RESUMO
PURPOSE: Oncogene addiction provides important therapeutic opportunities for precision oncology treatment strategies. To date the cellular circuitries associated with driving oncoproteins, which eventually establish the phenotypic manifestation of oncogene addiction, remain largely unexplored. Data suggest the DNA damage response (DDR) as a central signaling network that intersects with pathways associated with deregulated addicting oncoproteins with kinase activity in cancer cells. EXPERIMENTAL: DESIGN: We employed a targeted mass spectrometry approach to systematically explore alterations in 116 phosphosites related to oncogene signaling and its intersection with the DDR following inhibition of the addicting oncogene alone or in combination with irradiation in MET-, EGFR-, ALK- or BRAF (V600)-positive cancer models. An NSCLC tissue pipeline combining patient-derived xenografts (PDXs) and ex vivo patient organotypic cultures has been established for treatment responsiveness assessment. RESULTS: We identified an 'oncogene addiction phosphorylation signature' (OAPS) consisting of 8 protein phosphorylations (ACLY S455, IF4B S422, IF4G1 S1231, LIMA1 S490, MYCN S62, NCBP1 S22, P3C2A S259 and TERF2 S365) that are significantly suppressed upon targeted oncogene inhibition solely in addicted cell line models and patient tissues. We show that the OAPS is present in patient tissues and the OAPS-derived score strongly correlates with the ex vivo responses to targeted treatments. CONCLUSIONS: We propose a score derived from OAPS as a quantitative measure to evaluate oncogene addiction of cancer cell samples. This work underlines the importance of protein phosphorylation assessment for patient stratification in precision oncology and corresponding identification of tumor subtypes sensitive to inhibition of a particular oncogene.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Vício Oncogênico , Medicina de Precisão , Fosforilação , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação , Proteínas do CitoesqueletoRESUMO
Metabolism negotiates cell-endogenous requirements of energy, nutrients and building blocks with the immediate environment to enable various processes, including growth and differentiation. While there is an increasing number of examples of crosstalk between metabolism and chromatin, few involve uptake of exogenous metabolites. Solute carriers (SLCs) represent the largest group of transporters in the human genome and are responsible for the transport of a wide variety of substrates, including nutrients and metabolites. We aimed to investigate the possible involvement of SLC-mediated solutes uptake and cellular metabolism in regulating cellular epigenetic states. Here, we perform a CRISPR-Cas9 transporter-focused genetic screen and a metabolic compound library screen for the regulation of BRD4-dependent chromatin states in human myeloid leukaemia cells. Intersection of the two orthogonal approaches reveal that loss of transporters involved with purine transport or inhibition of de novo purine synthesis lead to dysfunction of BRD4-dependent transcriptional regulation. Through mechanistic characterization of the metabolic circuitry, we elucidate the convergence of SLC-mediated purine uptake and de novo purine synthesis on BRD4-chromatin occupancy. Moreover, adenine-related metabolite supplementation effectively restores BRD4 functionality on purine impairment. Our study highlights the specific role of purine/adenine metabolism in modulating BRD4-dependent epigenetic states.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Purinas/metabolismo , Proteínas Carreadoras de Solutos/metabolismo , Fatores de Transcrição/metabolismo , Adenina/metabolismo , Vias Biossintéticas , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana Transportadoras , Modelos Biológicos , Proteínas Carreadoras de Solutos/genética , Fatores de Transcrição/antagonistas & inibidores , Transcrição GênicaRESUMO
Solute carrier (SLC) membrane transporters remain a largely unexploited target class, despite their central roles in cell identity and metabolism. This gap is reflected in the lack of high-quality chemical ligands or probes and in the small number of compounds that have progressed toward clinical development. In this review, we discuss recent advancements in SLC ligand discovery as well as new candidates that have been added to the investigational toolkit, with a particular focus on first-in-class ligands and the cognate discovery strategies. The availability of new probes expands the opportunity to elucidate the functions of SLCs and their relevance in physiology and explores any future potential of SLC druggability.
Assuntos
Antineoplásicos/química , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Bibliotecas de Moléculas Pequenas/química , Proteínas Carreadoras de Solutos/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Membrana Celular/ultraestrutura , Descoberta de Drogas , Humanos , Ligantes , Doenças Metabólicas , Neoplasias/tratamento farmacológico , Neurotransmissores/metabolismo , Nutrientes/metabolismo , Ligação Proteica , Conformação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
This review aims to serve as an introduction to the solute carrier proteins (SLC) superfamily of transporter proteins and their roles in human cells. The SLC superfamily currently includes 458 transport proteins in 65 families that carry a wide variety of substances across cellular membranes. While members of this superfamily are found throughout cellular organelles, this review focuses on transporters expressed at the plasma membrane. At the cell surface, SLC proteins may be viewed as gatekeepers of the cellular milieu, dynamically responding to different metabolic states. With altered metabolism being one of the hallmarks of cancer, we also briefly review the roles that surface SLC proteins play in the development and progression of cancer through their influence on regulating metabolism and environmental conditions.
Assuntos
Transporte Biológico/genética , Proteínas de Membrana Transportadoras/genética , Neoplasias/genética , Proteínas Carreadoras de Solutos/genética , Membrana Celular/genética , HumanosRESUMO
The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR, and DNA-PK. ATM is missing or inactivated in the genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the double-strand break-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine-tuned dynamics between the PIKKs following genotoxic stress, such as DNA-PK-dependent attenuation of ATM. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics. The results highlight intricate relationships between these PIKKs in the DDR.
Assuntos
Dano ao DNA/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Fosfatidilinositol 3-Quinases/genética , Proteômica/métodos , Transdução de Sinais/genéticaRESUMO
With more than 450 members, the solute carrier (SLC) group of proteins represents the largest class of transporters encoded in the human genome. Their several-pass transmembrane domain structure and hydrophobicity contribute to the orphan status of many SLCs, devoid of known cargos or chemical inhibitors. We report that SLC proteins belonging to different families and subcellular compartments are amenable to induced degradation by heterobifunctional ligands. Engineering endogenous alleles via the degradation tag (dTAG) technology enabled chemical control of abundance of the transporter protein, SLC38A2. Moreover, we report the design of d9A-2, a chimeric compound engaging several members of the SLC9 family and leading to their degradation. d9A-2 impairs cellular pH homeostasis and promotes cell death in a range of cancer cell lines. These findings open the era of SLC-targeting chimeric degraders and demonstrate potential access of multi-pass transmembrane proteins of different subcellular localizations to the chemically exploitable degradation machinery.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Linhagem Celular , Feminino , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Masculino , Proteínas de Membrana Transportadoras/química , Domínios Proteicos , ProteóliseRESUMO
Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity-based phosphoproteomic discovery survey, we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using invivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene-driven proliferation and genomic stability.
Assuntos
Dano ao DNA , Epitélio/patologia , Mesoderma/patologia , Fosfoproteínas/metabolismo , Proteômica , Animais , Linhagem Celular Tumoral , Reparo do DNA/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Epitélio/efeitos da radiação , Feminino , Humanos , Mesoderma/efeitos da radiação , Camundongos , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos da radiação , Radiação Ionizante , Reprodutibilidade dos Testes , Especificidade por Substrato/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Consistent detection and quantification of protein post-translational modifications (PTMs) across sample cohorts is a prerequisite for functional analysis of biological processes. Data-independent acquisition (DIA) is a bottom-up mass spectrometry approach that provides complete information on precursor and fragment ions. However, owing to the convoluted structure of DIA data sets, confident, systematic identification and quantification of peptidoforms has remained challenging. Here, we present inference of peptidoforms (IPF), a fully automated algorithm that uses spectral libraries to query, validate and quantify peptidoforms in DIA data sets. The method was developed on data acquired by the DIA method SWATH-MS and benchmarked using a synthetic phosphopeptide reference data set and phosphopeptide-enriched samples. IPF reduced false site-localization by more than sevenfold compared with previous approaches, while recovering 85.4% of the true signals. Using IPF, we quantified peptidoforms in DIA data acquired from >200 samples of blood plasma of a human twin cohort and assessed the contribution of heritable, environmental and longitudinal effects on their PTMs.
Assuntos
Espectrometria de Massas/métodos , Peptídeos/sangue , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Algoritmos , Apolipoproteína A-I/química , Humanos , Fosfopeptídeos/sangue , Fosfopeptídeos/química , GêmeosRESUMO
The DNA of all organisms is constantly subjected to damaging agents, both exogenous and endogenous. One extremely harmful lesion is the double-strand break (DSB), which activates a massive signaling network - the DNA damage response (DDR). The chief activator of the DSB response is the ATM protein kinase, which phosphorylates numerous key players in its various branches. Recent phosphoproteomic screens have extended the scope of damage-induced phosphorylations beyond the direct ATM substrates. We review the evidence for the involvement of numerous other protein kinases in the DDR, obtained from documentation of specific pathways as well as high-throughput screens. The emerging picture of the protein phosphorylation landscape in the DDR broadens the current view on the role of this protein modification in the maintenance of genomic stability. Extensive cross-talk between many of these protein kinases forms an interlaced signaling network that spans numerous cellular processes. Versatile protein kinases in this network affect pathways that are different from those they have been identified with to date. The DDR appears to be one of the most extensive signaling responses to cellular stimuli.
Assuntos
Dano ao DNA , Proteínas Quinases/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The double-strand break (DSB) is a cytotoxic DNA lesion caused by oxygen radicals, ionizing radiation, and radiomimetic chemicals. Cells cope with DNA damage by activating the DNA damage response (DDR), which leads either to damage repair and cellular survival or to programmed cell death. The main transducer of the DSB response is the nuclear protein kinase ataxia telangiectasia mutated (ATM). We applied label-free quantitative mass spectrometry to follow the dynamics of DSB-induced phosphoproteome in nuclear fractions of the human melanoma G361 cells after radiomimetic treatment. We found that these dynamics are complex, including both phosphorylation and dephosphorylation events. In addition to identifying previously unknown ATM-dependent phosphorylation and dephosphorylation events, we found that about 40% of DSB-induced phosphorylations were ATM-independent and that several other kinases are potentially involved. Sustained activity of ATM was required to maintain many ATM-dependent phosphorylations. We identified an ATM-dependent phosphorylation site on ATM itself that played a role in its retention on damaged chromatin. By connecting many of the phosphorylated and dephosphorylated proteins into functional networks, we highlight putative cross talks between proteins pertaining to several cellular biological processes. Our study expands the DDR phosphorylation landscape and identifies previously unknown ATM-dependent and -independent branches. It reveals insights into the breadth and complexity of the cellular responses involved in the coordination of many DDR pathways, which is in line with the critical importance of genomic stability in maintenance of cellular homeostasis.