RESUMO
Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.
Assuntos
Aptâmeros de Nucleotídeos , Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Inativação de Genes , Linfócitos T , Humanos , Edição de Genes/métodos , Aptâmeros de Nucleotídeos/genética , Linfócitos T/metabolismo , Linfócitos T/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Técnicas de Introdução de Genes/métodos , TransgenesRESUMO
Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAßGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments.
Assuntos
Senescência Celular/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Marcadores Genéticos , Células HCT116 , Humanos , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase ß-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Proto-Oncogênicas/genética , UbiquitinaçãoRESUMO
Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Inativação Gênica , Pulmão , Células Epiteliais/metabolismoRESUMO
Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-pointTM that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.
Assuntos
Aptâmeros de Nucleotídeos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Edição de RNA , Animais , Bactérias/genética , Bactérias/metabolismo , Sistemas CRISPR-Cas , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Mutação INDEL , RNA Guia de Cinetoplastídeos/genética , Reparo de DNA por Recombinação , Sequenciamento do ExomaRESUMO
Bim(EL) the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim(EL). It has been proposed that the RING finger protein Cbl binds to Bim(EL) and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim(EL) is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim(EL) turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim(EL) is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim(EL) does not involve its tyrosine phosphorylation; indeed, Bim(EL) is not a tyrosine phosphoprotein. Furthermore, Bim(EL) fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim(EL) turnover proceeds normally in Cbl-containing or Cbl-/- fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim(EL) turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Fibroblastos/enzimologia , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação , Fosforilação , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-cbl/deficiência , Proteínas Proto-Oncogênicas c-cbl/genética , Serina/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Tirosina/metabolismo , Vanadatos/farmacologiaRESUMO
Studies to identify predictive biomarkers can be carried out in isogenic cancer cell lines, which enable interrogation of the effect of a specific mutation. We assessed the effects of four drugs, the PI3K-mammalian target of rapamycin inhibitor dactolisib, the PI3K inhibitor pictrelisib, and the MEK (MAPK/ERK Kinase) inhibitors PD 0325901 and selumetinib, in isogenic DLD1 parental, KRAS(+/-), KRAS(G13D/-), PIK3CA(+/-) and PIK3CA(E545K/-) colorectal carcinoma cell lines. Importantly, we found substantial differences in the growth of these cells and in their drug sensitivity depending on whether they were studied under 2D (standard tissue culture on plastic) or 3D (in vitro soft agar and in vivo xenograft) conditions. DLD1 KRAS(+/-) and DLD1 PIK3CA(+/-) cells were more sensitive to MEK inhibitors than parental, DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) cells under 2D conditions, whereas DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) xenografts were sensitive to 10 mg/kg daily ×14 PD 0325901 in vivo (p ≤ 0.02) but tumours derived from parental DLD1 cells were not. These findings indicate that KRAS and PIK3CA mutations can influence the response of DLD1 colorectal cancer cell lines to MEK and PI3K inhibitors, but that the effect is dependent on the experimental model used to assess drug sensitivity.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais , Neoplasias Colorretais/tratamento farmacológico , MAP Quinase Quinase Quinases/antagonistas & inibidores , Mutação , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Feminino , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The pro-apoptotic protein BIM(EL) is phosphorylated by ERK1/2 and this targets the protein for poly-ubiquitination and degradation by the proteasome as a survival mechanism. To define in greater detail the sequence determinants required for BIM(EL) turnover we have compared various BIM splice variants and truncation mutants. Of the naturally occurring splice variants BIMbeta1, which lacks the C-terminal hydrophobic domain, the BH3 domain and is cytosolic, exhibited the fastest turnover rate. Indeed, neither the C-terminus, the BH3 domain nor the DLC1 binding region was required for poly-ubiquitination and turnover of BIM. However, we demonstrate that a region consisting of the ERK1/2 docking domain, ERK1/2 phosphorylation sites and either of the two potential ubiquitin-acceptor lysine residues is sufficient to allow poly-ubiquitination and turnover of BIM. In the process we demonstrate that the C-terminal hydrophobic domain, previously suggested to be important in membrane localisation, is as important as the BH3 domain for BIM to induce cell death; similarly, the pro-death BH3-domain can also confer correct mitochondrial localisation in the absence of the C-terminus. These results refine the minimal sequence for ERK1/2-driven degradation and further define the functional importance of key regions within BIM(EL), highlighting the complexity of this pro-apoptotic protein.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitinação , Processamento Alternativo/genética , Sequência de Aminoácidos , Apoptose , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Ativação Enzimática , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mitocôndrias/metabolismo , Mutagênese , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The BCL-2 homology domain 3 (BH3)-only protein, B-cell lymphoma 2 interacting mediator of cell death (BIM) is a potent pro-apoptotic protein belonging to the B-cell lymphoma 2 protein family. In recent years, advances in basic biology have provided a clearer picture of how BIM kills cells and how BIM expression and activity are repressed by growth factor signalling pathways, especially the extracellular signal-regulated kinase 1/2 and protein kinase B pathways. In tumour cells these oncogene-regulated pathways are used to counter the effects of BIM, thereby promoting tumour cell survival. In parallel, a new generation of targeted therapeutics has been developed, which show remarkable specificity and efficacy in tumour cells that are addicted to particular oncogenes. It is now apparent that the expression and activation of BIM is a common response to these new therapeutics. Indeed, BIM has emerged from this marriage of basic and applied biology as an important mediator of tumour cell death in response to such drugs. The induction of BIM alone may not be sufficient for significant tumour cell death, as BIM is more likely to act in concert with other BH3-only proteins, or other death pathways, when new targeted therapeutics are used in combination with traditional chemotherapy agents. Here we discuss recent advances in understanding BIM regulation and review the role of BIM as a mediator of tumour cell death in response to novel oncogene-targeted therapeutics.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose , Autofagia , Proteínas de Membrana/fisiologia , Neoplasias/tratamento farmacológico , Oncogenes , Proteínas Proto-Oncogênicas/fisiologia , Animais , Proteína 11 Semelhante a Bcl-2 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Sobrevivência Celular , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Neoplasias/patologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de SinaisRESUMO
Bim (Bcl-2-interacting mediator of cell death) is a BH3-only protein (BOP), a pro-apoptotic member of the Bcl-2 protein family. The Bim mRNA undergoes alternate splicing to give rise to the short, long and extra long protein variants (Bim(S), Bim(L) and Bim(EL)). These proteins have distinct potency in promoting death and distinct modes of regulation conferred by their interaction with other proteins. Quite how Bim and other BOPs promote apoptosis has been the subject of some debate. Bim was isolated by it's interaction with pro-survival proteins such as Bcl-2 and it has been suggested that this is key to the ability of Bim to induce apoptosis. However, an alternative model argues that some forms of Bim can bind directly to the pro-apoptotic Bax and Bak proteins to initiate apoptosis. A new study may finally put this debate to rest as it provides strong evidence to suggest that Bim and other BOPs act primarily by binding to pro-survival Bcl-2 proteins, thereby releasing Bax or Bak proteins to promote apoptosis. The importance of the interaction between Bim and the pro-survival Bcl-2 proteins is underlined by our demonstration that it is regulated by ERK1/2-dependent phosphorylation of Bim(EL). ERK1/2-dependent dissociation of Bim(EL) from pro-survival proteins is the first step in a process by which the pro-survival ERK1/2 pathway promotes the destruction of this most abundant Bim splice variant. In this review we outline the significance of these new studies to our understanding of how BOPs such as Bim initiate apoptosis and how this process is regulated by growth factor-dependent signalling pathways.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2 , Sobrevivência Celular/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/genéticaRESUMO
The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim-/- fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-x(L) and Mcl-1. Upon serum withdrawal, newly expressed Bim(EL) associates with Bcl-x(L) and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed Bim(EL)/Mcl-1 and Bim(EL)/Bcl-x(L) complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for Bim(EL) and requires ERK1/2-dependent phosphorylation of Bim(EL) at Ser(65). Finally, ERK1/2-dependent dissociation of Bim(EL) from Mcl-1 and Bcl-x(L) may play a role in regulating Bim(EL) degradation, since mutations in the Bim(EL) BH3 domain that disrupt binding to Mcl-1 cause increased turnover of Bim(EL). These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.