RESUMO
Blocking the import of nutrients essential for cancer cell proliferation represents a therapeutic opportunity, but it is unclear which transporters to target. Here we report a CRISPR interference/activation screening platform to systematically interrogate the contribution of nutrient transporters to support cancer cell proliferation in environments ranging from standard culture media to tumours. We applied this platform to identify the transporters of amino acids in leukaemia cells and found that amino acid transport involves high bidirectional flux dependent on the microenvironment composition. While investigating the role of transporters in cystine starved cells, we uncovered a role for serotonin uptake in preventing ferroptosis. Finally, we identified transporters essential for cell proliferation in subcutaneous tumours and found that levels of glucose and amino acids can restrain proliferation in that environment. This study establishes a framework for systematically identifying critical cellular nutrient transporters, characterizing their function and exploring how the tumour microenvironment impacts cancer metabolism.
Assuntos
Proliferação de Células , Microambiente Tumoral , Humanos , Animais , Sistemas CRISPR-Cas , Nutrientes/metabolismo , Linhagem Celular Tumoral , Transporte Biológico , Glucose/metabolismo , Aminoácidos/metabolismo , Serotonina/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Camundongos , Repetições Palindrômicas Curtas Agrupadas e Regularmente EspaçadasRESUMO
The non-essential amino acid serine is a critical nutrient for cancer cells due to its diverse biosynthetic functions. While some tumors can synthesize serine de novo, others are auxotrophic and therefore reliant on serine uptake. Importantly, despite several transporters being known to be capable of transporting serine, the transporters that mediate serine uptake in cancer cells are not known. Here, we characterize the amino acid transporter ASCT2 (SLC1A5) as a major contributor to serine uptake in cancer cells. ASCT2 is well known as a glutamine transporter in cancer, and our work demonstrates that serine and glutamine compete for uptake through ASCT2. We further show that ASCT2-mediated serine uptake is essential for purine nucleotide biosynthesis and that estrogen receptor α (ERα) promotes serine uptake by directly activating SLC1A5 transcription. Collectively, our work defines an additional important role for ASCT2 as a serine transporter in cancer and evaluates ASCT2 as a potential therapeutic target.
Assuntos
Sistema ASC de Transporte de Aminoácidos , Antígenos de Histocompatibilidade Menor , Serina , Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Humanos , Serina/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Glutamina/metabolismo , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/genética , Animais , Transporte Biológico , Feminino , Células MCF-7RESUMO
Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.
Assuntos
Diferenciação Celular , Replicação do DNA , Replicação do DNA/genética , Animais , Humanos , Diferenciação Celular/genética , Camundongos , Nucleotídeos/metabolismo , Nucleotídeos/genética , Linhagem da Célula/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Fase S/genética , Transdução de SinaisRESUMO
We introduce an approach for detection of drug-protein interactions that combines a new yeast three-hybrid screening for identification of interactions with affinity chromatography for their unambiguous validation. We applied the methodology to the profiling of clinically approved drugs, resulting in the identification of previously known and unknown drug-protein interactions. In particular, we were able to identify off-targets for erlotinib and atorvastatin, as well as an enzyme target for the anti-inflammatory drug sulfasalazine. We demonstrate that sulfasalazine and its metabolites, sulfapyridine and mesalamine, are inhibitors of the enzyme catalyzing the final step in the biosynthesis of the cofactor tetrahydrobiopterin. The interference with tetrahydrobiopterin metabolism provides an explanation for some of the beneficial and deleterious properties of sulfasalazine and furthermore suggests new and improved therapies for the drug. This work thus establishes a powerful approach for drug profiling and provides new insights in the mechanism of action of clinically approved drugs.
Assuntos
Biopterinas/análogos & derivados , Saccharomyces cerevisiae/metabolismo , Sulfassalazina/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Anti-Infecciosos , Anti-Inflamatórios não Esteroides/farmacologia , Biopterinas/antagonistas & inibidores , Biopterinas/biossíntese , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos , Mesalamina , Métodos , Ligação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , SulfapiridinaRESUMO
The non-essential amino acid serine is a critical nutrient for cancer cells due to its diverse biosynthetic functions. While some tumors can synthesize serine de novo, others are auxotrophic for serine and therefore reliant on the uptake of exogenous serine. Importantly, however, the transporter(s) that mediate serine uptake in cancer cells are not known. Here, we characterize the amino acid transporter ASCT2 (coded for by the gene SLC1A5) as the primary serine transporter in cancer cells. ASCT2 is well-known as a glutamine transporter in cancer, and our work demonstrates that serine and glutamine compete for uptake through ASCT2. We further show that ASCT2-mediated serine uptake is essential for purine nucleotide biosynthesis and that ERα promotes serine uptake by directly activating SLC1A5 transcription. Together, our work defines an additional important role for ASCT2 as a serine transporter in cancer and evaluates ASCT2 as a potential therapeutic target in serine metabolism.
RESUMO
The identification of all protein targets of a given drug or bioactive molecule within the human body is a prerequisite for an understanding of its beneficial and deleterious activities. Current approaches to reveal protein targets often fail to reveal physiologically relevant interactions. Here we review a recently introduced yeast-based approach for the identification of the binding partners of small molecules. We discuss the advantages and limitations of the approach using the clinically approved drug sulfasalazine as an example.
Assuntos
Anti-Inflamatórios não Esteroides , Descoberta de Drogas/métodos , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas , Sulfassalazina , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Biopterinas/análogos & derivados , Biopterinas/biossíntese , Biotransformação , Descoberta de Drogas/tendências , Estrutura Molecular , Ligação Proteica , Proteínas/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfassalazina/química , Sulfassalazina/metabolismo , Sulfassalazina/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.
Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Teste para COVID-19 , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Humanos , RNA/metabolismo , RNA Viral/genética , RNA Viral/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Recombinases/metabolismo , SARS-CoV-2 , Saliva/virologia , Vírion/genéticaRESUMO
Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. We developed a molecular diagnostic test for SARS-CoV-2, FIND (Fast Isothermal Nucleic acid Detection), based on an enhanced isothermal recombinase polymerase amplification reaction. FIND has a detection limit on patient samples close to that of RT-qPCR, requires minimal instrumentation, and is highly scalable and cheap. It can be performed in high throughput, does not cross-react with other common coronaviruses, avoids bottlenecks caused by the current worldwide shortage of RNA isolation kits, and takes ~45 minutes from sample collection to results. FIND can be adapted to future novel viruses in days once sequence is available. ONE SENTENCE SUMMARY: Sensitive, specific, rapid, scalable, enhanced isothermal amplification method for detecting SARS-CoV-2 from patient samples.
RESUMO
Targeted inhibition of oncogenic pathways can be highly effective in halting the rapid growth of tumors but often leads to the emergence of slowly dividing persister cells, which constitute a reservoir for the selection of drug-resistant clones. In BRAFV600E melanomas, RAF and MEK inhibitors efficiently block oncogenic signaling, but persister cells emerge. Here, we show that persister cells escape drug-induced cell-cycle arrest via brief, sporadic ERK pulses generated by transmembrane receptors and growth factors operating in an autocrine/paracrine manner. Quantitative proteomics and computational modeling show that ERK pulsing is enabled by rewiring of mitogen-activated protein kinase (MAPK) signaling: from an oncogenic BRAFV600E monomer-driven configuration that is drug sensitive to a receptor-driven configuration that involves Ras-GTP and RAF dimers and is highly resistant to RAF and MEK inhibitors. Altogether, this work shows that pulsatile MAPK activation by factors in the microenvironment generates a persistent population of melanoma cells that rewires MAPK signaling to sustain non-genetic drug resistance.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Melanoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Melanoma/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/fisiologia , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Proteínas ras/genéticaRESUMO
Curative cancer therapies are uncommon and nearly always involve multi-drug combinations developed by experimentation in humans; unfortunately, the mechanistic basis for the success of such combinations has rarely been investigated in detail, obscuring lessons learned. Here, we use isobologram analysis to score pharmacological interaction, and clone tracing and CRISPR screening to measure cross-resistance among the five drugs comprising R-CHOP, a combination therapy that frequently cures Diffuse Large B-Cell Lymphomas. We find that drugs in R-CHOP exhibit very low cross-resistance but not synergistic interaction: together they achieve a greater fractional kill according to the null hypothesis for both the Loewe dose-additivity model and the Bliss effect-independence model. These data provide direct evidence for the 50 year old hypothesis that a curative cancer therapy can be constructed on the basis of independently effective drugs having non-overlapping mechanisms of resistance, without synergistic interaction, which has immediate significance for the design of new drug combinations.
Assuntos
Terapia Combinada/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Neoplasias/tratamento farmacológico , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Mutação , Neoplasias/genética , Fator de Transcrição CHOP/efeitos dos fármacosRESUMO
Bioconjugations often rely on adaptor molecules to cross-link different biomolecules. In this work, we introduce the molecular adaptor covalin, which is a protein chimera of two self-labeling proteins with nonoverlapping substrate specificity. Covalin permits a selective and covalent heteroconjugation of biomolecules displaying appropriate functional groups. Examples for the use of covalin include the specific heteroconjugation of a reporter enzyme to an antibody and of molecular probes to the surface of living cells. The efficiency and specificity of covalin-based bioconjugations together with the availability of a large variety of substrates create immediate and ubiquitous applications for covalin in bioconjugate chemistry.
Assuntos
Células , Reagentes de Ligações Cruzadas/química , Hidrolases/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Proteínas , Animais , Sítios de Ligação , Células/química , Células/metabolismo , Fluoresceínas/química , Hidrolases/química , O(6)-Metilguanina-DNA Metiltransferase/química , Proteínas/química , Proteínas/metabolismo , Espectrometria de Fluorescência , Coloração e Rotulagem , Especificidade por SubstratoRESUMO
The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives.
Assuntos
Guanina/análogos & derivados , O(6)-Metilguanina-DNA Metiltransferase/química , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Coloração e Rotulagem/métodos , Sítios de Ligação , Membrana Celular/metabolismo , Células Cultivadas , Escherichia coli/enzimologia , Escherichia coli/genética , Evolução Molecular , Guanina/química , Guanina/farmacologia , Modelos Moleculares , Peso Molecular , Mutagênese Sítio-Dirigida , O(6)-Metilguanina-DNA Metiltransferase/genética , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Phenotypic screens allow the identification of small molecules with promising anticancer activity, but the difficulty in characterizing the mechanism of action of these compounds in human cells often undermines their value as drug leads. Here, we used a loss-of-function genetic screen in human haploid KBM7 cells to discover the mechanism of action of the anticancer natural product ophiobolin A (OPA). We found that genetic inactivation of de novo synthesis of phosphatidylethanolamine (PE) mitigates OPA cytotoxicity by reducing cellular PE levels. OPA reacts with the ethanolamine head group of PE in human cells to form pyrrole-containing covalent cytotoxic adducts and these adducts lead to lipid bilayer destabilization. Our characterization of this unusual cytotoxicity mechanism, made possible by unbiased genetic screening in human cells, suggests that the selective antitumor activity displayed by OPA may be due to altered membrane PE levels in cancer cells.