Sidi Chen - US grants

To understand the evolutionary diversification of animals, the applicants will investigate the variation of phenotypes and their genetic basis in multiple species of fruitflies in the genus Drosophila. The connection to diverse phenotypic features has been mapped to protein-coding changes, cis-regulatory changes and lineage-specific genes. The applicants' recent discoveries showed that many young genes played essential roles in organism development. This research will (1) investigate the reproductive contribution of young genes using the evolutionary genetic analysis and gene silencing techniques, (2) detect their interactions with other genes in the genome using the gene-chip based techniques of experimental genomics, and (3) study their roles in the species divergence by comparative analysis of related species. These efforts will advance the understanding of fundamental problems of gene evolution and phenotypic evolution.

These studies will bring conceptual innovations in the understanding of biological systems. The applicants will also create new data and share with the public. Furthermore, the applicants will train undergraduate students, visiting students from other schools and different disciplines, to encourage more talented undergraduates to enter graduate schools and nurture cross-disciplinary investigation. The applicants will purposely initiate more collaborate with multiple research groups in various institutions both nationally and internationally. The knowledge on gene origination is important to the understanding of human evolution. The proposed project will be of general interest to the broad audience including scientists and non-scientists and the discoveries from this project will play a role in scientific education for the general public.

PROJECT SUMMARY/ABSTRACT: Core 2 We will establish a core facility for in vivo cancer modeling and screening. This core facility (core 2 in the cancer systems biology center) will consist of several sets of functional components, defined as modules, including vector design and genome-scale library construction, large-scale cell culture and virus production, tumor transplantation and in vivo pooled screening, CRISPR/Cas9 mediated somatic genome editing for invasive phenotype analysis. In preliminary studies, we have applied in vivo somatic genome editing to generate tumor models of specific driver genes in mice, and demonstrated in vivo screening in a lung metastasis animal model. We will build four modules in this core, including the following: Module 1. Facility for vector design and genome-scale library construction. This module supports functional investigations of the mammalian genome that can reveal how genetic alterations lead to changes in phenotype, for example cellular invasiveness phenotype described in the Projects 1 and 2. Module 2. Facility for large-scale cell culture, viral vector production and transduction. This will serve for two purposes: (1) generation of customized viral particles for in vivo studies involving animals models for Projects 1 and 2 as well as other collaborators; (2) high-throughput screening applications that requires transduction of a population of cells with highly complex libraries. Module 3. Facility for tumor transplantation and in vivo pooled screening. This module will setup a facility with two capacities: (1) tumor transplantation and (2) in vivo pooled screening. This will serve for the purpose of validating the genes discovered in the projects in mouse models, to discover new genes with invasive phenotypes using in vivo screens, or to enable collaborative research for discovery of new drug targets. Module 4. Facility for CRISPR/Cas9 mediated somatic genome editing, for somatic genome editing in various animals for virtually any loci in the mouse genome. We will establish this platform and utilize it for research described in the projects and for setting up collaborations with various Yale investigators. Specifically, we will utilize CRISPR library approach in conjunction with the Rapid Analysis of Cell migration Enhancement (RACE) system described in the Projects, to identify how genetic alterations lead to changes in cellular invasiveness phenotype. In addition, we will utilize these modules in the core to build novel models of WNK1, NKCC1 and their targets, as well as combinations of mutant ERK, AKT and downstream genes. In summary, this core will be tightly integrated into the U54 center, provide a powerful technology and resource platform for in vivo cancer systems biology, and support Research Projects in the center as well as other collaborators at Yale and the wider scientific community. Since there is no facility currently existing at Yale providing similar functions, this new core will facilitate the establishment of the first in vivo cancer modeling and screening core facility at Yale. We will also utilize this core facility for promoting education and training, serving as a base for education of cancer researchers, scientists, students, and the general public.

PROJECT SUMMARY: The evolution of human cancer is a complex process driven by multiple molecular and cellular events. Cancer cells often harbor numerous aberrations that can act in additive, parallel, antagonistic, epistatic or synergistic fashion. Those interactions contribute to tumorigenesis, progression, metastasis, drug resistance or other life-threatening features. While these interactions can be weakly inferred from analysis of tumor sequence data, elucidating genetic interactions in vivo is essential for rapidly building a robust map of cancer development and to accelerate therapeutic developments. However, there are currently few effective tools for precise multigenic manipulation of cancer in vivo, limiting our scope for accurately dissecting these interactions. We endeavored to harness single-effector RNA-guided endonucleases (RGNs) for genome editing, parallel screening and in vivo modeling of human cancer. Recently, we generated a platform to systematically interrogate several hundred loci directly in vivo. To overcome current limitations in multigene editing and achieve more accurate control of simultaneity and sequentiality of multi-allelic tumor modeling, we utilized Cpf1, an RGN that can edit its target simply with crRNAs independent of tracrRNA thus allowing simultaneous editing of multiple genes with a single crRNA array. We developed a preliminary Cpf1-based crRNA array screening (CCAS) system in mammalian cells, and applied it in mouse models of progression and metastasis. In our first aim, we will perform validation and optimization of CCAS for in vivo double-knockout phenotyping of cancer co-drivers. We will establish its technical rigor, efficiency and specificity for simultaneous editing, as well as developing a set of computational pipelines for accurate calling of statistically significant gene pairs. We will apply this approach to study the genetic interactions of tumor suppressors found in lung cancer patients at Yale Cancer Center and Hospital, and identify potential co-drivers of metastasis to vital organs. In the second aim, we will carry out validation and optimization of a Cpf1-Flip system for sequential mutagenesis of cancer targets. We will demonstrate its broader applicability by testing clinically relevant gene sets identified from public studies of the genomics of metastasis as well as a large multi-sample metastasis dataset gathered on Yale cancer patients. We will then apply this methodology as an unbiased depletion screen to identify targets that are essential for survival in specific oncogenic backgrounds. We will develop novel versatile transgenic mouse strains and companion viral vectors for direct modeling of multigenic tumorigenesis in mice. We will combine these tools to enable high-throughput genetic interaction screening in healthy cells directly in the native organ to identify causative mutation pairs that drive tumorigenesis. We anticipate that developing and establishing these tools will transform multigenic tumor modeling and pre-clinical studies of human cancer, directly addressing NCI Provocative Question 4. These powerful toolkits will enable scientists to target any gene pairs or combinations simultaneously or sequentially, assessing the phenotypic outcome of their in vivo interactions in tumor progression, metastasis, synthetic lethality, drug sensitivity or other processes in cancer evolution.

Summary The immune system is the foundation to maintain health. Enhanced ability to manipulate the immune system will enable us to precisely unleash the power of the immune system to combat against virtually all diseases. For example, cancer is one of the major health challenges in the US and around the world. Recently, major breakthroughs have been made by manipulating immune cells to fight cancer. However, these methods are currently imperfect, often encounter irresponsiveness to primary treatments, acquired resistance, or systemic toxicity due to failure to control the cytotoxic cell activity that damage host organs, or inflammation induced cytokine storms. Moreover, the immune system has enormous diversity and plasticity, which can go beyond control under pathological settings in an immunological disorder, or under artificial settings of immunotherapy. Many immunotherapy targets remain to be discovered in an unbiased manner. Identification and characterization of key immunomodulatory genes and pathways in vivo remains a critical bottleneck in the expansion of the immunotherapeutic armamentarium. My goal is to innovate new platforms to precisely engineer the genome and transcriptome of immune cells in a high-throughput manner, and apply them to identify and characterize new genes fundamental for T cell function and anti- tumor activity. To approach this goal my lab has recently developed a high-throughput screening system in CD8+ T cells, and successful performed T cell genetic screens in vivo, which identified multiple genes modulating the phenotypes of CD8+ T including cells trafficking, survival, tumor infiltration, and effector function. In the first program of this project, we will interrogate newly discovered immunotherapy targets including surface proteins, signaling or regulatory proteins, and metabolic enzymes, which will yield new insights on how they regulate T cell's function and anti-tumor activity. We will develop new agents and methods for pharmacological inhibition of the validated and clinically relevant targets as new routes of treatments and test them in pre-clinical settings. In the second program, we will further innovate next- generation immunogenetics tools. We will generate a genetic toolbox for better control of T cell gene expression, and couple it to high-throughput screening system for identification of new sets of immunotherapy targets that acts mainly through gene expression. Finally, we will engineer T cell signal transduction circuitry for more efficient activation of T cell function. These studies, if successful, will lead to new platforms for high-throughput in vivo immunogenetics, new targets to modulate T cell function and anti- tumor activity, as well as provisional concepts of first-in-class agents for immunotherapy.

Project Summary: Cancer is a major cause of death worldwide with outstanding challenges for a cure. Such challenges are primarily due to the nature of tumor heterogeneity and evolvability. Thus, the ability to generate unbiased, quantitative and causal maps of functional drivers and their combinations in native tumor microenvironment is a key to accelerate therapeutic discovery. To date, little has been done to comprehensively and combinatorially test which of the mutations identified in human patients can indeed functionally drive tumorigenesis of normal cells in native organs. The major barriers include accurate delivery, precise genome manipulation, efficient massively parallel perturbation, and unbiased, high-sensitivity quantitative readout, all of which have to be achieved simultaneously in the native tissue microenvironment. We recently established a novel approach named Pooled AAV Screen with Targeted Amplicon Sequencing (PASTAS) for direct in vivo screening of causative cancer drivers and combinations. This method generates precision models of cancer that (1) spontaneously develop from tumor-originating cells in the native organ microenvironment, (2) develop in fully immunocompetent animals and preserve the immune microenvironment, (3) genetically mimic significant mutations found in patients, (4) closely mimic the histopathology of human disease and clinical features, (5) encompass high degree of genetic and cellular heterogeneity, (6) offer flexibility to target any choice of target genes and rapidly scalable as pooled mutant screens, and (7) is easy to use by the community. In this study, we will conduct advanced development, robust validation and full establishment of this screening system. We will first establish technical parameters for optimal performance of this technology by quantitative measurements using independent patient cohorts with two lethal cancer types: glioblastoma and liver hepatocellular carcinoma. Then, we will extend the utility for causative driver discovery in therapeutic settings. Finally, we will advance the development of a lentiviral vector-based orthogonal approach to open up larger screening capabilities. Such screening systems and models will enable rapid identification of causative factors that directly drive transformation of healthy cells, tumor initiation, progression and therapeutic responses to treatments. More importantly, compared to existing alternatives, the fully immunocompetent setting allows robust pre-clinical testing of immunotherapies, in genetically matched animal avatars, as well as screening for genes that modulate the response to these therapies. Outcome and impact: This R33 will deliver optimized and validated PASTAS / PLeSTASS systems to link causative genes to oncogenesis in native TME; to enable autochthonous immunotherapy screen in fully immunocompetent setting for identification of targets that modulate the response to these life-saving drugs; and to share resources and protocols for the community to collectively yield novel and far-reaching insights in oncology.