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Background: Cold agglutinin disease (CAD) is a rare autoimmune haemolytic anaemia mediated by the classical complement pathway (CP). Sutimlimab selectively targets complement C1s inhibiting classical CP activation. In CADENZA Part A (26-weeks), a placebo-controlled study in patients without recent transfusion history, sutimlimab reduced haemolysis, anaemia, and fatigue, and was generally well tolerated. Methods: The CADENZA study (NCT03347422) started in March 2018 (Part A) and completed in December 2021 (Part B). All patients in Part B were eligible to receive sutimlimab for up to 1 year after the last patient completed Part A. Efficacy and safety was assessed throughout Part B, until the last on-treatment visit with available assessment (LV), and after a 9-week washout. Findings: In total, 32/39 patients completed Part B; median treatment duration: 99 weeks. Similar sustained improvements in haemolysis, anaemia, and quality of life were observed in patients switching to sutimlimab and those continuing sutimlimab. Mean LV values for the combined group (ie, placebo-to-sutimlimab group and sutimlimab-to-sutimlimab group) improved from baseline for haemoglobin (≥11.0 g/dL on-treatment vs 9.3 g/dL at baseline), bilirubin (≤20.0 µmol/L on-treatment vs 35.0 µmol/L at baseline), and FACIT-Fatigue scores. Following a 9-week washout, inhibition of CP activity was reversed, and haemolytic markers approached baseline levels. Overall, sutimlimab was generally well tolerated throughout the study. No patients developed systemic lupus erythematosus or meningococcal infections. During the 9-week washout, most adverse events could be attributed to recurrence of underlying CAD. Interpretation: The CADENZA Part B results support the sustained efficacy and safety of sutimlimab for treatment of CAD; however, upon discontinuation disease activity reoccurs. Funding: Sanofi.
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Problem: The spread of mis- and disinformation on mobile and messaging apps during the COVID-19 pandemic not only fuelled anxieties and mistrust in health authorities but also undermined the effectiveness of the overall public health response. Context: Mobile and messaging apps help users stay informed and connected to their families, friends, colleagues and communities. However, during the COVID-19 pandemic, these apps were also one of the primary channels where mis- and disinformation were circulated. Action: Recognizing the importance of including mobile and messaging apps in risk communication and emergency response strategies, the World Health Organization (WHO) and some countries in the WHO Western Pacific Region independently piloted initiatives to reach messaging app users, meet their evolving information needs, and streamline health ministry communication. Outcome: The enhanced use of mobile and messaging apps enabled consistent and timely communication and improved coordination during the COVID-19 pandemic. Leveraging their features also helped identify and potentially fill crucial information gaps, mitigating the harms of mis- and disinformation and fostering stronger trust in health authorities. Discussion: The findings from the work carried out by WHO and countries in the Western Pacific Region identified some promising innovative communication interventions using mobile and messaging apps. While these interventions should be further explored and evaluated, they have demonstrated that interventions need to be proactive, flexible, and able to adapt to changes in mis- and disinformation content being shared through messaging apps.
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COVID-19 , Aplicaciones Móviles , Pandemias , SARS-CoV-2 , Humanos , COVID-19/prevención & control , COVID-19/epidemiología , Pandemias/prevención & control , Comunicación , Envío de Mensajes de Texto , Islas del Pacífico/epidemiología , Comunicación en Salud/métodosRESUMEN
BACKGROUND: Once-weekly efanesoctocog alfa provides high sustained factor VIII activity with superior bleeding prevention as compared with prestudy factor VIII prophylaxis in previously treated patients 12 years of age or older with severe hemophilia A. Data on outcomes of efanesoctocog alfa treatment in children younger than 12 years of age with severe hemophilia A are limited. METHODS: We conducted a phase 3, open-label study involving previously treated patients younger than 12 years of age with severe hemophilia A. Patients received prophylaxis with once-weekly efanesoctocog alfa (50 IU per kilogram of body weight) for 52 weeks. The primary end point was the occurrence of factor VIII inhibitors (neutralizing antibodies against factor VIII). Secondary end points included annualized rates of treated bleeding episodes, bleeding treatment, safety, and pharmacokinetics. RESULTS: A total of 74 male patients were enrolled (38 with an age of <6 years and 36 with an age of 6 to <12 years). No factor VIII inhibitors developed. Most adverse events were nonserious. No serious adverse events that were assessed by the investigator as being related to efanesoctocog alfa were reported. In the 73 patients treated according to the protocol, the median and model-based mean annualized bleeding rates were 0.00 (interquartile range, 0.00 to 1.02) and 0.61 (95% confidence interval, 0.42 to 0.90), respectively. A total of 47 patients (64%) had no treated bleeding episodes, 65 (88%) had no spontaneous bleeding episodes, and 61 (82%) had no episodes of bleeding into joints. A total of 41 of 43 bleeding episodes (95%) resolved with one injection of efanesoctocog alfa. Mean factor VIII activity at steady state was more than 40 IU per deciliter for 3 days and more than 10 IU per deciliter for almost 7 days after dose administration. The geometric mean terminal half-life was 40.0 hours. CONCLUSIONS: In children with severe hemophilia A, once-weekly prophylaxis with efanesoctocog alfa provided high sustained factor VIII activity in the normal to near-normal range (>40 IU per deciliter) for 3 days and more than 10 IU per deciliter for almost 7 days after administration, leading to effective bleeding prevention. Efanesoctocog alfa was associated with mainly nonserious adverse events. (Funded by Sanofi and Sobi; XTEND-Kids ClinicalTrials.gov number, NCT04759131.).
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Factor VIII , Hemofilia A , Hemorragia , Proteínas Recombinantes , Niño , Preescolar , Humanos , Lactante , Masculino , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Esquema de Medicación , Factor VIII/administración & dosificación , Factor VIII/efectos adversos , Factor VIII/inmunología , Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Hemofilia A/complicaciones , Hemorragia/etiología , Hemorragia/prevención & control , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Artropatías/etiología , Calidad de VidaRESUMEN
Two-dimensional (2D) ferromagnets have attracted significant interest for their potential in spintronic device miniaturization, especially since the discovery of ferromagnetic ordering in monolayer materials such as CrI3 and Fe3GeTe2 in 2017. This study presents a detailed investigation into the effects of the Hubbard U parameter, biaxial strain, and structural distortions on the magnetic characteristics of Tâ³-phase VTe2. We demonstrate that setting the Hubbard U to 0 eV provides an accurate representation of the observed structural, magnetic, and electronic features for both bulk and monolayer Tâ³-phase VTe2. The application of strain reveals two distinct ferromagnetic states in the monolayer Tâ³-phase VTe2, each characterized by minor structural differences, but notably different magnetic moments. The Tâ³-1 state, with reduced magnetic moments, emerges under compressive strain, while the Tâ³-2 state, featuring increased magnetic moments, develops under tensile strain. Our analysis also compares the magnetic anisotropy between the T and Tâ³ phases of VTe2, highlighting that the periodic lattice distortion in the Tâ³-phase induces an in-plane anisotropy, which makes it a material with an easy-axis of magnetization. Monte Carlo simulations corroborate our findings, indicating a high Curie temperature of approximately 191 K for the Tâ³-phase VTe2. Our research not only sheds light on the critical aspects of the VTe2 system but also suggests new pathways for enhancing low-dimensional magnetism, contributing to the advancement of spintronics and straintronics.
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Problem: Communication is an integral component of an emergency response, including to the coronavirus disease (COVID-19) pandemic. Designing effective communication requires systematic measurement, evaluation and learning. Context: In the Western Pacific Region, the World Health Organization (WHO) responded to the COVID-19 pandemic by using the Communication for Health (C4H) approach. This included the development and application of a robust measurement, evaluation and learning (MEL) framework to assess the effectiveness of COVID-19 communication, and to share and apply lessons in real time to continuously strengthen the pandemic response. Action: MEL was applied during the planning, implementation and summative evaluation phases of COVID-19 communication, with evidence-based insights and recommendations continuously integrated in succeeding phases of the COVID-19 response. Lessons learned: This article captures good practices that helped WHO to implement MEL during the COVID-19 pandemic. It focuses on lessons from the evaluation process, including the importance of planning, data integration, collaboration, partnerships, piggybacking, using existing data and leveraging digital media. Discussion: Despite some limitations, the systematic application of MEL to COVID-19 communication shows its value in the planning and implementation of effective, evidence-based communication to address public health challenges. It enables the evaluation of outcomes and reflection on lessons identified to strengthen the response to the current pandemic and future emergencies.
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COVID-19 , Internet , Humanos , Pandemias/prevención & control , COVID-19/epidemiología , Comunicación , Salud PúblicaRESUMEN
Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.
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Replicación del ADN , Neoplasias , Humanos , Replicación del ADN/genética , ADN Helicasas/metabolismo , Repeticiones de Microsatélite , Daño del ADN , Neoplasias/tratamiento farmacológico , Neoplasias/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Helicasa del Síndrome de Werner/genética , Helicasa del Síndrome de Werner/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismoRESUMEN
Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN, knowledge that would be helpful for informing clinical development of WRN-targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system wherein the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We find that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we find no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provided the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggested a potential therapeutical rationale for dual targeting of WRN and ATR.
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Sutimlimab, a humanized monoclonal antibody targeting the classic complement pathway, is approved in the United States, Japan, and the European Union for the treatment of hemolytic anemia in adults with cold agglutinin disease. The objectives of this study were to support dose selection for phase 3 studies, assess dose recommendations, and establish the relationship between sutimlimab exposure and clinical outcome [hemoglobin (Hb) levels]. Clinically meaningful biomarkers were graphically analyzed and the exposure-response relationship was proposed. The pharmacokinetic (PK) characteristics of sutimlimab were best described by a two-compartment model with parallel linear and nonlinear clearance terms. Body weight was a significant covariate for the volume of distribution in the central compartment (Vc) and total body clearance of sutimlimab. Ethnicity (Japanese, non-Japanese) was a covariate on Vc and maximal nonlinear clearance. There were no PK differences between healthy participants and patients. After graphical exposure-response analysis for biomarkers, a pharmacokinetic-pharmacodynamic model was developed by integrating an indirect response/turnover model for Hb with a maximum effect (Emax) model, relating the Hb-elevating effect of sutimlimab to plasma exposure. Renal function and occurrence of blood transfusion were identified as covariates on Hb change from baseline. Simulations showed that Emax was attained with the approved dosing (6.5 g in patients <75 kg and 7.5 g in patients ≥75 kg), independent of covariate characteristics, and provided adequate sutimlimab exposure to maximize effects on Hb, bilirubin, and total complement component C4 levels. A change in Hb from baseline at steady state of 2.2 g/dl was projected, consistent with phase 3 study observations. SIGNIFICANCE STATEMENT: The final validated population pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) models confirm that the approved dosing regimen for sutimlimab (6.5 g in patients <75 kg and 7.5 g in patients ≥75 kg) is sufficient, without the need for further dose adjustments in populations of patients with cold agglutinin disease.
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Anemia Hemolítica Autoinmune , Adulto , Humanos , Anemia Hemolítica Autoinmune/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Biomarcadores , Peso CorporalRESUMEN
SAR445088 is an anti-C1s humanized monoclonal antibody that inhibits activated C1s in the proximal portion of the classical complement system and has the potential to provide clinical benefit in the treatment of complement-mediated diseases. A phase I, first-in-human, double-blind, randomized, placebo-controlled, dose-escalation trial of single and multiple doses of SAR445088 was conducted in 93 healthy participants to evaluate the safety, tolerability, and pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Single (intravenous [i.v.] and subcutaneous [s.c.]) ascending doses (SAD) and multiple (s.c.) ascending doses (MAD) of SAR445088 were well-tolerated. The PK of SAR445088 was characterized by slow absorption after the s.c. dose and a long half-life (mean terminal half-life [t1/2 ] 8-15 weeks). Two PD assays were used to measure inhibition of the classical complement pathway (CP): Wieslab CP and complement mediated hemolytic capacity (CH50). The estimated half-maximal inhibitory concentration (IC50 ) and 90% inhibitory concentration (IC90 ) for the Wieslab CP assay were 96.4 and 458 µg/ml, respectively, and 16.6 and 57.0 µg/ml, respectively, for the CH50 assay. In summary, SAR445088 was well-tolerated and had favorable PK and PD profiles after SAD (i.v. or s.c.) and MAD (s.c.) in humans. These findings warrant further clinical investigations in patients with classical complement-mediated disorders.
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Anticuerpos Monoclonales Humanizados , Vía Clásica del Complemento , Humanos , Administración Intravenosa , Método Doble Ciego , Anticuerpos Monoclonales Humanizados/farmacocinética , Relación Dosis-Respuesta a Droga , Voluntarios SanosRESUMEN
Neurons harbor high levels of single-strand DNA breaks (SSBs) that are targeted to neuronal enhancers, but the source of this endogenous damage remains unclear. Using two systems of postmitotic lineage specification-induced pluripotent stem cell-derived neurons and transdifferentiated macrophages-we show that thymidine DNA glycosylase (TDG)-driven excision of methylcytosines oxidized with ten-eleven translocation enzymes (TET) is a source of SSBs. Although macrophage differentiation favors short-patch base excision repair to fill in single-nucleotide gaps, neurons also frequently use the long-patch subpathway. Disrupting this gap-filling process using anti-neoplastic cytosine analogs triggers a DNA damage response and neuronal cell death, which is dependent on TDG. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage, a process that normally safeguards cell identity but can also provoke neurotoxicity after anticancer treatments.
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Roturas del ADN de Cadena Simple , Desmetilación del ADN , Reparación del ADN , Elementos de Facilitación Genéticos , Células Madre Pluripotentes Inducidas , Neuronas , Timina ADN Glicosilasa , Diferenciación Celular , Neuronas/enzimología , 5-Metilcitosina/metabolismo , Humanos , Transdiferenciación CelularRESUMEN
Sutimlimab, a first-in-class humanized immunoglobulin G4 (IgG4) monoclonal antibody that selectively inhibits the classical complement pathway at C1s, rapidly halted hemolysis in the single-arm CARDINAL study in recently transfused patients with cold agglutinin disease (CAD). CADENZA was a 26-week randomized, placebo-controlled phase 3 study to assess safety and efficacy of sutimlimab in patients with CAD without recent (within 6 months prior to enrollment) transfusion history. Forty-two patients with screening hemoglobin ≤10 g/dL, elevated bilirubin, and ≥1 CAD symptom received sutimlimab (n = 22) or placebo (n = 20) on days 0 and 7 and then biweekly. Composite primary endpoint criteria (hemoglobin increase ≥1.5 g/dL at treatment assessment timepoint [mean of weeks 23, 25, 26], avoidance of transfusion, and study-prohibited CAD therapy [weeks 5-26]) were met by 16 patients (73%) on sutimlimab, and 3 patients (15%) on placebo (odds ratio, 15.9 [95% confidence interval, 2.9, 88.0; P < .001]). Sutimlimab, but not placebo, significantly increased mean hemoglobin and FACIT-Fatigue scores at treatment assessment timepoint. Sutimlimab normalized mean bilirubin by week 1. Improvements correlated with near-complete inhibition of the classical complement pathway (2.3% mean activity at week 1) and C4 normalization. Twenty-one (96%) sutimlimab patients and 20 (100%) placebo patients experienced ≥1 treatment-emergent adverse event. Headache, hypertension, rhinitis, Raynaud phenomenon, and acrocyanosis were more frequent with sutimlimab vs placebo, with a difference of ≥3 patients between groups. Three sutimlimab patients discontinued owing to adverse events; no placebo patients discontinued. These data demonstrate that sutimlimab has potential to be an important advancement in the treatment of CAD. This trial was registered at www.clinicaltrials.gov as #NCT03347422.
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Anemia Hemolítica Autoinmune , Anticuerpos Monoclonales Humanizados , Anemia Hemolítica Autoinmune/sangre , Anemia Hemolítica Autoinmune/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Bilirrubina/sangre , Método Doble Ciego , Hemoglobinas/análisis , Humanos , Resultado del TratamientoRESUMEN
The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.
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Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Reparación del ADN/genética , Distonía/genética , Femenino , Factor C1 de la Célula Huésped/metabolismo , Proteínas Mad2/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Factor de Transcripción YY1/metabolismoRESUMEN
Recombinant factor VIII Fc fusion protein (rFVIIIFc) has been indicated for adults and children with hemophilia A. The objective of this article was to build a population pharmacokinetic (PK) model using adult and pediatric data sets and explore relevant dosing scenarios across all ages. The activity-time profiles of rFVIIIFc from 3 clinical studies (all trials registered at https://www.clinicaltrials.gov: NCT01027377, NCT01181128, and NCT01458106) were characterized, and covariates that determine variability of rFVIIIFc PK in children and adults were identified and implemented. Data sets were pooled to estimate population PK parameters. Simulations were conducted to generate activity-time profiles at steady state (SS). The proportion of subjects maintaining SS trough >1 and >3 IU/dL and time >10 IU/dL were estimated. The rFVIIIFc model was a two-compartment model that identified weight and von Willebrand factor as significant covariates. Model-predicted SS peaks and troughs of rFVIIIFc activity-time profiles confirmed the necessity of modifying dosing in pediatric subjects. The model also predicted that the average subject in the adult and adolescent group dosed with 40 IU/kg every 2 days maintained factor VIII activity >10 IU/dL for the entire duration. Children aged <6 years and aged 6 to <12 years receiving this dose maintained factor VIII activity of >10 IU/dL for nearly two-thirds and three-quarters of their time, respectively. In conclusion, these population PK analyses characterize activity-time profiles for rFVIIIFc among pediatric and adult subjects. The model was used for simulation of clinically relevant dosing scenarios, which can provide better protection and better clinical outcomes.
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Factor VIII/farmacocinética , Hemofilia A/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacocinética , Adolescente , Factores de Edad , Peso Corporal , Niño , Simulación por Computador , Cálculo de Dosificación de Drogas , Factor VIII/administración & dosificación , Semivida , Humanos , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Masculino , Tasa de Depuración Metabólica , Modelos Biológicos , Gravedad del Paciente , Proteínas Recombinantes de Fusión/administración & dosificación , Factor de von Willebrand/efectos de los fármacosRESUMEN
DNA double-strand breaks (DSBs) represent the most toxic form of DNA damage and can arise in either physiological or pathological conditions. If left unrepaired, these DSBs can lead to genome instability which serves as a major driver to tumorigenesis and other pathologies. Consequently, localizing DSBs and understanding the dynamics of break formation and the repair process are of great interest for dissecting underlying mechanisms and in the development of targeted therapies. Here, we describe END-seq, a highly sensitive next-generation sequencing technique for quantitatively mapping DNA double-strand breaks (DSB) at nucleotide resolution across the genome in an unbiased manner. END-seq is based on the direct ligation of a sequencing adapter to the ends of DSBs and provides information about DNA processing (end resection) at DSBs, a critical determinant in the selection of repair pathways. The absence of cell fixation and the use of agarose for embedding cells and exonucleases for blunting the ends of DSBs are key advances that contribute to the technique's increased sensitivity and robustness over previously established methods. Overall, END-seq has provided a major technical advance for mapping DSBs and has also helped inform the biology of complex biological processes including genome organization, replication fork collapse and chromosome fragility, off-target identification of RAG recombinase and gene-editing nucleases, and DNA end resection at sites of DSBs.
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Biología Computacional/métodos , Roturas del ADN de Doble Cadena , Reparación del ADN , Edición Génica/métodos , Desoxirribonucleasas/metabolismo , Exonucleasas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Programas Informáticos , Secuenciación Completa del GenomaRESUMEN
The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.
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Roturas del ADN de Doble Cadena , Expansión de las Repeticiones de ADN/genética , Repeticiones de Dinucleótido/genética , Neoplasias/genética , Helicasa del Síndrome de Werner/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Cromosomas Humanos/genética , Cromosomas Humanos/metabolismo , Cromotripsis , División del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Inestabilidad Genómica , Humanos , Recombinasas/metabolismoRESUMEN
Topoisomerase II (TOP2) relieves topological stress in DNA by introducing double-strand breaks (DSBs) via a transient, covalently linked TOP2 DNA-protein intermediate, termed TOP2 cleavage complex (TOP2cc). TOP2ccs are normally rapidly reversible, but can be stabilized by TOP2 poisons, such as the chemotherapeutic agent etoposide (ETO). TOP2 poisons have shown significant variability in their therapeutic effectiveness across different cancers for reasons that remain to be determined. One potential explanation for the differential cellular response to these drugs is in the manner by which cells process TOP2ccs. Cells are thought to remove TOP2ccs primarily by proteolytic degradation followed by DNA DSB repair. Here, we show that proteasome-mediated repair of TOP2cc is highly error-prone. Pre-treating primary splenic mouse B-cells with proteasome inhibitors prevented the proteolytic processing of trapped TOP2ccs, suppressed the DNA damage response (DDR) and completely protected cells from ETO-induced genome instability, thereby preserving cellular viability. When degradation of TOP2cc was suppressed, the TOP2 enzyme uncoupled itself from the DNA following ETO washout, in an error-free manner. This suggests a potential mechanism of developing resistance to topoisomerase poisons by ensuring rapid TOP2cc reversal.
Molecules of DNA contain the archive of a cell's genetic information and identity. DNA comprises two strands that twist together into a structure known as a double helix. Physical tension tends to build up in the double helix that can cause it to break apart. To avoid this, cells have an enzyme called Topoisomerase II (TOP2) that relieves the tension by attaching itself to DNA and breaking it in a controlled way before re-sealing the break. Drugs known as TOP2 poisons stop TOP2 from working and trap it on the DNA, which may lead to cells accumulating DNA breaks and eventually dying. Cancer cells are particularly prone to acquiring breaks in their DNA, and TOP2 poisons are therefore often used as part of chemotherapy treatments for cancer. However, it remains unclear why TOP2 poisons are more effective at killing some types of cancer cells than others. It is thought that a molecular machine, known as the proteasome, helps cells repair the damage caused by TOP2 poisons by removing the trapped TOP2 proteins and allowing DNA repair proteins access to the broken DNA underneath. Now, Sciascia et al. have used a genetic approach to study the relationship between the proteasome and DNA repair in mouse cells exposed to TOP2 poisons. The experiments found that when the proteasome removed TOP2 proteins that had become trapped on DNA, the subsequent DNA repair was prone to errors. Pre-treating mouse cells with another drug that inhibited the proteasome protected the cells from the effects of the TOP2 poison. Once the TOP2 poison had left the cells, the previously trapped TOP2 proteins correctly fixed the DNA and detached as they would normally. As a result, cells that had been treated with a proteasome inhibitor were more likely to survive treatment with TOP2 poisons. Since both TOP2 poisons and proteasome inhibitors are clinically approved drugs for treating cancer they can be, and already have been, tested for use together in combination drug therapies. However, these findings suggest that caution should be taken when using these drugs together, because instead of harming the cancer cells, the proteasome inhibitors may protect the cells from the toxic effects of TOP2 poisons.
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ADN-Topoisomerasas de Tipo II/metabolismo , ADN/metabolismo , Genoma , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Daño del ADN , Reparación del ADN , Genoma/genética , Humanos , Ratones Endogámicos C57BL , ProteolisisRESUMEN
53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.
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Recombinación Homóloga/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Envejecimiento/efectos de los fármacos , Envejecimiento/genética , Animales , Proteína BRCA1/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Recombinación Homóloga/efectos de los fármacos , Ratones , Mutación/efectos de los fármacos , Mutación/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN-Topoisomerasas de Tipo II/química , ADN/genética , Complejos Multiproteicos/química , Proteínas de Unión a Poli-ADP-Ribosa/química , Rotura Cromosómica , Cromosomas/genética , ADN/química , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Etopósido/química , Conversión Génica/genética , Células HCT116 , Humanos , Cinética , Complejos Multiproteicos/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Torsión Mecánica , Transcripción Genética , Translocación Genética/genéticaRESUMEN
BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.
Asunto(s)
Proteína BRCA1/genética , Cromatina/enzimología , Fibroblastos/enzimología , Haploinsuficiencia , Neoplasias/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Proteína BRCA2/genética , Línea Celular Tumoral , Cromatina/genética , Daño del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.