Linking Proteomic and Transcriptional Data through the Interactome and Epigenome Reveals a Map of Oncogene-induced Signaling

Cellular signal transduction generally involves cascades of post-translational protein modifications that rapidly catalyze changes in protein-DNA interactions and gene expression. High-throughput measurements are improving our ability to study each of these stages individually, but do not capture the connections between them. Here we present an approach for building a network of physical links among these data that can be used to prioritize targets for pharmacological intervention. Our method recovers the critical missing links between proteomic and transcriptional data by relating changes in chromatin accessibility to changes in expression and then uses these links to connect proteomic and transcriptome data. We applied our approach to integrate epigenomic, phosphoproteomic and transcriptome changes induced by the variant III mutation of the epidermal growth factor receptor (EGFRvIII) in a cell line model of glioblastoma multiforme (GBM). To test the relevance of the network, we used small molecules to target highly connected nodes implicated by the network model that were not detected by the experimental data in isolation and we found that a large fraction of these agents alter cell viability. Among these are two compounds, ICG-001, targeting CREB binding protein (CREBBP), and PKF118–310, targeting β-catenin (CTNNB1), which have not been tested previously for effectiveness against GBM. At the level of transcriptional regulation, we used chromatin immunoprecipitation sequencing (ChIP-Seq) to experimentally determine the genome-wide binding locations of p300, a transcriptional co-regulator highly connected in the network. Analysis of p300 target genes suggested its role in tumorigenesis. We propose that this general method, in which experimental measurements are used as constraints for building regulatory networks from the interactome while taking into account noise and missing data, should be applicable to a wide range of high-throughput datasets.National Science Foundation (U.S.) (DB1-0821391)National Institutes of Health (U.S.) (Grant U54-CA112967)National Institutes of Health (U.S.) (Grant R01-GM089903)National Institutes of Health (U.S.) (P30-ES002109

Imetajate pseudokinaasi TRIB3 funktsioonid ja regulatsioon

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Tribbles homoloog 3 (TRIB3) on imetajate geen, mille avaldumistase suureneb mitmesuguste rakustresside, näiteks glükoosi- või aminohappepuuduse, endoplasmaatilise retiikulumi stressi, hüpoksia või oksüdatiivse stressi korral. Selle geeni poolt kodeeritav valk TRIB3 on pseudokinaas – valk, mis primaarjärjestuselt sarnaneb proteiinikinaasile, kuid sisaldab asendusi katalüütiliselt kriitilistes aminohappejääkides. TRIB3 reguleerib rakus toimuvad protsesse valk-valk interaktsioonide kaudu. Kirjeldatud on tema seondumist mitmete transkriptsioonifaktoritega, kinaasidega, ubikvitiini ligaasidega ja muud tüüpi valkudega ning sel moel mõjutab TRIB3 raku stressivastust ja rakusurma, arengulisi protsesse, põletikku ja ainevahetust. Käesolevas doktoritöös uuriti mitmeid TRIB3 geeni toimimisega seotud küsimusi nii raku kui ka organismi tasemel, rakendades muuhulgas meie töörühma poolt loodud hiireliini, mille genoomist on eemaldatud Trib3 geen (Trib3−/− hiired). Töös saadud tulemused näitavad, et inimese maksakasvaja rakuliinis muutub rakustressi korral TRIB3 mRNA isovormide hulgas domineerivaks variant, millel on lühenenud 5′-liiderjärjestus, mis võimaldab efektiivsemat valgutootmist. Nuumrakud on immuunrakud, mis viivad ellu allergilisi reaktsioone. Uurides Trib3 rolli hiire nuumrakkude kultuurides, selgus, et Trib3 avaldumist nendes rakkudes suurendab kasvufaktor interleukiin-3 ning Trib3 puudumine vähendab nuumrakkude võimet teostada immunoloogilisi reaktsioone in vitro. Analüüsides Trib3 avaldumist hiire peaajus, tuvastati, et mRNA arvukus kasvab aju lootelise arengu käigus ja sööda tarbimisel, milles puudub asendamatu aminohape. Trib3−/− hiirte aju uurimisel ilmnes, et neil on suurenenud külgmised ajuvatsakesed, kuid muid olulisi erinevusi aju ehituses ei täheldatud ning käitumiskatsete tulemused näitasid, et Trib3−/− hiirtel on tüüpiline pikaajaline ruumimälu, hirmumälu ja võimekus tunnetada sööda aminohappelist koostist. Selgitamaks TRIB3 tähtsust glükoosipuuduse korral teostati ülegenoomne geeniekspressiooni uuring ning leiti, et TRIB3 pidurdab oluliselt IGFBP2 geeni avaldumise mahasurumist glükoosipuuduse käes kannatavates rakkudes, mis on varasemalt kirjeldamata mehhanism toitainepuudusest tingitud rakusurma takistamiseks.Tribbles homolog 3 (TRIB3) is a mammalian gene that is upregulated in response to several types of cellular stress, including glucose or amino acid deprivation, endoplasmic reticulum stress, hypoxia and oxidative stress. The TRIB3 protein is a pseudokinase, i.e., a protein that displays sequence similarity to protein kinases but contains substitutions at positions that are critical for catalytic activity in canonical protein kinases. TRIB3 is known to form protein–protein interactions with several transcription factors, protein kinases, ubiquitin ligases and other proteins, and, through these interactions, TRIB3 is implicated in the regulation of the cellular stress response, cell death, developmental processes, inflammation and metabolism. In this dissertation, several aspects of TRIB3 gene regulation and function were studied at the cell and organism levels, facilitated by the generation of a Trib3 knockout mouse line by our group. To obtain a better understanding of TRIB3 induction mechanisms, comparative quantification of different TRIB3 mRNA isoforms was performed in human hepatoma cells, revealing that mRNA isoforms containing a truncated 5′-UTR become predominant in stressful conditions, enhancing the translational potential of the TRIB3 mRNA pool. Studying the role of mouse Trib3 in mast cells, tissue-resident immune cells that mediate allergic responses, it is shown that the growth factor interleukin-3 positively regulates Trib3 expression in these cells, and a lack of Trib3 impairs the immunological functions of mast cells, implicating Trib3 in the modulation of the immune response. Analysis of Trib3 expression in the mouse brain uncovered upregulation during embryonic brain development and after the consumption of an amino acid-deficient diet. Trib3 knockout mice exhibited enlarged lateral ventricles in the brain; nevertheless, long-term spatial memory, fear memory and aversion to amino acid-imbalanced diet appear unaltered by a lack of Trib3. Finally, the role of TRIB3 in the cellular stress response to glucose deficiency was investigated using genome-wide gene expression profiling. Crucially, TRIB3 substantially alleviated the repression of IGFBP2 in glucose-deprived cells, which represents a novel mechanism of deferring cell death caused by nutrient deficiency

INTEGRATIVE ANALYSIS OF OMICS DATA IN ADULT GLIOMA AND OTHER TCGA CANCERS TO GUIDE PRECISION MEDICINE

Transcriptomic profiling and gene expression signatures have been widely applied as effective approaches for enhancing the molecular classification, diagnosis, prognosis or prediction of therapeutic response towards personalized therapy for cancer patients. Thanks to modern genome-wide profiling technology, scientists are able to build engines leveraging massive genomic variations and integrating with clinical data to identify “at risk” individuals for the sake of prevention, diagnosis and therapeutic interventions. In my graduate work for my Ph.D. thesis, I have investigated genomic sequencing data mining to comprehensively characterise molecular classifications and aberrant genomic events associated with clinical prognosis and treatment response, through applying high-dimensional omics genomic data to promote the understanding of gene signatures and somatic molecular alterations contributing to cancer progression and clinical outcomes. Following this motivation, my dissertation has been focused on the following three topics in translational genomics. 1) Characterization of transcriptomic plasticity and its association with the tumor microenvironment in glioblastoma (GBM). I have integrated transcriptomic, genomic, protein and clinical data to increase the accuracy of GBM classification, and identify the association between the GBM mesenchymal subtype and reduced tumorpurity, accompanied with increased presence of tumor-associated microglia. Then I have tackled the sole source of microglial as intrinsic tumor bulk but not their corresponding neurosphere cells through both transcriptional and protein level analysis using a panel of sphere-forming glioma cultures and their parent GBM samples.FurthermoreI have demonstrated my hypothesis through longitudinal analysis of paired primary and recurrent GBM samples that the phenotypic alterations of GBM subtypes are not due to intrinsic proneural-to-mesenchymal transition in tumor cells, rather it is intertwined with increased level of microglia upon disease recurrence. Collectively I have elucidated the critical role of tumor microenvironment (Microglia and macrophages from central nervous system) contributing to the intra-tumor heterogeneity and accurate classification of GBM patients based on transcriptomic profiling, which will not only significantly impact on clinical perspective but also pave the way for preclinical cancer research. 2) Identification of prognostic gene signatures that stratify adult diffuse glioma patientsharboring1p/19q co-deletions. I have compared multiple statistical methods and derived a gene signature significantly associated with survival by applying a machine learning algorithm. Then I have identified inflammatory response and acetylation activity that associated with malignant progression of 1p/19q co-deleted glioma. In addition, I showed this signature translates to other types of adult diffuse glioma, suggesting its universality in the pathobiology of other subset gliomas. My efforts on integrative data analysis of this highly curated data set usingoptimizedstatistical models will reflect the pending update to WHO classification system oftumorsin the central nervous system (CNS). 3) Comprehensive characterization of somatic fusion transcripts in Pan-Cancers. I have identified a panel of novel fusion transcripts across all of TCGA cancer types through transcriptomic profiling. Then I have predicted fusion proteins with kinase activity and hub function of pathway network based on the annotation of genetically mobile domains and functional domain architectures. I have evaluated a panel of in -frame gene fusions as potential driver mutations based on network fusion centrality hypothesis. I have also characterised the emerging complexity of genetic architecture in fusion transcripts through integrating genomic structure and somatic variants and delineating the distinct genomic patterns of fusion events across different cancer types. Overall my exploration of the pathogenetic impact and clinical relevance of candidate gene fusions have provided fundamental insights into the management of a subset of cancer patients by predicting the oncogenic signalling and specific drug targets encoded by these fusion genes. Taken together, the translational genomic research I have conducted during my Ph.D. study will shed new light on precision medicine and contribute to the cancer research community. The novel classification concept, gene signature and fusion transcripts I have identified will address several hotly debated issues in translational genomics, such as complex interactions between tumor bulks and their adjacent microenvironments, prognostic markers for clinical diagnostics and personalized therapy, distinct patterns of genomic structure alterations and oncogenic events in different cancer types, therefore facilitating our understanding of genomic alterations and moving us towards the development of precision medicine

Identifying therapeutic targets in glioma using integrated network analysis

Gliomas are the most common brain tumours in adult population with rapid progression and poor prognosis. Survival among the patients diagnosed with the most aggressive histopathological subtype of gliomas, the glioblastoma, is a mere 12.6 months given the current standard of care. While glioblastomas mostly occur in people over 60, the lower-grade gliomas afflict themselves upon individuals in their third and fourth decades of life. Collectively, the gliomas are one of the major causes of cancer-related death in individuals under fortyin the UK. Over the past twenty years, little has changed in the standard of glioma treatment and the disease has remained incurable. This study focuses on identifying potential therapeutic targets in gliomasusing systems-level approaches and large-scale data integration.I used publicly available transcriptomic data to identify gene co-expression networks associated with the progression of IDH1-mutant 1p/19q euploid astrocytomas from grade II to grade III and high-lighted hub-genes of these networks, which could be targeted to modulate their biological function. I also studied the changes in co-expression patterns between grade II and grade III gliomas and identified a cluster of genes with differential co-expression in different disease states (module M2). By data integration and adaptation of reverse-engineering methods, I elucidated master regulators of the module M2. I then sought to counteract the regulatory activity by using drug-induced gene expression dataset to find compounds inducing gene expression in the opposite direction of the disease signature. I proposed resveratrol as a potentially disease modifying compound, which when administered to patients with a low-grade disease could potentially delay glioma progression.Finally, I appliedanensemble-learning algorithm on a large-scale loss-of-function viability screen in cancer cell-lines with different genetic backgrounds to identify gene dependencies associated with chromosomal copy-number losses common intheglioblastomas. I propose five novel target predictions to be validated in future experiments.Open acces

Macrophage-Dependent Trafficking and Remodeling of Extracellular Matrix Barriers

Macrophages are evolutionarily-conserved immune cells distributed throughout all tissues in the body, which rapidly mobilize to defend against a range of insults. In executing events ranging from wound healing and host defense functions to regulating the tumor microenvironment, macrophages traverse and remodel extracellular matrix (ECM) barriers, i.e. the basement membrane and interstitial matrix. To date, the molecular mechanisms operative during macrophage migration and remodeling of ECM barriers have relied on non-physiologic in vitro constructs whose relevance to the in vivo environment remains unclear. As such, we have adopted an ex vivo native tissue model as well as a 3-dimensionsal type I collagen hydrogel model that retain structural crosslinks integral to the barrier characteristics of the in vivo ECM. Using primary mouse and human macrophages in conjunction with high-resolution confocal microscopy, we characterize a program wherein macrophages degrade the basement membrane and infiltrate the interstitial matrix. We find that of the dozens of proteases that macrophages express in response to immune stimuli, only the membrane-anchored metalloprotease, MT1-MMP, is absolutely required for basement membrane degradation. Furthermore, we discover a unique hybrid ability of macrophages to either degrade the basement membrane in an MT1-MMP-dependent fashion or alternatively, mobilize actomyosin-mediated mechanical forces to non-proteolytically traverse preformed portals that exist in the basement membrane. Though macrophages can transmigrate the basement membrane via either mechanism, the transcriptional program of tissue-invasive macrophages is alternatively regulated during proteinase-dependent versus independent invasion. Following basement membrane transmigration, macrophages then confront a high-density interstitial matrix that is dominated by type I collagen. Under these conditions, macrophages must again mobilize MT1-MMP to create passageways through the interstitial matrix that permit the transit of the rigid macrophage nucleus. Strikingly, in the absence of MT1-MMP activity, the macrophage is incapable of creating matrix tunnels that support efficient invasion. Instead, the macrophage traverses the matrix while the rigid nucleus remains trapped and distorted above the surface of the collagen matrix. These studies, together with preliminary data from mouse models of cancer, establish new paradigms for MT1-MMP-dependent macrophage trafficking and remodeling of physiologically-relevant ECM barriers.PHDCancer BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147550/1/jcbahr_1.pd

Molecular Targets of CNS Tumors

Molecular Targets of CNS Tumors is a selected review of Central Nervous System (CNS) tumors with particular emphasis on signaling pathway of the most common CNS tumor types. To develop drugs which specifically attack the cancer cells requires an understanding of the distinct characteristics of those cells. Additional detailed information is provided on selected signal pathways in CNS tumors

Synthetic Genetic Tracing of Molecular and Cellular Heterogeneity in Glioblastoma

Das Glioblastom (GBM) repräsentiert den am schwierigsten zu behandelnden primären soliden Tumor des Zentralnervensystems dar, trotz der intensiv wachsenden Zahl von Studien zu seinen molekularen und zellulären Eigenschaften. Obwohl die GBM-Therapie aggressiv ist und chirurgische Resektion, Strahlentherapie und Chemotherapie umfasst, ist ein Wiederauftreten des Tumors unvermeidlich. Die GBM-Behandlungsresistenz ist mit genetischer und zellulärer Heterogenität sowie phänotypischer Plastizität verbunden. Um das Verständnis der Heterogenität des Glioblastoms zu vertiefen, haben wir maßgeschneiderte genetische Tracing-Strategien für subtypspezifische Transkriptionszustände aus Glioblastom-Patientensignaturen entwickelt. In GBM-Zellen ermöglichte uns unsere neuartige Technologie, intrinsische und nicht-zellautonome Bestimmungsfaktoren von Zellzuständen zu identifizieren. In vitro und in vivo konnten wir zeigen, dass sich der mesenchymale GBM-Subtyp als adaptive Identität in Gegenwart von Mikroumgebungssignalen ausbildet und durch Entzündungs- und Differenzierungsprogramme reguliert wird. Wir haben gezeigt, dass die Ausbildung eines mesenchymalen Zellzustand adaptiv und reversibel ist und durch verschiedene Auslöser wie externer Signaltransduktion und ionisierende Strahlung mit teilweise überlappenden transkriptionellen Signaturen eingenommen werden kann. Insbesondere konnten wir mithilfe synthetischer Locus-Kontrollregionen (sLCRs) eine Interaktion zwischen Zellen des angeborenen Immunsystems und Glioma-Zellen aufdecken, wodurch die Tumorzellen in einen mesenchymalen Zustand versetzt wurden, der mit einer erhöhten Resistenz gegen Chemotherapie verbunden ist. Hier bauen wir auf diesem innovativen Ansatz auf, um Übergänge von Zellzuständen in komplexen biologischen Umgebungen zu verfolgen, mit einem Schwerpunkt auf der zellulären Wechselwirkung zwischen gesunden und Tumorzellen im Zusammenhang mit phänotypischer Plastizität und therapeutischer Resistenz. Darüber hinaus bietet diese Methode ein breites translationales Potenzial für die Anwendung auf andere Forschungsgebiete, einschließlich der Entwicklungsbiologie oder der regenerativen Medizin.Glioblastoma (GBM) remains the most difficult primary solid tumor of the central nervous system despite the intensively growing body of research on its molecular and cellular characteristics. Whereas GBM treatment is aggressive and involves surgical resection, radiotherapy, and chemotherapy, tumor recurrence is unavoidable. GBM treatment resistance is associated with genetic and cellular heterogeneity, as well as phenotypic plasticity. To improve understanding of Glioblastoma heterogeneity, we developed custom genetic tracing strategies for subtype-specific transcriptional states from Glioblastoma patient signatures. In GBM cells, our novel technology enabled us to identify intrinsic and non-cell autonomous determinants of cell fate commitment. In vitro and in vivo, we discovered that the mesenchymal GBM adapts in the presence of microenvironmental signaling and is regulated by inflammatory and differentiation programs. We demonstrated that cell fate commitment towards a mesenchymal state is adaptive and reversible and occurs through partially overlapping transcriptional responses, including external signaling and ionizing radiation. Importantly, using synthetic locus control regions (sLCRs), we were able to uncover crosstalk between innate immune cells and glioma-initiating cells, directing the tumor cells into a mesenchymal state linked to increased resistance to chemotherapy. Here, we build on this innovative approach to trace cell fate transitions in complex biological settings, with a focus on the cellular crosstalk between malignant and non-tumor cells in the context of phenotypic plasticity and therapeutic resistance. Beyond that, this method offers the broad translational potential to be applied to other fields of research, including developmental biology or regenerative medicine

The Role of Microenviroment in Glioblastoma Progression and Resistance Development

El glioblastoma (GBM) es un tumor cerebral primario altamente heterogéneo, con una tasa de supervivencia muy baja. Recientemente se ha demostrado que su microentorno tumoral complejo tiene un papel esencial en la progresión del tumor y la respuesta a la terapia. Por lo tanto, es crucial identificar todos los componentes y sus interacciones, e incorporarlos en modelos in vitro utilizados para estudios sobre GBM y el desarrollo de nuevas terapias. El desarrollo de nuevas tecnologías en las últimas décadas ha asegurado el progreso en ambos campos mencionados. Diferentes técnicas multiómicas permiten una caracterización detallada de las muestras de los pacientes. Por otro lado, la evolución de las técnicas de cultivo celular y los procesos de fabricación permiten la creación de sistemas in vitro más fisiológicos que el cultivo tradicional en placas de Petri (organ on chip). El principal objetivo de esta tesis fue estudiar el papel del microentorno en la respuesta del GBM al tratamiento con temozolomida (TMZ). Se modificaron dispositivos microfluídicos, desarrollados previamente dentro del grupo, para estudiar el impacto de la concentración de oxígeno en la progresión de GBM. Se demostró que la hipoxia es esencial para la formación del núcleo necrótico y protege a las células del efecto de TMZ. Además, se mejoró el diseño del dispositivo microfluídico para permitir la creación de un sistema más avanzado y controlable. Igualmente, el cultivo de esferoides nos proporcionó un modelo valioso para los estudios de desarrollo de quimio-resistencia. Tras la aplicación de dos ciclos de tratamiento clínico con TMZ, se observó la aparición de una población de esferoides resistentes. Morfológicamente, esos esferoides eran una combinación de esferoides control y esferoides tratados, que tenían un patrón de expresión génica específico. Por último, se utilizó una nueva técnica de transcriptómica espacial para caracterizar mejor las muestras de pacientes con GBM, correlacionando su expresión génica con la ubicación histológica. Esto permitió la identificación de clusters transcriptómicos diferenciales dentro de tejidos aparentemente homogéneos, confirmando la alta heterogeneidad de este tumor, no solo en el aspecto morfológico sino también molecular. Glioblastoma (GBM) is a highly heterogeneous primary brain tumor, with a very low survival rate. It has been shown recently that the complex tumor microenvironment has an essential role in tumor progression and therapy response. Hence, it is crucial to identify all the components and their interactions, and incorporate them in in vitro models used for GBM studies and therapy development. The development of new technologies in the last decades ensured progress in both mentioned fields. Different multiomics techniques allow detailed characterization of the patient samples. On the other hand, the evolution of cell culture techniques and fabrication processes enables the creation of more physiological in vitro systems than traditional Petri dish culture (organ on chip). The main aim of this thesis was to study the role of the microenvironment in the response of GBM to temozolomide (TMZ) treatment. Microfluidic devices, previously developed within the group, were modified to study the impact of oxygen concentration on GBM progression. Hypoxia was shown to be essential for the necrotic core formation and it protected cells from the TMZ effect. Moreover, the microfluidic device design was improved to enable the creation of a more advanced and controllable system. Furthermore, spheroid culture gave us a valuable model for chemoresistance development studies. After the application of two clinical TMZ treatment cycles, the presence of a population of resistant spheroids was observed. Morphologically, those spheroids were a combination of control and treated spheroids, and they had a specific gene expression pattern. Last but not least, a new spatial transcriptomics technique was used to characterize better GBM patient samples correlating their gene expression with the histological location. It enabled the identification of differential transcriptomic clusters within apparently homogeneous tissues, confirming the high heterogeneity of this tumor, not only in a morphological aspect but also molecularly.<br /