mycobiota on the skin was dominated by the class Malasseziomycetes, and the core community was composed of Malassezia restricta, M. globosa, and M. sympodialisat all skin sites. Over the period of 1 year, the intrapersonal mycobiota remained largely stable, with some fluctuations of low abundant non-Malasseziafungi. We conclude that despite fluctuations of low abundant classes, fungal skin communities form a temporally robust and individual fingerprint in healthy subjects.

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by skin blistering and wounds. To uncover changes in skin and mucosal microbiome related to age and disease progression, and microbiome impact on clinical and inflammatory laboratory parameters, swabs from wounded and unwounded skin, oral mucosa and stool samples of 28 children with RDEB and 28 healthy controls were subjected to 16S-rRNA gene sequencing.

Niemeyer: Celgene: Consultancy; Novartis: Consultancy.

Dyskeratosis congenita (DC) is a short telomere syndrome with bone marrow failure (BMF), mucocutaneous fragility and predisposition to malignancy due to inherited mutations in telomere associated genes. The genetic cause remains unknown in 10-20% of DC patients. Here, we describe a new DC candidate gene, RPA1, which encodes the largest subunit of the Replication Protein A (RPA) complex, a single stranded DNA (ssDNA) binding protein essential for DNA replication, damage repair and telomere maintenance. The patient presented at the age of 5 years with pancytopenia and hypocellular marrow with morphology resembling refractory cytopenia of childhood, mucocutaneous triad and a congenital retinal anomaly. Further workup revealed telomeres in blood below 1stpercentile, normal female karyotype and a negative chromosomal breakage test. After excluding known genetic causes for DC, family whole exome sequencing identified a de novo RPA1c.718G>A (p.E240K) mutation with a predicted pathogenic CADD score of 23 and absent in >140,000 gnomAD population controls. The mutation had 50% allelic frequency in fibroblasts in comparison to a reduced mutation burden of 27% in bone marrow (BM) cells. Using bulk and single cell sequencing of BM, we identified 2 somatic compensatory events that likely explain patient’s stable hematologic phenotype over a period of 20 years: i) an acquired RPA1p.E579X mutation (in ciswith p.E240K) was found at 10% frequency, which resulted in RNA degradation of the mutated allele as confirmed by RNA sequencing; ii) a founder clone with uniparental isodisomy on chromosome 17p encompassing RPA1locus causing loss of germline E240K mutation. Analysis of serial BM samples using SNP arrays and deep sequencing demonstrated UPD17p clonal expansion concurrent with a decline of germline p.E240K and increase of acquired p.E579X mutation. This is consistent with a toxic, likely gain-of-function (GOF) effect of germline p.E240K mutation leading to acquired loss-of-function (LOF) escape mechanisms.

This corrects the article DOI: 10.1038/nm.4484

Oncogenic JAK mutation sensitizes myeloproliferative neoplasms to immune checkpoint inhibition.

Familial myelodysplastic syndromes (MDS) arise from haploinsufficiency of genes involved in hematopoiesis, show variable penetrance and are primarily associated with MDS in younger patients. The same genes involved in familial MDS might also cause sporadic manifestation, e.g. in GATA2 deficiency where ~2/3 of identified germline mutations arise de novo. In this study we aimed to discover the genetic cause in 7 patients of 4 unrelated pedigrees (Fig. A). The phenotypic denominator was MDS with progressive or transient loss of chromosome 7 and remarkably, spontaneous long-lasting remission was observed in 2 cases. Dysmorphic features or neurological symptoms were absent. Median age at diagnosis was 2 (1-42) years, all patients presented with thrombocytopenia with or without additional cytopenias and a hypocellular marrow without increase of blasts. There was no spontaneous growth in an in vitroGM-CSF hypersensitivity assay performed in 2 patients.

The cover image, by Nurgazy Sulaimanov et al., is based on Advanced Review Understanding the mTOR signaling pathway via mathematical modeling, DOI: 10.1002/wsbm.1379. The cover image, by Nurgazy Sulaimanov et al., is based on Advanced Review Understanding the mTOR signaling pathway via mathematical modeling, DOI: 10.1002/wsbm.1379.

The mechanistic target of rapamycin (mTOR) is a central regulatory pathway that integrates a variety of environmental cues to control cellular growth and homeostasis by intricate molecular feedbacks. In spite of extensive knowledge about its components, the molecular understanding of how these function together in space and time remains poor and there is a need for Systems Biology approaches to perform systematic analyses. In this work, we review the recent progress how the combined efforts of mathematical models and quantitative experiments shed new light on our understanding of the mTORsignaling pathway. In particular, we discuss the modeling concepts applied in mTORsignaling, the role of multiple feedbacks and the crosstalk mechanisms of mTORwith other signaling pathways. We also discuss the contribution of principles from information and network theory that have been successfully applied in dissecting design principles of the mTORsignaling network. We finally propose to classify the mTORmodels in terms of the time scale and network complexity, and outline the importance of the classification toward the development of highly comprehensive and predictive models. WIREs Syst Biol Med2017, 9:e1379. doi: 10.1002/wsbm.1379 For further resources related to this article, please visit the WIREs website. Mathematical models closely linked to experiments are instrumental in dissecting the underlying molecular mechanisms of the mTOR signaling pathway. This review summarizes a recent progress in our understanding of mTOR signaling using different modelling formalisms in different timescales.

Claus: Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel Funding.

No relevant conflicts of interest to declare.

Dyskeratosis congenita (DC) is a rare telomere disease with pleiotropic manifestations and bone marrow failure (BMF) as a major cause of mortality. The genes involved in DC pathogenesis play a role in telomere maintenance but their function in other in biological systems has not been well characterized. Compound heterozygous missense mutations in TCAB1(WRAP53)were reported so far in two pedigrees with classic DC (Zhong et al, 2011). TCAB1 functions as a scaffold protein recruiting TERCto the sub-organelle Cajal bodies, thus contributing to the assembly of telomerase enzyme; among its other functions it regulates DNA double-strand break repair. Here we functionally characterize novel TCAB1mutations and describe transcriptome and proteome profiles in patient-derived lymphoblastoid cell lines (LCL). Among a cohort of 50 DC patients we identified three pedigrees with TCAB1mutations, BMF and telomeres below first percentile. Index 1 carried compound heterozygous mutations R155X/Y345C also present in the affected brother. Both siblings suffered from transfusion-dependent pancytopenia manifesting in the childhood and typical dyskeratotic features. Index 2 harbored a single mutation Q7TfsX27 and presented with hypocellular BMF. Although non-hematologic symptoms were initially absent, the patient suffered from mild restrictive lung disease after stem cell transplantation. Index 3 carried biallelic mutations Y345C/G435R. He was diagnosed with microcephaly, cerebellar hypoplasia, early onset BMF with immunodeficiency, consistent with Hoyeraal-Hreidarsson syndrome (HHS).

Introduction: In-depth knowledge about molecular pathogenesis of malignant diseases and rapidly increasing availability of targeted treatment options enables molecularly guided decision-making. We have established a Molecular Tumor Board (MTB) that focuses on patient management based on specific molecular data at the individual patient level.

The extent of phosphorylation of a guanine nucleotide exchange factor determines the cytotoxicity of natural killer cells.