STAT5B Mutations Research Articles

Drug Sensitivity Profiling Identifies Drugs for Targeting Constitutively Active Mutant STAT3 and Mutant STAT5B Positive Malignancies

Abstract Introduction: Constitutive hyperactivation of the STAT3 and STAT5B transcription factors is often observed in cancer. Lately, activating STAT3 mutations have been identified in hematological malignancies including large granular lymphocytic (LGL) leukemia (prevalence 40%), aplastic anemia (7%) and CD30+ T-cell lymphoma (17%). Furthermore, recent studies highlight the importance of STAT5B mutations in the pathogenesis and prognosis of T-cell malignancies such as T-cell prolymphocytic leukemia (36%), T-cell acute lymphoblastic leukemia (8%) and hepatosplenic T-cell lymphoma (33%). While STAT3 and STAT5B mutations lead to constitutive STAT3/STAT5B signaling, several other known gene mutations and mechanisms may also cause JAK/STAT-pathway activation. These findings indicate that inhibiting the JAK/STAT pathway with targeted drugs could be used as a treatment option. Here, we aimed to identify drugs that inhibit STAT3 or STAT5B function and determine if mutant STAT3/5B and wild type STAT3/5B cells respond differently to the tested drugs. In addition, we wished to ascertain if STAT3 inhibition is sufficient to induce apoptosis in patient derived LGL cells with constitutively active STAT3. Methods: High-throughput drug sensitivity testing was performed with a compound collection containing over 300 approved and investigational oncology drugs including many kinase inhibitors (such as those targeting JAK, SRC, VEGFR, mTOR, MEK, and CHK) and small molecule STAT3 inhibitors (Stattic, LLL12, Sta-21). All drugs were tested in 5-8 different concentrations over a 10,000-fold concentration range. Mutant STAT3 (Y640F) and mutant STAT5B (N642H) transformed Ba/F3 cells as well as HEK293 cells containing a STAT5 (pGL4.52[luc2P/STAT5 RE/Hygro]) or STAT3 specific luciferase reporter gene element (HEK-SIE) were used in the screens. In addition, drug sensitivities of five LGL leukemia patient samples were also assessed. Primary patient cells and the Ba/F3 cells were incubated in 384-well plates for three days with the drugs after which cell viability was measured with CellTiter-Glo. STAT3/5B induced luciferase activity in the HEK cells was analyzed after the cells were incubated for 6 or 24 hours with the drugs using the ONE-Glo luciferase assay system. Results: A significant decrease in luciferase activity was detected in STAT3 mutant Y640F, STAT5B mutant N642H and wild type STAT3 transfected HEK-SIE cells in the presence of PI3K/mTOR inhibitors such as PF-04691502 and INK128. In addition, PI3K/mTOR inhibitors significantly decreased the viability of mutant STAT3 and STAT5B transformed Ba/F3 cells compared to wild type cells. Interestingly, JAK inhibitors (e.g. ruxolitinib, gandotinib) did not inhibit mutant STAT3 activity in the HEK-SIE cells, whereas IL6-induced wild type STAT3 activity was completely blocked. A BET family inhibitor (JQ1+) and glucocorticoids (e.g. dexamethasone, methylprednisolone) showed specific and strong cytotoxicity to mutant STAT3 and STAT5B transformed Ba/F3 cells. Although JQ1+ inhibited luciferase activity of STAT5B N642H cells, no effect on the luciferase activity of STAT3 Y640F transfected HEK cells was detected, suggesting that JQ1+ may have a direct effect on mutant STAT5B function while the effect on mutant STAT3 transformed cells may be indirect. Cells from LGL leukemia patients showed high sensitivity against glucocorticoids, the histone deacetylase inhibitor quisinostat, JQ1+ and PF-04691502 when compared to healthy CD8+ T-cells. However, no increase in apoptosis was observed with JAK or other mTOR inhibitors. Conclusions: Our results suggest that JAK inhibitors lack efficacy in STAT3 mutated diseases. However, our ex vivo and in vitro drug screens highlight some other promising agents including PF-04691502 (PI3K/mTOR inhibitor) and JQ1+ (BET family inhibitor) that inhibited mutant STAT5B and STAT3 activity in the cell line models and were effective against primary LGL patient cells. Additional experiments are ongoing to determine how these drugs function to block STAT signaling and induce cell death. As the STAT3 and STAT5 pathways are activated in many other cancer types as well, the results may be applicable to a variety of different malignancies. Figure 1 Figure 1. Table 1. Drug sensitivity scores of the different cell line models and patient samples. DSS value range 0-50. (0 = no drug response with any conc., 50 = maximal drug response with every conc.) Disclosures Mustjoki: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Discovery of Novel Drug Sensitivities in T-Prolymphocytic Leukemia (T-PLL) By High-Throughput Ex Vivo Drug Testing and Genetic Profiling

IntroductionT-PLL is a mature post-thymic T-cell neoplasm with an aggressive clinical course (5-year overall survival 21%). Almost 75% of T-PLL cases harbor chromosome 14 translocations resulting in aberrant activation of the proto-oncogene TCL1A. Furthermore, in the majority of T-PLL cases the ATM gene is mutated or deleted, and recently it was reported that mutations in genes involved in the JAK-STAT pathway were found in 76% of T-PLL cases. Due to the rareness and aggressive nature of the disease, clinical trials are difficult to execute. By using a high-throughput ex vivo drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology drugs we systematically investigated the heterogeneity of drug responses in PLL-patients. As the impact of mutations on drug sensitivity is not well understood we aimed to identify relevant associations between the drug responses and genetic lesions in T-PLL patients.MethodsPrimary cells (MNCs) from seven T-PLL patients were obtained for drug screening. Samples were seeded in 384-well plates and 306 active substances were tested using a 10,000-fold concentration range resulting in a dose-response curve for each compound. Cell viability was measured after 72 h incubation and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were calculated by comparing patient samples to healthy donors. Hierarchical clustering of the drug responses was performed with Cluster 3.0 and Java Tree View. To assess the performance of the drug screening platform we also exchanged six samples with the German Cancer Research Center in Heidelberg for a comparison of results between two independent drug screening systems.To understand heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by exome sequencing of tumor and matched healthy cells to profile known recurrent genetic variants (ATM, STAT5b, IL2RG, JAK1, JAK3) as well as CNVs (TCL1A translocations, ATM deletions, recurrent chromosomal aberrations).ResultsFour out of seven patient samples showed high sensitivity to small molecule BCL2 inhibitors navitoclax (IC50: 10-68nM) and ABT-199 (IC50: 14-45nM) and to HDAC inhibitors panobinostat and belinostat (IC50: 2-65nM). Intriguingly, the CDK inhibitor SNS-032 was effective in 6/7 patient samples (IC50: 7-95nM). SNS-032 inhibits Cdk2, Cdk7 and Cdk9, which control transcription of anti-apoptotic proteins including MCL1 and XIAP. As the AKT1/MTOR pathway is activated in many T-PLL patients due to expression of the TCL1A oncoprotein, it was interesting to observe that patient samples did not show any response to AKT inhibitors (MK-2206 and GDC-0068 IC50 values >1000 nM) nor to MTOR inhibitors (rapalogs temsirolimus and everolimus). Similarly, T-PLL cells were insensitive to JAK-inhibitors.Clustering of drug responses from T-PLL patients with primary AML and ALL patient samples revealed the drug response profiles to be specific for T-PLL patients (Figure). 6/7 patients clustered together while the only patient (PLL4) in our cohort with confirmed mutations in the JAK-STAT pathway genes STAT5b (P702S) and IL2RG (K315E) exhibited a non-sensitive response pattern when compared to other samples (Figure). Interestingly, exome sequencing did not reveal any JAK mutations in our PLL-cohort (n=5) nor additional STAT5b or IL2RG mutations in other patients except in this unresponsive patient.In the comparison between the platforms the correlation of the censored IC50 values from the 60 overlapping drugs was r=0.75. Similar fits of dose-response curves were seen for most drugs, although there were notable exceptions, which may be due to divergent culture conditions and day of read-out.ConclusionsEx vivo drug testing of primary patient cells has the potential to provide novel personalized drug candidates (such as BCL2, HDAC and CDK inhibitors) for T-PLL. The drug response pattern was T-PLL specific warranting further clinical testing. Drug screening, mutation analysis and RNA sequencing of additional patients is currently ongoing (n=20) to validate whether drug responses can be predicted based on the mutation profile or aberrant gene expression. [Display omitted] DisclosuresKallioniemi:Medisapiens: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

The analysis of clonal diversity and therapy responses using STAT3 mutations as a molecular marker in large granular lymphocytic leukemia.

T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of natural killer cells are intriguing entities between benign and malignant lymphoproliferation. The molecular pathogenesis has partly been uncovered by the recent discovery of somatic activating STAT3 and STAT5b mutations. Here we show that 43% (75/174) of patients with T-cell large granular lymphocytic leukemia and 18% (7/39) with chronic lymphoproliferative disorder of natural killer cells harbor STAT3 mutations when analyzed by quantitative deep amplicon sequencing. Surprisingly, 17% of the STAT3-mutated patients carried multiple STAT3 mutations, which were located in different lymphocyte clones. The size of the mutated clone correlated well with the degree of clonal expansion of the T-cell repertoire analyzed by T-cell receptor beta chain deep sequencing. The analysis of sequential samples suggested that current immunosuppressive therapy is not able to reduce the level of the mutated clone in most cases, thus warranting the search for novel targeted therapies. Our findings imply that the clonal landscape of large granular lymphocytic leukemia is more complex than considered before, and a substantial number of patients have multiple lymphocyte subclones harboring different STAT3 mutations, thus mimicking the situation in acute leukemia.

Integrated genomic sequencing reveals mutational landscape of T-cell prolymphocytic leukemia

AbstractThe comprehensive genetic alterations underlying the pathogenesis of T-cell prolymphocytic leukemia (T-PLL) are unknown. To address this, we performed whole-genome sequencing (WGS), whole-exome sequencing (WES), high-resolution copy-number analysis, and Sanger resequencing of a large cohort of T-PLL. WGS and WES identified novel mutations in recurrently altered genes not previously implicated in T-PLL including EZH2, FBXW10, and CHEK2. Strikingly, WGS and/or WES showed largely mutually exclusive mutations affecting IL2RG, JAK1, JAK3, or STAT5B in 38 of 50 T-PLL genomes (76.0%). Notably, gain-of-function IL2RG mutations are novel and have not been reported in any form of cancer. Further, high-frequency mutations in STAT5B have not been previously reported in T-PLL. Functionally, IL2RG-JAK1-JAK3-STAT5B mutations led to signal transducer and activator of transcription 5 (STAT5) hyperactivation, transformed Ba/F3 cells resulting in cytokine-independent growth, and/or enhanced colony formation in Jurkat T cells. Importantly, primary T-PLL cells exhibited constitutive activation of STAT5, and targeted pharmacologic inhibition of STAT5 with pimozide induced apoptosis in primary T-PLL cells. These results for the first time provide a portrait of the mutational landscape of T-PLL and implicate deregulation of DNA repair and epigenetic modulators as well as high-frequency mutational activation of the IL2RG-JAK1-JAK3-STAT5B axis in the pathogenesis of T-PLL. These findings offer opportunities for novel targeted therapies in this aggressive leukemia.

Novel activating STAT5B mutations as putative drivers of T-cell acute lymphoblastic leukemia

Novel activating STAT5B mutations as putative drivers of T-cell acute lymphoblastic leukemia

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) and IPEX-related disorders

To summarize recent progress in our understanding of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) and IPEX-related disorders. A number of Mendelian disorders of immune dysregulation and autoimmunity have been noted to result from defects in T regulatory cell, development and function. The best characterized of these is IPEX, resulting from mutations affecting FOXP3. A number of other gene defects that affect T regulatory cell function also give rise to IPEX-related phenotypes, including loss-of-function mutations in CD25, STAT5b and ITCH. Recent progress includes the identification of gain-of-function mutations in STAT1 as a cause of an IPEX-like disease, emerging FOXP3 genotype/phenotype relationships in IPEX, and the elucidation of a role for the microbiota in the immune dysregulation associated with regulatory T cell deficiency. An expanding spectrum of genetic defects that compromise T regulatory cell function underlies human disorders of immune dysregulation and autoimmunity. Collectively, these disorders offer novel insights into pathways of peripheral tolerance and their disruption in autoimmunity.

Novel Activating STAT5B Mutations As Drivers Of T-ALL

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is caused by the cooperation of multiple oncogenic lesions. Recent evidence supports that IL-7 and its receptor IL-7R contribute to T-ALL development (Zenatti et al, 2011). The two main pathways induced by IL-7R are JAK/STAT5 and PI3K/Akt/mTOR. Activating mutations to IL7R, JAK1, JAK2 or JAK3 are estimated to occur in 20-30% of all T-ALL patients (Cools 2013). STAT5 plays an important role in many hematologic malignancies but constitutive STAT5 activation often is a secondary event. Mutations in STAT5B (N642H) were recently described in LGL-leukemia in patients with an unusually aggressive and fatal form of the disease (Rajala et al, 2013). In other cancers, including ALL, patients with mutations in STAT5B have not been described. Here we report novel activating STAT5B mutations as drivers of T-ALL. MethodsWe performed exome sequencing of bone marrow (BM) samples from an 18-year-old female with relapsed T-ALL. Targeted next-generation amplicon sequencing and Sanger sequencing was used to analyze the region encoding the STAT5B SRC homology 2 (SH2) domain including the N642, T648 and I704 codons in a cohort of 17 adult and pediatric T-ALL patients treated at HUCH 2008-2013. For functional studies STAT5B expression vectors with the N642H, T648S or I704L mutation and an expression vector with both N642H and T648S mutations were used to transiently transfect HEK293 cells. To investigate the effect on transcriptional activity we co-transfected the mutant constructs with a STAT5 luciferase reporter plasmid and used Western blot analysis to study the phosphorylation status of the generated constructs. For drug sensitivity of STAT5B mutated cells we performed ex vivo drug testing on primary blasts from the index patient using a comprehensive set of 202 oncology drugs (approved and in clinical development). Each drug was tested over a 10,000-fold concentration range. ResultsSequencing of the index patient revealed 3 different somatic missense mutations in STAT5B (T648S, N642H, I704L) and mutations in KRAS, WT1 and SUZ12. No mutations affecting the JAK genes or IL7R were detected. All STAT5B mutations were located in the SH2 domain, which mediates dimerization and activation by trans-phosphotyrosine binding. The same three STAT5B mutations were also found in the diagnostic sample and most likely represent founding events in leukemogenesis. The N642H and T648S mutations occurred on the same allele with tumor mutation frequencies of approximately 40% while the I704L mutation occurred on a different allele with a similar tumor mutation frequency. To investigate the prevalence of STAT5B mutations in T-ALL we sequenced 17 BM samples from T-ALL patients. In this cohort we could not detect any other patients carrying mutations in the STAT5B SH2 domain.Western blot analysis made with mutant constructs showed that the N642H and I704L mutations induced constitutive phosphorylation of STAT5B. Compared to wild type STAT5B the N642H and I704L mutants induced 47- and 6-fold increases in transcriptional activity, respectively, while T648S mutation had no effect in the assays. The construct with both the N642H and T648S mutations showed the highest amount of constitutive phosphorylation and induced a 56-fold increase in transcriptional activity compared to wild type STAT5B. Using ex vivo drug testing the STAT5B mutated blasts were resistant (EC50≥1 uM) to inhibitors of PI3K (e.g. idelalisib, XL147), dual inhibitors of PI3K/mTOR (PF-04691502, dactolisib) and mTOR inhibitors (temsirolimus, everolimus). Furthermore the blasts showed no response to AKT1 inhibitors (MK-2220) or JAK inhibitors (ruxolitinb, tofacitinib). In contrast, the cells were most sensitive to the BCL-2/BCL-XL inhibitor navitoclax (EC50 83 nM). SummarySTAT5B mutations are uncommon in T-ALL but their occurrence underlines the significance of the IL7R-JAK-STAT5 pathway in the pathogenesis of T-ALL. While STAT5B mutant blasts were not sensitive to inhibitors targeting JAK kinases, the cells were unusually sensitive to inhibitors of target molecules of STAT5B, including anti-apoptotic BCL-2 proteins. These results suggest that BCL-2/BCL-XL inhibitors such as navitoclax are novel candidate therapies for T-ALL patients. Disclosures:Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.

Frequent Activating Mutations Of JAK-STAT Pathway Genes In Natural Killer Cell Lymphomas

BackgroundNatural killer cell lymphomas (NKCLs) are rare diseases with poor prognosis. There are few studies that reported oncogenic mutations in this disease. Identifying mutations critical to the neoplastic transformation of NK cells is crucial for the development of targeted therapies. MethodsWe performed RNA sequencing (RNA-Seq) on NKCL cases (n=17), malignant NK cell lines (n=3) and resting or activated normal NK cells (n=3) to analyze the genome-wide mutation profile in NKCLs. All SNVs detected by RNA-Seq were validated by Sanger sequencing using the corresponding genomic DNA (gDNA). We expanded our analysis to include specific sequencing of the SH2 domains of STAT3 and STAT5B using 37 additional NKCL cases and 6 NK cell lines. Retroviral or lentiviral transduction was performed to express SOCS6 or STAT3 shRNA in NK cell lines, respectively. After transduction, cells were tracked by quantifying the % of GFP+ cells. Apoptosis was assessed by quantifying AnnexinV-PE stained cells. Western blot was performed on 6 NK cell lines using p-STAT3 (Tyr705) antibody. q-MSP and q-RT-PCR were used to detect SOCS6 promoter methylation and mRNA expression in NK cell lines, respectively. ResultsRNA-Seq showed frequent oncogenic mutations in JAK/STAT pathway members STAT3 (18%), STAT5B (6%), BRAF (6%) and MAP2K1 (6%). Targeted Sanger sequencing of 37 additional NKCL cases showed one patient with activating STAT3 and two patients with activating STAT5B mutations leading to 7.4% (4 of 54) and 6% (3 of 50) total mutation frequency, respectively. STAT3 and STAT5B mutations were located in the SH2 domain and three of four STAT3 mutations and all STAT5B mutations were previously observed in NK- or T-LGL leukemia cases. Oncogenic activities of two other JAK-STAT pathway genes, BRAF (G469A), MAP2K1 (K57N) have been reported in solid tumors and leukemias. The JAK/STAT pathway mutations were present in a total of 53% of the NKCL cases with RNA-Seq data available (n=17). Intriguingly, targeted sequencing revealed oncogenic STAT3 mutations in 50% of malignant NK cell lines (n=6), which were associated with p-STAT3 expression detected by western blot.STAT3 knock-down resulted in reduced growth in a NK cell line with STAT3 mutation. In agreement with Kimura et. al. Leuk Lymphoma 2013, we were not able to detect JAK3 mutations as reported previously. In an accompanying DNA methylation analysis, we observed epigenetic silencing of SOCS6, a negative regulator of JAK-STAT3 signaling, in NKCL samples. Reintroduction of SOCS6 showed negative selection pressure associated with increased apoptosis in limiting concentrations of IL2 in two SOCS6-null NK cell lines with activating STAT3 mutations suggesting a possible cooperation of oncogenic JAK-STAT pathway mutations and the epigenetic silencing of a negative regulator of this pathway. ConclusionsWe have identified a high incidence of activating mutations of STAT3, STAT5B and other oncogenic JAK-STAT pathway genes in NKCLs. SOCS6 was frequently methylated in NKCLs with corresponding low gene expression. There was evidence suggestive of cooperation of genetic and epigenetic mechanisms in the activation of the JAK-STAT pathway in NKCLs. This study suggests that JAK-STAT pathway inhibition may be a therapeutic option in NKCLs. Disclosures:No relevant conflicts of interest to declare.

Discovery of somatic STAT5b mutations in large granular lymphocytic leukemia

AbstractLarge 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.

STAT5b in LGL leukemia - a novel therapeutic target?

<i>STAT5b</i> in LGL leukemia - a novel therapeutic target?

Immunodeficiency with Autoimmunity: Beyond the Paradox

The association of immunodeficiency and autoimmunity may represent a paradox, yet it has been described in an increasing number of conditions. Use of unbiased genomic approach to identify novel forms of primary immunodeficiencies (PIDs), along with in-depth functional studies in biological samples from affected individuals continue to unravel novel mechanisms underlying immune dysregulation in patients with altered ability of fighting pathogens. In particular, it has been clearly established that genetic defects that affect T and B cell development compromise not just the ability to generate a diversified repertoire of lymphocytes capable of recognizing multiple pathogens, but also impinge on mechanisms of central and peripheral tolerance, hence favoring autoimmune and inflammatory manifestations. Yet, the diagnosis of autoimmune symptoms in the context of PIDs is troublesome, the prognosis unclear, and the treatment challenging. In the present collection of manuscripts, several experts in the field provide an overview of the spectrum of different forms of monogenic defects of the immune system manifesting also with autoimmunity, and discuss established and novel mechanisms involved in immune dysregulation. Studies on patients with Immunedysregulation-Polyendocrinopathy Enteropathy-X-linked (IPEX) Syndrome, have paved the way to understand the phenotype arising from impaired peripheral tolerance due to dysfunctional regulatory T cells (Treg) expressing mutated FOXP3. However, this important T cell subpopulation can also be affected in other forms of PID, such as Wiskott–Aldrich syndrome (WAS) and adenosine deaminase (ADA) deficiency. In these disorders, the underlying genetic defect affects multiple cell types, resulting in impaired immune defense, but also poor Treg function. Similarly, STAT5B mutations disrupt an essential intracellular transcriptional activator for Treg cells, causing reduction of Treg number in affected individuals. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is an autosomal recessive condition due to mutation of the Autoimmune regulator (AIRE) gene. Patients with APECED present with predominant organ specific autoimmunity and autoantibodies with multiple specificities. AIRE has been shown to play a critical role in allowing expression of self-antigens in the thymus, thereby permitting deletion of self-reactive T lymphocytes or their diversion to Treg cells. Thus APECED stands as the prototypic monogenic disorder of central T cell tolerance. While it is still questionable whether deficiency of AIRE also affects peripheral tolerance, recent data indicate that the autoimmune-associated tissue damage may not be primarily due to autoantibodies, but rather to autoreactive CD8+ T cells. Moreover, recent studies in patients affected with Common Variable Immunodeficiency, a condition in which proper specific antibody production is deficient in favor of pathogenic autoantibody secretion, have highlighted the importance of mechanisms that control B cell development and receptor editing in maintaining immune homeostasis. Finally, two manuscripts call the attention to the dual role of certain cell types and their ability to acquire different immunological functions depending on the environment in which they differentiate, as described for Th17 cells and dendritic cells, at the end of the Topic. Possibly, the future of medicine should aim to implement physiological plasticity and to empower epigenetics modifications in order to recover from inborn errors of Nature.

Discovery of STAT5b Mutations and Small Subclones of STAT3 Mutations in Large Granular Lymphocytic (LGL) Leukemia

AbstractAbstract ▪871▪This icon denotes a clinically relevant abstract Background:Large granular lymphocytic (LGL) leukemia is a rare lymphoproliferative disease, characterized by the clonal expansion of cytotoxic CD3+CD8+ T-cells or CD3-CD16/56+ natural killer (NK)-cells. It is often associated with autoimmune phenomena (e.g. cytopenias, rheumatoid arthritis). We recently identified somatic mutations in the STAT3 gene in 40% of monoclonal T-LGL cases (Koskela et al, NEJM, 2012). Here, we report the discovery and functional analysis of novel STAT5b mutations as well as small subclones of STAT3 mutations in other LGL patients, expanding the evidence implicating STAT activation in LGL. METHODS:In order to find novel LGL-leukemia associated mutations, exome sequencing was done from untreated STAT3 mutation negative T-LGL leukemia patients using CD8+ LGL-leukemic cells and matched CD4+ control cells. Samples from 158 T-LGL and 40 NK-LGL leukemia patients were further analyzed using both targeted Sanger sequencing and ultra-deep targeted next-gen amplicon sequencing with up to 10,000x coverage (MiSeq, Illumina). Functional analysis of mutated proteins was carried out in Hela cells by Western analysis and luciferase reporter assays. RESULTS:Exome sequencing revealed a novel somatic missense mutation Y665F in the STAT5b gene in two T-LGL patients diagnosed with WHO2009 criteria with a large CD8+ T-LGL clone (>90%). Only wild-type STAT5b was seen in the matched CD4+ control cells of these patients. Amplicon sequencing of exon 16 of STAT5b (corresponding to the Y665F site) in 158 T-LGL and 40 NK-LGL patients revealed an additional mutation (N642H) in one T-LGL and one NK-LGL patient, resulting in the 2% total frequency (4/198) of STAT5b mutations across all patients. The N642H and Y665F mutations were both located in the Src homology 2 (SH2) domain of STAT5b, which mediates dimerization and activation by trans-phosphotyrosine binding. STAT3 mutations previously reported in T-LGL patients were located in the corresponding domain.The transcriptional activity of wild-type and mutant STAT5b proteins (N642H and Y665F) was assayed in cells carrying a luciferase reporter with STAT5 binding elements. Luciferase activity of Hela cells transfected with the mutated STAT5b constructs was significantly increased compared to wild-type STAT5b. Furthermore, both the N642H and Y665F variants of STAT5b exhibited higher levels of tyrosine (Y694) phosphorylation than the wild type protein.The exon 21 in the SH2 domain of the STAT3 gene was also screened by ultra-deep next-gen amplicon sequencing, both from the original T-LGL patient cohort (n=77, patients with monoclonal disease) and 142 additional monoclonal/oligoclonal LGL patients. In the monoclonal cohort, a total of nine new STAT3 mutation positive patients were detected by amplicon sequencing, raising the total number of positive cases to 41 (53%) from the 32 identified by Sanger sequencing. Concomitant to the previously described Y640F, N647I, K658N, D661H, D661V and D661Y STAT3 mutations, several novel mutations in this gene were found: I659L, Q643H, G656C, K658H, K658R and D661I. In the oligoclonal LGL cohort, the mutation frequency was lower (31/142, 22%), suggesting that it may also include patients with reactive polyclonal LGL proliferation. CONCLUSIONS:Our mutational and functional data affirm that STAT family transcription factors play a critical role in the pathogenesis of LGL leukemia. In addition to the previously identified mutations in the STAT3 gene, we found recurrent somatic STAT5b mutations in LGL leukemia. Furthermore, our ultra-deep (10,000x) next-gen sequencing revealed small subclones of STAT3 mutations in patients with oligoclonal LGL. Both STAT3 and STAT5b mutations increased the phosphorylation and transcriptional activity of corresponding proteins. The detection of STAT mutations should be included in the diagnostic assessment of LGL leukemia. Disclosures:Koskela:Novartis: Honoraria; BMS: Honoraria; Janssen-Cilag: Honoraria. Kallioniemi:TEKES-FiDiPro: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

Severe Growth Deficiency is Associated with STAT5b Mutations that Disrupt Protein Folding and Activity

The first genetic defect in human signal transducer and activator of transcription (STAT)5b was identified in an individual with profound short stature and GH insensitivity, immune dysfunction, and severe pulmonary disease, and was caused by an alanine to proline substitution (A630P) within the Src homology-2 (SH2) domain. STAT5b(A630P) was found to be an inactive transcription factor based on its aberrant folding, diminished solubility, and propensity for aggregation triggered by its misfolded SH2 domain. Here we have characterized the second human STAT5b amino acid substitution mutation in an individual with similar pathophysiological features. This single nucleotide transition, predicted to change phenyalanine 646 to serine (F646S), also maps to the SH2 domain. Like STAT5b(A630P), STAT5b(F646S) is prone to aggregation, as evidenced by its detection in the insoluble fraction of cell extracts, the presence of dimers and higher-order oligomers in the soluble fraction, and formation of insoluble cytoplasmic inclusion bodies in cells. Unlike STAT5b(A630P), which showed minimal GH-induced tyrosine phosphorylation and no transcriptional activity, STAT5b(F646S) became tyrosine phosphorylated after GH treatment and could function as a GH-activated transcription factor, although to a substantially lesser extent than STAT5b(WT). Biochemical characterization demonstrated that the isolated SH2 domain containing the F646S substitution closely resembled the wild-type SH2 domain in secondary structure, but exhibited reduced thermodynamic stability and altered tertiary structure that were intermediate between STAT5b(A630P) and STAT5b(WT). Homology-based structural modeling suggests that the F646S mutation disrupts key hydrophobic interactions and may also distort the phosphopeptide-binding face of the SH2 domain, explaining both the reduced thermodynamic stability and impaired biological activity.

Genetic Analysis of Short Children with Apparent Growth Hormone Insensitivity

Background/Aims: In short children, a low IGF-I and normal GH secretion may be associated with various monogenic causes, but their prevalence is unknown. We aimed at testing GH1, GHR, STAT5B, IGF1, and IGFALS in children with GH insensitivity. Subjects and Methods: Patients were divided into three groups: group 1 (height SDS <–2.5, IGF-I <–2 SDS, n = 9), group 2 (height SDS –2.5 to –1.9, IGF-I <–2 SDS, n = 6) and group 3 (height SDS <–1.9, IGF-I –2 to 0 SDS, n = 21). An IGF-I generation test was performed in 11 patients. Genomic DNA was used for direct sequencing, multiplex ligation-dependent probe amplification and whole-genome SNP array analysis. Results: Three patients in group 1 had two novel heterozygous STAT5B mutations, in two combined with novel IGFALS variants. In groups 2 and 3 the association between genetic variants and short stature was uncertain. The IGF-I generation test was not predictive for the growth response to GH treatment. Conclusion: In severely short children with IGF-I deficiency, genetic assessment is advised. Heterozygous STAT5B mutations, with or without heterozygous IGFALS defects, may be associated with GH insensitivity. In children with less severe short stature or IGF-I deficiency, functional variants are rare.

Evidence for a Continuum of Genetic, Phenotypic, and Biochemical Abnormalities in Children with Growth Hormone Insensitivity

GH insensitivity (GHI) presents in childhood as growth failure and in its severe form is associated with dysmorphic and metabolic abnormalities. GHI may be caused by genetic defects in the GH-IGF-I axis or by acquired states such as chronic illness. This article discusses the former category. The field of GHI due to mutations affecting GH action has evolved considerably since the original description of the extreme phenotype related to homozygous GH receptor (GHR) mutations over 40 yr ago. A continuum of genetic, phenotypic, and biochemical abnormalities can be defined associated with clinically relevant defects in linear growth. The role and mechanisms of the GH-IGF-I axis in normal human growth is discussed, followed by descriptions of mutations in GHR, STAT5B, PTPN11, IGF1, IGFALS, IGF1R, and GH1 defects causing bioinactive GH or anti-GH antibodies. These defects are associated with a range of genetic, clinical, and hormonal characteristics. Genetic abnormalities causing growth failure that is less severe than the extreme phenotype are emphasized, together with an analysis of height and serum IGF-I across the spectrum of different types of GHR defects. An overall view of genotype and phenotype relationships is presented, together with an updated approach to the assessment of the patient with GHI, focusing on investigation of the GH-IGF-I axis and relevant molecular studies contributing to this diagnosis.

STAT5b Deficiency: An Unsuspected Cause of Growth Failure, Immunodeficiency, and Severe Pulmonary Disease

Over the past 20 years, more than 150 new genetic disorders related to primary immunodeficiencies have been described, most of which initially present in the pediatric population. 1 Pediatric researchers and medical personnel should be cognizant of signs and symptoms associated with diseases related to immunodeficiency. STAT5b deficiency is a recently identified disease entity that involves both severe growth hormone‐resistant growth failure and severe immunodeficiency. Due to the early growth failure and the key role of STAT5b in growth hormone (GH) action, most of the cases have been published in the endocrinology literature until now. However, the identification of rare, human STAT5Bmutations has highlighted the distinct and critical role of STAT5b in both growth and immunity. 2-5 The possibility of a STAT5b disorder in children should be considered by the general pediatrician when chronic infection and/or unexplained pulmonary disease are concomitant with growth failure. The aim of this review is to provide updated information toward recognition of possible STAT5b deficiencies and to help provide an appropriate workup to mitigate symptoms associated with the disease. In this way, careful monitoring, timely therapy, frequent health care visits, and prophylactic medicines can be initiated. The molecular etiology and clinical consequences of STAT5b deficiencies will be described. First Cases of STAT5b Deficiency The cardinal feature of GH insensitivity (GHI) is severe growth failure, associated with elevated serum concentrations of GH, resulting in a clinical phenotype that is essentially indistinguishable from that of congenital GH deficiency. The phenotype of GHI has been attributed to more than 70 mutations in the GHR gene, encoding the GH receptor (GHR). 7 GHI in the presence of normal GHR, in contrast to classic GHI, has remained largely uninvestigated. The molecular basis has been determined in only a few cases, including three reports of homozygous mutations in the IGF-I (insulin growth factor-I) gene. 3-5 Beginning in 2003, a series of GHI cases with normal GHR were identified 3-5 and found to be associated with mutations in the STAT5B gene. 3 The first STAT5B mutation was a homozygous autosomal recessive missense mutation identified in a female patient with extremely short stature (‐7.5 SD) and who had serum IGF-I levels less than 15% of normal, despite elevated serum GH concentrations.Shortly thereafter, several additional cases, each harboring novel mutations of STAT5B, were reported. All of these cases were characterized by severe growth failure and GHI. Together, these cases convincingly implicated STAT5B mutations as potential etiologies of the GHI syndrome and pointed to STAT5b playing an important role in normal postnatal human growth. The first cases of identified STAT5b deficiency, 3-5 distinct from GHI caused by mutations in the GHR or IGF1 genes, were also found to be associated with symptoms of severe infection, autoimmune diatheses, and lymphocytic interstitial pneumonitis. The combination of severe GH-resistant growth failure and chronic infection led to evaluation of potential disorders of cytokine receptor signaling. In the first described case, the patient, 3 with a missense mutation (p.A630P) in the STATB gene, had lymphocytic interstitial pneumonitis, chronic lung disease, hemorrhagic varicella, atopy, and autoimmune disease diagnosed at a young age. More detailed evaluations indicated that the patient had decreased numbers of regulatory CD4+CD25hi T cells (Treg). 8

Genetic Defects in the Growth Hormone-IGF-I Axis Causing Growth Hormone Insensitivity and Impaired Linear Growth.

Human genetic defects in the growth hormone (GH)–IGF-I axis affecting the IGF system present with growth failure as their principal clinical feature. This is usually associated with GH insensitivity (GHI) presenting in childhood as severe or mild short stature. Dysmorphic features and metabolic abnormalities may also be present. The field of GHI due to mutations affecting GH action has evolved rapidly since the first description of the extreme phenotype related to homozygous GH receptor (GHR) mutations in 1966. A continuum of genetic, phenotypic, and biochemical abnormalities can be defined associated with clinically relevant defects in linear growth. The mechanisms of the GH–IGF-I axis in the regulation of normal human growth is discussed followed by descriptions of mutations in GHR, STAT5B, IGF-I, IGFALS, IGF1R, and GH1 defects causing bio-inactive GH or anti-GH antibodies. These GH–IGF-I axis defects are associated with a range of clinical, and hormonal characteristics. An up-dated approach to the clinical assessment of the patient with GHI focusing on investigation of the GH–IGF-I axis and relevant molecular studies contributing to the identification of causative genetic defects is also discussed.

A novel STAT5B mutation causing GH insensitivity syndrome associated with hyperprolactinemia and immune dysfunction in two male siblings

GH insensitivity (GHI) syndrome caused by STAT5B mutations was recently reported, and it is characterized by extreme short stature and immune dysfunction. Treatment with recombinant human IGF1 (rhIGF1) is approved for patients with GHI, but the growth response to this therapy in patients with STAT5B mutations has not been reported. To report the clinical features, molecular findings, and the short-term growth response to rhIGF1 therapy in patients with STAT5B mutation. Hormonal and immunological evaluations were performed in two male siblings with GHI associated with atopic eczema, interstitial lung disease, and thrombocytopenic purpura. STAT5B genes were directly sequenced. The younger sibling was treated with rhIGF1 at a dose of 110 microg/kg BID. Both siblings had laboratory findings compatible with GHI associated with hyperprolactinemia. Lymphopenia and reduced number of natural killer cells without immunoglobulin abnormalities were observed. STAT5B sequence revealed a homozygous frameshift mutation (p.L142fsX161) in both siblings. The younger sibling (9.9 years of age) was treated with rhIGF1 at appropriate dosage, and he did not present any significant change in his growth velocity (from 2.3 to 3.0 cm/year after 1.5 years of therapy). The presence of a chronic illness could possibly be responsible for the poor result of rhIGF1 treatment. Further studies in patients with STAT5B defects are necessary to define the response to rhIGF1 treatment in this disorder. GHI associated with immune dysfunction, especially interstitial lung disease, and hyperprolactinemia is strongly suggestive of a mutation in STAT5B in both sexes.

Dispersed Chromosomal Stat5b-binding Elements Mediate Growth Hormone-activated Insulin-like Growth Factor-I Gene Transcription

The growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis regulates somatic growth during childhood and orchestrates tissue repair throughout the life span. Recently described inactivating mutations in Stat5b in humans with impaired growth have focused attention on this transcription factor as a key agent linking GH-stimulated signals to IGF-I gene expression, and several putative Stat5b sites have been identified in the IGF-I gene. Here, we define and characterize potential GH- and Stat5b-activated chromosomal enhancers that can regulate IGF-I gene transcription. Of 89 recognizable Stat5 sequences in 200 kb centering on the rat IGF-I gene, 22 resided within conserved regions and/or were identical among different species. Only 15 of these sites, organized into 7 distinct domains, were found to bind Stat5b by quantitative chromatin immunoprecipitation assays in liver chromatin of rats, but only after acute GH treatment. These sites could bind Stat5b in vitro, and individual domains could mediate GH- and Stat5b-stimulated IGF-I promoter activity in cultured cells. Further analyses revealed that four Stat5b domains possessed chromatin signatures of enhancers, including binding of co-activators p300 and Med1, and RNA polymerase II. These modifications preceded GH-stimulated recruitment of Stat5b, as did lysine 4 monomethylation of histone H3, which was enriched in 6/7 Stat5b-binding elements. In contrast, histone acetylation was induced by GH but was limited to Stat5b binding domains found within the IGF-I transcription unit. We conclude that GH stimulates recruitment of Stat5b to multiple dispersed regions within the igf1 locus, including several with properties consistent with long range transcriptional enhancers that collectively regulate GH-activated IGF-I gene transcription.

Growth hormone secretion and immunological function of a male patient with a homozygous STAT5b mutation

STAT5b is a component of the GH signaling pathway. Recently, we described a 31-year-old male patient (height, -5.9 SDS) with a novel homozygous inactivating mutation in the STAT5b gene. The purpose of this study is to describe the phenotype in detail, including GH secretion and immunological function. In addition, we report four family members of this patient, all heterozygous carriers of the mutation. Twenty-four hour GH and prolactin secretion characteristics were assessed by blood sampling at 10-min intervals. An IGF-I generation test was performed. Monocyte function was tested by stimulation of whole blood with lipopolysaccharide (LPS) in the presence or absence of Interferon-gamma (IFN-gamma). In addition, T cell function was determined by measuring proliferative responses of peripheral blood mononuclear cells (PBMC) after stimulation by various polyclonal activators and Interleukin-2 (IL-2). Clinical and biochemical characteristics were determined in the carriers of the mutation. GH secretory parameters were comparable with that of healthy male controls (mean fat percentage 25), but likely increased in relation to the patient's 40% body fat. The regularity of GH secretion was diminished. Prolactin secretion was increased by sixfold. The IGF-I generation test showed a small increase in IGF-I and IGF-binding protein-3 on lower GH doses and an increase in IGF-I to -2.4 SDS on the highest dose of GH. In vitro, IL-12p40, IL 10, and tumour necrosis factor-alpha (TNF-alpha) production rates by PBMC increased to values within the normal range upon stimulation of LPS. Heterozygous carriers of the mutation did not show abnormalities, although the height of the males was below the normal range. This report shows that GH and prolactin secretion were increased in this patient homozygous for a new STAT5b mutation. Although STAT5b plays a role in signaling within immune cells, clinical immunodeficiency is not an obligatory phenomenon of STAT5b deficiency per se. Heterozygous carriers of a STAT5b mutation show no signs of GH insensitivity.