RNA:RNA interaction in ternary complexes resolved by chemical probing.

RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.

Efficient 3'-pairing renders microRNA targeting less sensitive to mRNA seed accessibility.

MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2-g8 in the miRNA), but pairing beyond the seed (3'-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3'-pairing and secondary structures in mRNAs on repression efficiency. Using the interaction between miR-34a and its SIRT1 binding site as a model, we provide structural and functional evidence that 3'-pairing can compensate for low seed-binding site accessibility, enabling repression of sites that would otherwise be ineffective. We show that miRNA 3'-pairing regions can productively base-pair with nucleotides far upstream of the seed-binding site and that both hairpins and unstructured bulges within the target site are tolerated. We use SHAPE to show that sequences that overcome inaccessible seed-binding sites by strong 3'-pairing adopt the predicted structures and corroborate the model using luciferase assays and high-throughput modelling of 8177 3'-UTR targets for six miRNAs. Finally, we demonstrate that PHB2, a target of miR-141, is an inaccessible target rescued by efficient 3'-pairing. We propose that these results could refine predictions of effective target sites.

STE20-type kinases MST3 and MST4 promote the progression of hepatocellular carcinoma: Evidence from human cell culture and expression profiling of liver biopsies.

Hepatocellular carcinoma (HCC) is one of the most fatal and fastest growing malignancies. Recently, nonalcoholic steatohepatitis (NASH), characterized by liver steatosis, inflammation, cell injury (hepatocyte ballooning), and different stages of fibrosis, has emerged as a major catalyst for HCC. Because the STE20-type kinases, MST3 and MST4, have been described as critical molecular regulators of NASH pathophysiology, we here focused on determining the relevance of these proteins in human HCC. By analyzing public datasets and in-house cohorts, we found that hepatic MST3 and MST4 expression was positively correlated with the incidence and severity of HCC. We also found that the silencing of both MST3 and MST4, but also either of them individually, markedly suppressed the tumorigenesis of human HCC cells including attenuated proliferation, migration, invasion, and epithelial-mesenchymal transition. Mechanistic investigations revealed lower activation of STAT3 signaling in MST3/MST4-deficient hepatocytes and identified GOLGA2 and STRIPAK complex as the binding partners of both MST3 and MST4. These findings reveal that MST3 and MST4 play a critical role in promoting the progression of HCC and suggest that targeting these kinases may provide a novel strategy for the treatment of liver cancer.

Spatially segregated defects and IGF1-responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome.

BACKGROUND & AIMS: Alagille syndrome (ALGS) manifests with peripheral intrahepatic bile duct (IHBD) paucity, which can spontaneously resolve. In a model for ALGS, Jag1Ndr/Ndr mice, this occurs with distinct architectural mechanisms in hilar and peripheral IHBDs. Here, we investigated region-specific IHBD characteristics and addressed whether IGF1, a cholangiocyte mitogen that is downregulated in ALGS and in Jag1Ndr/Ndr mice, can improve biliary outcomes. METHODS: Intrahepatic cholangiocyte organoids (ICOs) were derived from hilar and peripheral adult Jag1+/+ and Jag1Ndr/Ndr livers (hICOs and pICOs, respectively). ICOs were grown in Matrigel or microwell arrays, and characterized using bulk RNA sequencing, immunofluorescence, and high throughput analyses of nuclear sizes. ICOs were treated with IGF1, followed by analyses of growth, proliferation, and death. CellProfiler and Python scripts were custom written for image analyses. Key results were validated in vivo by immunostaining. RESULTS: Cell growth assays and transcriptomics demonstrated that Jag1Ndr/Ndr ICOs were less proliferative than Jag1+/+ ICOs. IGF1 specifically rescued survival and growth of Jag1Ndr/Ndr pICOs. Jag1Ndr/Ndr hICOs were the least proliferative, with lower Notch signalling and an enrichment of hepatocyte signatures and IGF uptake/transport pathways. In vitro (Jag1Ndr/Ndr hICOs) and in vivo (Jag1Ndr/Ndr hilar portal tracts) analyses revealed ectopic HNF4a+ hepatocytes. CONCLUSIONS: Hilar and peripheral Jag1Ndr/Ndr ICOs exhibit differences in Notch signalling status, proliferation, and cholangiocyte commitment which may result in cholangiocyte-to-hepatocyte transdifferentiation. While Jag1Ndr/Ndr pICOs can be rescued by IGF1, hICOs are unresponsive, perhaps due to their hepatocyte-like state and/or expression of IGF transport components. IGF1 represents a potential therapeutic for peripheral bile ducts.

Acute-Phase Neurofilament Light and Glial Fibrillary Acidic Proteins in Cerebrospinal Fluid Predict Long-Term Outcome After Severe Traumatic Brain Injury.

BACKGROUND: This study investigated trajectory profiles and the association of concentrations of the biomarkers neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) in ventricular cerebrospinal fluid (CSF) with clinical outcome at 1 year and 10-15 years after a severe traumatic brain injury (sTBI). METHODS: This study included patients with sTBI at the Neurointensive Care Unit at Sahlgrenska University Hospital, Gothenburg, Sweden. The injury was regarded as severe if patients had a Glasgow Coma Scale ≤ 8 corresponding to Reaction Level Scale ≥ 4. CSF was collected from a ventricular catheter during a 2-week period. Concentrations of NfL and GFAP in CSF were analyzed with enzyme-linked immunosorbent assay. The Glasgow Outcome Scale (GOS) was used to assess the 1-year and 10-15-year outcomes. After adjustment for age and previous neurological diseases, logistic regression was performed for the outcomes GOS 1 (dead) or GOS 2-5 (alive) and GOS 1-3 (poor) or GOS 4-5 (good) versus the independent continuous variables (NfL and GFAP). RESULTS: Fifty-three patients with sTBI were investigated; forty-seven adults are presented in the article, and six children (aged 7-18 years) are described in Supplement 1. The CSF concentrations of NfL gradually increased over 2 weeks post trauma, whereas GFAP concentrations peaked on days 3-4. Increasing NfL and GFAP CSF concentrations increased the odds of GOS 1-3 outcome 1 year after trauma (odds ratio [OR] 1.73, 95% confidence interval [CI] 1.07-2.80, p = 0.025; and OR 1.61, 95% CI 1.09-2.37, p = 0.016, respectively). Similarly, increasing CSF concentrations of NfL and GFAP increased the odds for GOS 1-3 outcome 10-15 years after trauma (OR 2.04, 95% CI 1.05-3.96, p = 0.035; and OR 1.60, 95% CI 1.02-2.00, p = 0.040). CONCLUSIONS: This study shows that initial high concentrations of NfL and GFAP in CSF are both associated with higher odds for GOS 1-3 outcome 1 year and 10-15 years after an sTBI, implicating its potential usage as a prognostic marker in the future.

Genetic Ablation of STE20-Type Kinase MST4 Does Not Alleviate Diet-Induced MASLD Susceptibility in Mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its advanced subtype, metabolic dysfunction-associated steatohepatitis (MASH), have emerged as the most common chronic liver disease worldwide, yet there is no targeted pharmacotherapy presently available. This study aimed to investigate the possible in vivo function of STE20-type protein kinase MST4, which was earlier implicated in the regulation of hepatocellular lipotoxic milieu in vitro, in the control of the diet-induced impairment of systemic glucose and insulin homeostasis as well as MASLD susceptibility. Whole-body and liver-specific Mst4 knockout mice were generated by crossbreeding conditional Mst4fl/fl mice with mice expressing Cre recombinase under the Sox2 or Alb promoters, respectively. To replicate the environment in high-risk subjects, Mst4-/- mice and their wild-type littermates were fed a high-fat or a methionine-choline-deficient (MCD) diet. Different in vivo tests were conducted in obese mice to describe the whole-body metabolism. MASLD progression in the liver and lipotoxic damage to adipose tissue, kidney, and skeletal muscle were analyzed by histological and immunofluorescence analysis, biochemical assays, and protein and gene expression profiling. In parallel, intracellular fat storage and oxidative stress were assessed in primary mouse hepatocytes, where MST4 was silenced by small interfering RNA. We found that global MST4 depletion had no effect on body weight or composition, locomotor activity, whole-body glucose tolerance or insulin sensitivity in obese mice. Furthermore, we observed no alterations in lipotoxic injuries to the liver, adipose, kidney, or skeletal muscle tissue in high-fat diet-fed whole-body Mst4-/- vs. wild-type mice. Liver-specific Mst4-/- mice and wild-type littermates displayed a similar severity of MASLD when subjected to an MCD diet, as evidenced by equal levels of steatosis, inflammation, hepatic stellate cell activation, fibrosis, oxidative/ER stress, and apoptosis in the liver. In contrast, the in vitro silencing of MST4 effectively protected primary mouse hepatocytes against ectopic lipid accumulation and oxidative cell injury triggered by exposure to fatty acids. In summary, these results suggest that the genetic ablation of MST4 in mice does not mitigate the initiation or progression of MASLD and has no effect on systemic glucose or insulin homeostasis in the context of nutritional stress. The functional compensation for the genetic loss of MST4 by yet undefined mechanisms may contribute to the apparent discrepancy between in vivo and in vitro phenotypic consequences of MST4 silencing.

Knockout of STE20-type kinase TAOK3 does not attenuate diet-induced NAFLD development in mice.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD), the primary hepatic consequence of obesity, is affecting about 25% of the global adult population. The aim of this study was to examine the in vivo role of STE20-type protein kinase TAOK3, which has been previously reported to regulate hepatocellular lipotoxicity in vitro, in the development of NAFLD and systemic insulin resistance in the context of obesity. METHODS: Taok3 knockout mice and wild-type littermates were challenged with a high-fat diet. Various in vivo tests were performed to characterize the whole-body metabolism. NAFLD progression in the liver, and lipotoxic damage in adipose tissue, kidney, and skeletal muscle were compared between the genotypes by histological assessment, immunofluorescence microscopy, protein and gene expression profiling, and biochemical assays. Intracellular lipid accumulation and oxidative/ER stress were analyzed in cultured human and mouse hepatocytes where TAOK3 was knocked down by small interfering RNA. The expression of TAOK3-related STE20-type kinases was quantified in different organs from high-fat diet-fed Taok3-/- and wild-type mice. RESULTS: TAOK3 deficiency had no impact on body weight or composition, food consumption, locomotor activity, or systemic glucose or insulin homeostasis in obese mice. Consistently, Taok3-/- mice and wild-type littermates developed a similar degree of high-fat diet-induced liver steatosis, inflammation, and fibrosis, and we detected no difference in lipotoxic damage of adipose tissue, kidney, or skeletal muscle when comparing the two genotypes. In contrast, the silencing of TAOK3 in vitro markedly suppressed ectopic lipid accumulation and metabolic stress in mouse and human hepatocytes. Interestingly, the hepatic mRNA abundance of several TAOK3-related kinases, which have been previously implicated to increase the risk of NAFLD susceptibility, was significantly elevated in Taok3-/- vs. wild-type mice. CONCLUSIONS: In contrast to the in vitro observations, genetic deficiency of TAOK3 in mice failed to mitigate the detrimental metabolic consequences of chronic exposure to dietary lipids, which may be partly attributable to the activation of liver-specific compensation response for the genetic loss of TAOK3 by related STE20-type kinases.

Inhibition of MAP4K4 signaling initiates metabolic reprogramming to protect hepatocytes from lipotoxic damage.

The primary hepatic consequence of obesity is non-alcoholic fatty liver disease (NAFLD), affecting about 25% of the global adult population. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD characterized by liver lipid accumulation, inflammation, and hepatocyte ballooning, with a different degree of hepatic fibrosis. In the light of rapidly increasing prevalence of NAFLD and NASH, there is an urgent need for improved understanding of the molecular pathogenesis of these diseases. The aim of this study was to decipher the possible role of STE20-type kinase MAP4K4 in the regulation of hepatocellular lipotoxicity and susceptibility to NAFLD. We found that MAP4K4 mRNA expression in human liver biopsies was positively correlated with key hallmarks of NAFLD (i.e., liver steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis). We also found that the silencing of MAP4K4 suppressed lipid deposition in human hepatocytes by stimulating ß-oxidation and triacylglycerol secretion, while attenuating fatty acid influx and lipid synthesis. Furthermore, downregulation of MAP4K4 markedly reduced the glycolysis rate and lowered incidences of oxidative/endoplasmic reticulum stress. In parallel, we observed suppressed JNK and ERK and increased AKT phosphorylation in MAP4K4-deficient hepatocytes. Together, these results provide the first experimental evidence supporting the potential involvement of STE20-type kinase MAP4K4 as a component of the hepatocellular lipotoxic milieu promoting NAFLD susceptibility.

Modern therapeutic approaches to liver-related disorders.

The liver is a key production and processing site that is essential for health. Liver dysfunction can result in both systemic and liver-specific diseases. To combat these diseases, genetic approaches have been developed that have high liver tropism and are based on gene addition/editing or gene silencing. The gene addition/editing approach has yielded encouraging clinical data on the use of viral vectors in patients with haemophilia, as well as neuromuscular diseases, and has led to trials for liver-related disorders. However, the immune response and the long-term stability of exogenous expression remain important challenges. Gene editing and mRNA therapy have yielded first in-human proof-of-concept therapeutics and vaccines, but the road to the treatment of liver-related disorders remains long. Gene silencing is accomplished primarily via antisense oligonucleotides and small-interfering RNAs (siRNAs). siRNA modification with N-acetyl galactosamine results in hepatocellular-specific targeting and catapulted the liver to the centre of siRNA research. Several siRNA drugs for liver-related disorders have recently been approved, and the pipeline of drugs under investigation is crowded. Loss-of-function mutations might also be treated with enzyme substitution therapy. This review summarises current genetic approaches as well as key enzyme substitution therapies, focusing on recently approved compounds, potential adverse effects, and future challenges. Collectively, these recent advances place the liver at the forefront of precision medicine for metabolic and genetic diseases and are expected to transform the care and treatment of patients with both liver-specific and systemic diseases.

Circulating tumor DNA is a prognostic biomarker in metastatic melanoma patients treated with chemoimmunotherapy and BRAF inhibitor.

BACKGROUND: Detectable circulating tumor DNA (ctDNA) has been associated with worse prognosis in melanoma patients. MATERIAL AND METHODS: We studied plasma ctDNA as a prognostic biomarker in 19 patients with metastatic melanoma and a detectable tumor mutation (13 BRAF, 5 NRAS, and 1 KRAS). Patients had received chemotherapy, interferon-alpha, and vemurafenib in a prospective clinical trial. Mutant allele frequency (MAF %) was determined with droplet digital PCR from pretreatment and sequential plasma samples. RESULTS: Higher pretreatment plasma ctDNA levels (MAF ≥3%) and detectable plasma ctDNA levels (MAF >0%) at the time of radiologically confirmed best objective response were associated with poor prognosis even when accounting for other relevant prognostic factors including performance status, tumor mutation, metastasis stage, and lactate dehydrogenase levels in multivariable analysis. CONCLUSION: Higher pretreatment plasma ctDNA levels and sustained detectable plasma ctDNA levels during treatment indicated poor prognosis in metastatic melanoma patients.

Do as I say, Not(ch) as I do: Lateral control of cell fate.

Breaking symmetry in populations of uniform cells, to induce adoption of an alternative cell fate, is an essential developmental mechanism. Similarly, domain and boundary establishment are crucial steps to forming organs during development. Notch signaling is a pathway ideally suited to mediating precise patterning cues, as both receptors and ligands are membrane-bound and can thus act as a precise switch to toggle cell fates on or off. Fine-tuning of signaling by positive or negative feedback mechanisms dictate whether signaling results in lateral induction or lateral inhibition, respectively, allowing Notch to either induce entire regions of cell specification, or dictate binary fate choices. Furthermore, pathway activity is modulated by Fringe modification of receptors or ligands, co-expression of receptors with ligands, mode of ligand presentation, and cell surface area in contact. In this review, we describe how Notch signaling is fine-tuned to mediate lateral induction or lateral inhibition cues, and discuss examples from C.elegans, D. melanogaster and M. musculus. Identifying the cellular machinery dictating the choice between lateral induction and lateral inhibition highlights the versatility of the Notch signaling pathway in development.

The developmental biology of genetic Notch disorders.

Notch signaling regulates a vast array of crucial developmental processes. It is therefore not surprising that mutations in genes encoding Notch receptors or ligands lead to a variety of congenital disorders in humans. For example, loss of function of Notch results in Adams-Oliver syndrome, Alagille syndrome, spondylocostal dysostosis and congenital heart disorders, while Notch gain of function results in Hajdu-Cheney syndrome, serpentine fibula polycystic kidney syndrome, infantile myofibromatosis and lateral meningocele syndrome. Furthermore, structure-abrogating mutations in NOTCH3 result in CADASIL. Here, we discuss these human congenital disorders in the context of known roles for Notch signaling during development. Drawing on recent analyses by the exome aggregation consortium (EXAC) and on recent studies of Notch signaling in model organisms, we further highlight additional Notch receptors or ligands that are likely to be involved in human genetic diseases.

Multi-parametric single cell evaluation defines distinct drug responses in healthy hematologic cells that are retained in corresponding malignant cell types.

Innate drug sensitivity in healthy cells aids identification of lineage specific anti-cancer therapies and reveals off-target effects. To characterize the diversity in drug responses in the major hematopoietic cell types, we simultaneously assessed their sensitivity to 71 small molecules utilizing a multi-parametric flow cytometry assay and mapped their proteomic and basal signaling profiles. Unsupervised hierarchical clustering identified distinct drug responses in healthy cell subsets based on their cellular lineage. Compared to other cell types, CD19+/B and CD56+/NK cells were more sensitive to dexamethasone, venetoclax and midostaurin, while monocytes were more sensitive to trametinib. Venetoclax exhibited dose-dependent cell selectivity that inversely correlated to STAT3 phosphorylation. Lineage specific effect of midostaurin was similarly detected in CD19+/B cells from healthy, acute myeloid leukemia and chronic lymphocytic leukemia samples. Comparison of drug responses in healthy and neoplastic cells showed that healthy cell responses are predictive of the corresponding malignant cell response. Taken together, understanding drug sensitivity in the healthy cell-of-origin provides opportunities to obtain a new level of therapy precision and avoid off-target toxicity.

Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.

BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1. CONCLUSIONS: In mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development.

Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells.

Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1(-/-) mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1(-/-);Wnt5a(-/-) mice revealed a greater loss of Nurr1(+) cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson's disease.

Somatic STAT3 mutations in large granular lymphocytic leukemia.

BACKGROUND: T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias. METHODS: We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes. RESULTS: Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations. CONCLUSIONS: The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).

Discovery of somatic STAT5b mutations in large granular lymphocytic leukemia.

Large granular lymphocytic (LGL) leukemia is characterized by clonal expansion of cytotoxic T cells or natural killer cells. Recently, somatic mutations in the signal transducer and activator of transcription 3 (STAT3) gene were discovered in 28% to 40% of LGL leukemia patients. By exome and transcriptome sequencing of 2 STAT3 mutation-negative LGL leukemia patients, we identified a recurrent, somatic missense mutation (Y665F) in the Src-like homology 2 domain of the STAT5b gene. Targeted amplicon sequencing of 211 LGL leukemia patients revealed 2 additional patients with STAT5b mutations (N642H), resulting in a total frequency of 2% (4 of 211) of STAT5b mutations across all patients. The Y665F and N642H mutant constructs increased the transcriptional activity of STAT5 and tyrosine (Y694) phosphorylation, which was also observed in patient samples. The clinical course of the disease in patients with the N642H mutation was aggressive and fatal, clearly different from typical LGL leukemia with a relatively favorable outcome. This is the first time somatic STAT5 mutations are discovered in human cancer and further emphasizes the role of STAT family genes in the pathogenesis of LGL leukemia.