Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key ...KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients’ tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer.
The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a ...member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.
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•Genome-wide CRISPR screens for SARS-CoV-2 and seasonal coronavirus host factors•Identification of host factors and pathways with pan-coronavirus and discrete roles•Coronaviruses co-opt multiple biological pathways•TMEM41B is a critical pan-coronavirus host factor
Schneider et al. conducted parallel genome-wide CRISPR knockout screens with SARS-CoV-2 and three seasonal coronaviruses to identify pan-coronavirus and virus-specific host factor requirements. They identified an interconnected network of host factors required by these four viruses and validated TMEM41B as a pan-coronavirus host factor required for a post-entry step in the coronavirus life cycle.
Molecularly targeted therapies aim to obstruct cell autonomous programs required for tumor growth. We show that mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase 4/6 inhibitors act ...in combination to suppress the proliferation of KRAS-mutant lung cancer cells while simultaneously provoking a natural killer (NK) cell surveillance program leading to tumor cell death. The drug combination, but neither agent alone, promotes retinoblastoma (RB) protein-mediated cellular senescence and activation of the immunomodulatory senescence-associated secretory phenotype (SASP). SASP components tumor necrosis factor-α and intercellular adhesion molecule-1 are required for NK cell surveillance of drug-treated tumor cells, which contributes to tumor regressions and prolonged survival in a KRAS-mutant lung cancer mouse model. Therefore, molecularly targeted agents capable of inducing senescence can produce tumor control through non-cell autonomous mechanisms involving NK cell surveillance.
Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the ...heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater T cell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation.
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•Most small cell lung cancer (SCLC) tumors share a small PLCG2-high subpopulation•This PLCG2-high SCLC subpopulation is linked to metastasis and poor prognosis•SCLC is enriched in profibrotic and immunosuppressive monocytes/macrophages•The presence of myeloid cells is associated with the PLCG2-high SCLC subpopulation
Chan et al. use single-cell transcriptome sequencing and imaging techniques to study the heterogeneity and tumor microenvironment of clinical small cell lung cancer specimens. This analysis identifies a PLCG2-high-expressing subpopulation linked to metastasis and poor prognosis, and an enrichment of a monocyte/macrophage population with a profibrotic, immunosuppressive phenotype.
Small cell lung cancer (SCLC) is an exceptionally lethal malignancy for which more effective therapies are urgently needed. Several lines of evidence, from SCLC primary human tumours, patient-derived ...xenografts, cancer cell lines and genetically engineered mouse models, appear to be converging on a new model of SCLC subtypes defined by differential expression of four key transcription regulators: achaete-scute homologue 1 (ASCL1; also known as ASH1), neurogenic differentiation factor 1 (NeuroD1), yes-associated protein 1 (YAP1) and POU class 2 homeobox 3 (POU2F3). In this Perspectives article, we review and synthesize these recent lines of evidence and propose a working nomenclature for SCLC subtypes defined by relative expression of these four factors. Defining the unique therapeutic vulnerabilities of these subtypes of SCLC should help to focus and accelerate therapeutic research, leading to rationally targeted approaches that may ultimately improve clinical outcomes for patients with this disease.
The recently described clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has proven to be an exquisitely powerful and invaluable method of genetic manipulation and/or ...modification. As such, many researchers have realized the potential of using the CRISPR/Cas9 system as a novel screening method for the identification of important proteins in biological processes and have designed short guide RNA libraries for an in vitro screening. The seminal papers describing these libraries offer valuable information regarding methods for generating the short guide RNA libraries, creating cell lines containing these libraries, and specific details regarding the screening workflow. However, certain considerations are often overlooked that may be important when planning and performing a screen, including which CRISPR library to use and how to best analyze the resulting screen data. In this review, we offer suggestions to answer some of these questions that are not covered as deeply in the papers describing the available CRISPR libraries for an in vitro screening.
The recently discovered CRISPR/Cas9 technology allows for researchers to perform genome‐wide screens for many biological processes. However, for screens to generate high quality data, many decisions need to be considered during the design and progression of a CRISPR screen. In this review, we discuss important variables in the design, progression, and downstream steps of a CRISPR genome‐wide screen.
The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of ...function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.
Small cell lung cancer (SCLC) is a recalcitrant, aggressive neuroendocrine-type cancer for which little change to first-line standard-of-care treatment has occurred within the last few decades. ...Unlike nonsmall cell lung cancer (NSCLC), SCLC harbors few actionable mutations for therapeutic intervention. Lysine-specific histone demethylase 1A (LSD1 also known as KDM1A) inhibitors were previously shown to have selective activity in SCLC models, but the underlying mechanism was elusive. Here, we found that exposure to the selective LSD1 inhibitor ORY-1001 activated the NOTCH pathway, resulting in the suppression of the transcription factor ASCL1 and the repression of SCLC tumorigenesis. Our analyses revealed that LSD1 bound to the
locus, thereby suppressing NOTCH1 expression and downstream signaling. Reactivation of NOTCH signaling with the LSD1 inhibitor reduced the expression of ASCL1 and neuroendocrine cell lineage genes. Knockdown studies confirmed the pharmacological inhibitor-based results. In vivo, sensitivity to LSD1 inhibition in SCLC patient-derived xenograft (PDX) models correlated with the extent of consequential NOTCH pathway activation and repression of a neuroendocrine phenotype. Complete and durable tumor regression occurred with ORY-1001-induced NOTCH activation in a chemoresistant PDX model. Our findings reveal how LSD1 inhibitors function in this tumor and support their potential as a new and targeted therapy for SCLC.
The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. ...To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E HCoV-229E, HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks.
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•SARS-CoV-2 host protein interactome CRISPR screens for SARS-CoV-2 and three coronaviruses•Parallel CRISPR screens uncover unique and shared coronavirus host factors•Numbers of interacting host proteins and functional interactors are not proportional•Identified SARS-CoV-2 host factors are expressed in relevant cells in the human airway
Building upon a published SARS-CoV-2 protein interactome, Hoffmann et al. use a custom CRISPR library to determine which of these interacting host proteins are essential for infection by SARS-CoV-2 virus as well as three seasonal coronaviruses. These factors represent potential targets to combat COVID-19 and perhaps future coronavirus outbreaks.
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