Functional Toxicogenomics: Mechanism-Centered Toxicology

Traditional toxicity testing using animal models is slow, low capacity, expensive and assesses a limited number of endpoints. Such approaches are inadequate to deal with the increasingly large number of compounds found in the environment for which there are no toxicity data. Mechanism-centered high-throughput testing represents an alternative approach to meet this pressing need but is limited by our current understanding of toxicity pathways. Functional toxicogenomics, the global study of the biological function of genes on the modulation of the toxic effect of a compound, can play an important role in identifying the essential cellular components and pathways involved in toxicity response. The combination of the identification of fundamental toxicity pathways and mechanism-centered targeted assays represents an integrated approach to advance molecular toxicology to meet the challenges of toxicity testing in the 21st century

Multiplexed combinatorial drug screening using droplet-based microfluidics

The therapy of most cancers has greatly benefited from the use of targeted drugs. However, their effects are often short-lived since many tumors develop resistance against these drugs. Resistance of tumor cells against drugs can be adaptive or acquired and is often caused by genetic or non-genetic heterogeneity between tumor cells. A potential solution to overcome drug resistance is the use of drug combinations addressing multiple targets at once. Finding potent drug combinations against heterogeneous tumors is challenging. One reason is the high number of possible combinations. Another reason is the possibility of inter-patient heterogeneity in drug responses, making patient tailored treatments necessary. These require screens on patient material, which would drastically benefit from miniaturization, as it is the case in droplet-based microfluidics. However, drug screens in droplets against primary tumor cells have so far only been performed at a modest chemical complexity (55 treatment conditions) and with low content readouts. In this thesis we aimed at developing a droplet-based microfluidic workflow that allows the generation of high numbers of drug combinations in picolitre-sized droplets and their multiplexed analysis. To this end, we have established a pipeline to produce up to 420 drug combinations in droplets. We were able to significantly increase the number of possible combinations by building a microfluidic setup that comprises valve and micro-titer plate based injection of drugs into microfluidic devices for droplet generation Furthermore, we integrated a DNA-based barcoding approach to encode each treatment condition, enabling their multiplexed analyses since all droplets can be stored and processed together, which highly increases the throughput. With the established approach we can perform barcoding of each cells’ transcriptome according to the drugs it was exposed to in the droplet. Thereby, the effects of drug combinations on gene expression can be studied in a highly multiplexed way using RNA-Sequencing. We applied the developed approach to run combinatorial drug screens in droplets and analysed the effects of in total 630 drug combinations on gene expression in K562 cells. The low number of cells needed (max. 2 million cells) for such screens, could enable their application directly on tumor biopsies, thus paving the way for personalized therapy approaches. Since the established workflow is compatible with single cell readouts, we also envision its application to analyse drug resistances in heterogeneous tumor samples on the single cell level

Defining Protein Synthesis: New Technologies to Elucidate Translational Control

Protein translation has emerged as an important mediator of cellular activity. Here, we discuss efforts to develop and apply new technologies designed to gain insights into translational control. We begin with the application of ribosome profiling to a RiboTag Glioma mouse model which enables translational profiling of transformed cellular populations. This approach demonstrates a number of abnormalities of translation in transformed cells. We go on to report the development of an inexpensive and rapid library preparation methodology which enables high-throughput sequencing of ribosome-protected footprints from small amounts of input material. We apply this technique to a CAMKII RiboTag mouse model to make new insights into cell-type specific translation. Finally, we describe efforts to investigate translation regulatory networks through the development of a technique which couples large-scale perturbation with a genome-wide readout of translation. Molecular dissection of tissues through the ectopic expression of modified ribosomal proteins commonly relies on tissue-specific genes which act as drivers. In the case of glioma, a gene specific to transformed tissue, but not expressed in normal brain tissue, has not been identified. Chapter 2 focuses on efforts to bypass this through the development of a RiboTag Glioma mouse model which allows for concurrent transformation and the expression of an epitope-tagged ribosomal protein in virally infected cells. This model made possible the isolation of translating mRNA from transformed cellular populations and was used to demonstrate the existence of a number of translational abnormalities in transformed cells. Conventional ribosome profiling is a powerful tool which allows for the identification of ribosome-protected mRNA footprints. However, it is time-consuming, expensive, and difficult to implement. Based on our experiences with conventional ribosome profiling, we sought to develop a method which could decrease the overall number of enzymatic reactions and purification steps, thereby reducing the time and cost associated with the procedure; these efforts are discussed in Chapter 3. Utilizing a ligation-free library preparation process, which incorporates poly(A)-polymerase, template switching and bead-based purification, we reduced the time, costs and input requirements required to generate a ribosome profiling library while maintaining high library complexity. We applied our ligation-free ribosome profiling technique to a CAMKII RiboTag mouse model which enabled us to identify patterns of cell-type specific translation and the effects of mTOR inhibition in CAMKII-expressing excitatory neurons. Regulation of protein expression is an essential and highly complex cellular activity. Aberrations of translational control are central to a host of pathologies and have direct clinical relevance. However, our knowledge of the networks which control translation is limited. Chapter 4 details our efforts to develop a highly-scalable technology which enables the identification of gene-specific translational alteration in response to perturbation. Coupled with a large-scale perturbation screen, this technique could lead to the generation of a network for translational control, similar to efforts previously undertaken to understand transcriptional control. By combining the recently developed PLATE-Seq method, which utilizes unique barcode identifiers and pooled library construction, with a technique for the identification and isolation of ribosome associated mRNA, we are able to rapidly and inexpensively determine genome-wide translational states in a highly scalabl

LONGITUDINAL CLONAL LINEAGE DYNAMICS AND FUNCTIONAL CHARACTERIZATION OF PANCREATIC CANCER CHEMO-RESISTANCE AND METASTASIZATION

In recent years, technological advancements, such as next-generation sequencing and single-cell interrogation techniques, have enriched our understanding in tumor heterogeneity. By dissecting tumors and characterizing clonal lineages, we are better understanding the intricacies of tumor evolution. Tumors are represented by the presence of and dynamic interactions amongst clonal lineages. Each lineage and each cell contributes to tumor dynamics through intrinsic and extrinsic mechanisms, and the variable responses of clones to perturbations in the environment, especially therapeutics, underlie disease progression and relapse. Thus, there exists a pressing need to understand the molecular mechanisms that determine the functional heterogeneity of tumor sub-clones to improve clinical outcomes. Clonal replica tumors (CRTs) is an in vivo platform created specifically to enable robust tracing and functional study of clones within a tumor. The establishment of CRTs is built upon our current concept of tumor heterogeneity, intrinsic cancer cell hierarchy and clonal self-renewal properties. The model allows researchers to create large cohorts of tumors in different animals that are identical in their clonal lineage composition (clonal correlation amongst tumors \u3e0.99). CRTs allow simultaneously tracking of tens of thousands of clonal lineages in different animals to provide a high level of resolution and biological reproducibility. CRTs are comprised of barcoded cells that can be identified and quantified. A critical feature is that we have developed a systematic method to isolate and expand essentially any of the clonal lineages present within a CRT in their naïve state; that is, we can characterize each sub-clonal lineage at the molecular and functional levels and correlate these findings with the behavior of the same lineage in vivo and in response to drugs. Here, based on the CRT model and its concept, we studied differential chemo-resistance among clones, where we identified pre-existing upregulation in DNA repair as a mechanism for chemo-resistance. Furthermore, through stringent statistical testing, we demonstrated orthotopic CRTs to be a powerful and robust model to quantitatively track clonal evolution. Specifically, we longitudinally tracked clones in models of pancreatic ductal adenocarcinoma (PDAC) from primary tumor expansion through metastasization, where we captured unexpected clonal dynamics and “alternating clonal dominance” naturally occurring in unperturbed tumors. Moreover, by characterizing pro- and none-metastasizing clones, we were able to identified key clonal intrinsic factors that determined the nature of tumor metastases. Finally, I will discuss distinct clonal evolution patterns that emerged under different environmental pressures, leading to the hypothesis of “tumor clonal fingerprint”, where the characteristic of a tumor could be defined by actively maintained ratio of different tumor lineages, which could provide measurable insights to how we approach treatments

Principles of Massively Parallel Sequencing for Engineering and Characterizing Gene Delivery

The advent of massively parallel sequencing and synthesis technologies have ushered in a new paradigm of biology, where high throughput screening of billions of nucleid acid molecules and production of libraries of millions of genetic mutants are now routine in labs and clinics. During my Ph.D., I worked to develop data analysis and experimental methods that take advantage of the scale of this data, while making the minimal assumptions necessary for deriving value from their application. My Ph.D. work began with the development of software and principles for analyzing deep mutational scanning data of libraries of engineered AAV capsids. By looking at not only the top variant in a round of directed evolution, but instead a broad distribution of the variants and their phenotypes, we were able to identify AAV variants with enhanced ability to transduce specific cells in the brain after intravenous injection. I then shifted to better understand the phenotypic profile of these engineered variants. To that end, I turned to single-cell RNA sequencing to seek to identify, with high resolution, the delivery profile of these variants in all cell types present in the cortex of a mouse brain. I began by developing infrastructure and tools for dealing with the data analysis demands of these experiments. Then, by delivering an engineered variant to the animal, I was able to use the single-cell RNA sequencing profile, coupled with a sequencing readout of the delivered genetic cargo present in each cell type, to define the variant’s tropism across the full spectrum of cell types in a single step. To increase the throughput of this experimental paradigm, I then worked to develop a multiplexing strategy for delivering up to 7 engineered variants in a single animal, and obtain the same high resolution readout for each variant in a single experiment. Finally, to take a step towards translation to human diagnostics, I leveraged the tools I built for scaling single-cell RNA sequencing studies and worked to develop a protocol for obtaining single-cell immune profiles of low volumes of self-collected blood. This study enabled repeat sampling in a short period of time, and revealed an incredible richness in individual variability and time-of-day dependence of human immune gene expression. Together, my Ph.D. work provides strategies for employing massively parallel sequencing and synthesis for new biological applications, and builds towards a future paradigm where personalized, high-resolution sequencing might be coupled with modular, customized gene therapy delivery.</p

Estimating the time-dependent RNA kinetic rates in the cell cycle

Die Menge an RNA in Eukaryonten wird durch ihre kinetischen Transkriptions-, Verarbeitungs- und Abbauraten bestimmt. Diese kinetischen Raten wurden bereits ausführlich in Zellpopulationen untersucht, allerdings unter der Annahme, dass diese in verschiedenen Zelltypen identisch sind. Die Genexpression ist jedoch während biologischer Prozesse wie z.B der Zellproliferation, Zelldifferenzierung und Zellteilung hochdynamisch. Die Untersuchung der RNA- Kinetikraten in Einzelzellen, die sich in verschiedenen Phasen desselben dynamischen Prozesses befinden, kann uns ein umfangreicheres Bild davon geben, wie RNA-Kinetikraten die Genexpression zeitabhängig koordinieren. In diesem Projekt, Wir haben die Methode der RNA- Stoffwechselmarkierung und der biochemischen Nukleosidkonversion mit der Einzelzell-RNA- Sequenzierung kombiniert. Wir leiteten ein zeitabhängiges kinetisches Geschwindigkeitsmodell ab und schätzten RNA-Transkriptions- und - Abbauraten über den zeitlichen Verlauf des Zellzyklus ab. Dabeiverwendeten wir Näherungen basierend auf der Lösung des resultierenden Differentialgleichungssystems. Wir fanden heraus, dass Transkriptions- und Abbauraten der meisten zyklischen Gene hochdynamisch sind. Unterschiedliche kinetische Regulationsmuster formen spezifische Genexpressionsprofile. Etwa 89 % der 377 von uns analysierten zyklischen Gene werden durch dynamische Transkriptions- und Abbauraten reguliert. Während der dynamischen Transkriptionsrate beobachteten wir auch, dass einige zyklische Gene durch dynamische Zerfallsraten angetrieben wurden. Unsere Studie bekräftigt die Bedeutung der zeitlichen Regulation von der Genexpression durch Produktion und Zerfall. Darüber hinaus hat die von uns entwickelte Methode das Potenzial, an verschiedene biologische Prozesse angepasst zu werden. Unser Ansatz in dieser Studie kann die Untersuchung der zeitlichen Genexpressionsregulation und der RNS- Kinetikraten voranbringen.RNA abundance in eukaryotes is determined by its kinetic rates of transcription, processing and degradation. Each of the kinetic rates has been extensively studied in bulk cell populations assuming they are equal in different cells. However, gene expression is highly dynamic during biological processes such as cell proliferation, cell differentiation, and cell division. Investigation of RNA kinetic rates in individual cells which are in different phases of the same dynamic process can give us a more comprehensive picture of how RNA kinetic rates coordinate gene expression in a time-dependent manner. In this project, we adapted the RNA metabolic labeling and biochemical nucleoside conversion method to droplet- based single-cell RNA sequencing. We derived a time- dependent kinetic rate model and estimated RNA transcription and degradation rates over the time course of the cell cycle using approximations based on the solution of the resulting system of differential equations. We found that transcription and degradation rates of most cycling genes are highly dynamic. Different kinetic regulation patterns shape specific gene expression profiles. Around 89% of the 377 cycling genes we analyzed are regulated by dynamic transcription and degradation rates. While dynamic transcription rate was prevalent, we also observed some cycling genes were driven by dynamic decay rates. Our study underscores the importance of temporal gene expression regulation by both production and decay. Moreover, the method we developed has the potential to be adapted to different biological processes. We suggest that our approach can advance the study of temporal gene expression regulation and RNA kinetic rates

Chemical-genetic interrogation of small molecule mechanism of action in S. cerevisiae

The budding yeast S. cerevisiae is widely used as a model organism to study biological processes that are conserved among eukaryotes. Di fferent genomic approaches have been applied successfully to interrogate the mode of action of small molecules and their combinations. In this thesis, these technologies were applied to di fferent sets of chemical compounds in the context of two collaborative projects. In addition to insight into the mode of action of these molecules, novel approaches for analysis of chemical-genetic pro files to integrate GO annotation, genetic interactions and protein complex data have been developed. The fi rst project was motivated by a pressing need to design novel therapeutic strategies to combat infections caused by opportunistic fungal pathogens. Systematic screens of 1180 FDA approved drugs identifi ed 148 small molecules that exhibit synergy in combination with uconcazole, a widely used anti-fungal drug (Wright lab, McMaster University, Canada). Genome-wide chemical-genetic profiles for 6 of these drugs revealed two di fferent modes of action of synergy. Five of the compounds a ffected membrane integrity; these chemical-genetic interactions were supported by microscopy analysis and sorbitol rescue assays. The sixth compound targets a distinct membrane-associated pathway, sphingolipid biosynthesis. These results not only give insight into the mechanism of the synergistic interactions, they also provide starting points for the prediction of synergistic anti-fungal combinations with potential clinical applications. The second project characterised compounds that aff ected melanocytes in a chemical screen in zebra fish (Patton lab, Edinburgh). Chemical-genetic screens in S.cerevisiae enabled us to show that melanocyte pigmentation reducing compounds do so by interfering with copper metabolism. Further, we found that defects in intracellular AP1 and AP3 trafficking pathways cause sensitivity to low copper conditions. Surprisingly, we observed that the widely-used MAP-kinase inhibitor U0126 a ffects copper metabolism. A nitrofuran compound was found to speci fically promote melanocyte cell death in zebrafi sh. This enabled us to study off -target eff ects of these compounds that are used to treat trypanosome infections. Nifurtimox is a nitrofuran prodrug that is activated by pathogen-specifi c nitroreductases. Using yeast and zebra fish we were able to show that nitrofurans are also bioactivated by host-specifi c aldehyde dehydrogenases suggesting that a combination therapy with an aldehyde dehydrogenase inhibitor might reduce side e ffects associated with nifurtimox

Functional Screens Identify Vulnerabilities in Acute Leukemia

Acute leukemia refers to a group of aggressive hematological malignancies of myeloid and lymphoid lineages termed acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) respectively. Acute leukemia is characterized by the presence of underlying genetic aberrations which alter the biology of normal hematopoietic cells resulting in the accumulation of immature abnormally differentiated blast cells. In this thesis, we have used advanced molecular techniques to identify vulnerabilities in acute leukemia.In paper I, we performed an in vivo CRISPR-Cas9 screen targeting cell surface genes in murine AML stem cells and showed that CXCR4 is a top cell surface regulator of AML cell growth and survival. Notably, loss of CXCR4 signaling in leukemia cells leads to oxidative stress and differentiation in vivo. In contrast, the CXCR4 ligand CXCL12 is dispensable for leukemia development in recipient mice.To identify key regulators of AML, in paper II, we performed an ex vivo cytokine screen on arrayed molecularly barcoded murine AML cells with a competitive in vivo read-out of their leukemia-initiating capacity. We identified TNFSF13 as a positive regulator of leukemia-initiating cells. We confirmed that TNFSF13 supports leukemia initiation under physiological conditions using Tnfsf13-/- mice. We further showed that TNFSF13 suppresses apoptosis and promotes AML cell proliferation in an NF-κB dependent manner.DUX4-rearranged BCP-ALL is a recently identified molecular subtype characterized by the expression of the IGH- DUX4 fusion gene. With the aim of identifying biological dependencies of this subtype, in paper III, we performed a genome-wide CRISPR-Cas9 screen in the NALM6 cell line, driven by the IGH-DUX4 fusion gene, and two control cell lines. We showed that FNIP1, IRF4 and SYNCRIP are selectively important for the growth and survival of NALM6 cells and that their expression is under the control of the IGH-DUX4 fusion gene. While the deletion of FNIP1 led to the enrichment of transcriptional signatures associated with metabolic dysregulation, loss of IRF4 resulted in upregulation of genes involved in differentiation and apoptosis of NALM6 cells. Moreover, disruption of SYNCRIP caused downregulation of the TGFβ-SMAD signaling pathways in NALM6 cells.In paper IV, we explored the immune-mediated anti-leukemic activity of the cytokine interleukin 4 (IL4) in a murine AML model. Overexpression of IL4 in AML cells resulted in a strong anti-leukemic effect accompanied by an expansion of macrophages in the bone marrow and spleen of the recipient mice. Depletion of macrophages in vivo eliminated the antileukemic effect of IL4. In addition, IL4 directly activates murine macrophages resulting in enhanced phagocytosis of AML cells in vitro. Interestingly, IL4 also induced Stat6-dependent upregulation of CD47 in AML cells thereby inhibiting phagocytosis. Consistent with this finding, IL4 stimulation combined with CD47 blockade enhanced macrophage-mediated phagocytosis of AML cells.Taken together, the studies included in this thesis employed high-throughput functional screens using CRISPR- Cas9 and molecular barcoding techniques to identify key regulators of AML cells. These findings improve our understanding of the disease and may translate into the development of new therapies for acute leukemia