Human cancers harbor complex genomic alterations. Understanding the functional consequences of these alterations is key to developing personalized therapeutics for patients. However, tumors are composed of not only cancer cells but also various infiltrating cell types, and these intercellular interactions dictate numerous facets of tumor biology. Thus, functional cancer genomics necessitates the study of cancer in vivo, within the native micro-environmental context.
Our lab focuses on cancer immunology, genetics and systems biology. We develop and utilize a wide variety of modern tools, including in vivo gene editing and tumor modeling, genome-wide and focused CRISPR screens, immune engineering, high-density and high-dimensional genetic manipulations, and systems-level profiling. Using these tools, we seek to interrogate the genetic, epigenetic and immunological bases of cancer progression, metastasis, immunity and treatment.
Currently, our work follows these directions:
0. Development of MAEGI and other novel viral based immune-gene therapy:
The major challenge in cancer prevention and treatment is to devise a therapy that potently and specifically targets tumor cells without harming normal cells. Major types of immunotherapy include checkpoint blockade, adoptive cell transfer, human recombinant cytokines, and cancer vaccines. While checkpoint blockade immunotherapies and adoptive cell therapies such as CAR-Ts have yielded significant clinical benefits across a broad spectrum of cancer types, however, only a fraction of patients show sustained clinical responses, with many patients suffering from major or even life-threatening toxicities. These challenges urge for new types of immunotherapies that are more potent and potentially less toxic. Very recently, we have developed CRISPRa-mediated Multiplexed Activation of Endogenous Genes as an Immunotherapy (MAEGI) (Wang*, Chow* et al. 2019 Nature Immunology). While neoantigen-targeting approaches have demonstrated the concept of leveraging personalized neoantigens as cancer treatments, and are based on delivery of synthetic mutant peptides or transcripts. However, the efficacy and scalability of these approaches is limited. The CRISPR activation (CRISPRa) system uses a catalytically inactive Cas9 (dCas9), enabling simple and flexible gene expression regulation through dCas9-transcriptional activators paired with single guide RNAs (sgRNAs). This enables precise targeting of large gene pools of endogenous genes in a flexible manner. We demonstrate that MAEGI has therapeutic efficacy across three tumor types. Mechanistically, our preliminary work showed that MAEGI treatment elicits anti-tumor immune responses by recruiting effector T cells and remodeling the tumor microenvironment. We will perform advanced development, characterization and optimization of MAEGI, as a novel immune-gene therapy approach to elicit a potent and specific immune response to tumors based on their unique genetic composition.
Wang G*, Chow RD*, Bai Z, Zhu L, Errami Y, Dai X, Dong MB, Ye L, Zhang X, Renauer RA, Park JJ, Shen L, Ye H, Fuchs CS, and Chen S†. Multiplexed activation of endogenous genes by CRISPRa elicits potent anti-tumor immunity.
Nature Immunology (2019)
1. Systems-level cancer immunology and immunotherapy
Immunotherapy, which harnesses the body’s own immune system to combat the disease, has been strikingly effective in inducing durable responses across multiple cancer types. However, only a subset of the patients responds to immunotherapy such as checkpoint blockade or adoptive T cell transfer. Our lab is interested in utilizing a combinatorial approach including gene editing and animal models to better understand tumor immunity for improved immunotherapy.
CD8 T cells play essential roles in anti-tumor immune responses. We recently performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings and identified regulators of tumor infiltration and degranulation (Dong et al. 2019 Cell). The in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. We discovered an RNA helicase Dhx37 as a key regulator of CD8 T cell function and anti-tumor immunity, thereby servicing as a new immunotherapy target. The high-throughput genetic screens open new venues for immunotherapy target discovery in primary T cells in vivo.
Systematic Immunotherapy Target Discovery Using Genome-Scale In vivo CRISPR Screens in CD8 T Cells
Dong MB*, Wang G*, Chow RD*, Ye L*, Zhu L, Dai X, Park JJ, Kim HR, Errami Y, Guzman CD, Zhou X, Chen KY, Renauer PA, Du Y, Shen J, Lam SZ, Zhou JJ, Lannin DR, Herbst RS, Chen S. Systematic Immunotherapy Target Discovery Using Genome-Scale In vivo CRISPR Screens in CD8 T Cells. Cell 2019. doi: 10.1016/j.cell.2019.07.044.
AAV-based in vivo T cell gene editing and high-throughput CRISPR screening of immunotherapy in GBM models
Ye L*, Park JJ*, Dong MB*, Yang Q, Chow RD, Peng L, Guo J, Dai X, Wang G, Errami Y, and Chen S†. In vivo CRISPR screening in CD8 T cells with AAV–Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma. Nature Biotechnology (2019)
Evading immune destruction is a key for resistance to immunotherapy, we leveraged in vivo screening approaches to identify and interrogate of tumor-intrinsic immune modulators in vivo (Codina et al. 2019Cell Systems). Our genome-scale in vivo CRISPR screens robustly identified multiple tumor-intrinsic factors that alter the ability of cells to grow as tumors across different levels of immunocompetence. Functional interrogation of top hits showed that Prkar1a loss greatly altered the transcriptome and proteome involved in inflammatory and immune responses and tumor-intrinsic mutations in Prkar1aled to drastic alterations in the genetic program of cancer cells, thereby remodeling the tumor microenvironment.
Codina A*,Renauer P*, Wang G*, Chow RD*, Park JJ, Ye H, Zhang K, Dong M, Gassaway B, Ye L, Errami Y, Shen L, Chang A, Jain D, Herbst RS, Bosenberg M, Rinehart J, Fan R and Chen S†. Convergent identification and interrogation of tumor-intrinsic factors that modulate cancer immunity in vivo.Cell Systems 2019 Feb 27;8(2):136-151.e7. doi: 10.1016/j.cels.2019.01.004. (Cover Story) PMID: 30797773. Highlighted in Yale News.
2. Immune engineering and chimeric antigen receptor T cells (CAR-T)
Chimeric antigen receptor T cell is a transformative class of cell therapy, which has recently been FDA-approved for hematopoietic malignancies. However, current CAR-T therapy faces many hurdles especially in solid tumors, where the T cell-mediated cytotoxicity against cancer can be abolished by multiple cancer-immune mechanisms, such as reduced or lost antigen presentation, generation of an immune-suppressive tumor environment, heightened expression of immune checkpoint proteins, lack of T cell persistence, and T cell exhaustion. Engineering more sophisticated CAR-T cells with precision control and other desired features requires a highly efficient platform. Harnessing the Cas12a/Cpf1 systems with AAV, we have recently built a novel system that enables stable CAR-T with HDR knockin and immune checkpoint knockout (KIKO CAR-T) generation at high efficiency in one step (Dai et al. 2019 Nature Methods). The modularity of AAV-Cpf1 KIKO enables flexible and efficient generation of multiple different CARs in the same T cell, opening new capabilities of therapeutic cellular engineering with simplicity and precision.
Dai X*, Park JJ*, Du Y, Kim RK, Wang G, Errami Y and Chen S†. One-step generation of modular CAR-T with AAV-Cpf1. Nature Methods (2019) Mar;16(3):247-254. doi: 10.1038/s41592-019-0329-7. PMID: 30804551. Highlighted in Yale News, BioArt, Yale College News, MedicalXpress, Naked Science, Mendeley, ScienceBlog.com, Scitech Daily, etc.
3. Precision cancer modeling and in vivo CRISPR screening to map functional cancer drivers
We previously developed a CRISPR-based genetically engineered mouse model (CGEMM) of several cancer types. By co-targeting combinations of key tumor suppressor genes and oncogenes, we developed methods to induce liver cancer (Xue*, Chen*, Yin* et al. 2014 Nature) and lung adenocarcinoma (Platt*, Chen* et al. 2014 Cell). Cancer genomics initiatives have charted the genomic landscapes of human cancers. While some mutations were found in classical oncogenes and tumor suppressors, many others have not been previously implicated in cancer. We developed direct high-throughput in vivo mapping of functional variants in an autochthonous mouse model of cancer and direct identification of novel functional drivers in vivo (Chow et al. 2017 Nature Neuroscience; Wang et al. 2018 Science Advances). The most devastating hallmark of the cancer cells is that they evolve to become invasive and metastatic. Understanding how cancer cells become metastatic, how they disseminate through circulation, and how the circulating tumor cells seed new micro-tumors is a key to treat the disease. Our approach is to perform systematic genetic screens in mouse models to identify metastasis regulators (Chen*, Sanjana* et al. 2015 Cell). We summarize the tools and problems of cancer CRISPR screens in vivo (Chow and Chen, 2018, Trends In Cancer).
Chow RD*, Guzman CD*, Wang G*, Schmidt F*, Youngblood MW, Ye L, Errami Y, Dong MB, Martinez MA, Zhang S, Renauer P, Bilguvar K, Gunel M, Sharp PA, Zhang F, Platt RJ @, Chen S @.AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma. Nature Neuroscience, 20, 1329–1341 (2017) doi:10.1038/nn.4620) Aug 14. PMID: 28805815
Wang G*, Chow RD*, Ye L, Guzman CD, Dai X, Dong MB, Zhang F, Sharp PA, Platt RJ@, and Chen S@.Mapping a Functional Cancer Genome Atlas of Tumor Suppressors in Mouse Liver Using AAV-CRISPR Mediated Direct in vivo Screening. (2018) Science Advances. Feb 28;4(2):eaao5508. doi: 10.1126/sciadv.aao5508. PMID: 29503867
Chow RD and Chen S@. Cancer CRISPR screens in vivo. Trends In Cancer. 2018 May;4(5):349-358. doi: 10.1016/j.trecan.2018.03.002.. Review. PMID: 29709259 (Cover story)
Chow RD and Chen S@. Sno-derived RNAs are prevalent molecular markers of cancer immunity. Oncogene, 2018DOI – 10.1038/s41388-018-0420-z
Chen S*,Sanjana NE*, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song J, Pan JQ, Weissleder R, Lee H, Zhang F, Sharp PA. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell. 2015 Mar 12;160(6):1246-60. doi: 10.1016/j.cell.2015.02.038. PMID: 25748654; (* = co-first authors) (Selected as Best of Cell 2015)
Xue W*, Chen S*, Yin H*, Tammela T, Papagiannakopoulos T, Joshi NS, Cai W, Yang G, Bronson R, Crowley DG, Zhang F, Anderson DG, Sharp PA, Jacks T. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature. 2014 Oct 16;514(7522):380-4. doi: 10.1038/nature13589. PMID: 25119044
Platt RJ*, Chen S*,Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014 Oct 9;159(2):440-55. doi: 10.1016/j.cell.2014.09.014. PMID: 25263330; (Cover story)
4. Development of novel biotechnologies
The lab also exerts strong interests in development of novel technologies to enable new paths of discoveries, such as new ways to manipulate the genome, the transcriptome, the proteome, as well as control of cellular behaviors in vivo. Examples below demonstrated creative works by lab members. Lab members are welcomed as new innovators and develop their own creative ideas in the lab.
Chow RD, Wang G, Ye L, Codina A, Kim HR, Shen L, Dong MB, Errami Y, Chen S. In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens. Nature Methods. 2019 May;16(5):405-408. doi: 10.1038/s41592-019-0371-5. Epub 2019 Apr 8. PMID: 30962622
Ye L, Wang C, Hong L, Sun N, Chen D, Chen S, Han F. Programmable DNA repair with CRISPRa/i enhanced homology-directed repair efficiency with a single Cas9. Cell Discov. 2018 Jul 24;4:46. doi: 10.1038/s41421-018-0049-7. eCollection 2018. PubMed PMID: 30062046
Chow RD, Kim HR, Chen S. Programmable sequential mutagenesis by inducible Cpf1 crRNA array inversion. Nat Commun. 2018 May 15;9(1):1903. doi: 10.1038/s41467-018-04158-z. PubMed PMID: 29765043
Pyzocha NK, Chen S. Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications. ACS Chem Biol. 2018 Feb 16;13(2):347-356. doi: 10.1021/acschembio.7b00800. Epub 2017 Dec 5. PubMed PMID: 29121460.
5. Other on-going directions in cancer immunology, immune engineering and immunotherapy immunity
- Tumor-intrinsic factors that modulate checkpoint blockade efficacy
- Genetic regulation of T cell function
- Immune components and regulation in the microenvironment of brain tumors, such as GBM
- Innate immune cells in oncology, such as macrophage and dendritic cells
- Engineering of immune cells and the immunological machinery
- Development of new classes of immunotherapies