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Drake, Joshua, Anna Nichenko, Orion Wiloughby, Matt Brisendine, Garrett Hays, Grace DiGirolamo, Zach Weingrad i Ryan McMillan. "PHOSPHORYLATION OF ULK1 AT S555 IS REQUIRED FOR METABOLIC ADAPTATIONS TO CALORIC RESTRICTION". Innovation in Aging 6, Supplement_1 (1.11.2022): 424–25. http://dx.doi.org/10.1093/geroni/igac059.1668.
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Abstract Unc-51 Like Autophagy Activating Kinase 1 (Ulk1) is responsible for initiating selective degradation of damaged/dysfunctional mitochondria (mitophagy) once phosphorylated at S555 in response to energetic stress. Mitophagy is integral for mitochondrial health and Ulk1 has been implicated to be important for metabolic adaptation to exercise. Caloric restriction (CR), which extends lifespan and healthspan, has profound metabolic benefits, including improved mitochondrial health. However, the contribution of Ulk1 in adaptation to CR is unknown. To decipher a functional role of Ulk1(S555) in adaptations to CR we used CRISPR-Cas9 generated, loss-of-function Ulk1(S555A) mice, in which Ulk1 cannot be phosphorylated at S555. 6-month-old, male and female homozygous Ulk1(S555A) mice and C57BL6/J (wild type, WT) mice were placed on a 40% CR diet for 8 weeks. Body mass in both male and female Ulk1(S555A) and WT mice was reduced with CR (p < 0.001), however female Ulk1(S555A) were heavier than their WT counterparts (p=0.02). Via nuclear magnetic resonance (NMR), male and female Ulk1(S555A) mice did not lose fat mass during CR. In addition, periovarian (female) and epididymal (male) fat mass was greater in Ulk1(S555A) compared to WT mice post-CR (p < 0.001). Furthermore, fasting blood glucose increased in male and female Ulk1(S555A) post-CR (p < 0.0001), suggesting altered substrate metabolism. In support of this notion, glucose oxidation in both quadriceps muscle and liver of male mice increased in WT following CR but not in Ulk1(S555A) mice (interaction effect p< 0.002). In sum, these data suggest that phosphorylation of Ulk1 at S555 is required for metabolic adaptations to CR.
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Guan, Yuntian, Mei Zhang, Christie Lacy, Soham Shah, Frederick H. Epstein i Zhen Yan. "Endurance Exercise Training Mitigates Diastolic Dysfunction in Diabetic Mice Independent of Phosphorylation of Ulk1 at S555". International Journal of Molecular Sciences 25, nr 1 (3.01.2024): 633. http://dx.doi.org/10.3390/ijms25010633.
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Millions of diabetic patients suffer from cardiovascular complications. One of the earliest signs of diabetic complications in the heart is diastolic dysfunction. Regular exercise is a highly effective preventive/therapeutic intervention against diastolic dysfunction in diabetes, but the underlying mechanism(s) remain poorly understood. Studies have shown that the accumulation of damaged or dysfunctional mitochondria in the myocardium is at the center of this pathology. Here, we employed a mouse model of diabetes to test the hypothesis that endurance exercise training mitigates diastolic dysfunction by promoting cardiac mitophagy (the clearance of mitochondria via autophagy) via S555 phosphorylation of Ulk1. High-fat diet (HFD) feeding and streptozotocin (STZ) injection in mice led to reduced endurance capacity, impaired diastolic function, increased myocardial oxidative stress, and compromised mitochondrial structure and function, which were all ameliorated by 6 weeks of voluntary wheel running. Using CRISPR/Cas9-mediated gene editing, we generated non-phosphorylatable Ulk1 (S555A) mutant mice and showed the requirement of p-Ulk1at S555 for exercise-induced mitophagy in the myocardium. However, diabetic Ulk1 (S555A) mice retained the benefits of exercise intervention. We conclude that endurance exercise training mitigates diabetes-induced diastolic dysfunction independent of Ulk1 phosphorylation at S555.
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Stockklausner, Clemens, Christin Maria Duffert, Nicole Dickemann, Anne Christine Klotter, Isabelle Nadine Kuhlee, Carolin Kerber i Andreas E. Kulozik. "The Predominant Activation of Alternative Signaling Pathways Explains the Differential Propensity of Different c-Mpl Gain-of-Function Mutations to Promote Myeloproliferative Malignancies". Blood 124, nr 21 (6.12.2014): 1578. http://dx.doi.org/10.1182/blood.v124.21.1578.1578.
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Abstract Thrombopoiesis is tightly regulated by the binding of thrombopoietin (THPO) to its receptor c-Mpl that leads to the clearance of THPO from the plasma thus establishing a negative feedback loop. Several mutations in the c-Mpl receptor gene have been linked to a gain-of-function resulting in thrombocytosis. We focused on a comparison of gain-of-function mutation in the extracellular part of the c-Mpl receptor, where ligand binding and receptor dimerization occur, with the S505N, W515K and W515L mutations in the transmembrane and juxtamembrane region, respectively. Interestingly, the latter mutations are known to also promote myeloproliferative malignancies and AML, whereas the P106L mutation causes hereditary thrombocytosis without a known predisposition to hematologic malignancies. We have now performed functional analyses of these gain-of-function mutations to address the question of how the different propensity to induce malignancy can be explained. We first analyzed the post-translational processing of the normal and the P106L mutated receptor in comparison to the receptors carrying the S505N, W515K and W515L mutations in transfected HeLa and BA/F3 cells. The normal and the S505N, W515K and W515L mutated c-Mpl receptors were properly glycosylated during their transport through the Golgi apparatus, whereas the P106L mutated receptor did not enter the Golgi and was not fully glycosylated. The plasma membrane expression, assayed by confocal microscopy and FACS, of the S515N, W515K and W515L mutated receptors was comparable to the normal receptor, whereas the P106L mutated receptor was not detectable on the cell surface. Functional analyses of the THPO/c-Mpl signaling pathways in THPO stimulated c-Mpl transfected BA/F3 cells showed activation of the ERK1/2 pathway in all mutants but only weaker activation of the PI3K/m-TOR and Stat3/5 signaling pathways for the P106L mutant. By contrast, cells transfected with the normal receptor gene and the S505N, W515K and W515L c-Mpl mutants showed predominant up-regulation of the PI3K/m-TOR and Stat3/5 pathways. These results show that (1) the activation of c-Mpl by THPO does not absolutely require surface expression of the receptor (2) different c-Mpl gain-of-function mutations activate separable downstream pathways and (3) the predominant activation of the PI3K/m-TOR and Stat3/5 pathways correlates with the propensity to induce hematopoietic malignancy. Further, it is interesting to note that the c-Mpl P106L gain-of-function mutant is known to cause 10 to 20-fold elevated thrombopoietin (THPO) plasma levels in patients and is shown here not to be properly glycosylated and transported to the cell surface. By contrast, patients with the other gain-of-function mutations analyzed here show normal or even reduced THPO plasma concentrations and normal post-translational processing and cell surface expression. In conclusion, we propose that c-Mpl gain-of-function mutations exert their effect by predominantly activating either the PI3K/m-TOR and Stat3/5 or the ERK signaling pathways and that the predominant activation of PI3K/m-TOR and Stat3/5 correlates with the propensity to induce malignancy. Furthermore, THPO clearance and maintenance of the negative feedback loop regulating THPO plasma levels, but not signaling activity of the c-Mpl receptor necessarily require proper glycosylation, intracellular trafficking and cell surface expression. Disclosures No relevant conflicts of interest to declare.
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Levy, Gabriel, Serge Carillo, Benjamin Papoular, Bruno Cassinat, Jean-Marc Zini, Emilie Leroy, Leila N. Varghese i in. "MPL mutations in essential thrombocythemia uncover a common path of activation with eltrombopag dependent on W491". Blood 135, nr 12 (19.03.2020): 948–53. http://dx.doi.org/10.1182/blood.2019003240.
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Abstract Mutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.
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Porret, Naomi A., Elisabeth Oppliger Leibundgut, Serge Carillo, Bruno Cassinat, Francois Girodon, Éric Lippert, Valérie Ugo i in. "Interlaboratory Quality Control Round of MPL Mutation Detection in Fourteen European Laboratories: A MPN&MPNr-EuroNet Study",. Blood 118, nr 21 (18.11.2011): 3859. http://dx.doi.org/10.1182/blood.v118.21.3859.3859.
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Abstract Abstract 3859 The MPL gene is located on chromosome 1p34 and encodes the thrombopoietin receptor. It includes 12 exons and is a key factor for growth and survival of megacaryocytes. Acquired mutations in this gene activate the thrombopoietin receptor constitutively. MPL515 somatic mutations are stem cell-derived events that involve both myeloid and lymphoid progenitors. Two distinct exon 10 mutations are found in 15% of JAK2-V617F negative myeloproliferative neoplasms (MPN),), i.e. 3% of essential thrombocythemia (ET) and 10% of primary myelofibrosis (PMF).: W515L, W515K and the rare W515A variant. A hereditary mutation, S505N, is associated with familial thrombocytosis. MPL mutation detection is a helpful new tool to detect clonality in JAK2 V617F negative MPN and to establish the diagnosis of MPN. As many laboratories use very different methods and interpretations, standardization is highly warranted. Particularly the methodology is diverse and the results need to be comparable, requiring comprehensive testing. This quality control project was established within workgroup 2 of the MPN&MPNr-EuroNet network, (www.mpneuronet.eu, supported by the European program COST (CoOperation in Science and Technology)). The lab from the ‘Hopital H. Mondor AP-HP Paris' provided 29 samples containing randomized concentrations (between 100% and 1%) of the four mutations MPL W515L, W515K, W515A and S505N. The plasmids used for this quality control experiment spanned exons 9, intron 9 and exon10 of the MPL with S505N, W515L, W515K and W515A mutants diluted first with wild-type plasmid gene and then diluted in human genomic DNA. Thirteen European laboratories tested these 29 samples, each using their own chosen methods (14 altogether). The following methods were used: Mutascreen W515L/K Kit (Ipsogen, France):(n=4), allelic discrimination real-time PCR (n=2), high resolution melting (HRM) (n=7) and sequencing (n=2, 1 Sanger, 1 pyrosequencing)). There were no false positive results in any of the labs. All labs using the Mutascreen W515L/K Kit detected all W515L and W515K mutations, from 100% mutated down to 1% mutated plasmids. The allelic discrimination assays which were also designed for W515L and W515K only, detected the mutations down to 2%. The HRM methods were all designed differently. All except one (which did not recognize S505N) detected all 4 mutations with a sensitivity, down to 5% mutated plasmids, with few exceptions detecting either lower or higher amounts. The Sanger sequencing and pyrosequencing assays had a detection limit of 5–10%, with the pyrosequencing assay not being designed for the S505N mutation. All participating labs detected the most frequent MPL mutations in MPN W515L and W515K, with many designs not including W515A and S505N. Achieved sensitivities differed between methods with cutoffs of 1% to 10% (1.5% for the Ipsogen kit). Most laboratories reported the results as either positive or negative. However, the percentages of mutated alleles reported by a few labs differed greatly from each other and from the original dilutions (range 2–50 times) In conclusion, these results show that the diverse methods for MPL mutation detection used by different European labs yielded comparable specificity with varying sensitivity. Smaller clones might be missed by the less sensitive methods, and quantification of mutated alleles should be interpreted very carefully until standardised reference material for MPL mutation testing will be available. More extensive interlaboratory testing including patient samples is needed to identify the most robust assays suitable for diagnostic mutation detection and particularly for quantification of mutated alleles. Disclosures: No relevant conflicts of interest to declare.
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Stockklausner, Clemens, Christin Maria Duffert, Ziwei Zhou, Anne Christine Klotter, Isabelle Nadine Kuhlee i Andreas E. Kulozik. "Mpl Gain-of-Function Mutations Can be Classified By Differential Subcellular Processing, Molecular Mechanisms, Mode of Inheritance and Clinical Impact". Blood 126, nr 23 (3.12.2015): 1634. http://dx.doi.org/10.1182/blood.v126.23.1634.1634.
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Abstract The interaction between the c-Mpl receptor and its ligand thrombopoietin (TPO) on the cell surface is crucial for the regulation of thrombopoiesis. Several mutations in the c-Mpl receptor gene have been linked to a gain-of-function resulting in thrombocytosis. We have analyzed the known gain-of-function mutations in the extracellular part of the Mpl receptor, K39N and P106L, as well as the S505N, W515K and W515L mutations in the transmembrane and juxtamembrane region, respectively. Interestingly, the latter mutations can occur as autosomal dominant and/or as somatic mutations and are known to be associated with myeloproliferative malignancies and AML, whereas the abundant K39N and the P106L mutations are the cause of autosomal recessive hereditary thrombocytosis without a known predisposition to hematologic malignancies. To date, these differences in clinical impact and mode of inheritance are poorly understood. Starting from these clinical observations, we have performed functional analyses of the described gain-of-function mutations to address the key functional properties that might explain the observed clinical differences. Three crucial stages of the c-Mpl receptor life cycle were addressed: (1) post-translational processing of the immature receptor protein and its subcellular distribution, (2) membranous expression of the mature receptor and (3) receptor internalization upon stimulation with its ligand TPO. We first analyzed the post-translational processing of the normal, the K39N and the P106L mutated receptor in comparison with receptors carrying the S505N, the W515K and W515L mutations in a HeLa cell culture model. The normal, the K39N, S505N, W515K and W515L mutated c-Mpl receptors were properly glycosylated during their transport through the Golgi apparatus, whereas the P106L mutated receptor did not enter the Golgi and was not fully glycosylated. The K39N mutant was fully glycosylated but did show different running behavior on the SDS Gel, most likely caused by post-translational modifications different from glycosylation. The S505N, the W515K and the W515L mutated receptors displayed stable surface expression in confocal microscopy and FACS analysis, whereas the P106L mutated receptor was not detectable on the cell surface. After stimulation with TPO, a decrease in mean receptor surface protein could be observed for the wild type and all mutants that were expressed on the surface, namely S505N, W515K and W515L, however not significant (p>0.05). Interestingly, our functional analyses of the TPO/c-Mpl signaling pathways in TPO stimulated c-Mpl transfected BA/F3 cells showed activation of the ERK1/2 pathway in all mutants but only weaker activation of the PI3K/m-TOR and Stat3/5 signaling pathways for the P106L mutant. By contrast, cells transfected with the wild type, the S505N, W515K and W515L c-Mpl mutants showed predominant up-regulation of the PI3K/m-TOR and Stat3/5 pathways. These results show that first, both impaired and regular receptor glycosylation and correlating subcellular distribution may occur in c-Mpl gain-of function mutants. Second, the c-Mpl gain-of-function mutants differ substantially in surface expression levels. Third, our results suggest differences in the maintenance of the TPO negative feedback loop across c-Mpl gain-of-function mutants. Indeed, in contrast to P106L, it seems likely that the TPO negative feedback-loop is preserved in the S505N, the W515K and the W515L mutants. In line with this, highly elevated TPO serum levels have only been described for P106L, but not for the other gain-of-function mutations. We hypothesize that maintenance of the TPO negative feedback-loop is sufficient to prevent dysregulation of TPO levels but not transmission of a harmful c-Mpl gain-of-function effect. Instead, the predominant activation of the PI3K/m-TOR and Stat3/5 pathways might explain the different propensity to induce hematopoietic malignancy. In summary, our findings suggest the existence of different disease causing molecular mechanisms behind the mutations' respective clinical correlates and provide the basis for an important extension to the current classification of c-Mpl mutations that is primordially based on clinical observations. Disclosures No relevant conflicts of interest to declare.
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Ardicli, Sena, Hale Samli, Deniz Dincel, Bahadir Soyudal i Faruk Balci. "Individual and combined effects of <i>CAPN1</i>, <i>CAST</i>, <i>LEP</i> and <i>GHR</i> gene polymorphisms on carcass characteristics and meat quality in Holstein bulls". Archives Animal Breeding 60, nr 3 (8.09.2017): 303–13. http://dx.doi.org/10.5194/aab-60-303-2017.
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Abstract. The objective of this study was to determine the association of single nucleotide polymorphisms (SNPs) with carcass characteristics and meat quality traits in selected candidate genes in Holstein bulls. Five SNPs in four genes, i.e. calpain 1 (CAPN1), calpastatin (CAST), leptin (LEP) and growth hormone receptor (GHR), were genotyped in 400 purebred bulls using PCR-RFLP. Statistically significant associations were as follows: CAPN1 G316A with live weight, carcass weight, back fat thickness, m. longissimus thoracis et lumborum area and carcass measurements; CAPN1 V530I with pH and L∗; CAST S20T with live weight, inner chest depth and b∗ value; and GHR with ph, a∗ and h∗. In addition, significant genotypic interactions were observed for dressing percentage (LEP A80V × CAST S20T), pH (CAPN1 V530I × GHR S555G and LEP A80V × GHR S555G) and rump width (CAPN1 V530I × CAST S20T). There was no association between the LEP A80V marker and any of the traits evaluated, nor was there any association of the tested SNPs with chest width, C∗ and marbling score. The present results could therefore be indicative for future studies on meat yield and quality.
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Bridgford, Jessica L., Su Min Lee, Christine M. M. Lee, Paola Guglielmelli, Elisa Rumi, Daniela Pietra, Stephen Wilcox i in. "Novel drivers and modifiers of MPL-dependent oncogenic transformation identified by deep mutational scanning". Blood 135, nr 4 (23.01.2020): 287–92. http://dx.doi.org/10.1182/blood.2019002561.
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Abstract The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F− essential thrombocythemia and primary myelofibrosis patients, respectively, have “canonical” MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other “noncanonical” MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.
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Kim, Ung-Jun, Ho-Jong Lee, In-Sun Choi, Seong-Ho Kang, Sook-Jin Jang, Dae-Soo Moon i Geon Park. "Primary Myelofibrosis with MPL S505N Mutation: The First Case Reported in Korea". Laboratory Medicine Online 8, nr 4 (2018): 167. http://dx.doi.org/10.3343/lmo.2018.8.4.167.
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Aung, Lynn, Tommy Petros, Jessica Geiger i Sara Graham. "Agree to Disagree? Equianalgesic Consensus Amongst Hospice and Palliative Providers (S555)". Journal of Pain and Symptom Management 63, nr 5 (maj 2022): 936–37. http://dx.doi.org/10.1016/j.jpainsymman.2022.02.178.
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Martinez-Aviles, Luz Maria, Carlos Besses, Alberto Alvarez-Larran, Aina Pons, Sergi Serrano i Beatriz Bellosillo. "TET2 Mutations in a Cohort of Patients with Myeloproliferative Neoplasms Lacking a Molecular Marker." Blood 114, nr 22 (20.11.2009): 3912. http://dx.doi.org/10.1182/blood.v114.22.3912.3912.
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Abstract Abstract 3912 Poster Board III-848 Background The myeloproliferative neoplasms (MPNs) include different diseases presenting several mutations in variable frequency. The JAK2V617F mutation is present in 90-95% Polycythemia Vera (PV), 50-60% Essential Thrombocythemia (ET) and 50-60% Primary Myelofibrosis (PMF) patients. In addition, JAK2 exon 12 mutations are observed in 1-3% of PV patients and mutations in the thrombopoietin receptor gene (c-MPL) (S505N, W515K/L) are present in a 5-9% of PMF and in a 1-4% of ET patients. Recently, acquired mutations of the Ten-Eleven Translocation (TET) 2 gene, have been reported in about 14% of sporadic MPNs. TET2 mutations may precede the acquisition of JAK2V617F predisposing to a MPN development. However, the incidence of TET2 mutations in patients lacking the JAK2V617F mutation has not been extensively studied. Aim To analyze the incidence of TET2 mutations in a cohort of MPN patients negative for JAK2V617F, JAK2 exon 12 mutations and c-MPL exon 10 mutations (S505N or W515K/L). Patients and methods From a whole cohort of 241 patients with MPN (93 PV, 16 PMF and 132 ET) we excluded those patients positive for JAK2V617F (determined by quantitative allele-specific PCR), JAK2 exon 12 mutations or c-MPL exon 10 mutations (S505N or W515K/L) (analyzed by direct sequencing). We analyzed the TET2 gene in 5 PV, 5 PMF and 53 ET patients lacking any of the aforementioned molecular markers. The mutational analysis of the coding sequence of TET2 was performed by direct sequencing using cDNA from granulocytes. In 13 patients, DNA from T lymphocytes was obtained to indentify the presence of single nucleotide polymorphisms (SNPs) in germline DNA. Results Sixty-three patients were screened for mutations in the whole coding sequence of the TET2 gene. Only 3 ET patients (4.7%) presented deleterious mutations in the TET2 gene. The three distinct TET2 mutations were: Q706X, S1848X and V1395_R1400delinsR. In 48 out of 63 (76.1%) patients we observed a total of 13 different missense mutations and 2 silent mutations in the coding sequence of the gene. The most frequent missense mutation was the I1762V which was detected in 27 patients. In 13 patients whose matched normal DNA was available, we analyzed the presence of missense mutations being all of them present in the control DNA suggesting that they were SNPs and not acquired mutations. The two nonsense mutations (Q706X and S1848X), were not present in matched normal tissue indicating that these mutations were somatically acquired in myeloid cells. Conclusions TET2 pathogenic mutations are infrequent (<5%) in myeloproliferative neoplasms negative for JAKV617F, JAK2 exon 12 and c-MPL (S505, W515K/L) gene mutations. The role and the biological significance of missense mutations in the coding sequence of TET2 has to be elucidated. Acknowledgments Fellowship FI2008 (AGAUR) to LMM-A, FIS EC07/90791 Disclosures: No relevant conflicts of interest to declare.
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Hendrickson, John A. "Probability, Random Signals, and Statistics". Technometrics 43, nr 1 (luty 2001): 103–4. http://dx.doi.org/10.1198/tech.2001.s555.
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Liu, K., M. Martini, B. Rocca, C. I. Amos, L. Teofili, F. Giona, J. Ding, H. Komatsu, L. M. Larocca i R. C. Skoda. "Evidence for a founder effect of the MPL-S505N mutation in eight Italian pedigrees with hereditary thrombocythemia". Haematologica 94, nr 10 (16.07.2009): 1368–74. http://dx.doi.org/10.3324/haematol.2009.005918.
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Waqar, Maham, Muhammad Kamal, Umar Hayat, Fnu Faheela i Muhammad Haseeb. "S555 Impact of Constipation on Resource Utilization in Patients with Parkinson’s Disease: A Nationwide Analysis". American Journal of Gastroenterology 117, nr 10S (październik 2022): e392-e393. http://dx.doi.org/10.14309/01.ajg.0000858860.23950.61.
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Wang, Edwin, Neha Naidoo, Christina Tran, Zhaoyi Wang, Elaine Chen, Jenny Jung Sun Park, Vy Nguyen i Yutaka Tomizawa. "S555 Racial Differences of Neoplastic Progression of Barrett’s Esophagus: A Large Multi-Ethnic North American Cohort". American Journal of Gastroenterology 118, nr 10S (październik 2023): S409—S410. http://dx.doi.org/10.14309/01.ajg.0000951860.58517.35.
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Ardicli, Sena, Deniz Dincel, Hale Samli i Faruk Balci. "Effects of polymorphisms at <i>LEP</i>, <i>CAST</i>, <i>CAPN1</i>, <i>GHR</i>, <i>FABP4</i> and <i>DGAT1</i> genes on fattening performance and carcass traits in Simmental bulls". Archives Animal Breeding 60, nr 2 (11.04.2017): 61–70. http://dx.doi.org/10.5194/aab-60-61-2017.
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Abstract. The aim of this study was to investigate the effects of single nucleotide polymorphisms (SNPs) at six candidate genes (LEP, CAST, CAPN1, GHR, FABP4 and DGAT1) on fattening performance and carcass traits of Simmental bulls in Turkey. The analysis covered a total of 81 Simmental bulls grown on a private farm that were randomly selected for their fattening period for use in this study. Genotyping was performed using the PCR-RFLP method. The S20T polymorphism at the CAST gene and the G316A polymorphism at the CAPN1 gene were associated with variation in final weight, fattening period, weight gain and average daily gain (P < 0.05). In addition, LEP A80V had a significant effect on hot and chilled carcass weight and dressing percentage (P < 0.05). There was no association between GHR S555G, FABP4 V110M and DGAT1 K232A markers with the traits analysed. These results suggested that focusing on the novel effects of LEP, CAST and CAPN1 gene polymorphisms on meat production traits might be useful for marker-assisted selection in Simmental cattle.
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Defour, Jean-Philippe, Gabriel Levy, Emilie Leroy, Steven O. Smith i Stefan N. Constantinescu. "The S505A thrombopoietin receptor mutation in childhood hereditary thrombocytosis and essential thrombocythemia is S505N: single letter amino acid code matters". Leukemia 33, nr 2 (11.01.2019): 563–64. http://dx.doi.org/10.1038/s41375-018-0356-x.
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Williams, J. D., i F. Moosdeen. "Antibiotic Resistance in Haemophilus influenzae: Epidemiology, Mechanisms, and Therapeutic Possibilities". Clinical Infectious Diseases 8, Supplement_5 (1.11.1986): S555—S561. http://dx.doi.org/10.1093/clinids/8.supplement_5.s555.
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Dermawan, Budi, Tsuko Nakamura i Fumi Yoshida. "Subaru Lightcurve Observations of Sub-km-Sized Main-Belt Asteroids". Publications of the Astronomical Society of Japan 63, sp2 (25.03.2011): S555—S576. http://dx.doi.org/10.1093/pasj/63.sp2.s555.
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Markowitz, Lauri E., i Phillip Nieburg. "The Burden of Acute Respiratory Infection Due to Measles in Developing Countries and the Potential Impact of Measles Vaccine". Clinical Infectious Diseases 13, Supplement_6 (1.05.1991): S555—S561. http://dx.doi.org/10.1093/clinids/13.supplement_6.s555.
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Ding, Jianmin, Hirokazu Komatsu, Shinsuke Iida, Asahi Ito, Hiroki Yano, Masaki Ri, Atsushi Inagaki i in. "Mutational Analysis of Thrombopietin, c-MPL and Jak2 Genes in Essential Thrombothethymia Patients - Identification of Concurrent Mutations of c-MPL and Jak2." Blood 110, nr 11 (16.11.2007): 4658. http://dx.doi.org/10.1182/blood.v110.11.4658.4658.
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Abstract The molecular pathogenesis in chronic myeloproliferative disorder including essential thrombocythemia (ET) has been recently clarified. We previously identified a novel activating mutation of c-MPL gene (S505N) as a cause of familial ET. The Jak2 mutation (Jak2V617F) is also reported most frequently as a constitutive activating mutation in ET. Here we screened the mutations in the open reading frames of thrombopoietin (TPO), c-MPL and Jak2 in the 26 Japanese (sporadic) ET patients. None had any somatic mutations in TPO. We found Jak2V617F mutation in 7 patients. Interestingly, one of the patients had the c-MPL somatic mutation (W515L). The c-MPL W515L transfectants presented the autonomous STAT5 and MEK1/2 phosphorylation in the absence of the ligand, which show the c-MPL W515L is a constitutive activating mutation for signalling. The concurrent mutations of Jak2V617F and c-MPL W515L are likely to contribute to the pathogenesis of the thrombocythemia in this patient. Now we are focusing on the issue whether the single clone with both of two mutations occupy or the two clones with either Jak2 or c-MPL mutation coexist in the bone marrow of this ET patient (Single cell PCR is on going with the megakaryocytes of this patient).
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Gupta, Sudhir Kumar, Ashwini Charpe, Sunita Koul, Kumble Vinod Prabhu i Qazi Mohd Rizwanul Haq. "Development and validation of molecular markers linked to an Aegilops umbellulata–derived leaf-rust-resistance gene, Lr9, for marker-assisted selection in bread wheat". Genome 48, nr 5 (1.10.2005): 823–30. http://dx.doi.org/10.1139/g05-051.
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An Aegilops umbellulata–derived leaf-rust-resistance gene, Lr9, was tagged with 3 random amplified polymorphic DNA (RAPD) markers, which mapped within 1.8 cM of gene Lr9 located on chromosome 6BL of wheat. The markers were identified in an F2 population segregating for leaf-rust resistance, which was generated from a cross between 2 near-isogenic lines that differed in the alien gene Lr9 in a widely adopted agronomic background of cultivar 'HD 2329'. Disease phenotyping was done in controlled environmental conditions by inoculating the population with the most virulent pathotype, 121 R63-1 of Puccinia triticina. One RAPD marker, S5550, located at a distance of 0.8 ± 0.008 cM from the Lr9 locus, was converted to sequence-characterized amplified region (SCAR) marker SCS5550. The SCAR marker was validated for its specificity to gene Lr9 against 44 of the 50 known Lr genes and 10 wheat cultivars possessing the gene Lr9. Marker SCS5550 was used with another SCAR marker, SCS73719, previously identified as being linked to gene Lr24 on a segregating F2 population to select for genes Lr9 and Lr24, respectively, demonstrating the utility of the 2 markers in marker-assisted gene pyramiding for leaf-rust resistance in wheat.Key words: wheat, leaf rust resistance, Lr9, Lr24, RAPD, SCAR.
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Yamamoto, Yukiya, Sachiko Iba, Akihiro Abe i Nobuhiko Emi. "Elongation of MPL Transmembrane Domain Is a Novel Activating-Mutation in Essential Thrombocythemia". Blood 126, nr 23 (3.12.2015): 1628. http://dx.doi.org/10.1182/blood.v126.23.1628.1628.
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Abstract Essential thrombocythemia (ET) is a clonal disease of hematopoietic stem cell. Somatic gene mutations including JAK2 or CALR are hallmark of diagnosis and molecular targets for developing novel therapies. However, non-mutated ET cases within JAK2 and CALR still exist. The third mutated gene of ET is MPL, which is known as encoding thrombopoietin receptor. MPL W515 and S505 are hot spot for missense mutations leading to constitutive active MPL signaling. These missense mutations are located within transmembrane or juxtamembrane domain of MPL. Here, we present a novel activating-mutation of MPL in an ET patient. The patient was a 54-year-old woman with occasional headache. A full blood count showed a hemoglobin level of 12.2 g/dL, a white blood cell count of 11.2 x 103/mL and a platelet count of 164 x 104/mL. A bone marrow sample was hypercellular, containing increased megakaryocytes. Chromosomal analysis showed normal karyotype. Further genetic analysis did not detect JAK2 V617F or CALR mutation. Finally, we directly sequenced MPL exon 10. The result showed MPL c.1496-1497AT>TGGGCCTCAGCTGGGCG (Figure 1). This mutation has been considered as an in-frame mutation, indicating MPL p.H499LGLSWA (reference: NP_005364). The amino acids insertion in transmembrane domain of MPL belongs to hydrophobic family, suggesting that MPL H499 mutation (H499ins) might construct stable structure. To investigate whether MPL H499ins is functionally active, we established stable BaF3/MPL H499ins cell lines. In contrast to BaF3/MPL wild-type, BaF3/MPL H499ins cells proliferate without WEHI3-conditioned medium as well as BaF3/MPL W515L or S505N. Western blot analysis showed BaF3/MPL H499ins cells constitutionally activate downstream signaling including JAK-STAT, MAPK and AKT. Furthermore, we established stable BaF3 cell lines with MPL H499 LGLSWALGLSWA (H499 insx2), MPL H499del and others. In contrast to BaF3/H499del, H499L, H499LG and H499insx3, BaF3/MPL H499insx2 cells proliferate without WEHI3-conditioned medium. This result suggests that elongation of MPL transmembrane domain is a novel oncogenic mechanism leading to constitutive active MPL signaling. Phosphorylation of MPL Y626 has significant role to transduce MPL signaling. To explore if constitutive activation of MPL H499ins depends on phosphorylation of MPL Y626, we established stable BaF3 cell lines with MPL H499insY626F. The BaF3 cells could not proliferate without WEHI3-conditioned medium. This result clearly shows phosphorylation of MPL Y626 has a pivotal role for constitutive activation of MPL H499ins. Finally, we examined potential effect to inhibit constitutive MPL signaling with JAK1/2 inhibitor, Ruxolitinib. In contrast to K562, growth of BaF3/MPL H499ins cells were inhibited with Ruxolitinib at half maximal effective concentration 113 nM as well as MPL W515L or S505N (Figure 2). In summary, elongation of MPL transmembrane domain is a novel oncogenic mechanism in ET patients. Ruxolitinib is also a potent inhibitor against MPL activating mutations as well as JAK2 V617F-associated myeloproliferative neoplasm. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.
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Badar, Nazish, Aamer Ikram, Muhammad Salman, Muhammad Masroor Alam, Massab Umair, Yasir Arshad, Nighat Mushtaq i in. "Chikungunya virus: Molecular epidemiology of nonstructural proteins in Pakistan". PLOS ONE 16, nr 12 (23.12.2021): e0260424. http://dx.doi.org/10.1371/journal.pone.0260424.
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Chikungunya virus (CHIKV) is considered a public health problem due to its rapid spread and high morbidity. In 2016–2017 an outbreak of CHIKV was occurred in Pakistan but the data regarding the genomic diversity of CHIKV was not reported. Hence, the current study aimed to determine the genetic diversity of CHIKVs in Pakistan. A cross sectional study was carried out using sera of infected CHIKV patients (n = 1549) during the outbreak in Pakistan (2016–2018). Nucleotide sequencing of non-structural genes of CHIKV from eight isolates were performed followed by phylogenetic analysis using Bayesian method. Phylogenetic analysis suggested that the Pakistani CHIKV strains belonged to Indian Ocean Lineage (IOL) of genotype ECSA and C1.3a clade. Furthermore, the Pakistani isolates showed several key mutations (nsP2-H130Y, nsP2-E145D, nsP4-S55N and nsP4- R85G) corresponding to mutations reported in 2016 Indian strains of CHIKV. The molecular analysis revealed high evolutionary potential of CHIKV strains as well as better understanding of enhanced virulence and pathogenesis of this outbreak. The study highlights the need to continue surveillance in order to understand viral diversity over time and to devise preventive measures to limit diseases transmission in the region.
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Yassin, Mohamed A., Nader I. Al-Dewik, Hanadi ElAyoubi i Bruno Cassinat. "Familial Essential Thrombocythemia Among Qatari Tribes". Blood 122, nr 21 (15.11.2013): 5244. http://dx.doi.org/10.1182/blood.v122.21.5244.5244.
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Abstract Essential thrombocythaemia (ET) is a chronic myeloproliferative syndrome due to sustained proliferation of megakaryocytes,which results in elevated numbers of circulating platelets, thrombotic or haemorrhagic episodes and occasional leukaemic transformation.Familial essential thrombocythemia,also called hereditary or inherited thrombocythemia or thrombocytosis, is a rare disorder transmitted in an autosomal dominant with clinical presentation and complications resembling sporadic essential thrombocythaemia (ET).Mutations in the thrombopoietin (TPO) gene causing overproduction of TPO and elevated TPO serum levels familial essential thrombocythemia is a heterogeneous disorder as families have been described with an activating mutation in the gene for the TPO receptor (c-MPL), the thrombopoietin gene, a germline mutation in JAK2 V617I or without a mutation in any of these genes Occurrence of Essential thrombocythemia among arab population never been reported before. In this study, essential thrombocythemia is documented in 25 families from 5 major tribes in Qatar Familial cases were defined when two or more individuals within the same pedigree were affected with MPNs. Clinical records of affected relatives were reviewed systematically to confirm MPNs diagnosis. Patients without family history of were defined as having sporadic. MPNs Diagnosis of MPNs was made in accordance with the WHO criteria 2008 Ninety eight members of both sexes from 18 to 62 years of age in three successive generations. The propositus had a persistent elevation of the platelet count, splenomegaly, a normal hemoglobin level, a normal white blood cell count, and abnormal platelet aggregation. Platelet arachidonic acid metabolites assayed by high-performance liquid chromatography and serum thrombopoietin levels were normal. Megakaryocytes were increased in number and size. Both mature and early immature megakaryocytes, but no atypical megakaryocytes, were identified by surface immunofluorescence. Bone marrow cultures showed normal myeloid and erythroid colony formation, and chromosome studies revealed a normal The analysis of mutations of JAK2 and MPL may improve our ability to identify these conditions. In a consecutive series of patients observed in our Institution from January 2011 to June 2013 total No of 300 MPNs patients were diagnosed, out of 300 patient, 119 had PV, 160 had ET, 15 had PMF and six patient had unclassified atypical MPN case. Out of 119 PV pts, 116 (97.4%) were positive for the JAK2 V617F mutation and 3 cases met the WHO criteria for PV but were negative for JAK2 V617F and JAK2 exon 12 mutations. Out of 160 ET pts, 80 (50%) were positive for JAK2 V617F, one had MPL S505N mutation and 79 cases met the WHO criteria for ET but were negative for both JAK2 617F and MPL mutations. Out of 15 PMF pts 5 (33.3%) were positive for JAK2 V617F and the unclassified case which was characterized by Deep Vein Thrombosis had JAK2 exon 13 mutation (R564L). 25 Arab families were described as having familial ET (5 major tribes were screened )20 were having JAK2V617F positive in three consequetive generations ,4 families were having Familial ET in three generations but both Jak2v617F and MPL were negative MPL S505N mutation was found in one family( Father and daughter).there was no difference in clinical presentation and response to therapy in sporadic versus familial cases. Conclusion In our series of familial cases clinical presentation, therapeutic approach type and severity of complications were comparable to that of sporadic cases. Furthermore, the present study indicates the relevant possibility of familial MPNs, thus suggesting the opportunity of a detailed family history as part of the initial work-up of patients with MPNs; in addition it also suggests the usefulness of an accurate biological studies Disclosures: Yassin: qatar national research fund: Patents & Royalties, Research Funding. Al-Dewik:qatar national research fund: Patents & Royalties, Research Funding. Cassinat:qatar national research fund: Patents & Royalties, Research Funding.
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Mittal, Anmol, Mansi Patel i Sushil Ahlawat. "S555 Trends in Evaluation of Obese Patients Admitted for Abdominal Pain Using Modalities Including Esophagogastroduodenoscopy (Egd), Computer Tomography (Ct), or Symptom Management Without Diagnostic Testing". American Journal of Gastroenterology 116, nr 1 (październik 2021): S253—S254. http://dx.doi.org/10.14309/01.ajg.0000774696.14983.d9.
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Ara, Anjuman, Aizhang Xu, Khawaja Ashfaque Ahmed, Scot C. Leary, Md Fahmid Islam, Zhaojia Wu, Rajni Chibbar i Jim Xiang. "The Energy Sensor AMPKα1 Is Critical in Rapamycin-Inhibition of mTORC1-S6K-Induced T-cell Memory". International Journal of Molecular Sciences 23, nr 1 (21.12.2021): 37. http://dx.doi.org/10.3390/ijms23010037.
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Energy sensors mTORC1 and AMPKα1 regulate T-cell metabolism and differentiation, while rapamycin (Rapa)-inhibition of mTORC1 (RIM) promotes T-cell memory. However, the underlying pathway and the role of AMPKα1 in Rapa-induced T-cell memory remain elusive. Using genetic and pharmaceutical tools, we demonstrate that Rapa promotes T-cell memory in mice in vivo post Listeria monocytogenesis rLmOVA infection and in vitro transition of effector T (TE) to memory T (TM) cells. IL-2- and IL-2+Rapa-stimulated T [IL-2/T and IL-2(Rapa+)/T] cells, when transferred into mice, differentiate into short-term IL-7R−CD62L−KLRG1+ TE and long-lived IL-7R+CD62L+KLRG1− TM cells, respectively. To assess the underlying pathways, we performed Western blotting, confocal microscopy and Seahorse-assay analyses using IL-2/T and IL-2(Rapa+)/T-cells. We determined that IL-2(Rapa+)/T-cells activate transcription FOXO1, TCF1 and Eomes and metabolic pAMPKα1(T172), pULK1(S555) and ATG7 molecules and promote mitochondrial biogenesis and fatty-acid oxidation (FAO). We found that rapamycin-treated AMPKα-deficient AMPKα1-KO IL-2(Rapa+)/TM cells up-regulate transcription factor HIF-1α and induce a metabolic switch from FAO to glycolysis. Interestingly, despite the rapamycin treatment, AMPKα-deficient TM cells lost their cell survival capacity. Taken together, our data indicate that rapamycin promotes T-cell memory via transcriptional FOXO1-TCF1-Eomes programs and AMPKα1-ULK1-ATG7 metabolic axis, and that AMPKα1 plays a critical role in RIM-induced T-cell memory.
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Hua, Chunting, Qiaoli Zheng, Jiang Zhu, Siji Chen, Yinjing Song, Stijn van der Veen i Hao Cheng. "Human Papillomavirus Type 16 Early Protein E7 Activates Autophagy through Inhibition of Dual-Specificity Phosphatase 5". Oxidative Medicine and Cellular Longevity 2022 (10.03.2022): 1–19. http://dx.doi.org/10.1155/2022/1863098.
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Consistent high-risk human papillomavirus (HPV) infection leads to various malignant cancers. Autophagy can promote cancer progression by helping cancer cells survive under stress or induce oncogenic effects when mutations or abnormalities occur. Mitogen activated protein kinases (MAPKs) can transduce various external or intrinsic stimuli into cellular responses, including autophagy, and dual-specificity phosphates (DUSPs) contribute to the direct regulation of MAPK activities. Previously, we showed that expression of DUSP5 was repressed in HPV16 E7-expressing normal human epidermal keratinocytes (NHEKs). Here we show that clinical HPV16 E7-positive precancerous and cancerous tissues also demonstrate low DUSP5 levels compared with control tissues, indicating that the inverse correlation between HPV16 E7 and DUSP5 is clinically relevant. We furthermore investigated the autophagy response in both DUSP5-deficient and HPV16 E7-expressing NHEKs. Confocal microscopy and Western analysis showed induction of LC3-II levels, autophagosome formation and autophagy fluxes in DUSP5-deficient NHEKs. Furthermore, Western analysis demonstrated specific induction of phosphorylated ERK in DUSP5-deficient and HPV16 E7-expressing NHEKs, indicating that HPV16 E7-mediated repression of DUSP5 results in induced MAPK/ERK signaling. Finally, phosphorylated mTOR and ULK (S757) were reduced in DUSP5-deficient NHEKs, while phosphorylated ULK (S555) and AMPK were increased, thereby inducing canonical autophagy through the mTOR and AMPK pathways. In conclusion, our results demonstrate that HPV16 E7 expression reduces DUSP5 levels, which in turn results in active MAPK/ERK signaling and induction of canonical autophagy through mTOR and MAPK regulation. Given its demonstrated inverse correlation with clinical cancerous tissues, DUSP5 may serve as a potential therapeutic target for cervical cancer.
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Ara, Anjuman, i Jim Xiang. "A critical role of energy sensor AMPKα1 in rapamycin dependent memory CD8+ T cell survival". Journal of Immunology 208, nr 1_Supplement (1.05.2022): 55.08. http://dx.doi.org/10.4049/jimmunol.208.supp.55.08.
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Abstract Energy sensors mTORC1 and AMPKα1 regulate T-cell metabolism and differentiation, while rapamycin (Rapa), an inhibitor of mTORC1, supports T-cell memory. However, the underlying pathway, the exact mechanisms, and the role of AMPKα1 in Rapa-induced T-cell memory is not well understood. Using the Listeria monocytogenes (rLmOVA) infection model and genetic and pharmaceutical tools, we demonstrate that Rapa promotes T-cell memory in mice post-rLmOVA infection. When T-cells stimulated in vitro in the presence of IL-2 without rapamycin (IL-2/T) or with rapamycin (IL-2+Rapa /T) and transferred into mice, it differentiated into short-term effector T (TE) [IL-7R−CD62L−KLRG1+] and long-lived memory T (TM) [IL-7R+CD62L+KLRG1−] cells, respectively. We determined that rapamycin-treated T cells activated transcriptional factors, FOXO1, TCF1 and Eomes and metabolic pAMPKα1(T172), pULK1(S555), and ATG7 molecules and promoted mitochondrial biogenesis and oxidative phosphorylation (OXPHOS). Using Seahorse-real time metabolic analyzer, we found that rapamycin-treated AMPKα-deficient TM cells up-regulated transcription factor HIF-1α and induced a metabolic switch from OXPHOS to glycolysis. Interestingly, despite the rapamycin treatment, AMPKα-deficient TM cells lost their cell survival capacity. Altogether, our data provide a mechanistic explanation for enhanced T-cell survival after rapamycin treatment and suggest that rapamycin promotes T-cell memory via transcriptional FOXO1-TCF1-Eomes programs and AMPKα1-ULK1-ATG7 metabolic axis. And AMPKα1 plays a critical role in rapamycin-induced increased survival and metabolic switching of CTLs during the transition of effector to memory T cells. Supported by Canadian Institutes of Health Research grant (409228). Saskatchewan Health Research Foundation (SHRF) postdoctoral fellowship.
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Braverman, Erica Lynne, Andrea Dobbs, Darlene A. Monlish i Craig Byersdorfer. "Increasing AMPK Activity in Human T Cells Enhances Memory Subset Formation without Sacrificing in Vitro Expansion". Blood 136, Supplement 1 (5.11.2020): 38–39. http://dx.doi.org/10.1182/blood-2020-141605.
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BACKGROUND: While chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL), treatment failures continue to occur. In studying therapeutic T cell function, it has become clear that achieving a memory-like phenotype is ideal for CAR-T production. This is likely related to the enhanced oxidative metabolic potential of this subset, which allows for improved persistence and enhanced anti-leukemia activity in vivo. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. Finding methods to improve the yield of memory-like cells without sacrificing T cell expansion has been challenging. AMP-activated protein kinase (AMPK) is a key metabolic regulator responsible for promoting mitochondrial biogenesis and oxidative metabolism, and is more active in memory T cells at baseline. It is similarly induced by TCR ligation, making it unlikely that it would significantly detract from proliferation. These properties make activation of AMPK a potential candidate pathway for improving the yield of more functional T cells for CAR-T cell therapy. METHODS: AMPK is a heterotrimeric protein complex consisting of alpha, beta, and gamma domains. Functionally, the alpha subunit contains the kinase domain, which is activated by phosphorylation. The gamma subunit controls the phosphorylation, and therefore the activity, of the alpha domain. To increase AMPK signaling in T cells, we cloned the gamma subunit into a lentiviral plasmid containing the elongation factor 1a (EF1a) promoter and a green fluorescent protein (GFP) tag. An empty vector, containing GFP only, served as a negative control. Human T cells were isolated from three separate donors, transduced with our lentiviral construct, and expanded in vitro in the presence of IL-2. AMPK activity was assessed by phosphorylation of Thr172 on the AMPKα subunit as well as phosphorylation of S555 on downstream target Unc-51-like autophagy activating kinase (ULK1) using western blot densitometry, normalized to the total protein amounts. Memory marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes for in vitro expansion were calculated by adjusting manual cell counts to reflect GFP positivity and CD4+/CD8+ surface staining. RESULTS: The AMPK gamma subunit was efficiently transduced and expressed by human T cells as measured by GFP expression, qRT-PCR, and western blot analysis. Further, AMPK activity increased in GFP+ cells as indicated by the phosphorylation of AMPKα Thr172 (1.93 +/- 0.05 vs 0.6 +/- 0.09, p<0.001) and ULK1 S555 (1.28 +/- 0.11 vs 0.67 +/- 0.08, p<0.01). Cells transduced with AMPK augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (Figure 1). In addition, AMPK-transduced T cells showed a statistically significant increase in mitochondrial density along with notable enhancement of SRC and maximal oxygen consumption rates (Figure 2A,B). Furthermore, the rate of expansion of AMPK-transduced T cells did not differ significantly from Empty-transduced controls, and in fact trended towards increased in both CD4+ and CD8+ cells (Figure 3A). Indeed, the improved rate of expansion in AMPK-transduced CD4+ T cells led to a measurable increase in CD4+ T cell percentages by flow cytometry (Figure 3B). DISCUSSION: Here we present an efficient and direct method to increase AMPK activity in human T cells and demonstrate that increased AMPK activity endows T cells with a variety of characteristics ideal for CAR-T cell therapy. These features include increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.
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Lazebnaya, I. V., A. V. Perchun, B. B. Lhasaranov, O. E. Lazebny i Yu A. Stolpovskiy. "Analysis of GH1, GHR and PRL gene polymorphisms for estimation of the genetic diversity of Buryat and Altai cattle breeds". Vavilov Journal of Genetics and Breeding 22, nr 6 (27.09.2018): 734–41. http://dx.doi.org/10.18699/vj18.417.
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Small and unique Buryat and Altai cattle breeds of TuranoMongolian origin are well adapted to harsh conditions of the continental climate to be their habitat. However, the population-genetic structure of the breeds has been poorly studied. This paper presents the results of analysis of polymorphisms GH1 (AC_000176.1: BTA 19, exon 5, rs41923484, g.2141C>G, L127V), GHR (AC_000177.1: BTA 20, exon 10, rs109300983, g.257A>G, S555G) and PRL (AC_000180.1: BTA 23, exon 3, g.35108342A>G) in the samples of Buryat cattle breed of Russia, China and Mongolia, and indigenous Altai cattle breed (Russia) that belong to TuranoMongolian cattle. The Russian sample of Buryat breed was differentiated from the Mongolian sample based on pairwise G-test and FST values for the PRL-RsaI polymorphism and from the Chinese sample – based on pairwise G-test values for the GH1-AluI polymorphism. All the three samples of Buryat breed clearly differed from the sample of Altai breed based on pairwise G-test and FST values for the GHR-AluI polymorphism as well as on the base of FST values for the joint polymorphism of the three genes. Nei’s genetic distances calculated from the three gene polymorphisms also confirmed the difference between the two breeds. The results of AMOVA demonstrated that GHR gene variability (16 %) gave the largest contribution to the differentiation that was confirmed by FST values (0.12–0.27). The STRUCTURE software enabled us to reveal four clusters, with a specific ratio for each sample, in the Chinese and Mongolian samples of Buryat breed, and in the sample of Altai breed, while the Russian sample of Buryat breed had only three clusters. The differences within the breed level were determined based on the GH1-AluI and PRL-RsaI polymorphisms, while at the inter-breed level – based on the GHR-AluI polymorphism. Linkage disequilibrium analysis demonstrated significant linkage of the following pairs of genes in the Buryat breed: GH1-GHR, GH1-PRL, GHR-PRL.
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Yun, Jiwon, Jung-Ah Kim, Byungjin Hwang, Hee Sue Park, Kyongok Im, Sung-Min Kim, Dajeong Jeong, Kyu Min Lim, Duhee Bang i Dong Soon Lee. "Triple-Negative Myeloproliferative Neoplasms Vs. Calr, JAK2 or MPL-Mutated Myeloproliferative Neoplasms: Distinct Molecular Characteristics". Blood 132, Supplement 1 (29.11.2018): 1772. http://dx.doi.org/10.1182/blood-2018-99-118013.
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Abstract Background: We compared the clinical, cytogenetic, molecular features, and telomere lengths of patients with triple negative MPN and MPN with any of CALR, JAK2 or MPL mutations. Methods: Target capture sequencing of 87 genes and molecular cytogenetic studies were performed in 61 Korean patients with MPN. Also, we searched the newly reported mutations and novel mutations in triple negative MPN [JAK2-G335D (germline), JAK2-F556V, JAK2-G571S (germline), JAK2-V625F (germline), MPL-T119I, MPL-S204F/P, MPL-E230G, MPL-V285E (germline), MPL-R321W (germline), MPL-Y591D, MPL-S505N and MPL-W515R]. We compared clinical and molecular characteristics between two groups. Additionally, we performed telomere quantitative FISH for 48 patients' samples and measured telomere/centromere ratios of them. Results: Among 61 patients, 13 patients showed mutations in CALR, 34 in JAK2, and 3 in MPL. All of JAK2 mutation and CALR mutation site were reported sites, but 2 among 5 mutation site of MPL were novel mutation [D128N, D261Y]. Twelve patients showed triple negative (7 of PMF 7, 2 of ET, and 3 of MPN-U) - they showed 8 different mutation sites among 6 different genes (ASXL1, DNMT3A, NPM1, POLG, SRSF2, and ZMYM3). NPM1, POLG, and ZMYM3 mutations were seen only in triple negative patients. NPM1 mutation was significantly higher in triple negative MPN (P=0.0301). In telomere length study, there was no statistical difference between triple negative group (T/C ratio mean 12.5) and CALR, JAK2 or MPL mutated group (T/C ratio mean 10.0). Although, MPN patients with telomere length shorter than normal control's lower 10% (7.04) showed poor prognosis (P=0.0045). Conclusions: Patients with triple negative MPN are characterized by high frequency of NPM1 mutation and less number of somatic mutations. Since the mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR at present, search for JAK2 and MPL mutations in other sites are essential in triple negative MPNs. Keywords: Myeloproliferative neoplasms, next-generation sequencing, triple negative MPN, chromosome, FISH, telomere Disclosures No relevant conflicts of interest to declare.
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Nunez, Felipe, Maria Garcia-Fabiani, Padma Kadiyala, Hanna Hong, Andrea Comba, Costas Lyssiotis, Shi-Yuan Cheng, Pedro Lowenstein i Maria Castro. "RDNA-03. AUTOPHAGY AS A NOVEL THERAPEUTIC TARGET IN MUTANT IDH1 GLIOMAS". Neuro-Oncology 21, Supplement_6 (listopad 2019): vi207. http://dx.doi.org/10.1093/neuonc/noz175.863.
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Abstract Mutated Isocitrate dehydrogenase 1 (IDH1R132H; mIDH1) is found in 50% of all adult gliomas and in 80% of lower-grade gliomas. IDH1R132H promotes 2-hydroxyglutarate production which inhibits histone and DNA demethylases, inducing epigenetic reprogramming of the tumor transcriptome. We recently demonstrated that epigenetic reprogramming enhances DNA-damage response and confers radioresistance in mIDH1 glioma, harboring p53 and ATRX loss. Furthermore, several studies indicate that IDH1R132H triggers cellular metabolism reprogramming. In this study, RNA-seq data revealed that mIDH1 glioma neurospheres (mIDH1-NS) have downregulated gene ontologies related to mitochondrial metabolism. Analysis of mitochondrial and glycolytic activity, using a Seahorse Analyzer, showed that mIDH1-NS have decreased levels of oxygen consumption and extracellular acidification rates, characteristic of quiescent state. Nevertheless, mIDH1 glioma cells can grow and generate tumors, suggesting that they might maintain self-renewal via autophagy. To investigate this hypothesis, we studied autophagy in wild-type and mIDH1 glioma cells analyzing LC3 conversion, p62 expression, and ULK1 protein phosphorylation. Mouse and human mIDH1 glioma cells exhibited increased expression of pULK1-S555, enhanced conversion of LC3I to LC3II, and decreased expression of p62, indicating that mIDH1 cells have an augmented autophagy. Also, key proteins involved in autophagy, including MST4 and UVRAG, are upregulated in mIDH1-NS. We also studied autophagy flux using LC3-mCherry-GFP reporter to identify LC3 recruitment at the autophagosome membranes and the autolysosome activity. Our results showed that mIDH1 cells have increased LC3 recruitment and enhanced autolysosome activity. Interestingly, autophagy has been related with glioma radioresistance. In our study, mouse and human mIDH1 glioma cells are sensitive to autophagy inhibition. In addition, autophagy inhibition restored radiosensitivity in mouse and human mIDH1 glioma cells, and in our mIDH1 glioma model, enhancing median survival compared with radiation alone. In conclusion, our results indicate that mIDH1 glioma cells maintain self-renewal via activation of autophagy, which represents a novel therapeutic target.
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Xi, Gang, Clifford J. Rosen i David R. Clemmons. "IGF-I and IGFBP-2 Stimulate AMPK Activation and Autophagy, Which Are Required for Osteoblast Differentiation". Endocrinology 157, nr 1 (1.01.2016): 268–81. http://dx.doi.org/10.1210/en.2015-1690.
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Abstract IGF-I/insulin-like growth factor binding protein 2 (IGFBP-2) coordinately stimulate osteoblast differentiation but the mechanisms by which they function have not been determined. AMP-activated protein kinase (AMPK) is induced during differentiation and AMPK knockout mice have reduced bone mass. IGF-I modulates AMPK in other cell types; therefore, these studies determined whether IGF-I/IGFBP-2 stimulate AMPK activation and the mechanism by which AMPK modulates differentiation. Calvarial osteoblasts and MC-3T3 cells expressed activated AMPK early in differentiation and AMPK inhibitors attenuated differentiation. However, expression of constitutively activated AMPK inhibited differentiation. To resolve this discrepancy we analyzed the time course of AMPK induction. AMPK activation was required early in differentiation (day 3–6) but down-regulation of AMPK after day 9 was also necessary. IGF-I/IGFBP-2 induced AMPK through their respective receptors and blocking-receptor activation blocked AMPK induction. To determine the mechanism by which AMPK functioned we analyzed components of the autophagosome. Activated AMPK stimulated ULK-1 S555 phosphorylation as well as beclin-1 and microtubule-associated protein 1A/1B light-chain phosphatidylethanolamine conjugate (LC3II) induction. Inhibition of AMPK attenuated these changes and direct inhibition of autophagy inhibited differentiation. Conversely, expression of activated AMPK was associated with persistence of these changes beyond day 9 and inhibited differentiation. Blocking AMPK activation after day 9 down-regulated these autophagosome components and rescued differentiation. This allowed induction of mechanistic target of rapamycin and AKT, which suppressed autophagy. The results show that early induction of AMPK in response to IGF-I/IGFBP-2 followed by suppression is required for osteoblast differentiation. AMPK functions through stimulation of autophagy. The findings suggest that these early catabolic changes are important for determining the energy source for osteoblast respiration and down-regulation of these components may be required for induction of glycolysis, which is required during the final anabolic stages of differentiation.
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Ostankova, Yu V., D. E. Valutite, E. B. Zueva, E. N. Serikova, A. N. Shchemelev, S. Boumbaly, T. A. L. Balde i A. V. Semenov. "Primary HCV Drug Resistance Mutations in Patients with Newly Diagnosed HIV Infection". Problems of Particularly Dangerous Infections, nr 3 (22.10.2020): 97–105. http://dx.doi.org/10.21055/0370-1069-2020-3-97-105.
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Objective of our work was to assess prevalence of the primary HCV drug resistance mutations in the NS5b gene in patients with newly diagnosed HIV infection.Materials and methods. The study material was 196 blood plasma samples from patients living in the North-Western Federal District with newly diagnosed HIV. Samples were examined for the anti-HCV antibodies and HCV RNA presence. If HCV RNA was detected, amplification was performed using three primers pairs that co-flanked the NS5b gene. After sequencing the indicated gene nucleotide sequence, the virus subtype was determined and drug resistance mutations were detected.Results and discussion. Antibodies to HCV were detected in 18.87 % of HIV-infected individuals. HCV RNA was detected in 18.36 % of the patients, including 89.18 % anti-HCV-positive and 1.88 % anti-HCV-negative. It was shown that co-infection is more common in men (77.8 %) compared to women (22.2 %) – χ2 = 3.996 at p = 0.0456, df = 2. The difference in the HIV viral load between the groups with HIV monoinfection and with HIV + HCV coinfection was demonstrated (χ2 = 6.284 at p = 0.0432, df = 2). A significant difference between the groups by the CD4 + lyphocytes number was shown. In the phylogenetic analysis, the HCV subtypes are distributed as follows: HCV 1b – 47.2 %, HCV 3a – 30.6 %, HCV 1a – 13.9 %, HCV 2a – 5.5 % and only one sample was defined as HCV 2k – 2.8 %, respectively. Nine samples (25 %) presented NS5b mutations in the positions related to the development of drug resistance of HCV, including two samples among HCV genotypes 1a and 3a (i.e., 5.6 % of the total HIV + HCV group), as well as five samples among HCV 1b (13.9 % of the total group). Mutations among HCV 1a were C316Y and N444D substitutions. Among HCV 1b, C316N, C451S, S556N/G substitutions were identified. Among patients with HCV 3a, 2 samples (5.6 %) with a D310N mutation associated with an unfavorable disease prognosis were found. The introduction of direct sequencing of HCV nucleotide sequences into the routine laboratory diagnostics will allow us to estimate the primary drug resistance mutations prevalence in risk groups to predict the HCV life-threatening complications development – fibrosis, cirrhosis, hepatocellular carcinoma, as well as the outcome of antiviral therapy prognosis. The data obtained can be rationally used to assess the dynamics of the HCV primary pharmacoresistance prevalence among HIV-infected individuals.
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Ma, Wanlong, XI Zhang, Xiuqiang Wang, Zhong Zhang, Chen-Hsiung Yeh, Anthony Sferruzza, Jennifer Uyeji i Maher Albitar. "MPL Mutation Profile in JAK2 Mutation-Negative Patients with Myeloproliferative Neoplasms." Blood 114, nr 22 (20.11.2009): 1891. http://dx.doi.org/10.1182/blood.v114.22.1891.1891.
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Abstract Abstract 1891 Poster Board I-914 Myeloproliferative neoplasms (MPNs) are multipotent hematopoietic stem cell neoplasms characterized by excess production of various blood cells. Mutations in the thrombopoietin receptor gene (MPL) have been reported in JAK2 V617F-negative patients with MPNs. We evaluated the prevalence of MPL mutations relative to those of JAK2 mutations at V617 or in exons 12 through 15 in a large number of patients with suspected MPNs. A total of 2790 patients with suspected MPNs referred to our institution were first screened for JAK2 V617F and exon 12–15 mutations. Patients with no JAK2 mutations detected were then tested for MPL mutations in exons 10 and 11 by means of a sensitive MPL reverse-transcription-PCR-based assay with direct bidirectional sequencing. All JAK2 and MPL mutation assays were performed on plasma RNA, rather than DNA isolated from blood or bone marrow cells. Of the 2790 patients, 529 (18.96%) had a V617F mutation; 12 (0.43%) had small insertion/deletions in exon 12; and 7 (0.25%) had other JAK2 mutations, including point mutations in exons 13–15 and an exon 14 splice mutation. MPL mutations were identified in 66 of the 2242 (2.94%) JAK2 mutation-negative patients (2.37% of all tested patients). W515L was the dominant MPL mutant detected in 46 patients (70%) including two patients with homozygous W515L mutation. The other W515 variants (W515K, W515R, W515S, W515G, W515A, W515*) comprised 16% (N=11) of the MPL mutations. The remaining MPL mutations (n =9, 14%) were detected at other locations in exon 10 and 11. Two of these were novel exon 10 deletion/insertion mutations and two were unreported exon 11 point mutations. The exon 10 T496-A497ALVI (4AA) homozygous insertion was detected in one patient with a confirmed diagnosis of idiopathic myelofibrosis and the W515-P518 del/ins (KT) mutation was detected in another patient with unspecified MPN. The two novel MPL exon 11 point mutations were D545G and D545N. The S505N mutation (n=2) and V507I (n=1), R514K (n=1), and A519V (n=1) were also detected. Furthermore, three unreported silent polymorphisms/mutations (T496, L543, and D534) were detected. In conclusion, our results demonstrate that for every 100 V617F mutations detected in patients with MPNs, there are 2.3 JAK2 exon 12 mutations, 1.3 JAK2 exon 13–15 mutations, and 12.5 MPL mutations. Thus, MPL mutation detection should be performed on all JAK2 V617F-negative patients with suspected MPNs. In addition, our findings indicate that >20% of MPL mutations would have been missed if only W515L and W515K were analyzed; thus, sequencing of both exon 10 and 11 may be beneficial. Disclosures: No relevant conflicts of interest to declare.
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Gundabolu, Krishna, Catalina C. Amador, Allison M. Cushman-Vokoun, Vijaya R. Bhatt, Lori J. Maness i Timothy C. Greiner. "Concurrent Somatic Mutations in Exon 14 of Janus KINASE2 (JAK2) and Exon 10 of Myeloprolifeative Leukemia Virus Oncogene (MPL) in Myeloproliferative Neoplasms (MPN) and Myelodysplastic Syndromes (MDS)/MPN". Blood 126, nr 23 (3.12.2015): 5211. http://dx.doi.org/10.1182/blood.v126.23.5211.5211.
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Abstract INTRODUCTION: JAK2 p.V617F mutations are common in Polycythemia Vera (>90%), Essential Thrombocythemia (50%; ET) and Primary Myelofibrosis (30-50%; PMF). Somatic mutations of codons 515 or 505 in MPL Exon 10 are present in 5% of non-JAK2-mutated ET and 1% of PMF. MPL p.W515L mutation has also been identified in patients with Refractory Anemia with Ring Sideroblasts associated with Thrombocytosis (RARS-T). MPL mutation in Exon 10 codon 505 (S505N) was originally identified as a germ line mutation associated with hereditary thrombocytopenia, but was subsequently reported in ET and PMF. Most current evidence suggests that JAK2 and MPL mutations are mutually exclusive. Pardanani et al had reported 6 cases out of 1182 patients with concurrent JAK2 and MPL mutations, using assays of different sensitivities. In the reported cases the more frequent allele mutated is MPL and less frequent allele is JAK2 as a possible second hit. Here, we report 2 cases with concurrent JAK2 and MPL mutations. METHODS: DNA was extracted and subjected to library preparation using the 50 gene Ion AmpliSeq™ Cancer Hotspot Panel v2, clonal amplification and Ion Sphere™ Particle enrichment on the Ion One Touch™ 2 and One Touch™ ES, and Next Generation Sequencing (NGS) on the Ion Torrent PGM System™. Data was analyzed using Variant Caller Software v.4.0 and NextGENe software v.2.3.4. The original target region BED file for the Ion AmpliSeq™ Cancer Hotspot Panel v2 was modified to include only MPL exon 10 and JAK2 exon 14 amplicons. Assay coverage averaged approximately 3000X. RESULTS: From February 2015, JAK2/MPL NGS was performed on 105 specimens to determine somatic mutations in possible MPN and MDS/MPN cases. 2/105 (1.9%) of the specimens harbored concurrent JAK2 and MPL mutations compared to 6/1182 patients reported previously (0.5%). One patient had RARS-T and one had PMF. Mean age (69 and 74 years) was 71 years, hemoglobin (8.3 and 10.3 gm/dL) 9.3 gm/dL, hematocrit (25 and 26%) 25%, Mean Corpuscular Volume (92 and 92 fL) 92 fL, platelet count (171 and 1320 x 10E3/cmm) 745 x 10E3/cmm, White blood count-WBC (6.2 and 10.3 x 10E3/UL) 8.25 x10E3/cmm, monocytes (3-5%) 4%, Basophils (1 and 2%) 2%, Neutrophils (61 and 66%) 64%, Bands (3and 15%) 9%, Eosinophils (0 and 2%) 1%, peripheral blood blasts (0 and 4%) 2%, bone marrow fibrosis grade of 2 in a grading scale of 1-3. Both were males. Spleen was not palpable at diagnosis in either patient. The case with RARS-T had 4.5% MPL p.W515L mutation allele frequency and 22.7% JAK2 p.V617F mutation allele frequency. The case with PMF had 3.3% MPL p.S505N mutation allele frequency and 2.4% JAK2 p.V617F mutation allele frequency (detected at the follow up 6 months bone marrow in May 2015). They were treated with EPO analogues/Anagrelide and Ruxolitinib respectively. None had thrombosis or bleeding complications. At a mean follow-up of 366 days (113 and 620 day), both patients are alive. CONCLUSION: MPL (codons 505 and 515) and JAK2 p.V617F mutations are not always mutually exclusive. NGS panels with dual gene coverage and high sensitivity identify coexistent mutations that would not be identified by single gene assays. The clinical significance of concurrent mutations remains unclear. Disclosures No relevant conflicts of interest to declare.
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Etheridge, Leah, Lana M. Corbo, Kenneth Kaushansky, Edward Chan i Ian S. Hitchcock. "A Novel Activating JAK2 Mutation, JAK2R564Q, Causes Familial Essential Thrombocytosis (fET) Via Mechanisms Distinct From JAK2V617F". Blood 118, nr 21 (18.11.2011): 123. http://dx.doi.org/10.1182/blood.v118.21.123.123.
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Abstract Abstract 123 Activating mutations in JAK2 are responsible for the majority of myeloproliferative diseases (MPDs) by stimulating aberrant signaling and hyperproliferation of one or more cell lineages. Although JAK2V617F is the most common activating mutation, a number of other point mutations also appear to have clinical relevance. Here we describe JAK2R564Q, a novel mutation in the pseudokinase domain that causes fET and determine its biological function in vitro, compared to JAK2V617F. A 6 year old male was referred for evaluation of thrombocytosis (initial platelet count 961k/mcL). The patient was otherwise asymptomatic with no past medical history, no recent illnesses and on no medications. The family history, review of systems and physical examination were unremarkable and workup for secondary thrombocytosis was negative. While the patient did not have the JAK2V617F, MPLW515L/K or S505N mutations, we discovered a novel JAK2 mutation, R564Q. His mother and sister also presented with elevated platelet counts (500–600k/mcL) and were also found to have the JAK2R564Q mutation, whereas the father presented with a normal platelet count and displayed two wild type (WT) JAK2 alleles. The arginine residue at 564 is highly evolutionarily conserved in the autoinhibitory domain of JAK2. To determine the biological significance of JAK2R564Q and compare it to JAK2V617F, we stably expressed WT, R564Q, V617F and R564Q+V617F JAK2 in Ba/F3 cells stably expressing the thrombopoietin receptor c-Mpl (BaF-Mpl). These cells express comparable levels of both JAK2 and c-Mpl. TPO-dependent proliferation assays demonstrated striking differences between the different cell types. JAK2R564Q exhibits significant increases in cell survival in the absence of TPO and at low concentrations compared to WT, while cells expressing JAK2V617F and R564Q+V617F are growth factor independent. Interestingly, the double mutant (R564Q+V617F) exhibits higher maximal cell proliferation than V617F alone, suggesting that R564Q is functioning through alternative mechanisms to that of V617F. Next, we analyzed annexin V expression following growth factor withdrawal to determine the effects of mutated JAK2 on apoptosis. Concurrent with our proliferation assays, JAK2R564Q inhibited apoptosis compared to WT, while JAK2V617F and R564Q+V617F exhibited even less apoptosis. These data suggest that JAK2R564Q is important for cell survival in the absence of cytokines, but it does not elicit the proliferation-promoting effects of JAK2V617F. To elucidate the mechanisms through which JAK2R564Q and JAK2V617F mediate their actions we determined their effects on intracellular signaling. Cells were starved prior to stimulation with TPO. Interestingly, we found that cells expressing JAK2R564Q have considerably higher levels of phospho-JAK2 (Y1007/8) and phospho-STAT5, signals which are normally associated with proliferation, than WT, V617F alone and the double mutant. We also observed differences in the phosphorylation of several other JAK2 tyrosine residues that are important for regulating its activity. Intriguingly, hyperphosphorylation of the negative regulator JAK2Y570, was by far the most robust in JAK2R564Q mutants, which could potentially contribute to the reduced factor-independent proliferation observed, compared to JAK2V617F. Interestingly, we also found differential phosphorylation of JAK2 at Y831, which positively regulates JAK2 signaling via interactions with SH2-Bβ. JAK2Y831 was also hyperphosphorylated in R564Q mutants compared to V617F mutants, especially in the absence of cytokines. Levels of phospho-ERK1/2 and phospho-Akt were comparable in all JAK2 mutants and significantly reduced compared to WT cells, characteristic of cells that fail to undergo starvation induced cell cycle arrest. Taken together, these data demonstrate that the JAK2R564Q mutation causes fET most likely by inhibiting apoptosis in hematopoietic stem cells and megakaryocytic progenitors. Importantly, even though this mutation is localized in the same pseudokinase domain as V617F, its effect on cell survival and signaling in response to TPO is significantly different. This work provides an insight into the functionality of alternative, clinically-relevant JAK2 mutations and how they have separate and additive effects on cell growth and survival. Disclosures: No relevant conflicts of interest to declare.
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Braverman, Erica, Andrea Dobbs, Darlene Monlish i Craig Byersdorfer. "106 Increasing AMPK activity in human T cells enhances memory subset formation without sacrificing in vitro expansion". Journal for ImmunoTherapy of Cancer 8, Suppl 3 (listopad 2020): A117—A118. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0106.
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BackgroundThe ideal adoptive cell therapy consists of memory-like T cells with enhanced oxidative potential. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. AMP-activated protein kinase (AMPK), whose activity increases in memory cells, is a key regulator of mitochondrial biogenesis and oxidative metabolism, making AMPK activation an attractive candidate to improve adoptive T cell function.MethodsTo increase AMPK activity, AMPKγ, which controls the phosphorylation status of AMPKa and therefore activity of the AMPK complex, was cloned into a lentiviral plasmid downstream of the elongation factor 1a (EF1a) promoter and upstream of green fluorescent protein (GFP). An empty vector, containing GFP only, served as a negative control. Human T cells were transduced and expanded in vitro in the presence of IL-2. AMPK activity was assessed via immunoblot for phosphorylation of AMPKa on Thr172 and S555 on downstream target Unc-51-like kinase 1 (ULK1). Memory-marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes of in vitro expansion were calculated by adjusting manual cell counts for GFP positivity and CD4+/CD8+ staining.ResultsAMPKγ was efficiently transduced and expressed by human T cells, which significantly increased AMPK activity (AMPKa phosphorylation 1.93 ± 0.05 vs 0.6 ± 0.09, p<0.001, ULK1 phosphorylation 1.28 ± 0.11 vs 0.67 ± 0.08, p<0.01). AMPKγ-overexpressing T cells augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (figure 1). Mitochondrial density, SRC, and maximal oxygen consumption rates were similarly increased in AMPKγ-transduced cells (figure 2A,B). Further, while enhanced memory cell production is often linked with reduced proliferation, T cells with increased AMPK activity maintained and even trended towards increased rates of expansion compared to empty-transduced controls (figure 3A), with a measurable increase in CD4+ T cell percentages by flow cytometry (figure 3B).Abstract 106 Figure 1AMPK-transduced T cells increase expression of memory surface markers. Human T cells were transduced with AMPK-GFP or GFP-only control (Empty). Memory markers were assessed by flow cytometry on Days 7–14 of in vitro culture following expansion with IL-2. Plots are representative of 3 separate donorsAbstract 106 Figure 2AMPK-transduced T cells show enhanced mitochondrial density and SRC. (A) Human T cells transduced with AMPK-GFP or GFP-only (Empty) were stained with Mitotracker Red and fluorescence intensity compared between transduced cells and GFP- controls within the same culture to account for variability in Mitotracker dye staining. (B) AMPK and Empty transduced T cells were assessed via Seahorse Metabolic Analyzer using the Mito Stress Test. Results are representative of 3 separate donors. OCR = O2 consumption rateAbstract 106 Figure 3Proliferation is maintained in AMPK-transduced T cells, with enhanced recovery of CD4+ T cells. (A) Primary human T cells transduced with AMPK-GFP or GFP-only (Empty) were expanded in vitro in the presence of IL-2. Cells were manually counted and the ratio of day 7 to day 5 cell counts calculated to assess fold expansion over time. (B) At the same, CD4+ and CD8+ percentages were measured in GFP+ cells by flow cytometryConclusionsIncreasing AMPK activity endows T cells with a variety of characteristics ideal for adoptive cell therapy, including increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo.
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Alimam, Sam, William Villiers, Richard Dillon, Michael Simpson, Alexander E. Smith, Paul Lavender, Prodromos Chatzikyriakou, Ghulam J. Mufti, Donal P. McLornan i Claire N. Harrison. "Molecular, Epigenetic and Gene Expression Profiling of Triple Negative Essential Thrombocythaemia". Blood 134, Supplement_1 (13.11.2019): 308. http://dx.doi.org/10.1182/blood-2019-126008.
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Introduction The majority of patients with Essential Thrombocythaemia (ET) have mutations in JAK2, MPL, and CALR, causing activation of the JAK/STAT pathway; but 10-15% of ET patients lack detectable mutations in these genes, so-called 'Triple Negative' (TN). We applied a systematic approach to investigate mutational status and epigenetic signatures in a cohort of TN ET patients. Methods and Results We investigated 46 patients (72% female), median age at diagnosis 35 years (range 8-77 years) including a father and son. All patients were TNusing standard diagnostic assays. We applied deep, error corrected, next generationsequencing (NGS) of 24 genes using the HaloPlexHS platform to peripheral blood samples. Whole exome sequencing was also performed in 23 patients using skin as constitutional control. Overall we identified somatic mutations in 10/46 patients including MPL(3 patients, W515R, W515G, W515S, R537W, VAF 0.02-0.1) JAK2V617F (4 patients, VAF 0.02-0.08); and germline MPLmutations in a further 3 patients (P453R, S505N); including the father and son pair. We selected patients lacking somatic or germline mutations ("true TN") to analyse gene expression using RNA-seq and DNA methlyation status using 850K Epic Arrays. Patients with JAK2V617Fand CALRmutations and healthy donors (HC) were included as controls. Concerning RNA-seq data, we performed multiple differential analysis of HC vs TN, CALRand JAK2V671F; as well as HC vs all ET samples (adjusted for subtype). Each HC comparison highlighted clear differences between gene expression profiles of HC and disease (Figure 1A). The differentially expressed genes (DEGs) in each comparison overlapped significantly, suggesting that all ET samples have consistent gene expression differences to HC samples regardless of their driver mutation status. In total 1444 differentially expressed genes (ET vs HC) were highlighted (figure 1B). Functional analysis identified significant enrichment for genes involved in the MAPK pathway. Addtionally, we noted upregulation of GATA1,ITGA2B and GP6 genes, not previously reported to be dysregulated in ET. Correlation of gene expression data with DNA methylation status identified a consistent signature of 306 hypomethylated genes, showing significant enrichment for genes involved in transcriptional misregulation and upregulation of inflammatory regulators such as TNF and NFκB signaling pathways. Next, we identified which transcription factor motifs preferentially bind within these methylation blocks. The blocks showed an enrichment for 6 key transcriptional regulators: ATF3, ATF4, CEBPA, CEBPB, MAX, and RARA. All 6 were significantly upregulated in all ET samples. To validate the motifs, we processed ChIP-seq data from the K562 cell line and identified a significant proportion of the hypo methylated regions are bound by these, transcription factors: 374/410 (91%) regions; 43/410 (10%) are bound by all 6 transcription factors. Conclusions A significant proportion (22%) of patients assigned as 'TN' ET via traditional diagnostic techniques in fact harbor known driver mutations at a low allele frequency, suggesting that error corrected NGS approaches may be diagnostically useful in this setting. Additionally, for a group of "true" TN ET patients we demonstrate that patterns of gene expression and DNA methylation are more similar to patients with ET with known driver mutations than healthy controls. Among the upregulated genes are key platelet regulatory genes: GP6, GP1BB, ACTN1and ITGA2B. Furthermore, we identify consistently hypomethylated genes with increased expression across all molecular subtypes of ET which are highly enriched for genes involved in proinflammatory pathways and show that binding of 6 key transcription factorsmay underlie these changes regardless of driver mutation status. Our observations suggest that the ET disease phenotype may, at least in part, be driven by transcriptional misregulation and may be propagated downstream via the MAPK, TNF and NFKappa pathways in addition to activation of JAK/STAT pathways. These findings identify novel mechanisms of disease initiation which require further evaluation. Disclosures Dillon: Novartis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; TEVA: Consultancy, Honoraria. Mufti:Cellectis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. McLornan:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria. Harrison:Shire: Speakers Bureau; CTI: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; AOP: Honoraria; Janssen: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria; Promedior: Honoraria; Gilead: Speakers Bureau; Sierra Oncology: Honoraria; Incyte: Speakers Bureau.
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Girodon, Francois, Eric Lippert, Sylvie Hermouet i Serge Carillo. "High-Resolution Melting (HRM) Curve Analysis Is a Reliable and Relevant Tool In JAK2V617F-Negative Myeloproliferative Neoplasms". Blood 122, nr 21 (15.11.2013): 4077. http://dx.doi.org/10.1182/blood.v122.21.4077.4077.
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Abstract Introduction The JAK2V617F mutation is observed in more than 95% of patients with Polycythemia Vera (PV), and 50–60% of patients with Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and Refractory Anaemia with ringed Sideroblasts with marked Thrombocytosis (RARS-T). As a consequence, for JAK2V617F-negative cases it is worth looking for other mutations in the JAK2 and MPL genes as they are an important diagnostic aid for the myeloproliferative neoplasms (MPN). For example, half of JAK2V617F-negative PV patients carry mutations in the exon 12 of JAK2. To date, 40 different JAK2 exon 12 mutants have been reported. All are located between aa536 and aa547 and include deletions, insertions, and duplications. In the same way, nearly 5% and 10% of JAK2V617F-negative ET and PMF carry mutations in exon 10 of MPL, mainly affecting codons S505 and W515. Techniques such as allele-specific polymerase chain reaction (AS-PCR) are inappropriate for screening and sequencing, including Next Generation Sequencing (NGS), is time consuming as a first screening. High-resolution melting (HRM) curve analysis allows a rapid and whole exon mutation scanning approach associated with excellent sensitivity. The goal of our study was to evaluate the relevance of HRM in JAK2V617F-negative MPN and RARS-T in 3 French hospitals. Methods HRM is based on the analysis of fusion curves of a short amplification product of PCR. This procedure, which derives PCR in real time and uses a fluorescent agent intercalated DNA, makes it possible to obtain a good resolution and to detect different types of mutations. The analyses were carried out on DNA from purified blood granulocytes. Diagnosis of PV, ET, PMF and RARS-T was established according to the 2008 WHO criteria. All patients were tested for their JAK2V617F status. To evaluate the efficiency of HRM in a routine diagnostic setting, we investigated 1997 patients with suspicion for a MPN from 3 French university hospitals. Samples with abnormal HRM curves were sequenced in order to confirm and characterize the mutations. Results For 37/881 (4.2%) JAK2V617F-negative IE and PV patients, the HRM curve raised suspicion of the presence of a mutation in JAK2 exon 12, all confirmed by sequencing: the most frequent mutations were H538Q K539L (n=6), R541 E543 delins K (n=6), N542 E543 del (n=5), F537–I546 dup 10_F547L (n=4). For 2 additional PV cases, an abnormal HRM curve was obtained for JAK2 exon 14. Sequencing analysis revealed a V617F mutation with additional mutation of base 1831 (1831T>G), changing leucine 611 for a valine (L611V). For the second PV patient, a silent mutation corresponding to a substitution of a cytosine by a thymine in position 1848 of exon 14 of JAK2 was observed (C616), also associated with a V617F mutation. In both cases the JAK2V617F mutation had not been detected because the primers of the JAK2V617F AS-qPCR assay covered the sequence coding for L611 and C616. In addition, mutations in JAK2 exons 13 and 15 were tested on 33 PV-suspected samples: only one PV with a JAK2 exon 15 mutation was noted. Regarding the detection of mutations in exon 10 of MPL, an abnormal HRM curve was noted for 59/1116 (5.3%) MPN suspected. The mutations found were W515L (31 ET, 4 PMF), W515K (8 ET, 3 PMF), W515A (2 ET), W515R (1 ET, 1 PMF), W515S (1 ET), S505N (4 ET, 2 PMF, 1 RARS-T), V501A (1 ET). Conclusion In ET and PMF, the HRM approach offers the advantage of quickly and reliably assessing patients for W515 and S505 mutations. In JAK2V617F-negative PV, due to the large number of possible mutations, HRM is a highly potent screening technique and allows a rapid scan of the whole exon 12, leading secondarily to sequence only a small number of HRM positive cases. Moreover, in 2 PV, HRM analysis of JAK2 exon 14 allowed to detect a V617F mutation hidden by a second mutation that inhibited primer hybridization and subsequent DNA amplification in the routine AS-qPCR assay. In summary, HRM is a highly relevant technique, cheaper than NGS, for routine molecular diagnosis of JAK2V617F-negative MPN, which typically carry a large variety of mutations, expressed at low levels. Disclosures: No relevant conflicts of interest to declare.
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Al-Dewik, Nader I., Bruno Cassinat, Jean-Jacques Kiladjian, Alexander Knuth i Mohamed A. Yassin. "Targeted Exome Sequencing Identifies Novel Mutations in Familial Myeloproliferative Neoplasms Patients in the State of Qatar". Blood 124, nr 21 (6.12.2014): 5570. http://dx.doi.org/10.1182/blood.v124.21.5570.5570.
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Abstract Background: Myeloproliferative Neoplasms (MPNs) are clonal hematopoietic disorders characterized by excessive proliferation of one or more myeloid cell lineages. Philadelphia negative MPNs include Polycythemia Vera (PV), Essential Thrombocytosis (ET) & Primary Myelofibrosis (PMF). MPNs are associated with the presence JAK2 V617F mutation in 95% of PV & 50% of ET & PMF patients. Several molecular techniques such as RQ-PCR, HRM & Sequencing are currently used to detect common mutations. However, there are still significant numbers of MPNs that are negative to the most common genetic anomalies & many mutations are still unknown. The advent of Next Generation Sequencing (NGS) gives the opportunity to study relevant mutations in several genes. Aim: Utilizing NGS to identify potential genetic anomalies causing familial MPNs patients in Qatar. Methods: 6 MPNs patients from consanguineous families & 5 healthy individuals were consented into the study & peripheral blood samples were collected. gDNA was extracted & used for multiplex PCR amplification of amplicons targeting cancer associated mutations in 28 key genes (JAK2, MPL, THPO, CBL, LNK, SH2B3, NF1, SOCS1/2/3, TP53, NRAS/KRAS, NF1, IDH1/2, EZH2, ASXL1, TET2, ATM, KIT, RB, TP53, IKZF1, RUNX1, PDGFRB, TERT & CALR) using the Ion AmpliSeq Kit. NGS was performed via the Ion Torrent using the 318 chip & data was analyzed with the Torrent Suite Software. Mutation details were obtained from COSMIC database. A hg 19 sequence was used as reference. The confirmation of NGS data was performed using RQ-PCR or Sequencing. Results: 11 samples were successfully sequenced, with a mean depth of 1500 reads & the FASTQC plugin indicated good quality sequencing metrics. JAK2 V617F, JAK2 exon 12-15 & MPL (S505N, W515 L/K) negative samples tested before via RQ-PCR, HRM & sequencing were called negative by NGS. NGS identified novel deleterious mutations in MPNs patients. Out of 6 familial cases, 5 patients (P1- P5) were ET & 1 patient (P6) was PV. P1 had JAK2 V617F, ASXL1 T600P, CBFB G180S, THPO S184R &ITGA2R76Q, P2 had JAK2 V617F, MPL A554G & ATM F582L, the other three Patients (P3, P4 & P5) had CLAR K385fs*47 & one PV patient (P6) had TYK2 E1163G, ASXL1 P808H, PDGFRB P4L & TERT G300fs. Among the patients & healthy individuals, mutations/SNVs such as MPL P106L, K553N, SH2B3 L476F, ATM F1036F KIT N564S & TET2 T730R were also found Discussion & conclusion: Initial screening of known common genes (JAK2 V617F, JAK2 exon 12-15 & MPL W515 L/K) mutations did not reveal the causative mutations in 3% of 180 PV patients, 52% of 200 ET patients & 77% of 20 PMF patients. In this study, several deleterious somatic & germ-line mutations & SNVs were identified using Targeted Exome Sequencing approach. A complex combination of mutations in JAK2, THPO, ITGA2 & MPL genes occurred in ET patients & coexistence of several oncogenic events in TYK2, ASXL1, PDGFRB & TERT occurred in PV patient. This finding may also suggest that the MPNs phenotype may depend on presence of other mutations. It is worth mentioning that the presence of ATM variant in P2 is associated with increased risk of CLL. Somatic CALR type-2 mutation was identified in 3 ET (nonmutated JAK2 or MPL) patients. This mutation is 5-bp TTGTC insertion in exon 9 that generates a mutant protein with a novel C-terminal (p.K385fs*47). In patients & healthy individuals, a heterozygous germ-line mutation in exon 3 of the MPL gene (MPL P106L) has been observed. it has previously been described as a rare autosomal-dominant disorder. However, this mutation is considered to be frequent in Arabic populations, leading to severe thrombocytosis in homozygotes & occasionally to mild thrombocytosis in heterozygotes. In addition, several unreported variants of uncertain significance were identified. Our preliminary results suggested that MPNs patients in Qatar have several potential disease- associated variants & mutations. Evidences show that there exists a possibility of the disease arising out of the accumulation of genetic alterations & not as the consequence of a single genetic-hit event. This could possibly be due to the high rate of consanguineous marriages in Qatar i.e. the "Founder Effect". Our results recommended carrying out WES to explore & identify mutations which will be crucial to characterize many cases of MPNs with unknown molecular causes, gain a deep understanding of genotype-phenotype correlations & MPNs pathogenesis. Disclosures Al-Dewik: Qatar National Research Fund: Patents & Royalties, Research Funding. Yassin:Qatar National research fund: Patents & Royalties, Research Funding.
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Alzahrani, Musa, Areej Al Mugairi, Saeed Al Turki, Waleed Al Rajban, Fatimah Alshalati, Khadega A. Abuelgasim, Bader Alahmari i in. "Clinicopathological Description from the Largest Series of Patients with the Novel Pro106Leu MPL gene Mutation Associated with Hereditary Thrombocythemia". Blood 136, Supplement 1 (5.11.2020): 7–8. http://dx.doi.org/10.1182/blood-2020-136077.
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Background: Hereditary thrombocythemia (HT) has been reported in Japanese and African populations in association with S505N, and N35K c-Mpl mutations, respectively. A novel Pro106Leu germ-line mutation in the c-Mpl gene has recently been shown to be associated with HT in Arabic population. Clinical and bone marrow (BM) features of Pro106Leu mutation are largely unknown. Methods: The molecular genetic databases at two tertiary hospitals in Riyadh, King Abdulaziz medical city (KAMC) and King Saud University medical city (KSUMC), were searched to identify all patients (pts) with MPL Pro106Leu mutation. Clinical and pathological data were retrospectively collected. BM aspiration and biopsy were independently reviewed retrospectively by two consultant hematopathologists and agreement was reached by a consensus review. Simple descriptive statistics were used to summarize the results. A univariate subgroup analysis, comparing the hematologic parameters between the homozygous and heterozygous genotypes was conducted using Pearson Chi-Square and t-tests. Results: A total of 115 pts with Pro106Leu MPL mutation were included, 86 (75%) from KAMC and 29 (25%) from KSUMC. All pts were ethnically Arabs. Median age was 33 years (yrs) (range: 0.4-68), 65 (56.5%) were female, and 31 (27%) were pediatric pts (age <18 yrs). MPL Pro106Leu mutation was homozygous in 87 (75.7%) pts, and heterozygous in 28 (24.3%). Spleen was enlarged in 3 (3%) pts, not documented in 15 (13%), and normal in 97 (84%). History of bleeding was documented among 11 (10%) pts. Thrombosis history was positive in 5 (4%) pts only, unavailable in 6 (5%), and negative in 104 (90%). Family history of thrombocytosis was reported in 46 (40%), but family history was not documented in 20 (17%). Common comorbidities include: autoimmune disease in 33 (29%), diabetes 21 (18%), and hypertension 20 (17%). Reasons for MPL testing was: abnormal routine blood work 79 (69%), family history of thrombocytosis 23 (20%), or others 13 (11%). Thrombocytosis [platelet (plt) counts > 450 x109/L] was documented in 107 (93%) pts, normal in 4 (3.5%), and low in 4 (3.5%) at the time of diagnosis of Pro106Leu mutation. See figure 1. The median plt count at the time of diagnosis of MPL Pro106Leu mutation was 667 x 109/L (range: 13-1473). The median mean plt volume was 8 fL (range 6.1-10.2), white blood cell count 8.4 x 109/L (2.46-68.35), absolute neutrophil count 5 x 109/L (1.01- 21.19), hemoglobin 132 gm/L (85-148), mean corpuscular volume 84.1 fL (57-117.3), mean corpuscular hemoglobin 27.7 pg (18-37.5), mean corpuscular hemoglobin concentration 327 g/dL (300-351), and red cell distribution width 13.6 (10.9-22.8). Ferritin less than 30 was seen in 40 (35%) pts, 27 of whom were women. No ferritin done in 14 pts. Iron stores (based on bone marrow, ferritin and iron saturation) were adequate in 56 (49%), inadequate in 49 (43%), and not documented in 10 (9%). BCR-ABL, JAK2 and CALR were only detected in 1 pt each. One pt was not tested for CALR mutation. All other pts were negative for the three mutations. Out of all 115 pts, 33 (29%) had an evaluable BM. BM cellularity ranged from 20-100 %, 12/33 (36%) were hypocellular, 17/33 (52%) normocellular, and 4/33 (12%) hypercellular. Megakaryocyte (meg) morphology revealed dysplastic changes in 20 (60%) (hypolobated megs or with separated lobes), only 7 (21%) of cases had cloudlike megs but none had staghorn or giant megs as described in essential thrombocythemia. BM megs were increased in 29 (87%). Small size megs were seen in 15/33 (45%). Clustering of megs was seen in the majority of the cases 30/33 (90%) of whom, 29(87%) had loose and 20 (60%) dense clusters. On univariate analysis (see tables 1-2), homozygous genotype was associated with higher plt count. Total of 65 (57%) pts were prescribed aspirin, and 16 (14%) hydroxyurea. At the time of last follow-up 114 (99%) of pts were alive. The median follow-up was 7.8 yrs from the time of thrombocytosis (ranged from 0-24.8). No case developed disease progression to myelofibrosis. One pt was diagnosed with T-lymphoblastic lymphoma and later died from treatment complications and another pt was diagnosed with CML. Conclusion: Pro106Leu mutation is associated with marked thrombocytosis at a younger age with a low risk of thrombosis. Homozygous genotype is associated with a significantly higher plt count. BM usually shows either normo- or hypocellular marrow with increased megs proliferation, and clustering. Disclosures No relevant conflicts of interest to declare.
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Guan, Yuntian, Hannah Spaulding, Yu Qing, Mei Zhang, Orion Willoughby, Joshua C. Drake i Zhen Yan. "Ulk1 Phosphorylation at S555 is Not Required in Exercise Training-mediated Improvements in Endurance and Metabolic Capacity in Healthy Mice". Journal of Applied Physiology, 20.06.2024. http://dx.doi.org/10.1152/japplphysiol.00742.2023.
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Endurance exercise training improves exercise capacity as well as skeletal muscle and whole-body metabolism, which are hallmarks of high quality-of-life and healthy aging. However, its mechanisms are not yet fully understood. Exercise-induced mitophagy has merged as an important step in mitochondrial remodeling. ULK1, specifically its activation by phosphorylation at serine 555, was discovered as an autophagy driver and to be important for energetic stress-induced mitophagy in skeletal muscle, making it a potential mediator the benefit of exercise on mitochondrial remodeling. Here, we employed CRISPR/Cas9-mediated gene editing and generated knock-in mice with a serine-to-alanine mutation of Ulk1. We now report that these mice displayed normal endurance capacity and cardiac function at baseline with a mild impairment of energy metabolism as indicated by accelerated increase of respiratory exchange ratio (RER) during acute exercise stress; however, this was completely corrected by 8 weeks of voluntary running. Ulk1-S555A mice also completely retained the exercise-mediated improvements of endurance capacity. We conclude that Ulk1 phosphorylation at S555 is not required for exercise-mediated improvements of endurance and metabolic capacity in healthy mice.
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Han, Yiran, Pritha Bagchi i C. Chris Yun. "Regulation of the intestinal Na+/H+ exchanger NHE3 by AMP-activated kinase is dependent on phosphorylation of NHE3 at S555 and S563". American Journal of Physiology-Cell Physiology, 4.12.2023. http://dx.doi.org/10.1152/ajpcell.00540.2023.
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Electroneutral NaCl transport by Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and decreased NHE3 activity contributes to diarrhea. Patients with diabetes often experience gastrointestinal adverse effects and medications are often a culprit for chronic diarrhea in type 2 diabetes (T2D). We have shown previously that metformin, the most widely prescribed drug for treatment of T2D, induces diarrhea by inhibition of NHE3 in rodent models of T2D. Metformin was shown to activate AMP-activated protein kinase (AMPK), but AMPK-independent glycemic effects of metformin are also known. The current study is undertaken to determine whether metformin inhibits NHE3 by activation of AMPK and the mechanism by which NHE3 is inhibited by AMPK. Inhibition of NHE3 by metformin was abolished by knockdown of AMPK-α1 or AMPK-α2. AMPK activation by 5- aminoimidazole-4-carboxamide ribonucleoside (AICAR) phosphorylated NHE3 at S555. S555 is the primary site of phosphorylation by protein kinase A (PKA), but AMPK phosphorylated S555 independent of PKA. Using Mass spectrometry, we found S563 as a newly recognized phosphorylation site in NHE3. Altering either S555 or S563 to Ala was sufficient to block the inhibition of NHE3 activity by AMPK. NHE3 inhibition is dependent on ubiquitination by the E3 ubiquitin ligase Nedd4-2 and metformin was shown to induces NHE3 internalization via Nedd4-2-mediated ubiquitination. AICAR did not increase NHE3 ubiquitination when S555 or S563 was mutated. We conclude that AMPK activation inhibits NHE3 activity and NHE3 inhibition is associated with phosphorylation of NHE3 at S555 and S563.
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"Review article on COVID-19 and Guillain-Barré syndrome". Frontiers in Bioscience-Scholar 13, nr 1 (2021): 97. http://dx.doi.org/10.52586/s555.
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Defour, Jean-Philippe, Emilie Leroy, Sharmila Dass, Thomas Balligand, Gabriel Levy, Ian C. Brett, Nicolas Papadopoulos i in. "Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers". eLife 12 (20.06.2023). http://dx.doi.org/10.7554/elife.81521.
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Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of 6 amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together these data allow us to develop a general model for activation of TpoR and to explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.
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Trelford, Charles B., i Gianni M. Di Guglielmo. "Canonical and Non-canonical TGFβ Signaling Activate Autophagy in an ULK1-Dependent Manner". Frontiers in Cell and Developmental Biology 9 (25.10.2021). http://dx.doi.org/10.3389/fcell.2021.712124.
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The mechanism(s) in which transforming growth factor beta 1 (TGFβ) modulates autophagy in cancer remain unclear. Here, we characterized the TGFβ signaling pathways that induce autophagy in non-small cell lung cancer cells, using cells lines stably expressing GFP-LC3-RFP-LC3ΔG constructs that measure autophagic flux. We demonstrated that TGFβ1 increases Unc 51-like kinase 1 (ULK1) protein levels, 5′ adenosine monophosphate-activated protein kinase (AMPK)-dependent ULK1 phosphorylation at serine (S) 555 and ULK1 complex formation but decreases mechanistic target of rapamycin (mTOR) activity on ULK1. Further analysis revealed that the canonical Smad4 pathway and the non-canonical TGFβ activated kinase 1/tumor necrosis factor receptor-associated factor 6/P38 mitogen activated protein kinase (TAK1-TRAF6-P38 MAPK) pathway are important for TGFβ1-induced autophagy. The TAK1-TRAF6-P38 MAPK pathway was essential for downregulating mTOR S2448 phosphorylation, ULK1 S555 phosphorylation and autophagosome formation. Furthermore, although siRNA-mediated Smad4 silencing did not alter mTOR-dependent ULK1 S757 phosphorylation, it did reduce AMPK-dependent ULK1 S555 phosphorylation and autophagosome formation. Additionally, Smad4 silencing and inhibiting the TAK1-TRAF6-P38 MAPK pathway decreased autophagosome-lysosome co-localization in the presence of TGFβ. Our results suggest that the Smad4 and TAK1-TRAF6-P38 MAPK signaling pathways are essential for TGFβ-induced autophagy and provide specific targets for the inhibition of TGFβ in tumor cells that utilize autophagy in their epithelial-mesenchymal transition program.
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Halle, Jessica L., Brittany R. Counts, Hector G. Paez, Dryden R. Baumfalk, Quan Zhang, Junaith S. Mohamed, Evan S. Glazer i in. "Recovery from FOLFOX Chemotherapy-induced Systemic and Skeletal Muscle Metabolic Dysfunction in Mice". American Journal of Physiology-Endocrinology and Metabolism, 28.06.2023. http://dx.doi.org/10.1152/ajpendo.00096.2023.
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FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is used to treat colorectal cancer and can acutely induce metabolic dysfunction. However, the lasting effects on systemic and skeletal muscle metabolism after treatment cessation are poorly understood. Therefore, we investigated the acute and lasting effects of FOLFOX chemotherapy on systemic and skeletal muscle metabolism in mice. Direct effects of FOLFOX in cultured myotubes were also investigated. Male C57BL/6J mice completed four cycles (acute) of FOLFOX or PBS. Subsets were allowed to recover for 4-wks or 10-wks. CLAMS metabolic measurements were performed for 5-days prior to study endpoint. C2C12 myotubes were treated with FOLFOX for 24-hrs. Acute FOLFOX attenuated body mass and body fat accretion independent of food intake or cage activity. Acute FOLFOX decreased blood glucose, oxygen consumption (VO2), carbon dioxide production (VCO2), energy expenditure, and carbohydrate (CHO) oxidation. Deficits in VO2 and energy expenditure remained at 10-wks. CHO oxidation remained disrupted at 4-wks but returned to control levels after 10-wks. Acute FOLFOX reduced muscle COXIV enzyme activity, AMPK(T172), ULK1(S555), and LC3BII protein expression. Muscle LC3BII/I ratio was associated with altered CHO oxidation (r=0.75, p=0.03). In vitro, FOLFOX suppressed myotube AMPK(T172), ULK1(S555), and autophagy flux. Recovery for 4-wks normalized skeletal muscle AMPK and ULK1 phosphorylation. Our results provide evidence that FOLFOX disrupts systemic metabolism, which is not readily recoverable after treatment cessation. FOLFOX effects on skeletal muscle metabolic signaling did recover. Further investigations are warranted to prevent and treat FOLFOX-induced metabolic toxicities that negatively impact cancer patient survival and life quality.
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Palliative Care Research 11, Supplement (2016): S555—S569. http://dx.doi.org/10.2512/jspm.11.s555.
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