المؤلفون: Silvia Fibi-Smetana, Camila Inglis, Daniela Schuster, Nina Eberle, José Luis Granados-Soler, Wen Liu, Saskia Krohn, Christian Junghanss, Ingo Nolte, Leila Taher, Hugo Murua Escobar

المصدر: Frontiers in Veterinary Science, Vol 10 (2023)

مصطلحات موضوعية: targeted next-generation sequencing, canine lymphoma, genomic variants, comparative oncology, personalized oncology, patient stratification, Veterinary medicine, SF600-1100

الوصف: Targeted next-generation sequencing (NGS) enables the identification of genomic variants in cancer patients with high sensitivity at relatively low costs, and has thus opened the era to personalized human oncology. Veterinary medicine tends to adopt new technologies at a slower pace compared to human medicine due to lower funding, nonetheless it embraces technological advancements over time. Hence, it is reasonable to assume that targeted NGS will be incorporated into routine veterinary practice in the foreseeable future. Many animal diseases have well-researched human counterparts and hence, insights gained from the latter might, in principle, be harnessed to elucidate the former. Here, we present the TiHoCL targeted NGS panel as a proof of concept, exemplifying how functional genomics and network approaches can be effectively used to leverage the wealth of information available for human diseases in the development of targeted sequencing panels for veterinary medicine. Specifically, the TiHoCL targeted NGS panel is a molecular tool for characterizing and stratifying canine lymphoma (CL) patients designed based on human non-Hodgkin lymphoma (NHL) research outputs. While various single nucleotide polymorphisms (SNPs) have been associated with high risk of developing NHL, poor prognosis and resistance to treatment in NHL patients, little is known about the genetics of CL. Thus, the ~100 SNPs featured in the TiHoCL targeted NGS panel were selected using functional genomics and network approaches following a literature and database search that shielded ~500 SNPs associated with, in nearly all cases, human hematologic malignancies. The TiHoCL targeted NGS panel underwent technical validation and preliminary functional assessment by sequencing DNA samples isolated from blood of 29 lymphoma dogs using an Ion Torrent™ PGM System achieving good sequencing run metrics. Our design framework holds new possibilities for the design of similar molecular tools applied to other diseases for which limited knowledge is available and will improve drug target discovery and patient care.

وصف الملف: electronic resource

العلاقة: https://www.frontiersin.org/articles/10.3389/fvets.2023.1301536/fullTest; https://doaj.org/toc/2297-1769Test

الوصول الحر: https://doaj.org/article/25e9204043184b0a9c92b84f47e11d65Test

المؤلفون: Andrea Daniela Hobeck, Sophia Wendt, Saskia Krohn, Gudrun Knuebel, Stephan Bartels, Elisa Schipper, Christian Junghanss, Hugo Murua Escobar

المصدر: International Journal of Molecular Sciences, Vol 25, Iss 6, p 3534 (2024)

مصطلحات موضوعية: next-generation sequencing, OncomineTM Myeloid Panel, amplicon sequencing performance, FFPE material, fresh blood/bone marrow material, Biology (General), QH301-705.5, Chemistry, QD1-999

الوصف: Next-generation sequencing is a vital tool for personalized diagnostics and therapies in cancer. Despite numerous advantages, the method depends on multiple parameters regarding the sample material, e.g., sample fixation. A panel’s ability to ensure balanced pre-amplification of the regions of interest is challenging, especially in targeted sequencing approaches, but of significant importance to its applicability across hematological malignancies and solid tumors. This study comparatively evaluated the technical performance of the commercially available OncomineTM Myeloid Panel in fresh and Formalin-fixed paraffin-embedded (FFPE) material by using an Ion Torrent™ Personal Genome Machine™ System and Ion GeneStudio S5 System platform. In total, 114 samples were analyzed, including 55 fresh materials and 59 FFPE samples. Samples were sequenced with a minimum of one million reads. Amplicons with coverage below 400 reads were classified as underperforming. In fresh material, 49/526 amplicons were identified as performing insufficiently, corresponding with 18 genes. Using FFPE material, 103/526 amplicons underperformed. Independent of input material, regions in 27 genes, including ASXL1, BCOR and BRAF, did not match quality parameters. Subsequently, exemplary mutations were extracted from the Catalogue of Somatic Mutations in Cancer database. This technical evaluation of the OncomineTM Myeloid Panel identified amplicons that do not achieve adequate coverage levels and which need to be considered when interpreting sequencing.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/1422-0067/25/6/3534Test; https://doaj.org/toc/1661-6596Test; https://doaj.org/toc/1422-0067Test

الوصول الحر: https://doaj.org/article/106ea3a6cf1a4990b7dec074c307514cTest

المؤلفون: Fatima Efendic, Saskia Krohn, Hugo Murua Escobar, Sunita Venkateswaran, Steffany A.L. Bennett, Andreas Hermann, Moritz J. Frech

المصدر: Stem Cell Research, Vol 71, Iss , Pp 103178- (2023)

مصطلحات موضوعية: Biology (General), QH301-705.5

الوصف: Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a hereditary neurodegenerative disease caused by mutations in the FA2H gene. Patients show a wide range of neurological symptoms and an abnormal myelination. Here we describe the generation of the human induced pluripotent stem cell (hiPSC) lines AKOSi011-A and AKOSi012-A, derived from FAHN-patient fibroblasts, carrying the compound heterozygous mutation p.Pro65Ser/p.Asp35Tyr and the homozygous mutation p.Tyr231His, respectively. The hiPSC lines were generated using a non-integrating Sendai virus. The obtained hiPSCs show an unobtrusive karyotype, carry the mutations of the original fibroblasts, express pluripotency markers and can differentiate into cells of the three germ layers.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S1873506123001642Test; https://doaj.org/toc/1873-5061Test

الوصول الحر: https://doaj.org/article/7da70861230c483f9f2814d6e658b33eTest

المؤلفون: Anna Richter, Sandra Lange, Clemens Holz, Luisa Brock, Thomas Freitag, Anett Sekora, Gudrun Knuebel, Saskia Krohn, Rico Schwarz, Burkhard Hinz, Hugo Murua Escobar, Christian Junghanss

المصدر: Cell Death Discovery, Vol 8, Iss 1, Pp 1-11 (2022)

مصطلحات موضوعية: Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282, Cytology, QH573-671

الوصف: Abstract Dysregulation of the intrinsic BCL-2 pathway-mediated apoptosis cascade is a common feature of hematological malignancies including acute B-lymphoblastic leukemia (B-ALL). The KMT2A-rearranged high-risk cytogenetic subtype is characterized by high expression of antiapoptotic protein BCL-2, likely due to the direct activating binding of KMT2A fusion proteins to the BCL2 gene. The BCL-2 inhibitor venetoclax (VEN) has proven great clinical value in other blood cancers, however, data on B-ALL is sparse and past studies have not so far described the effects of VEN on gene and protein expression profiles. Using cell lines and patient-derived in vivo xenograft models, we show BCL-2 pathway-mediated apoptosis induction and decelerated tumor cell counts in KMT2A-rearranged B-ALL but not in other cytogenetic subtypes. VEN treatment of cell line- and patient-derived xenografts reduced blast frequencies in blood, bone marrow, and spleen, and tumor cell doubling times were increased. Growth rates are further correlated with VEN concentrations in blood. In vitro incubation with VEN resulted in BCL-2 dephosphorylation and targeted panel RNA sequencing revealed reduced gene expression of antiapoptotic pathway members BCL2, MCL1, and BCL2L1 (BCL-XL). Reinforced translocation of BAX proteins towards mitochondria induced caspase activation and cell death commitment. Prolonged VEN application led to upregulation of antiapoptotic proteins BCL-2, MCL-1, and BCL-XL. Interestingly, the extrinsic apoptosis pathway was strongly modulated in SEM cells in response to VEN. Gene expression of members of the tumor necrosis factor signaling cascade was increased, resulting in canonical NF-kB signaling. This possibly suggests a previously undescribed mechanism of BCL-2-independent and NF-kB-mediated upregulation of MCL-1 and BCL-XL. In summary, we herein prove that VEN is a potent option to suppress tumor cells in KMT2A-rearranged B-ALL in vitro and in vivo. Possible evasion mechanisms, however, must be considered in subsequent studies.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2058-7716Test

الوصول الحر: https://doaj.org/article/f965715f9603445aaf305e682578afd1Test

المؤلفون: Fatima Efendic, Christin Völkner, Saskia Krohn, Hugo Murua Escobar, Sunita Venkateswaran, Steffany Bennett, Andreas Hermann, Moritz J. Frech

المصدر: Stem Cell Research, Vol 63, Iss , Pp 102863- (2022)

مصطلحات موضوعية: Biology (General), QH301-705.5

الوصف: Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a rare childhood onset neurodegenerative disease caused by mutations in the FA2H gene. Patients display abnormal myelination, cerebellar atrophy and some have iron deposition in the central nervous system. Here we describe the generation of AKOSi010-A, a human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a female patient carrying the compound heterozygous p.Gly45Arg/p.His319Arg, using non-integrating Sendai virus. The generated iPSCs express pluripotency markers, can differentiate into cell types of the three germ layers and show a normal karyotype. This cell line displays a unique source to study the pathophysiology of FAHN.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S1873506122002124Test; https://doaj.org/toc/1873-5061Test

الوصول الحر: https://doaj.org/article/aab543386ef64e7b8f4316cf757dbd93Test

المؤلفون: Luise Grunwald, Christina Grosse-Thie, Sina Sender, Gudrun Knuebel, Saskia Krohn, Catrin Roolf, Christian Junghanss, Larissa Henze, Hugo Murua Escobar

المصدر: Biomarker Research, Vol 8, Iss 1, Pp 1-4 (2020)

مصطلحات موضوعية: Molecular genetics, Blood coagulation, Myeloproliferative neoplasms, Splanchnic vein thrombosis, Next generation sequencing, Ultradeep targeted sequencing, Therapeutics. Pharmacology, RM1-950

الوصف: Abstract Myeloproliferative neoplasms are characterized by mutations in JAK2, MPL and CALR genes. Commonly in diagnostics and previous studies mainly sequencing and common PCR techniques under conventional detection limits are used. Splanchnic vein thromboses are rare, but often appear associated with myeloproliferative neoplasms and represent serious complications. Herein, blood from patients with abdominal vein thromboses in Mecklenburg-West Pomerania (federal district of northern Germany), included in an ongoing prospective prevalence study, was analyzed by next generation sequencing representing the complete protein coding regions of JAK2, MPL and CALR genes with a coverage of > 2000 reads, therefore an ultradeep targeting approach. JAK2 V617F mutations were detected in 11/44 patients. In four of these cases allele frequencies ranged below the conventional cut off of 2%. MPL W515R was detected in 3/44 cases in low frequencies. Very low allele frequencies of JAK2 and MPL variants in patients with abdominal vein thromboses may indicate early manifestations of myeloproliferative neoplasms.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2050-7771Test

الوصول الحر: https://doaj.org/article/cdfa19447b6e45e0b7b5beb463afe6caTest

المؤلفون: Clemens Holz, Sandra Lange, Anett Sekora, Gudrun Knuebel, Saskia Krohn, Hugo Murua Escobar, Christian Junghanss, Anna Richter

المصدر: International Journal of Molecular Sciences, Vol 24, Iss 2, p 1359 (2023)

مصطلحات موضوعية: acute lymphoblastic leukemia, B-ALL, BCL-2, venetoclax, apoptosis, PI3K/AKT inhibition, Biology (General), QH301-705.5, Chemistry, QD1-999

الوصف: Numerous hematologic neoplasms, including acute B-lymphoblastic leukemia (B-ALL), are characterized by overexpression of anti-apoptotic BCL-2 family proteins. Despite the high clinical efficacy of the specific BCL-2 inhibitor venetoclax in acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL), dose limitation and resistance argue for the early exploration of rational combination strategies. Recent data indicated that BCL-2 inhibition in B-ALL with KMT2A rearrangements is a promising intervention option; however, combinatorial approaches have not been in focus so far. The PI3K/AKT pathway has emerged as a possible target structure due to multiple interactions with the apoptosis cascade as well as relevant dysregulation in B-ALL. Herein, we demonstrate for the first time that combined BCL-2 and PI3K/AKT inhibition has synergistic anti-proliferative effects on B-ALL cell lines. Of note, all tested combinations (venetoclax + PI3K inhibitors idelalisib or BKM-120, as well as AKT inhibitors MK-2206 or perifosine) achieved comparable anti-leukemic effects. In a detailed analysis of apoptotic processes, among the PI3K/AKT inhibitors only perifosine resulted in an increased rate of apoptotic cells. Furthermore, the combination of venetoclax and perifosine synergistically enhanced the activity of the intrinsic apoptosis pathway. Subsequent gene expression studies identified the pro-apoptotic gene BBC3 as a possible player in synergistic action. All combinatorial approaches additionally modulated extrinsic apoptosis pathway genes. The present study provides rational combination strategies involving selective BCL-2 and PI3K/AKT inhibition in B-ALL cell lines. Furthermore, we identified a potential mechanistic background of the synergistic activity of combined venetoclax and perifosine application.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/1422-0067/24/2/1359Test; https://doaj.org/toc/1661-6596Test; https://doaj.org/toc/1422-0067Test

الوصول الحر: https://doaj.org/article/f7717778f5544ab8a8bbb6b1b39740c6Test

المؤلفون: Janine Petters, Christin Völkner, Saskia Krohn, Hugo Murua Escobar, Jörn Bullerdiek, Ulrike Reuner, Moritz J. Frech, Andreas Hermann, Jan Lukas

المصدر: Stem Cell Research, Vol 49, Iss , Pp 102079- (2020)

مصطلحات موضوعية: Biology (General), QH301-705.5

الوصف: Wilson disease (WD) is a rare, monogenic disorder caused by mutations in the gene ATP7B. A loss of function of the expressed protein leads to excessive hepatic and cerebral copper storage. In this study, we present the generation of two induced pluripotent stem cell (iPSC) lines derived from fibroblasts of a clinically asymptomatic, chelator treated female WD patient carrying the common missense mutation p.H1069Q and an age-matched female healthy control subject. The generated iPSC lines expressed pluripotency markers, showed differentiation potential and retained their parental genotype. Therefore, these cells provide a valuable resource to understand the pathophysiology of WD and can be used as model systems for drug testing.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S1873506120303809Test; https://doaj.org/toc/1873-5061Test

الوصول الحر: https://doaj.org/article/ae78d5daff104f98916aeae139f7fd34Test

المؤلفون: Anna Richter, Catrin Roolf, Anett Sekora, Gudrun Knuebel, Saskia Krohn, Sandra Lange, Vivien Krebs, Bjoern Schneider, Johannes Lakner, Christoph Wittke, Christoph Kiefel, Irmela Jeremias, Hugo Murua Escobar, Brigitte Vollmar, Christian Junghanss

المصدر: Cells, Vol 11, Iss 1, p 150 (2022)

مصطلحات موضوعية: acute lymphoblastic leukemia, PDX, biobanking, serial transplantation, engraftment site, proliferation kinetics, Cytology, QH573-671

الوصف: In acute lymphoblastic leukemia (ALL), conventional cell lines do not recapitulate the clonal diversity and microenvironment. Orthotopic patient-derived xenograft models (PDX) overcome these limitations and mimic the clinical situation, but molecular stability and engraftment patterns have not yet been thoroughly assessed. We herein describe and characterize the PDX generation in NSG mice. In vivo tumor cell proliferation, engraftment and location were monitored by flow cytometry and bioluminescence imaging. Leukemic cells were retransplanted for up to four passages, and comparative analyses of engraftment pattern, cellular morphology and genomic hotspot mutations were conducted. Ninety-four percent of all samples were successfully engrafted, and the xenograft velocity was dependent on the molecular subtype, outcome of the patient and transplantation passage. While BCR::ABL1 blasts were located in the spleen, KMT2A-positive cases had higher frequencies in the bone marrow. Molecular changes appeared in most model systems, with low allele frequency variants lost during primary engraftment. After the initial xenografting, however, the PDX models demonstrated high molecular stability. This protocol for reliable ALL engraftment demonstrates variability in the location and molecular signatures during serial transplantation. Thorough characterization of experimentally used PDX systems is indispensable for the correct analysis and valid data interpretation of preclinical PDX studies.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2073-4409/11/1/150Test; https://doaj.org/toc/2073-4409Test

الوصول الحر: https://doaj.org/article/21fdd92f54984c47a5294b6e0d5b6d24Test

المؤلفون: Christin Völkner, Franziska Peter, Maik Liedtke, Saskia Krohn, Iris Lindner, Hugo Murua Escobar, Chiara Cimmaruta, Jan Lukas, Andreas Hermann, Moritz J. Frech

المصدر: Stem Cell Research, Vol 41, Iss , Pp - (2019)

مصطلحات موضوعية: Biology (General), QH301-705.5

الوصف: Niemann-Pick disease Type C (NPC) is a rare progressive neurodegenerative disorder with an incidence of 1:120,000 caused by mutations in the NPC1 or NPC2 gene. Only 5% of NPC patients suffer from mutations of the NPC2 gene. Here we demonstrate the generation of a Niemann–Pick disease Type C2 (NPC2) patient-derived induced pluripotent stem cell line. This cell line is capable to differentiate into derivatives of the neuronal lineage, providing a valuable tool to study pathogenic mechanisms of NPC2.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S1873506119302363Test; https://doaj.org/toc/1873-5061Test

الوصول الحر: https://doaj.org/article/f019f925d49f4d1897c661f7dc354fb9Test