RESUMEN
Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.
Asunto(s)
ADP-Ribosilación , Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ADP-Ribosilación/efectos de los fármacos , Secuencia de Aminoácidos , Anticuerpos/metabolismo , Bencimidazoles/farmacología , Línea Celular Tumoral , Técnicas de Visualización de Superficie Celular , Daño del ADN , Glicósido Hidrolasas/metabolismo , Histonas/metabolismo , Humanos , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/química , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Tirosina/metabolismoRESUMEN
Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.
Asunto(s)
Animales Modificados Genéticamente/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de los Músculos , Rabdomiosarcoma , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/inmunología , Femenino , Xenoinjertos , Humanos , Células K562 , Masculino , Neoplasias de los Músculos/tratamiento farmacológico , Neoplasias de los Músculos/inmunología , Neoplasias de los Músculos/metabolismo , Neoplasias de los Músculos/patología , Trasplante de Neoplasias , Ftalazinas/farmacología , Piperazinas/farmacología , Rabdomiosarcoma/tratamiento farmacológico , Rabdomiosarcoma/inmunología , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Temozolomida/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/genética , Pez Cebra/inmunologíaRESUMEN
KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Piperazinas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Quinazolinas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Células HCT116 , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación del Acoplamiento Molecular , Mutación , Piperazinas/química , Piperazinas/uso terapéutico , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinazolinas/química , Quinazolinas/uso terapéuticoRESUMEN
PARTNER is a prospective, phase II-III, randomized controlled clinical trial that recruited patients with triple-negative breast cancer1,2, who were germline BRCA1 and BRCA2 wild type3. Here we report the results of the trial. Patients (n = 559) were randomized on a 1:1 basis to receive neoadjuvant carboplatin-paclitaxel with or without 150 mg olaparib twice daily, on days 3 to 14, of each of four cycles (gap schedule olaparib, research arm) followed by three cycles of anthracycline-based chemotherapy before surgery. The primary end point was pathologic complete response (pCR)4, and secondary end points included event-free survival (EFS) and overall survival (OS)5. pCR was achieved in 51% of patients in the research arm and 52% in the control arm (P = 0.753). Estimated EFS at 36 months in the research and control arms was 80% and 79% (log-rank P > 0.9), respectively; OS was 90% and 87.2% (log-rank P = 0.8), respectively. In patients with pCR, estimated EFS at 36 months was 90%, and in those with non-pCR it was 70% (log-rank P < 0.001), and OS was 96% and 83% (log-rank P < 0.001), respectively. Neoadjuvant olaparib did not improve pCR rates, EFS or OS when added to carboplatin-paclitaxel and anthracycline-based chemotherapy in patients with triple-negative breast cancer who were germline BRCA1 and BRCA2 wild type. ClinicalTrials.gov ID: NCT03150576 .
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Terapia Neoadyuvante , Ftalazinas , Piperazinas , Neoplasias de la Mama Triple Negativas , Adulto , Anciano , Femenino , Humanos , Persona de Mediana Edad , Antraciclinas/uso terapéutico , Antraciclinas/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carboplatino/administración & dosificación , Carboplatino/uso terapéutico , Genes BRCA1 , Genes BRCA2 , Paclitaxel/administración & dosificación , Paclitaxel/uso terapéutico , Respuesta Patológica Completa , Ftalazinas/administración & dosificación , Ftalazinas/uso terapéutico , Piperazinas/administración & dosificación , Piperazinas/uso terapéutico , Supervivencia sin Progresión , Estudios Prospectivos , Análisis de Supervivencia , Factores de Tiempo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/cirugía , Adolescente , Adulto JovenRESUMEN
The dopamine transporter has a crucial role in regulation of dopaminergic neurotransmission by uptake of dopamine into neurons and contributes to the abuse potential of psychomotor stimulants1-3. Despite decades of study, the structure, substrate binding, conformational transitions and drug-binding poses of human dopamine transporter remain unknown. Here we report structures of the human dopamine transporter in its apo state, and in complex with the substrate dopamine, the attention deficit hyperactivity disorder drug methylphenidate, and the dopamine-uptake inhibitors GBR12909 and benztropine. The dopamine-bound structure in the occluded state precisely illustrates the binding position of dopamine and associated ions. The structures bound to drugs are captured in outward-facing or inward-facing states, illuminating distinct binding modes and conformational transitions during substrate transport. Unlike the outward-facing state, which is stabilized by cocaine, GBR12909 and benztropine stabilize the dopamine transporter in the inward-facing state, revealing previously unseen drug-binding poses and providing insights into how they counteract the effects of cocaine. This study establishes a framework for understanding the functioning of the human dopamine transporter and developing therapeutic interventions for dopamine transporter-related disorders and cocaine addiction.
Asunto(s)
Benzotropina , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Inhibidores de Captación de Dopamina , Dopamina , Metilfenidato , Modelos Moleculares , Piperazinas , Humanos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Dopamina/metabolismo , Metilfenidato/farmacología , Metilfenidato/metabolismo , Metilfenidato/química , Inhibidores de Captación de Dopamina/farmacología , Inhibidores de Captación de Dopamina/metabolismo , Inhibidores de Captación de Dopamina/química , Piperazinas/farmacología , Piperazinas/metabolismo , Piperazinas/química , Benzotropina/farmacología , Benzotropina/metabolismo , Benzotropina/química , Sitios de Unión , Unión Proteica , Apoproteínas/metabolismo , Apoproteínas/química , Cocaína/farmacología , Cocaína/metabolismo , Conformación ProteicaRESUMEN
Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.
Asunto(s)
Carbolinas/farmacología , Muerte Celular/efectos de los fármacos , Glutatión Peroxidasa/antagonistas & inhibidores , Piperazinas/farmacología , Animales , Carcinoma de Células Renales/tratamiento farmacológico , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Glutatión/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Xenoinjertos , Humanos , Linfoma de Células B/tratamiento farmacológico , Ratones , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Fosfolípido Hidroperóxido Glutatión PeroxidasaRESUMEN
Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems.
Asunto(s)
Membrana Celular/química , Sistema Enzimático del Citocromo P-450/genética , Citocromo-B(5) Reductasa/genética , Ácidos Grasos Insaturados/metabolismo , Ferroptosis/genética , NADP/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Concanavalina A/farmacología , Sistema Enzimático del Citocromo P-450/deficiencia , Citocromo-B(5) Reductasa/deficiencia , Transporte de Electrón/efectos de los fármacos , Ferroptosis/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Oxígeno/metabolismo , Compuestos de Fenilurea/farmacología , Piperazinas/farmacología , Piridinas/farmacología , Sorafenib/farmacologíaRESUMEN
Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Eicosanoides , Leucocitos Mononucleares , Ratones , Compuestos Orgánicos , Oxazoles , Piperazinas , Poliésteres , Prostaglandinas , Glicoproteína de la Espiga del Coronavirus , SulfonamidasRESUMEN
Cancer progression is driven in part by genomic alterations1. The genomic characterization of cancers has shown interpatient heterogeneity regarding driver alterations2, leading to the concept that generation of genomic profiling in patients with cancer could allow the selection of effective therapies3,4. Although DNA sequencing has been implemented in practice, it remains unclear how to use its results. A total of 1,462 patients with HER2-non-overexpressing metastatic breast cancer were enroled to receive genomic profiling in the SAFIR02-BREAST trial. Two hundred and thirty-eight of these patients were randomized in two trials (nos. NCT02299999 and NCT03386162) comparing the efficacy of maintenance treatment5 with a targeted therapy matched to genomic alteration. Targeted therapies matched to genomics improves progression-free survival when genomic alterations are classified as level I/II according to the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT)6 (adjusted hazards ratio (HR): 0.41, 90% confidence interval (CI): 0.27-0.61, P < 0.001), but not when alterations are unselected using ESCAT (adjusted HR: 0.77, 95% CI: 0.56-1.06, P = 0.109). No improvement in progression-free survival was observed in the targeted therapies arm (unadjusted HR: 1.15, 95% CI: 0.76-1.75) for patients presenting with ESCAT alteration beyond level I/II. Patients with germline BRCA1/2 mutations (n = 49) derived high benefit from olaparib (gBRCA1: HR = 0.36, 90% CI: 0.14-0.89; gBRCA2: HR = 0.37, 90% CI: 0.17-0.78). This trial provides evidence that the treatment decision led by genomics should be driven by a framework of target actionability in patients with metastatic breast cancer.
Asunto(s)
Neoplasias de la Mama , Toma de Decisiones Clínicas , Genoma Humano , Genómica , Metástasis de la Neoplasia , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Toma de Decisiones Clínicas/métodos , Análisis Mutacional de ADN , Progresión de la Enfermedad , Femenino , Genes BRCA1 , Genes BRCA2 , Genoma Humano/genética , Humanos , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Ftalazinas/uso terapéutico , Piperazinas/uso terapéuticoRESUMEN
Young mammals possess a limited regenerative capacity in some tissues, which is lost upon maturation. We investigated whether cellular senescence might play a role in such loss during liver regeneration. We found that following partial hepatectomy, the senescence-associated genes p21, p16Ink4a, and p19Arf become dynamically expressed in different cell types when regenerative capacity decreases, but without a full senescent response. However, we show that treatment with a senescence-inhibiting drug improves regeneration, by disrupting aberrantly prolonged p21 expression. This work suggests that senescence may initially develop from heterogeneous cellular responses, and that senotherapeutic drugs might be useful in promoting organ regeneration.
Asunto(s)
Compuestos de Bifenilo/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/fisiología , Nitrofenoles/farmacología , Regeneración/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Piperazinas/farmacologíaRESUMEN
KRAS gain-of-function mutations are frequently observed in sporadic arteriovenous malformations. The mechanisms underlying the progression of such KRAS-driven malformations are still incompletely understood, and no treatments for the condition are approved. Here, we show the effectiveness of sotorasib, a specific KRAS G12C inhibitor, in reducing the volume of vascular malformations and improving survival in two mouse models carrying a mosaic Kras G12C mutation. We then administered sotorasib to two adult patients with severe KRAS G12C-related arteriovenous malformations. Both patients had rapid reductions in symptoms and arteriovenous malformation size. Targeting KRAS G12C appears to be a promising therapeutic approach for patients with KRAS G12C-related vascular malformations. (Funded by the European Research Council and others.).
Asunto(s)
Malformaciones Arteriovenosas , Proteínas Proto-Oncogénicas p21(ras) , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Malformaciones Arteriovenosas/diagnóstico , Malformaciones Arteriovenosas/diagnóstico por imagen , Malformaciones Arteriovenosas/tratamiento farmacológico , Malformaciones Arteriovenosas/genética , Modelos Animales de Enfermedad , Mutación con Ganancia de Función , Mutación , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridinas/uso terapéutico , Pirimidinas , Fármacos Cardiovasculares/uso terapéutico , Adulto JovenRESUMEN
Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.
Asunto(s)
Muerte Celular , Hierro/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Ciclohexilaminas/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/citología , Humanos , Técnicas In Vitro , Metabolismo de los Lípidos , Neoplasias/patología , Fenilendiaminas/farmacología , Piperazinas/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.
Asunto(s)
Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Glicina en la Membrana Plasmática/química , Glicina/metabolismo , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Cristalografía , Humanos , Modelos Moleculares , Piperazinas/química , Piperazinas/farmacología , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Anticuerpos de Dominio Único , Sulfonas/química , Sulfonas/farmacología , SincrotronesRESUMEN
The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclina D/metabolismo , Adenocarcinoma del Pulmón/genética , Animales , División Celular , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Genes Supresores de Tumor , Humanos , Neoplasias Pulmonares/genética , Ratones , Piperazinas/farmacología , Piridinas/farmacología , Células U937 , UbiquitinaciónRESUMEN
Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/genética , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Acetonitrilos/farmacología , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular , Estudios de Cohortes , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Piperazinas/farmacología , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.
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Proteínas de Ciclo Celular/metabolismo , Resistencia a Antineoplásicos , Proteínas F-Box/metabolismo , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN , Resistencia a Antineoplásicos/genética , Proteínas F-Box/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones Endogámicos NOD , Ratones SCID , Fosforilación , Proteolisis , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The CDK4/6 inhibitor palbociclib blocks cell cycle progression in Estrogen receptor-positive, human epidermal growth factor 2 receptor-negative (ER+/HER2-) breast tumor cells. Despite the drug's success in improving patient outcomes, a small percentage of tumor cells continues to divide in the presence of palbociclib-a phenomenon we refer to as fractional resistance. It is critical to understand the cellular mechanisms underlying fractional resistance because the precise percentage of resistant cells in patient tissue is a strong predictor of clinical outcomes. Here, we hypothesize that fractional resistance arises from cell-to-cell differences in core cell cycle regulators that allow a subset of cells to escape CDK4/6 inhibitor therapy. We used multiplex, single-cell imaging to identify fractionally resistant cells in both cultured and primary breast tumor samples resected from patients. Resistant cells showed premature accumulation of multiple G1 regulators including E2F1, retinoblastoma protein, and CDK2, as well as enhanced sensitivity to pharmacological inhibition of CDK2 activity. Using trajectory inference approaches, we show how plasticity among cell cycle regulators gives rise to alternate cell cycle "paths" that allow individual tumor cells to escape palbociclib treatment. Understanding drivers of cell cycle plasticity, and how to eliminate resistant cell cycle paths, could lead to improved cancer therapies targeting fractionally resistant cells to improve patient outcomes.
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Neoplasias de la Mama , Piperazinas , Piridinas , Humanos , Femenino , Ciclo Celular , División Celular , Piperazinas/farmacología , Piperazinas/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
SLC7A11 is a cystine transporter and ferroptosis inhibitor. How the stability of SLC7A11 is coordinately regulated in response to environmental cystine by which E3 ligase and deubiquitylase (DUB) remains elusive. Here, we report that neddylation inhibitor MLN4924 increases cystine uptake by causing SLC7A11 accumulation, via inactivating Cullin-RING ligase-3 (CRL-3). We identified KCTD10 as the substrate-recognizing subunit of CRL-3 for SLC7A11 ubiquitylation, and USP18 as SLC7A11 deubiquitylase. Upon cystine deprivation, the protein levels of KCTD10 or USP18 are decreased or increased, respectively, contributing to SLC7A11 accumulation. By destabilizing or stabilizing SLC7A11, KCTD10, or USP18 inversely regulates the cystine uptake and ferroptosis. Biologically, MLN4924 combination with SLC7A11 inhibitor Imidazole Ketone Erastin (IKE) enhanced suppression of tumor growth. In human breast tumor tissues, SLC7A11 levels were negatively or positively correlated with KCTD10 or USP18, respectively. Collectively, our study defines how SLC7A11 and ferroptosis is coordinately regulated by the CRL3KCTD10/E3-USP18/DUB axis, and provides a sound rationale of drug combination to enhance anticancer efficacy.
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Cistina , Ferroptosis , Pirimidinas , Ubiquitina Tiolesterasa , Animales , Femenino , Humanos , Ratones , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ciclopentanos/metabolismo , Ciclopentanos/farmacología , Cistina/metabolismo , Células HEK293 , Piperazinas/farmacología , Pirimidinas/farmacología , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear whether APC/C maintains all types of arrest. Here, by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves CDKs acting in an atypical order to inactivate retinoblastoma-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability.
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Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Humanos , Quinasa 6 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/genética , Línea Celular Tumoral , Fase S/efectos de los fármacos , Piridinas/farmacología , Piperazinas/farmacología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Factores de Transcripción E2F/metabolismo , Factores de Transcripción E2F/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Ciclinas/metabolismo , Ciclinas/genética , Proteínas F-BoxRESUMEN
BACKGROUND: Adagrasib, an oral small-molecule inhibitor of mutant KRAS G12C protein, has shown clinical activity in pretreated patients with several tumor types, including colorectal cancer. Preclinical studies suggest that combining a KRAS G12C inhibitor with an epidermal growth factor receptor antibody could be an effective clinical strategy. METHODS: In this phase 1-2, open-label, nonrandomized clinical trial, we assigned heavily pretreated patients with metastatic colorectal cancer with mutant KRAS G12C to receive adagrasib monotherapy (600 mg orally twice daily) or adagrasib (at the same dose) in combination with intravenous cetuximab once a week (with an initial loading dose of 400 mg per square meter of body-surface area, followed by a dose of 250 mg per square meter) or every 2 weeks (with a dose of 500 mg per square meter). The primary end points were objective response (complete or partial response) and safety. RESULTS: As of June 16, 2022, a total of 44 patients had received adagrasib, and 32 had received combination therapy with adagrasib and cetuximab, with a median follow-up of 20.1 months and 17.5 months, respectively. In the monotherapy group (43 evaluable patients), a response was reported in 19% of the patients (95% confidence interval [CI], 8 to 33). The median response duration was 4.3 months (95% CI, 2.3 to 8.3), and the median progression-free survival was 5.6 months (95% CI, 4.1 to 8.3). In the combination-therapy group (28 evaluable patients), the response was 46% (95% CI, 28 to 66). The median response duration was 7.6 months (95% CI, 5.7 to not estimable), and the median progression-free survival was 6.9 months (95% CI, 5.4 to 8.1). The percentage of grade 3 or 4 treatment-related adverse events was 34% in the monotherapy group and 16% in the combination-therapy group. No grade 5 adverse events were observed. CONCLUSIONS: Adagrasib had antitumor activity in heavily pretreated patients with metastatic colorectal cancer with mutant KRAS G12C, both as oral monotherapy and in combination with cetuximab. The median response duration was more than 6 months in the combination-therapy group. Reversible adverse events were common in the two groups. (Funded by Mirati Therapeutics; KRYSTAL-1 ClinicalTrials.gov number, NCT03785249.).