The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of ...human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.
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•Identification of 5-fold more fitness genes in human cells than previously observed•Core fitness genes are highly enriched for ancient protein complexes•Context-specific fitness genes illuminate biological differences between cell types•Distinct genetic signatures can be used to predict differential drug response
CRISPR knockout screens for essential genes reveal oncogenic drivers specific to different cancer cell lines as well as unexpected metabolic and signaling dependencies that may guide future therapeutic targeting.
Nintedanib (formerly known as BIBF 1120) is an intracellular inhibitor that targets multiple tyrosine kinases. A phase 2 trial suggested that treatment with 150 mg of nintedanib twice daily reduced ...lung-function decline and acute exacerbations in patients with idiopathic pulmonary fibrosis.
We conducted two replicate 52-week, randomized, double-blind, phase 3 trials (INPULSIS-1 and INPULSIS-2) to evaluate the efficacy and safety of 150 mg of nintedanib twice daily as compared with placebo in patients with idiopathic pulmonary fibrosis. The primary end point was the annual rate of decline in forced vital capacity (FVC). Key secondary end points were the time to the first acute exacerbation and the change from baseline in the total score on the St. George's Respiratory Questionnaire, both assessed over a 52-week period.
A total of 1066 patients were randomly assigned in a 3:2 ratio to receive nintedanib or placebo. The adjusted annual rate of change in FVC was -114.7 ml with nintedanib versus -239.9 ml with placebo (difference, 125.3 ml; 95% confidence interval CI, 77.7 to 172.8; P<0.001) in INPULSIS-1 and -113.6 ml with nintedanib versus -207.3 ml with placebo (difference, 93.7 ml; 95% CI, 44.8 to 142.7; P<0.001) in INPULSIS-2. In INPULSIS-1, there was no significant difference between the nintedanib and placebo groups in the time to the first acute exacerbation (hazard ratio with nintedanib, 1.15; 95% CI, 0.54 to 2.42; P=0.67); in INPULSIS-2, there was a significant benefit with nintedanib versus placebo (hazard ratio, 0.38; 95% CI, 0.19 to 0.77; P=0.005). The most frequent adverse event in the nintedanib groups was diarrhea, with rates of 61.5% and 18.6% in the nintedanib and placebo groups, respectively, in INPULSIS-1 and 63.2% and 18.3% in the two groups, respectively, in INPULSIS-2.
In patients with idiopathic pulmonary fibrosis, nintedanib reduced the decline in FVC, which is consistent with a slowing of disease progression; nintedanib was frequently associated with diarrhea, which led to discontinuation of the study medication in less than 5% of patients. (Funded by Boehringer Ingelheim; INPULSIS-1 and INPULSIS-2 ClinicalTrials.gov numbers, NCT01335464 and NCT01335477.).
Large-scale genomic studies have identified multiple somatic aberrations in breast cancer, including copy number alterations and point mutations. Still, identifying causal variants and emergent ...vulnerabilities that arise as a consequence of genetic alterations remain major challenges. We performed whole-genome small hairpin RNA (shRNA) “dropout screens” on 77 breast cancer cell lines. Using a hierarchical linear regression algorithm to score our screen results and integrate them with accompanying detailed genetic and proteomic information, we identify vulnerabilities in breast cancer, including candidate “drivers,” and reveal general functional genomic properties of cancer cells. Comparisons of gene essentiality with drug sensitivity data suggest potential resistance mechanisms, effects of existing anti-cancer drugs, and opportunities for combination therapy. Finally, we demonstrate the utility of this large dataset by identifying BRD4 as a potential target in luminal breast cancer and PIK3CA mutations as a resistance determinant for BET-inhibitors.
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•We screened 77 breast cancer lines using a genome-wide pooled shRNA library•We developed an algorithm (siMEM) to improve identification of context-dependent genes•Integrating screen results with genomic data reveals potential “drivers”•BRD4 is essential for luminal cancer, and mutant PIK3CA confers BET-I resistance
Pooled shRNA screens of a large panel of breast cancer cell lines, coupled with an improved analytical tool, siMEM, and integration with genomic and proteomic data, identify general and context-dependent essential genes in breast cancer. This study constitutes the largest functional characterization of breast cancer to date.
Acute exacerbation of idiopathic pulmonary fibrosis has been defined as an acute, clinically significant, respiratory deterioration of unidentifiable cause. The objective of this international ...working group report on acute exacerbation of idiopathic pulmonary fibrosis was to provide a comprehensive update on the topic. A literature review was conducted to identify all relevant English text publications and abstracts. Evidence-based updates on the epidemiology, etiology, risk factors, prognosis, and management of acute exacerbations of idiopathic pulmonary fibrosis are provided. Finally, to better reflect the current state of knowledge and improve the feasibility of future research into its etiology and treatment, the working group proposes a new conceptual framework for acute respiratory deterioration in idiopathic pulmonary fibrosis and a revised definition and diagnostic criteria for acute exacerbation of idiopathic pulmonary fibrosis.
Technological advancement has opened the door to systematic genetics in mammalian cells. Genome‐scale loss‐of‐function screens can assay fitness defects induced by partial gene knockdown, using RNA ...interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by off‐target effects of perturbation reagents and poorly quantified error rates in large‐scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold‐standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods.
Synopsis
This study provides a gold‐standard set for essential and nonessential human genes in cancer cell lines. The ‘Daisy model’ for core versus context‐specific essentiality provides a method to evaluate data quality in genome‐scale RNAi and CRISPR screens.
Gold‐standard reference sets of human essential and nonessential genes are leveraged to improve analyses of RNAi and CRISPR screens.
Characteristics of human essential genes are derived from the cumulative analysis of RNAi screens.
The Daisy model of gene essentiality is derived from the difference between core and context‐specific cell line essentials.
A computational framework is presented for the prediction of human essential genes from reverse genetic screening data.
This study provides a gold‐standard set for essential and nonessential human genes in cancer cell lines. The ‘Daisy model’ for core versus context‐specific essentiality provides a method to evaluate data quality in genome‐scale RNAi and CRISPR screens.
Definitive evidence of clinical efficacy in a Phase 3 trial is best shown by a beneficial impact on a clinically meaningful endpoint-that is, an endpoint that directly measures how a patient feels ...(symptoms), functions (the ability to perform activities in daily life), or survives. In idiopathic pulmonary fibrosis (IPF), we believe the endpoints that best meet these criteria are all-cause mortality and all-cause nonelective hospitalization. There are no validated measures of symptoms or broader constructs such as health status or functional status in IPF. A surrogate endpoint is defined as an indirect measure that is intended to substitute for a clinically meaningful endpoint. Surrogate endpoints can be appropriate outcome measures if validated. However, validation requires substantial evidence that the effect of an intervention on a clinically meaningful endpoint is reliably predicted by the effect of an intervention on the surrogate endpoint. For patients with IPF, there are currently no validated surrogate endpoints.
Preclinical data have suggested that nintedanib, an intracellular inhibitor of tyrosine kinases, inhibits processes involved in the progression of lung fibrosis. Although the efficacy of nintedanib ...has been shown in idiopathic pulmonary fibrosis, its efficacy across a broad range of fibrosing lung diseases is unknown.
In this double-blind, placebo-controlled, phase 3 trial conducted in 15 countries, we randomly assigned patients with fibrosing lung disease affecting more than 10% of lung volume on high-resolution computed tomography (CT) to receive nintedanib at a dose of 150 mg twice daily or placebo. All the patients met criteria for progression of interstitial lung disease in the past 24 months despite treatment and had a forced vital capacity (FVC) of at least 45% of the predicted value and a diffusing capacity of the lung for carbon monoxide ranging from 30 to less than 80% of the predicted value. Randomization was stratified according to the fibrotic pattern (a pattern of usual interstitial pneumonia UIP or other fibrotic patterns) on high-resolution CT. The primary end point was the annual rate of decline in the FVC, as assessed over a 52-week period. The two primary populations for analysis were the overall population and patients with a UIP-like fibrotic pattern.
A total of 663 patients were treated. In the overall population, the adjusted rate of decline in the FVC was -80.8 ml per year with nintedanib and -187.8 ml per year with placebo, for a between-group difference of 107.0 ml per year (95% confidence interval CI, 65.4 to 148.5; P<0.001). In patients with a UIP-like fibrotic pattern, the adjusted rate of decline in the FVC was -82.9 ml per year with nintedanib and -211.1 ml per year with placebo, for a difference of 128.2 ml (95% CI, 70.8 to 185.6; P<0.001). Diarrhea was the most common adverse event, as reported in 66.9% and 23.9% of patients treated with nintedanib and placebo, respectively. Abnormalities on liver-function testing were more common in the nintedanib group than in the placebo group.
In patients with progressive fibrosing interstitial lung diseases, the annual rate of decline in the FVC was significantly lower among patients who received nintedanib than among those who received placebo. Diarrhea was a common adverse event. (Funded by Boehringer Ingelheim; INBUILD ClinicalTrials.gov number, NCT02999178.).
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of ...therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour
kg
, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.
Idiopathic pulmonary fibrosis (IPF) remains a truly idiopathic fibrotic disease, with a modest genetic predilection and candidate triggers but no overall explanation for the development of disease in ...non-familial cases. Agreement on terminology has contributed to major clinical and translational advances since the millennium. It is likely that the entity currently captured by the term "IPF" will be radically reclassified over the next decade, either through "splitting" (into IPF subgroups responding selectively to individual disease-modifying agents) or through "lumping" of IPF with other forms of progressive fibrotic lung disease (with shared pathogenetic mechanisms and IPF-like disease behaviour). In this perspective, we summarise the clinical and pathogenetic justification for a focus on "the progressive fibrotic phenotype" in future clinical and translational research. By this means, we can hope to address the needs of non-IPF patients with inexorably progressive fibrotic disease, currently disenfranchised by lack of access to agents that are efficacious in IPF. In this regard, ongoing trials of anti-fibrotic therapies in non-IPF patients with progressive fibrosis may be highly influential. Future revision of IPF nomenclature may be warranted if there are major conceptual changes but without compelling justification, the benefits of renaming IPF are likely to be outweighed by the resulting confusion.
This document is an international evidence-based guideline on the diagnosis and management of idiopathic pulmonary fibrosis, and is a collaborative effort of the American Thoracic Society, the ...European Respiratory Society, the Japanese Respiratory Society, and the Latin American Thoracic Association. It represents the current state of knowledge regarding idiopathic pulmonary fibrosis (IPF), and contains sections on definition and epidemiology, risk factors, diagnosis, natural history, staging and prognosis, treatment, and monitoring disease course. For the diagnosis and treatment sections, pragmatic GRADE evidence-based methodology was applied in a question-based format. For each diagnosis and treatment question, the committee graded the quality of the evidence available (high, moderate, low, or very low), and made a recommendation (yes or no, strong or weak). Recommendations were based on majority vote. It is emphasized that clinicians must spend adequate time with patients to discuss patients' values and preferences and decide on the appropriate course of action.
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