James T. Neal

A key challenge of the modern genomics era is developing empirical data-driven representations of gene function. Here we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-wide genotype–phenotype maps comprising CRISPR–Cas9-based knockouts of >20,000 genes in >30 million cells. Our optical pooled cell profiling platform (PERISCOPE) combines a destainable high-dimensional phenotyping panel (based on Cell Painting) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This perturbation atlas comprises high-dimensional phenotypic profiles of individual cells with sufficient resolution to cluster thousands of human genes, reconstruct known pathways and protein–protein interaction networks, interrogate subcellular processes and identify culture media-specific responses. Using this atlas, we identify the poorly characterized disease-associated TMEM251/LYSET as a Golgi-resident transmembrane protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes. In sum, this perturbation atlas and screening platform represents a rich and accessible resource for connecting genes to cellular functions at scale.

A key challenge of the modern genomics era is developing data-driven representations of gene function. Here, we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-scale genotype-phenotype maps comprising >20,000 single-gene CRISPR-Cas9-based knockout experiments in >30 million cells. Our optical pooled cell profiling approach (PERISCOPE) combines a de-stainable high-dimensional phenotyping panel (based on Cell Painting ^1,2 ) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This approach provides high-dimensional phenotypic profiles of individual cells, while simultaneously enabling interrogation of subcellular processes. Our atlas reconstructs known pathways and protein-protein interaction networks, identifies culture media-specific responses to gene knockout, and clusters thousands of human genes by phenotypic similarity. Using this atlas, we identify the poorly-characterized disease-associated transmembrane protein TMEM251/LYSET as a Golgi-resident protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes, showing the power of these representations. In sum, our atlas and screening technology represent a rich and accessible resource for connecting genes to cellular functions at scale.

Sequencing has revealed hundreds of millions of human genetic variants, and continued efforts will only add to this variant avalanche. Insufficient information exists to interpret the effects of most variants, limiting opportunities for precision medicine and comprehension of genome function. A solution lies in experimental assessment of the functional effect of variants, which can reveal their biological and clinical impact. However, variant effect assays have generally been undertaken reactively for individual variants only after and, in most cases long after, their first observation. Now, multiplexed assays of variant effect can characterise massive numbers of variants simultaneously, yielding variant effect maps that reveal the function of every possible single nucleotide change in a gene or regulatory element. Generating maps for every protein encoding gene and regulatory element in the human genome would create an ‘Atlas’ of variant effect maps and transform our understanding of genetics and usher in a new era of nucleotide-resolution functional knowledge of the genome. An Atlas would reveal the fundamental biology of the human genome, inform human evolution, empower the development and use of therapeutics and maximize the utility of genomics for diagnosing and treating disease. The Atlas of Variant Effects Alliance is an international collaborative group comprising hundreds of researchers, technologists and clinicians dedicated to realising an Atlas of Variant Effects to help deliver on the promise of genomics.

We established a genome-wide compendium of somatic mutation events in 3949 whole cancer genomes representing 19 tumor types. Protein-coding events captured well-established drivers. Noncoding events near tissue-specific genes, such as ALB in the liver or KLK3 in the prostate, characterized localized passenger mutation patterns and may reflect tumor-cell-of-origin imprinting. Noncoding events in regulatory promoter and enhancer regions frequently involved cancer-relevant genes such as BCL6 , FGFR2 , RAD51B , SMC6 , TERT , and XBP1 and represent possible drivers. Unlike most noncoding regulatory events, XBP1 mutations primarily accumulated outside the gene’s promoter, and we validated their effect on gene expression using CRISPR-interference screening and luciferase reporter assays. Broadly, our study provides a blueprint for capturing mutation events across the entire genome to guide advances in biological discovery, therapies, and diagnostics.

Genes required for tumor proliferation and survival (dependencies) are challenging to predict from cancer genome data, but are of high therapeutic value. We developed an algorithm (network purifying selection [NPS]) that aggregates weak signals of purifying selection across a gene’s first order protein-protein interaction network. We applied NPS to 4,742 tumor genomes to show that a gene’s NPS score is predictive of whether it is a dependency and validated 58 NPS-predicted dependencies in six cancer cell lines. Importantly, we demonstrate that leveraging NPS predictions to execute targeted CRISPR screens is a powerful, highly cost-efficient approach for identifying and validating dependencies quickly, because it eliminates the substantial experimental overhead required for whole-genome screening.

The advent of microenvironment directed cancer treatments such as antiangiogenic agents and recent immunotherapies have produced a pressing need for robust and systematic characterization of patient tumors and corresponding stromal populations. However, in vitro cultures of tumor biopsies do not typically preserve both the tumor epithelium and tumor microenvironment as an intact syngeneic unit. To address these limitations, we developed organoid cultures of surgically resected patient cancer biopsies that intrinsically retain diverse tumor microenvironmental cellular components without requiring reconstitution. We compared transcriptome profiles of fresh tumor cell populations to organoid cultures by droplet based single cell 5' RNA sequencing (scRNA-seq). Analysis of >50k single cell transcriptome profiles revealed tumor and stromal cell populations including tumor epithelia, fibroblasts and immune cells in fresh tumors. scRNA-seq of organoid cultures revealed a similar composition of native cancer cells and stromal components in both immune and non immune populations. In particular, 5' scRNA-seq enabled simultaneous characterization of paired T cell receptor alpha and beta chains as well as generalized gene expression in individual cells. We observed clonotype expansion of cytotoxic T cells in both fresh tumors and organoid cultures. The faithful recapitulation of tumor microenvironmental diversity within human organoid cultures should facilitate the in vitro exploration of immunotherapeutic agents and modeling of associated patient-specific responses. Citation Format: Ameen A. Salahudeen, Junjie Zhu, Jihang Ju, Arpit Batish, Ken Sutha, James T. Neal, Valeria Giangarra, Luz Montesclaros, Jerald Sapida, Osman Sharifi, Josephine Lee, Grace X. Zheng, Dhananjay A. Wagh, John A. Coller, Joel W. Neal, Sukhmani K. Padda, Chiara Sabatti, Calvin J. Kuo. Organoid-based characterization of patient tumors and microenvironments at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 987.

Cancer models strive to recapitulate the incredible diversity inherent in human tumors. A key challenge in accurate tumor modeling lies in capturing the panoply of homo- and heterotypic cellular interactions within the context of a three-dimensional tissue microenvironment. To address this challenge, researchers have developed organotypic cancer models (organoids) that combine the 3D architecture of in vivo tissues with the experimental facility of 2D cell lines. Here we address the benefits and drawbacks of these systems, as well as their most recent advances. In particular, we focus on the application of such models to the discovery of novel cancer drivers, the study of tumor biology, and the development of novel therapeutic approaches for the treatment of cancer.

Modeling and documenting malignant progression in vitro without the need for in vivo transplantation represents a clear step forward for cancer investigation. Using an air-liquid interface methodology, Xingnan Li and colleagues show they can robustly model a range of gastrointestinal malignancies from pancreas, stomach and colon in primary epithelial/mesenchymal organoid culture. This setup is able to generate detailed histologic endpoints for oncogenic transformation in vitro and demonstrate in vivo tumorigenicity when the organoids are transplanted. The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (KrasG12D), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.