The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 ...EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We propose the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method for prioritizing single-guide RNAs, genes and pathways in genome-scale CRISPR/Cas9 knockout screens. MAGeCK ...demonstrates better performance compared with existing methods, identifies both positively and negatively selected genes simultaneously, and reports robust results across different experimental conditions. Using public datasets, MAGeCK identified novel essential genes and pathways, including EGFR in vemurafenib-treated A375 cells harboring a BRAF mutation. MAGeCK also detected cell type-specific essential genes, including BCR and ABL1, in KBM7 cells bearing a BCR-ABL fusion, and IGF1R in HL-60 cells, which depends on the insulin signaling pathway for proliferation.
The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation ...(CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.
Abstract
Immune checkpoint blockade (ICB) benefits only a small subset of patients with small cell lung cancer (SCLC), yet the mechanisms driving benefit are poorly understood. To identify predictors ...of clinical benefit to ICB, we performed immunogenomic profiling of tumor samples from patients with relapsed SCLC. Tumors of patients who derive clinical benefit from ICB exhibit cytotoxic T-cell infiltration, high expression of antigen processing and presentation machinery (APM) genes, and low neuroendocrine (NE) differentiation. However, elevated Notch signaling, which positively correlates with low NE differentiation, most significantly predicts clinical benefit to ICB. Activation of Notch signaling in a NE human SCLC cell line induces a low NE phenotype, marked by increased expression of APM genes, demonstrating a mechanistic link between Notch activation, low NE differentiation and increased intrinsic tumor immunity. Our findings suggest Notch signaling as a determinant of response to ICB in SCLC.
Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Children's Oncology Group, and ...Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma.
This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention.
We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. ...Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.
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•Neuroblastomas differentially express high levels of GPC2 versus normal tissues•MYCN and gain of chromosome 7q mediate neuroblastoma GPC2 differential expression•GPC2 is integral in neuroblastoma cell proliferation•A GPC2-directed antibody-drug conjugate is cytotoxic to neuroblastoma cells
Bosse et al. show that GPC2 is expressed at high levels on most neuroblastomas (NB) but not at appreciable levels in normal childhood tissues and that GPC2 is critical for NB maintenance. They develop a GPC2-directed antibody-drug conjugate that is cytotoxic to GPC2-expressing NB cells in vitro and in vivo.
Survival from malignant mesothelioma, particularly pleural mesothelioma, is very poor. For patients with breast, ovarian, or prostate cancers, overall survival is associated with increased ...sensitivity to platinum chemotherapy due to loss-of-function mutations in DNA repair genes. The goal of this project was to evaluate, in patients with malignant mesothelioma, the relationship between inherited loss-of-function mutations in DNA repair and other tumor suppressor genes and overall survival following platinum chemotherapy. Patients with histologically confirmed malignant mesothelioma were evaluated for inherited mutations in tumor suppressor genes. Survival was evaluated with respect to genotype and site of mesothelioma. Among 385 patients treated with platinum chemotherapy, median overall survival was significantly longer for patients with loss-of-function mutations in any of the targeted genes compared with patients with no such mutation (P = 0.0006). The effect of genotype was highly significant for patients with pleural mesothelioma (median survival 7.9 y versus 2.4 y, P = 0.0012), but not for patients with peritoneal mesothelioma (median survival 8.2 y versus 5.4 y, P = 0.47). Effect of patient genotype on overall survival, measured at 3 y, remained independently significant after adjusting for gender and age at diagnosis, two other known prognostic factors. Patients with pleural mesothelioma with inherited mutations in DNA repair and other tumor suppressor genes appear to particularly benefit from platinum chemotherapy compared with patients without inherited mutations. These patients may also benefit from other DNA repair targeted therapies such as poly-ADP ribose polymerase (PARP) inhibitors.
Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which ...PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the
-regulatory landscape by inducing
super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy.
PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma.
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High-throughput CRISPR screens have shown great promise in functional genomics. We present MAGeCK-VISPR, a comprehensive quality control (QC), analysis, and visualization workflow for CRISPR screens. ...MAGeCK-VISPR defines a set of QC measures to assess the quality of an experiment, and includes a maximum-likelihood algorithm to call essential genes simultaneously under multiple conditions. The algorithm uses a generalized linear model to deconvolute different effects, and employs expectation-maximization to iteratively estimate sgRNA knockout efficiency and gene essentiality. MAGeCK-VISPR also includes VISPR, a framework for the interactive visualization and exploration of QC and analysis results. MAGeCK-VISPR is freely available at http://bitbucket.org/liulab/mageck-vispr .
To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that ...takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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