Abstract
Mutational outcomes following CRISPR-Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and, in certain cases, skewed toward single genotypes. However, the ...ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.
Somatic copy number gains are pervasive across cancer types, yet their roles in oncogenesis are insufficiently evaluated. This inadequacy is partly due to copy gains spanning large chromosomal ...regions, obscuring causal loci. Here, we employed organoid modeling to evaluate candidate oncogenic loci identified via integrative computational analysis of extreme copy gains overlapping with extreme expression dysregulation in The Cancer Genome Atlas. Subsets of “outlier” candidates were contextually screened as tissue-specific cDNA lentiviral libraries within cognate esophagus, oral cavity, colon, stomach, pancreas, and lung organoids bearing initial oncogenic mutations. Iterative analysis nominated the kinase DYRK2 at 12q15 as an amplified head and neck squamous carcinoma oncogene in p53−/− oral mucosal organoids. Similarly, FGF3, amplified at 11q13 in 41% of esophageal squamous carcinomas, promoted p53−/− esophageal organoid growth reversible by small molecule and soluble receptor antagonism of FGFRs. Our studies establish organoid-based contextual screening of candidate genomic drivers, enabling functional evaluation during early tumorigenesis.
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•Integrative analysis of extreme somatic copy number gains and extreme expression dysregulation•Screening of “outlier” oncogenes in diverse organoid models of early tumorigenesis•Validation of DYRK2 and FGF3 as oncogenic candidates in oral and esophageal squamous cancers•Demonstration of FGFR inhibitor efficacy in engineered esophageal squamous cancers with FGF3 copy gain
Salahudeen et al. utilize organoid models to evaluate candidate outlier oncogenic loci within somatic copy number gain regions in TCGA datasets of esophagus, oral cavity, colon, stomach, pancreas, and lung cancer. Contextual screening within tissue-matched organoids validated oncogenic candidates, including DYRK2 and FGF3 in oral and esophageal cancer, respectively.
Ovarian cancer and triple-negative breast cancer (TNBC) are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Here we show that CD24 can be the ...dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a new, promising target for cancer immunotherapy. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins, called “don’t eat me” signals, including CD47
1
, programmed cell death ligand 1 (PD-L1)
2
, and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)
3
. Monoclonal antibodies which antagonize the interaction of “don’t eat me” signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers
4
–
5
. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown, “don’t eat me” signals. Here we demonstrate a novel role for tumor-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor, Sialic Acid Binding Ig Like Lectin 10 (Siglec-10), expressed by tumor-associated macrophages (TAMs). We observe that many tumors overexpress CD24 and that TAMs express high levels of Siglec-10. Both genetic ablation of CD24 or Siglec-10, and monoclonal antibody blockade of the CD24–Siglec-10 interaction, robustly augment the phagocytosis of all CD24-expressing human tumors tested. Genetic ablation as well as therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumor growth and extension of survival,
in vivo
. These data highlight CD24 as a highly-expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24-blockade as cancer immunotherapy.
Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combining analysis of rare coding variants from the UK Biobank and genome-scale ...CRISPR-Cas9 knockout and activation screening, we substantially improve the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake. We use co-essentiality-based gene module analysis to show that dysfunction of the RAB10 vesicle transport pathway leads to hypercholesterolemia in humans and mice by impairing surface LDL receptor levels. Further, we demonstrate that loss of function of OTX2 leads to robust reduction in serum LDL-C levels in mice and humans by increasing cellular LDL-C uptake. Altogether, we present an integrated approach that improves our understanding of the genetic regulators of LDL-C levels and provides a roadmap for further efforts to dissect complex human disease genetics.
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•Genome-scale CRISPR screens identify 490 genes that alter liver cell LDL-C uptake•21 LDL-C uptake-altering genes enriched in rare coding variants in UK Biobank exomes•Dysfunction in the RAB10 exocytosis pathway raises LDL-C levels in mice and humans•OTX2 loss of function robustly reduces serum LDL-C in mice and humans
By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, Hamilton et al. improve the identification of genes whose disruption alters serum LDL-C levels. They show that RAB10 vesicle transport pathway dysfunction leads to hypercholesterolemia and that OTX2 disruption reduces serum LDL-C levels.
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