Identification of novel regulators of STAT3 activity

STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705). Here, we show that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. To find novel and potentially targetable STAT3 regulators we performed a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3. The screen identified cell division cycle 7-related protein kinase (CDC7), casein kinase 2, alpha 1 (CSNK2), discoidin domain-containing receptor 2 (DDR2), cyclin-dependent kinase 8 (CDK8), phosphatidylinositol 4-kinase 2-alpha (PI4KII), C-terminal Src kinase (CSK) and receptor-type tyrosine-protein phosphatase H (PTPRH) as potential STAT3 regulators. Using small molecule inhibitors targeting these proteins, we confirmed dose and time dependent inhibition of STAT3-mediated transcription, suggesting that inhibition of these kinases may provide strategies for dampening STAT3 activity in cancers.Peer reviewe

Functional Characterization of the Infection-Inducible Peptide Edin in Drosophila melanogaster

Drosophila is a well-established model organism for studying innate immunity because of its high resistance against microbial infections and lack of adaptive immunity. In addition, the immune signaling cascades found in Drosophila are evolutionarily conserved. Upon infection, activation of the immune signaling pathways, Toll and Imd, leads to the expression of multiple immune response genes, such as the antimicrobial peptides (AMPs). Previously, we identified an uncharacterized gene edin among the genes, which were strongly induced upon stimulation with Escherichia coli in Drosophila S2 cells. Edin has been associated with resistance against Listeria monocytogenes, but its role in Drosophila immunity remains elusive. In this study, we examined the role of Edin in the immune response of Drosophila both in vitro and in vivo. We report that edin expression is dependent on the Imd-pathway NF-κB transcription factor Relish and that it is expressed upon infection both in vitro and in vivo. Edin encodes a pro-protein, which is further processed in S2 cells. In our experiments, Edin did not bind microbes, nor did it possess antimicrobial activity to tested microbial strains in vitro or in vivo. Furthermore, edin RNAi did not significantly affect the expression of AMPs in vitro or in vivo. However, edin RNAi flies showed modestly impaired resistance to E. faecalis infection. We conclude that Edin has no potent antimicrobial properties but it appears to be important for E. faecalis infection via an uncharacterized mechanism. Further studies are still required to elucidate the exact role of Edin in the Drosophila immune response

Long-term follow up of families with pathogenic NFKB1 variants reveals incomplete penetrance and frequent inflammatory sequelae

Publisher Copyright: © 2022 The AuthorsNuclear factor κ light-chain enhancer of activated B cells (NF-κB) family of evolutionarily conserved transcription factors are involved in key cellular signaling pathways. Previously, hypogammaglobulinemia and common variable immunodeficiency (CVID)-like phenotypes have been associated with NFKB1 variants and loss-of-function NFKB1 variants have been reported as the most common monogenic cause for CVID among Europeans. Here, we describe a Finnish cohort of NFKB1 carriers consisting of 31 living subjects in six different families carrying five distinct heterozygous variants. In contrast to previous reports, the clinical penetrance was not complete even with advancing age and the prevalence of CVID/hypogammaglobulinemia was significantly lower, whereas (auto)inflammatory manifestations were more common (42% of the total cohort). At current stage of knowledge, routine genetic screening of asymptomatic individuals is not recommended, but counseling of potential adult carriers seems necessary.Peer reviewe

Loss-of-function mutation in IKZF2 leads to immunodeficiency with dysregulated germinal center reactions and reduction of MAIT cells

Peer reviewe

Loss-of-function mutation in IKZF2 leads to immunodeficiency with dysregulated germinal center reactions and reduction of MAIT cells

Peer reviewe

Loss of DIAPH1 causes SCBMS, combined immunodeficiency, and mitochondrial dysfunction

Background: Homozygous loss of DIAPH1 results in seizures, cortical blindness, and microcephaly syndrome (SCBMS). We studied 5 Finnish and 2 Omani patients with loss of DIAPH1 presenting with SCBMS, mitochondrial dysfunction, and immunodeficiency. Objective: We sought to further characterize phenotypes and disease mechanisms associated with loss of DIAPH1. Methods: Exome sequencing, genotyping and haplotype analysis, B- and T-cell phenotyping, in vitro lymphocyte stimulation assays, analyses of mitochondrial function, immunofluorescence staining for cytoskeletal proteins and mitochondria, and CRISPR-Cas9 DIAPH1 knockout in heathy donor PBMCs were used. Results: Genetic analyses found all Finnish patients homozygous for a rare DIAPH1 splice-variant (NM_005219:c.68411G>A) enriched in the Finnish population, and Omani patients homozygous for a previously described pathogenic DIAPH1 frameshift-variant (NM_005219:c.2769delT;p.F923fs). In addition to microcephaly, epilepsy, and cortical blindness characteristic to SCBMS, the patients presented with infection susceptibility due to defective lymphocyte maturation and 3 patients developed B-cell lymphoma. Patients' immunophenotype was characterized by poor lymphocyte activation and proliferation, defective B-cell maturation, and lack of naive T cells. CRISPR-Cas9 knockout of DIAPH1 in PBMCs from healthy donors replicated the T-cell activation defect. Patient-derived peripheral blood T cells exhibited impaired adhesion and inefficient microtubule-organizing center repositioning to the immunologic synapse. The clinical symptoms and laboratory tests also suggested mitochondrial dysfunction. Experiments with immortalized, patient-derived fibroblasts indicated that DIAPH1 affects the amount of complex IV of the mitochondrial respiratory chain. Conclusions: Our data demonstrate that individuals with SCBMS can have combined immune deficiency and implicate defective cytoskeletal organization and mitochondrial dysfunction in SCBMS pathogenesis.Peer reviewe

Enrichment of rare variants in population isolates : single AICDA mutation responsible for hyper-IgM syndrome type 2 in Finland

Antibody class-switch recombination and somatic hypermutation critically depend on the function of activation-induced cytidine deaminase (AID). Rare variants in its gene AICDA have been reported to cause autosomal recessive AID deficiency (autosomal recessive hyper-IgM syndrome type 2 (HIGM2)). Exome sequencing of a multicase Finnish family with an HIGM2 phenotype identified a rare, homozygous, variant (c.416T > C, p.(Met139Thr)) in the AICDA gene, found to be significantly enriched in the Finnish population compared with other populations of European origin (38.56-fold, P <0.001). The population history of Finland, characterized by a restricted number of founders, isolation and several population bottlenecks, has caused enrichment of certain rare disease-causing variants and losses of others, as part of a phenomenon called the Finnish Disease Heritage. Accordingly, rare founder mutations cause the majority of observed Finnish cases in these mostly autosomal recessive disorders that consequently are more frequent in Finland than elsewhere. Screening of all currently known Finnish patients with an HIGM2 phenotype showed them to be homozygous for p.(Met139Thr). All the Finnish p.(Met139Thr) carriers with available data on their geographic descent originated from the eastern and northeastern parts of Finland. They were observed to share more of their genome identity by descent (IBD) than Finns in general (P <0.001), and they all carried a 207.5-kb ancestral haplotype containing the variant. In conclusion, the identified p.(Met139Thr) variant is significantly enriched in Finns and explains all thus far found AID deficiencies in Finland.Peer reviewe

In vivo characterization of two novel regulators of the JAK/STAT pathway in Drosophila melanogaster

Background and aims: The JAK/STAT pathway is a well-conserved signalling pathway that takes part in many developmental and functional processes in both humans and flies. In both organisms, defects in this pathway have been found to result in the development of pathological conditions such as cancer and immune diseases. Therefore, there is great interest in identifying new genes that take part in the regulation of signalling through the JAK/STAT pathway. Drosophila melanogaster is a well-suited model organism for screening and studying new regulators of JAK/STAT signalling, as unlike mammals, it lacks redundancy in the key components of the pathway. The aim of this project was to study the effect of overexpression of two genes, et and not4, found in an RNAi screen for new JAK/STAT pathway regulators in Drosophila, and to verify their function as JAK/STAT pathway regulators. Methods: The UAS-GAL4 system was used for directed gene expression in Drosophila melanogaster in vivo. UAS-not4 and UAS-et transgenic lines were obtained from the Best Gene Inc. and balanced against suitable balancers. Balanced lines were crossed over the drivers C564-GAL4 or eyeless-GAL4 to study the effects of not4 and et overexpression on the stress response or eye-development of the fly in vivo, respectively. Gene expression was assayed with quantitative RT-PCR. Results: Overexpression of not4 in the fatbody of the fly caused an increase in the expression of the stress response genes TotA and TotM, which are known JAK/STAT pathway targets. not4 expression was, however, not induced by septic injury, which is known to activate JAK/STAT signalling. et expression in turn was strongly induced by septic injury. Despite previous results, et overexpression caused only mild changes in the expression of Tot genes and could not prevent their induction following septic injury. However, as has also been previously shown elsewhere, et knock-down caused dramatic upregulation of Tot gene expression. Overexpression of not4 in the eye-discs had no visible effect on the development or morphology of the fly eye. Conclusions: Based on previous knowledge, Et seems to function as a negative regulator of JAK/STAT signalling, but in our assay the overexpression of et could not prevent the transcription of pathway targets in vivo. In any case, since the expression of et is induced by septic injury, Et seems to be involved in the stress response of Drosophila. not4 overexpression evidently increases the expression of the JAK/STAT targets TotA and TotM, which in conjunction with evidence from other experiments suggests that Not4 functions as a positive regulator of JAK/STAT signalling. However, the mechanisms of function of both Not4 and Et still require further studying

In vivo characterization of two novel regulators of the JAK/STAT pathway in Drosophila melanogaster

Background and aims: The JAK/STAT pathway is a well-conserved signalling pathway that takes part in many developmental and functional processes in both humans and flies. In both organisms, defects in this pathway have been found to result in the development of pathological conditions such as cancer and immune diseases. Therefore, there is great interest in identifying new genes that take part in the regulation of signalling through the JAK/STAT pathway. Drosophila melanogaster is a well-suited model organism for screening and studying new regulators of JAK/STAT signalling, as unlike mammals, it lacks redundancy in the key components of the pathway. The aim of this project was to study the effect of overexpression of two genes, et and not4, found in an RNAi screen for new JAK/STAT pathway regulators in Drosophila, and to verify their function as JAK/STAT pathway regulators. Methods: The UAS-GAL4 system was used for directed gene expression in Drosophila melanogaster in vivo. UAS-not4 and UAS-et transgenic lines were obtained from the Best Gene Inc. and balanced against suitable balancers. Balanced lines were crossed over the drivers C564-GAL4 or eyeless-GAL4 to study the effects of not4 and et overexpression on the stress response or eye-development of the fly in vivo, respectively. Gene expression was assayed with quantitative RT-PCR. Results: Overexpression of not4 in the fatbody of the fly caused an increase in the expression of the stress response genes TotA and TotM, which are known JAK/STAT pathway targets. not4 expression was, however, not induced by septic injury, which is known to activate JAK/STAT signalling. et expression in turn was strongly induced by septic injury. Despite previous results, et overexpression caused only mild changes in the expression of Tot genes and could not prevent their induction following septic injury. However, as has also been previously shown elsewhere, et knock-down caused dramatic upregulation of Tot gene expression. Overexpression of not4 in the eye-discs had no visible effect on the development or morphology of the fly eye. Conclusions: Based on previous knowledge, Et seems to function as a negative regulator of JAK/STAT signalling, but in our assay the overexpression of et could not prevent the transcription of pathway targets in vivo. In any case, since the expression of et is induced by septic injury, Et seems to be involved in the stress response of Drosophila. not4 overexpression evidently increases the expression of the JAK/STAT targets TotA and TotM, which in conjunction with evidence from other experiments suggests that Not4 functions as a positive regulator of JAK/STAT signalling. However, the mechanisms of function of both Not4 and Et still require further studying

Constant B cell lymphocytosis since early age in a patient with CARD11 mutation : A 20-year follow-up

Non peer reviewe