Two Alleles of NF-κB in the Sea Anemone Nematostella vectensis Are Widely Dispersed in Nature and Encode Proteins with Distinct Activities

BACKGROUND. NF-κB is an evolutionarily conserved transcription factor that controls the expression of genes involved in many key organismal processes, including innate immunity, development, and stress responses. NF-κB proteins contain a highly conserved DNA-binding/dimerization domain called the Rel homology domain. METHODS/PRINCIPAL FINDINGS. We characterized two NF-κB alleles in the sea anemone Nematostella vectensis that differ at nineteen single-nucleotide polymorphisms (SNPs). Ten of these SNPs result in amino acid substitutions, including six within the Rel homology domain. Both alleles are found in natural populations of Nematostella. The relative abundance of the two NF-κB alleles differs between populations, and departures from Hardy-Weinberg equilibrium within populations indicate that the locus may be under selection. The proteins encoded by the two Nv-NF-κB alleles have different molecular properties, in part due to a Cys/Ser polymorphism at residue 67, which resides within the DNA recognition loop. In nearly all previously characterized NF-κB proteins, the analogous residue is fixed for Cys, and conversion of human RHD proteins from Cys to Ser at this site has been shown to increase DNA-binding ability and increase resistance to inhibition by thiol-reactive compounds. However, the naturally-occurring Nematostella variant with Cys at position 67 binds DNA with a higher affinity than the Ser variant. On the other hand, the Ser variant activates transcription in reporter gene assays more effectively, and it is more resistant to inhibition by a thiol-reactive compound. Reciprocal Cys<->Ser mutations at residue 67 of the native Nv-NF-κB proteins affect DNA binding as in human NF-κB proteins, e.g., a Cys->Ser mutation increases DNA binding of the native Cys variant. CONCLUSIONS/SIGNIFICANCE. These results are the first demonstration of a naturally occurring and functionally significant polymorphism in NF-κB in any species. The functional differences between these alleles and their uneven distribution in the wild suggest that different genotypes could be favored in different environments, perhaps environments that vary in their levels of peroxides or thiol-reactive compounds.National Institutes of Health (CA047763); National Science Foundation (FP-91656101-0); Environmental Protection Agency (F5E11155); Conservation International Marine Management Area Science Program; Boston University (SPRInG grant); Postdoctoral Scholar Program at the Woods Hole Oceanographic Institution; The Beacon Institute for Rivers and Estuaries; the J Seward Johnson Fund; Boston University (5 P42 ES07381

The NF-KAPPAB signaling pathway of the sea anemone Nematostella vectensis: functional characterization of core elements and two naturally occuring polymorphisms

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [email protected]. Thank you.The NF-κB family of eukaryotic transcription factors is activated in response to many environmental and biological stresses and is required for several important developmental processes. The sea anemone Nematostella vectensis (Nv) is the leading developmental and genomic model for the phylum Cnidaria, which includes anemones, hydras, jellyfish and corals. Sequencing of the Nematostella genome revealed sequences encoding NF-κB pathway proteins, including homologs of NF-κB, IκB, Bcl-3 and IKK. The goal ofthis research was to characterize molecular and biological functions of the Nv-NF-κB pathway proteins, in part because Nematostella is a model organism in which a framework for understanding the evolutionary origins of NF-κB signaling could be established. In this thesis, cDNAs for Nv-NF-κB pathway proteins were cloned and characterized. Nv-NF-κB is shown to bind to κB sites and activate transcription of reporter genes containing multimeric κB sites. Nv-NF-κB is localized in the nucleus of cells in developing Nematostella embryos and is expressed in the cytoplasm of a subset of ectodermal cells in juvenile and adult Nematostella. When over-expressed in human A293 cells, Nv-IκB and Nv-Bcl-3 can both interact with Nv-NF-κB and block its ability to activate reporter gene expression. Nv-IKK is most similar to human IKKc/TBK kinases, and can phosphorylate Nv-IκB in vitro. These results indicate that the core NF-κB pathway proteins in Nematostella are similar to their vertebrate homologs. This thesis also describes two geographically dispersed, naturally occurring NF-κB alleles in Nematostella. Proteins encoded by these two alleles, Nv-NF-κB-C and Nv-NF-κB-S, are polymorphic at key amino acids and have differing activities. Polymorphic residue 67 contributes to the difference between the two Nv-NF-κB variants in reporter gene transactivation. Computational modeling and electrophoretic mobility shift assays indicate that polymorphic positions 67 and 269 are responsible for differences in DNA binding of the two NF-KB variants. Additionally, the DNA-binding activity of Nv-NF-κB-C is more sensitive to redox conditions and thiol-reactive compounds than is the activity of Nv-NF-κB-S. These results suggest that environmental conditions, such as oxidative stress, have driven the divergence of the two alleles

Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis

International audienceWe describe a protocol for microinjection of embryos for an emerging model system, the cnidarian sea anemone, Nematostella vectensis. In addition, we provide protocols for carrying out overexpression and knockdown of gene function through microinjection of in vitro-translated mRNAs or gene-specific oligonucleotide morpholinos (MOs), respectively. Our approach is simple, and it takes advantage of the natural adherence properties of the early embryo to position them in a single layer on a polystyrene dish. Embryos are visualized on a dissecting microscope equipped with epifluorescence and injected with microinjection needles using a picospritzer forced-air injection system. A micromanipulator is used to guide the needle to impale individual embryos. Injection takes ∼1.5 h, and an experienced researcher can inject ∼2,000 embryos in a single session. With the availability of the published Nematostella genome, the entire protocol, including cloning and transcription of mRNAs, can be carried out in ∼1 week

Characterization of the Core Elements of the NF-κB Signaling Pathway of the Sea Anemone Nematostella vectensis ▿ ‡

The sea anemone Nematostella vectensis is the leading developmental and genomic model for the phylum Cnidaria, which includes anemones, hydras, jellyfish, and corals. In insects and vertebrates, the NF-κB pathway is required for cellular and organismal responses to various stresses, including pathogens and chemicals, as well as for several developmental processes. Herein, we have characterized proteins that comprise the core NF-κB pathway in Nematostella, including homologs of NF-κB, IκB, Bcl-3, and IκB kinase (IKK). We show that N. vectensis NF-κB (Nv-NF-κB) can bind to κB sites and activate transcription of reporter genes containing multimeric κB sites or the Nv-IκB promoter. Both Nv-IκB and Nv-Bcl-3 interact with Nv-NF-κB and block its ability to activate reporter gene expression. Nv-IKK is most similar to human IKKɛ/TBK kinases and, in vitro, can phosphorylate Ser47 of Nv-IκB. Nv-NF-κB is expressed in a subset of ectodermal cells in juvenile and adult Nematostella anemones. A bioinformatic analysis suggests that homologs of many mammalian NF-κB target genes are targets for Nv-NF-κB, including genes involved in apoptosis and responses to organic compounds and endogenous stimuli. These results indicate that NF-κB pathway proteins in Nematostella are similar to their vertebrate homologs, and these results also provide a framework for understanding the evolutionary origins of NF-κB signaling

Abstract LB565: Efficacy of a highly potent and selective KIT V654A inhibitor for treatment of imatinib resistant GIST

Abstract Gastrointestinal stromal tumor (GIST) is the most common type of sarcoma, with approximately 5,000 patients diagnosed per year in the US. Approximately 80% of patients with GIST present with mutations in the c-KIT oncogene at exon 9 or 11, which leads to constitutive, ligand-independent activation of the KIT receptor tyrosine kinase. For patients with metastatic GIST, frontline therapy with imatinib is effective, with a response rate of approximately 51-54% and median progression-free survival (PFS) of 19-23 months, in a molecularly unselected population. Other agents are approved for advanced GIST, without molecular selection, after progression on imatinib, including sunitinib, regorafenib, and ripretinib; however, response rates are less than 10% with PFS of approximately 5-6 months. Notably, patients who progress on imatinib and other tyrosine kinase inhibitors may develop a variety of on-target resistance mutations in the KIT oncogene, such as those in exon 17 (including at amino acids D816 and D820, activation loop mutation), exon 13 (V654A, ATP-binding region mutation), and less frequently in exon 14 (T670I, gatekeeper mutation). Several KIT inhibitors have been developed to potently target the exon 17 resistance mutations (avapritinib and ripretinib); however, there remains an important medical need in 2nd- and 3rd-line therapy in a molecularly unselected population of imatinib-resistant GIST. This suggests more broad-spectrum KIT inhibition is likely required, a hypothesis supported by the observation of large inter- and intra-patient heterogeneity of KIT secondary mutations across hundreds of samples obtained from patients with GIST treated with avapritinib. Sequencing data from the NAVIGATOR phase 1 trial (NCT02508532) revealed that patients with KIT mutant GIST and with the KIT V654A secondary resistance mutation had a poor response to treatment with avapritinib. To address this, we developed a highly potent and selective inhibitor of KIT V654A. This inhibitor showed dose-dependent modulation of downstream pharmacodynamic markers and induced tumor regression in a mastocytoma xenograft model driven by an exon 11 plus 13 V654A resistance mutation. Importantly, this inhibitor was generally well-tolerated and showed high selectivity over wild-type KIT. These findings suggest this novel KIT inhibitor has the potential to be used as a single agent or combination therapy for patients with imatinib-resistant GIST harboring the KIT V654A mutation. Citation Format: Alexandra R. Grassian, Joseph Kim, Omar Ahmad, Kevin Barvian, Alison Davis, Tom Dineen, Wei Hu, Ebby Job, Ludivine Moine, Kate Newberry, Maria Roche, Doug Shorten, Yeon Sook Choi, Francis Wolenski, Sebastian Bauer, Cesar Serrano, Jonathan Trent, Suzanne George. Efficacy of a highly potent and selective KIT V654A inhibitor for treatment of imatinib resistant GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB565

Additional file 7: Figure S6. of The beagle dog MicroRNA tissue atlas: identifying translatable biomarkers of organ toxicity

Top 20 expressed kidney miRNA. Top 20 expressed kidney miRNA found in the dog miRNA tissue atlas compared to previously published miRNA expression data from macro-dissection studies of the cortex and medulla of the cat and dog (Ichii et al). Shaded boxes indicate that the dog miRNA was among the top 10 expressed kidney miRNAs in the dog atlas; bolded miRNAs indicate that the miRNA was among the top 20. Comparison between dog whole kidney (dog altas) and data from dog cortex and medulla (Ichii et al) show good correlation with 7/10 miRNAs and 8/10 miRNAs expressed in the top 10 miRNAs, respectively, when compared to kidney expression observed in the dog atlas. Similarly, a comparison of dog whole kidney data (dog atlas) to cat cortex and medulla (Ichii et al) show good correlation as well with 7/10 and 6/10 miRNAs expressed in the top 10 miRNAs, respectively, when compared to dog whole kidney data (dog atlas). (PDF 41 kb