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1
McArtney, Steven, Duane Greene, Terence Robinson, and James Wargo. "Evaluation of GA4+7 plus 6-Benzyladenine as a Frost-rescue Treatment for Apple." HortTechnology 24, no. 2 (April 2014): 171–76. http://dx.doi.org/10.21273/horttech.24.2.171.
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Freeze events during bloom can be a relatively frequent occurrence in many apple (Malus ×domestica) production areas in the United States that significantly reduce orchard productivity and profitability. This study investigated the potential for a proprietary mixture of gibberellin A4 + A7 and 6-benzyladenine (GA4+7 plus 6-BA) to increase fruit set and cropping of apple following freeze events at three locations across the United States during bloom in 2012. GA4+7 plus 6-BA increased fruit set in two of five experiments, and increased fruit number and yield per tree in three of five experiments. GA4+7 plus 6-BA increased fruit set and yield of ‘Taylor Spur Rome’ following freezes on two consecutive days during bloom when the minimum temperature reached 23.9 and 28.4 °F. Fruit set was increased due to a stimulation of parthenocarpic fruit growth. Using locally obtained market prices, GA4+7 plus 6-BA treatments increased the crop value of ‘Taylor Spur Rome’, ‘Ginger Gold’, and ‘Jonagold’ by $3842, $977, and $6218 per acre, respectively. Although GA4+7 plus 6-BA application(s) after a freeze increased fruit set and cropping in some instances, tree yields were well below the average yields previously obtained in the test orchards.
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Watanabe, Eiichiro, Yusuke Kawashima, Wataru Suda, Tomo Kakihara, Shinya Takazawa, Daisuke Nakajima, Ren Nakamura, et al. "Discovery of Candidate Stool Biomarker Proteins for Biliary Atresia Using Proteome Analysis by Data-Independent Acquisition Mass Spectrometry." Proteomes 8, no. 4 (November 27, 2020): 36. http://dx.doi.org/10.3390/proteomes8040036.
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Biliary atresia (BA) is a destructive inflammatory obliterative cholangiopathy of the neonate that affects various parts of the bile duct. If early diagnosis followed by Kasai portoenterostomy is not performed, progressive liver cirrhosis frequently leads to liver transplantation in the early stage of life. Therefore, prompt diagnosis is necessary for the rescue of BA patients. However, the prompt diagnosis of BA remains challenging because specific and reliable biomarkers for BA are currently unavailable. In this study, we discovered potential biomarkers for BA using deep proteome analysis by data-independent acquisition mass spectrometry (DIA–MS). Four patients with BA and three patients with neonatal cholestasis of other etiologies (non-BA) were recruited for stool proteome analysis. Among the 2110 host-derived proteins detected in their stools, 49 proteins were significantly higher in patients with BA and 54 proteins were significantly lower. These varying stool protein levels in infants with BA can provide potential biomarkers for BA. As demonstrated in this study, the deep proteome analysis of stools has great potential not only in detecting new stool biomarkers for BA but also in elucidating the pathophysiology of BA and other pediatric diseases, especially in the field of pediatric gastroenterology.
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Luan, Siliang, Qingfang Yang, Huxing Zhou, Zhongtai Jiang, Wei Wang, Zhuorui Wang, and Ruijuan Chu. "The HSABA for Emergency Location-Routing Problem." Mathematical Problems in Engineering 2019 (July 31, 2019): 1–12. http://dx.doi.org/10.1155/2019/5391687.
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This article presents a Location-Routing Problem (LRP) model to assist decision makers in emergency logistics. The model attempts to consider the relationship between the location of warehouses and the delivery routes in order to maximize the rescue efficiency. The objective function of the minimization of time and cost is established in the single-stage LRP model considering different scenarios. The hybrid self-adaptive bat algorithm (HSABA) is an improved nature-inspired algorithm for solving this LRP model, hard optimization problem. The HSABA with self-adaptation mechanism and hybridization mechanism effectively improves the defect of the original BA, that is, trapping into the local optima easily. An example is provided to prove the effectiveness of our model. The studied example shows that the single-stage LRP model can effectively select supply locations and plan rescue routes faced with different disasters and the HSABA outperforms the basic BA.
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Nohara, Kazunari, Travis Nemkov, Angelo D’Alessandro, Seung-Hee Yoo, and Zheng Chen. "Coordinate Regulation of Cholesterol and Bile Acid Metabolism by the Clock Modifier Nobiletin in Metabolically Challenged Old Mice." International Journal of Molecular Sciences 20, no. 17 (September 1, 2019): 4281. http://dx.doi.org/10.3390/ijms20174281.
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Cholesterol and bile acid (BA) homeostasis plays a central role in systemic metabolism. Accumulating evidence suggests a key regulatory function of the circadian clock, our biological timer, in lipid metabolism, particularly cholesterol and bile acid flux. Previously, we showed that Nobiletin (NOB), a natural compound targeting the ROR (Retinoic acid receptor-related orphan receptor) nuclear receptors in the circadian oscillator, strongly protects lipid homeostasis, including normal serum cholesterol levels in high-fat (HF) fed mice at both young and old ages. In this study, we further examined the role of NOB in cholesterol metabolism in HF-fed aged mice, and found that NOB lowered the serum LDL/VLDL cholesterol levels and consequently the LDL/HDL ratio. BA levels in the serum were markedly reduced in the HF.NOB group, and examination of additional hepatic markers further indicate a protective role of NOB in the liver. At the molecular level, whereas HF feeding downregulated hepatic expression of several ROR target genes involved in bile acid synthesis, NOB treatment (HF.NOB) was able to rescue it. In accordance, fecal BA excretion was enhanced by NOB, and microbial 16S sequencing revealed alteration of several taxa known to be involved in secondary BA production in the gut. Together, these results demonstrate concerted effects of the clock-modulating compound NOB in cholesterol and BA metabolism, suggesting pharmacological manipulation of the clock as a novel therapeutic strategy against metabolic disorders and age-related decline.
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Farchi, M. U. "(A65) Stress & Trauma Studies Program (STSP): Theoretical & Practical Emergency Mental Health Interventions Studies for BA Social Work Students." Prehospital and Disaster Medicine 26, S1 (May 2011): s18. http://dx.doi.org/10.1017/s1049023x11000720.
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The Tel Hai college Department of Social Work established this program as part of its community commitment to ensure that persons with skills in emergency mental health / trauma intervention will be available to the community as first responders when needed. The main goal of the STSP: Training Social work students As First Responders with Very High Professional Standards of Emergency as well as Long Term Mental Health Interventions Qualifications. This program enables the students to integrate between theory and hands-on basic and advanced skills in stress & trauma interventions – from the help to a single traumatized person to mass disasters involving more complex interventions. In addition, program underlines and empowers the students self efficacy and resilience. The studies are carried out in 4 main channels: A. Academic studies and advanced professional workshops. B. Outdoor drills with other help and rescue units: MDA (EMS), IDF, Police, Israel fire and rescue services, local and national rescue units) C. Volunteering in community trauma / first responder units D. Emergency mental health interventions during real time events (Last one: Emergency interventions among the evacuated families during the mount Carmel bushfire) Student's Skills Acquired During the STSP • Theoretical & practical knowledge of the stress & trauma development process. • Differentional diagnosis of the trauma stages (From ASR to C-PTSD). • Identifying all sources of resilience and coping strategies. • Basic & advanced crisis and disaster intervention methods. • Crisis & disaster management & command • Professional self confidence, Independency & Creativity, leadership and leading capabilities. The program, its benefits and latest drills and real time intervention will be discussed as well as demonstrated with videos.
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Дудченко, Лейла, Leila Dudchenko, Валентин Савченко, and Valentin Savchenko. "MATHEMATICAL MODEL OF SELECTION OF PHENOTYPES OF BRONCHIAL ASTHMA AT THE RESORT STAGE OF MEDICAL REHABILITATION." Bulletin physiology and pathology of respiration 1, no. 68 (June 7, 2018): 17–22. http://dx.doi.org/10.12737/article_5b188e929239f4.95375481.
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The aim of the research is to develop a mathematical model of selection of phenotypes of bronchial asthma (BA) at a resort stage of medical rehabilitation. Materials and methods: 300 patients with BA who arrived at the climatic resort for medical rehabilitation; values of 64 indices of the research describing features of emergence and a course of a disease; the current clinic-functional state of patients; the discriminant analysis for the creation of a mathematical model. Results of the research: by the discriminant analysis there were created 7 statistically significant linear discriminant functions for the discernment of BA phenotypes; 23 indices of the research were under study: gender, asthma control, quantity of not allergic associated diseases, coughing during the day, character of the sputum, expressiveness of symptoms of asthma, use of rescue medication, the number of dry rattles in lungs, severity of hypostases, systolic and diastolic arterial blood pressure, shortness of breath, wheezing, reaction to the change of weather, allergic reactions, existence of symptoms of intoxication and feature of treatment of the last exacerbation, a dose of inhaled glucocorticosteroids, presence of emphysema and fibrosis in lungs at the X-ray examination, expressiveness of electrographic changes, FEF25-75%, 6-minute walk test. Conclusion: the procedure of the selection of 7 BA phenotypes at the resort stage of medical rehabilitation can be carried out by the use of the mathematical model consisting of 7 linear discriminant functions including 23 indices; the accuracy of the selection of the offered BA phenotypes of the developed mathematical model in general is 89.0%.
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Klčová, L., and M. Gubišová. "Evaluation of different approaches to buckwheat (Fagopyrum esculentum Moench.) micropropagation." Czech Journal of Genetics and Plant Breeding 44, No. 2 (June 27, 2008): 66–72. http://dx.doi.org/10.17221/2677-cjgpb.
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Plant regeneration by different techniques was evaluated in three buckwheat cultivars: indirect regeneration from cotyledons and hypocotyls, direct regeneration by nodal segment cultivation and induction of multiple shoots from seedling apices. Regenerated shoots were obtained in all procedures. The effects of BA (6-benzylaminopurine), media composition and gelling agent were tested. The regeneration efficiency of shoot apex culture was 2.65–3.33 nodal segments/explant. Cotyledon and hypocotyl segments produced 1.25–2.44 shoots per explant plated. Nodal segment cultivation yielded 4.1–4.8 new nodal segments/explant in 4 weeks. Eighty percent of shoots rooted on the basal medium. Rooting was improved (up to 95.6%) by IBA (3-indolebutyric acid) addition to the culture medium. Regenerated plantlets were transferred to the soil. The most efficient and simple micropropagation of buckwheat was nodal segment cultivation on MS medium solidified by agar with the addition of 1 mg/l BA. This method is advisable for rapid multiplication, in vitro conservation or rescue of genetic resources.
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Liu, S. M., S. R. Sykes, and P. R. Clingeleffer. "Improved in ovulo embryo culture for stenospermocarpic grapes (Vitis vinifera L.)." Australian Journal of Agricultural Research 54, no. 9 (2003): 869. http://dx.doi.org/10.1071/ar03053.
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In ovulo embryo rescue techniques have been used to recover new hybrids from seedless × seedless grape crosses. This study was conducted to increase efficiency by investigating effects of genotype, medium, and ovule removal age on ovule elongation, embryo recovery, growth, and plantlet formation. Ovules from self-pollinated berries of seedless varieties Sunmuscat, Merbein Seedless, and Marroo Seedless were cultured at 30, 43, 60, and 70 days after flowering (DAF) in a range of media, some of which were supplemented with gibberellic acid (GA3) and indole-3-acetic acid (IAA). The effect of activated charcoal (AC) in media on rescued embryos was also investigated. Ovules exhibited continuous growth in vivo and in vitro. The most vigorous growth was observed for ovules cultured at 30 and 43 DAF, but more embryos were recovered from ovules cultured at 60 and 70 DAF. Ovule growth and embryo production in vitro were improved in Bouquet and Davis (BD) and Nitsch and Nitsch (NN) media. Supplementation with GA3 increased embryo recovery rates. Highest embryo recovery rates were 18.1%, 9.6%, and 12.2% for Sunmuscat, Merbein Seedless, and Marroo Seedless, respectively, when ovules were excised and cultured at 60 or 70 DAF in either BD or NN media. In vitro embryo survival and plantlet formation were higher for torpedo-shaped embryos, and improved greatly in 6-benzyladenine (BA)-supplemented woody plant (WP) medium containing 0.3% AC. Embryo recovery was improved by excising and culturing ovules at 60 DAF in BD or NN media and then by transferring embryos to WP medium supplemented with BA and AC.
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S, Chang, Li J, Sun W, Lin F, and Xu B. "A method to rescue the polluted explant in banana (Musa spp) tissue culture." South Asian Journal of Experimental Biology 2, no. 4 (September 2, 2012): 157–61. http://dx.doi.org/10.38150/sajeb.2(4).p157-161.
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Plant tissue culture is an important commercial tool in banana production. Sterilization is the first key step in plant tissue culture. When explants were found infected, they were always discarded. But if the materials were precious and rare, once they were cast away, it will be very difficult to get them again. No paper has been found to resolve this problem. In this paper, two precious banana plantlets were polluted during the process of tissue culture. The leaves and the stem were cut from the polluted seedlings. Only the pseudostem was left. The black and brown part of the pseudostem were also cut and discarded. The modified pseudostem was soaked in 6% NaClO solution 10 minutes first, and then, it was kept in 70% ethanol for 1.5 minutes. After that, the pseudostem was cultured on MS basal medium supplemented with sucrose, 7 g Lâ€1 agar and 3 mg/L 6â€Benzylaminopurine (6â€BA). Most of the explants grow healthily during the following time.
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Educational Research Institute, War, Konrad, Radosław Kaczan, and Małgorzata Rękosiewicz. "Off-time higher education as a risk factor in identity formation." Polish Psychological Bulletin 44, no. 3 (September 1, 2013): 299–309. http://dx.doi.org/10.2478/ppb-2013-0033.
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Abstract One of the important determinants of development during the transition to adulthood is the undertaking of social roles characteristic of adults, also in the area of finishing formal education, which usually coincides with beginning fulltime employment. In the study discussed in this paper, it has been hypothesized that continuing full-time education above the age of 26, a phenomenon rarely observed in Poland, can be considered as an unpunctual event that may be connected with difficulties in the process of identity formation. Relationships between identity dimensions and identity statuses, and age and educational context were analyzed. 693 individuals aged 19-35 took part in the study. The participants attended three types of educational institutions: (1) full-time university studies (BA or MA level), (2) part-time university studies (BA or MA level), and (3) full-time post-secondary school (certificate courses such as: medical rescue, massage therapy, cosmetology, occupational therapy). Among the students of full-time university studies predictable dependencies, also in respect of highlevels of indicators of identity crisis and a high frequency of diffused identity occurrence, were observed. Such dependencies were not found in the group of full-time post-secondary school students.
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Bai, Ren-Yuan, Peter Dieter, Christian Peschel, Stephan W. Morris, and Justus Duyster. "Nucleophosmin-Anaplastic Lymphoma Kinase of Large-Cell Anaplastic Lymphoma Is a Constitutively Active Tyrosine Kinase That Utilizes Phospholipase C-γ To Mediate Its Mitogenicity." Molecular and Cellular Biology 18, no. 12 (December 1, 1998): 6951–61. http://dx.doi.org/10.1128/mcb.18.12.6951.
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ABSTRACT Large-cell anaplastic lymphoma is a subtype of non-Hodgkin’s lymphoma characterized by the expression of CD30. More than half of these lymphomas have a chromosomal translocation, t(2;5), that leads to the expression of a hybrid protein comprised of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK). Here we show that transfection of the constitutively active tyrosine kinase NPM-ALK into Ba/F3 and Rat-1 cells leads to a transformed phenotype. Oncogenic tyrosine kinases transform cells by activating the mitogenic signal transduction pathways, e.g., by binding and activating SH2-containing signaling molecules. We found that NPM-ALK binds most specifically to the SH2 domains of phospholipase C-γ (PLC-γ) in vitro. Furthermore, we showed complex formation of NPM-ALK and PLC-γ in vivo by coimmunoprecipitation experiments in large-cell anaplastic lymphoma cells. This complex formation leads to the tyrosine phosphorylation and activation of PLC-γ, which can be corroborated by enhanced production of inositol phosphates (IPs) in NPM-ALK-expressing cells. By phosphopeptide competition experiments, we were able to identify the tyrosine residue on NPM-ALK responsible for interaction with PLC-γ as Y664. Using site-directed mutagenesis, we constructed a comprehensive panel of tyrosine-to-phenylalanine NPM-ALK mutants, including NPM-ALK(Y664F). NPM-ALK(Y664F), when transfected into Ba/F3 cells, no longer forms complexes with PLC-γ or leads to PLC-γ phosphorylation and activation, as confirmed by low IP levels in these cells. Most interestingly, Ba/F3 and Rat-1 cells expressing NPM-ALK(Y664F) also show a biological phenotype in that they are not stably transformed. Overexpression of PLC-γ can partially rescue the proliferative response of Ba/F3 cells to the NPM-ALK(Y664F) mutant. Thus, PLC-γ is an important downstream target of NPM-ALK that contributes to its mitogenic activity and is likely to be important in the molecular pathogenesis of large-cell anaplastic lymphomas.
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Aichberger, Karl J., Karoline V. Gleixner, Irina Mirkina, Sabine Cerny-Reiterer, Barbara Peter, Veronika Ferenc, Michael Kneidinger, et al. "Identification of proapoptotic Bim as a tumor suppressor in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs." Blood 114, no. 26 (December 17, 2009): 5342–51. http://dx.doi.org/10.1182/blood-2008-08-175190.
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Abstract Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases, neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816–induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore, midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally, a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression, or by BH3-mimetics such as obatoclax, may be an attractive therapy concept in SM.
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Prawestri, Apriliana Dyah, Indira Riastiwi, Resa Sri Rahayu, Tri Handayani, Aryani Leksonowati, and Yuyu Suryasari Poerba. "Hydropriming Improves Germination and Plant Recovery During Embryo Rescue of Wild Banana Musa acuminata var. tomentosa." Jurnal Biodjati 6, no. 1 (June 1, 2021): 11–22. http://dx.doi.org/10.15575/biodjati.v6i1.10528.
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Wild bananas are believed to have genes for resistance to biotic and abiotic stress in nature, making them potential genetic resources for creating superior varieties. Wild banana seeds, such as Musa acuminata var. tomentosa are generally difficult to germinate in vivo, so that in vitro embryo culture technique is needed. This study aimed to increase embryo germination and regeneration of wild banana M. acuminata var. tomentosa by soaking the seeds as hydropriming. The treatment comprised of soaking the seeds in sterile distilled water for four periods of time: 0 (control), 1, 4, and 7 days. A total of 45 embryos for each treatment were planted on petri dishes containing MS + 0.5 mg/L BA + 1 mg/L biotin + 1 mg/L proline. The results showed that hydropriming increased the rate of embryo germination and regeneration. Seeds soaked for 1, 4, and 7 days successfully resulted in embryo germination percentages of 87%, 62%, and 62%, respectively, while the control unsoaked seeds germinated with a lower percentage of 42%. One-day soaking treatment was the most optimal treatment to increase the rate of germination and regeneration as well as obtained the best vigor as demonstrated by the highest average height of plantlets, number of leaves, and roots than other treatments. Thus, 1-day seed hydropriming is the best treatment for embryo rescue and regeneration of wild banana M. acuminata var. tomentosa
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Mohamed, Ehab Mahmoud, Sherief Hashima, Abdallah Aldosary, Kohei Hatano, and Mahmoud Ahmed Abdelghany. "Gateway Selection in Millimeter Wave UAV Wireless Networks Using Multi-Player Multi-Armed Bandit." Sensors 20, no. 14 (July 16, 2020): 3947. http://dx.doi.org/10.3390/s20143947.
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Recently, unmanned aerial vehicle (UAV)-based communications gained a lot of attention due to their numerous applications, especially in rescue services in post-disaster areas where the terrestrial network is wholly malfunctioned. Multiple access/gateway UAVs are distributed to fully cover the post-disaster area as flying base stations to provide communication coverage, collect valuable information, disseminate essential instructions, etc. The access UAVs after gathering/broadcasting the necessary information should select and fly towards one of the surrounding gateways for relaying their information. In this paper, the gateway UAV selection problem is addressed. The main aim is to maximize the long-term average data rates of the UAVs relays while minimizing the flights’ battery cost, where millimeter wave links, i.e., using 30~300 GHz band, employing antenna beamforming, are used for backhauling. A tool of machine learning (ML) is exploited to address the problem as a budget-constrained multi-player multi-armed bandit (MAB) problem. In this setup, access UAVs act as the players, and the arms are the gateway UAVs, while the rewards are the average data rates of the constructed relays constrained by the battery cost of the access UAV flights. In this decentralized setting, where information is neither prior available nor exchanged among UAVs, a selfish and concurrent multi-player MAB strategy is suggested. Towards this end, three battery-aware MAB (BA-MAB) algorithms, namely upper confidence bound (UCB), Thompson sampling (TS), and the exponential weight algorithm for exploration and exploitation (EXP3), are proposed to realize gateways selection efficiently. The proposed BA-MAB-based gateway UAV selection algorithms show superior performance over approaches based on near and random selections in terms of total system rate and energy efficiency.
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Mendes, Rita Ferreira Rocha, and Darcy Ribeiro Castro. "DIVERSIDADE VEGETAL DO RIACHO CANABRAVA NO MUNICÍPIO DE UIBAÍ/BA, A PARTIR DO ETNOCONHECIMENTO DA COMUNIDADE LOCAL." Revista Conhecimento Online 1 (January 2, 2020): 168. http://dx.doi.org/10.25112/rco.v1i0.1641.
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Este estudo trata da diversidade vegetal do riacho Canabrava no município de Uibaí, estado da Bahia. O mesmo buscou compreender o etnoconhecimento da comunidade local sobre a diversidade vegetal do riacho Canabrava, levando em consideração seus aspectos sociais, físicos e culturais, tendo em vista contribuir com o desenvolvimento de ações de preservação e recuperação da flora local. Para tal, foram realizadas entrevistas com 21 moradores da localidade com diferentes faixas etárias (crianças/adolescentes, adultos e idosos). Para a coleta dos dados, utilizou-se um questionário estruturado com oito perguntas, sendo as repostas dos entrevistados categorizadas e discutidas, conforme os referenciais teóricos apresentados. Com isso, foi possível tecer um paralelo entre a diversidade pertencente à localidade em tempos passados e o atual modelo, indicando espécies da flora típicas do local como gameleira, juá, jurema, aroeira, quixaba, embaúba, entre outras, e aquelas que estão ameaçadas de extinção, como a canabrava, o murici, o puçá, a maniçoba, o araçá, a sucupira, entre outras. Esse estudo pode contribuir para o resgate da cultura popular relacionada à diversidade vegetal mediante trabalho de parceria evolvendo a escola e a comunidade.Palavras-chave: Flora local. Preservação. Cultura popular.ABSTRACTThis study deals with the plant diversity in the Canabrava stream in the municipality of Uibaí, Bahia. The same sought to understand the ethnic knowledge of the local community about the plant diversity in the Canabrava stream, taking into account their social, physical and cultural aspects, in order to contribute to the development of conservation actions and recovery of the local flora. To this end, interviews were conducted with 21 local residents with different age groups (children / teenagers, adults and seniors). To collect the data, we used a structured questionnaire with eight questions, and the answers of respondents categorized and discussed, as the theoretical frameworks presented. Therefore, it was possible to make a parallel between the diversity belonging to the town in times past and the current model, indicating species of typical local flora as gameleira, juá, jurema, mastic, quixaba, embaúba, among others and those who are threatened extinction as the canabrava, murici, the dip net, maniçoba, the guava, the sucupira, among others. This study may contribute to the rescue of popular culture related to plant diversity through working partnership evolving the school and the community.Keywords: Local flora. Preservation. Popular culture.
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Kampa, Kerstin M., Tanja Grandl, Sandra Mueller, Emmanuel Normant, Michael Walter, Wolfgang Schuetz, Lothar Kanz, Michael C. Heinrich, and Marcus M. Schittenhelm. "KIT Inhibition Affects Heat Shock Protein (HSP) Activity: A Potential Rescue Mechanism towards KIT Tyrosine Kinase Inhibition and a Rationale for Dual KIT/HSP Inhibition." Blood 114, no. 22 (November 20, 2009): 2753. http://dx.doi.org/10.1182/blood.v114.22.2753.2753.
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Abstract Abstract 2753 Poster Board II-729 Activating mutations of the KIT class III receptor tyrosine kinases are associated with core binding factor leukemias (CBF AML), systemic mastocytosis (SM), gastrointestinal stromal tumors (GIST), melanomas, seminoma/dysgerminoma and sinonasal natural killer/T-cell non-Hodgkin lymphoma. Despite the encouraging therapeutic potential of KIT-tyrosine kinase inhibitors (TKI), resistance leading to disease progression occurs in many patients, specifically after TKI monotherapy. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways which override TKI inhibition. To explore the downstream signaling pathways of class III receptor tyrosine kinases, we performed unbiased phoshoproteomic analyses of mutant FLT3 or KIT leukemia and mastocytosis cell lines before and after TKI treatment. Tantalizingly, immunoaffinity purification of phosphopeptides followed by tandem mass spectrometry following KIT-inhibition with Imatinib at IC90 (100nM) revealed a significant upregulation of phosphorylation levels of peptides identified as members of the heat shock protein (HSP) family. Of interest, mRNA GeneChip® Array analysis of hematopoietic Ba/F3 cells transfected with either a mutant KIT isoform (D816V) or a mutant FLT3 isoform (ITD) and treated with TKI revealed significant downregulation of HSP family members in the FLT3 model – but stable mRNA levels in the KIT model. Taken together, our phosphoproteome and mRNA data suggest a protective function of HSP in mutant-KIT tumors treated with TKI. Next we studied the antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504, a 17-AGG-derivative, in mutant-KIT cell models. IPI-504 potently inhibited proliferation and induced apoptosis with an IC50 of 0.5 up to 5μM depending on the KIT isoform. Importantly, combination of IPI-504 with TKIs resulted in potentiation of the antiproliferative and proapoptotic effects achieved by either drug alone. Antitumor efficacy in combination therapy was observed even at HSP90 inhibitor concentrations that did not display antitumor activity if administered alone. In conclusion, our model suggests that inhibition of KIT affects heat shock protein activity serving to stabilize the functionality of targeted autoactivated receptor tyrosine kinases, which provides a potential mechanism for resistance to TKI therapy. Importantly, we provide a rationale to combine TKI with (low-dose) HSP-inhibitors such as IPI-504 to optimize TKI therapy. Disclosures: Normant: Infinity: Employment.
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Cleyrat, Cédric, Romain Girard, Éric Jeziorski, Thierry Lavabre-Bertrand, Sylvie Hermouet, Serge Carillo, and Bridget S. Wilson. "In Vitro Functional Rescue of a Double MPL K39N/W272R Mutant Associated with Congenital Amegakaryocytic Thrombocytopenia (CAMT) Using Crispr/Cas9." Blood 126, no. 23 (December 3, 2015): 1207. http://dx.doi.org/10.1182/blood.v126.23.1207.1207.
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Abstract Introduction: Thrombopoietin (Tpo) and its receptor, Mpl, are the principal regulators of early and late thrombopoiesis. Mutations in MPL can drastically impair its function and be a contributing factor in chronic thrombocytosis and in congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is a rare inherited syndrome characterized by thrombocytopenia at birth, progressing to bone marrow failure and pancytopenia. The functional impact of CAMT mutations on Mpl signaling/trafficking is yet to be determined and could be relevant to multiple hematologic malignancies. Here we report unique familial cases of CAMT presenting with a previously unreported MPL mutation: T814C (W272R) in the background of the MPL K39N (Baltimore mutation), known to cause hereditary thrombocytosis (HT). Patients: Consanguineous parents and their eldest daughter, all heterozygous for Mpl K39N/W272R, do not present any signs of disease. Their monozygotic twin daughters presented at birth with severe thrombocytopenia (platelet counts: 12,000/L and 14,000/L), low hemoglobin levels (10.4 mg/dL and 7.8 mg/dL) and very high Tpo levels (3,650 pg/mL and 3115 pg/mL). Bone marrow smears performed 19 days after birth showed severe megakaryocytopenia (only 1 megakaryocyte (MK) seen). Bone marrow colony formation assays yielded 3 and 9 MK colonies vs 84 MK colonies/105 mononuclear cells for the control, leading to a diagnosis of CAMT type I. Whole blood sequencing revealed the presence of a homozygous double MPL K39N/W272R mutation. One of the twins died after bone marrow transplant. A younger male sibling, homozygous for MPL K39N/W272R mutation, has also been diagnosed with CAMT type I. Objectives: This study focuses on the functional characterization of the novel MPL W272R and K39N/W272R mutations and in vitro genetic engineering as a potential therapeutic option for CAMT. Methods: Human megakaryoblastic UT-7 and murine Ba/F3 cells stably expressing human wild-type (WT) Mpl or K39N, W272R or doubly mutated K39N/W272R Mpl fused to mNeonGreen were used as models. Confocal microscopy, proliferation and surface biotinylation assays, as well as co-immunoprecipitation (co-IP) and western blotting analysis, were used to elucidate the function and trafficking of Mpl mutants. CRISPR/Cas9 genetic engineering was used to repair mutant MPL and rescue its function. Results: Confocal microscopy shows that a significant fraction of chimeric WT Mpl protein reaches the cell surface. Significant surface expression is also noted for Mpl K39N. In contrast, the chimeric Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, showed no detectable surface expression of the Tpo receptor. These results were confirmed by surface biotinylation assay. Co-expression of WT CALR fused to RFP, used as an ER marker, showed significantly higher co-localization (Pearson's R value) with mutant Mpl than with WT Mpl, evidence that the large majority of receptors were retained within the ER. We also evaluated Tpo signaling through the JAK/STAT, MAPK and PI3K pathways and Tpo-induced proliferation. Both WT and K39N-mutated Mpl were competent for signaling, while single or double mutants bearing W272R were unresponsive to Tpo. Tpo-induced signaling was partially restored via GRASP55 over-expression (forcing ER-trapped Mpl to traffic to the cell surface). Genetic engineering performed on cells carrying the W272R mutation restored the WT sequence and the response to Tpo, with similar cell proliferation as WT Mpl cells. In addition, co-IP studies indicate that Jak2 associates strongly with WT Mpl and Mpl K39N but not with Mpl W272R. Conclusion: We report a new mutation of Mpl (W272R) present in cis with HT-causing K39N mutation in the context of CAMT. The absence of symptoms in the Mpl K39N/W272R-mutated parents (found to be heterozygous) can be explained by the opposite (apparently neutralizing) effects of the two mutations on the trafficking and signaling of Mpl, as shown by confocal microscopy, western blotting and proliferation assays. In children homozygous for Mpl K39N/W272R, the W272R mutation prevented Mpl binding of Jak2 and expression at cell surface, rendering Tpo signaling impossible despite the presence of the K39N mutation. Function of the deficient Mpl receptor could be rescued using two separate approaches: CRISPR/Cas9 genetic engineering and GRASP55 over-expression. Cell-permeable Tpo analogs will also be tested. Disclosures No relevant conflicts of interest to declare.
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Akahane, Koshi, Zhaodong Li, Julia Etchin, Alla Berezovskaya, Evisa Gjini, Craig E. Masse, Wenyan Miao, et al. "Anti-Leukemic Activity of the TYK2 Selective Inhibitor Ndi-031301 in T-Cell Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 1596. http://dx.doi.org/10.1182/blood.v128.22.1596.1596.
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Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from the transformation of T-cell progenitors. Although the prognosis of this disease has substantially improved due to the introduction of intensified chemotherapy, the clinical outcome of T-ALL patients with primary resistant or relapsed disease remains poor, indicating that further therapeutic improvement is urgently needed. We have previously demonstrated that activation of tyrosine kinase 2 (TYK2) contributes to aberrant survival of human T-ALL cells. TYK2 is a member of the Janus-activated kinase (JAK) tyrosine kinase family and our report was the first to implicate TYK2 in T-ALL pathogenesis. Indeed, our gene knockdown experiments showed TYK2 dependency in 14 of 16 (88%) T-ALL cell lines and 5 of 8 (63%) patient-derived T-ALL cells tested, suggesting that inhibition of TYK2 would be beneficial in most patients with T-ALL. Based on these findings, we investigated the therapeutic potential of a novel small-molecule TYK2 kinase inhibitor NDI-031301 in T-ALL. We found that NDI-031301 shows potent and selective inhibitory activity against TYK2 in a cellular context, because this compound strongly inhibited the growth of TYK2-transfomed Ba/F3 cells when compared to the JAK inhibitors tofacitinib and baricitinib, whereas Ba/F3 cells transformed by other tyrosine kinases showed decreased sensitivity to NDI-031301. NDI-031301 induced robust growth inhibition in each of 4 human T-ALL cell lines representing different molecular subtypes of the disease (DU.528, KOPT-K1, HPB-ALL and SKW-3), with IC50 values of 0.8186 - 2.380 μM after 72 hours of exposure. NDI-031301 treatment of human T-ALL cell lines resulted in induction of apoptosis that was not observed with tofacitinib and baricitinib. To elucidate the mechanism of apoptosis induced by NDI-031301 in T-ALL cells, we next investigated cellular signaling pathways that are associated with cell survival and specifically affected by TYK2 inhibition with NDI-031301. Western blotting analysis demonstrated that treatment with 3 μM of NDI-031301 resulted in reduction of STAT1 Tyr-701 phosphorylation and BCL2 levels in KOPT-K1 cells, consistent with our previous finding that TYK2 phosphorylates STAT1 and upregulates BCL2 expression in most T-ALL cells. Surprisingly, the treatment also uniquely led to activation of three mitogen-activated protein kinases (MAPKs), resulting in phosphorylation of ERK, SAPK/JNK and p38 MAPK coincident with PARP cleavage, which was not observed with tofacitinib and baricitinib. NDI-031301-mediated activation of SAPK/JNK and p38 MAPK pathways are likely mediated through inhibition of TYK2, because increased phosphorylation levels of SAPK/JNK and p38 MAPK were observed in the cells transfected with TYK2-targeting shRNAs, while the levels of ERK1/2 phosphorylation were not upregulated. Further investigation revealed that activation of p38 MAPK occurred within 1 hour of NDI-031301 treatment and was responsible for NDI-031301-induced T-ALL cell death, as pharmacologic inhibition of p38 MAPK by SB203580 partially rescued apoptosis induced by TYK2 inhibitor, while inhibition of ERK or SAPK/JNK showed no rescue effects. Finally, we found that daily oral administration of NDI-031301 at 100mg/kg BID to immunodeficient mice engrafted with KOPT-K1 T-ALL cells was well tolerated, and led to decreased tumor burden and a significant survival benefit. After 29 days of treatment, the mice receiving NDI-031301 had marked reductions in infiltration of leukemia cells into spleen and bone marrow by comparison with controls. Thus, our findings clearly support TYK2 inhibition with NDI-031301 or a related compound as a potential therapeutic strategy for patients with T-ALL, and also raise the possibility that enhancing p38 MAPK activation in T-ALL cells may be an approach to accentuate its anti-leukemic activity. Disclosures Masse: Nimbus Therapeutics: Employment. Miao:Nimbus Therapeutics: Employment. Rocnik:Nimbus Therapeutics: Employment. Kapeller:Nimbus Therapeutics: Employment.
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Liu, Qiang, Olga I. Gan, Gabriela Krivdova, Aaron Trotman-Grant, Stephanie M. Dobson, Karin G. Hermans, Mark D. Minden, et al. "Mir-125b Regulates the Self-Renewal of Acute Myeloid Leukemia Stem Cells through PTPN18 and GSK3." Blood 136, Supplement 1 (November 5, 2020): 16–17. http://dx.doi.org/10.1182/blood-2020-141733.
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Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor survival, especially in older patients. Despite high remission rates after chemotherapy, relapse and death are frequent due to persistence of leukemia stem cells (LSCs), which possess properties linked to therapy resistance. Thus, there is an urgent need for a deeper understanding of the unique properties of LSCs. MicroRNAs (miRNAs) are non-coding RNAs that decrease expression of their target mRNAs by post-translational silencing. miRNA profiling of human AML samples fractionated based on LSC activity revealed that miR-125b is expressed at significantly higher levels on cell fractions enriched in LSCs. To evaluate the role of miR-125b in LSCs, expression of miR-125b was enforced in a hierarchical AML model cell line (OCI-AML-8227). miR-125b overexpression (OE) resulted in a significantly lower percentage of CD14+CD15+ differentiated myeloblasts (Figure 1A) and enhanced clonogenic potential in vitro (Figure 1B). Xenotransplantation of four AML patient samples with miR-125b OE revealed a significant increase in the proportion of CD117+ cells, a marker of hematopoietic and leukemic progenitors (Figure 1C). Secondary transplantation of cells harvested from primary engrafted mice at limiting dilution demonstrated a marked increase in LSC frequency with miR-125b OE compared to controls for the two AML samples tested (Figure 1D). Together, these data strongly suggest that miR-125b enhances the self-renewal of LSCs. To investigate the mechanisms by which miR-125b enhances self-renewal, proteomic analysis of miR-125b-OE Ba/F3 cells as well as in silico target prediction were performed and identified PTPN18 as a top putative target for miR-125b. PTPN18 is a tyrosine phosphatase that has been reported to dephosphorylate auto-phosphorylated kinases such as Her2 and Abl to prevent their activation. To evaluate whether PTPN18 OE can rescue the effects miR-125b on LSCs, we carried out transduction of an AML patient sample with control, miR-125b OE, PTPN18 OE, or both miR-125b and PTPN18 OE vectors followed by xenotransplantation. Similar to previous findings, miR-125b OE alone significantly reduced the frequency of CD11b+CD15+ differentiated myeloblasts. Co-transduction of miR-125b/PTPN18 OE vectors resulted in generation of significantly more CD11b+CD15+ cells compared to miR-125b OE alone (Figure 1E), suggesting that suppression of PTPN18 contributes to miR-125b-mediated enhancement of LSC self-renewal. To identify putative phosphotyrosines that might be altered through the miR-125b-PTPN18 signalling axis, we performed immunoprecipitation of phosphotyrosines followed by mass spectrometry in miR-125b-OE Ba/F3 cells and identified increased GSK3 tyrosine phosphorylation as a top target. Additionally, miR-125b OE was confirmed to enhance GSK3 tyrosine phosphorylation, whereas PTPN18 OE reduced it (Figure 1F), together strongly suggesting that miR-125b could enhance tyrosine phosphorylation of GSK3 by silencing PTPN18. GSK3A and GSK3B (GSK3A/B) are paralogous genes that share a high degree of sequence homology and belong to the glycogen synthase kinase 3 (GSK3) family. Tyrosine phosphorylation activates the kinase activity of GSK3, whereas serine phosphorylation inactivates it. We recently identified GSK inhibitors as top candidates targeting LSCs in a stemness-based drug screen using OCI-AML-8227 cells (data not shown). Treatment of OCI-AML-8227 cells with two selective inhibitors of GSK3 selectively reduced the proportion of CD34+ cells while concomitantly increasing expression of myeloid markers CD14 and CD15 (Figure 1G). Overall, our results support an important functional role for PTPN18 and GSK3 in LSC function, and present a potential novel therapeutic target against LSCs. This study highlights the importance of understanding the role of miRNAs and may identify a new druggable vulnerability in LSCs that could lead to the development of new treatment options for AML patients. Figure 1 Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium Therapeutics: Patents & Royalties.
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Krysiak, Kilannin, Justin Tibbitts, and Matthew J. Walter. "Reduced Hspa9 Expression Alters IL-7 Signaling In B-Cells." Blood 122, no. 21 (November 15, 2013): 1569. http://dx.doi.org/10.1182/blood.v122.21.1569.1569.
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Abstract Myeloid and erythroid differentiation defects and cytopenias are most commonly described in myelodysplastic syndromes (MDS), however, a reduction in B-cell progenitors exists. The genetic events contributing to this reduction are poorly understood. Interstitial deletion or loss of one copy of the long arm of chromosome 5 (del5q) is the most common cytogenetic abnormality associated with MDS. Two commonly deleted regions on del(5q) have been described and no biallelic mutations have been identified implicating haploinsufficiency of genes on this interval as a driving mechanism. We, and others, have identified several del(5q) candidate genes, including RPS14, EGR1, CTNNA1, APC, NPM1, DIAPH1, miR145, miR146a, and HSPA9. Consistent with haploinsufficiency, HSPA9 mRNA levels are 50% reduced in del(5q) patients. We previously showed that knockdown of Hspa9by shRNA in a murine bone marrow transplant model resulted in a significant reduction in murine B-cells in the bone marrow, spleen and peripheral blood. To further characterize the role of Hspa9 in hematopoiesis, we created Hspa9 heterozygous mice (Hspa9+/-). Heterozygotes express 50% less Hspa9 protein and are born at normal Mendelian frequencies (N>100). No significant differences in mature lineage markers, complete blood counts, and hematopoietic organ cellularity, have been identified up to 12 months of age. However, as early as 2 months of age, Hspa9+/- mice show a significant reduction in CFU-PreB colonies compared to their wild-type littermates, indicating B-cell progenitor defects (14 vs. 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p<0.001). Following long-term engraftment of transplanted bone marrow cells from Hspa9+/-or littermate controls into lethally irradiated recipients, we also observed a 5.8-fold reduction in bone marrow CFU-PreB colonies (N=7-9 mice/genotype, p=0.002), confirming the B-cell progenitor defect is hematopoietic cell-intrinsic. Despite the reduction in CFU-PreB colony numbers, frequencies of freshly isolated early B-cell progenitor and precursor populations in the bone marrow and spleen of Hspa9+/- mice are not different than wild-type littermate controls when assessed by flow cytometry (common lymphoid progenitor, Hardy fractions A-F). We hypothesized that these mice were able to compensate for B-cell alterations caused by loss of Hspa9 in vivo. Consistent with our hypothesis, the reduction in CFU-PreB colony numbers was partially rescued by increasing the concentration of IL-7 in the media. Hspa9+/- colony numbers increased 1.8 fold when the IL-7 concentration was increased from 10ng/mL to 50ng/mL compared to 0.80 fold for wild-type littermates (p=0.03, N=6 mice/genotype). This effect was unique to IL-7. Adding increasing concentrations of Flt-3 ligand, another cytokine that contributes to early B-cell development, did not alter CFU-PreB colony formation. We isolated B220+ cells from Day 7 CFU-PreB cultures for gene expression array analysis and observe reduced expression of genes promoting B-cell proliferation and activation in Hspa9+/- compared to Hspa9+/+ cells. Since IL-7 is the only supportive cytokine in the methylcellulose media, can partially rescue the reduced CFU-PreB phenotype, and is required for early B-cell development and survival, we hypothesized that Hspa9 haploinsufficiency inhibits transduction of IL-7 signaling. We tested this hypothesis using an IL-7 dependent mouse B-cell line (B7 cells; Ba/F3 cells that stably express the IL-7 receptor). Knockdown of Hspa9 by siRNAs resulted in a 8-fold reduction in cell number after 4 days in culture (p=0.004, confirmed with two independent siRNAs) and was associated with an increase in apoptosis and reduction in cells in S-phase of the cell cycle. Knockdown of Hspa9 in B7 cells resulted in reduced levels of phosphorylated Stat5, an immediate downstream target of IL-7 receptor stimulation, compared to cells treated with a non-targeting siRNA (measured at 5, 10, 15 and 30 minutes following 10ng/mL IL-7 stimulation, p≤0.03). Ongoing studies will further interrogate the effects of Hsap9 knockdown on Jak-Stat signaling. Collectively, these data implicate that loss of HSPA9 alters IL-7 signaling, potentially contributing to the reduction of B-cell progenitors observed in patients with del(5q)-associated MDS. Disclosures: No relevant conflicts of interest to declare.
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Brack, Alexandra R., Johannes M. Dijkstra, Harald Granzow, Barbara G. Klupp, and Thomas C. Mettenleiter. "Inhibition of Virion Maturation by Simultaneous Deletion of Glycoproteins E, I, and M of Pseudorabies Virus." Journal of Virology 73, no. 7 (July 1, 1999): 5364–72. http://dx.doi.org/10.1128/jvi.73.7.5364-5372.1999.
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ABSTRACT Glycoprotein M (gM), the product of the UL10 gene of pseudorabies virus (PrV), is one of the few nonessential glycoproteins conserved throughout the Herpesviridae. In contrast to wild-type PrV strains, the UL10 gene product of the attenuated PrV vaccine strain Bartha (PrV-Ba) is not modified by N-glycans due to a mutation in the DNA sequence encoding the consensus N-glycosylation motif. To assay function of the UL10 protein in PrV-Ba, a UL10-deletion mutant (PrV-Ba-UL10−) was isolated. Surprisingly, in contrast to gM-deleted wild-type PrV, PrV-Ba-UL10− was severely impaired in plaque formation, inducing only foci of very few infected RK13, Vero, and PSEK cells and tiny plaques on MDBK cells. Since this effect was significantly more dramatic than in wild-type PrV, additional mutations known to be present in PrV-Ba were analyzed for their contribution to this phenotype.trans-complementation of the mutated PrV-Ba UL21 or gC protein by the wild-type version had no influence on the observed phenotype. In contrast, complementation of the gE/gI deletion rescued the phenotype. The synergistic effect of deletions in gE/gI and gM on plaque size was verified by construction of a gE/I/M triple mutant derived from wild-type PrV which exhibited the same phenotype. The dramatic effect of deletion of gM on plaque size in a gE/I− virus background was mainly attributable to a function of gM, and not of the gM/gN complex, as shown by analysis of a gE/I/N triple mutant. Interestingly, despite the strong effect on plaque size, penetration was not significantly impaired. In noncomplementing cells infected with the gE/I/M triple mutant, electron microscopy showed absence of secondary envelopment in the cytoplasm but occurrence of intracytoplasmic accumulations of nucleocapsids in association with electron dense material, presumably tegument proteins. These structures were not observed after infection of cells expressing either gE/I or gM. We suggest that gE/I and gM are required for late stages in virion morphogenesis prior to final envelopment in the cytoplasm.
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Hazera, Alejandro, Carmen Quirvan, and Salvador Marin-Hernandez. "The impact of guaranteed bailout assistance on bank loan overstatement." International Journal of Managerial Finance 12, no. 2 (April 4, 2016): 177–210. http://dx.doi.org/10.1108/ijmf-04-2014-0046.
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Purpose – The purpose of this paper is to highlight how the basic binomial option pricing model (BOPM) might be used by regulators to help formulate rules, prior to financial crisis, that help prevent loan overstatement by banks in emerging market economies undergoing financial crises. Design/methodology/approach – The paper draws on the theory of soft budget constraints (SBC) to construct a simple model in which banks overstate loans to minimize losses. The model is used to illustrate how guarantees of bailout assistance (BA) (to banks) by crisis stricken countries’ financial authorities may encourage banks to overstate loans and delay the implementation of IFRS for loan valuation. However, the model also illustrates how promises of BA may be depicted as binomial put options which provide banks with the option of either: reporting loan values on poor projects accurately and receiving the loans’ liquidation values; or, overstating loans and receiving the guaranteed BA. An illustration is also provided of how authorities may use this representation to help minimize bank loan overstatement in periods of financial crisis. In order to provide an illustration of how the option value of binomial assistance may evolve during a financial crisis, the model is generalized to the Mexican financial crisis of the late 1990s. During this period, Mexican authorities’ guarantees of BA to the nation’s largest banks encouraged those institutions to overstate loans and delay the implementation of (previously adopted) international “best practices” based loan valuation standards. Findings – Application of the model to the Mexican financial crisis provides evidence that, in spite of Mexico’s “official” 1997 adoption of international “best accounting practices” for banks, “iron clad” guarantees of BA by the country’s financial authorities to Mexico’s largest banks provided those institutions with an incentive to knowingly overstate loans in the late 1990s and early 2000s. Research limitations/implications – The model is compared against only one country in which the BA was directly infused into banks’ loan portfolios. Thus, as conceived, it is directly applicable to crisis countries in which the bailout took this form. However, the many quantitative variations of SBC models as well as recent studies which have applied the binomial model to other forms of bailout (e.g. direct purchases of bank shares by authorities) suggest that the model could be modified to accommodate different bailout scenarios. Practical implications – The model and application show that guaranteed BA can be viewed as a put option and that ex-ante regulatory policies based on the correct valuation of the BA as a binomial option might prevent banks from overstating loans. Social implications – Use of the binomial or similar approaches to valuing BA may help regulators to determine the level of BA that will not encourage banks to overstate the value of their loans. Originality/value – Recent research has used the BOPM to value, on an ex-post basis, the BA which appears on the balance sheet of institutions which have been rescued. However, little research has advocated the use of this type of model to help prevent, on an ex-ante basis, the overstatement of loans on poor projects.
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Chhunchha, Bhavana, Prerna Singh, Dhirendra Singh, and Eri Kubo. "Ginkgolic Acid Rescues Lens Epithelial Cells from Injury Caused by Redox Regulated-Aberrant Sumoylation Signaling by Reviving Prdx6 and Sp1 Expression and Activities." International Journal of Molecular Sciences 19, no. 11 (November 8, 2018): 3520. http://dx.doi.org/10.3390/ijms19113520.
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Sumoylation is a downstream effector of aging/oxidative stress; excess oxidative stress leads to dysregulation of a specificity protein1 (Sp1) and its target genes, such as Peroxiredoxin 6 (Prdx6), resulting in cellular damage. To cope with oxidative stress, cells rely on a signaling pathway involving redox-sensitive genes. Herein, we examined the therapeutic efficacy of the small molecule Ginkgolic acid (GA), a Sumoylation antagonist, to disrupt aberrant Sumoylation signaling in human and mouse lens epithelial cells (LECs) facing oxidative stress or aberrantly expressing Sumo1 (small ubiquitin-like modifier). We found that GA globally reduced aberrant Sumoylation of proteins. In contrast, Betulinic acid (BA), a Sumoylation agonist, augmented the process. GA increased Sp1 and Prdx6 expression by disrupting the Sumoylation signaling, while BA repressed the expression of both molecules. In vitro DNA binding, transactivation, Sumoylation and expression assays revealed that GA enhanced Sp1 binding to GC-boxes in the Prdx6 promoter and upregulated its transcription. Cell viability and intracellular redox status assays showed that LECs pretreated with GA gained resistance against oxidative stress-driven aberrant Sumoylation signaling. Overall, our study revealed an unprecedented role for GA in LECs and provided new mechanistic insights into the use of GA in rescuing LECs from aging/oxidative stress-evoked dysregulation of Sp1/Prdx6 protective molecules.
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Sigurdsson, Valgardur, Hajime Takei, Svetlana Soboleva, Visnja Radulovic, Roman Galeev, Kavitha Siva, L. M. Fredrik Leeb-Lundberg, Takashi Iida, Hiroshi Nittono, and Kenichi Miharada. "Bile Acids Protect Expanding Hematopoietic Stem Cells from Unfolded Protein Stress in Fetal Liver." Blood 126, no. 23 (December 3, 2015): 897. http://dx.doi.org/10.1182/blood.v126.23.897.897.
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Abstract Hematopoietic stem cells (HSCs) are effectively expanded in fetal liver (FL), while they are maintained in a dormant state in adult bone marrow (BM). However, developmental mechanisms allowing this have not been fully explained. BM-HSCs have the lowest protein synthesis rate within the blood hierarchy, even under forced self-renewal divisions. In addition, HSCs are vulnerable to and quickly activate endoplasmic reticulum (ER) stress responses fueled by accumulation of unfolded / misfolded proteins (Miharada et al., Cell Rep. 2014). Of note, we have seen that FL-HSCs have low levels of ER stress related genes despite their high proliferation status without an increase in heat shock protein levels, strongly indicating that other factor(s) block ER stress elevation. This raises the question how HSCs deal with the higher protein-folding requirement during expansion in the FL. Here we demonstrate that bile acids (BAs) are required to eliminate ER stress in the FL and are essential for proper expansion of FL-HSCs. Measurement of protein synthesis rate using OP-puro incorporation revealed that protein synthesis was enhanced in FL-HSCs, whereas BM-HSCs have half the rate of other populations in BM. Mass spectrometry analyses showed that BAs in the FL were all taurine conjugated while 30% of BA in the adult liver was taurine-conjugated, and the main proportion was taurocholic acid (TCA) that is known for its low toxicity. In the FL we also detected secondary BAs (e.g. TDCA), requiring intestinal bacteria in the production process, suggesting that FL BAs are a mixture of fetal and maternal BAs. Reduction of BA levels using GW4064, a chemical inhibitor of BA synthesis, significantly decreased the number of HSCs (6.6 fold decrease compared to vehicle treatment). This decrease was due to increased apoptosis caused by elevated ER stress levels. Similarly, dual deletion of Cyp27a1, a key BA synthetic enzyme, in both mother and fetus severely decreased total cellularity (2.0 fold decrease compared to littermate heterozygotes) and number of HSCs (6.8 fold decrease) in FL due to increased ER stress and subsequent apoptosis. Interestingly, FL of homozygotes grown in heterozygous mothers did not show any significant differences compared to littermate heterozygotes, suggesting that the contribution of maternal BA in FL is critical for HSCs. In both models, ER stress-oriented apoptosis and reduction in cellularity were most pronounced within the HSC population, indicating that stem cells are particularly sensitive to BA levels during development in FL. Importantly, injection of TCA or Salubrinal, an ER stress inhibitor, rescued the effects of BA reduction in both models. These data strongly suggest that BAs are required to block ER stress elevation in expanding FL-HSCs. ER stress and protein aggregation are closely linked together in number of pathological diseases like AlzheimerÕs- and HuntingtonÕs disease. Quantification of aggregated proteins (aggresomes) revealed that Cyp27a1 KO FL-HSCs from homozygote mothers contained significantly higher amount of aggresomes (2.0 fold), while KO FL-HSCs from heterozygote mothers showed no increase. Higher levels of aggregated proteins were most pronounced within the HSC population and BA suppressed formation of aggresomes during in vitro culture. This leads to reduction of ER stress and the maintenance of functional HSCs. Finally, transplantation assay showed that TCA can support functional HSCs ex vivo for up to 14 days. These findings propose a novel role for BA as a critical part of fetal hematopoiesis supporting expansion of HSC. Maternal and fetal BA coordinately contribute to this natural chaperone regulation. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.
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Wasag, Bartosz, Els Lierman, Peter Meeus, Jan Cools, and Peter Vandenberghe. "The Kinase Inhibitor TKI258 Is Active Against the Novel CUX1-FGFR1 Fusion Detected In a Patient with T-Lymphoblastic Leukemia/Lymphoma and t(7;8)(q22;p11)." Blood 116, no. 21 (November 19, 2010): 2715. http://dx.doi.org/10.1182/blood.v116.21.2715.2715.
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Abstract Abstract 2715 The 8p11 myeloproliferative syndrome (EMS) is an aggressive, atypical stem cell disorder associated with chromosome translocations that constitutively activate FGFR1 by fusion to diverse partner genes. Here, we describe a case with a clinical and hematological diagnosis of T-lymphoblastic leukemia/lymphoma and with a t(7;8)(q22;p11) on cytogenetic analysis. We identified the fusion partner involved and characterized this translocation functionally in vitro using the interleukin 3 (IL3) dependent Ba/F3 cell line. The translocation was analyzed in more detail by FISH using FGFR1 flanking probes. We could confirm the 8p11 breakpoint and 7q as the partner chromosome. Using 5'-RACE CUX1 (7q22) was identified as the fusion partner of FGFR1 in this patient with T-lymphoblastic leukemia/lymphoma. CUX1 is a homeobox family DNA binding protein not previously described as a fusion partner in hematological malignancies. To evaluate the transforming potential of this novel fusion, the CUX1-FGFR1 fusion was cloned and used to transform Ba/F3 cells. CUX1-FGFR1 expressing Ba/F3 cells displayed IL3 independent proliferation thus demonstrating the oncogenic character of this fusion protein. Western blotting of the transformed Ba/F3 cells showed activation of FGFR1 as well as its downstream target STAT5. Treatment of the CUX1-FGFR1 expressing Ba/F3 cells with the kinase inhibitors PKC412 and TKI258 significantly inhibited cell growth with an IC50 of 483 and 489 nM respectively. With western blotting a direct effect of both inhibitors on FGFR1 kinase activity as well as on different downstream effectors was proven. Furthermore using an annexinV/propidium iodide-based apoptosis assay, we could show that PKC412 and TKI258 both induced apoptosis followed by cell death in inhibitor treated CUX1-FGFR1 transformed Ba/F3 cells. The antiproliferative effect of the inhibitors could be rescued by addition of IL3 for the TKI258 treated but not for PKC412 treated CUX1-FGFR1 expressing cells. This observation indicates a selective action of TKI258 on FGFR1 signaling at the concentrations used. In contrast, for PKC412 non-specific cytotoxicity is also contributing to the antiproliferative effect. In summary, we identified a novel CUX1-FGFR1 fusion in a case with EMS and a novel t(7;8)(q22;p11), and demonstrated the oncogenic potential of CUX1-FGFR1 in the Ba/F3 cell line. This new fusion partner CUX1 contains a potential coiled coil domain that can explain the observed constitutive FGFR1 activation, as has been elaborately demonstrated for other oncogenic kinase fusions. The in vitro data presented here using the inhibitor TKI258 support the use of this compound for the treatment of the novel CUX1-FGFR1 fusion as well as other constitutively active FGFR1 fusion proteins. Disclosures: No relevant conflicts of interest to declare.
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Jensen, Lasse D., Masaki Nakamura, Lars Bräutigam, Xuri Li, Yizhi Liu, Nilesh J. Samani, and Yihai Cao. "VEGF-B-Neuropilin-1 signaling is spatiotemporally indispensable for vascular and neuronal development in zebrafish." Proceedings of the National Academy of Sciences 112, no. 44 (October 19, 2015): E5944—E5953. http://dx.doi.org/10.1073/pnas.1510245112.
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Physiological functions of vascular endothelial growth factor (VEGF)-B remain an enigma, and deletion of the Vegfb gene in mice lacks an overt phenotype. Here we show that knockdown of Vegfba, but not Vegfbb, in zebrafish embryos by specific morpholinos produced a lethal phenotype owing to vascular and neuronal defects in the brain. Vegfba morpholinos also markedly prevented development of hyaloid vasculatures in the retina, but had little effects on peripheral vascular development. Consistent with phenotypic defects, Vegfba, but not Vegfaa, mRNA was primarily expressed in the brain of developing zebrafish embryos. Interestingly, in situ detection of Neuropilin1 (Nrp1) mRNA showed an overlapping expression pattern with Vegfba, and knockdown of Nrp1 produced a nearly identically lethal phenotype as Vegfba knockdown. Furthermore, zebrafish VEGF-Ba protein directly bound to NRP1. Importantly, gain-of-function by exogenous delivery of mRNAs coding for NRP1-binding ligands VEGF-B or VEGF-A to the zebrafish embryos rescued the lethal phenotype by normalizing vascular development. Similarly, exposure of zebrafish embryos to hypoxia also rescued the Vegfba morpholino-induced vascular defects in the brain by increasing VEGF-A expression. Independent evidence of VEGF-A gain-of-function was provided by using a functionally defective Vhl-mutant zebrafish strain, which again rescued the Vegfba morpholino-induced vascular defects. These findings show that VEGF-B is spatiotemporally required for vascular development in zebrafish embryos and that NRP1, but not VEGFR1, mediates the essential signaling.
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Jin, Liqing, Haruhiko Asano, and C. Anthony Blau. "Stimulating Cell Proliferation Through the Pharmacologic Activation of c-kit." Blood 91, no. 3 (February 1, 1998): 890–97. http://dx.doi.org/10.1182/blood.v91.3.890.
Full textAbstract:
Abstract Previous studies have shown that expression of a membrane targeted chimeric protein containing the erythropoietin receptor (EpoR) cytoplasmic domain fused to the FK506-binding peptide FKBP12 allowed Ba/F3 cells to be rescued from interleukin-3 (IL-3) deprivation using a dimeric form of FK506, called FK1012. In this report, a similar approach is applied to the c-kit receptor. Expression of a membrane targeted fusion protein containing the c-kit receptor linked to one or more copies of FKBP12 allowed Ba/F3 cells to be switched from IL-3 dependence to FK1012-dependence. Similar results were obtained using an alternative dimerizer of FKBP12 domains called AP1510. Pharmacologic dimerization of chimeric proteins containing only a single FKBP12 domain confirmed that receptor dimerization is sufficient for proliferative signaling. Interestingly, while the proliferative effects of both FK1012 and AP1510 were reversible, FK1012-driven proliferation persisted for several days after drug withdrawal. Furthermore, much higher concentrations of FK506 were required to inhibit FK1012-mediated proliferation than were required to inhibit AP1510-mediated proliferation. The persistence of FK1012's effect appeared to be specific to clones expressing c-kit–containing fusion proteins. These results suggest that pharmacologically-responsive fusion proteins containing c-kitmay be useful for specifically and reversibly expanding genetically modified hematopoietic cell populations.
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Jin, Liqing, Haruhiko Asano, and C. Anthony Blau. "Stimulating Cell Proliferation Through the Pharmacologic Activation of c-kit." Blood 91, no. 3 (February 1, 1998): 890–97. http://dx.doi.org/10.1182/blood.v91.3.890.890_890_897.
Full textAbstract:
Previous studies have shown that expression of a membrane targeted chimeric protein containing the erythropoietin receptor (EpoR) cytoplasmic domain fused to the FK506-binding peptide FKBP12 allowed Ba/F3 cells to be rescued from interleukin-3 (IL-3) deprivation using a dimeric form of FK506, called FK1012. In this report, a similar approach is applied to the c-kit receptor. Expression of a membrane targeted fusion protein containing the c-kit receptor linked to one or more copies of FKBP12 allowed Ba/F3 cells to be switched from IL-3 dependence to FK1012-dependence. Similar results were obtained using an alternative dimerizer of FKBP12 domains called AP1510. Pharmacologic dimerization of chimeric proteins containing only a single FKBP12 domain confirmed that receptor dimerization is sufficient for proliferative signaling. Interestingly, while the proliferative effects of both FK1012 and AP1510 were reversible, FK1012-driven proliferation persisted for several days after drug withdrawal. Furthermore, much higher concentrations of FK506 were required to inhibit FK1012-mediated proliferation than were required to inhibit AP1510-mediated proliferation. The persistence of FK1012's effect appeared to be specific to clones expressing c-kit–containing fusion proteins. These results suggest that pharmacologically-responsive fusion proteins containing c-kitmay be useful for specifically and reversibly expanding genetically modified hematopoietic cell populations.
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Benke, Ashwini P., Ram Krishna, Roshni R. Samarth, Shweta S. Dhumal, Waquar A. Ansari, Poonam V. Shelke, Somnath S. Dukare, and Major Singh. "Development of an embryo germination protocol for shy-seeded grape (Vitis vinifera L.)." Plant Genetic Resources: Characterization and Utilization 19, no. 3 (June 2021): 252–60. http://dx.doi.org/10.1017/s1479262121000307.
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AbstractAcquisition and germination of seeds are the most desired targets for the improvement of vegetatively propagated crops. In the present study, we developed a potential embryo germination protocol for the Red Globe grape cultivar having a low seed germination rate. Three grape berries at different developmental stages, viz. 50, 60 and 70 days after flowering (DAF), were selected for in-vitro embryo germination. Three growth media, namely Emershad and Ramming (ER), Nitsch and Nitsch (NN) and Murashige and Skoog (MS), and plant growth regulators (benzyl amino purine (BA), 0.5, 0.7 and 0.9 mg/l; indole butyric acid (IBA), 1.0, 1.5 and 2.0 mg/l; and gibberellic acid (GA), 0.1, 0.3 and 0.9 mg/l) were screened individually in different combinations with three amino acids, namely cysteine, glutamine and proline (2.0 μmol/l each). The maximum embryos germination percentage recorded at 70 DAF was 63.33, 47.78 and 45.56% in ER, NN and MS media, respectively, supplemented with 0.9 mg/l BA, 2.0 mg/l IBA, 0.9 mg/l GA and 2.0 μmol glutamine. Glutamine was found to have the most significant impact, and it improved the rescued embryos germination. The present study provides a potential recipe for a medium that can facilitate efficient germination of grape embryos.
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AL Assaf, Carla, Petros Papadopoulos, Laura Guttierez, Sanne Smits, Carlos Graux, Jan Emmerechts, Els Lierman, Timothy Devos, Lucienne Michaux, and Peter Vandenberghe. "MPL p.S204P Is a Recurrent Mutation in Essential Thrombocythemia." Blood 126, no. 23 (December 3, 2015): 2837. http://dx.doi.org/10.1182/blood.v126.23.2837.2837.
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Abstract Introduction: The JAK2 p.V617F, MPL p.W515K/L and CALR indels occur in a mutually exclusive pattern in 80-90% of cases with Essential Thrombocythemia (ET), but the driver mutations are unknown in the remaining 10-20%. In this study we aimed to identify driver mutations in the latter group of triple negative (TN) ET by exome sequencing of 10 such cases. Results: We found 27 somatic variants, including indels, in 6 out of 10 TN ET patients (range: 1-10 mutations/case; mean: 2,7 mutations/case), none of which were recurrent. In one case, we found a MPL c.610T>C (p.S204P) mutation, which is located in the extracellular domain of the MPLreceptor. By Sanger sequencing of MPL exon 4 in 20 additional TN ET cases, an additional patient with the MPL S204P mutation was identified. Moreover, this mutation was previously reported in one case with idiopathic myelofibrosis1. In order to study the effect of this mutation on the function of MPL, we produced stable Ba/F3 cell lines expressing MPL S204P, MPL W515K or MPL WT, and assessed the dependence of their growth on exogenous thrombopoietin (TPO). Only MPL W515K transduced Ba/F3 cells proliferated in the absence of TPO, but growth of MPL S204P Ba/F3 and of MPL WT Ba/F3 could be rescued by exogenous TPO, indicating the proper surface expression and the functionality of the transduced receptors. The levels of phospho-JAK2 and phospho-STAT5 were low in cytokine-deprived MPL S204P cells but increased upon TPO stimulation. In contrast, phospho-JAK2 and phospho-STAT5 were detectable in MPL W515K transduced Ba/F3 in the absence of cytokines as assessed by Western blotting. Culture of MPL S204P transduced Ba/F3 in the presence of TPO over a range of concentrations (0,01-10 ng/ml) yielded growth curves comparable with MPL WT transduced Ba/F3. Using flow cytometry, we also explored cell surface marker expression on peripheral blood platelets from the two MPL S204P ET patients. Data were compared with healthy donors or ET patients with JAK2 or CALR mutations. MPL S204P ET platelets displayed higher expression of CD61 than platelets from healthy donors or from JAK2 or CALR mutated ET (p<0,01). In addition, there was a trend for higher expression of KIT, CD36 and CD42b on platelets from the MPL S204P ET cases. Moreover, following platelet activation through the protease activated receptor 1, the degranulation response of platelets from MPL S204P ET was decreased in comparison with JAK2 or CALR mutated ET. Conclusion: The MPL S204P mutation is a recurrent mutation in TN ET, with a frequency of 7% (2/30) in this series, but this mutation does not induce TPO-independent growth nor increased TPO-sensitivity in Ba/F3 cells. However, preliminary phenotypic and functional evidence supports the notion that MPL S204P platelets display specific characteristics as compared with JAK2 or CALR mutated ET. The mechanisms by which the MPL S204P mutation influences megakaryopoiesis and platelet function remain to be elucidated. 1. Williams DM, et al. Phenotypic variations and new mutations in JAK2 V617F-negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis. Exp Hematol 2007; 35: 1641. Disclosures Graux: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Sigurdsson, Valgardur, Hajime Takei, Svetlana Soboleva, Takashi Iida, Hiroshi Nittono, and Kenichi Miharada. "Taurine-Conjugated Bile Acids Protect Expanding Hematopoietic Stem/Progenitor Cells from Unfolded Protein Stress As Natural Chaperones." Blood 124, no. 21 (December 6, 2014): 4318. http://dx.doi.org/10.1182/blood.v124.21.4318.4318.
Full textAbstract:
Abstract Hematopoietic stem cells (HSCs) give rise to all lineages of hematopoietic cells in the body for entire life span and are thus protected from risk factors by multiple defense systems. We have recently discovered that HSCs are highly susceptible to stress caused by accumulation of mis-/un-folded proteins, so called endoplasmic reticulum (ER) stress upon enhanced growth conditions, and addition of a specific type of bile acid (BA), Tauroursodeoxycholic acid (TUDCA), known as a chemical chaperone can maintain functional murine HSCs for 2 weeks in vitro, by reducing ER stress (Miharada et al., Cell Rep. 2014). This work depicts the importance of proper protein quality control in HSC maintenance, particularly during the expansion. HSCs are kept in dormant state in the adult body, but actively expanding in the fetal liver. BAs are synthesized from cholesterol in the liver. Interestingly, bile acid synthesis is highly up-regulated in the fetal liver during embryogenesis and the composition of fetal BAs gradually reduces after birth. In addition, composition of bile acids in the fetus is different from adult liver, with the vast majority of fetal BAs are of Taurine-conjugated form that is more stable and non-toxic. Of note, hematopoietic cells and hepatocytes producing BAs are in close contact in the fetal liver and HSCs are therefore exposed to BAs, whereas the adult liver has anatomically isolated bile duct structures that separate blood flow and bile flow. However the role for these fetal BAs has been unknown. Here we report that bile acids support expansion of hematopoietic stem and progenitor cells (HSPCs) in the fetal liver and ex vivo. Since TUDCA is a rare component in human and mouse BAs, even in the fetal liver, we sought analogue(s) that similarly function as ER stress inhibitors. We identified that Taurocholic acid (TCA), one of the main components of fetal BA, and Tauro-alpha-muricholic acid (TαMCA) that is a rodent specific BA have a potential to reduce ER stress, similar to TUDCA. Mouse HSCs cultured with TCA or TαMCA in vitro for 2 weeks showed a robust increase in the reconstitution level compared to non-treated cells (14-fold, n=14, p<0.001 and 13-fold, n=9, p<0.05, respectively), which has comparable or even better potential to support HSC function than TUDCA. To study physiological roles of BA in ER stress reduction and HSC expansion in the fetal liver, an inhibitor of BA synthesis, GW4064 (an agonist of a nuclear receptor FxR that negatively regulates key enzymes in the BA biosynthesis, CYP7A1 and CYP8B1), was intraperitoneally injected into pregnant mice. E16.5 fetuses derived from GW4064-injected pregnant mice showed severe decrease in the number of HSPCs (0.40-fold, n=28-33, p<0.001) in the fetal liver, due to increased apoptosis triggered by elevated ER stress levels. Importantly, co-injection of TCA or Salubrinal (inhibitor of the ER stress-induced apoptosis signal) rescued the effects of GW4064 on cellularity of the fetal liver and levels of ER stress, confirming that the phenotype seen here is due to increased ER stress resulting from lowered levels of BA. Analyses of CYP27A1 knockout (KO) mice that have reduced BA synthesis observed decreased HSC number and increased ER stress in the fetal liver, whereas CYP8B1 KO mice that have increased Tα/βMCA synthesis instead of lack of TCA didn’t show any difference. These findings strongly suggest that fetal BA, particularly TCA and TαMCA, supports the expansion of HSPCs in the fetal liver and the ex vivo culture as chemical chaperones by lowering ER stress levels. Our findings propose a new role of bile acids in hematopoiesis as natural chaperones and provide a novel connection between hematopoiesis and fetal liver. Disclosures No relevant conflicts of interest to declare.
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Inoue, Daichi, Chew Guo-Liang, Bo Liu, Stanley C. Lee, Brittany C. Michel, Joey Pangallo, Andrew R. D'Avino, et al. "Spliceosomal Disruption of the Non-Canonical SWI/SNF Chromatin Remodeling Complex in SF3B1 Mutant Leukemias." Blood 134, Supplement_1 (November 13, 2019): 637. http://dx.doi.org/10.1182/blood-2019-124616.
Full textAbstract:
Mutations in the RNA splicing factor SF3B1 are common in MDS and other myeloid malignancies. SF3B1 mutations promote expression of mRNAs that use an aberrant, intron proximal 3' splice site (ss). Despite the consistency of this finding, linking aberrant splicing changes to disease pathogenesis has been a challenge. Here we identify aberrant splicing and downregulated expression of BRD9, a member of the recently described ATP-dependent non-canonical BAF (ncBAF) chromatin remodeling complex, across SF3B1 mutant leukemias. In so doing, we identify a novel role for altered ncBAF function in hematopoiesis and MDS. To systematically identify functionally important aberrant splicing events created by mutant SF3B1, we integrated differential splicing events in SF3B1 mutant versus wild-type MDS with a positive enrichment CRISPR screen mimicking splicing changes induced by mutant SF3B1 that promote NMD (non-sense mediated mRNA decay). We tested whether loss of any gene functionally inactivated by SF3B1 mutations promoted transformation of Ba/F3 and 32D cells. This identified a specific NMD-inducing aberrant splicing event in BRD9 which promoted cytokine independence (Fig. A) and exhibited striking aberrant splicing across CLL and MDS and across all mutational hotspots in SF3B1 (Fig. B). SF3B1 mutations cause exonization of a normally intronic sequence in BRD9, resulting in inclusion of a poison exon that interrupts BRD9's reading frame (Fig. C) and reduced BRD9 mRNA and protein expression through NMD (Fig. D). We confirmed that mutant SF3B1 suppressed full-length BRD9 levels without generating truncated BRD9 protein. Loss of BRD9 impaired ncBAF complex formation as indicated by abolished interaction between the ncBAF specific component GLTSCR1 and the ATPase subunit BRG1 upon chemical or spliceosomal BRD9 ablation (Fig. D). Given that prior work has linked mutant SF3B1 to use of aberrant 3' ss, we sought to understand the molecular basis for aberrant exon inclusion in BRD9 by mutant SF3B1. Lariat sequencing of SF3B1 mutant versus WT K562 cells and BRD9 minigene analyses identified use of a deep intronic branchpoint adenosine by mutant SF3B1 to promote BRD9 poison exon inclusion (Fig. E). The data above suggest a role for BRD9 downregulation in SF3B1 mutant leukemia. While prior work indicated that BRD9 is required in MLL-rearranged AML (Hohmman et al. Nature Chemical Biology 2016), the role of BRD9 in normal hematopoiesis or other subtypes of myeloid neoplasms has not been evaluated. Genetic downregulation of BRD9 in normal human hematopoietic progenitors from cord blood promoted myelopoiesis while impairing megakaryopoiesis. Interestingly and unexpectedly, BRD9 loss in CD34+ cells promoted terminal erythroid differentiation in vitro. To further evaluate BRD9's role in hematopoiesis in vivo, we also generated mice with inducible knockout of the bromodomain of BRD9 (required for BRD9 function) and generation of a frameshift transcript resulting in reduced Brd9 expression (Fig. F). Loss of Brd9 resulted in macrocytosis with bone marrow erythroid dysplasia in a dosage-dependent manner, along with impaired lymphopoiesis and myeloid skewing. Moreover, competitive transplantation of hematopoietic precursors from these mice revealed that ablation of Brd9 function impaired lymphoid reconstitution while promoting advantage of myeloid cells and hematopoietic precursors (Fig. G-I). In myeloid leukemia cells, introduction of SF3B1K700E or downregulation of BRD9 resulted in increased chromatin accessibility at promoters with a significant overlap in commonly upregulated genes. This finding suggests shared epigenetic effects of SF3B1K700E mutations and BRD9 loss (Fig. J). These data identify aberrant splicing of BRD9 across the spectrum of SF3B1 mutant cancers and identify a novel role for downregulation of ncBAF function in MDS pathogenesis. Consistent with human genetic data, genetic ablation of BRD9 function in mouse and human hematopoietic cells resulted in myeloid skewing and dyserythropoiesis. These data suggest that targeted correction of aberrant BRD9 splicing might serve as a novel therapeutic approach for SF3B1-mutant leukemias. Of note, treatment with drugs impairing the binding of mutant SF3B1 to RNA resulted in a dose-dependent rescue of aberrant BRD9 splicing in vitro (Fig. K) and in treatment of an SF3B1 mutant AML patient-derived xenograft in vivo. Figure Disclosures Kadoch: Foghorn Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Aguilar Jiménez, Daniel, and José Luis Rodríguez De la O. "Micropropagación y aclimatación de Maguey Pitzometl (Agave marmorata Roezl) en la Mixteca Poblana." Revista Colombiana de Biotecnología 20, no. 2 (July 1, 2018): 124–31. http://dx.doi.org/10.15446/rev.colomb.biote.v20n2.77084.
Full textAbstract:
Agave marmorata R. es una planta que se adapta a terrenos someros y fertilidad baja, también sirve para la retención y conservación de agua de lluvia reduciendo la erosión del suelo. Actualmente se encuentra de forma silvestre y escasa en la Mixteca Poblana (México) donde es intensamente aprovechada en gastronomía y como planta medicinal. Por ello, se propuso la propagación in vitro como estrategia de rescate y conservación a partir de brotes in vitro de Agave marmorata en un medio de cultivo básico de Murashige y Skoog (1962) 100 %, suplementado con azúcar de caña 3 %, myo-inositol 100 mg·L-1, agar 0.7 % y 0.40 mg·L-1 de tiamina-HCl, donde se agregaron por separado diferentes concentraciones de 6-Bencil-adenina (BA) y ácido indol-3-acético (AIA) mediante un arreglo factorial 5x5. Se evaluó el número y longitud de brotes y de raíces obtenidos in vitro. Para las plantas en aclimatación sólo se evaluó el porcentaje de sobrevivencia en diferentes sustratos. Los datos de las variables se sometieron a un análisis de varianza y a una prueba de medias de Tukey (p=0.05). La mejor respuesta para longitud de brotes, número y longitud de raíces fue en el medio MS 100 % adicionando 10 mg·L-1 de AIA y la proliferación de nuevos brotes fue promovida con la adición de BA y AIA en igual concentración. Finalmente, se obtuvo el 100 % de sobrevivencia de las plántulas en los sustratos de peat moss más agrolita y peat moss más arena de río.
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Zhang, Linsheng, Florence B. Fried, Hong Guo, and Alan D. Friedman. "Cyclin-dependent kinase phosphorylation of RUNX1/AML1 on 3 sites increases transactivation potency and stimulates cell proliferation." Blood 111, no. 3 (February 1, 2008): 1193–200. http://dx.doi.org/10.1182/blood-2007-08-109702.
Full textAbstract:
Abstract RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and stimulates G1 cell-cycle progression. Within RUNX1, S48, S303, and S424 fit the cyclin-dependent kinase (cdk) phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cdks on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now use an in vitro kinase assay, phosphopeptide-specific antiserum, and the cdk inhibitor roscovitine to demonstrate that S48 and S424 are also phosphorylated by cdk1 or cdk6 in hematopoietic cells. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell-cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation of the cdk sites to the partially phosphomimetic aspartic acid mildly reduced DNA affinity while progressively increasing transactivation of a model reporter. Mutation to alanine increased DNA affinity, suggesting that in other gene or cellular contexts phosphorylation of RUNX1 by cdks may reduce transactivation. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Together these findings indicate that cdk phosphorylation of RUNX1 potentially couples stem/progenitor proliferation and lineage progression.
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Tran, Thai Hoa, Jonathan Van Nguyen, Catherine C. Smith, Kathryn G. Roberts, Charles G. Mullighan, Neil P. Shah, and Mignon L. Loh. "Identifying Drug-Resistant Mutations in Ebf1-Pdgfrb Ph-like Acute Lymphoblastic Leukemia." Blood 126, no. 23 (December 3, 2015): 1423. http://dx.doi.org/10.1182/blood.v126.23.1423.1423.
Full textAbstract:
Abstract Background: Advances in cancer genomics have recently identified a particular group of patients who display a gene expression profile (GEP) similar to that of Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) patients in approximately 15% of children and over 25% of young adults with B-ALL; thus known as Ph-like ALL. The latter has a worse prognosis compared to those without the Ph-like GEP with conventional chemotherapy. Recent studies have unraveled the genomic landscape of Ph-like ALL which is characterized by genetic alterations activating kinase signaling pathways predicted to respond to tyrosine kinase inhibitors (TKIs). In light of the remarkable outcomes of Ph+ALL patients through incorporation of TKI, the Children's Oncology Group (COG) ALL Committee is actively working to incorporate dasatinib for Ph-like ALL patients harboring ABL-class kinase fusions (ABL1, ABL2, PDGFRB, CSF1R) and eventually, ruxolitinib for those with lesions that are predicted to respond to JAK inhibition. While it is hoped that many of these patients will be cured with the addition of relevant TKIs to chemotherapy, we hypothesize that a proportion of patients will develop resistance to TKI, similar to adults with chronic myeloid leukemia who have been treated with long-term TKI. Hence, investigating the underlying mechanisms governing therapy resistance in Ph-like ALL becomes critical for proactively developing novel therapeutic strategies in the relapsed setting. Objectives: To identify the full spectrum of mutations conferring resistance to clinically-active TKIs in Ph-like ALL and to characterize their relative biochemical resistance to different TKIs. Methods: We first focused on the EBF1-PDGFRB rearrangement since this is the most common recurrent kinase-activating fusion genes in pediatric Ph-like ALL. We used a previously validated in-vitro saturation mutagenesis screen to predict the full spectrum of EBF1-PDGFRB drug-resistant mutations. In brief, EBF1-PDGFRB plasmid was propagated into DNA-repair-deficient E. Coli strain XL-1 Red to generate a library of random mutants. Mutagenized EBF1-PDGFRB plasmid was transfected into 293T cells. Viral supernatants were collected and used to infect Ba/F3 cells. Transduced Ba/F3 cells were plated in 1.2% Bacto-agar and exposed to different TKIs (imatinib, dasatinib) at various concentrations. Genomic DNA from IL-3 independent and TKI-resistant colonies was isolated. The PDGFRB kinase domain was amplified and bidirectional sequencing was performed. Results: Our in-vitro screens showed that the vast majority of drug-resistant clones harbor a kinase domain (KD) point mutation. The predominant KD point mutation conferring resistance to imatinib (94%; 168/178 colonies) or dasatinib (81%; 338/416 colonies) was T681I, which is analogous to BCR-ABL1 T315I gatekeeper mutation. N666S was the second most common KD mutation (6%; 18/321 colonies). The full panel of KD mutations recovered is shown in Table 1. Ba/F3 cells harboring mutant EBF1-PDGFRB T681I was 100 times more resistant to dasatinib compared to wild-type and could be rescued by ponatinib, as predicted (Figure 1). Conclusion: Our screens suggest that KD point mutations may represent the primary mechanism of acquired TKI resistance in EBF1-PDGFRB Ph-like ALL. T681I was the most common KD point mutation in EBF1-PDGFRB upon exposure to imatinib or dasatinib. Future efforts should focus on targeting the T681I gatekeeper mutation with ponatinib or alternative agents for relapsed Ph-like ALL patients harboring these mutations. Figure 1. Cell proliferation profile of Ba/F3 cells harboring EBF1-PDGFRB wild-type and mutant T681I treated with dasatinib or ponatinib. Figure 1. Cell proliferation profile of Ba/F3 cells harboring EBF1-PDGFRB wild-type and mutant T681I treated with dasatinib or ponatinib. Figure 2. Figure 2. Disclosures Smith: Astellas: Research Funding; Plexxikon: Research Funding. Mullighan:Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding. Shah:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Plexxikon Inc.: Research Funding.
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Kondo, Rudin, Matthias Mayerhofer, Karoline Gleixner, Anja Vales, Maria-Theresa Krauth, Sophia Derdak, Alexander Gruze, et al. "Heme Oxygenase-1 (HO-1): A Novel KIT D816V-Dependent Target in Neoplastic Human Mast Cells (HMC-1)." Blood 106, no. 11 (November 16, 2005): 3521. http://dx.doi.org/10.1182/blood.v106.11.3521.3521.
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Abstract Systemic mastoyctosis is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC). Most patients express the D816V-mutated variant of c-KIT, which mediates resistance against most available tyrosine kinase inhibitors. Therefore, current research is focusing on novel targets in MC. We analyzed expression and function of the survival factor heme oxygenase-1 (HO-1) in neoplastic MC. As assessed by Northern blotting and RT-PCR, the human MC line HMC-1 that exhibits KIT D816V, was found to express HO-1 mRNA. Expression of the HO-1 protein was demonstrable by immunocytochemistry and Western blotting. To examine the role of mutated KIT in expression of HO-1, Ba/F3 cells with doxycycline-inducible expression of c-KIT D816V were employed. In these experiments, KIT D816V was found to induce HO-1 promoter activity and expression of HO-1 mRNA and HO-1 protein in these cells. Moreover, the KIT-D816V-targeting compound PKC412 was found to downregulate expression of HO-1 in HMC-1 cells. The KIT D816V-induced upregulation of HO-1 in Ba/F3 cells was completely blocked by a combination of LY294002 (PI3-kinase inhibitor) and PD89059 (MEK inhibitor), but not by single agents, suggesting involvement of multiple signaling pathways. We next examined whether targeting of HO-1 is associated with decreased survival. As assessed by 3H-thymidine uptake, the pegylated HO-1 inhibitor zinc-protoporphyrin (PEG-ZnPP) reduced proliferation of HMC-1 cells in a dose-dependent manner (IC50: 5 μM). The PEG-ZnPP-induced inhibition of growth was found to be associated with induction of apoptosis (control: 1±0.6 vs PEG-ZnPP, 5 μM: 55±5% apoptotic cells, p<0.05). The HO-1 inductor hemin (10 μM) increased the expression of HO-1 in HMC-1 cells and partly rescued these cells from PKC412-induced growth-inhibition (PKC412, 1 μM: 39±8% vs PKC412 + hemin: 23±11%). Finally, PKC412 and PEG-ZnPP were found to produce cooperative (additive) growth-inhibitory effects on HMC-1 cells. In summary, our data show that HO-1 is a novel survival factor and interesting target in neoplastic human MC exhibiting the D816V-mutated variant of KIT.
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Oaks, Joshua J., A. Mukhopadhyay, Ramasamy Santhanam, S. A. Saddoughi, Christopher Walker, Paolo Neviani, Jason G. Harb, et al. "Pharmacologic Restoration of PP2A Activity and Interference with the SET-PP2A Interplay by FTY720 and Its Non-Immunosuppressive Derivative as a Novel and Efficient Therapy for Ph-Negative Myeloproliferative Disorders." Blood 116, no. 21 (November 19, 2010): 775. http://dx.doi.org/10.1182/blood.v116.21.775.775.
Full textAbstract:
Abstract Abstract 775 We have shown (Oaks JJ et al. ASH 2009) that the tumor suppressor Protein Phosphatase 2A (PP2A) is functionally inactivated by Jak2V617F in cell line models of Jak2V617F myeloproliferative disorders (MPD) and Jak2V617F-transduced primary mouse bone marrow cells. Inhibition of Jak2 (600 nM Jak Inhibitor I; 50 μM AG490; 10h) or treatment with the PP2A activator FTY720 (2.5μM, 24 hours) restored PP2A activity that caused loss of Jak2V617F protein/activity, impaired Jak2V617F-driven colony formation, and induced apoptosis of Jak2V617F+ but not normal myeloid cells. Notably, FTY720 is a sphingosine analog suggested by the FDA to treat patients with Multiple Sclerosis due to its immunosuppressive activity when phosphorylated by sphingosine kinase 2 (SPHK2). Here we show that FTY720 treatment of CD34+ primary bone marrow cells from JakV617F+ PV patients (n=3) also rescued PP2A activity, induced Jak2 downregulation and significantly impaired cytokine-dependent clonogenic potential. Thus, FTY720 could be used as an alternative to Jak2 inhibitors, as in vitro and in animal assays showed that FTY720 (2.5μM) is not toxic against normal human myeloid progenitors while decreasing survival of CD34+ progenitors from MPD patients. To find out whether FTY720 uses the same mechanism to exert its immunosuppressive and anti-leukemic activities, we determined if the conversion of FTY720 into its phosphorylated form is important for rescuing PP2A activity in Jak2V617F-expressing cells. Impaired FTY720-P conversion by exposure to the SPHK inhibitor dimethylsphingosine (2.5μM, 6 hours) did not affect the ability of FTY720 to activate PP2A. Also, a synthetically phosphorylated FTY720 (FTY720-P, 2.5μM, 6 hours) was unable to activate PP2A or exert any anti-leukemic activity, suggesting that the anti-proliferative and pro-apoptotic effects of FTY720 are independent of its phosphorylation and interaction with the S1PR1 receptor. We found that activation of S1PR1 through the specific agonist SEW2871 (10μM), FTY720-P (2.5μM), or sphingosine-1-phosphate (100nM) markedly suppresses (~60% inhibition) rather than activates PP2A in normal myeloid progenitors. As expected, knockdown of S1PR1 had no effect on FTY720-mediated PP2A activation in Jak2V617F-transformed cells. Mechanistically we found that Jak2V617F and PP2Ac were found in a ternary complex with the PP2A inhibitor SET. SET knockdown by shRNA restored PP2A activity in Jak2V617F+ Ba/F3 cells to levels similar to those found in non-transformed cells, and led to an 84% decrease in Jak2V617F+-driven colony formation. In addition, co-immunoprecipitation assays revealed that FTY720 (10μM) disrupts Jak2-PP2A, PP2A-SET and Jak2-SET interactions, suggesting that SET may be the target of FTY720. Consistently, affinity chromatography showed that FTY720 efficiently interferes with the ability of C6-ceramide (10μM) to bind SET as the amount of SET eluted from the biotin-labeled C6-ceramide was significantly reduced by exposure of the cell lysate to FTY720. As well, lentiviral-mediated expression of wild type or K209D SET mutant (ceramide binding deficient) in Ba/F3 cells impaired PP2A activity (≥80% decrease), which could be totally rescued by FTY720 only in cells transduced with wild type but not K209D SET. The formal demonstration that FTY720 activates PP2A by displacing SET came when we found SET in anti-NBD immunoprecipitates from Jak2V617F-expressing Ba/F3 cells treated with FTY720-phenoxy-NBD (10μM; 30 min). Together, our data show that FTY720 has the potential to be an effective therapeutic agent for MPD patients by virtue of its low toxicity and ability to activate PP2A by displacing SET; however, FTY720 still retains the ability to become phosphorylated and inhibit, at least in part, PP2A. Thus, we developed non-phosphorylatable FTY720 derivatives and assessed them for their ability to: activate PP2A; induce downregulation/inactivation of targeted kinases (e.g. Jak2, BCR-ABL1, Akt); act as anti-proliferative and pro-apoptotic agent to leukemic but not normal myeloid/lymphoid progenitors; do not interact with S1PR1; and show no in vivo effects on B220+/CD19+ and CD4 or CD8 cellular compartments. These FTY720 derivatives were found to be not immunosuppressive but able to mirror FTY720 in terms of inducing Jak2V617F downregulation and cell killing while retaining the parent compound's minimal toxicity towards untransformed cells. Disclosures: Verstovsek: Incyte Corporation: Research Funding.
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Jahn, Thomas, Petra Seipel, Susanne Urschel, Christian Peschel, and Justus Duyster. "Role for the Adaptor Protein Grb10 in the Activation of Akt." Molecular and Cellular Biology 22, no. 4 (February 15, 2002): 979–91. http://dx.doi.org/10.1128/mcb.22.4.979-991.2002.
Full textAbstract:
ABSTRACT Grb10 is a member of the Grb7 family of adapter proteins lacking intrinsic enzymatic function and encodes functional domains including a pleckstrin homology (PH) domain and an SH2 domain. The role of different Grb10 splice variants in signal transduction of growth factors like insulin or insulin-like growth factor has been described as inhibitory or stimulatory depending on the presence of a functional PH and/or SH2 domain. Performing a yeast two-hybrid screen with the c-kit cytoplasmic tail fused to LexA as a bait and a mouse embryo cDNA library as prey, we found that the Grb10 SH2 domain interacted with the c-kit receptor tyrosine kinase. In the course of SCF-mediated activation of c-kit, Grb10 is recruited to the c-kit receptor in an SH2 domain- and phosphotyrosine-dependent but PH domain-independent manner. We found that Akt and Grb10 form a constitutive complex, suggesting a role for Grb10 in the translocation of Akt to the cell membrane. Indeed, coexpression studies revealed that Grb10 and c-kit activate Akt in a synergistic manner. This dose-dependent effect of Grb10 is wortmannin sensitive and was also seen at a lower level in cells in which c-kit was not expressed. Expression of a Grb10 mutant lacking the SH2 domain as well as a mutant lacking the PH domain did not influence Akt activity. Grb10-induced Akt activation was observed without increased phosphatidylinositol 3-kinase (PI3-kinase) activity, suggesting that Grb10 is a positive regulator of Akt downstream of PI3-kinase. Significantly, deficient activation of Akt by a constitutively activated c-kit mutant lacking the binding site for PI3-kinase (c-kitD814V/Y719F) could be fully compensated by overexpression of Grb10. In Ba/F3 cells, the incapacity of c-kitD814V/Y719F to induce interleukin-3 (IL-3)-independent growth could be rescued by overexpression of Grb10. In contrast, expression of the SH2 deletion mutant of Grb10 together with c-kitD814V/Y719F did not render Ba/F3 cells independent of IL-3. In summary, we provide evidence that Grb10 is part of the c-kit signaling pathway and that the expression level of Grb10 critically influences Akt activity. We propose a model in which Grb10 acts as a coactivator for Akt by virtue of its ability to form a complex with Akt and its SH2 domain-dependent translocation to the cell membrane.
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Friedman, Alan D., Linsheng Zhang, and Florence Bernardin Fried. "Cyclin-Dependent Kinase Phosphorylation of RUNX1/AML1 on Three Sites Increases Trans-Activation Potency and Stimulates Cell Proliferation." Blood 110, no. 11 (November 16, 2007): 3351. http://dx.doi.org/10.1182/blood.v110.11.3351.3351.
Full textAbstract:
RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and also stimulates G1 cell cycle progression. CBFβ-SMMHC or AML1-ETO dominantly inhibit RUNX1 and slow G1 progression in hematopoietic cell lines or in murine or human marrow progenitors, cdk4, cyclin D2, or c-Myc overcome inhibition of proliferation by these CBF oncoproteins, exogenous RUNX1 stimulates G1 progression, and stimulation of G1 via deletion of p16INK4a or expression of E7 cooperates with CBFβ-SMMHC or TEL-AML1 to induce acute leukemia in mice. Induction of cdk4 or cyclin D3 transcription may underlie stimulation of G1 progression by RUNX1. Remarkably, the C. elegans ortholog of RUNX1, RNT-1, also stimulates G1 progression and couples stem cell proliferation with differentiation. Not only does RUNX1 regulate cell cycle progression, but in addition RUNX1 levels increase as hematopoietic cells progress from G1 to S and from S to G2/M. Within RUNX1, S48, S303, and S424 fit the cdk phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cyclin dependent kinases on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now find that S48 and S424 are also phosphorylated by cdk1 or cdk6. S48, S303, or S424 phosphopeptide antiserum that we developed specifically recognized kinased GST-RUNX1 and interacted with RUNX1 expressed in 293T cells or in the Ba/F3 hematopoietic cell line. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation to alanine or to the partially phosphomimetic aspartic acid progressively diminished or increased trans-activation, such that the tripleA mutant activated a RUNX1 reporter 5-fold less potently than the tripleD mutant. Aspartic acid does not perfectly mimic serine phosphorylation, as illustrated by the much greater affinity of our antisera for wild-type RUNX1 versus RUNX1(tripleD), suggesting that the biologic effect of RUNX1 cdk phosphorylation is even more significant. The p300 co-activator retained interaction with the tripleA variant. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Cdk phosphorylation of RUNX1 on three sites increases its ability to active transcription and to stimulate proliferation, potentially coupling entry of stem/progenitors into cycle with induction of genes required for hematopoietic lineage progression, such as those encoding myeloperoxidase, neutrophil elastase, the M-CSF receptor, and PU.1.
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Krosl, Gorazd, Gang He, Martin Lefrancois, Frédéric Charron, Paul-Henri Roméo, Paul Jolicoeur, Ilan R. Kirsch, Mona Nemer, and Trang Hoang. "Transcription Factor SCL Is Required for c-kit Expression and c-Kit Function in Hemopoietic Cells." Journal of Experimental Medicine 188, no. 3 (August 3, 1998): 439–50. http://dx.doi.org/10.1084/jem.188.3.439.
Full textAbstract:
In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34+ hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony–stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit–dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL– or dn-SCL– expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro–B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.
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Nonami, Atsushi, Martin Sattler, Ellen L. Weisberg, Liu Qingsong, Jianming Zhang, Guillaume Adelmant, Jarrod A. Marto, et al. "Deciphering the Critical Pathways of Mutant N-RAS in AML Using Small Molecule Inhibitors." Blood 120, no. 21 (November 16, 2012): 2455. http://dx.doi.org/10.1182/blood.v120.21.2455.2455.
Full textAbstract:
Abstract Abstract 2455 Activating mutations in the small GTPase N-RAS occur in about 10% of acute myeloid leukemia (AML) cases. Active N-RAS is thought to drive the disease and is therefore a potential target for drug development. There have been numerous unsuccessful efforts to target RAS itself with small molecules, and blocking post-translational modifications of RAS proteins, such as through inhibition of farnesyl transferase, has similarly not proven useful. In addition, efficacy of targeting critical downstream effectors has been limited by the complexity of RAS signaling, such as redundancy of signaling pathways and feedback mechanisms. While targeting RAS is challenging, it was our hypothesis that inhibiting the right combination of downstream pathways in a particular lineage with small molecules could be effective. Initially, we created a Ba/F3 cell line that was completely dependent on oncogenic N-RAS-G12D for growth and survival. Growth was suppressed >99% by shRNA for N-RAS, but could be rescued entirely by interleukin-3 (IL-3), which does not require N-RAS signaling in these cells. Using this cell line, we performed a high-throughput chemical screen with a large library of multi-targeted kinase inhibitors. The lead compound (NRAS1) showed a 70-fold difference in the EC50 for growth inhibition between BaF3-NRAS G12D cells cultured in the absence (0.01μM) or presence (0.77μM) of IL-3. Importantly, this compound showed selectivity towards several leukemia cell lines that were shown to be dependent on mutant N-RAS by shRNA compared to cells expressing wild-type N-RAS (p=0.02). Also, in a xenotransplant model using NRAS-G12D+ OCI-AML3 cells, this compound significantly reduced tumor burden (P=0.005) and prolonged survival (P=0.002) compared to controls. Next, we sought to identify the targets of NRAS1, Interestingly, the compound did not suppress MEK or ERK, which are classical targets of RAS signaling in epithelial cells. NRAS1 profoundly reduced AKT and RPS6 phosphorylation. Kinase selectivity profiling of this compound (1μM) in OCI-AML3 cells (EC50: 0.3μM) identified 13 major binding partners with more than 85% efficacy. The targets consisted mainly of SRC family proteins (SRC, FGR, and LYN etc.) and MAPK family proteins (MAP4K2, 3, 5, and p38 etc.) and others (ZAK and BTK etc), but not MEK and ERK, and AKT was not detected in this assay. In preliminary studies, most of these target kinases were knocked-down by shRNA and, as expected, no single kinase was found to be responsible for mediating growth inhibition. Using a phospho-antibody microarray, the most significantly de-phosphorylated kinases were p38, AKT and SRC, which supports our preliminary findings. To validate the significance of these results, we treated Ba/F3-N-RAS cells with combinations of kinase inhibitors. Combining the AKT inhibitor MK2206 and Dasatinib (SRC family inhibitor) revealed marked synergy, while neither had activity individually. Also, the combination of MK2206 and a cleaner SRC family inhibitor, AZD0530, also synergized, although to lesser extent. In both examples, however, the inhibition of N-RAS transformed cells by NRAS1 proved superior, suggesting that one or more additional targets are required for inhibition of NRAS signaling. To identify additional critical targets of our compound we generated several derivatives with different potency. In particular, one less potent analog of NRAS1 (analog 6, 1% EC50 of original compound) showed a loss of binding activity towards the MAP4K family of proteins, especially MAP4K2. Observed synergy between the selective MAP4K2 inhibitor NG25 and selective inhibitors of MK2206 and Dasatinib in Ba/F3-NRAS G12D cells further points toward MAP4K2 as being of additional significance for oncogenic RAS signaling. Together with the previous data, we propose AKT and MAP4K2 as critical targets of NRAS1. In conclusion, we have identified a novel and selective kinase inhibitor of the N-RAS signaling pathway by chemical screen using Ba/F3-N-RAS G12D cells. By combination of signaling study, kinase selectivity profiling and phosphoproteomics, the main functional targets were found to be AKT, and MAP4K2, and additional functional targets will be elucidated. Our approach also could be applied for other type of oncogenes, and it could help to find therapeutic compound and also help to decipher signaling mechanisms of the oncogenes which are thus far undruggable. Disclosures: No relevant conflicts of interest to declare.
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Nonami, Atsushi, Martin Sattler, Ellen L. Weisberg, Jianming Zhang, Qingsong Liu, Matthew Patricell, Hojong Yoon, et al. "Using Small Molecules To Identify Critical Signaling Pathways Of Mutant N-RAS In Acute Leukemia Cells." Blood 122, no. 21 (November 15, 2013): 169. http://dx.doi.org/10.1182/blood.v122.21.169.169.
Full textAbstract:
Abstract Activating point mutations in NRAS are detected in more than 10% of AML patients, making NRAS an important therapeutic target. Using small molecules to directly target NRAS or inhibit post-translational modification, such as farnesylation, have been extensively investigated. The potential of strategies focused on targeting downstream effectors of RAS, such as RAF or MEK, has been limited by the complexity of RAS signaling, including redundancy and feedback loops. Large-scale RNAi screens have been used to identify genes (TBK1, STK33 and GATA2, for example) that are synthetically lethal with RAS mutations and these are being explored as therapeutic targets. Recognizing the complexity of RAS signaling, we tested the notion that small molecule screens designed to simultaneously inhibit multiple signaling pathways might identify combinations of pathways that are critical for NRAS signaling in leukemic cells. Initially, we created an experimental Ba/F3 cell line model that was completely dependent on oncogenic N-RAS-G12D for growth and survival. Knockdown of NRAS suppressed growth >95%, but could be rescued by interleukin-3 (IL-3). A chemical screen using panels of multi-targeted small molecule kinase inhibitors against BaF3-NRAS-G12D cells revealed a lead compound, NRAS1 (N-(4-methyl-3-(1-methyl-7-(6-methylpyridin-3-ylamino)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)phenyl)-3-(trifluoromethyl)benzamide), with high selectivity and sensitivity toward leukemia cell lines with NRAS mutations in vitro. A number of studies were then performed to investigate the targets of this compound. Transcriptional profiling before and after treatment of two AML cell lines with NRAS mutation (OCI-AML3 and KO52 cells, respectively) showed profiles similar to that obtained by knocking down NRAS, supporting the hypothesis that this compound suppressed NRAS signaling. Biochemical studies demonstrated that NRAS1 did not inhibit several classical targets of RAS signaling, including, RAF, MEK and ERK. In contrast, NRAS1 was found to substantially reduce AKT and RPS6 phosphorylation. Over-expression of a constitutively active allele of AKT, myrAKT, in Ba/F3-NRAS G12D cells conferred strong resistance to NRAS1, confirming that suppression of phospho-AKT may be important for the function of NRAS1. However, direct inhibition of AKT only partially recapitulated the effects of NRAS1. Kinase selectivity profiling of NRAS1 (1μM) in OCI-AML3 cells (EC50: 0.3μM) identified 13 major binding partners with more than 85% efficacy. The targets consisted mainly of SRC family proteins (ie SRC, FGR, and LYN) and MAPK family proteins (ie GCK, KSH, and p38), but not MEK1/2, ERK1/2 or AKT1-3. A series of analogs of NRAS1 was synthesized and structure/function studies were carried out. One compound, (LKB-0304601, 1% EC50 of original compound) lost the ability to bind to the MAP4K family of proteins, especially GCK (MAPK4K2). A combination effect was observed between a known GCK inhibitor, NG25, and a known allosteric AKT inhibitor, MK-2206, against mutant NRAS-expressing cells. This finding supports the hypothesis that simultaneous inhibition of GCK and AKT has suppressive activity against leukemia cells transformed by NRAS. Furthermore, a putative gate-keeper mutation introduced into GCK (GCK G96S) resulted in partial resistance to growth suppression by NG25 or NRAS1. Growth suppression of NRAS-transformed leukemic cells was further induced by knock-down of GCK by shRNAs in cells with mutant NRAS, THP-1 cells and MOLT-3, and this effect could be rescued by over-expression of GCK. Finally, in a xenotransplant model using NRAS-mutant-expressing OCI-AML3 cells and MOLT-3 cells, NRAS1 significantly reduced tumor burden and prolonged survival compared to controls. Overall, by using a chemical screen designed to inhibit multiple signaling pathways simultaneously in oncogene-addicted cells, followed by signaling studies, cell biological studies and kinase selectivity profiling, we found that simultaneous inhibition of AKT and GCK, by either NRAS1 or selective inhibitors, exhibits activity against NRAS-transformed leukemia cells. Disclosures: Griffin: Novartis Pharmaceuticals: Research Funding.
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Gorantla, Sivahari P., Tobias Dechow, Rebekka Grundler, Christian Peschel, and Justus Duyster. "Molecular Mechanisms of JAK2V617F Oncogenic Activation: The JAK2- V617F Mutant Utilizes the SH2 Domain for Constitutive Kinase Activity in the Presence of Unstimulated Heterodimeric Cytokine Receptor." Blood 112, no. 11 (November 16, 2008): 175. http://dx.doi.org/10.1182/blood.v112.11.175.175.
Full textAbstract:
Abstract The JAK2-V617F mutation has been reported in the majority of MPDs including PV, ET, and IMF. This mutation leads to the constitutive activation of the JAK2 tyrosine kinase activity and overexpression of JAK2V617F renders hematopoietic cell lines growth factor-independent. However, the molecular mechanism leading to constitutive activation of JAK2V617F is largely unclear and the requirement of homodimeric or heterodimeric cytokine receptors needs to be determined. Here we show that oncogenic JAK2-V617F requires an intact SH2 domain for constitutive kinase activity. To this end we mutated the conserved arginine 426 within the SH2 domain to a lysine. Ba/F3 cells expressing JAK2V617F grew IL-3-independent and showed constitutive activation of JAK2, STAT5, and ERK1/2. In contrast, introduction of the SH2 mutation in JAK2V617F abrogated both transformation as well as constitutive activation of downstream signaling pathways. Accordingly, reconstitution of JAK2 mutants in a JAK2-negative cell line with IL-3R co-expression revealed reduced activation of JAK2 when the SH2 domain was mutated. It has been reported that JAK2 binding to homodimeric type I cytokine receptor may facilitate JAK2V617F-mediated transformation. Interestingly, co-expression of the homodomeric EpoR with SH2 mutated JAK2V617F rescues the phenotype indicating that the SH2 domain is required for JAK2 signaling in the presence of heterodimeric but not homodimeric cytokine receptors. Membrane localization studies showed equal membrane distribution of SH2-mutated and unmutated JAK2-V617F indicating that the SH2 domain mutation does not affect subcellular distribution of JAK2. However, co-IP experiments revealed a possible role for the SH2 domain in the dimerization and transphosphorylation of JAK2. Consequently, reduced transphosphorylation was seen in IL-3R- but not in EpoR-expressing cells. In a BM transplantation model we found that an intact SH2 domain in JAK2V617F was required for the induction of a MPD-like disease. Thus, our results points to an important role of the SH2 domain for the constitutive activation of JAK2V617F in cells expressing heterodimeric cytokine receptors.
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Placke, Theresa, Katrin Faber, Atsushi Nonami, Helmut R. Salih, Stephen M. Sykes, David E. Root, David A. Barbie, et al. "Requirement For CDK6 In MLL-Rearranged Acute Myeloid Leukemia." Blood 122, no. 21 (November 15, 2013): 3782. http://dx.doi.org/10.1182/blood.v122.21.3782.3782.
Full textAbstract:
Abstract Chromosomal rearrangements involving the H3K4 methyltransferase MLL trigger aberrant gene expression programs in hematopoietic stem and progenitor cells and give rise to an aggressive subtype of acute myeloid leukemia (AML) that is associated with intermediate or poor survival. Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies in the clinic, in part because mutant MLL is difficult to target directly and it is incompletely understood which of the genes affected by altered epigenetic regulation in MLL-rearranged AML are responsible for malignant transformation. To search for essential signaling pathways in MLL-rearranged AML that might serve as new therapeutic targets, we performed loss-of-function RNA interference (RNAi) screens in 5 AML cell lines (NOMO-1, THP-1, OCI-AML3, HL-60, U937) using a lentiviral short hairpin RNA (shRNA) library, and observed that the cell cycle regulator CDK6, but not its functional homolog CDK4, was preferentially required by MLL-AF9pos NOMO-1 and THP-1 cells. The enhanced CDK6 dependence of MLL-rearranged cells was confirmed in an expanded panel of AML cell lines (MLL-rearranged, n=6; MLL wildtype [WT], n=4) that also included cell lines harboring other MLL fusions (MLL-AF4 and MLL-AF6), and the RNAi-induced phenotype was countered by overexpression of an shRNA-resistant CDK6 cDNA. Stable knockdown of MLL-AF9 in MLL-AF9pos cell lines and overexpression of MLL-AF9 in WT MLL-expressing cell lines, normal human CD34pos cells, or Ba/F3 murine pro-B cells led to concordant changes in CDK6 mRNA and protein levels that resembled those of HOXA9, a known MLL-AF9 target, indicating that CDK6 is rendered essential via transcriptional activation by truncated MLL. Analysis of cell cycle distribution, apoptosis induction, and myeloid differentiation demonstrated that the differential growth-inhibitory effect of CDK6 suppression was mainly attributable to myeloid differentiation, as MLL-AF9pos cell lines upregulated CD11b expression and assumed a more mature, macrophage-like morphology upon CDK6 knockdown, effects not observed in WT MLL-expressing cells. Furthermore, the immature phenotype of NOMO-1 cells could be rescued by overexpression of an shRNA-resistant CDK6 cDNA. Consistent with the observations in AML cell lines, knockdown of Cdk6 also impaired the proliferation and in vitro clonogenic activity of primary murine bone marrow (BM) cells stably transduced with MLL-AF9, whereas cells expressing another leukemogenic fusion gene (MOZ-TIF2) and Ba/F3 cells were largely unaffected. We also expressed MLL-AF9 in unfractionated BM derived from Cdk6 knockout mice and observed that colony numbers were gradually reduced in cultures initiated with Cdk6+/- and Cdk6-/- BM compared to WT BM. Furthermore, most of the colonies obtained were small and displayed loose morphology in contrast to the large, dense, blast-like colonies seen in cultures initiated with transduced WT BM. We are currently investigating whether Cdk6 is also required for AML development and propagation in vivo using a murine BM transplantation model of MLL-AF9-induced leukemia. The context-dependent effects of lowering CDK6 expression could be recapitulated in cell lines and primary human AML specimens using palbociclib (also known as PD-0332991), a small-molecule inhibitor of CDK4 and CDK6 enzymatic activity that is in clinical development as an anticancer agent. We are currently devising strategies to combine this compound with cytotoxic chemotherapy as well as other targeted therapeutics, such as small-molecule bromodomain inhibitors, to maximize killing of MLL-rearranged AML cells. Together, our data (1) identify CDK6 as a critical and potentially “actionable” effector of MLL fusion proteins in leukemogenesis, (2) link the catalytic activity of CDK6 to arrested myeloid differentiation in MLL-rearranged AML, and (3) underscore that cell cycle regulators thought to normally act redundantly may have distinct functions in different genetic contexts. Disclosures: No relevant conflicts of interest to declare.
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Fink, Emma C., Jan Krönke, Slater N. Hurst, Namrata D. Udeshi, Tanya Svinkina, Rebekka K. Schneider, Marie E. McConkey, et al. "Lenalidomide Induces Ubiquitination and Degradation of CSNK1A1 in MDS with Del(5q)." Blood 124, no. 21 (December 6, 2014): 4. http://dx.doi.org/10.1182/blood.v124.21.4.4.
Full textAbstract:
Abstract The immunomodulatory (IMiD) drug lenalidomide is a highly effective treatment for multiple myeloma and myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Recently, we and others demonstrated that lenalidomide activates the CRBN-CRL4 E3 ubiquitin ligase to ubiquitinate IKZF1 and IKZF3. Degradation of these lymphoid transcription factors explains lenalidomide’s growth inhibition of multiple myeloma cells and increased IL-2 release from T cells. However, it is unlikely that degradation of IKZF1 and IKZF3 accounts for lenalidomide’s activity in MDS with del(5q). Instead, we hypothesized that ubiquitination of a distinct CRBN substrate in myeloid cells explains the efficacy of lenalidomide in del(5q) MDS. Applying quantitative proteomics in the myeloid cell line KG-1, we identified a novel target, casein kinase 1A1 (CSNK1A1), that had increased ubiquitination and decreased protein abundance following lenalidomide treatment. CSNK1A1 is encoded in the del(5q) commonly deleted region and is thus a potential lenalidomide target in del(5q) MDS. Previous studies have demonstrated that Csnk1a1 is a therapeutic target in a murine model of acute myeloid leukemia. We validated that lenalidomide treatment decreased CSNK1A1 protein levels in multiple human cell lines in a dose-dependent manner without altering CSNK1A1 mRNA levels. Moreover, lenalidomide treatment increased ubiquitination of CSNK1A1 in cell lines. The decrease in CSNK1A1 protein levels in response to lenalidomide was abrogated by treatment with the proteasome inhibitor MG132 and by Cullin-RING ubiquitin ligase inhibition with MLN4924. CSNK1A1 co-immunoprecipitated with CRBN in the presence of lenalidomide, demonstrating direct interaction of CSNK1A1 with the substrate adaptor for the ubiquitin ligase. Homozygous genetic inactivation of the CRBN gene by CRISPR/Cas9 genome editing in 293T cells eliminated lenalidomide-induced degradation of CSNK1A1. In aggregate, these experiments demonstrate that CSNK1A1 is a CRBN-CRL4 substrate that is ubiquitinated and degraded in the presence of lenalidomide. We next explored how degradation of CSNK1A1 might explain the specificity of lenalidomide for cells with del(5q). ShRNA-mediated knockdown of CSNK1A1 sensitized primary human CD34+ cells to lenalidomide treatment, indicating that haploinsufficiency for CSNK1A1 might increase lenalidomide sensitivity in del(5q) hematopoietic cells. We sought to further validate this finding in a genetically defined Csnk1a1 conditional knockout mouse model. While murine cells are resistant to the effects of IMiDs, murine Ba/F3 cells overexpressing human CRBN (hCRBN), but not murine CRBN, degraded CSNK1A1 in response to lenalidomide. To examine the effect of Csnk1a1 haploinsufficiency on lenalidomide sensitivity, we isolated hematopoietic stem and progenitor cells from Csnk1a1+/- and Csnk1a1+/+ mice and transduced them with a retroviral vector expressing hCRBN. When treated with lenalidmide, Csnk1a1+/- cells expressing hCRBN were depleted over time relative to wild-type controls. The enhanced sensitivity of Csnk1a1+/- cells to lenalidomide was associated with induction of p21 and was rescued by heterozygous deletion of p53, demonstrating a critical downstream role for p53 consistent with clinical observations that TP53 mutations confer lenalidomide resistance. In aggregate, these studies demonstrate that lenalidomide induces the ubiquitination and consequent degradation of CSNK1A1 by the CRBN-CRL4 E3 ubiquitin ligase. del(5q) cells have only one copy of CSNK1A1, so they are selectively depleted over wild-type cells, explaining lenalidomide’s clinical efficacy in del(5q) MDS. Although the idea that heterozygous deletions could be cancer vulnerabilities was first proposed 20 years ago, lenalidomide provides the first example of an FDA-approved and clinically effective drug that derives its therapeutic window from specifically targeting a haploinsufficient gene. Disclosures Ebert: Celgene: Research Funding; Genoptix: Consultancy.
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Wagner, Marie-Christine, Marek Dziadosz, Tina Schnoeder, Florian H. Heidel, Thomas Fischer, and Daniel B. Lipka. "A Novel Paradigm In Pharmacodynamics of Tyrosine Kinase Inhibitors: Pulse Treatment Induced Apoptosis Is Mediated by Intracellular Retention." Blood 116, no. 21 (November 19, 2010): 1828. http://dx.doi.org/10.1182/blood.v116.21.1828.1828.
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Abstract Abstract 1828 Small molecule tyrosine kinase inhibitors (TKI) have become a valuable therapeutic tool in a variety of malignancies. It has been widely accepted that continuous target inhibition is a prerequisite for clinically effective TKI treatment. This paradigm has been questioned by evidence obtained in a clinical trial for chronic myeloid leukemia (CML) using the second generation BCR-ABL inhibitor dasatinib: Once daily dosing (QD) resulted in only transient inhibition of BCR-ABL kinase activity. Nevertheless, similar clinical activity was demonstrated for the QD schedule as compared to twice daily dosing (BID). Recent in vitro data demonstrated that pulse treatment with 50–100 nM dasatinib for 20 min to 4h induced apoptosis in BCR-ABL positive cells. Pulse treatment with imatinib using standard concentrations (1-7,5 μM) was not effective. However, if pulses with high doses of imatinib (32,5 μM) are applied, induction of apoptosis was observed. So far, the underlying molecular mechanism for this effect remains elusive. Our study aims to elucidate the underlying molecular mechanism of induction of apoptosis upon TKI pulse treatment in a variety of hematopoietic cells dependent on oncogenic kinases. We employed the human K562, and MV4-11 cell lines, as well as cellular reconstitution models of murine hematopoietic Ba/F3 cells stably transfected with BCR-ABL(p210), JAK2-V617F, or FLT3-ITD, respectively. Human primary cells from healthy controls and CML patients were obtained after written informed consent. Cells were incubated either with dasatinib or imatinib (BCR-ABL inhibition), JAK-Inhibitor I (JAK2-V617F inhibition) or with PKC412 (FLT3-ITD inhibition). Concentrations used were 100 nM dasatinb, 25 μM imatinib, 10 μM Jak-Inhibitor I or 3,5 μM PKC412, respectively, for 2–24h, followed by thorough washing steps. Cells were then incubated in medium without TKI. Upon a total incubation period of 24h, apoptosis was measured using flow cytometry and Western blotting (caspase 3 cleavage). In parallel, inhibition of intracellular signaling pathways (pSTAT5, pERK, pCrkl) was determined by Western blotting. To test for intracellular retention of TKI, untreated cells were incubated with cell culture supernatant obtained after incubation of previously washed TKI treated cells for 2h in TKI-free media. Again, apoptosis was measured at 24h and inhibition of signaling pathways was analyzed at various time points. Drug levels of imatinib and dasatinib in the cell culture supernatants before and after the washing procedures were quantified by HPLC. TKI pulse treatment with each inhibitor for a period of 2h was sufficient to effectively induce apoptosis independent of the transforming oncogene and of cellular origin (mouse or human). To test for possible cellular retention upon TKI pulse treatment, we introduced additional washing steps and applied the supernatants to previously untreated cells. This analysis revealed: 1) Cells were almost completely rescued from apoptosis when additional washing steps were applied and 2) supernatants induced high levels of apoptosis in previously untreated cells. To confirm that the nature of this effect is indeed cellular TKI retention, imatinib and dasatinib concentrations in cellular supernatants were monitored at various time points (0, 30, 60, and 120min). This revealed a dramatic, time-dependent increase in either imatinib or dasatinib concentrations reaching levels well above the IC50 concentration of each inhibitor. These results were confirmed using normal primary human CD34+ cells and peripheral blood CML-MNCs. To further confirm that the TKI is being stored intracellularly upon TKI pulse treatment, we lysed pulse-treated cells immediately after completion of the first washing procedure. By HPLC we were able to detect relevant TKI concentrations in the cell lysate, thus confirming that TKIs are indeed retained intracellularly. Taken together, using three different oncogene-dependent cellular reconstitution models, our data demonstrate that cellular TKI retention and prolonged inhibition of signal transduction is the common molecular mechanism in induction of apoptosis upon TKI pulse treatment. This suggests that both in-vitro and in-vivo analysis of pharmacokinetics and pharmacodynamics are required to determine optimal dosing schedules in future clinical trials. Disclosures: No relevant conflicts of interest to declare.
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Agarwal, Anupriya, Ryan J. Meckenzie, Raffaella Pippa, Christopher A. Eide, Jessica Oddo, Jeffrey W. Tyner, Rosalie Sears, et al. "OP449, a Novel SET Antagonist, Is Cytotoxic To Leukemia Cells and Enhances Efficacy Of Tyrosine Kinase Inhibitors In Drug-Resistant Myeloid Leukemias." Blood 122, no. 21 (November 15, 2013): 2511. http://dx.doi.org/10.1182/blood.v122.21.2511.2511.
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Abstract Background The SET oncoprotein, an inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia cells, preventing PP2A from performing its regulatory role in deactivating signaling proteins by dephosphorylation. Restoration of PP2A activity in both chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells to normal levels through shRNA-mediated knockdown of SET results in reduced leukemogenesis. Given the central role of PP2A and SET in regulating various kinase-dependent and -independent downstream signaling pathways, we evaluated the efficacy of SET antagonism in CML and AML cell lines as well as primary patient cells using OP449, a novel, specific, cell-penetrating SET antagonist. Results Treatment of human and murine CML cells with OP449 resulted in dose-dependent increase in PP2A activity and selective inhibition of cell growth (IC50: 0.60 to 1.11 μM), while parental Ba/F3 cells exhibited no measurable cytotoxicity. OP449-mediated decrease in the viability of leukemia cells was significantly rescued by co-treatment with okadaic acid, a PP2A inhibitor, confirming efficacy is mediated through PP2A activation. OP449 was also 3 to 8-fold more potent than FTY720 (a known activator of PP2A) and induced dephosphorylation/degradation of BCR-ABL1, AKT, and STAT5. Importantly, OP449 demonstrated activity against the ABL1 tyrosine kinase inhibitor-resistant BCR-ABL1T315I mutant and the BCR-ABL1E255V/T315I compound mutant (IC50: 1.62 and 1.97 μM, respectively). Consistent with cell line findings, OP449 also inhibited growth of primary cells from CML blastic phase patients harboring either wildtype BCR-ABL1 or BCR-ABL1T315I while normal CD34+ cells exhibited minimal effect. Further, treatment of CML cell lines and primary CD34+ CML cells with OP449 in combination with the ABL1 tyrosine kinase inhibitors showed significantly increased cytotoxicity as compared to each compound alone. For example, treatment of primary CD34+ CML cells with 2.5 μM OP449 or 200 nM nilotinib alone each resulted in a 50% reduction in colony formation, while combination of OP449 and nilotinib at these concentrations reduced colony formation by approximately 87%, suggesting synergistic reduction of clonogenicity (combination index: 0.195). Similar to our findings in CML cells, OP449 increased PP2A activity and suppressed growth in a dose-dependent manner in AML cell lines and primary patient samples harboring various different genetic lesions including FLT3-ITD, CSF1R overexpression, NRASQ61L, and JAK3A572V. Additionally, synergistic inhibition of these cells was observed when OP449 was combined with relevant tyrosine kinase inhibitors and chemotherapy. For example, treatment of MOLM-14 cells (FLT3-ITD) with 2.5 μM OP449 or 1 nM AC220 alone reduced cell viability by 58% and 75%, respectively; combined treatment reduced cell growth 96% (combination index: 0.723). Similarly, treatment of HL-60 cells (NRASQ61L) with 1 μM OP449 or 250 nM cytarabine alone reduced cell viability by 40% and 60%, respectively, whereas combined treatment led to a 94% reduction in viability (combination index: 0.630). Mechanistically, AML patient samples showed significantly increased SET expression compared to normal CD34+ cells, and treatment of AML cells with OP449 reduced phosphorylation of downstream ERK, STAT5, AKT and S6 ribosomal protein signaling. Finally, to evaluate OP449 antitumor efficacy in vivo, we tested OP449 (5 mg/kg intraperitoneally every 3 days) in xenograft mice bearing human HL-60 cell derived tumors. OP449 significantly inhibited tumor growth measured over time and resulted in a >2-fold reduction in tumor burden at the end of the experiment compared to vehicle-treated controls (Day 18: 1.14±0.06 g vs. 0.45±0.08 g, respectively; p<0.001). These results demonstrate the in vivo efficacy of OP449 in a murine leukemia model. Conclusions SET antagonism is selectively cytotoxic to CML and AML cells harboring various genetic lesions and drug-resistant mutations. Our results demonstrate that combined targeting of SET and tyrosine kinases provides more efficient and selective inhibition of leukemia cell growth for a broad range of oncogenic lesions as compared to normal cells. Taken together, our findings suggest a novel therapeutic paradigm of SET antagonism in combination with tyrosine kinase inhibitors for the treatment of CML and AML patients with drug resistance. Disclosures: Agarwal: Oncotide Pharmaceuticals: Research Funding. Tyner:Incyte Corporation: Research Funding. Vitek:Oncotide Pharmaceuticals: Employment. Christensen:Oncotide Pharmaceuticals: Employment. Druker:Ambit Biosciences: Consultancy, PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb/Novartis: Currently PI or co-I on Novartis & Bristol-Myers Squibb clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. He does not derive salary, nor does his lab Other; Oncotide Pharmaceuticals: Research Funding, Subaward from NIH STTR, Subaward from NIH STTR Other.
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Moosmann, Julia, Ariawan Purbojo, Susanne Eder, and Sven Dittrich. "Case Report: Trans-axillary Artery Access for Rescue Stent Implantation in an Infant With Retrograde Non-passable Aortic Coarctation." Frontiers in Pediatrics 9 (April 8, 2021). http://dx.doi.org/10.3389/fped.2021.625011.
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Primary surgical repair remains the traditional treatment for patients with critical duct-dependent coarctation of the aorta (CoA). Initial surgical repair might not be possible or associated with higher risks if additional comorbidities arise in small infants and neonates. Balloon angioplasty (BA) has been described as a rescue strategy for these children. We describe the feasibility of a palliative BA and rescue stent implantation via an alternative antegrade right-axillary artery approach in an initially inoperable infant with pneumonia and respiratory failure and severe CoA, where the stenosis was not passable by traditional retrograde femoral access. This case adds new aspects to the therapy of critical CoA: Stent implantation provides a bridge to surgery in critically ill infants and does not preclude successful surgical repair. Further, if the classic retrograde approach is not possible, the right axillary artery access should be considered as an alternative to pass the stenosis.
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Mendonça, Nuno, David Rodríguez-Luna, Sandra Boned-Riera, Marta Rubiera, Marc Ribó, Jorge Pagola, Socorro Piñeiro, et al. "Abstract 2226: Predictors Of tPA Non-responders According To Location Of Vessel Occlusion." Stroke 43, suppl_1 (February 2012). http://dx.doi.org/10.1161/str.43.suppl_1.a2226.
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Background and purpose: Information on the clinical and hemodynamic profile of IV tPA non-responders, at different location of arterial occlusion, may improve the selection of candidates for rescue reperfusion therapies. Therefore, we aimed to investigate predictors of failing IV tPA therapy according to occluded vessel and location of clot. Methods: We prospectively evaluated consecutive patients with an acute ischemic stroke admitted within the first 6 hours of onset. Five hundred and forty-eight patients with documented intracranial occlusion were included. Patients were categorized according to site of vessel occlusion into 4 distinct groups: proximal MCA occlusion (n=251), distal MCA occlusion (n=194), ICA T occlusion (n=61) and BA occlusion (n=42). Recanalization was assessed on TCD at 1 hour of tPA bolus. Results: Among patients with proximal MCA occlusion, the presence of severe extracranial ICA stenosis or occlusion (OR 2.36, 95% CI 1.15-4.84, p=0.02) and age >74 years (OR 1.84, 95% CI 1.02-3.31, p=0.04) independently predicted no recanalization (NR). No independent predictors of NR were identified in patients with distal MCA occlusion. In patients with ICA T occlusion, history of hypertension (OR 12.77, 95% CI 2.12-76.88, p=0.05) and absence of atrial fibrillation (OR 0.12, 95% CI 0.02-0.71, p=0.02) emerged as independent predictors of NR. Similarly, among patients with BA occlusion, atrial fibrillation was as an independent predictor of NR (OR 0.13, 95% CI 0.03-0.72, p=0.02). Conclusions: Absence of atrial fibrillation independently predicts persistent occlusion at 1-h after tPA bolus in patients with ICA T and BA occlusions. The use of relevant predictors of NR and a rapid neurovascular evaluation may improve the selection of patients for more aggressive rescue strategies.
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Chen, Zhiwei, Yinyin Xie, Dan Liu, Ping Liu, Fei Li, Zhanglin Zhang, Mengmeng Zhang, et al. "Downregulation of miR-142a Contributes to the Enhanced Anti-Apoptotic Ability of Murine Chronic Myelogenous Leukemia Cells." Frontiers in Oncology 11 (July 27, 2021). http://dx.doi.org/10.3389/fonc.2021.718731.
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BackgroundLeukemic stem cell (LSC) is thought to be responsible for chronic myelogenous leukemia (CML) initiation and relapse. However, the inherent regulation of LSCs remains largely obscure. Herein, we integratedly analyzed miRNA and gene expression alterations in bone marrow (BM) Lin-Sca1+c-Kit+ cells (LSKs) of a tet-off inducible CML mouse model, Scl/tTA-BCR/ABL (BA).MethodsScl/tTA and TRE-BA transgenic mice were crossed in the presence of doxycycline to get double transgenic mice. Both miRNA and mRNA expression profiles were generated from BM LSKs at 0 and 3 weeks after doxycycline withdrawal. The target genes of differentially expressed miRNAs were predicted, followed by the miRNA-mRNA network construction. In vitro and in vivo experiments were further performed to elucidate their regulation and function in CML progression.ResultsAs a result of the integrated analysis and experimental validation, an anti-apoptotic pathway emerged from the fog. miR-142a was identified to be downregulated by enhanced ERK-phosphorylation in BA-harboring cells, thereby relieving its repression on Ciapin1, an apoptosis inhibitor. Moreover, miR-142a overexpression could partially rescue the abnormal anti-apoptotic phenotype and attenuate CML progression.ConclusionTaken together, this study explored the miRNA-mRNA regulatory networks in murine CML LSKs and demonstrated that ERK-miR-142a-Ciapin1 axis played an essential role in CML pathogenesis.