RESUMEN
We studied DNA methylation profiles in four different cell populations from a unique constellation of monozygotic triplets in whom two had developed Hodgkin Lymphoma (HL). We detected shared differences in DNA methylation signatures when comparing the two HL-affected triplets with the non-affected triplet. The differences were observed in naïve B-cells and marginal zone-like B-cells. DNA methylation differences were also detected when comparing each of the HL-affected triplets against each other. Even though we cannot determine whether treatment and/or disease triggered the observed differences, we believe our data are important on behalf of forthcoming studies, and that it might provide important clues for a better understanding of HL pathogenesis.
RESUMEN
The activating-mutation of JAK2V617F drives the development of myeloproliferative neoplasms (MPNs). Several JAK2 inhibitors such as ruxolitinib and gandotinib (LY2784544) currently in clinical trials and, provide improvements in MPNs including myelofibrosis. However, JAK2 inhibitors are non-curative and murine experiments show that JAK2 inhibitors don't eradicate MPN stem cells and it is currently unclear how they escape. We thus determined the effect of the specific JAK2V617F inhibitor LY2784544 on leukemic stem (CD34+) cells (LSCs) using the JAK2V617F-bearing erythroleukemia cell line HEL. The LY2784544 treatment caused a transient proliferation inhibition and apoptosis of HEL cells, but a recovery occurred within a week. Thereafter, the continuous LY2784544 exposure induced the accumulation of CD34+ LSCs, and the CD34+ cells increased from 2% to >90% by week 9, which was accompanied by increased clonogenic potentials. LY2784544 was capable of stimulating CD34 expression even in CD34- HEL cells, which indicated cellular de-differentiation. A significantly enhanced expression of the stem cell factor KLF4 was observed in LY2784544-treated HEL cells. Inhibiting KLF4 expression attenuated LY2784544-mediated accumulation of CD34+ LSCs. Moreover, the telomerase inhibitor GRN163L abolished the LY2784544-effect. JAK2 inhibitors thus cause enrichment of LSCs and are unlikely to cure MPN as a monotherapy. Simultaneously targeting JAK2V617F and KLF4 or telomerase may be a novel strategy for MPN therapy, which should be of significance both biologically and clinically.
Asunto(s)
Imidazoles/farmacología , Leucemia/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Oligonucleótidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridazinas/farmacología , Telomerasa/antagonistas & inhibidores , Antígenos CD34/análisis , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Factor 4 Similar a Kruppel , Leucemia/patología , Células Madre Neoplásicas/patologíaRESUMEN
Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Enfermedad de Hodgkin , Linfocitos B , Células Cultivadas , Herpesvirus Humano 4 , Enfermedad de Hodgkin/genética , HumanosRESUMEN
The original version of this article contained a mistake. The name of Magnus Björkhom should have been Magnus Björkholm.
RESUMEN
The somatic mutation of FLT3 occurs in 30% of acute myeloid leukemia (AML), with the majority of mutations exhibiting internal tandem duplication (ITD). On the other hand, the induction of telomerase reverse transcriptase (hTERT) and the activation of telomerase is a key step in AML development. Here, we sought to determine whether FLT3ITD regulates hTERT expression in AML cells and whether hTERT expression affects FLT3 inhibitors' therapeutic efficacy on AML. FLT3ITD-harboring AML cell lines and primary cells treated with the FLT3 inhibitor PKC412 displayed a rapid decline in the levels of hTERT mRNA and telomerase activity. Moreover, PKC412 inhibited hTERT gene transcription in a c-MYC-dependent manner. The ectopic expression of hTERT significantly attenuated the apoptotic effect of PKC412 on AML cells. Mechanistically, hTERT enhanced the activity of FLT3 downstream effectors or alternative RTK signaling, thereby enhancing AKT phosphorylation, in AML cells treated with PKC412. Collectively, PKC412 downregulates hTERT expression and telomerase activity in a MYC-dependent manner and this effect is required for its optimal anti-AML efficacy, while hTERT over-expression confers AML cells resistance to a targeted therapeutic agent PKC412. These findings suggest that the functional interplay between FLT3ITD and hTERT contributes to the AML pathogenesis and interferes with the efficacy of FLT3ITD-targeted therapy.
Asunto(s)
Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-myc/fisiología , Telomerasa/genética , Anciano , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Células HeLa , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , Resultado del Tratamiento , Células Tumorales Cultivadas , Adulto Joven , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidoresRESUMEN
The telomerase reverse transcriptase (TERT) gene rs2736100_C allele has recently been shown to be associated with an increased risk for myeloproliferative neoplasms (MPNs) among Caucasians. However, it is unknown if this association is present in other ethnical populations and whether rs2736100 allele frequencies mirror the incidence of MPNs in a population. Here we genotyped TERT rs2736100 variants in 126 Swedish and 101 Chinese MPN patients and their age-, sex-, and ethnically-matched healthy controls. Healthy Chinese adults had a higher frequency of the A allele and lower frequencies of the C allele compared to Swedish counterparts (57.4 vs 47.0 % for A, 42.6 vs 53.0 % for C, P = 0.006). Both Swedish and Chinese patients harbored significantly higher C allele frequency than their controls (62.7 vs 53.0 % and 57.4 vs 42.6 % for Swedish and Chinese, respectively, P = 0.004). Swedes and Chinese bearing the CC genotype had a significantly increased risk of MPN compared to AA carriers (OR = 2.47; 95 % CI: 1.33-4.57, P = 0.003, for Swedes, and OR = 3.45; 95 % CI: 1.52-7.85, P = 0.005, for Chinese). Further analyses showed that rs2736100_CC was associated with robustly enhanced risk in males only (CC vs AA, OR = 5.11; 95 % CI: 2.19-11.92, P < 0.0001). The CC-carrying MPN patients exhibited significantly higher TERT expression than patients with the AC genotype. Collectively, the rs2736100_C is a risk allele for MPNs in Swedish and Chinese males, and the lower incidence of MPNs in the Chinese population is correlated with a lower rs2736100_C risk allele frequency.
Asunto(s)
Pueblo Asiatico/genética , Trastornos Mieloproliferativos/genética , Polimorfismo de Nucleótido Simple , Telomerasa/genética , Población Blanca/genética , Anciano , Alelos , Estudios de Casos y Controles , China , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Trastornos Mieloproliferativos/etnología , Riesgo , Suecia , Homeostasis del TelómeroRESUMEN
The WNT receptors of the Frizzled family comprise ten mammalian isoforms, bind WNT proteins and mediate downstream signaling to regulate stem cell fate, neuronal differentiation, cell survival and more. WNT-induced signaling pathways are either ß-catenin-dependent or -independent, thereby dividing the 19 mammalian WNT proteins into two groups. So far hardly any quantitative, pharmacological information is available about WNT-FZD interaction profiles, affinities or mechanisms of signaling specification through distinct WNT/FZD pairings. This lack of knowledge originates from difficulties with WNT purification and a lack of suitable assays, such as ligand binding assays and FZD activity readouts. In order to minimize this gap, we employ fluorescence recovery after photobleaching (FRAP) to investigate WNT effects on the lateral mobility of FZD6-GFP in living cells. Pharmacological uncoupling of heterotrimeric G proteins by pertussis toxin and N-ethylmaleimide argues that changes in FZD6 mobility are related to putative precoupling of heterotrimeric Gi/o proteins to FZD6. We show that recombinant WNT-1, -2, 3A, -4, -5A, -7A, -9B and -10B affect FZD6 surface mobility and thus act on this receptor. WNT-5B and WNT-11, on the other hand, have no effect on FZD6 mobility and we conclude that they do not act through FZD6. We introduce here a novel way to assess WNT-FZD interaction by live cell imaging allowing further mapping of WNT-FZD interactions and challenging previous experimental limitations. Increased understanding of WNT-FZD selectivity provides important insight into the biological function of this crucial signaling system with importance in developmental biology, stem cell regulation oncogenesis, and human disease.
Asunto(s)
Membrana Celular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Receptores Frizzled/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas Wnt/metabolismo , Etilmaleimida/farmacología , Receptores Frizzled/agonistas , Receptores Frizzled/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HEK293 , Humanos , Toxina del Pertussis/toxicidad , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/genética , Proteína Wnt-5aRESUMEN
Microglia activation is central to the neuroinflammation associated with neurological and neurodegenerative diseases, particularly because activated microglia are often a source of proinflammatory cytokines. Despite decade-long research, the molecular cascade of proinflammatory transformation of microglia in vivo remains largely elusive. Here, we report increased ß-catenin expression, a central intracellular component of WNT signaling, in microglia undergoing a proinflammatory morphogenic transformation under pathogenic conditions associated with neuroinflammation such as Alzheimer's disease. We substantiate disease-associated ß-catenin signaling in microglia in vivo by showing age-dependent ß-catenin accumulation in mice with Alzheimer's-like pathology (APdE9). In cultured mouse microglia expressing the WNT receptors Frizzled FZD(4,5,7,8) and LDL receptor-related protein 5/6 (LRP5/6), we find that WNT-3A can stabilize ß-catenin. WNT-3A dose dependently induces LRP6 phosphorylation with downstream activation of disheveled, ß-catenin stabilization, and nuclear import. Gene-expression profiling reveals that WNT-3A stimulation specifically increases the expression of proinflammatory immune response genes in microglia and exacerbates the release of de novo IL-6, IL-12, and tumor necrosis factor α. In summary, our data suggest that the WNT family of lipoglycoproteins can instruct proinflammatory microglia transformation and emphasize the pathogenic significance of ß-catenin-signaling networks in this cell type.