Low miR-150-5p and miR-320b Expression Predicts Reduced Survival of COPD Patients

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk of death, reducing life expectancy on average between 5 and 7 years. The survival time after diagnosis, however, varies considerably as a result of the heterogeneity of COPD. Therefore, markers that predict individual survival of COPD patients are of great value. We analyzed baseline molecular profiles and collected 54 months of follow-up data of the cohort study "COPD and SYstemic consequences-COmorbidities NETwork" (COSYCONET). Genome-wide microRNA signatures from whole blood collected at time of the inclusion in the study were generated for 533 COPD patients including patients that deceased during the 54-month follow-up period (n = 53) and patients that survived this period (n = 480). We identified two blood-born microRNAs (miR-150-5p and miR-320b) that were highly predictive for survival of COPD patients. The expression change was then confirmed by RT-qPCR in 245 individuals. Ninety percent of patients with highest expression of miR-150-5p survived the 54-month period in contrast to only 50% of patients with lowest expression intensity. Moreover, the abundance of the oncogenic miR-150-5p in blood of COPD patients was predictive for the development of cancer. Thus, molecular profiles measured at the time of a COPD diagnosis have a high predictive power for the survival of patients

Low miR-150-5p and miR-320b Expression Predicts Reduced Survival of COPD Patients

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk of death, reducing life expectancy on average between 5 and 7 years. The survival time after diagnosis, however, varies considerably as a result of the heterogeneity of COPD. Therefore, markers that predict individual survival of COPD patients are of great value. We analyzed baseline molecular profiles and collected 54 months of follow-up data of the cohort study “COPD and SYstemic consequences-COmorbidities NETwork” (COSYCONET). Genome-wide microRNA signatures from whole blood collected at time of the inclusion in the study were generated for 533 COPD patients including patients that deceased during the 54-month follow-up period (n = 53) and patients that survived this period (n = 480). We identified two blood-born microRNAs (miR-150-5p and miR-320b) that were highly predictive for survival of COPD patients. The expression change was then confirmed by RT-qPCR in 245 individuals. Ninety percent of patients with highest expression of miR-150-5p survived the 54-month period in contrast to only 50% of patients with lowest expression intensity. Moreover, the abundance of the oncogenic miR-150-5p in blood of COPD patients was predictive for the development of cancer. Thus, molecular profiles measured at the time of a COPD diagnosis have a high predictive power for the survival of patients

LncRNAs signature defining major subtypes of B-cell acute lymphoblastic leukemia

Introduction: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most prevalent heterogeneous cancer in children and adults, with multiple subtypes. Emerging evidence suggests that long non-coding RNAs (lncRNAs) might play a key role in the development and progression of leukemia. Thus, we performed a transcriptional and DNA methylation survey to explore the lncRNA landscape on three BCP-ALL subtypes (82 samples) and demonstrated their functions and epigenetic profile. Methodology: The primary BCP-ALL samples from bone marrow material were collected from diagnosis (ID) and relapse (REL) stages of adult (n = 21) and pediatric (n = 24) BCP-ALL patients, using RNA-seq and DNA methylation array technology. The subtype-specific and relapse-specific lncRNAs were analyzed by differential expression (DE) analysis method using LIMMA Voom. By analyzing the co-expression of the subtype-specific lncRNAs and protein-coding (PC) genes from all subtypes, we inferred potential functions of these lncRNAs by applying “guilt-by-association” approach. Additionally, we validated our subtype-specific lncRNAs on an independent cohort of 47 BCP-ALL samples. The epigenetic regulation of subtype-specific lncRNAs were identified using the Bumphunter package. The correlation analysis was performed between DM and DE lncRNAs from three subtypes to determine the epigenetically facilitated and silenced lncRNAs. Results: We present a comprehensive landscape of lncRNAs signatures which classifies three molecular subtypes of BCP-ALL on DNA methylation and RNA expression levels. The principle component analysis (PCA) on most variable lncRNAs on RNA and DNA methylation level confirmed robust separation of DUX4, Ph-like and NH-HeH BCP-ALL subtypes. Using integrative bioinformatics analysis, subtype-specific and relapse-specific lncRNAs signature together determine 1564 subtype-specific and 941 relapse-specific lncRNAs from three subtypes. The unsupervised hierarchical clustering on these subtype-specific lncRNAs validated their specificity on the independent validation cohort. For the first time, our study demonstrates that BCP-ALL subtype specific as well as relapse-specific lncRNAs may contribute to the activation of key pathways including TGF-β, PI3K-Akt, mTOR and activation of JAK-STAT signaling pathways from DUX4 and Ph-like subtypes. Finally, the significantly hyper-methylated and hypo-methylated subtype-specific lncRNAs were profiled. In addition to that, we identified 23 subtypes specific lncRNAs showing hypo and hyper-methylation pattern in their promoter region that significantly correlates with their diminished and increased expression in respective subtypes. Conclusions: Overall, our work provides the most comprehensive analyses for lncRNAs in BCP-ALL subtypes. Our findings suggest a wide range of biological functions associated with lncRNAs and epigenetically facilitated lncRNAs in BCP-ALL and provide a foundation for functional investigations that could lead to novel therapeutic approaches.Einführung: Die B-Vorläufer akute lymphatischen Leukämie (BCP-ALL) ist eine heterogene Krebserkrankung mit mehreren definierten Subgruppen. Neue Daten deuten darauf hin, dass lange nicht-kodierende RNAs (long noncoding RNAs - lncRNAs) eine Schlüsselrolle bei der Entwicklung und Progression der BCP-ALL spielen könnten. Daher führten wir eine Transkriptions- und DNA-Methylierungsstudie durch, um die lncRNA-Landschaft von drei BCP-ALL-Subgruppen (82 Proben) zu charakterisieren und potentielle regulative Konsequenzen zu analysieren. Methodik: Material wurde zum Zeitpunkt der Erstdiagnose (ID) und im Rezidiv (REL) von erwachenen (n = 21) und pädiatrischen (n = 24) BCP-ALL-Patienten entnommen und unter Verwendung von RNA-Seq und DNA-Methylierungs-Array-Technologien untersucht. Die Subgruppen-spezifischen und rezidiv-spezifischen lncRNAs wurden durch differentielle Expressions (DE) Analysen mit LIMMA Voom analysiert. Durch die Analyse der Koexpression von lncRNAs mit Protein-kodierenden (PC) Genen aus allen Subgruppen schlossen wir unter Verwendung eines ‚Guilt-by-association‘ -Ansatzes auf potentielle Funktionen der DE lncRNAs. Zudem haben wir die Subgruppen-spezifischen lncRNAs auf einem unabhängigen Datenset von 47 BCP-ALL-Proben validiert. Die epigenetische. Die epigenetische Regulation von Subgruppen-spezifischen lncRNAs wurde durch eine differentielle Methylierungs (DM) analyse identifiziert. Die Korrelation zwischen DM und DE lncRNAs aus drei Subgruppen wurde ermittelt, um den Einfluss der epigenetischen Regulation auf die Expression von lncRNAs zu analysieren. Ergebnisse: Wir präsentieren eine umfassende Landschaft von lncRNA-Signaturen, die drei molekulare Subtypen von BCP-ALL auf DNA-Methylierungs- und RNA-Expressionslevel klassifiziert. Die Hauptkomponentenanalyse (PCA) auf den top variablen lncRNAs auf RNA und DNA-Methylierungsniveau bestätigte eine robuste Trennung von Ph-like, DUX4 und NH-NeH BCP-ALL Subtypen. Mit integrativer bioinformatischer Analyse, zusammen 1564 subtyp-spezifische und 941 rezidiv-spezifische lncRNAs aus den drei Subtypen. Das unüberwachte hierarchische Clustering auf diesen Subtyp-spezifischen lncRNAs validierte ihre Spezifität in der unabhängigen Validierungskohorte. Unsere Studie zeigt erstmals, dass BCP-ALL-Subtyp-spezifische sowie Rezidiv-spezifische lncRNAs zur Aktivierung von Signalwegen wie TGF-β, PI3K-Akt, mTOR und Aktivierung von JAK-STAT-Signalwegen von DUX4 und Ph-like Subtypen. Endlich wurden die signifikant DM subtyp-spezifische lncRNAs profiliert. Darüber hinaus identifizierten wir 23 Subtyp-spezifische lncRNAs, die ein Hypo- und Hypermethylierungsmuster in ihrer Promotorregion zeigen, das signifikant mit ihrer verringerten und erhöhten Expression in den jeweiligen Subtypen korreliert. Schlussfolgerungen: Insgesamt liefert unsere Arbeit die umfassendsten Analysen für lncRNAs in BCP-ALL-Subtypen. Unsere Ergebnisse weisen auf eine Vielzahl von biologischen Funktionen im Zusammenhang mit lncRNAs und epigenetisch erleichterten lncRNAs in BCP-ALL hin und bieten eine Grundlage für funktionelle Untersuchungen, die zu neuen therapeutischen Ansätzen führen könnten

BCAT1 is a novel target of MLL fusions and essential for leukemic stem cell transformation

In cancer, the branched-chain amino acid (BCAA) metabolism is frequently activated through the increased uptake (second only to glutamine) of valine, leucine, and isoleucine (Jain et al., 2012), as well as the overexpression of branched-chain amino acid transaminase 1 (BCAT1), the cytoplasmic BCAA transaminase. We and others showed that proliferation, migration, and chemoresistance of a variety of cancer entities, such as glioblastoma, breast cancer, and myeloid leukemia, are heavily reliant on BCAT1 expression (Hattori et al., 2017; Raffel et al., 2017; Thewes et al., 2017; Tönjes et al., 2013). Epigenetic gene regulation and metabolism are highly intertwined, as many histone and DNA modifiers rely on substrates and cofactors provided by various metabolic reactions. For example, in cancer, the upregulation of BCAT1 results in a decrease of α-KG and a reduced activity of α-KG dependent enzymes, such as EGLN1 (Raffel et al., 2017). Furthermore, DNA hypermethylation is observed upon BCAT1 suppression in acute myeloid leukemia (AML), suggesting an α-KG-dependent effect on the TET-family DNA demethylases (Raffel et al., 2017). In an attempt to unravel the interdependencies of BCAT1 and chromatin-modifying enzymes, I discovered that the upregulation of BCAT1 in mammary carcinoma xenografts influences histone modifications in an α-KG-independent manner. Global upregulation of gene expression mediated by altered chromatin modifications in BCAT1 knockdown breast cancer xenografts led to a comparison between BCAT1 and histone modifiers in expression data sets of breast cancer and AML patients. H3K4 methyltransferase MLL was found to be the most significantly positively correlating modifier in both cancer entities. MLL has been extensively studied in leukemia in which recurring translocations lead to gain-of-function rearrangements, such as MLL-AF9 and MLL-ENL fusion. These oncogenic fusions are driver gene mutations, and I was able to identify BCAT1 as one of their targets regulating its expression. MLL-AF9 and MLL-ENL were able to transform hematopoietic stem and progenitor cells (HSPCs) into leukemic stem cells (LSCs). However, loss of Bcat1 or its transaminase activity results in loss of self-renewal and immortalization, making Bcat1 activity essential for MLL fusion-mediated tumor development. Further expression and histone modification profiling of transformed wildtype HSPCs, as well as Bcat1 knockout HSPCs and their rescues, revealed that the lack of Bcat1 initiates the inhibition of DNA replication and cell cycle arrest missing in Bcat1 wildtype cells. BCAT1s limited expression in most healthy tissues makes it an interesting target for cancer therapy. Furthermore, this and other studies implicate that the inhibition of BCAT1’s transaminase activity can eradicate LSCs and may prevent relapse. Additionally, controlling BCAT1 has the potential to reduce tumor development, relevant especially for patients harboring clonal hematopoiesis of intermediate potential (CHIP)

Transcriptional Regulation of the Metabolic Response to Therapy in Leukemia

Cancer cells are under constant stress due to their uncontrolled growth, oncogenic signaling, and the metabolic insufficiencies of their microenvironments. Under various stresses, cells activate the integrated stress response (ISR), a transcriptional program to restore cellular homeostasis. Activating transcription factor 4 (ATF4) acts as the master transcriptional regulator of the ISR by promoting the transcription of genes that mitigate stress or promote cell death if the stress remains unresolved. Despite being the common mediator of various stress response and metabolic pathways, ATF4 generates tailored transcriptional outputs to distinct cellular stresses by cooperating with other transcriptional machinery. The precise mechanisms by which ATF4 activates an appropriate transcriptional program in response to metabolic stresses, however, remain unclear. In this work, we used forward genetic screens, metabolic profiling, and biochemical approaches to identify transcriptional regulators required for the cellular response to various metabolic stress conditions. This work revealed that ATF4 is universally required under amino acid starvation, but identified the transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as a critical regulator of the response to asparagine deprivation in acute lymphoblastic leukemia (ALL). We found that under asparagine depleted conditions ZBTB1 enables cellular proliferation by promoting the synthesis of asparagine from aspartate. Mechanistically, ZBTB1 binds directly to a sequence within the promoter of asparagine synthetase (ASNS), the enzyme responsible for the synthesis of asparagine from aspartate. Loss of ZBTB1 results in a dramatic reduction in the transcription of ASNS, and, subsequently, a reduced capacity for cells to synthesize asparagine from aspartate. ZBTB1 knockout T-ALLs are not only sensitive to asparagine deprivation in vitro but are also sensitive to treatment with L-asparaginase, a chemotherapy that reduces serum asparagine, in in vivo xenograft models of ALL. Additionally, this work clarifies the metabolic stress induced by CPI-613, a lipoic acid analog designed to inhibit the function of Pyruvate Dehydrogenase (PDH), the enzyme responsible for the decarboxylation of pyruvate to acetyl-CoA. In line with the proposed mechanism of CPI-613, genetic screens suggested a synthetic lethal relationship between electron transport chain or TCA cycle enzymes and CPI-613. Unexpectedly, however, glycerolipid synthesis genes were found to be essential for the cellular response to CPI-613-induced stress. Further work revealed a substantial incorporation of CPI-613 into glycerolipid species, a finding that correlates with sensitivity to the drug. Altogether, our work defines novel metabolic and transcriptional mechanisms of the response of acute leukemias to metabolic stresses. In acute lymphoblastic leukemia, we have identified a critical transcriptional regulator of the cellular response to asparagine deprivation. The role of ZBTB1 in the transcriptional regulation of ASNS in parallel with ATF4 has direct relevance to the therapeutic response of ALLs to Lasparaginase. We have also determined a novel mechanism of action of CPI-613, a first-in-class PDH inhibitor currently in phase III clinical trials for acute myeloid leukemia (AML). This work revealed the incorporation of CPI-613 into glycerolipid species which may be relevant to toxicity of the drug. Altogether this work provides a framework for investigating the metabolic and transcriptional mechanisms by which leukemias respond to cellular stresses such as those induced by metabolically targeted therapies

The Essential Role of the Non-Essential Amino Acid Asparagine in Lymphoid Malignancies

Indiana University-Purdue University Indianapolis (IUPUI)Cancer cells display increased metabolic demands to support their proliferation and biosynthetic needs. It has been extensively shown in cancers, that amino acids have functions beyond the role of mRNA translation. The breadth of functions makes amino acid restriction an effective strategy for cancer therapy; hence an important line of research involves targeting amino acid acquisition and metabolism therapeutically. Currently, asparagine depletion via L-Asparaginase in acute lymphoblastic leukemia (ALL) remains the only clinically approved therapy to date. In the first project, we showed that ALL cells are auxotrophic for asparagine and rely on exogenous sources for this non-essential amino acid. However, sensitivity to L-Asparaginase therapy is mitigated by the expression of the enzyme asparagine synthetase (ASNS), involved in de novo asparagine biosynthesis. We showed that this adaptive response requires two essential steps; demethylation of the ASNS promoter and recruitment of activating transcription factor 4 (ATF4) to the promoter to drive ASNS transcription. Our follow-up study in ALL cells showed that asparagine bioavailability (through de novo biosynthesis or exogenous sources) is essential to maintain the expression of the critical oncogene c-MYC. c-MYC is a potent transcription factor and is dysregulated in over 60% of cancers, including hematopoietic malignancies. We showed that this regulation by asparagine is primarily at the translation level and c-MYC expression is rescued only when exogenous asparagine is available or when cells can undertake de novo biosynthesis. At the biochemical level, asparagine depletion also causes an induction of ATF4 mediated stress response and suppression of global translation mediated by decreased mammalian target of rapamycin complex 1 (mTORC1) activity. However, we found that neither inhibition of the stress response or rescuing global translation rescued c-MYC protein expression. We also extended this observation to c-MYC-driven lymphomas using cell lines and orthotopic in vivo models. We showed that genetic inhibition of ASNS or pharmacological inhibition of asparagine production can significantly limit c-MYC protein and tumor growth when environmental asparagine is limiting. Overall, our work shows an essential role for asparagine in lymphoid cancers and has expanded on the usage of L-Asparaginase to resistant leukemias and lymphomas

Meta-analysis of heterogeneous Down Syndrome data reveals consistent genome-wide dosage effects related to neurological processes

<p>Abstract</p> <p>Background</p> <p>Down syndrome (DS; trisomy 21) is the most common genetic cause of mental retardation in the human population and key molecular networks dysregulated in DS are still unknown. Many different experimental techniques have been applied to analyse the effects of dosage imbalance at the molecular and phenotypical level, however, currently no integrative approach exists that attempts to extract the common information.</p> <p>Results</p> <p>We have performed a statistical meta-analysis from 45 heterogeneous publicly available DS data sets in order to identify consistent dosage effects from these studies. We identified 324 genes with significant genome-wide dosage effects, including well investigated genes like <it>SOD1</it>, <it>APP</it>, <it>RUNX1 </it>and <it>DYRK1A </it>as well as a large proportion of novel genes (N = 62). Furthermore, we characterized these genes using gene ontology, molecular interactions and promoter sequence analysis. In order to judge relevance of the 324 genes for more general cerebral pathologies we used independent publicly available microarry data from brain studies not related with DS and identified a subset of 79 genes with potential impact for neurocognitive processes. All results have been made available through a web server under <url>http://ds-geneminer.molgen.mpg.de/</url>.</p> <p>Conclusions</p> <p>Our study represents a comprehensive integrative analysis of heterogeneous data including genome-wide transcript levels in the domain of trisomy 21. The detected dosage effects build a resource for further studies of DS pathology and the development of new therapies.</p

Regulation of Mammalian Nucleotide Metabolism and Biosynthesis

Nucleotides are required for a wide variety of biological processes and are constantly synthesized de novo in all cells. When cells proliferate, increased nucleotide synthesis is necessary for DNA replication and for RNA production to support protein synthesis at different stages of the cell cycle, during which these events are regulated at multiple levels. Therefore the synthesis of the precursor nucleotides is also strongly regulated at multiple levels. Nucleotide synthesis is an energy intensive process that uses multiple metabolic pathways across different cell compartments and several sources of carbon and nitrogen. The processes are regulated at the transcription level by a set of master transcription factors but also at the enzyme level by allosteric regulation and feedback inhibition. Here we review the cellular demands of nucleotide biosynthesis, their metabolic pathways and mechanisms of regulation during the cell cycle. The use of stable isotope tracers for delineating the biosynthetic routes of the multiple intersecting pathways and how these are quantitatively controlled under different conditions is also highlighted. Moreover, the importance of nucleotide synthesis for cell viability is discussed and how this may lead to potential new approaches to drug development in diseases such as cancer