Littérature scientifique sur le sujet « Ink/Arf »

Abstract GATA-3 plays a crucial role in T cell development, however its role and collaboration with the Notch signaling pathway in the induction of T cell specification and commitment have not been fully elucidated. Work in our laboratory using both, in vitro and in vivo models of T cell development, has shown that GATA-3 deficiency arrests this development at the DN2 stage but does not result in B-lymphopoiesis in the presence of Notch ligand Delta-like 1 (Dll1). These GATA-3-/- progenitors express T-lineage associated genes but are unable to survive the T-specification event as indicated by their increased rate of apoptosis. Cell cycle regulation genes, mainly p19Arf and p16Ink4a (Arf/Ink), are upregulated in these cells as indicated by gene expression microarray and qRT-PCR analyses. We have thus sought to address the possibility that GATA-3 represses Arf/Ink following T cell specification/commitment in the presence of Notch signals. We are employing ChIP assay to show whether potential GATA-3 recognition sequences present in the Arf/Ink promoter region are occupied and regulated by GATA-3. Finally, using GATA-3-/-Arf-Ink-/- bone marrow progenitors, we will address a possible rescue of the early T cell developmental block observed in the absence of GATA-3. These experiments will help to identify a potential mechanism of how GATA-3 and the Notch signaling pathway collaborate in regulating T-lineage specification, commitment and survival during T cell development.

Abstract The p53 protein exerts a significant role in growth control of cells and impaired function of p53 by mutation or otherwise is regarded to be important for development of many human cancers. The p53 protein is strongly regulated by the E3-ligase HDM-2 that specifically binds to p53 and causes proteosomal degradation. P14ARF encoded from the INK 4a/ARF gene locus of chromosome 9p, activates the p53 pathway by binding to and inhibiting HDM-2. The objective was to study if the level of mRNA for p14ARF in leukemic cells from patients with AML was related to in vitro drug resistance and clinical outcome. Leukemic cells (>90% pure) isolated from 46 adult patients with normal karyotype de novo AML were incubated with antileukemic drugs (daunorubicin, mitoxantrone, etoposide and Ara-C) cultured for 4 days. The effect was determined by bioluminiscence measuring of intracellular ATP compared to an untreated control. We also tested the activity against PRIMA (p53-dependent reactivation and induction of massive apoptosis), a novel, small molecule shown to activate down regulated p53. mRNA for p14ARF was determined by real time PCR. Results: Patients whose leukemic cells expressed high level of p14ARF mRNA in the leukemic cells (≥0.2 compared to the housekeeping gene) had a significantly better survival compared to those with low level (<0.02). Median survival not obtained compared to 9 months. The mean activity of all tested conventional antileukemic drugs was higher on leukemic cell samples expressing p14ARF mRNA ≥0.2 compared to those with low levels. In contrast, PRIMA exerted significantly higher in vitro effect on leukemic cell samples with low levels of p14ARF mRNA. We conclude that low levels of p14ARF in leukemic cells from patients with normal karyotype AML is a marker for poor prognosis, which may depend on impaired p53 activity causing resistance against conventional antileukemic drugs. Treatment with p53 targeting drugs as PRIMA may be a future possibility to improve the outcome for these patients.

Abstract Abstract 5049 Expression of Proteins P16INK 4a, P53 and Bmi-1 in Hematopoietic Stem Cells of Patients with MDS. The Role of Cellular Senescence. Irene Constandinidou1, Eleni Kalivioti1,Vasilios Fertakis1, Costas Dallas1, Polyxeni Lampropoulou1, Evangelia Tzouvara1, Panagiotis Zoumboulis2, Nicholas Zoumbos.1 1Hematology Division, University of Patras, Medical School, Patras, Greece 2Orthopaedic Division, University Hospital of Patras, Patras, Greece Introduction The myelodysplastic syndromes (MDS) comprise a spectrum of heterogeneous clonal stem/progenitor cell disorders characterized by marrow failure. One potential reason, apart from apoptosis, for hematopoietic stem cells (HSCs) functional failure is cellular senescence, which is believed to have evolved as a tumor suppressor mechanism capable of arresting growth to reduce risk of malignancy. It can be activated by both telomere-dependent (telomere shortening) and telomere-independent pathways (DNA damage, oncogenic or oxidative stress). There are two widely recognized major tumor suppressor pathways p16/pRb and p53-p14/ARF that regulate cellular senescence. Activation of either pathway is profoundly associated with cellular senescence. Bmi-1, on the other hand, is a transcriptional repressor that plays an essential role in the self-renewal of HSCs and leukemic stem cells as well. One of its major targets is the INK/ARF locus, which encodes p16 and p53 independently. Aim To study the role of cellular senescence in the pathogenesis of MDS through the expression of p16, p53, phospho p53 (activated form of p53, phosphorylated at Ser-15) and bmi-1. Patients and methods We examined the expression level of p16INK4a, Bmi-1, p53 and phospho p53 by flow cytometry (direct and indirect staining) in CD34+ bone marrow mononuclear cells (BMMNCs). Furthermore, we analyzed the relative telomere length (RTL) in BMMNCs by quantitative fluorescence in situ hybridization assay using flow cytometry (flow-FISH).We verified the presence of p16 by SDS PAGE western blotting. We studied 36 samples of MDS patients (11 RA, 6 RARS, 3 RCMD, 4 5q- and 12 RAEB I-II), 17 samples of age-matched healthy controls after informed consent and 8 cord blood samples. All MDS diagnoses were histologically confirmed by bone marrow examination and categorized into low and high risk according to the international prognostic scoring system (IPSS). Results Expression of p16 is significantly increased in low risk MDS (mean± SD=8,7±12) when compared to healthy controls (p=0,02) and high risk patients(p=0,004). Patients with 5q- syndrome express lower level of p16 in comparison with the other low risk group patients. Increased level of bmi-1 expression is noticed in high risk group (mean± SD=30,3 ±33,9) when compared to low risk group (mean± SD=25±24,6) and healthy controls (mean± SD=18 ±16,4), while 5q- syndrome patients appear to express higher level of bmi-1 than any other risk group. Significantly increased expression of p53 (p=0,03) is noticed in high risk group (mean ±SD=64,3±27) when compared to low risk (mean±SD=34,2±28,8). Patients with 5q- syndrome express increased level of p53 (mean± SD=57,4±26,3) similar to that of high risk group. Expression of phospho p53 seams increased (p=0,055) in low risk group(mean± SD=7,1± 13,9) in comparison to high risk group (mean± SD=0,09± 0,18) and healthy controls (mean± SD=0,19± 0,63). Phospho p53 is expressed in lower level in 5q- syndrome patients. Regarding the RTL there is a significant difference (p=0,0018) between MDS (mean± SD=9,7± 2,7) and cord blood samples (mean± SD=13,8± 1,9). Conclusions Increased levels of p16 and phospho p53 in low risk MDS suggest that cellular senescence may contribute to the ineffective hematopoiesis of MDS and probably in a telomere independent way. Low expression level of of phospho p53 in high risk MDS raises the question of genetic integrity of p53 in this group of patients and when combined with high levels of bmi-1 and low expression of p16 may play a role in the disease progression to AML. Further investigation of bmi-1, especially in the 5q- group - and other markers of cellular senescence (SA- b galactosidase, SAHF) is needed in order to clarify the impact of cellular senescence in different risk groups of MDS. Disclosures No relevant conflicts of interest to declare.

Abstract p15INK4b is a cyclin-dependent kinase inhibitor known to regulate the G1-to-S transition of the cell cycle and to be involved in negatively regulating myeloid progenitor cell production. DNA hypermethylation leading to transcriptional silencing of p15INK4b has been reported in up to 70% of acute myeloid leukemia (AML) patient samples. In our study we sought to determine if p15INK4b DNA methylation in AML is accompanied by repressive histone modifications that contribute to the transcriptional repression of the gene at the chromatin level. Chromatin immunoprecipitation and DNA tiling microarrays (ChIP-on-chip) with 20bp resolution were utilized to assess the distribution of histone modifications over a 1.2 megabase region of human chromosome 9 including p15INK4b and adjacent tumor suppressor genes p14ARF and p16INK4a. We found that AML cell lines with p15INK4b hypermethylation (Kasumi-1, KG-1, and KG-1a) had high levels of the repressive histone modification trimethylation of lysine 27 of histone H3 (H3K27me3). Remarkably, this modification spanned the entire INK4b-ARF-INK4a region while little binding was observed in adjacent regions of chromosome 9. Binding of EZH2, the polycomb associated H3K27 histone methyltransferase, co-localized with H3K27me3 distribution over the INK locus. H3K27me3 was not identified at this region in AML cell lines without p15INK4b DNA methylation (U937 and HL-60). In contrast, histone modifications associated gene activation, trimethylation of lysine 4 of H3 (H3K4me3) and acetylation of lysine 9 (H3K9Ac), were found at the p15INK4b promoter in these cells. Enrichment of another repressive histone modification, trimethylation of histone H3 on lysine 9 (H3K9me3), did not correlate with the DNA methylation status of p15INK4b and appeared highest in exons 2 and 3 of p16INK4a in most cell lines. Since p15INK4b reactivation has been described as a component of a patient’s response to epigenetic therapies in AML treatment, we sought to determine the dynamics of histone modifications following treatment with the DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine and histone deacetylase (HDAC) inhibitor tricostatin A. In KG-1 cells, a reduction in p15INK4b DNA methylation was observed following treatment with DNMT inhibitors. Unexpectedly, treatment with HDAC inhibitors alone was also capable of reducing p15INK4b DNA methylation suggesting that a repressive chromatin structure contributes to the DNA methylation in this cell line. Loss of DNA methylation was not sufficient for reactivation of p15INK4b expression as detectible expression was only observed following the combined treatment of DNMT and HDAC inhibitors. Reactivation was associated with an increase in the activation-associated histone modifications H3K4me3 and H3K9Ac at the promoter region and, unexpectedly, maintenance of the repressive modification H3K27me3. This “bivalent” histone modification pattern is characteristic of many developmentally poised genes in embryonic stem cells and correlates with the histone methylation status of p15INK4b we observed in CD34+ bone marrow progenitor/ stem cells. This data indicates that optimal epigenetic therapies targeted to reactivate p15INK4b expression should be designed to induce activating histone modifications in addition to reducing DNA methylation.

Abstract Abstract 2534 Background: Allogeneic hematopoietic stem cell transplant is performed for high risk acute lymphoblastic leukemia (ALL). Complex allogeneic immune responses can produce graft versus host disease which is associated with antileukemia effects. The prevailing paradigm is that these antileukemia effects are mediated by allogeneic T cells. Our laboratory has been studying factors associated with long term disease free survival in murine models of allogeneic transplantation for ALL. Design/Method: We employed a well characterized murine model of MHC-matched, multiple minor histocompatibility mismatched transplantation in which C3.SW mice are donors and C57BL/6 mice are recipients. The ALL model is a recently derived C57BL/6 murine pre-B ALL driven by a bcr/abl p210 oncogene and an Ink/Arf region deletion; such lesions frequently present in human ALL. Recipients underwent myeloablation with 5-FU and total body irradiation and received hematopoietic grafts comprised of donor marrow and spleen cells. Recipients were challenged with ALL cells three weeks after transplant and followed for survival up to 3 months. Some groups of mice were treated with vaccines comprised of irradiated leukemia cells, Freund's adjuvant and GM-CSF. Serum samples were collected prior to leukemia challenge, three weeks after leukemia challenge and at the conclusion of the experiments. T cell responses were measured with ELISPOT assays specific for minor histocompatibility antigens and leukemia antigen specific responses. Antibody responses were measured by flow cytometry and ELISA. Leukemia was measured by flow cytometry. Results: Across all experiments approximately 35% of animals challenged with ALL were long term, leukemia free survivors. Interferon-gamma ELISPOT T cell responses specific for minor histocompatibilty antigens were found in none of the survivors and ELISPOT responses to leukemia antigens were seen in only 20% of long term survivors and these were very modest in magnitude. In contrast, IgM and IgG antibodies that bound leukemia cells were found in the vast majority of survivors. Antibody responses were not specific for the ALL with which the animals were challenged. Cross reactivity with other C57BL/6 ALL and AML leukemia lines were observed. In addition, lower levels of antibody cross reactivity were observed against normal C57BL/6 and C3.SW hematopoietic cells. Titers of the antibodies ranged from 1:40 to > 1:100. Antibody responses were observed in mice that had not received post-transplant vaccination. However, post-transplant vaccination significantly increased the titers of leukemia reactive antibodies. Statistical analysis was performed to determine if antibody levels at any point in the animal's course had any relationship with the risk of later death from leukemia or GVHD. Outcomes for each animal were categorized as alive, death from leukemia, death from GVHD or death of unspecified cause. Antibody levels for each animal were categorized as above or below the median for the entire experimental population. Chi square analysis demonstrated a statistically significant relationship between outcome and antibody level at the time of leukemia challenge. Few animals with high antibody levels died of leukemia (p = 0.0197). In comparison of death from leukemia with death from GVHD, higher antibody levels were associated with death from GVHD (p = 0.0099). A statistically significant difference was not observed between survivors and death from GVHD; however, the death from GVHD group was small, limiting the power of the analysis. Conclusion: Antibody responses to ALL cells were consistently observed in survivors. These studies do not prove the antibodies play a role in control of leukemia, but suggest the hypothesis that antibody responses may contribute to GVL effects. Current experiments in B cell deficient mice are underway to test the mechanistic role of antibody responses in the GVL effect. Disclosures: No relevant conflicts of interest to declare.

Le cellule staminali tumorali (CSC) sono una sottopopolazione di cellule tumorali con caratteristiche di cellule staminali, ovvero autorinnovamento (capacità di riformare un tumore dello stesso tipo) e capacità di "differenziarsi" in cellule che costituiscono la massa tumorale. Questi segni distintivi li rendono responsabili di eventi come recidiva del tumore, metastasi e resistenza ai farmaci. Per questo è molto importante capire quali sono i «fattori» fondamentali per il loro mantenimento. È interessante notare che gli stessi fattori di trascrizione possono essere responsabili del mantenimento sia delle cellule staminali normali che delle cellule staminali tumorali. In particolare sappiamo che il fattore di trascrizione "staminale" Sox2, un importante regolatore delle cellule staminali neurali, è sovraespresso anche nei tumori cerebrali. Nei gliomi, Sox2 è essenziale per mantenere CSC. Nel glioma di alto grado di topo (pHGG), la delezione di Sox2 causa l'arresto della proliferazione cellulare e l'incapacità di riformare i tumori in vivo; 134 geni sono significativamente derepressi. Per identificare i geni che mediano gli effetti della delezione di Sox2, ho overespresso nelle cellule pHGG nove tra i geni più upregolati e ho identificato quattro geni, Cdkn2b, Ebf1, Zfp423 e Hey2, che hanno ridotto fortemente la proliferazione cellulare in vitro e la tumorigenesi cerebrale in vivo. Mediante la mutagenesi CRISPR / Cas9, o inibizione farmacologica, di ciascuno di questi geni, individualmente, ho dimostrato che la loro attività è essenziale per l'arresto della proliferazione causato dalla delezione di Sox2. Questi antioncogeni inibiti da Sox2 hanno anche inibito la clonogenicità in linee di cellule staminali tumorali primarie derivate dal glioblastoma umano. Questi esperimenti identificano fattori anti-oncogenici critici la cui inibizione da parte di Sox2 è coinvolta nel mantenimento della CSC, definendo nuovi potenziali bersagli terapeutici per i gliomi. (Barone et al., 2020; Barone et al., 2018) Oltre a questo lavoro, che costituisce la parte principale del mio lavoro di tesi, ho contribuito a comprendere la funzione di Sox2 nelle cellule staminali neurali normali derivate dal cervello. Qui, studi genome-wide (ChIA-PET; ChIPseq; RNAseq) ci hanno portato a capire che Sox2 agisce nella regolazione genica, a livello genome-wide, mantenendo e regolando una rete genome-wide di interazioni a lungo raggio nella cromatina, collegare promotori genici a stimolatori distanti (Bertolini et al, 2019). Questa nuova prospettiva sulla funzione molecolare di Sox2 ci ha permesso di identificare nuovi geni regolati da Sox2, identificando il legame di SOX2 con esaltatori distanti (ChIPseq), permettendoci di capire quale gene controllano questi potenziatori, attraverso le nostre mappe di interazione a lungo Dati RNAseq). Questo ci ha portato a identificare nuovi importanti mediatori a valle della funzione Sox2 nell'auto-rinnovamento delle cellule staminali neurali (Pagin et al, in corso di revisione).
Cancer Stem Cells (CSCs) are a tumor cell sub-population with stem-cell features, i.e. self- renewal (ability to re-form a tumor of the same type) and the ability to “differentiate” into cells constituting the tumor bulk. These hallmarks make them responsible for events such as tumor relapse, metastasis and drug resistance. For this reason it is very important to understand which are the «factors» fundamental for their maintenance. Interestingly, the same transcription factors may be responsible of the maintenance of both normal stem cells and cancer stem cells. In particular we know that the “stemness” transcription factor Sox2, a major regulator in neural stem cells, is also overexpressed in brain tumors. In gliomas, Sox2 is essential to maintain CSC. In mouse high-grade glioma pHGG, Sox2 deletion causes cell proliferation arrest and inability to reform tumors in vivo; 134 genes are significantly derepressed. To identify genes mediating the effects of Sox2 deletion, I overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Cdkn2b, Ebf1, Zfp423 and Hey2, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. By CRISPR/Cas9 mutagenesis, or pharmacological inactivation, of each of these genes, individually, I showed that their activity is essential for the proliferation arrest caused by Sox2 deletion. These Sox2-inhibited antioncogenes also inhibited clonogenicity in primary human glioblastoma-derived cancer stem-like cell lines. These experiments identify critical anti-oncogenic factors whose inhibition by Sox2 is involved in CSC maintenance, defining new potential therapeutic targets for gliomas (Barone et al, Glia, under revision; Barone et al, 2018). Further to this work, constituting the main part of my thesis work, I contributed to understand Sox2 function in normal, brain-derived neural stem cells. Here, genome-wide studies (ChIA- PET; ChIPseq; RNAseq) led us to understand that Sox2 acts in gene regulation, at the genome- wide level, by maintaining and regulating a genome-wide network of long-range interactions in chromatin, connecting gene promoters to distant enhancers (Bertolini et al, 2019). This new perspective on Sox2 molecular function allowed us to identify novel Sox2-regulated genes, by identifying SOX2 binding to distant enhancers (ChIPseq), enabling us to understand which gene these enhancers control, through our long-range interaction maps (ChIA-PET and RNAseq data). This led us to identify important new downstream mediators of Sox2 function in neural stem cell self-renewal (Pagin et al, under revision).