RESUMEN
Hereditary defects in several genes have been shown to disturb the normal immune response to EBV and to give rise to severe EBV-induced lymphoproliferation in the recent years. Nevertheless, in many patients, the molecular basis of fatal EBV infection still remains unclear. The Fanconi anemia-associated protein 24 (FAAP24) plays a dual role in DNA repair. By association with FANCM as component of the FA core complex, it recruits the FA core complex to damaged DNA. Additionally, FAAP24 has been shown to evoke ATR-mediated checkpoint responses independently of the FA core complex. By whole exome sequencing, we identified a homozygous missense mutation in the FAAP24 gene (cC635T, pT212M) in two siblings of a consanguineous Turkish family who died from an EBV-associated lymphoproliferative disease after infection with a variant EBV strain, expressing a previously unknown EBNA2 allele.In order to analyze the functionality of the variant FAAP24 allele, we used herpes virus saimiri-transformed patient T cells to test endogenous cellular FAAP24 functions that are known to be important in DNA damage control. We saw an impaired FANCD2 monoubiquitination as well as delayed checkpoint responses, especially affecting CHK1 phosphorylation in patient samples in comparison to healthy controls. The phenotype of this FAAP24 mutation might have been further accelerated by an EBV strain that harbors an EBNA2 allele with enhanced activities compared to the prototype laboratory strain B95.8. This is the first report of an FAAP24 loss of function mutation found in human patients with EBV-associated lymphoproliferation.
Asunto(s)
Proteínas de Unión al ADN/genética , Trastornos Linfoproliferativos/diagnóstico , Trastornos Linfoproliferativos/genética , Mutación , Hermanos , Sustitución de Aminoácidos , Ciclo Celular , Codón , Consanguinidad , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Resultado Fatal , Femenino , Genotipo , Homocigoto , Humanos , Recuento de Linfocitos , Trastornos Linfoproliferativos/virología , Masculino , Linaje , Fenotipo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Intercambio de Cromátides Hermanas , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ubiquitinación , Secuenciación del ExomaAsunto(s)
Reparación del ADN , Mononucleosis Infecciosa/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Inmunodeficiencia Combinada Grave/genética , Adolescente , Ciclo Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/patología , Herpesvirus Humano 4/patogenicidad , Herpesvirus Humano 4/fisiología , Humanos , Mononucleosis Infecciosa/inmunología , Mononucleosis Infecciosa/patología , Masculino , Mitomicina/farmacología , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteína Recombinante y Reparadora de ADN Rad52/química , Proteína Recombinante y Reparadora de ADN Rad52/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/patologíaRESUMEN
ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches. Cancer Res; 77(16); 4365-77. ©2017 AACR.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Histona Demetilasas/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/microbiología , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genéticaRESUMEN
Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are not commonly used in clinical practice for treatment of B-cell lymphomas, although a subset of patients with refractory or relapsed B-cell lymphoma achieved partial or complete remissions. Therefore, the purpose of this study was to identify molecular features that predict the response of B-cell lymphomas to SAHA treatment. We designed an integrative approach combining drug efficacy testing with exome and captured target analysis (DETECT). In this study, we tested SAHA sensitivity in 26 B-cell lymphoma cell lines and determined SAHA-interacting proteins in SAHA resistant and sensitive cell lines employing a SAHA capture compound (CC) and mass spectrometry (CCMS). In addition, we performed exome mutation analysis. Candidate validation was done by expression analysis and knock-out experiments. An integrated network analysis revealed that the Src tyrosine kinase Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog (FGR) is associated with SAHA resistance. FGR was specifically captured by the SAHA-CC in resistant cells. In line with this observation, we found that FGR expression was significantly higher in SAHA resistant cell lines. As functional proof, CRISPR/Cas9 mediated FGR knock-out in resistant cells increased SAHA sensitivity. In silico analysis of B-cell lymphoma samples (n = 1200) showed a wide range of FGR expression indicating that FGR expression might help to stratify patients, which clinically benefit from SAHA therapy. In conclusion, our comprehensive analysis of SAHA-interacting proteins highlights FGR as a factor involved in SAHA resistance in B-cell lymphoma.