RESUMO
Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.
Assuntos
Glicina/análogos & derivados , Proteínas de Ligação a RNA/química , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Cristalografia por Raios X , Dimerização , Glicina/administração & dosagem , Glicina/química , Glicina/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Neoplasias Pancreáticas/tratamento farmacológico , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Sulfonas/administração & dosagem , Sulfonas/química , Proteínas ras/metabolismo , Quinase 1 Polo-LikeRESUMO
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer consisting of a catalytic subunit, p110α, and a regulatory subunit, p85α, and is referred to as PI3Kα. The catalytic subunit p110α is frequently mutated in cancer. The mutations induce a gain of function and constitute a driving force in cancer development. About 80% of these mutations lead to single-amino-acid substitutions in one of three sites of p110α: two in the helical domain of the protein (E542K and E545K) and one at the C-terminus of the kinase domain (H1047R). Here, we report the cryo-electron microscopy structures of these mutants in complex with the p110α-specific inhibitor BYL-719. The H1047R mutant rotates its sidechain to a new position and weakens the kα11 activation loop interaction, thereby reducing the inhibitory effect of p85α on p110α. E542K and E545K completely abolish the tight interaction between the helical domain of p110α and the N-terminal SH2 domain of p85α and lead to the disruption of all p85α binding and a dramatic increase in flexibility of the adaptor-binding domain (ABD) in p110α. Yet, the dimerization of PI3Kα is preserved through the ABD-p85α interaction. The local and global structural features induced by these mutations provide molecular insights into the activation of PI3Kα, deepen our understanding of the oncogenic mechanism of this important signaling molecule, and may facilitate the development of mutant-specific inhibitors.
Assuntos
Neoplasias , Fosfatidilinositol 3-Quinases , Humanos , Microscopia Crioeletrônica , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Domínio Catalítico/genética , Neoplasias/genéticaRESUMO
Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.
Assuntos
Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase , Classe I de Fosfatidilinositol 3-Quinases/química , Classe Ia de Fosfatidilinositol 3-Quinase/química , Humanos , Espectrometria de Massas/métodos , Anticorpos de Domínio ÚnicoRESUMO
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases essential for growth and metabolism. Their aberrant activation is associated with many types of cancers. Here we used single-particle cryoelectron microscopy (cryo-EM) to determine three distinct conformations of full-length PI3Kα (p110α-p85α): the unliganded heterodimer PI3Kα, PI3Kα bound to the p110α-specific inhibitor BYL-719, and PI3Kα exposed to an activating phosphopeptide. The cryo-EM structures of unbound and of BYL-719-bound PI3Kα are in general accord with published crystal structures. Local deviations are presented and discussed. BYL-719 stabilizes the structure of PI3Kα, but three regions of low-resolution extra density remain and are provisionally assigned to the cSH2, BH, and SH3 domains of p85. One of the extra density regions is in contact with the kinase domain blocking access to the catalytic site. This conformational change indicates that the effects of BYL-719 on PI3Kα activity extend beyond competition with adenosine triphosphate (ATP). In unliganded PI3Kα, the DFG motif occurs in the "in" and "out" positions. In BYL-719-bound PI3Kα, only the DFG-in position, corresponding to the active conformation of the kinase, was observed. The phosphopeptide-bound structure of PI3Kα is composed of a stable core resolved at 3.8 Å. It contains all p110α domains except the adaptor-binding domain (ABD). The p85α domains, linked to the core through the ABD, are no longer resolved, implying that the phosphopeptide activates PI3Kα by fully releasing the niSH2 domain from binding to p110α. The structures presented here show the basal form of the full-length PI3Kα dimer and document conformational changes related to the activated and inhibited states.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase/química , Tiazóis/química , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/ultraestrutura , Microscopia Crioeletrônica , Conformação Proteica , Células Sf9 , SpodopteraRESUMO
MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proliferação de Células/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/genética , Proteínas Repressoras/metabolismo , Aptâmeros de Nucleotídeos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteína 9 Associada à CRISPR/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Regiões Promotoras Genéticas , Domínios Proteicos , RNA Guia de Cinetoplastídeos , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transcrição GênicaRESUMO
We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.
Assuntos
Desenvolvimento de Medicamentos , Corantes Fluorescentes/farmacologia , Ensaios de Triagem em Larga Escala , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-AtividadeRESUMO
Small, noncoding RNAs are short untranslated RNA molecules, some of which have been associated with cancer development. Recently we showed that a class of small RNAs generated during the maturation process of tRNAs (tRNA-derived small RNAs, hereafter "tsRNAs") is dysregulated in cancer. Specifically, we uncovered tsRNA signatures in chronic lymphocytic leukemia and lung cancer and demonstrated that the ts-4521/3676 cluster (now called "ts-101" and "ts-53," respectively), ts-46, and ts-47 are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that ts-101 and ts-53 can associate with PiwiL2, a protein involved in the silencing of transposons. In this study, we extended our investigation on tsRNA signatures to samples collected from patients with colon, breast, or ovarian cancer and cell lines harboring specific oncogenic mutations and representing different stages of cancer progression. We detected tsRNA signatures in all patient samples and determined that tsRNA expression is altered upon oncogene activation and during cancer staging. In addition, we generated a knocked-out cell model for ts-101 and ts-46 in HEK-293 cells and found significant differences in gene-expression patterns, with activation of genes involved in cell survival and down-regulation of genes involved in apoptosis and chromatin structure. Finally, we overexpressed ts-46 and ts-47 in two lung cancer cell lines and performed a clonogenic assay to examine their role in cell proliferation. We observed a strong inhibition of colony formation in cells overexpressing these tsRNAs compared with untreated cells, confirming that tsRNAs affect cell growth and survival.
Assuntos
Neoplasias/metabolismo , Pequeno RNA não Traduzido/metabolismo , Células A549 , Estudos de Casos e Controles , Células HEK293 , Humanos , OncogenesRESUMO
MYC is a key transcriptional regulator involved in cellular proliferation and has established roles in transcriptional elongation and initiation, microRNA regulation, apoptosis, and pluripotency. Despite this prevalence, functional chemical probes of MYC function at the protein level have been limited. Previously, we discovered 5a, that binds to MYC with potency and specificity, downregulates the transcriptional activities of MYC and shows efficacy in vivo. However, this scaffold posed intrinsic pharmacokinetic liabilities, namely, poor solubility that precluded biophysical interrogation. Here, we developed a screening platform based on field-effect transistor analysis (Bio-FET), surface plasmon resonance (SPR), and a microtumor formation assay to analyze a series of new compounds aimed at improving these properties. This blind SAR campaign has produced a new lead compound of significantly increased in vivo stability and solubility for a 40-fold increase in exposure. This probe represents a significant advancement that will not only enable biophysical characterization of this interaction and further SAR, but also contribute to advances in understanding of MYC biology.
Assuntos
Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/síntese química , Piridinas/química , Pirimidinas/síntese química , Pirimidinas/química , Solubilidade , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
We have compared the proteome, transcriptome, and metabolome of two cell lines: the human breast epithelial line MCF-10A and its mutant descendant MCF-10A-H1047R. These cell lines are derived from the same parental stock and differ by a single amino acid substitution (H1047R) caused by a single nucleotide change in one allele of the PIK3CA gene, which encodes the catalytic subunit p110α of PI3K (phosphatidylinositol 3-kinase). They are considered isogenic. The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse, with examples in structural protein levels, the DNA repair machinery, and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.
Assuntos
Neoplasias da Mama/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , RNA Neoplásico/genética , Substituição de Aminoácidos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA/métodos , Reparo do DNA/genética , Feminino , Humanos , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Neoplásico/metabolismoRESUMO
HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The "shock and kill" strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a "hotspot" for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a "functional cure" of HIV/AIDS.
Assuntos
Sistemas CRISPR-Cas , HIV-1/fisiologia , Complexos Multiproteicos/metabolismo , Ativação Viral , Latência Viral , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Regulação Viral da Expressão Gênica , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/genética , Humanos , NF-kappa B/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , RNA Guia de Cinetoplastídeos/genética , Ativação TranscricionalRESUMO
Expression of the regulatory subunit p85ß of PI3K induces oncogenic transformation of primary avian fibroblasts. The transformed cells proliferate at an increased rate compared with nontransformed controls and show elevated levels of PI3K signaling. The oncogenic activity of p85ß requires an active PI3K-TOR signaling cascade and is mediated by the p110α and p110ß isoforms of the PI3K catalytic subunit. The data suggest that p85ß is a less effective inhibitor of the PI3K catalytic subunit than p85α and that this reduced level of p110 inhibition accounts for the oncogenic activity of p85ß.
Assuntos
Oncogenes , Fosfatidilinositol 3-Quinase/metabolismo , Animais , Transformação Celular Neoplásica , Embrião de Galinha , Fosfatidilinositol 3-Quinase/química , Transdução de SinaisRESUMO
In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The Kd of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.
Assuntos
Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Antineoplásicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Células Cultivadas , Embrião de Galinha , Avaliação Pré-Clínica de Medicamentos , Feminino , Polarização de Fluorescência , Genes myc , Humanos , Interferometria , Camundongos , Camundongos Nus , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/química , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/química , Piridinas/química , Pirimidinas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cells transformed by the p110α-H1047R mutant of PI3K show increased tyrosine phosphorylation of Stat3. This activation of Stat3 is important for the transformation process, because a dominant-negative mutant of Stat3 interferes with PI3K-induced oncogenesis. GDC-0941, a specific inhibitor of PI3K reduces the level of Stat3 phosphorylation. The effect of PI3K on Stat3 appears to be mediated by a member of the Tec kinase family. The Tec kinase inhibitor LFM-A13 blocks Stat3 phosphorylation in H1047R-transformed cells. The Janus kinase inhibitor AG490 and the Src kinase inhibitor Src-1, as well as rapamycin, have no effect on Stat3 phosphorylation in H1047R-transformed cells. The H1047R-transformed cells also release a factor that induces Stat3 phosphorylation in normal cells with possible effects on the cellular microenvironment. In some human tumor cell lines, the enhanced phosphorylation of Stat3 is inhibited by both PI3K and by Tec kinase inhibitors, suggesting that the link between PI3K and Stat3 is significant in human cancer.
Assuntos
Transformação Celular Neoplásica/patologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Fator de Transcrição STAT3/metabolismo , Amidas/farmacologia , Animais , Linhagem Celular Tumoral , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Genes Dominantes/genética , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Nitrilas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Cancer-specific mutations in the iSH2 (inter-SH2) and nSH2 (N-terminal SH2) domains of p85alpha, the regulatory subunit of phosphatidylinositide 3-kinase (PI3K), show gain of function. They induce oncogenic cellular transformation, stimulate cellular proliferation, and enhance PI3K signaling. Quantitative determinations of oncogenic activity reveal large differences between individual mutants of p85alpha. The mutant proteins are still able to bind to the catalytic subunits p110alpha and p110beta. Studies with isoform-specific inhibitors of p110 suggest that expression of p85 mutants in fibroblasts leads exclusively to an activation of p110alpha, and p110alpha is the sole mediator of p85 mutant-induced oncogenic transformation. The characteristics of the p85 mutants are in agreement with the hypothesis that the mutations weaken an inhibitory interaction between p85alpha and p110alpha while preserving the stabilizing interaction between p85alpha iSH2 and the adapter-binding domain of p110alpha.
Assuntos
Transformação Celular Neoplásica/genética , Mutação , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Adenina/análogos & derivados , Adenina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Domínio Catalítico/genética , Linhagem Celular , Proliferação de Células , Células Cultivadas , Embrião de Galinha , Classe I de Fosfatidilinositol 3-Quinases , Dioxóis/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imunoprecipitação , Morfolinas/farmacologia , Neoplasias/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pirimidinonas/farmacologia , Quinazolinas/farmacologia , Tiazolidinedionas/farmacologia , TransfecçãoRESUMO
In this article, we discuss a class of MYC-interacting lncRNAs (long non-coding RNAs) that share the following criteria: They are direct transcriptional targets of MYC. Their expression is coordinated with the expression of MYC. They are required for sustained MYC-driven cell proliferation, and they are not essential for cell survival. We refer to these lncRNAs as "MYC facilitators" and discuss two representative members of this class of lncRNAs, SNHG17 (small nuclear RNA host gene) and LNROP (long non-coding regulator of POU2F2). We also present a general hypothesis on the role of lncRNAs in MYC-mediated transcriptional regulation.
RESUMO
MYC controls most of the non-coding genome. Several long noncoding transcripts were originally identified in the human B cell line P496-3 and then shown to be required for MYC-driven proliferation of Burkitt lymphoma-derived RAMOS cells. In this study, we used RAMOS cells exclusively as a representative of the human B cell lineage. One of the MYC-controlled lncRNAs required for RAMOS cell proliferation is ENSG00000254887 which we will term LNROP (long non-coding regulator of POU2F2). In the genome, LNROP is located in close proximity of POU2F2, the gene encoding OCT2. OCT2 is a transcription factor with important roles in sustaining the proliferation of human B cells. Here we show that LNROP is a nuclear RNA and a direct target of MYC. Downregulation of LNROP attenuates the expression of OCT2. This effect of LNROP on the expression of OCT2 is unidirectional as downregulation of OCT2 does not alter the expression of LNROP. Our data suggest that LNROP is a cis-acting regulator of OCT2. To illustrate the downstream reach of LNROP, we chose a prominent target of OCT2, the tyrosine phosphatase SHP-1. Downregulation of OCT2 elevates the expression of SHP-1. Our data suggest the following path of interactions: LNROP enables the proliferation of B cells by positively and unidirectionally regulating the growth-stimulatory transcription factor OCT2. In actively proliferating B cells, OCT2 attenuates the expression and anti-proliferative activity of SHP-1.
Assuntos
Linfoma de Burkitt , RNA Longo não Codificante , Humanos , Linfócitos B , Proliferação de Células/genética , RNA Longo não Codificante/genética , Fatores de TranscriçãoRESUMO
Recent cryo-electron microscopic (cryo-EM) investigations have succeeded in the analysis of various structural conformations and functional states of PI3Kα, a dimer consisting of the catalytic subunit p110α and the regulatory subunit p85α of class IA of phosphoinositide 3-kinase. High resolution structures have been obtained of the unliganded and of BYL-719-bound PI3Kα. The latter provides information on excessively flexible domains of p85α that are then further analyzed with nanobodies and CXMS (chemical cross-linking, digestion and mass spectrometry). Analysis of p110α helical and kinase domain mutations reveals mutant-specific features that can be linked to the gain of function in enzymatic and signaling activities.
Assuntos
Fosfatidilinositol 3-Quinase , Fosfatidilinositol 3-Quinases , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Microscopia Crioeletrônica , Mutação , Domínio Catalítico/genéticaRESUMO
The catalytic and regulatory subunits of class I phosphoinositide 3-kinase (PI3K) have oncogenic potential. The catalytic subunit p110α and the regulatory subunit p85 undergo cancer-specific gain-of-function mutations that lead to enhanced enzymatic activity, ability to signal constitutively, and oncogenicity. The ß, γ, and δ isoforms of p110 are cell-transforming as overexpressed wild-type proteins. Class I PI3Ks have the unique ability to generate phosphoinositide 3,4,5 trisphosphate (PIP(3)). Class II and class III PI3Ks lack this ability. Genetic and cell biological evidence suggests that PIP(3) is essential for PI3K-mediated oncogenicity, explaining why class II and class III enzymes have not been linked to cancer. Mutational analysis reveals the existence of at least two distinct molecular mechanisms for the gain of function seen with cancer-specific mutations in p110α; one causing independence from upstream receptor tyrosine kinases, the other inducing independence from Ras. An essential component of the oncogenic signal that is initiated by PI3K is the TOR (target of rapamycin) kinase. TOR is an integrator of growth and of metabolic inputs. In complex with the raptor protein (TORC1), it controls cap-dependent translation, and this function is essential for PI3K-initiated oncogenesis.
Assuntos
Neoplasias/etiologia , Fosfatidilinositol 3-Quinases/fisiologia , Animais , Transformação Celular Neoplásica , Humanos , Isoenzimas/fisiologia , Mutação , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genéticaRESUMO
Tumor formation is generally linked to the acquisition of two or more driver genes that cause normal cells to progress from proliferation to abnormal expansion and malignancy. In order to understand genetic alterations involved in this process, we compared the transcriptomes of an isogenic set of breast epithelial cell lines that are non-transformed or contain a single or double knock-in (DKI) of PIK3CA (H1047R) or KRAS (G12V). Gene set enrichment analysis revealed that DKI cells were enriched over single mutant cells for genes that characterize a MYC target gene signature. This gene signature was mediated in part by the bromodomain-containing protein 9 (BRD9) that was found in the SWI-SNF chromatin-remodeling complex, bound to the MYC super-enhancer locus. Small molecule inhibition of BRD9 reduced MYC transcript levels. Critically, only DKI cells had the capacity for anchorage-independent growth in semi-solid medium, and CRISPR-Cas9 manipulations showed that PIK3CA and BRD9 expression were essential for this phenotype. In contrast, KRAS was necessary for DKI cell migration, and BRD9 overexpression induced the growth of KRAS single mutant cells in semi-solid medium. These results provide new insight into the earliest transforming events driven by oncoprotein cooperation and suggest BRD9 is an important mediator of mutant PIK3CA/KRAS-driven oncogenic transformation.