ABSTRACT
The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.
Subject(s)
AMP-Activated Protein Kinase Kinases , Phosphatidylinositol 3-Kinases , Protein Serine-Threonine Kinases , Spheroids, Cellular , Humans , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , AMP-Activated Protein Kinase Kinases/metabolism , AMP-Activated Protein Kinase Kinases/genetics , Spheroids, Cellular/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/genetics , Cell Proliferation , Cell Line, Tumor , CRISPR-Cas Systems , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/geneticsABSTRACT
The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1- mediated growth suppression we developed a spheroid-based cell culture assay to study LKB1- dependent growth. Using this assay, along with genome-wide CRISPR screens and validation with orthogonal methods, we discovered that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase, which promotes the internalization of wild-type EGFR. Our findings reveal a new mechanism of regulation of EGFR, which may have implications for the treatment of LKB1 -mutant LUAD.
ABSTRACT
KRAS is the most frequently mutated oncogene in human lung adenocarcinomas (hLUAD), and activating mutations frequently co-occur with loss-of-function mutations in TP53 or STK11/LKB1. However, mutation of all three genes is rarely observed in hLUAD, even though engineered comutation is highly aggressive in mouse lung adenocarcinoma (mLUAD). Here, we provide a mechanistic explanation for this difference by uncovering an evolutionary divergence in the regulation of triosephosphate isomerase (TPI1). In hLUAD, TPI1 activity is regulated via phosphorylation at Ser21 by the salt inducible kinases (SIK) in an LKB1-dependent manner, modulating flux between the completion of glycolysis and production of glycerol lipids. In mice, Ser21 of TPI1 is a Cys residue that can be oxidized to alter TPI1 activity without a need for SIKs or LKB1. Our findings suggest this metabolic flexibility is critical in rapidly growing cells with KRAS and TP53 mutations, explaining why the loss of LKB1 creates a liability in these tumors. SIGNIFICANCE: Utilizing phosphoproteomics and metabolomics in genetically engineered human cell lines and genetically engineered mouse models (GEMM), we uncover an evolutionary divergence in metabolic regulation within a clinically relevant genotype of human LUAD with therapeutic implications. Our data provide a cautionary example of the limits of GEMMs as tools to study human diseases such as cancers. This article is highlighted in the In This Issue feature, p. 799.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Triose-Phosphate Isomerase , Animals , Humans , Mice , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mutation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Triose-Phosphate Isomerase/genetics , Triose-Phosphate Isomerase/metabolismABSTRACT
Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.
Subject(s)
Lung Neoplasms/pathology , Oxidative Stress/physiology , Oxidoreductases/metabolism , Thioredoxin-Disulfide Reductase/metabolism , Glutathione/metabolism , Humans , Lung/metabolism , Lung/pathology , Lung Neoplasms/metabolism , Reactive Oxygen Species/metabolism , Thioredoxins/metabolismABSTRACT
Small-molecule kinase inhibitors represent a major group of cancer therapeutics, but tumor responses are often incomplete. To identify pathways that modulate kinase inhibitor response, we conducted a genome-wide knockout (KO) screen in glioblastoma cells treated with the pan-ErbB inhibitor neratinib. Loss of general control nonderepressible 2 (GCN2) kinase rendered cells resistant to neratinib, whereas depletion of the GADD34 phosphatase increased neratinib sensitivity. Loss of GCN2 conferred neratinib resistance by preventing binding and activation of GCN2 by neratinib. Several other Food and Drug Administration (FDA)-approved inhibitors, such erlotinib and sunitinib, also bound and activated GCN2. Our results highlight the utility of genome-wide functional screens to uncover novel mechanisms of drug action and document the role of the integrated stress response (ISR) in modulating the response to inhibitors of oncogenic kinases.
Subject(s)
Adenosine Triphosphate/metabolism , Antineoplastic Agents/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Protein Kinase Inhibitors/pharmacology , Quinolines/pharmacology , CRISPR-Cas Systems , Cell Line, Tumor , Drug Delivery Systems , Gene Deletion , Glioblastoma/drug therapy , Humans , Protein Kinase Inhibitors/chemistryABSTRACT
Synthetic lethality results when mutant KRAS and EGFR proteins are co-expressed in human lung adenocarcinoma (LUAD) cells, revealing the biological basis for mutual exclusivity of KRAS and EGFR mutations. We have now defined the biochemical events responsible for the toxic effects by combining pharmacological and genetic approaches and to show that signaling through extracellular signal-regulated kinases (ERK1/2) mediates the toxicity. These findings imply that tumors with mutant oncogenes in the RAS pathway must restrain the activity of ERK1/2 to avoid toxicities and enable tumor growth. A dual specificity phosphatase, DUSP6, that negatively regulates phosphorylation of (P)-ERK is up-regulated in EGFR- or KRAS-mutant LUAD, potentially protecting cells with mutations in the RAS signaling pathway, a proposal supported by experiments with DUSP6-specific siRNA and an inhibitory drug. Targeting DUSP6 or other negative regulators might offer a treatment strategy for certain cancers by inducing the toxic effects of RAS-mediated signaling.
Subject(s)
Adenocarcinoma of Lung/genetics , Dual Specificity Phosphatase 6/genetics , Genes, erbB-1/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Dual Specificity Phosphatase 6/antagonists & inhibitors , Enzyme Inhibitors/administration & dosage , Gene Expression Regulation, Neoplastic/drug effects , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Mutation/genetics , Phosphorylation/drug effects , RNA, Small Interfering/genetics , Synthetic Lethal Mutations/geneticsABSTRACT
Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent knock-in alleles of U2af1(S34F), the murine version of the most common mutant allele of U2AF1 encountered in human cancers. Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. In an attempt to identify U2af1(S34F)-cooperating changes that promote leukemogenesis, we combined U2af1(S34F) with Runx1 deficiency in mice and further treated the mice with a mutagen, N-ethyl-N-nitrosourea (ENU). Overall, 3 of 16 ENU-treated compound transgenic mice developed AML. However, AML did not arise in mice with other genotypes or without ENU treatment. Sequencing DNA from the three AMLs revealed somatic mutations homologous to those considered to be drivers of human AML, including predicted loss- or gain-of-function mutations in Tet2, Gata2, Idh1, and Ikzf1 However, the engineered U2af1(S34F) missense mutation reverted to WT in two of the three AML cases, implying that U2af1(S34F) is dispensable, or even selected against, once leukemia is established.
Subject(s)
Hematopoiesis/genetics , Leukemia/genetics , Splicing Factor U2AF/metabolism , Alleles , Animals , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Ethylnitrosourea/toxicity , Gene Expression Regulation/drug effects , Genetic Predisposition to Disease , Genotype , Humans , Mice , Mice, Transgenic , Mutation , Myelodysplastic Syndromes/genetics , RNA Splicing , Splicing Factor U2AF/geneticsABSTRACT
Spl574 MLV (murine leukemia virus) is a variant of Moloney ecotropic MLV (MoMLV) that is cytopathic in Mus dunni cells and restricted by other mouse cells. Its host range and cytopathicity are due to a mutation, S82F, at a site critical for binding to the CAT-1 receptor. To identify residues that affect affinity for receptor variants, virus with S82F was passed in restrictive cells. The env genes of the adapted viruses contained 18 novel mutations, including one, E114G, present in 6 of 30 sequenced envs. MoMLV-E114G efficiently infected all mouse cells as well as ecotropic MLV resistant Chinese hamster cells. Virus with E114G and S82F induced large multinucleated syncytia in NIH 3T3 and SC-1 cells as well as M. dunni cells. Inoculation of Mo-S82F,E114G into mice produced lymphomas typical of MoMLV. Residues at env position 114 are thus important determinants of host range, and E114G suppresses host range restriction due to S82F, but does not affect S82F-governed cytopathicity.
Subject(s)
Genes, env , Leukemia, Experimental/virology , Moloney murine leukemia virus/genetics , Retroviridae Infections/virology , Tumor Virus Infections/virology , Animals , Animals, Newborn , Cricetinae , Cricetulus , Giant Cells/virology , Host Specificity , Host-Pathogen Interactions , Leukemia, Experimental/mortality , Mice , Models, Molecular , Moloney murine leukemia virus/pathogenicity , Mutation , NIH 3T3 Cells , Retroviridae Infections/mortality , Sequence Analysis, DNA , Tumor Virus Infections/mortalityABSTRACT
Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIH 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.