The Impact of 3D Chromosomal Topology in Acute Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-15-SCI-15
Author(s):  
Iannis Aifantis
Keyword(s):  
Survival Rate ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Tumor Formation ◽  
Cohesin Complex ◽  
Self Renewal ◽  
Treatment Regimens ◽  
The Impact

Abstract Acute myeloid leukemia (AML) is the most common adult leukemia characterized by excessive proliferation of abnormal myeloid progenitors. AML continues to have a dismal survival rate amongst all subtypes of leukemia (<50% five-year overall survival rate), which can largely be attributed to limited advances in treatment regimens that, for the last decades, have relied on the use of two non-targeted cytotoxic drugs: cytarabine and anthracycline. Large-scale sequencing efforts have shed new light on genetic and epigenetic determinants of AML. Interestingly, these studies identified a frequent co-occurrence of somatic mutation between genes encoding cohesin complex subunits (such as STAG2, SMC1A, RAD21 and SMC3) and well-characterized AML oncogenic triggers, such as FLT3-ITD, TET2, and NPM1. Recent work has demonstrated an important role for the cohesin complex in normal stem/progenitor self-renewal and differentiation, gene regulation, and suppression of myeloproliferative neoplasms and AML, despite the precise mechanisms underlying these functions remaining poorly understood. It is believed that cohesin may suppress tumor formation by regulating chromatin looping at loci critical for self-renewal and myeloid progenitor differentiation. Utilizing established models of murine and human AML, this we will focus on the molecular mechanisms of cohesin-dependent myeloid tumor-suppression, with an emphasis on understanding novel treatment approaches that can exploit these functions. Using established protocols for identifying genome-wide changes in chromatin topology and gene expression, we propose undertaking an extensive characterization of cohesin-regulated chromatin changes driving AML. Furthermore, we investigate the application of targeted agents in cohesin-deficient AML whilst extensively mapping the mechanisms-of-action underlying these specific treatments. Ultimately, our work aims to generate novel, pre-clinical disease models of cohesin-mutated AML with strong mechanistic insights into the tumor-suppressive function of this complex. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Cohesin Subunit Rad21 Is a Negative Regulator of Hematopoietic Self-Renewal through Epigenetic Repression of HoxA7 and HoxA9

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1708-1708
Author(s):  
Joseph B. Fisher ◽  
Jonathan Peterson ◽  
Michael Reimer ◽  
Cary Stelloh ◽  
Kirthi Pulakanti ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Gene Array ◽  
Negative Regulator ◽  
Myelogenous Leukemia ◽  
Epigenetic Mechanism ◽  
Cohesin Complex ◽  
Self Renewal ◽  
Hematopoietic Malignancy

Abstract Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy with a poor prognosis. Developing novel treatments for AML has been difficult due to the heterogeneity of genetic mutations observed across patients. Recent genomic sequencing of AML patients have identified recurrent somatic mutations in cohesin complex genes in a subset of patients (10-20%). The canonical function of the cohesin complex is to maintain sister chromatid cohesion permitting proper segregation of genomic information to each daughter cell in mitosis. Interestingly, somatic cohesin mutations in AML are uniformly heterozygous and not associated with dramatic changes in chromosomal number, implying that cohesin mutations promote AML through a function independent of their role during mitosis. Recent studies have demonstrated that haploinsufficiency of the cohesin complex leads to enhanced self-renewal in Hematopoeitic Stem and Progenitor Cells (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal since this represents an early step of oncogenic transformation. In the present study we confirmed previous studies showing that reduced cohesin levels elicit enhanced self-renewal of murine HPSCs in vitro in serial-replating assays. Gene arrays and RT-qPCR identified significant increases in the HSPC self-renewal factors Hoxa7 and Hoxa9. We confirmed the gene array data by RT-PCR and observed that Hoxa9 levels increase within 24 hours following Rad21 depletion. Consistent with these data, multiple approaches demonstrated a high correlation between Rad21 depletion and Hoxa9 target gene expression programs. We next sought to identify the mechanism that leads to this abberant expression of Hoxa9. GSEA identified derepression of Polycomb Repressive Complex 2 (PRC2) target genes in Rad21-depleted HSPCs. Given the well-described silencing of Hox clusters by the PRC2 complex, we examined if Rad21-depletion-induced HoxA9 gene activation could be mediated by epigenetic changes. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, indicating that Rad21 plays a role in proper PRC2 mediated silencing of these genes. Using immunoprecipitation experiments we further demonstrate that cohesin and PRC2 interact and are bound in close proximity to Hoxa7 and Hoxa9, arguing that Hoxa7 and Hoxa9 derepression is directly mediated by Rad21-targeting of PRC2. Consistent with this finding we observed reduced global levels of PRC2's repressive histone mark H3K27me3 arguing that PRC2 requires the cohesin complex for proper targeting. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying both are critical downstream effectors of reduced cohesin levels. Interestingly, inhibition of Dot1L function also reduced the enhanced self-renewal capacity elicited by cohesin depletion, consistent with the Hoxa locus being regulated by H3K79 methylation. Our data demonstrate that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis and provide a possible therapeutic treatment for AML patients harboring cohesin mutations. Disclosures No relevant conflicts of interest to declare.

scholarly journals Interferon β-Mediated Protective Functions of Microglia in Central Nervous System Autoimmunity

2019 ◽  
Vol 20 (1) ◽  
pp. 190 ◽  
Author(s):  
Stefanie Scheu ◽  
Shafaqat Ali ◽  
Ritu Mann-Nüttel ◽  
Lisa Richter ◽  
Volker Arolt ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Molecular Mechanisms ◽  
Chronic Inflammatory Disease ◽  
Clinical Severity ◽  
Interferon Β ◽  
Treatment Regimens ◽  
Cns Autoimmunity ◽  
And Function ◽  
The Impact

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to demyelination and axonal damage. It often affects young adults and can lead to neurological disability. Interferon β (IFNβ) preparations represent widely used treatment regimens for patients with relapsing-remitting MS (RRMS) with therapeutic efficacy in reducing disease progression and frequency of acute exacerbations. In mice, IFNβ therapy has been shown to ameliorate experimental autoimmune encephalomyelitis (EAE), an animal model of MS while genetic deletion of IFNβ or its receptor augments clinical severity of disease. However, the complex mechanism of action of IFNβ in CNS autoimmunity has not been fully elucidated. Here, we review our current understanding of the origin, phenotype, and function of microglia and CNS immigrating macrophages in the pathogenesis of MS and EAE. In addition, we highlight the emerging roles of microglia as IFNβ-producing cells and vice versa the impact of IFNβ on microglia in CNS autoimmunity. We finally discuss recent progress in unraveling the underlying molecular mechanisms of IFNβ-mediated effects in EAE.

Private or Public Law Enforcement? The Case of Digital Anti-Piracy Policies with Illegal Non-Monitored Behaviors

2016 ◽  
Vol 15 (4) ◽  
Author(s):  
Eric Darmon ◽  
Thomas Le Texier
Keyword(s):  
Large Scale ◽  
Conflicts Of Interest ◽  
Public Law ◽  
Social Planner ◽  
Private Enforcement ◽  
Digital Piracy ◽  
Public And Private ◽  
The Social ◽  
Public And Private Enforcement ◽  
The Impact

AbstractShould rights be publicly or privately enforced in the case of digital piracy? The emergence of large-scale anti-piracy laws and the existence of illegal non-monitored channels raise important issues for the design of anti-piracy policies. We study the impact of these demand-side policies in two enforcement settings (namely, public and private enforcement settings) with an outside adoption option for users of an illegal non-monitored channel. Our results show that public enforcement generates higher monitoring and lower price levels, and also higher legal welfare than private enforcement. However, we identify potential conflicts of interest between the legal seller and the social planner when the efficiency of the illegal non-monitored channel is low. Introducing supply-side policies, i.e. policies targeted to suppliers of illegal content, we find that they may have unexpected impacts and can damage legal welfare. We also identify situations in which the two policies are substitutes or complements.

Gonadal Function in Survivors After Hodgkin Lymphoma (HL) Treatment within the German Hodgkin Study Group (GHSG) HD13–15 Trials.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2636-2636
Author(s):  
Karolin Behringer ◽  
Horst Mueller ◽  
Helen Goergen ◽  
Indra Thielen ◽  
Angelika Eibl ◽  
...  
Keyword(s):  
Menstrual Cycle ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Observation Time ◽  
Advanced Stage ◽  
Gonadal Function ◽  
Treatment Regimens ◽  
Male Survivors ◽  
One Year ◽  
The Impact

Abstract Abstract 2636 Purpose: To improve fertility advice in HL patients before treatment and counseling during survivorship, detailed information on the impact of chemotherapy is needed. Therefore, we analyzed gonadal function in survivors after treatment of early favorable, early unfavorable and advanced stage HL. Methods: Women <40 years and men <50 years at diagnosis in ongoing remission at least one year after treatment within the GHSG HD13–15 trials were included. Hormone parameters, menstrual cycle, symptoms of hypogonadism, measures to preserve fertility, pregnancies, and offspring were evaluated. Results: A total of 1,323 (55%) of 2,412 contacted female and male survivors were evaluable for the current analysis. In women and men, mean age at fertility assessment was 32 and 38 years and mean observation time from the end of treatment was 46 and 48 months, respectively. Comparison of the participating and non-participating patients qualifying for our analysis showed no relevant differences. Hormone levels correlated significantly with therapy intensity (p<.001). After 6–8 cycles BEACOPP (bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, prednisone), mean Anti-Muellerian hormone (AMH) levels in females were 0μg/l and 88.8% of males had Follicle-stimulating hormone (FSH) and inhibin B levels corresponding to oligospermia. Furthermore, low birth rates were observed in survivors after advanced-stage treatment within the observation time (women: 6.5%, men: 3.3%). Regular menstrual cycle was reported by >90% of early-stage HL female survivors and time to resumption of menstrual activity was mostly reached within one year. After advanced-stage treatment, menstrual activity was strongly related to age. 82% of women younger than 30 years had a regular cycle, compared to only 45% in the older age group (p<.001) and time to recovery was considerably longer than in early-stage patients. 34% of women >30 years suffered severe menopausal symptoms (3–4 fold more frequently than expected). In contrast, male survivors had mean levels of testosterone within the normal range and reported no increased symptoms of hypogonadism. Conclusions: The present analysis in a large group of female and male HL survivors provides well-grounded information on gonadal toxicity of the currently used treatment regimens. Accordingly, the results allow a risk adapted planning of fertility preservation before therapy and a comprehensive support during survivorship. Disclosures: No relevant conflicts of interest to declare.

Inhibition of Related JAK/STAT Pathways with Molecular Targeted Drugs Shows Strong Synergy with Ruxolitinib in Chronic Myeloproliferative Neoplasms

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5054-5054
Author(s):  
Santiago Barrio ◽  
Miguel Gallardo ◽  
Alicia Arenas ◽  
Rosa M. Ayala ◽  
Inmaculada Rapado ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Western Blot ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Targeted Drugs ◽  
Antitumor Effects ◽  
Chronic Myeloproliferative Neoplasms ◽  
The Stability ◽  
And Control

Abstract Abstract 5054 Objectives: In the present study we have determined the antitumor effects, molecular mechanisms of action and potential synergies between Ruxolitinib and the molecular targeted drugs Sorafenib, KNK437, Dasatinib and Perifosine, in Philadelphia negative chronic myeloproliferative neoplasms (MPN). Materials and methods: The cytotoxic and cytostatic effects of the different compounds were determined in the JAK2V617F positive cell lines HEL and Ba/F3 JAK2V617F EPOR, and in mononuclear and bone marrow CD34 positive cells from 19 MPN patients. The effects of different drugs at the molecular level were analyzed by flow cytometry and western blot. The IC50 and the synergy combination index (CI) were determined with GrafPath Prism software and Calcusyn software respectively. Results: Ruxolitinib (IC50PV=15nM), as well as Sorafenib (IC50PV=8uM), KNK437 (IC50PV=100uM) and Perifosine (IC50PV=15uM), was able to inhibit proliferation in cell line models and in cells from MPN patients. Dasatinib (IC50PV=12nM), however, was only effective in patient samples, with similar effects in both MPN patients and control donors. Moreover, Dasatinib, KNK437 and Sorafenib showed a strong synergistic effect in combination with Ruxolitinib (CIPV<0. 3, table 1). The Western blot analysis confirmed that Sorafenib inhibited the activation of ERK and P38, and, as a consequence, of STAT5. Ruxolitinib blocked the ERK and STAT5 activation, Dasatinib blocked SRC and STAT5, and KNK437 decreased the stability of the protein JAK2, reducing its expression. Conclusions: The blockage of JAK2 related proliferative pathways has the potential to inhibit cell proliferation in MPNs. Furthermore, the combination of Ruxolitinib with inhibitors targeted against these pathways has a strong synergistic effect. This may be related to a decrease in the activation of the final common effector STAT5. Disclosures: No relevant conflicts of interest to declare.

Regulation of Erythropoiesis by Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin A (DZNep)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 982-982
Author(s):  
Tohru Fujiwara ◽  
Haruka Saitoh ◽  
Yoko Okitsu ◽  
Noriko Fukuhara ◽  
Yasushi Onishi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Wide Range ◽  
The Impact ◽  
Chip Analysis

Abstract Abstract 982 Background. EZH2, a core component of Polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through mediating trimethylation of histone H3 at lysine 27 (H3K27), and is involved in various biological processes, including hematopoiesis. Overexpression of EZH2 has been identified in a wide range of solid tumors as well as hematological malignancies. Recent studies indicated that 3-deazaneplanocin A (DZNep), an inhibitor of EZH2, preferentially induces apoptosis in cancer cells, including acute myeloid leukemia and myelodysplastic syndromes, implying that EZH2 may be a potential new target for epigenetic treatment. On the other hand, whereas PRC2 complex has been reported to participate in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation (Yu et al. Mol Cell 2009; Ross et al. MCB 2012), the impact of DZNep on erythropoiesis has not been evaluated. Method. The K562 erythroid cell line was used for the analysis. The cells were treated with DZNep at doses of 0.2 and 1 microM for 72 h. Quantitative ChIP analysis was performed using antibodies to acetylated H3K9 and GATA-1 (Abcam). siRNA-mediated knockdown of EZH2 was conducted using Amaxa nucleofection technology™ (Amaxa Inc.). For transcription profiling, SurePrint G3 Human GE 8 × 60K (Agilent) and Human Oligo chip 25K (Toray) were used for DZNep-treated and EZH2 knockdown K562 cells, respectively. Gene Ontology was analyzed using the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/). Results. We first confirmed that DZNep treatment decreased EZH2 protein expression without significantly affecting EZH2 mRNA levels, suggesting that EZH2 was inhibited at the posttranscriptional level. We also confirmed that DZNep treatment significantly inhibited cell growth. Interestingly, the treatment significantly induced erythroid differentiation of K562 cells, as determined by benzidine staining. Transcriptional profiling with untreated and DZNep-treated K562 cells (1 microM) revealed that 789 and 698 genes were upregulated and downregulated (> 2-fold), respectively. The DZNep-induced gene ensemble included prototypical GATA-1 targets, such as SLC4A1, EPB42, ALAS2, HBA, HBG, and HBB. Concomitantly, DZNep treatment at both 0.2 and 1 microM upregulated GATA-1 protein level as determined by Western blotting, whereas the effect on its mRNA levels was weak (1.02- and 1.43-fold induction with 0.2 and 1 microM DZNep treatment, P = 0.73 and 0.026, respectively). Furthermore, analysis using cycloheximide treatment, which blocks protein synthesis, indicated that DZNep treatment could prolong the half-life of GATA-1 protein, suggesting that DZNep may stabilize GATA-1 protein, possibly by affecting proteolytic pathways. Quantitative ChIP analysis confirmed significantly increased GATA-1 occupancy as well as increased acetylated H3K9 levels at the regulatory regions of these target genes. Next, to examine whether the observed results of DZNep treatment were due to the direct inhibition of EZH2 or hitherto unrecognized effects of the compound, we conducted siRNA-mediated transient knockdown of EZH2 in K562 cells. Quantitative RT-PCR analysis demonstrated that siRNA-mediated EZH2 knockdown had no significant effect on the expression of GATA-1 as well as erythroid-lineage related genes. Furthermore, transcription profiles of the genes in the quantitative range of the array were quite similar between control and EZH2 siRNA-treated K562 cells, with a correlation efficient of 0.977. Based on our profiling results, we are currently exploring the molecular mechanisms by which DZNep promotes erythroid differentiation of K562 cells. Conclusion. DZNep promotes erythroid differentiation of K562 cells, presumably through a mechanism not directly related to EZH2 inhibition. Our microarray analysis of DZNep-treated K562 cells may provide a better understanding of the mechanism of action of DZNep. Disclosures: No relevant conflicts of interest to declare.

Tet2 Loss Accelerates The Myeloproliferative Neoplasm (MPN) Phenotype Of Jak2V617F Knockin Mice But Is Insufficient To Cause Leukemic Transformation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4095-4095
Author(s):  
Edwin Chen ◽  
Lawrence J Breyfogle ◽  
Rebekka K. Schneider ◽  
Luke Poveromo ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Conditional Knockout ◽  
The Other ◽  
Myelogenous Leukemia ◽  
Leukemic Transformation ◽  
The Impact

Abstract TET2 mutations are early somatic events in the pathogenesis of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) and are one of the most common genetic lesions found in these diseases. In MPN, TET2 mutations are enriched within more advanced disease phenotypes such as myelofibrosis and leukemic transformation and often co-occur with the JAK2V617F mutation, which is present in the majority of MPN patients. We have developed and characterized a Jak2V617F conditional knockin mouse (Jak2VF/+), the phenotype of which closely recapitulates the features of human MPN. To determine the impact of Tet2 loss on Jak2V617F-mediated MPN, we crossed Tet2 conditional knockout mice with Jak2VF/+ knockin and Vav-Cre transgenic mice and backcrossed the compound mutant animals. We then characterized the effects of heterozygous and homozygous loss of Tet2 on the phenotype of Jak2VF/+ mice. We assessed peripheral blood counts, histopathology, hematopoietic differentiation using flow cytometry, colony formation and re-plating capacity. We also evaluated the effects of Tet2 loss on the transcriptome of the HSC compartment using gene expression microarrays and on HSC function using competitive bone marrow transplantation assays. Similar to Jak2VF/+/VavCre+ mice, Tet2+/-/Jak2VF/+/VavCre+ and Tet2-/-/Jak2VF/+/VavCre+ mice develop leukocytosis, elevated hematocrits (HCT) and thrombocytosis. Tet2-/-/Jak2VF/+/VavCre+ mice demonstrate enhanced leukocytosis and splenomegaly compared to the other groups. All groups demonstrate myeloid expansion, erythroid hyperplasia and megakaryocytic abnormalities consistent with MPN in the bone marrow and spleen, while more prominent myeloid expansion and megakaryocytic morphological abnormalities are observed in Tet2-/-/Jak2VF/+/VavCre+ mice as compared to the other groups. Notably, we do not see the development of acute myelogenous leukemia (AML) in Tet2-/-/Jak2VF/+/VavCre+ mice at 6 months. We see enhanced expansion of lineagelowSca1+cKithigh (LSK) cells (enriched for HSC) most prominently in the spleens of Tet2+/-/Jak2VF/+/VavCre+ and Tet2-/-/Jak2VF/+/VavCre+ mice as compared to Jak2VF/+/VavCre+ mice. In colony forming assays, we find that Tet2-/-/Jak2VF/+/VavCre+ LSK cells have enhanced re-plating activity compared to Jak2VF/+/VavCre+ LSK cells and that Tet2-/-/Jak2VF/+/VavCre+ LSK cells form more colonies that Tet2-/-/Jak2+/+/VavCre+ cells. Gene expression analysis demonstrates enrichment of a HSC self-renewal signature inTet2-/-/Jak2VF/+/VavCre+ LSK cells. Concordant with this, we find that Tet2-/-/Jak2VF/+/VavCre+ LSK cells have enhanced competitive repopulation at 16 weeks as compared to Jak2VF/+/VavCre+ and Tet2+/-/Jak2VF/+/VavCre+ LSK cells. In aggregate these findings demonstrate that Tet2 loss promotes disease progression in MPN but is insufficient to drive full leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

Ph-Negative Chronic Myeloproliferative Neoplasms – Population Analysis, a Single Center 10-years’ Experience

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5556-5556
Author(s):  
Vasily Shuvaev ◽  
Irina Martynkevich ◽  
Alla Abdulkadyrova ◽  
Vera Udaleva ◽  
Tatyana Zamotina ◽  
...  
Keyword(s):  
Overall Survival ◽  
Risk Stratification ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Risk Group ◽  
Myeloproliferative Neoplasms ◽  
Population Analysis ◽  
Myelogenous Leukemia ◽  
Rank Test ◽  
Chronic Myeloproliferative Neoplasms

Abstract Objectives and background. Nowadays chronic myeloproliferative neoplasms (MPN) other than chronic myelogenous leukemia undergo renaissance of interest. It results from advances in decryption of molecular mechanisms of pathogenesis and invention of target drugs. Epidemiological information is needed to assess potential effect and additional costs of new diagnostic and therapeutic techniques. The objective of our study was to review experience of MPN diagnostic and treatment in our center for past ten years. Methods. Our institution serves as primary hematological outpatient department for a half of Saint-Petersburg city with about 2 million inhabitants. We reviewed patients' charts to obtain information about incidence, symptoms, diagnostic test results, treatment options and relationship to prognostic factors. Statistical methods included descriptive statistics, nonparametric ANOVA for frequencies comparisons and Kaplan-Meyer method with log-rank test for survival comparisons in Statistica 7.0 package. Results. Since 2004 to 2013 there were 570 newly diagnosed MPN patients (pts) in our center. This group consisted of primary myelofibrosis (PMF) (203 pts; 126 female, 77 male; median age 63 years, range 16-83 years), essential thrombocythemia (ET) (201 pts; 146 female, 55 male; median age 58 years, range 23-78 years), polycythemia vera (PV) (166 pts; 96 female, 70 male; median age 57 years, range 20-85 years). The incidence rates were stable during study period: PMF incidence varied from 0.65 to 1.35 with mean of 1.01 new patient per 100 000 inhabitants per year; ET had incidence from 0.60 to 2.1 with mean of 1.00 and PV had incidence from 0.5 to 1.15 with mean of 0.83. The most prevalent symptoms of disease were: splenomegaly (65.5%), constitutional symptoms (fever, night sweats, weight loss) (31.0%), anemia (36.3%) thrombosis (24.1%) for PMF; fatigue (33.2%), headache and dizziness (25.6%), arthralgia (21.8%), erythromelalgia (15.8%) for ET; plethora (82.5%), headache and dizziness (52.4%), fatigue (31.3%) for PV. JAK2V617F was detected in 49.7% of PMF pts, 57.8% of ET pts and in 97.7% of PV pts. Thrombosis rates according WHO IPSET-thrombosis system risks` groups of ET and PV pts were: low-risk group 3.33% (3/90), intermediate-risk group 11.1% (13/117) and 39.4% (63/160) in high-risk group with highly significant (p<0.0001) differences between risks' groups. There were 169 lethal outcomes in the analysed group (102 PMF; 31 ET; 36 PV). Ten-years overall survival rates were 49.8% in PMF pts, 84.6% in ET pts and 78.3% in PV pts. (fig.1). Overall survival in PMF was significantly influenced by risk stratification as IPSS, DIPSS and DIPSS+. Survival curves according DIPSS+ groups are presented in fig.1. Conclusions. Patients with MPN are presented in substantial number; therefore need much finance for novel therapy introduction. Risk stratification systems has high predictive value. Innovative drugs treatment results should be evaluated in comparison with historical control. Figure1 Overall survival in PMF patients according to DIPPS+ stratification groups. Figure1. Overall survival in PMF patients according to DIPPS+ stratification groups. Disclosures No relevant conflicts of interest to declare.

scholarly journals Enforcing Post-Transcriptional Circuitries to Achieve Human Hematopoietic Stem Cell Expansion

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-46-SCI-46
Author(s):  
Kristin Hope
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Self Renewal ◽  
Control Of Gene Expression ◽  
Hematopoietic Stem

Abstract The balance between hematopoietic stem cell (HSC) differentiation and self-renewal is central to clinical regenerative paradigms. Unravelling the precise molecular mechanisms that govern HSC fate choices will thus have far reaching consequences for the development of effective therapies for hematopoietic and immunological disorders. There is an emerging recognition that beyond transcription, HSC homeostasis is subject to post-transcriptional control by RNA-binding proteins (RBPs) that ensure precise control of gene expression by modulating mRNA splicing, polyadenylation, localization, degradation or translation. RBPs can synchronously regulate the fates of operationally similar RNAs, in what have been termed RNA regulons. We have used a variety of functional approaches, in combination with unbiased genome- and proteome-scale, methods to define the tenets that govern this regulation and to determine key downstream circuitries of stem cell-regulating RBPs whose targeting could provide the basis for novel regenerative treatments. Through loss-of-function efforts, we have identified the RBP, MSI2, as a required factor for human HSC maintenance. By contrast, at supraphysiological levels, MSI2 has a profound positive effect on human HSC self-renewal decisions. Using a combination of global profiling, including mapping MSI2's targets through cross-linking immunoprecipitation (CLIP)-seq, we show that MSI2 achieves its ex vivo self-renewal-promoting effects by directing a co-ordinated post-transcriptional repression of key targets within the aryl hydrocarbon receptor (AHR) pathway. We are currently exploring the "rules" by which MSI2 influences its downstream effects on target RNAs and how it functions, in combination with other protein interactors, to instill a putative RBP regulatory code in HSCs. HSCs exhibit highly unique epigenomes, transcriptomes and proteomes and it is this distinctive molecular landscape that provides the canvas upon which MSI2, and indeed any other HSC-specific RBP exert their post-transcriptional influence over stem cell function. As such, to decipher the bona fide RNA networks that RBPs function upon in HSCs and to understand how they influence this network to enforce self-renewal, we are working towards performing systematic studies of RBP regulons in these cells specifically. In turn these approaches are elucidating a host of RBPs and post-transcriptional control mechanisms previously unappreciated for their role in HSC control. When modulated appropriately, we can successfully tailor these post-transcriptional regulons to enforce desired HSC outputs ex vivo. In summary, approaches to elucidate key HSC-regulatory RBPs and their protein and RNA interactomes provide valuable insights into a layer of HSC control previously not well understood, and one that can be capitalized on to achieve successful HSC expansion. Disclosures No relevant conflicts of interest to declare.

scholarly journals Incorporation of Clinical Information to Decipher Driver Mutations in Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5489-5489
Author(s):  
Murtadha K. Al-Khabori ◽  
Shoaib Al-Zadjali ◽  
Mohamed Al-Rawahi ◽  
Iman Al Noumani ◽  
Khalil Al Farsi ◽  
...  
Keyword(s):  
Platelet Count ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Clinical Information ◽  
Primary Myelofibrosis ◽  
Mutation Load ◽  
Female Ratio ◽  
Epigenetic Regulators ◽  
The Impact

Abstract Introduction: Mutations in the epigenetic regulators are commonly found in myeloid disorders including Myeloproliferative Neoplasm (MPN). Primary myelofibrosis, dysplastic changes and severity of the disease were associated with the mutation load. Most of the studies had a limited number of targeted genes and included a mixture of JAK2 positive and negative disease. The objective of this study is to assess the impact of mutations in the epigenetic regulators on the presentation of patients with JAK2 V617F positive MPN. Methods: We retrieved the clinical and laboratory information on 61 consecutive eligible patients. Mutation analysis of the entire coding regions of ASXL1, ASXL2, CBL, CEBPA, CSF3R, DNMT3A, EZH2, IDH2, TET1 and TET2 genes was performed using next generation sequencing (NGS; Ion PGM Sequencer; Thermo Fisher ScientificÒ). The library was constructed and the templates were prepared using the PGM tool. The variants were annotated using the ClinVar database and the prediction from the Scale-Invariant Feature Transform (SIFT) and or Polymorphism Phenotyping (Polyphen) algorithms. Alignment, variant filtering and annotation were performed using Ion Torrent Suite. Standard descriptive and analytical statistics were used as appropriate to describe and compare different groups. The MPN subtype, bleeding, thrombosis, hemoglobin, platelet count, White Cell Count (WBC), Lactate Dehydrogenase (LDH) and erythropoietin level were compared for each candidate variant. An alpha threshold of 0.05 was used with no adjustment for multiple comparisons as the analyses were considered exploratory. All statistics were performed using R program. Variants were selected for further experimentation based on their frequency and association with the clinical information at diagnosis. Results: Sixty-one patients were included (Polycythemia Vera: 29, Essential Thrombocythemia: 21, Primary Myelofibrosis: 9, MPN unclassifiable: 2) with a median age of 62 years (Interquartile Range [IQR]: 44 - 70). Male to female ratio was 35:26. The median hemoglobin, WBC, platelet count, LDH and erythropoietin were 14.6 g/dL (IQR: 12.8 - 16.8), 11.5 *109/L (IQR: 11.5 - 14.4), 507 *109/L (IQR: 391 - 779), 265 mU/mL (IQR: 231 - 409) and 1.2 mU/mL (IQR: 1.0 - 4.8) respectively. At presentation, 54% had splenomegaly, 23% had an arterial or venous thrombosis, and 5% had bleeding. Sixty-three variants were found in the samples tested. The median mutation load was 13 variants (Range: 11-14). Patients with higher than the median mutation load had higher mean erythropoietin (7.8 vs. 0.9 g/dL; p = 0.02016). ASXL1 p.Leu815Pro variant was found in all patients. Only three variants were found in the ClinVar database. Seven variants were predicted to be pathogenic (ASXL1: 1, EZH2: 1, IHD1: 1, TET1: 1 and TET2: 3). Patients with TET2: p.Leu1721Trp variant had 6.4 higher odds of bleeding (p = 0.04345). Patients with TET2: p.His1778Arg variant had a lower WBC (9.1 vs. 13.9 *109/L; p = 0.01699) and LDH (213 vs. 348 mU/mL; p = 0.0006528) while those with TET1: p.Lle1123Met variant had a higher WBC (13.5 vs. 8.3 *109/L; p = 0.02756). None of the remaining comparisons were statistically significant. Conclusions: Incorporation of clinical information facilitates the prioritization of variants from DNA sequencing experiments. In MPN, we recommend ASXL1: p.Leu815Pro (expressed in all patients), TET2: p.Leu1721Trp (associated with bleeding), TET2: p.His1778Arg (correlated with the WBC and LDH) and TET1: p.Lle1123Met (correlated with LDH) for further functional experimentation. Disclosures No relevant conflicts of interest to declare.

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