Genome-wide CRISPR-cas9 knockout screening identifies GRB7 as a driver for MEK inhibitor resistance in KRAS mutant colon cancer.
Ontology highlight
ABSTRACT:
SUBMITTER: Yu C
PROVIDER: S-EPMC8732282 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Oncogenic KRAS impairs antitumor immune responses. As effective strategies to combine KRAS inhibitors and immunotherapies have so far proven elusive, a better understanding of the mechanisms by which oncogenic KRAS drives immune evasion is needed to identify approaches that could sensitize KRAS-mutant lung cancer to immunotherapy. In vivo CRISPR-Cas9 screening in an immunogenic murine lung cancer model identified mechanisms by which oncogenic KRAS promotes immune evasion, most notably via upregulation of immunosuppressive COX2 in cancer cells. Oncogenic KRAS potently induced COX2 in both mouse and human lung cancer, which was suppressed using KRAS inhibitors. COX2 acted via prostaglandin E2 (PGE2) to promote resistance to immune checkpoint blockade (ICB) in lung adenocarcinoma. Targeting COX2/PGE2 remodeled the tumor microenvironment by inducing proinflammatory polarization of myeloid cells and influx of activated cytotoxic CD8+ T cells, which increased the efficacy of ICB. Restoration of COX2 expression contributed to tumor relapse after prolonged KRAS inhibition. These results provide the rationale for testing COX2/PGE2 pathway inhibitors in combination with KRASG12C inhibition or ICB in patients with KRAS-mutant lung cancer. Significance: COX2 signaling via prostaglandin E2 is a major mediator of immune evasion driven by oncogenic KRAS that promotes immunotherapy and KRAS-targeted therapy resistance, suggesting effective combination treatments for KRAS-mutant lung cancer.
| S-EPMC11247323 | biostudies-literature
Project description:Mutant KRAS represents one of the most frequently observed oncogenes in NSCLC, yet no therapies are approved for tumors that express activated KRAS variants. While there is strong rationale for the use of MEK inhibitors to treat tumors with activated RAS/MAPK signaling, these have proven ineffective clinically. We therefore implemented a CRISPR screening approach to identify novel agents to sensitize KRAS mutant NSCLC cells to MEK inhibitor treatment. This approach identified multiple components of the canonical RAS/MAPK pathway consistent with previous studies. In addition, we identified MAPK7 as a novel, strong hit and validated this finding using multiple orthogonal approaches including knockdown and pharmacological inhibition. We show that MAPK7 inhibition attenuates the re-activation of MAPK signaling occurring following long-term MEK inhibition, thereby illustrating that MAPK7 mediates pathway reactivation in the face of MEK inhibition. Finally, genetic knockdown of MAPK7 combined with the MEK inhibitor cobimetinib in a mutant KRAS NSCLC xenograft model to mediate improved tumor growth inhibition. These data highlight that MAPK7 represents a promising target for combination treatment with MEK inhibition in KRAS mutant NSCLC.
| S-EPMC6005515 | biostudies-literature
Project description:KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.
| S-EPMC5848616 | biostudies-literature
Project description:Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression.
| S-EPMC3567858 | biostudies-other
Project description:BackgroundBrain tumours are the leading cause of cancer-related death in children, and there is no effective treatment. A growing body of evidence points to deregulated epigenetics as a tumour driver, particularly in paediatric cancers as they have relatively few genomic alterations, and key driver mutations have been identified in histone 3 (H3). Cancer stem cells (CSC) are implicated in tumour development, relapse and therapy resistance and thus particularly important to target. We therefore aimed to identify novel epigenetic treatment targets in CSC derived from H3-mutated high-grade glioma (HGG) through a CRISPR-Cas9 knockout screen.ResultsThe knockout screen identified more than 100 novel genes essential for the growth of CSC derived from paediatric HGG with H3K27M mutation. We successfully validated 12 of the 13 selected hits by individual knockout in the same two CSC lines, and for the top six hits we included two additional CSC lines derived from H3 wild-type paediatric HGG. Knockout of these genes led to a significant decrease in CSC growth, and altered stem cell and differentiation markers.ConclusionsThe screen robustly identified essential genes known in the literature, but also many novel genes essential for CSC growth in paediatric HGG. Six of the novel genes (UBE2N, CHD4, LSM11, KANSL1, KANSL3 and EED) were validated individually thus demonstrating their importance for CSC growth in H3-mutated and wild-type HGG. These genes should be further studied and evaluated as novel treatment targets in paediatric HGG.
| S-EPMC10170782 | biostudies-literature
Project description:Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-β (TGF-β) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.
| S-EPMC6681705 | biostudies-literature
Project description:A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.
| S-EPMC7018052 | biostudies-literature
Project description:In honey bees, complementary sex determiner (csd) is the primary signal of sex determination. Its allelic composition is heterozygous in females, and hemizygous or homozygous in males. To explore the transcriptome differences after sex differentiation between males and females, with genetic differences excluded, csd in fertilized embryos was knocked out by CRISPR/Cas9. The diploid mutant males at 24 h, 48 h, 72 h, and 96 h after egg laying (AEL) and the mock-treated females derived from the same fertilized queen were investigated through RNA-seq. Mutations were detected in the target sequence in diploid mutants. The diploid mutant drones had typical male morphological characteristics and gonads. Transcriptome analysis showed that several female-biased genes, such as worker-enriched antennal (Wat), vitellogenin (Vg), and some venom-related genes, were down-regulated in the diploid mutant males. In contrast, some male-biased genes, such as takeout and apolipophorin-III-like protein (A4), had higher expressions in the diploid mutant males. Weighted gene co-expression network analysis (WGCNA) indicated that there might be interactions between csd and fruitless (fru), feminizer (fem) and hexamerin 70c (hex70c), transformer-2 (tra2) and troponin T (TpnT). The information provided by this study will benefit further research on the sex dimorphism and development of honey bees and other insects in Hymenoptera.
| S-EPMC8396534 | biostudies-literature
Project description:Rationale: KRAS is one of the most frequently mutated oncogenes in cancers. The protein's picomolar affinity for GTP/GDP and smooth protein structure resulting in the absence of known allosteric regulatory sites makes its genomic-level activating mutations a difficult but attractive target. Methods: Two CRISPR systems, genome-editing CRISPR/SpCas9 and transcription-regulating dCas9-KRAB, were developed to deplete the KRAS G12S mutant allele or repress its transcription, respectively, with the goal of treating KRAS-driven cancers. Results: SpCas9 and dCas9-KRAB systems with a sgRNA targeting the mutant allele blocked the expression of the mutant KRAS gene, leading to an inhibition of cancer cell proliferation. Local adenoviral injections using SpCas9 and dCas9-KRAB systems suppressed tumor growth in vivo. The gene-depletion system (SpCas9) performed more effectively than the transcription-suppressing system (dCas9-KRAB) on tumor inhibition. Application of both Cas9 systems to wild-type KRAS tumors did not affect cell proliferation. Furthermore, through bioinformatic analysis of 31555 SNP mutations of the top 20 cancer driver genes, the data showed that our mutant-specific editing strategy could be extended to a reference list of oncogenic mutations with high editing potentials. This pipeline could be applied to analyze the distribution of PAM sequences and survey the best alternative targets for gene editing. Conclusion: We successfully developed both gene-depletion and transcription-suppressing systems to specifically target an oncogenic KRAS mutant allele that led to significant tumor regression. These findings show the potential of CRISPR-based strategies for the treatment of tumors with driver gene mutations.
| S-EPMC7163449 | biostudies-literature
Project description:BackgroundThe development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance.MethodsGenome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging.ResultsWe found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors.ConclusionsThrough systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.
| S-EPMC8760697 | biostudies-literature