RESUMEN
Importance: Multimodal generative artificial intelligence (AI) methodologies have the potential to optimize emergency department care by producing draft radiology reports from input images. Objective: To evaluate the accuracy and quality of AI-generated chest radiograph interpretations in the emergency department setting. Design, Setting, and Participants: This was a retrospective diagnostic study of 500 randomly sampled emergency department encounters at a tertiary care institution including chest radiographs interpreted by both a teleradiology service and on-site attending radiologist from January 2022 to January 2023. An AI interpretation was generated for each radiograph. The 3 radiograph interpretations were each rated in duplicate by 6 emergency department physicians using a 5-point Likert scale. Main Outcomes and Measures: The primary outcome was any difference in Likert scores between radiologist, AI, and teleradiology reports, using a cumulative link mixed model. Secondary analyses compared the probability of each report type containing no clinically significant discrepancy with further stratification by finding presence, using a logistic mixed-effects model. Physician comments on discrepancies were recorded. Results: A total of 500 ED studies were included from 500 unique patients with a mean (SD) age of 53.3 (21.6) years; 282 patients (56.4%) were female. There was a significant association of report type with ratings, with post hoc tests revealing significantly greater scores for AI (mean [SE] score, 3.22 [0.34]; P < .001) and radiologist (mean [SE] score, 3.34 [0.34]; P < .001) reports compared with teleradiology (mean [SE] score, 2.74 [0.34]) reports. AI and radiologist reports were not significantly different. On secondary analysis, there was no difference in the probability of no clinically significant discrepancy between the 3 report types. Further stratification of reports by presence of cardiomegaly, pulmonary edema, pleural effusion, infiltrate, pneumothorax, and support devices also yielded no difference in the probability of containing no clinically significant discrepancy between the report types. Conclusions and Relevance: In a representative sample of emergency department chest radiographs, results suggest that the generative AI model produced reports of similar clinical accuracy and textual quality to radiologist reports while providing higher textual quality than teleradiologist reports. Implementation of the model in the clinical workflow could enable timely alerts to life-threatening pathology while aiding imaging interpretation and documentation.
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Inteligencia Artificial , Servicios Médicos de Urgencia , Humanos , Femenino , Persona de Mediana Edad , Masculino , Estudios Retrospectivos , Servicio de Urgencia en Hospital , RadiólogosRESUMEN
The surfaceome is critical because surface proteins provide a gateway for internal signals and transfer of molecules into cells, and surfaceome differences can influence therapy response. We have used a surfaceome analysis method, based on comparing RNA-seq data between normal and abnormal cells (Surfaceome DataBase Mining or Surfaceome DBM), to identify sets of upregulated cell surface protein mRNAs in an LMO2-mediated T-ALL mouse model and corroborated by protein detection using antibodies. In this model the leukemia initiating cells (LICs) comprise pre-leukaemic, differentiation inhibited thymocytes allowing us to provide a profile of the LIC surfaceome in which GPR56, CD53 and CD59a are co-expressed with CD25. Implementation of cell surface interaction assays demonstrates fluid interaction of surface proteins and CD25 is only internalized when co-localized with other proteins. The Surfaceome DBM approach to analyse cancer cell surfaceomes is a way to find targetable surface biomarkers for clinical conditions where RNA-seq data from normal and abnormal cell are available.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas con Dominio LIM/metabolismo , Leucemia Linfoide/genética , Proteínas Proto-Oncogénicas/metabolismo , Transcriptoma , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Biomarcadores de Tumor/genética , Antígenos CD59/genética , Antígenos CD59/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Proteínas con Dominio LIM/genética , Leucemia Linfoide/metabolismo , Leucemia Linfoide/patología , Ratones , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas/genética , RNA-Seq , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Tetraspanina 25/genética , Tetraspanina 25/metabolismoRESUMEN
Heterogeneous upregulation of multiple prosurvival pathways underlies resistance to damage-induced apoptosis in acute lymphoblastic leukemia (ALL) cells despite normal p53 responses. Here, we show that the dual combination of insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1/R) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition using AG1024â¯+â¯U0126 can sensitize apoptosis-resistant ALL cells to ionizing radiation-induced DNA damage irrespective of effect of single pathway inhibition in vitro. This AG1024â¯+â¯U0126 combination also significantly potentiates the ability of the core chemotherapy compounds vincristine, dexamethasone, and daunorubicin to kill ALL cells in vitro. Evidence of the synergistic action of AG1024â¯+â¯U0126 in samples with variable basal levels of phosphorylated IGF1/Rß and ERK1/2 suggested additional targets of this drug combination. Consistent with this, gene expression profiling identified 32 "synergy genes" differentially targeted by IGF1/Râ¯+â¯MEK inhibition and, among these, Signal transducer and activator of transcription 6 (STAT6) and platelet-derived growth factor-associated protein 1 (PDAP1) were the most differentially downregulated cluster. Pearson correlation analysesrevealed that STAT6 and PDAP1 display significant expression codependency and a common expression pattern linked with other key "synergy" genes, supporting their predicted role in an STAT6-ERK-nuclear factor kappa beta (NF-κB) network. Knockdown studies revealed that loss of STAT6, but not PDAP1, impinges on the cell cycle, causing reduced numbers of viable cells. In combination with daunorubicin, STAT6 loss has an additive effect on cell killing, whereas PDAP1 loss is synergistic, indicating an important role of PDAP1 in the cellular response to this anthracycline. Inhibition of STAT6 or PDAP1 may therefore represent a potential novel therapeutic strategy for resistant ALL by enhancing sensitivity to chemotherapy.
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Butadienos/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/fisiología , Quinasa 1 de Quinasa de Quinasa MAP/antagonistas & inhibidores , Nitrilos/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Factor de Transcripción STAT6/fisiología , Tirfostinos/farmacología , Ciclo Celular/efectos de los fármacos , Cromonas/farmacología , Daunorrubicina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Morfolinas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Factor de Transcripción STAT6/biosíntesis , Factor de Transcripción STAT6/genética , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Vincristina/farmacologíaRESUMEN
Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL.
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Evolución Clonal/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Adolescente , Adulto , Alelos , Animales , Línea Celular Tumoral , Niño , Preescolar , Modelos Animales de Enfermedad , Estudio de Asociación del Genoma Completo , Xenoinjertos , Humanos , Hibridación Fluorescente in Situ , Lactante , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Proteínas de Fusión Oncogénica/metabolismo , Fosfohidrolasa PTEN/genética , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Análisis de la Célula Individual , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismo , Adulto JovenRESUMEN
INTRODUCTION: Emergency department (ED) crowding is associated with detrimental effects on ED quality of care. Triage liaison providers (TLP) have been used to mitigate the effects of crowding. Prior studies have evaluated attending physicians and advanced practice providers as TLPs, with limited data evaluating resident physicians as TLPs. This study compares operational performance outcomes between resident and attending physicians as TLPs. METHODS: This retrospective cohort study compared aggregate operational performance at an urban, academic ED during pre- and post-TLP periods. The primary outcome was defined as cost-effectiveness based upon return on investment (ROI). Secondary outcomes were defined as differences in median ED length of stay (LOS), median door-to-provider (DTP) time, proportion of left without being seen (LWBS), and proportion of "very good" overall patient satisfaction scores. RESULTS: Annual profit generated for physician-based collections through LWBS capture (after deducting respective salary costs) equated to a gain (ROI: 54%) for resident TLPs and a loss (ROI: -31%) for attending TLPs. Accounting for hospital-based collections made both profitable, with gains for resident TLPs (ROI: 317%) and for attending TLPs (ROI: 86%). Median DTP time for resident TLPs was significantly lower (p<0.0001) than attending or historical control. Proportion of "very good" patient satisfaction scores and LWBS was improved for both resident and attending TLPs over historical control. Overall median LOS was not significantly different. CONCLUSION: Resident and attending TLPs improved DTP time, patient satisfaction, and LWBS rates. Both resident and attending TLPs are cost effective, with residents having a more favorable financial profile.
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Centros Médicos Académicos/economía , Servicio de Urgencia en Hospital/economía , Internado y Residencia/economía , Triaje/economía , Centros Médicos Académicos/organización & administración , Centros Médicos Académicos/normas , Competencia Clínica , Análisis Costo-Beneficio , Aglomeración , Servicio de Urgencia en Hospital/organización & administración , Servicio de Urgencia en Hospital/normas , Humanos , Tiempo de Internación , Cuerpo Médico de Hospitales/economía , Pacientes Desistentes del Tratamiento , Satisfacción del Paciente , Estudios Retrospectivos , Factores de Tiempo , Triaje/organización & administración , Triaje/normas , Población Urbana , Flujo de Trabajo , Recursos HumanosRESUMEN
Prevention of central nervous system (CNS) relapse is critical for cure of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite this, mechanisms of CNS infiltration are poorly understood, and the timing, frequency, and properties of BCP-ALL blasts entering the CNS compartment are unknown. We investigated the CNS-engrafting potential of BCP-ALL cells xenotransplanted into immunodeficient NOD.Cg- ITALIC! Prkdc (ITALIC! scid) ITALIC! Il2rg (ITALIC! tm1Wjl)/SzJ mice. CNS engraftment was seen in 23 of 29 diagnostic samples (79%): 2 of 2 from patients with overt CNS disease and 21 of 27 from patients thought to be CNS negative by diagnostic lumbar puncture. Histologic findings mimic human pathology and demonstrate that leukemic cells transit the blood-cerebrospinal fluid barrier situated close to the dural sinuses, the site of recently discovered CNS lymphatics. Retrieval of blasts from the CNS showed no evidence for chemokine receptor-mediated selective trafficking. The high frequency of infiltration and lack of selective trafficking led us to postulate that CNS tropism is a generic property of leukemic cells. To test this, we performed serial dilution experiments which showed CNS engraftment in 5 of 6 mice after transplant of as few as 10 leukemic cells. Clonal tracking techniques confirmed the polyclonal nature of CNS-infiltrating cells, with multiple clones engrafting in both the CNS and periphery. Overall, these findings suggest that subclinical seeding of the CNS is likely to be present in most BCP-ALL patients at original diagnosis, and efforts to prevent CNS relapse should concentrate on effective eradication of disease from this site rather than targeting entry mechanisms.
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Barrera Hematoencefálica/patología , Movimiento Celular/fisiología , Sistema Nervioso Central/patología , Infiltración Leucémica/patología , Leucocitos/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Animales , Células Cultivadas , Neoplasias del Sistema Nervioso Central/líquido cefalorraquídeo , Neoplasias del Sistema Nervioso Central/secundario , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Trasplante de Neoplasias , Recurrencia , Trasplante HeterólogoRESUMEN
Inactivation of the Ataxia Telangiectasia Mutated gene in chronic lymphocytic leukemia results in resistance to p53-dependent apoptosis and inferior responses to treatment with DNA damaging agents. Hence, p53-independent strategies are required to target Ataxia Telangiectasia Mutated-deficient chronic lymphocytic leukemia. As Ataxia Telangiectasia Mutated has been implicated in redox homeostasis, we investigated the effect of the Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia genotype on cellular responses to oxidative stress with a view to therapeutic targeting. We found that in comparison to Ataxia Telangiectasia Mutated-wild type chronic lymphocytic leukemia, pro-oxidant treatment of Ataxia Telangiectasia Mutated-null cells led to reduced binding of NF-E2 p45-related factor-2 to antioxidant response elements and thus decreased expression of target genes. Furthermore, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia cells contained lower levels of antioxidants and elevated mitochondrial reactive oxygen species. Consequently, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia, but not tumors with 11q deletion or TP53 mutations, exhibited differentially increased sensitivity to pro-oxidants both in vitro and in vivo. We found that cell death was mediated by a p53- and caspase-independent mechanism associated with apoptosis inducing factor activity. Together, these data suggest that defective redox-homeostasis represents an attractive therapeutic target for Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , Homocigoto , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Mutación , Oxidantes/metabolismo , Fenotipo , Animales , Antioxidantes/metabolismo , Apoptosis , Caspasas/metabolismo , Modelos Animales de Enfermedad , Regulación Leucémica de la Expresión Génica , Humanos , Mitocondrias/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Elementos de Respuesta , Superóxidos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: Cluster randomized trials (CRTs) are increasingly used to evaluate quality improvement interventions aimed at health care providers. In trials testing emergency department (ED) interventions, migration of emergency physicians (EPs) between hospitals is an important concern, as contamination may affect both internal and external validity. We hypothesized that geographically isolating EDs would prevent migratory contamination in a CRT designed to increase ED delivery of tissue plasminogen activator (tPA) in stroke (the INSTINCT trial). METHODS: INSTINCT was a prospective, cluster randomized, controlled trial. Twenty-four Michigan community hospitals were randomly selected in matched pairs for study. Contamination was defined at the cluster level, with substantial contamination defined a priori as greater than 10% of EPs affected. Nonadherence, total crossover (contamination+nonadherence), migration distance, and characteristics were determined. RESULTS: Three hundred seven EPs were identified at all sites. Overall, 7 (2.3%) changed study sites. One moved between control sites, leaving 6 (2.0%) total crossovers. Of these, 2 (0.7%) moved from intervention to control (contamination); and 4 (1.3%) moved from control to intervention (nonadherence). Contamination was observed in 2 of 12 control sites, with 17% and 9% contamination of the total site EP workforce at follow-up, respectively. Average migration distance was 42 miles for all EPs moving in the study and 35 miles for EPs moving from intervention to control sites. CONCLUSION: The mobile nature of EPs should be considered in the design of quality improvement CRTs. Increased reporting of contamination in CRTs is encouraged to clarify thresholds and facilitate CRT design.
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Fibrinolíticos/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Activador de Tejido Plasminógeno/uso terapéutico , Adulto , Anciano , Análisis por Conglomerados , Servicio de Urgencia en Hospital/estadística & datos numéricos , Humanos , Masculino , Michigan , Persona de Mediana Edad , Médicos/estadística & datos numéricos , Proyectos de InvestigaciónRESUMEN
Ataxia telangiectasia patients, with constitutional bi-allelic ATM mutations, have a marked risk of lymphoid tumors and ATM mutation carriers have a smaller risk of cancer. Sporadic ATM mutations occur in 10-20% of chronic lymphocytic leukemia and are often associated with chromosome 11q deletions which cause loss of an ATM allele. The role of constitutional ATM mutations in the pathogenesis of chronic lymphocytic leukemia is unknown. Here we investigated the frequency of constitutional ATM mutations in either of two chronic lymphocytic leukemia cohorts, those with and without a chromosome 11q deletion. We found that in comparison to controls, constitutional pathogenic ATM mutations were increased in patients with chromosome 11q deletions (6 of 140 vs. 0 of 281, P = 0.001) but not in those without 11q deletions (2 of 178 vs. 0 of 281, P = 0.15). These results suggest that ATM germline heterozygosity does not play a role in chronic lymphocytic leukemia initiation but rather influences rapid disease progression through ATM loss.
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Alelos , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Mutación de Línea Germinal , Heterocigoto , Leucemia Linfocítica Crónica de Células B/genética , Pérdida de Heterocigocidad , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Anciano , Proteínas de la Ataxia Telangiectasia Mutada , Deleción Cromosómica , Cromosomas Humanos Par 11 , Humanos , Persona de Mediana Edad , Modelos BiológicosRESUMEN
The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.
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Antineoplásicos/uso terapéutico , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma de Células del Manto/tratamiento farmacológico , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Animales , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Daño del ADN/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Linfoma de Células del Manto/genética , Ratones , Ratones SCID , Mutación , Ftalazinas/farmacología , Piperazinas/farmacologíaRESUMEN
Recently a mouse model of T/natural killer acute lymphoblastic leukemia was used to assess global promoter methylation across the mouse genome using the restriction landmark genomic scanning technique. One of the methylated mouse genes identified in this way was Slit2. There are three mammalian SLIT genes (SLIT1, SLIT2, SLIT3), that belong to a highly conserved family of axon guidance molecules. We have previously demonstrated that SLIT2 is frequently inactivated in lung, breast, colorectal and glioma tumors by hypermethylation of a CpG island in its promoter region, whilst inactivating somatic mutations are rare. Furthermore, we demonstrated that SLIT2 acts as a tumor suppressor gene in breast and colorectal cancer cells. In this report we determined the methylation status of the SLIT2 gene in leukemias (CLL and ALL). SLIT2 was methylated in all ten leukemia cell lines analyzed (eight completely and two partially methylated). SLIT2 expression was restored after treating ALL lines with 5-aza-2dC. In primary ALL and CLL samples, SLIT2 was also frequently methylated, 58% (30/52) B-ALL; 83% (10/12) T-ALL and in 80% (24/30) CLL. Whilst DNA from peripheral blood and bone marrow from healthy control samples showed no SLIT2 methylation. Methylation results in leukemia cell lines and ALL and CLL primary samples were confirmed by direct sequencing of bisulfite modified DNA. Our results demonstrate that methylation of the SLIT2 5' CpG island is conserved between mice and humans, and therefore is likely to be of functional importance.
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Metilación de ADN , Péptidos y Proteínas de Señalización Intercelular/genética , Leucemia Linfocítica Crónica de Células B/genética , Proteínas del Tejido Nervioso/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Médula Ósea/metabolismo , Línea Celular Tumoral , Islas de CpG/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoRESUMEN
The molecular basis of different outcomes in pediatric acute lymphoblastic leukemia (ALL) remains poorly understood. We addressed the clinical significance and mechanisms behind in vitro cellular responses to ionizing radiation (IR)-induced DNA double-strand breaks in 74 pediatric patients with ALL. We found an apoptosis-resistant response in 36% of patients characterized by failure to cleave caspase-3, -7, -9, and PARP1 by 24 hours after IR and an apoptosis-sensitive response with the cleavage of the same substrates in the remaining 64% of leukemias. Resistance to IR in vitro was associated with poor early blast clearance at day 7 or 15 and persistent minimal residual disease (MRD) at day 28 of induction treatment. Global gene expression profiling revealed abnormal up-regulation of multiple prosurvival pathways in response to IR in apoptosis-resistant leukemias and differential posttranscriptional activation of the PI3-Akt pathway was observed in representative resistant cases. Importantly, pharmacologic inhibition of selected prosurvival pathways sensitized apoptosis-resistant ALL cells to IR in vitro. We suggest that abnormal prosurvival responses to DNA damage provide one of the mechanisms of primary resistance in ALL, and that they should be considered as therapeutic targets in children with aggressive disease.
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Roturas del ADN de Doble Cadena , Regulación Leucémica de la Expresión Génica , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Apoptosis/efectos de la radiación , Crisis Blástica/genética , Crisis Blástica/patología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Caspasa 9/metabolismo , Células Cultivadas , Niño , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Neoplasia Residual/genética , Neoplasia Residual/patología , Neoplasia Residual/terapia , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Proteínas Proto-Oncogénicas c-akt/metabolismo , Radiación Ionizante , Transducción de SeñalRESUMEN
B-cell chronic lymphocytic leukemia (B-CLL) is a clinically variable disease where mutations in DNA damage response genes ATM or TP53 affect the response to standard therapeutic agents. The in vitro cytotoxicity of a novel cyclin-dependent kinase inhibitor, CYC202, was evaluated in 26 B-CLLs, 11 with mutations in either the ATM or TP53 genes, and compared with that induced by ionizing radiation and fludarabine. CYC202 induced apoptosis within 24 hours of treatment in all 26 analyzed tumor samples independently of ATM and TP53 gene status, whereas 6 of 26 B-CLLs, mostly ATM mutant, showed marked in vitro resistance to fludarabine-induced apoptosis. Compared with B-CLLs, normal T and B lymphocytes treated with CYC202 displayed reduced and delayed apoptosis. Using global gene expression profiling, we found that CYC202 caused a significant down-regulation of genes involved in regulation of transcription, translation, survival, and DNA repair. Furthermore, induction of apoptosis by CYC202 was preceded by inhibition of RNA polymerase II phosphorylation, leading to down-regulation of several prosurvival proteins. We conclude that CYC202 is a potent inducer of apoptosis in B-CLL regardless of the functional status of the p53 pathway, and may be considered as a therapeutic agent to improve the outcome of resistant B-CLL tumors.
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Apoptosis/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Regulación hacia Abajo/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/patología , Purinas/farmacología , Transcripción Genética/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Evaluación de Medicamentos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Persona de Mediana Edad , Mutación , Proteínas Serina-Treonina Quinasas/genética , ARN Polimerasa II/metabolismo , Roscovitina , Transcripción Genética/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
To investigate possible causes of the variable response to treatment in pediatric B-precursor acute lymphoblastic leukemia (ALL) and to establish potential novel therapeutic targets, we used ionizing radiation (IR) exposure as a model of DNA damage formation to identify tumors with resistance to p53-dependent apoptosis. Twenty-one of 40 ALL tumors responded normally to IR, exhibiting accumulation of p53 and p21 proteins and cleavage of caspases 3, 7, and 9 and of PARP1. Nineteen tumors exhibited apoptotic resistance and lacked PARP1 and caspase cleavage; although 15 of these tumors had normal accumulation of p53 and p21 proteins, examples exhibited abnormal expression of TRAF5, TRAF6, and cIAP1 after IR, suggesting increased NF-kappaB prosurvival signaling as the mechanism of apoptotic resistance. The presence of a hyperactive PARP1 mutation in one tumor was consistent with such increased NF-kappaB activity. PARP1 inhibition restored p53-dependent apoptosis after IR in these leukemias by reducing NF-kappaB DNA binding and transcriptional activity. In the remaining 4 ALL tumors, apoptotic resistance was associated with a TP53 mutation or with defective activation of p53. We conclude that increased NF-kappaB prosurvival signaling is a frequent mechanism by which B-precursor ALL tumors develop apoptotic resistance to IR and that PARP1 inhibition may improve the DNA damage response of these leukemias.
Asunto(s)
Apoptosis/efectos de la radiación , Linfocitos B , FN-kappa B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Apoptosis/fisiología , Niño , Preescolar , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Daño del ADN , Perfilación de la Expresión Génica , Humanos , Lactante , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Radiación Ionizante , Transducción de Señal/inmunología , Transducción de Señal/efectos de la radiación , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The highly homologous Rac1 and Rac2 GTPases are co-expressed in cells of haematopoietic origin and are likely to show some functional redundancy. While disruption of the Rac2 gene in mice has provided insight into some of its functions, Rac1 null mice are embryonic lethal and only recently has conditional gene disruption been possible. Consequently, two articles1,2 have recently elucidated some overlapping and unique key roles of Rac1 and Rac2 in haematopoietic processes including specialized roles in innate and humoral immunity.
Asunto(s)
Hematopoyesis/fisiología , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Linfocitos B/metabolismo , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/metabolismo , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/genética , Proteína RCA2 de Unión a GTPRESUMEN
The ATM/p53-dependent DNA damage response pathway plays an important role in the progression of lymphoid tumors. Inactivation of the ATM or TP53 gene is frequent in B-cell lymphocytic leukemia (B-CLL) and leads to aggressive disease. Although the ATM and p53 pathways overlap, they are not congruent, and it is unclear how the mechanism of tumor progression differs between ATM- and p53-deficient tumors. Using microarray analysis of ATM-mutant, TP53-mutant, and ATM/TP53 wild-type B-CLLs, we show that after exposure to DNA damage transcriptional responses are entirely dependent on ATM function. The p53 proapoptotic responses comprise only a part of ATM-regulated transcription; additionally, ATM regulates prosurvival responses independently of p53. Consequently, the greater severity of the TP53-mutant B-CLLs compared with ATM-mutant B-CLLs is consistent with the additive effect of defective apoptotic and elevated survival responses after DNA damage in these tumors. We also show that transcription expression profiles of ATM-deficient, TP53-deficient, and wild-type B-CLLs are indistinguishable before irradiation. Therefore, damage-induced transcriptional fingerprinting can be used to stratify tumors according to their biologic differences and simultaneously identify potential targets for treating refractory tumors.