ABSTRACT
Reducing disparities is vital for equitable access to precision treatments in cancer. Socioenvironmental factors are a major driver of disparities, but differences in genetic variation likely also contribute. The impact of genetic ancestry on prioritization of cancer targets in drug discovery pipelines has not been systematically explored due to the absence of pre-clinical data at the appropriate scale. Here, we analyze data from 611 genome-scale CRISPR/Cas9 viability experiments in human cell line models to identify ancestry-associated genetic dependencies essential for cell survival. Surprisingly, we find that most putative associations between ancestry and dependency arise from artifacts related to germline variants. Our analysis suggests that for 1.2-2.5% of guides, germline variants in sgRNA targeting sequences reduce cutting by the CRISPR/Cas9 nuclease, disproportionately affecting cell models derived from individuals of recent African descent. We propose three approaches to mitigate this experimental bias, enabling the scientific community to address these disparities.
Subject(s)
CRISPR-Cas Systems , Germ-Line Mutation , Humans , Gene Editing/methods , RNA, Guide, CRISPR-Cas Systems/genetics , Germ Cells/metabolism , Genetic Variation , Neoplasms/genetics , False Negative Reactions , Genome, Human , Cell Line, Tumor , Cell LineABSTRACT
Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.
Subject(s)
Hydrazines , Kidney Neoplasms , Triazoles , Wilms Tumor , Humans , Exportin 1 Protein , Active Transport, Cell Nucleus , Karyopherins/genetics , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Cell Line, Tumor , Apoptosis , Neoplasm Recurrence, Local , Doxorubicin/pharmacology , Wilms Tumor/drug therapy , Wilms Tumor/genetics , Kidney Neoplasms/drug therapy , Kidney Neoplasms/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , Cell Cycle Proteins/metabolismABSTRACT
SUMMARY: Here, we define a future of cancer team science adopting "radical collaboration"-in which six "Hallmarks of Cancer Collaboration" are utilized to propel cancer teams to reach new levels of productivity and impact in the modern era. This commentary establishes a playbook for cancer team science that can be readily adopted by others.
Subject(s)
Cooperative Behavior , Neoplasms , Humans , Interdisciplinary Research , Neoplasms/therapyABSTRACT
BACKGROUND: Hundreds of functional genomic screens have been performed across a diverse set of cancer contexts, as part of efforts such as the Cancer Dependency Map, to identify gene dependencies-genes whose loss of function reduces cell viability or fitness. Recently, large-scale screening efforts have shifted from RNAi to CRISPR-Cas9, due to superior efficacy and specificity. However, many effective oncology drugs only partially inhibit their protein targets, leading us to question whether partial suppression of genes using RNAi could reveal cancer vulnerabilities that are missed by complete knockout using CRISPR-Cas9. Here, we compare CRISPR-Cas9 and RNAi dependency profiles of genes across approximately 400 matched cancer cell lines. RESULTS: We find that CRISPR screens accurately identify more gene dependencies per cell line, but the majority of each cell line's dependencies are part of a set of 1867 genes that are shared dependencies across the entire collection (pan-lethals). While RNAi knockdown of about 30% of these genes is also pan-lethal, approximately 50% have selective dependency patterns across cell lines, suggesting they could still be cancer vulnerabilities. The accuracy of the unique RNAi selectivity is supported by associations to multi-omics profiles, drug sensitivity, and other expected co-dependencies. CONCLUSIONS: Incorporating RNAi data for genes that are pan-lethal knockouts facilitates the discovery of a wider range of gene targets than could be detected using the CRISPR dataset alone. This can aid in the interpretation of contrasting results obtained from CRISPR and RNAi screens and reinforce the importance of partial gene suppression methods in building a cancer dependency map.
Subject(s)
CRISPR-Cas Systems , Neoplasms , Humans , Neoplasms/genetics , Genetic Techniques , RNA Interference , Cell LineABSTRACT
Despite advances in precision medicine, the clinical prospects for patients with ovarian and uterine cancers have not substantially improved. Here, we analyzed genome-scale CRISPR-Cas9 loss-of-function screens across 851 human cancer cell lines and found that frequent overexpression of SLC34A2-encoding a phosphate importer-is correlated with sensitivity to loss of the phosphate exporter XPR1, both in vitro and in vivo. In patient-derived tumor samples, we observed frequent PAX8-dependent overexpression of SLC34A2, XPR1 copy number amplifications and XPR1 messenger RNA overexpression. Mechanistically, in SLC34A2-high cancer cell lines, genetic or pharmacologic inhibition of XPR1-dependent phosphate efflux leads to the toxic accumulation of intracellular phosphate. Finally, we show that XPR1 requires the novel partner protein KIDINS220 for proper cellular localization and activity, and that disruption of this protein complex results in acidic "vacuolar" structures preceding cell death. These data point to the XPR1-KIDINS220 complex and phosphate dysregulation as a therapeutic vulnerability in ovarian cancer.
Subject(s)
Membrane Proteins , Nerve Tissue Proteins , Ovarian Neoplasms , Female , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Phosphates/pharmacology , Receptors, G-Protein-Coupled/genetics , Receptors, Virus/genetics , Xenotropic and Polytropic Retrovirus Receptor/genetics , Xenotropic and Polytropic Retrovirus Receptor/metabolismABSTRACT
Most variants in most genes across most organisms have an unknown impact on the function of the corresponding gene. This gap in knowledge is especially acute in cancer, where clinical sequencing of tumors now routinely reveals patient-specific variants whose functional impact on the corresponding genes is unknown, impeding clinical utility. Transcriptional profiling was able to systematically distinguish these variants of unknown significance as impactful vs. neutral in an approach called expression-based variant-impact phenotyping. We profiled a set of lung adenocarcinoma-associated somatic variants using Cell Painting, a morphological profiling assay that captures features of cells based on microscopy using six stains of cell and organelle components. Using deep-learning-extracted features from each cell's image, we found that cell morphological profiling (cmVIP) can predict variants' functional impact and, particularly at the single-cell level, reveals biological insights into variants that can be explored at our public online portal. Given its low cost, convenient implementation, and single-cell resolution, cmVIP profiling therefore seems promising as an avenue for using non-gene specific assays to systematically assess the impact of variants, including disease-associated alleles, on gene function.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Adenocarcinoma of Lung/genetics , Alleles , Humans , Lung Neoplasms/genetics , Microscopy , PhenotypeABSTRACT
BACKGROUND: Oncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood. METHODS: We use an in vivo expression screen of known cancer-associated somatic mutations in mouse syngeneic tumor models treated with checkpoint blockade to identify oncogenes that promote immune evasion. We then validated candidates from this screen in vivo and analyzed the tumor immune microenvironment of tumors expressing mutant protein to identify mechanisms of immune evasion. RESULTS: We found that expression of a catalytically active mutation in phospho-inositol 3 kinase (PI3K), PIK3CA c.3140A>G (H1047R) confers a selective growth advantage to tumors treated with immunotherapy that is reversed by pharmacological PI3K inhibition. PIK3CA H1047R-expression in tumors decreased the number of CD8+ T cells but increased the number of inhibitory myeloid cells following immunotherapy. Inhibition of myeloid infiltration by pharmacological or genetic modulation of Ccl2 in PIK3CA H1047R tumors restored sensitivity to programmed cell death protein 1 (PD-1) checkpoint blockade. CONCLUSIONS: PI3K activation enables tumor immune evasion by promoting an inhibitory myeloid microenvironment. Activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy. Our data suggest that some oncogenes promote tumorigenesis by enabling tumor cells to avoid clearance by the immune system. Identification of those mechanisms can advance rational combination strategies to increase the efficacy of immunotherapy.
Subject(s)
Neoplasms , Tumor Microenvironment , Animals , CD8-Positive T-Lymphocytes/metabolism , Carcinogenesis , Class I Phosphatidylinositol 3-Kinases/genetics , Disease Models, Animal , Humans , Immune Evasion , Inositol , Mice , Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolismABSTRACT
Genome sequencing studies have identified millions of somatic variants in cancer, but it remains challenging to predict the phenotypic impact of most. Experimental approaches to distinguish impactful variants often use phenotypic assays that report on predefined gene-specific functional effects in bulk cell populations. Here, we develop an approach to functionally assess variant impact in single cells by pooled Perturb-seq. We measured the impact of 200 TP53 and KRAS variants on RNA profiles in over 300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison with orthogonal assays. We discovered that KRAS variants did not merely fit into discrete functional categories, but spanned a continuum of gain-of-function phenotypes, and that their functional impact could not have been predicted solely by their frequency in patient cohorts. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, with potential applications in multiple disease settings.
Subject(s)
Lung Neoplasms , Proto-Oncogene Proteins p21(ras) , Chromosome Mapping , Humans , Lung Neoplasms/genetics , Phenotype , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
In high-throughput functional genomic screens, each gene product is commonly assumed to exhibit a singular biological function within a defined protein complex or pathway. In practice, a single gene perturbation may induce multiple cascading functional outcomes, a genetic principle known as pleiotropy. Here, we model pleiotropy in fitness screen collections by representing each gene perturbation as the sum of multiple perturbations of biological functions, each harboring independent fitness effects inferred empirically from the data. Our approach (Webster) recovered pleiotropic functions for DNA damage proteins from genotoxic fitness screens, untangled distinct signaling pathways upstream of shared effector proteins from cancer cell fitness screens, and predicted the stoichiometry of an unknown protein complex subunit from fitness data alone. Modeling compound sensitivity profiles in terms of genetic functions recovered compound mechanisms of action. Our approach establishes a sparse approximation mechanism for unraveling complex genetic architectures underlying high-dimensional gene perturbation readouts.
Subject(s)
Genomics , Genomics/methods , HumansABSTRACT
BACKGROUND: Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer-associated genes as an efficient approach to characterizing patient samples (compared to whole-exome and whole-genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost-effective method for genomic assessment of patient-derived cell lines to evaluate fidelity to initial patient tumor samples. METHODS: We developed a prostate cancer-specific targeted next-generation sequencing (NGS) panel to detect alterations in 62 prostate cancer-associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient-derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors. RESULTS: The prostate cancer-specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell-free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient-derived cell lines; however, no prostate cancer-specific mutations were identified in the tumor-derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells. CONCLUSIONS: We evaluated a prostate cancer-specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh-frozen tumors, cell-free DNA, CTCs, and cell lines. Targeted sequencing of patient-derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer-specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.
Subject(s)
Cell-Free Nucleic Acids , Prostatic Neoplasms , Cell Line , Credentialing , High-Throughput Nucleotide Sequencing/methods , Humans , Male , Mutation , Prostatic Neoplasms/geneticsABSTRACT
CRISPR loss of function screens are powerful tools to interrogate biology but exhibit a number of biases and artifacts that can confound the results. Here, we introduce Chronos, an algorithm for inferring gene knockout fitness effects based on an explicit model of cell proliferation dynamics after CRISPR gene knockout. We test Chronos on two pan-cancer CRISPR datasets and one longitudinal CRISPR screen. Chronos generally outperforms competitors in separation of controls and strength of biomarker associations, particularly when longitudinal data is available. Additionally, Chronos exhibits the lowest copy number and screen quality bias of evaluated methods. Chronos is available at https://github.com/broadinstitute/chronos .
Subject(s)
CRISPR-Cas Systems , Computational Biology , Genome , Population Dynamics , Algorithms , Biomarkers, Tumor/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Knockout Techniques , Gene Library , Humans , Neoplasms/geneticsABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in K-12 schools was rare during in 2020-2021; few studies included Centers for Disease Control and Prevention (CDC)-recommended screening of asymptomatic individuals. We conduct a prospective observational study of SARS-CoV-2 screening in a mid-sized suburban public school district to evaluate the incidence of asymptomatic coronavirus disease 2019 (COVID-19), document frequency of in-school transmission, and characterize barriers and facilitators to asymptomatic screening in schools. Staff and students undergo weekly pooled testing using home-collected saliva samples. Identification of >1 case in a school prompts investigation for in-school transmission and enhancement of safety strategies. With layered mitigation measures, in-school transmission even before student or staff vaccination is rare. Screening identifies a single cluster with in-school staff-to-staff transmission, informing decisions about in-person learning. The proportion of survey respondents self-reporting comfort with in-person learning before versus after implementation of screening increases. Costs exceed $260,000 for assays alone; staff and volunteers spend 135-145 h per week implementing screening.
Subject(s)
COVID-19/diagnosis , Mass Screening , Schools , Adolescent , Adult , COVID-19/transmission , Child , Educational Personnel , Humans , Prospective Studies , Students , United StatesABSTRACT
Gene fusions frequently result from rearrangements in cancer genomes. In many instances, gene fusions play an important role in oncogenesis; in other instances, they are thought to be passenger events. Although regulatory element rearrangements and copy number alterations resulting from these structural variants are known to lead to transcriptional dysregulation across cancers, the extent to which these events result in functional dependencies with an impact on cancer cell survival is variable. Here we used CRISPR-Cas9 dependency screens to evaluate the fitness impact of 3,277 fusions across 645 cell lines from the Cancer Dependency Map. We found that 35% of cell lines harbored either a fusion partner dependency or a collateral dependency on a gene within the same topologically associating domain as a fusion partner. Fusion-associated dependencies revealed numerous novel oncogenic drivers and clinically translatable alterations. Broadly, fusions can result in partner and collateral dependencies that have biological and clinical relevance across cancer types. SIGNIFICANCE: This study provides insights into how fusions contribute to fitness in different cancer contexts beyond partner-gene activation events, identifying partner and collateral dependencies that may have direct implications for clinical care.
Subject(s)
Cell Survival/genetics , Gene Fusion/genetics , Neoplasms/genetics , HumansABSTRACT
Parent scientists lead a journey to bring surveillance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing to public schools across the state of Massachusetts and beyond.
Subject(s)
COVID-19/diagnosis , Laboratory Personnel , Parents , COVID-19/virology , COVID-19 Testing , Cooperative Behavior , Education , Humans , Massachusetts , SARS-CoV-2/physiologyABSTRACT
Exciting therapeutic targets are emerging from CRISPR-based screens of high mutational-burden adult cancers. A key question, however, is whether functional genomic approaches will yield new targets in pediatric cancers, known for remarkably few mutations, which often encode proteins considered challenging drug targets. To address this, we created a first-generation pediatric cancer dependency map representing 13 pediatric solid and brain tumor types. Eighty-two pediatric cancer cell lines were subjected to genome-scale CRISPR-Cas9 loss-of-function screening to identify genes required for cell survival. In contrast to the finding that pediatric cancers harbor fewer somatic mutations, we found a similar complexity of genetic dependencies in pediatric cancer cell lines compared to that in adult models. Findings from the pediatric cancer dependency map provide preclinical support for ongoing precision medicine clinical trials. The vulnerabilities observed in pediatric cancers were often distinct from those in adult cancer, indicating that repurposing adult oncology drugs will be insufficient to address childhood cancers.
Subject(s)
Chromosome Mapping/methods , Gene Expression Regulation, Neoplastic , Genome, Human , Mutation , Neoplasm Proteins/genetics , Neoplasms/genetics , Adult , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , Cell Line, Tumor , Child , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Editing , Gene Expression Profiling , Genetic Predisposition to Disease , Humans , Neoplasm Proteins/classification , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Neoplasms/pathology , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolismSubject(s)
Biomedical Research/organization & administration , Biomedical Research/trends , Goals , International Cooperation , Molecular Targeted Therapy/trends , Neoplasms/drug therapy , Neoplasms/genetics , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , Biomedical Research/economics , CRISPR-Cas Systems/genetics , Cell Survival/drug effects , DNA Mutational Analysis , Datasets as Topic , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Information Dissemination , Machine Learning , Mutation , Neoplasms/metabolism , Neoplasms/pathology , Pilot Projects , Precision Medicine , Reproducibility of ResultsABSTRACT
Cell lines are key tools for preclinical cancer research, but it remains unclear how well they represent patient tumor samples. Direct comparisons of tumor and cell line transcriptional profiles are complicated by several factors, including the variable presence of normal cells in tumor samples. We thus develop an unsupervised alignment method (Celligner) and apply it to integrate several large-scale cell line and tumor RNA-Seq datasets. Although our method aligns the majority of cell lines with tumor samples of the same cancer type, it also reveals large differences in tumor similarity across cell lines. Using this approach, we identify several hundred cell lines from diverse lineages that present a more mesenchymal and undifferentiated transcriptional state and that exhibit distinct chemical and genetic dependencies. Celligner could be used to guide the selection of cell lines that more closely resemble patient tumors and improve the clinical translation of insights gained from cell lines.
Subject(s)
Gene Expression Profiling , Neoplasms/genetics , Cell Line, Tumor , Databases, Genetic , Epithelial-Mesenchymal Transition/genetics , Humans , Integrins/metabolismABSTRACT
Few therapies target the loss of tumor suppressor genes in cancer. We examine CRISPR-SpCas9 and RNA-interference loss-of-function screens to identify new therapeutic targets associated with genomic loss of tumor suppressor genes. The endosomal sorting complexes required for transport (ESCRT) ATPases VPS4A and VPS4B score as strong synthetic lethal dependencies. VPS4A is essential in cancers harboring loss of VPS4B adjacent to SMAD4 on chromosome 18q and VPS4B is required in tumors with co-deletion of VPS4A and CDH1 (E-cadherin) on chromosome 16q. We demonstrate that more than 30% of cancers selectively require VPS4A or VPS4B. VPS4A suppression in VPS4B-deficient cells selectively leads to ESCRT-III filament accumulation, cytokinesis defects, nuclear deformation, G2/M arrest, apoptosis, and potent tumor regression. CRISPR-SpCas9 screening and integrative genomic analysis reveal other ESCRT members, regulators of abscission, and interferon signaling as modifiers of VPS4A dependency. We describe a compendium of synthetic lethal vulnerabilities and nominate VPS4A and VPS4B as high-priority therapeutic targets for cancers with 18q or 16q loss.