Combined treatment with crizotinib and temsirolimus is an effective strategy in mantle cell lymphoma and can overcome acquired resistance to temsirolimus.

Constitutive activation of the PI3K/AKT/mTOR-pathway plays an important role in the pathogenesis of mantle cell lymphoma (MCL), leading to approval of the mTOR inhibitor temsirolimus for relapsed or refractory MCL. Yet, despite favorable initial response rates, early relapses under treatment have been observed. Therefore, understanding the underlying mechanisms of temsirolimus resistance and developing strategies to overcome it is highly warranted. Here, we established a new temsirolimus-resistant MCL cell line to evaluate the molecular background of resistance to this drug. Transcriptome profiling and gene set enrichment analysis comparing temsirolimus-sensitive and -resistant cell lines showed significant upregulation of PI3K/AKT/mTor-, RAS signaling- and the RTK-dependent PDGFR-, FGFR-, Met- and ALK-signaling-pathways in the resistant cells. Furthermore, MET, known as important proto-oncogene and mediator of drug resistance, was among the most upregulated genes in the resistant cells. Importantly, Met protein was overexpressed in both, MCL cells with acquired as well as intrinsic temsirolimus resistance, but could not be detected in any of the temsirolimus sensitive ones. Combined pharmacological inhibition of mTOR and Met signaling with temsirolimus and the RTK inhibitor crizotinib significantly restored sensitivity to temsirolimus. Furthermore, this combined treatment proved to be synergistic in all MCL cell lines investigated and was also active in primary MCL cells. In summary, we showed for the first time that overexpression of MET plays an important role for mediating temsirolimus resistance in MCL and combined treatment with temsirolimus and crizotinib is a very promising therapeutic approach for MCL and an effective strategy to overcome temsirolimus resistance.

Sequence dependent UV damage of complete pools of oligonucleotides.

Understanding the sequence-dependent DNA damage formation requires probing a complete pool of sequences over a wide dose range of the damage-causing exposure. We used high throughput sequencing to simultaneously obtain the dose dependence and quantum yields for oligonucleotide damages for all possible 4096 DNA sequences with hexamer length. We exposed the DNA to ultraviolet radiation at 266 nm and doses of up to 500 absorbed photons per base. At the dimer level, our results confirm existing literature values of photodamage, whereas we now quantified the susceptibility of sequence motifs to UV irradiation up to previously inaccessible polymer lengths. This revealed the protective effect of the sequence context in preventing the formation of UV-lesions. For example, the rate to form dipyrimidine lesions is strongly reduced by nearby guanine bases. Our results provide a complete picture of the sensitivity of oligonucleotides to UV irradiation and allow us to predict their abundance in high-UV environments.

OCT4/POU5F1 is indispensable for the lineage differentiation of the inner cell mass in bovine embryos.

The mammalian blastocyst undergoes two lineage segregations, that is, formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) as the remaining pluripotent lineage. To clarify the expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9, and 12 blastocysts completely produced in vivo by staining for OCT4, NANOG, SOX2 (EPI), and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4-deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4-deficient cells. Our data show that OCT4 is required cell autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos.

Large-scale analysis of Drosophila core promoter function using synthetic promoters.

The core promoter plays a central role in setting metazoan gene expression levels, but how exactly it "computes" expression remains poorly understood. To dissect its function, we carried out a comprehensive structure-function analysis in Drosophila. First, we performed a genome-wide bioinformatic analysis, providing an improved picture of the sequence motifs architecture. We then measured synthetic promoters' activities of ~3,000 mutational variants with and without an external stimulus (hormonal activation), at large scale and with high accuracy using robotics and a dual luciferase reporter assay. We observed a strong impact on activity of the different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, nucleosome positioning, and flanking sequences. A linear combination of the individual motif features largely accounts for the combinatorial effects on core promoter activity. These findings shed new light on the quantitative assessment of gene expression in metazoans.

Differential Effects of Insulin-Deficient Diabetes Mellitus on Visceral vs. Subcutaneous Adipose Tissue-Multi-omics Insights From the Munich MIDY Pig Model.

Adipose tissue (AT) is no longer considered to be responsible for energy storage only but is now recognized as a major endocrine organ that is distributed across different parts of the body and is actively involved in regulatory processes controlling energy homeostasis. Moreover, AT plays a crucial role in the development of metabolic disease such as diabetes. Recent evidence has shown that adipokines have the ability to regulate blood glucose levels and improve metabolic homeostasis. While AT has been studied extensively in the context of type 2 diabetes, less is known about how different AT types are affected by absolute insulin deficiency in type 1 or permanent neonatal diabetes mellitus. Here, we analyzed visceral and subcutaneous AT in a diabetic, insulin-deficient pig model (MIDY) and wild-type (WT) littermate controls by RNA sequencing and quantitative proteomics. Multi-omics analysis indicates a depot-specific dysregulation of crucial metabolic pathways in MIDY AT samples. We identified key proteins involved in glucose uptake and downstream signaling, lipogenesis, lipolysis and ß-oxidation to be differentially regulated between visceral and subcutaneous AT in response to insulin deficiency. Proteins related to glycogenolysis, pyruvate metabolism, TCA cycle and lipogenesis were increased in subcutaneous AT, whereas ß-oxidation-related proteins were increased in visceral AT from MIDY pigs, pointing at a regionally different metabolic adaptation to master energy stress arising from diminished glucose utilization in MIDY AT. Chronic, absolute insulin deficiency and hyperglycemia revealed fat depot-specific signatures using multi-omics analysis. The generated datasets are a valuable resource for further comparative and translational studies in clinical diabetes research.

Circadian clocks guide dendritic cells into skin lymphatics.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies.

Hematopoietic stem-cell senescence and myocardial repair - Coronary artery disease genotype/phenotype analysis of post-MI myocardial regeneration response induced by CABG/CD133+ bone marrow hematopoietic stem cell treatment in RCT PERFECT Phase 3.

BACKGROUND: Bone marrow stem cell clonal dysfunction by somatic mutation is suspected to affect post-infarction myocardial regeneration after coronary bypass surgery (CABG). METHODS: Transcriptome and variant expression analysis was studied in the phase 3 PERFECT trial post myocardial infarction CABG and CD133+ bone marrow derived hematopoetic stem cells showing difference in left ventricular ejection fraction (∆LVEF) myocardial regeneration Responders (n=14; ∆LVEF +16% day 180/0) and Non-responders (n=9; ∆LVEF -1.1% day 180/0). Subsequently, the findings have been validated in an independent patient cohort (n=14) as well as in two preclinical mouse models investigating SH2B3/LNK antisense or knockout deficient conditions. FINDINGS: 1. Clinical: R differed from NR in a total of 161 genes in differential expression (n=23, q<0•05) and 872 genes in coexpression analysis (n=23, q<0•05). Machine Learning clustering analysis revealed distinct RvsNR preoperative gene-expression signatures in peripheral blood acorrelated to SH2B3 (p<0.05). Mutation analysis revealed increased specific variants in RvsNR. (R: 48 genes; NR: 224 genes). 2. Preclinical:SH2B3/LNK-silenced hematopoietic stem cell (HSC) clones displayed significant overgrowth of myeloid and immune cells in bone marrow, peripheral blood, and tissue at day 160 after competitive bone-marrow transplantation into mice. SH2B3/LNK-/- mice demonstrated enhanced cardiac repair through augmenting the kinetics of bone marrow-derived endothelial progenitor cells, increased capillary density in ischemic myocardium, and reduced left ventricular fibrosis with preserved cardiac function. 3. VALIDATION: Evaluation analysis in 14 additional patients revealed 85% RvsNR (12/14 patients) prediction accuracy for the identified biomarker signature. INTERPRETATION: Myocardial repair is affected by HSC gene response and somatic mutation. Machine Learning can be utilized to identify and predict pathological HSC response. FUNDING: German Ministry of Research and Education (BMBF): Reference and Translation Center for Cardiac Stem Cell Therapy - FKZ0312138A and FKZ031L0106C, German Ministry of Research and Education (BMBF): Collaborative research center - DFG:SFB738 and Center of Excellence - DFG:EC-REBIRTH), European Social Fonds: ESF/IV-WM-B34-0011/08, ESF/IV-WM-B34-0030/10, and Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany. Japanese Ministry of Health : Health and Labour Sciences Research Grant (H14-trans-001, H17-trans-002) TRIAL REGISTRATION: ClinicalTrials.gov NCT00950274.

Clinical presentation and differential splicing of SRSF2, U2AF1 and SF3B1 mutations in patients with acute myeloid leukemia.

Previous studies demonstrated that splicing factor mutations are recurrent events in hematopoietic malignancies with both clinical and functional implications. However, their aberrant splicing patterns in acute myeloid leukemia remain largely unexplored. In this study, we characterized mutations in SRSF2, U2AF1, and SF3B1, the most commonly mutated splicing factors. In our clinical analysis of 2678 patients, splicing factor mutations showed inferior relapse-free and overall survival, however, these mutations did not represent independent prognostic markers. RNA-sequencing of 246 and independent validation in 177 patients revealed an isoform expression profile which is highly characteristic for each individual mutation, with several isoforms showing a strong dysregulation. By establishing a custom differential splice junction usage pipeline, we accurately detected aberrant splicing in splicing factor mutated samples. A large proportion of differentially used junctions were novel, including several junctions in leukemia-associated genes. In SRSF2(P95H) mutants, we further explored the possibility of a cascading effect through the dysregulation of the splicing pathway. Furthermore, we observed a validated impact on overall survival for two junctions overused in SRSF2(P95H) mutants. We conclude that splicing factor mutations do not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes.

The secretome of skin cancer cells activates the mTOR/MYC pathway in healthy keratinocytes and induces tumorigenic properties.

Cutaneous squamous cell carcinoma (cSCC) is the most prominent tumor of non-melanoma skin cancers and the most aggressive tumor among keratinocyte carcinoma of the skin, showing a high potential for local invasion and metastasis. The cSCC incidences increased dramatically in recent years and the disease occurs more commonly than any other malignancy. The secretome of cancer cells is currently the focus of many studies in order to identify new marker proteins for different types of cancer and to investigate its influence on the tumor microenvironment. In our study we evaluated whether the secretome of cSCC cells has an impact on keratinocytes, the surrounding tissue cells of cSCC. Therefore, we analyzed and compared the secretome of human A431 cancer cells and of HaCaT keratinocytes by mass spectrometry. In a second experiment, keratinocytes were exposed to the secretome of A431 cells and vice versa and the transcriptome was analyzed by next-generation sequencing. HaCaT cells incubated with A431 conditioned medium revealed a significantly activated mammalian target of rapamycin pathway with a concomitant increase in proliferation and migration. In conclusion, our data demonstrate the impact of the secretome of cancer cells on the transcription machinery of the cells surrounding the tumor, leading to a tumorigenic cell fate.

A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol.

Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies1-3. Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells4,5. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress6. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown1,2,7. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.

Sex-specific programming effects of parental obesity in pre-implantation embryonic development.

BACKGROUND: Obesity is a global rising problem with epidemiological dimension. Obese parents can have programming effects on their offspring leading to obesity and associated diseases in later life. This constitutes a vicious circle. Epidemiological data and studies in rodents demonstrated differential programming effects in male and female offspring, but the timing of their developmental origin is not known. METHODS: This study investigated if sex-specific programming effects of parental obesity can already be detected in the pre-implantation period. Diet-induced obese male or female mice were mated with normal-weight partners and blastocysts were recovered. RESULTS: Gene expression profiling revealed sex-specific responses of the blastocyst transcriptome to maternal and paternal obesity. The changes in the transcriptome of male blastocysts were more pronounced than those of female blastocysts, with a stronger impact of paternal than of maternal obesity. The sperm of obese mice revealed an increased abundance of several miRNAs compared with lean mice. CONCLUSIONS: Our study indicates that sex-specific programming effects of parental obesity already start in the pre-implantation period and reveals specific alterations of the sperm miRNA profile as mechanistic link to programming effects of paternal obesity.

Allelic Imbalance of Recurrently Mutated Genes in Acute Myeloid Leukaemia.

The patho-mechanism of somatic driver mutations in cancer usually involves transcription, but the proportion of mutations and wild-type alleles transcribed from DNA to RNA is largely unknown. We systematically compared the variant allele frequencies of recurrently mutated genes in DNA and RNA sequencing data of 246 acute myeloid leukaemia (AML) patients. We observed that 95% of all detected variants were transcribed while the rest were not detectable in RNA sequencing with a minimum read-depth cut-off (10x). Our analysis focusing on 11 genes harbouring recurring mutations demonstrated allelic imbalance (AI) in most patients. GATA2, RUNX1, TET2, SRSF2, IDH2, PTPN11, WT1, NPM1 and CEBPA showed significant AIs. While the effect size was small in general, GATA2 exhibited the largest allelic imbalance. By pooling heterogeneous data from three independent AML cohorts with paired DNA and RNA sequencing (N = 253), we could validate the preferential transcription of GATA2-mutated alleles. Differential expression analysis of the genes with significant AI showed no significant differential gene and isoform expression for the mutated genes, between mutated and wild-type patients. In conclusion, our analyses identified AI in nine out of eleven recurrently mutated genes. AI might be a common phenomenon in AML which potentially contributes to leukaemogenesis.

Multi-omics insights into functional alterations of the liver in insulin-deficient diabetes mellitus.

OBJECTIVE: The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically. METHODS: We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics. RESULTS: Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples. CONCLUSIONS: The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets.

Regulation and function of H3K36 di-methylation by the trithorax-group protein complex AMC.

The Drosophila Ash1 protein is a trithorax-group (trxG) regulator that antagonizes Polycomb repression at HOX genes. Ash1 di-methylates lysine 36 in histone H3 (H3K36me2) but how this activity is controlled and at which genes it functions is not well understood. We show that Ash1 protein purified from Drosophila exists in a complex with MRG15 and Caf1 that we named AMC. In Drosophila and human AMC, MRG15 binds a conserved FxLP motif near the Ash1 SET domain and stimulates H3K36 di-methylation on nucleosomes. Drosophila MRG15-null and ash1 catalytic mutants show remarkably specific trxG phenotypes: stochastic loss of HOX gene expression and homeotic transformations in adults. In mutants lacking AMC, H3K36me2 bulk levels appear undiminished but H3K36me2 is reduced in the chromatin of HOX and other AMC-regulated genes. AMC therefore appears to act on top of the H3K36me2/me3 landscape generated by the major H3K36 methyltransferases NSD and Set2. Our analyses suggest that H3K36 di-methylation at HOX genes is the crucial physiological function of AMC and the mechanism by which the complex antagonizes Polycomb repression at these genes.

OCT4/POU5F1 is required for NANOG expression in bovine blastocysts.

Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 in bovine preimplantation development, we generated OCT4 knockout (KO) fibroblasts by CRISPR-Cas9 and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In OCT4 KO morulae (day 5), ∼70% of the nuclei were OCT4 positive, indicating that maternal OCT4 mRNA partially maintains OCT4 protein expression during early development. In contrast, OCT4 KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of OCT4 KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to Oct4-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supports bovine embryogenesis as a model for early human development and exemplifies a general strategy for studying the roles of specific genes in embryos of domestic species.

Two different pathways of phosphatidylcholine synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate cellular triacylglycerol storage.

BACKGROUND: Lipids are stored within cells in lipid droplets (LDs). They consist of a core of neutral lipids surrounded by a monolayer of phospholipids, predominantly phosphatidylcholine (PC). LDs are very dynamic and can rapidly change in size upon lipid uptake or release. These dynamics require a fast adaptation of LD surface. We have recently shown that two Lands cycle PC synthesizing enyzmes, LPCAT1 and LPCAT2 can localize to the LD surface. RESULTS: Here, we show that knock-down of both enzymes leads to an increase in LD size without changes in the total amount of neutral lipids, while interference with the de-novo Kennedy pathway PC biosynthesis is associated with changes in triacylglyceride synthesis. We show that function of LPCAT1 and 2 is conserved in Drosophila melanogaster by the ortholog CG32699. Furthermore we demonstrate that modulation of the LD pool by LPCAT1 influences the release of lipoprotein from liver cells. CONCLUSION: Activity of the Kennedy pathway regulates the balance between phospholipids and neutral lipids, while the Lands cycle regulates lipid droplet size by regulating surface availability and influencing surface to volume ratio. Differences in lipid droplet size may account for differences in lipid dynamics and be relevant to understand lipid overload diseases.

Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells.

The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system from Streptococcus pyogenes has been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage by cas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factor lig4. Using cultured Drosophila melanogaster S2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5'-extensions. This enables generation of U6-promoter fusion templates by overlap-extension PCR with a standardized protocol. We present a series of PCR template vectors for C-terminal protein tagging and clonal Drosophila S2 cell lines with stable expression of a myc-tagged cas9 protein. The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated.

Monoubiquitination of ancient ubiquitous protein 1 promotes lipid droplet clustering.

Lipid droplets, the intracellular storage organelles for neutral lipids, exist in a wide range of sizes and of morphologically distinct organization, from loosely dispersed lipid droplets to tightly packed lipid droplet clusters. We show that the lipid droplet protein AUP1 induces cluster formation. A fraction of AUP1 is monoubiquitinated at various lysine residues. This process depends on its internal CUE domain, which is a known ubiquitin-binding domain. AUP1 with a deleted or point mutagenized CUE domain, as well as a lysine-free mutant, are not ubiquitinated and do not induce lipid droplet clustering. When such ubiquitination deficient mutants are fused to ubiquitin, clustering is restored. AUP1 mutants with defective droplet targeting fail to induce clustering. Also, another lipid droplet protein, NSDHL, with a fused ubiquitin does not induce clustering. The data indicate that monoubiquitinated AUP1 on the lipid droplet surface specifically induces clustering, and suggest a homophilic interaction with a second AUP1 molecule or a heterophilic interaction with another ubiquitin-binding protein.