Letteratura scientifica selezionata sul tema "Somatic gain of mutation STAT3"

Abstract Abstract 936 BACKGROUND: T-cell large granular lymphocyte (LGL) leukemia is an uncommon lymphoproliferative disorder characterized in most cases by expansion of mature, clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs). The pathogenesis of LGL-leukemia is unknown, and leukemic cells closely resemble normal terminally differentiated effector memory CTLs. While resistance to apoptotic pathways (Fas/Fas ligand, sphingolipid) and activation of survival signaling pathways (Ras) have been implicated in LGL leukemia, the underlying genetic defects have not yet been elucidated. We aimed to identify somatic mutations in LGL leukemia by whole exome sequencing of leukemic and matched healthy control cells. METHODS: Our index patient is a 70 year-old male with untreated CD8+ LGL leukemia diagnosed in 2009 with a clonal rearrangement in the T-cell receptor (TCR) delta and gamma gene. He has been asymptomatic with grade 2 neutropenia and an absolute lymphocyte count of 4–15 ×109/L. The patient had one large predominant T-cell clone: 94% of CD8+ cells consisted of a single Vβ16 clone, as assessed by flow cytometry. No clonal expansions were observed in the CD4+ fraction. DNA was extracted from FACS-sorted CD8+ (leukemic) and CD4+ (control) cells and sequenced by exome capture using an Agilent SureSelect All exon 50 MB capture kit and the Illumina GAII sequencing platform. Candidate somatic mutations were identified with a bioinformatics pipeline consisting of BWA for sequence alignment, Samtools for alignment filtering and Varscan for somatic mutation calling. Mutations were manually reviewed in IGV for alignment artifacts and validated by capillary sequencing. DNA samples from 8 additional untreated LGL-leukemia patients were used for further screening of confirmed somatic mutations by capillary sequencing. From six of these patients DNA was extracted from CD8 sorted cells and from two patients from whole blood. RESULTS: Whole exome sequencing of CD8+ leukemic DNA from the index patient identified a missense mutation in the STAT3 gene (D661V), which was subsequently confirmed by capillary sequencing. As STAT3 signaling has been associated with LGL leukemia pathogenesis previously, we next designed primers for the secondary screening of the six exomes of STAT3 SH2 region from the remaining patients. Another recurrent somatic missense mutation (STAT3 Y640F) was identified in two additional patients. Thus, three out of nine LGL patients (33%) showed evidence of mutations in the STAT3 SH2 region. Both missense mutations found (D661V and Y640F) were located in the area of the SH2 domain known to mediate STAT3 protein dimerization and activation. The Y640F mutation alters a conserved tyrosine residue leading to a hyperactivating STAT protein (Scarzello et al. Mol Biol Cell, 2007) and was recently found in a human inflammatory hepatocellular adenoma causing cytokine-independent tyrosine phosphorylation and activation as well as cytokine-dependent hyperactivation of STAT3 (Pitali et al., J Exp Med, 2011). The D661V mutation has not been described previously. CONCLUSIONS: Our data imply for the first time that STAT3 is a common mutational target in LGL leukemia, revealing insights to the molecular pathogenesis of this rare disease. Known structural and functional data on STAT biology imply that the mutations are leading to STAT3 hyperactivation and could also confer ligand-independent signaling. While confirmatory data from a larger series of patients are necessary, our results pinpoint STAT3 mutations and aberrations in the STAT3 pathway as key pathogenetic events in true clonal LGL leukemia. Detection of STAT3 mutations could therefore be applied in the diagnostic assessment, disease stratification and therapeutic monitoring of LGL patients. Disclosures: Koskela: Novartis: Honoraria. Kuittinen:Roche: Consultancy. Porkka:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Mustjoki:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

Abstract T-large granular lymphocyte leukemia (T-LGLL) is a clonal lymphoproliferative disorder of cytotoxic T-cells (CTL) that is associated with cytopenias, predominantly neutropenia and reticulocytopenic anemia. From a scientific point of view, T-LGLL provides a natural model to study the dynamics of CTL responses; the heterogeneity of the disorder allows for examining the diversity of CTL responses in both autoimmune disorders and putatively chronic reactive conditions. A proportion of patients may have an extreme reactive process that mimics an indolent neoplastic lymphoproliferation. NGS and deep T-cell repertoire (TCR) sequencing provide insight into the clonal dynamics at work in T-LGLL patients. A large proportion of T-LGLL patients present with a bona-fide low-grade leukemia; this notion is supported by the discovery of recurrent somatic STAT3 mutations in some patients. STAT3 clonal burden represents an excellent marker that can be serially monitored along with clinical milestones to ultimately gain a more comprehensive understanding of disease etiology and natural history. We collected a cohort of 183 LGLL patients and screened them via deep NGS for mutation status of STAT3. In 36% of patients, 4 distinct somatic mutations (Y640F, N647I, D661V, D661Y) were identified in the SH2 domain of STAT3. In patients with wildtype STAT3, no somatic mutation was implicated in clonal expansion except for a small minority with STAT5 mutations present. We performed a longitudinal analysis of 20 representative STAT3-mutated T-LGLL patients with up to 10-year follow-up and an average of 7 analyzed blood samples per case. All serial samples were deep-sequenced to detect and determine the VAF of the known STAT3 mutations. Overall, STAT3 mutation VAF had a significant, inverse relationship to both hemoglobin and absolute neutrophil count (ANC) (both p<=0.001). In 7/11 cases harboring the Y640F mutation, chemotherapy led to remission accompanied by a decrease in VAF; 3 were asymptomatic and received no treatment. In patients with D661V or D661Y, 6/9 achieved remission with treatment. Only 1/3 cases with N647I entered remission. This longitudinal cohort can be sub-categorized into distinct patterns of clonal dynamics: 1) emerging STAT3 mutation in 20% of patients with a decrease in ANC as VAF of STAT3 clones expand; 2) an opposite trend in 40% of patients where VAF decreased due to therapeutic manipulations; 3) stable VAF in 20% of patients with little change over time; 4) codominant or dominant/secondary STAT3 mutations with distinct subclonal burden in 20% of patients. We performed deep TCR NGS on a representative subset of 9 patients to explore how STAT3 mutations correlated with T-cell clonal expansions. The data were processed by an extensive bioanalytic pipeline to quantify the relative abundance of each CDR3 rearrangement within a patient's TCR. Our cohort had an average of over 53,000 CDR3 templates per sample and was compared with 587 healthy controls. Our results demonstrate multiple patterns of clonal dynamics over the course of T-LGLL. Within each case, the immunodominant clones in serial samples were identified and correlated with STAT3 VAF burden over time. When patients were in remission, both STAT3 VAF and clonality were typically low. Interestingly, functional remission occurred in 2 cases despite increases in both clonality and STAT3 VAF. In 5/9 cases, the T-LGLL process involved 1 STAT3 mutation and 1 corresponding pathogenic clonotype displaying similar dynamics over time. In patients with 2 mutations, multiple high-frequency clonotypes were observed. Most significantly, comparison of STAT3 VAF and the dominant clonotype(s) revealed that STAT3 mutation can arise within a pre-existing clonal expansion that may harbor 2 branching mutations in extreme cases. Identification of CDR3 rearrangement sequences allowed for analysis of the distribution of clonotypes among patients and controls. The pathogenic clonotypes found in T-LGLL patients were detected in a high proportion of controls but at extremely low frequencies. This suggests that these potentially autoimmune clones exist in normal individuals but are effectively suppressed. No pathogenic clonotypes were shared among disease patients. In sum, analysis of clonal dynamics suggests that STAT3 mutations can occur in the context of pre-existing oligoclonal responses and involve otherwise low-frequency clonal specificities. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Mustjoki:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding.

Chronic lymphoproliferative disorder of NK-cells (CLPD-NK) predominantly occurs in adults with a median age of diagnosis at 60 years. It is characterized by a persistent increase (≥2 x 109/L, for &gt; 6 months) of mature NK-cells in peripheral blood with an indolent clinical course similar to T-cell large granular lymphocytic leukemia (T-LGL). Somatic gain-of-function (GOF) mutations in STAT3 have been identified in approximately one-third of patients with CLPD-NK. On the other hand, somatic GOF mutations in JAK3 recurrently occur in various types of T-cell neoplasms and exert a GOF effect, unlike biallelic germline loss-of-function mutations found in severe combined immunodeficiency (Figure 1). Here we report on the discovery of a germline GOF JAK3 mutation as a first germline cause of CLPD-NK. Two individuals from one nonconsanguineous family (mother and son) presented at ages 35 and 12 years old with NK cell lymphoproliferation, lymphadenopathy, splenomegaly and autoimmune symptoms. The mother had history of vasculitis while the son was diagnosed with CVID, recurrent multilineage autoimmune cytopenia and subsequently developed psoriasis at 18 years old. The immunological phenotype was assessed in depth in the son and revealed hypogammaglobulinemia with normal vaccine response, expanded NK cells (between 40-60% of total lymphocytes), decreased FOXP3 expression in regulatory T cells and B cell subsets showing decreased total and isotype-switched memory B cells. Flow cytometry revealed expanded population of aberrant NK cells with normal KIR panel. Marrow studies revealed normal karyotype, cellularity and maturation but prominent large granular lymphocytes with benign cytology. Genomic studies identified a novel germline heterozygous JAK3 variant (c.1520A&gt;C/p.Q507P) located at the linker between SH2 and pseudokinase domain (Figure 1). No additional somatic mutations were found. The JAK3 variant was not present in gnomAD database but previously reported as somatic mutation in a patient with T cell prolymphocytic leukemia (Bergmann, Genes Chromosomes Cancer 2014) and predicted to exert a GOF effect. It is well known that JAK3 activation promotes STAT signaling, a known key player in lymphoproliferation. To better understand the biological effect in patient cells, we performed pSTAT5 phosphorylation assay in primary blood lymphocytes after IL2 stimulation, revealing increased pSTAT5 phosphorylation in patient's NK cells. The IL3-dependent BaF3 cell line (containing human wild type JAK3) has been previously used as a robust model to study the effect of JAK3 mutations (Elliott et al. Blood 2011). We therefore introduced the p.Q507P mutation using CRISPR/Cas9 system and used known GOF mutation p.A572V as positive control. While untransduced BaF3 cells died without IL3, p.Q507P-mutant BaF3 cells survived and rapidly expanded without IL3, showing comparable results to positive control. Finally, using western blot we identified constitutive phosphorylation as expected mechanism underlying the lymphoproliferation p.Q507P-mutant cells. In summary, we identify JAK3 as the first germline cause underlying familial CLPD-NK and describe a novel primary immune dysregulatory disorder characterized by non-malignant NK lymphoproliferation with CVID and autoimmune dysregulation. These findings broaden the genetic spectrum of primary immunodeficiency and immune dysregulatory conditions. Disclosures Takemoto: Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: DSMB Aplastic Anemia Trial. Nichols:Incyte corporation: Research Funding.

Abstract Background Large granular lymphocytic (LGL) leukemia is a clonal disease of mature cytotoxic T- or natural killer (NK)-cells, which was recently characterized by gain-of-function somatic STAT3 mutations in 40-70% of patients. Most of the T-LGL leukemia patients harbor one major Vbeta clone corresponding even up to 90% of total CD8+ T-cell population. Interestingly though, in a small proportion of T-LGL leukemia patients we have detected multiple mutations in the STAT3 gene suggesting the presence of subclones. Here, we aimed to study the clonal architecture and mutation spectrum of expanded lymphocytes with deep sequencing method and to follow the clones during immunosuppressive treatment. Methods DNA samples from 228 LGL leukemia patients were available for STAT3 mutation analysis. Additional flow cytometry-based sorting was done from 12 STAT3 mutation-positive patients, 6 of which had multiple STAT3 mutations in peripheral blood mononuclear cells (PBMNCs). First, frozen live PBMNCs were sorted into CD4+, CD8+ T-cell, and NK-cell fractions using antibodies for CD3, CD4, CD8, and CD16/56. Then CD8+ T-cell population was further sorted into clonal/non-clonal cells based on the flow cytometry analysis of T-cell receptor beta chain expression (Vbeta). STAT3 exon 21 was sequenced using Illumina Miseq platform with coverage aim over 10,000. The data was analyzed using an in-house bioinformatics pipeline: mutations were considered to be true if variant allele frequency (VAF) was over 1%, and false-positives were filtered out by comparing the VAF with calculated error rate of the amplicon. Results In total, 12/228 patients had multiple STAT3 mutations (16% of all STAT3 mutation positive cases). In all studied patients with multiple mutations (Table 1, 4 cases presented), the VAF was 30-50% in the purified major Vbeta clone suggesting that the whole population belonged to the same clone harboring a heterozygous STAT3 mutation (the clone size can be estimated to be twice the VAF). In addition to the major clone, STAT3 mutations were also discovered in smaller Vbeta expansions and in some cases in the non-clonal CD8+ population (Table 1). Interestingly, one patient diagnosed with T-LGL leukemia did not have STAT3 mutations in the major Vbeta expansion (65% of CD8+ cells) but harbored a D661Y mutation with 38% VAF in the NK-cell fraction (Patient 5 in Table 1). The follow-up samples during the treatment were available from 4 patients. In patients 1 and 2 (Table 1), the size of the clone was unchanged during 32 and 37 months follow-up despite of the treatment with methotrexate and cyclophosphamide. In patient 3 (Table 1), the Vb7.1+ clone carrying Y640F mutation decreased from 10% to 3% of CD8+ cells, whereas the Vb5.1+ clone (D661Y) was unchanged during the methotrexate treatment. In patient 5 complete remission was achieved with cyclophosphamide treatment and that was accompanied with the disappearance of D661Y-mutated NK-cells. Discussion Our preliminary results provide evidence that the STAT3 mutations are not only restricted to the significantly expanded lymphocyte clone in LGL leukemia patients, but they can also be found in smaller subclones mimicking the situation in acute leukemia. The actual cause of the mutations is unknown, but the results suggest the presence of a strong initial immune activation, which predisposes existing lymphocyte clones to somatic mutagenesis during cell proliferation. Considering the effects of treatment on STAT3-mutated clones, the only complete remission seen was connected to the disappearance of the mutated clone, which warrants STAT3-inhibitor trials in the future. Disclosures: Porkka: BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

Abstract (PS and UF contributed equally to this work.) Introduction: The Autoimmune Lymphoproliferative Syndrome (ALPS) is caused by inefficient clearing of T lymphocytes. Patients are thus characterized by lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias and an elevated number of double negative T cells (CD3+, TCRα/β+, CD4-, CD8-). Patients suffering from ALPS typically harbor germline or somatic mutations in genes involved in the apoptotic FAS death receptor signaling pathway (FAS, FASLG or CASP10). For 20-30% of patients, however, the genetic cause is still unknown. Methods: The objective of this study was to identify novel gene candidates underlying ALPS of unknown genetic cause. To this end, 25 patients with clinical ALPS symptoms, but without classical mutations were analyzed by whole-exome sequencing. The list of potential candidates was narrowed down using an in-house developed bioinformatic analysis pipeline for patient-based gene prioritization based on protein-protein interaction networks. Resulting candidates were validated by Sanger sequencing and their impact on Fas signaling was studied. Results We identified a de novo germline mutation of the Signal Transducer And Activator Of Transcription 3 (STAT3, c.833G>A, p.R278H) in one of the analyzed patients. The patient presented at the age of nine with Coombs positive hemolytic anemia, thrombocytopenia, generalized progressive, non-infectious, non-malignant lymphadenopathy and splenomegaly. Immunophenotyping revealed increased numbers of double negative T cells (20% in peripheral blood) and over time the patient developed panhypogammaglobulinemia. We performed immunoblot analyses and could demonstrate that the level of phosphorylated STAT3 (pSTAT3-Tyr705) was elevated in the patient's lymphocytes. This finding indicated that the mutation leads to constitutive activation of STAT3. Consistently, we detected an increased expression of STAT3 target genes (including SOCS3, MMP7 and the anti-apoptotic factors BCL2 and BCL2L1) compared to wild-type controls using quantitative real-time PCR. We could also show a decreased expression of the pro-apoptotic genes BAK1 and BAX that is in accordance with the known negative regulation by STAT3. Thus, in the analyzed patient we found that the balance of pro- and anti-apoptotic factors inside the cell was skewed towards apoptosis resistance. Consistently, we could induce apoptosis in vitro applying recombinant Fas ligand, IL21 or staurosporine efficiently in cells derived from healthy controls, but only to a significantly lesser extent in cells from the patient. Moreover, in healthy cells we observed a concurrent downregulation of anti-apoptotic BCL2/BCL2L1 and an upregulation of pro-apoptotic BAX/BAK1 expression upon treatment that was completely absent in the patient's cells. Next, we tried to rescue the effect of constitutively activated STAT3 by application of a STAT3 specific inhibitor: S3I-201. When we treated the patient's lymphocytes with S3I-201 the expression levels of pro- and anti-apoptotic genes were similar to healthy controls and the sensitivity to apoptosis was restored. Conclusion: We report here a novel germline dominant STAT3 gain-of-function mutation that caused a clinical phenotype mimicking ALPS. Recent studies indicated that dominant germline STAT3 gain-of-function mutations lead to autoimmunity, hypogammaglobulinemia, and lymphoproliferation. STAT3 gain-of-function patients therefore share some clinical characteristics with ALPS patients. The clinical presentation of the patient described here differed from the phenotypes previously reported and thus extends the spectrum of STAT3 -associated diseases. The mechanism underlying the clinical symptoms of STAT3 gain-of-function patients has not yet been determined. Here, we demonstrate increased activation of STAT3 and STAT3 target genes, leading to a skewed balance of pro- and anti-apoptotic factors and apoptosis evasion as a cause for lymphocyte accumulation and resulting autoimmunity in a STAT3 gain-of-function patient. Similar to ALPS patients, diminished responsiveness of lymphocytes to apoptosis seems to be a major characteristic. The clinical phenotype may differ because mutations in STAT3 or Fas signaling genes, respectively, affect overlapping, but also distinct signaling pathways. Disclosures No relevant conflicts of interest to declare.

Large granular lymphocyte (LGL) leukemia arises spontaneously in elderly Fischer (F344) rats. This rodent model has been shown to emulate many aspects of the natural killer (NK) variant of human LGL leukemia. Previous transplantation of leukemic material into young F344 rats resulted in several strains of rat NK (RNK) primary leukemic cells. One strain, RNK-16, was adapted into the RNK-16 cell line and established as an aggressive NK-LGL leukemia model. Whole genome sequencing of the RNK-16 cell line identified 255,838 locations where the RNK16 had an alternate allele that was different from F344, including a mutation in Jak1. Functional studies showed Jak1 Y1034C to be a somatic activating mutation that mediated increased STAT signaling, as assessed by phosphoprotein levels. Sanger sequencing of Jak1 in RNK-1, -3, -7, and -16 found only RNK-16 to harbor the Y1034C Jak1 mutation. In vivo studies revealed that rats engrafted with RNK-16 primary material developed leukemia more rapidly than those engrafted with RNK-1, -3, and -7. Additionally, ex vivo RNK-16 spleen cells from leukemic rats exhibited increased STAT1, STAT3, and STAT5 phosphorylation compared to other RNK strains. Therefore, we report and characterize a novel gain-of-function Jak1 mutation in a spontaneous LGL leukemia model that results in increased downstream STAT signaling.

Abstract Introduction: T-cell large granular lymphocyte (T-LGL) leukemia is a rare lymphoproliferative disorder with recurrent somatic STAT3 mutations. It has been suggested that viral antigens act as the initial stimuli resulting in clonal expansion of CD8+ cells in the disease. However, less is known whether chronic exposure to viral antigens is associated with acquisition of somatic mutations in CD8+ T cells among individuals without clinically detectable lymphoproliferations. Human T-cell leukemia virus type 2 (HTLV-2) antibody positivity has been detected in patients with T-LGL leukemia. Here, we examined whether CD8+ T cells from HTLV-2 positive healthy blood donors harbor somatic mutations in STAT3 or other immune-associated genes, potentially identifying individuals at risk of subsequent lymphoproliferative diseases. Methods: We analyzed HTLV-2 infected (n=30) and uninfected (n=35) healthy blood donor samples obtained from University of California San Francisco and Vitalant Research Institute, which were enrolled in the United States-based HTLV Outcomes Study (HOST) cohort. All cases had serologic evaluation for HTLV-2 infection at the time of sampling. We examined somatic mutations of STAT3 in CD4+ and CD8+ T-cell populations using ultra-deep targeted amplicon sequencing. In addition, we applied a custom sequencing panel covering the coding regions of 2,533 immune-related genes to characterize a larger spectrum of somatic mutations in CD8+ T cells. Results: Somatic STAT3 mutations were detected in CD8+ but not in CD4+ T cells of four (13.3%, 4/30) HTLV-2 positive healthy blood donors (p.Y640F, p.N647I, p.D661Y, and p.Y657_K658insY with variant allele frequencies of 11.9%, 0.5%, 4.9%, and 1.2%, respectively) using amplicon sequencing. The detected STAT3 mutations have been previously described and reported in T-LGL leukemia. Total white blood cell and lymphocyte counts were similar between STAT3 mutated and non-mutated cases. No STAT3 mutations were discovered in HTLV-2 negative donors with amplicon sequencing. Of the 28 HTLV-2 positive cases, 19 had at least one somatic variant in CD8+ T cells based on the immunogene panel sequencing (n=28). 8 cases had variants in genes previously identified in T-LGLL (STAT3, KMT2D, TYRO3, DIDO1, BCL11B, CACNB2, KRAS, LRBA and FANCA), and 5 cases had variants in genes involved in JAK-STAT signaling (NFKBIA, PIK3R5, MAPK14, EP300, MPL, IFNAR1, IL6ST and IL20RA). Three recurrently mutated genes were detected: VWF, SMAD7 and MXRA5. The four HTLV-2 positive blood donors harboring STAT3 mutations had more somatic mutations (median=6) than HTLV-2 positive donors without STAT3 mutations (median=1, p=0.06). Conclusion: We report the presence of somatic gain-of-function STAT3 mutations in CD8+ T cells of 13% of HTLV-2 positive healthy blood donors. We identified additional somatic mutations in genes associated with JAK-STAT signaling, immune regulation and lymphoproliferation in CD8+ T cells of HTLV-2 positive cases. While STAT3 activation, with or without mutations, is considered as a hallmark of T-LGLL, our results reveal the presence of STAT3 mutations in CD8+ T cells of healthy blood donors harboring HTLV-2 without clinical history of lymphoproliferative disease. Additional research is warranted to elucidate whether HTLV-2 carriers harboring STAT3 and other mutations are at increased risk of subsequent T-LGL leukemia or other lymphoproliferative diseases. Disclosures Mustjoki: Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding; Janpix: Research Funding.

Abstract Patients with severe congenital neutropenia (SCN) have a markedly increased risk of developing myelodysplasia (MDS) or acute myeloid leukemia (AML). Though the genetic basis for this increased susceptibility is unknown, gain-of-function mutations of the G-CSF receptor (G-CSFR) have been found in the great majority of patients with SCN who develop MDS/AML. These mutations are somatic and produce a truncated G-CSFR that, though remaining ligand-dependent, transmits a hyperproliferative signal. We and others have shown that targeted transgenic mice expressing a representative G-CSFR mutation (d715) have markedly exaggerated neutrophil responses to G-CSF treatment. Based on these observations, it has been suggested that these gain-of-function G-CSFR mutations contribute to leukemogenesis. However, direct evidence supporting this hypothesis is scant. Moreover, it is unclear how hematopoietic cells expressing the mutant G-CSFR gain clonal dominance. We previously showed that expression of the d715 G-CSFR results in a strong competitive advantage at the hematopoietic stem cell (HSC) level, but only in the presence of an increased concentration of G-CSF. Herein, we describe studies to characterize the cellular and molecular mechanisms responsible for the clonal dominance of HSC expressing the d715 G-CSFR. At baseline, the percentage of cycling c-Kit+ lineage− Sca+ (KLS) cells was similar in WT (8.9±2.0%) and d715 G-CSFR mice (10±3.0%). However, 24 hours after a single injection of G-CSF, a significantly greater percentage of cycling KLS was observed in d715 G-CSFR compared with WT mice (34.4±2.4% versus 20±3.8%; p &lt; .05). We next harvested KLS cells from WT or d715 G-CSFR mice 3 hours after treatment with a single injection of G-CSF or saline alone and performed RNA expression profiling. 14 genes were identified that were consistently differentially regulated by G-CSF in d715 G-CSFR versus WT KLS cells. A striking features shared by most of these genes is their regulation by STAT3 or STAT5. These data suggested the hypothesis that activation of STAT3 and/or STAT5 transduces the signal leading to HSC clonal dominance. To test this hypothesis, we first directly measured STAT activation by G-CSF in KLS cells using a flow cytometry-based method. These data showed that STAT3 and to a lesser extent STAT5 are activated by G-CSF in WT KLS cells. d715 G-CSFR KLS cells displayed significantly increased STAT5 activation by G-CSF, but STAT3 activation was slightly decreased, suggesting that STAT5 may play a key role in HSC activity. To further test this hypothesis, mice carrying a targeted mutation of their G-CSFR in which the sole remaining tyrosine in d715 G-CSFR is mutated to phenylalanine (termed d715F) were analyzed. Importantly, G-CSF induced STAT3 and STAT5 in KLS cells from d715F mice was markedly attenuated. Competitive repopulation experiments showed that the d715F G-CSFR did not confer a clonal advantage and may, in fact, confer a competitive disadvantage. Collectively, these data suggest that the d715 G-CSFR confers a clonal advantage at the HSC level that may be mediated by accentuated STAT5 activation.

Abstract Background T-cell receptor signaling along with intermittent homeostatic proliferation governed by STAT3/5-associated common γ chain (γc) cytokines IL-6, IL-2, IL-7 and IL-15 are the principal source of new T-cells after thymic involution. Persistent chronic infections and cancer lead to T-cell exhaustion characterized by a number of “exhaustion”-associated phenotypic changes and activation of a specific gene expression profile associated with negative regulation of cytokine signaling response over time. Lymphoproliferation, as seen in large granular lymphocyte leukemia, results from excessive proliferation in response to one or more of these cytokines leading to in vivo expansion. We showed previously using electrophoretic mobility shift assays (EMSA) that most LGL leukemia patients display constitutively active STAT3 DNA binding activity. Recently, an international group discovered that 30-40 % of patients have somatic mutations in STAT3. This provides new molecular insight into the basis of LGL leukemia, but does not resolve some key questions about the biology. First, leukemic LGL cells are not fully immortalized by the activation or mutation of STAT3 suggesting that their survival and expansion may be microenvironmentally regulated. Second, it is unclear if the STAT3 mutation is acquired within the clonal cell population that invariably possesses an “exhaustion-associated” CD57+/CD8+/CD28-/CD62L-/CD27- memory phenotype. Here, we determined the role of cytokine signaling in LGL leukemia. Methods To gain insight into the disease, we examined primary human T-cells from healthy controls (N=28) and LGL leukemia patients (N=28). In the LGL leukemia cohort, 14 patients harbored one of the recurring STAT3 mutations within the SH2 domain (Y640F, D661Y, D661H, D661V). The remaining LGL leukemia patients had no evidence of genomic abnormalities. Using these primary cells, we examined proliferation potential and pSTAT3/5 activation by flow cytometry (phosFlow) using methods published previously by our group. Signaling was determined after culture with 0.01, 0.1, and 1 ng/ml of IL-6 to activate STAT3, and similar doses of IL-2, IL-7 and IL-15 to activate STAT5. Results First, LGL leukemia cells showed hyperproliferative responses to the lowest dose of IL-15. Comparing CD57+ and CD57- T-cells in healthy individuals, CD57+ T-cells were hyporesponsive to all cytokines with regard to proliferation and phosphorylation in line with their exhausted phenotype. Next, we examined cytokine-mediated phosphorylation of STAT3 and STAT5 in LGL cases. STAT3 was normally phosphorylated in response to IL-6 in LGL leukemia T-cells. In contrast, hypersensitive STAT5 phosphorylation in response to the lowest dose of IL-6 (p=0.05), IL-2 (p=0.04), IL-7 (p=0.01), and IL-15 (p=0.02) occurred within CD57- T-cells. This occurred equally in both STAT3 mutant and non-mutant patient groups. The traditional CD57+ leukemic T-cell population was also hypersensitive to 100 fold-lower doses of IL-7 and IL-15 compared to healthy donors and again this difference was independent of the STAT3 mutation status. Conclusion LGL leukemia T-cells bypass protective mechanisms associated with T-cell exhaustion and display proliferation potential and hypersensitivity to STAT3/5-associated common γ chain (γc) cytokines. Similar differences in signaling were observed in both STAT3 mutant and non-mutant cases suggesting that it is a unifying abnormality. Although STAT3 was not hyperphosphorylated, it is possible that STAT3 is activated by enhanced dimerization capacity (STAT3-SH2 domain mutation) or through lack of negative regulators such as a deficiency in SOCS proteins. Our data, however, introduces a novel concept in this disease and suggests that the driving event lies within the CD57- population, where hypersensensitive STAT5 phosphorylation was evident in response to all cytokines. These results suggest that the driver mutation or event possibly resides within the common lymphoid progenitor population and that the presence of clonally-expanded CD57+ cells may result from incomplete exhaustion reprogramming following excessive cytokine-mediated proliferation within the lymphoid population. Disclosures: No relevant conflicts of interest to declare.

Abstract The SH2-JH2 linker domain of JAK2 has been implicated in the negative regulation of JAK2 activity. In 2 patients with myeloproliferative neoplasms (MPNs), we identified and characterized the novel JAK2 mutation S523L, which occurs in a key residue in the linker region. In 1 case, acquisition of JAK2S523L was associated with thrombocytosis and bone marrow megakaryocytic hyperplasia, and there were no other somatic alterations in this patient. The second patient with JAK2S523Lmutation presented with increased hematocrit and had concurrent mutations in RUNX1 and BCORL1. Consistent with the genetic and clinical data, expression of JAK2S523L causes interleukin-3–independent growth in Ba/F3 cells transduced with the erythropoietin receptor by constitutively active Jak2/Stat5 signaling.

La maladie cœliaque réfractaire de type II (MCRII), autrement appelé lymphome intraépithélial, est une complication rare mais sévère de la maladie cœliaque caractérisée par une expansion clonale d'une population particulière de lymphocytes intraépithéliaux (LIE) innés, présents dans l'intestin normal chez l'Homme comme chez la souris. Notre laboratoire a montré que cette population particulière de LIE innés partage des caractéristiques communes à celles des lymphocytes T et des cellules NK. Ces « LIE iCD3+ innés » sont caractérisées par une expression de CD3 au niveau intracellulaire mais pas à la surface, de récepteurs NK et présentent des réarrangements des gènes codant le récepteur T. En outre, le laboratoire a montré que ces cellules se développent dans l'épithélium intestinal à partir de précurseurs de la moëlle osseuse en réponse à une combinaison de signaux induits à travers la voie NOTCH et l'interleukine 15. Durant la lymphomagénèse, les LIE iCD3+ innés acquièrent des mutations somatiques gain-de-fonction dans JAK1et/ou STAT3. Ces mutations pourraient favoriser l'expansion clonale des LIE iCD3+ mutés aux dépens des lymphocytes T normaux résidents en leur conférant une sensibilité accrue à l'interleukine 15 (IL-15), une cytokine surexprimée dans l'intestin des patients. Ainsi, notre hypothèse est que ces mutations ont un rôle central dans l'initiation de la lymphomagénèse dans un contexte de production chronique d'IL-15 et, de ce fait, représentent une cible thérapeutique. Le premier objectif de ma thèse a été d'étudier l'intérêt des inhibiteurs de la voie JAK/STAT dans le traitement de la MCRII. Dans un premier temps, nous avons testé in vitro différents inhibiteurs de JAK/STAT sur des lignées cellulaires IL-15-dépendantes issues soit de LIE de MCRII soit de LIE T normaux. Nous avons démontré que ces drogues inhibent la prolifération et la phosphorylation de STAT3 et augmentent l'apoptose cellulaire aussi bien dans les LIE MCRII que dans les LIE T normaux. Dans un second temps, nous avons généré un modèle de xénogreffe en injectant des cellules issues de biopsies intestinales ou du sang d'un patient MCRII dans des souris immunodéficientes surexprimant l'IL-15 humaine dans l'épithélium intestinal (Rag-/-Gc-/-IL-15TgE ou IRGC) afin de tester l'efficacité des inhibiteurs de JAK/STAT in vivo. Le traitement des souris xénogreffées par le ruxolitinib, inhibiteur de JAK1/JAK2, a permis une diminution de la fréquence et du nombre ainsi que de l'activité cytotoxique des cellules tumorales humaines et une amélioration de l'état général des souris. Ces résultats encourageants restent à confirmer. Le second objectif de ma thèse a été de vérifier si la mutation pD661V de STAT3 était suffisante pour induire le développement de la MCRII dans un contexte de surproduction d'IL-15 dans des souris IRGC. Nous avons généré avec succès les LIE iCD3+ innés murins semblables aux LIE iCD3+ innés humaines à partir de précurseurs communs aux cellules lymphoïdes (CLP) en combinant un signal NOTCH et IL-15. Nous avons ensuite transduit les CLP avec un vecteur rétroviral contenant Stat3 sauvage ou muté (D661V). Les cellules transduites ont alors été injectées chez des souris IRGC suivies pendant 8 semaines. Les résultats préliminaires ont montré que les LIE iCD3+ innés se logent préférentiellement dans l'intestin mais aucun développement d'un lymphome intraépithélial n'a été observé au bout de 8 semaines suggérant que la mutation pD661V de STAT3 seule ne suffit pas en présence d'IL-15 à induire in vivo un lymphome intraépithélial. Ces résultats préliminaires sont toutefois à reproduire et à confirmer. Le modèle mise en place pour l'étude de STAT3 va désormais être utilisé afin d'évaluer la contribution respective de mutations canoniques de JAK1 et STAT3 et des autres mutations récurrentes retrouvées dans le lymphome intraépithélial
Refractory coeliac disease type II (RCDII), also called intraepithelial lymphoma, is a rare but severe complication of coeliac disease characterized by the clonal expansion of a small subset of innate intraepithelial lymphocytes (IEL), present in the normal human and murine intestine. Our lab has shown that this population displays shared features between T and natural killer (NK) cells. These so-called iCD3+ innate IEL are mainly characterized by intracellular expression of CD3, which is not detected at the cell surface, expression of NK receptors as well as DNA rearrangement of T cell receptor genes. Our lab has also shown that iCD3+ innate IEL originate from bone marrow precursors through coordinated NOTCH1 and interleukin (IL)-15 signals. During lymphomagenesis, iCD3+ innate IEL of most RCDII patients were shown to have acquired somatic gain-of-function mutations in JAK1 and/or STAT3 that confer increased sensitivity to interleukin-15, a cytokine overexpressed in the intestine of coeliac patients, thereby promoting their clonal expansion. Thus, our hypothesis is that JAK1/STAT3 mutations play a key role in initiating lymphomagenesis associated to coeliac disease in an IL-15-rich environment and that they could represent an attractive therapeutic target.The first objective of my thesis was to study the interest of JAK/STAT inhibitors for RCDII treatment. First, we have tested in vitro different JAK/STAT inhibitors on IL-15-dependent RCDII or normal IEL-T cell lines. We have shown that these inhibitors decrease the proliferation and phosphorylation of STAT3 and increase cellular apoptosis in both RCDII and normal T cell lines. Secondly, we have established a xenograft model based on the injection of cells derived from biopsy or blood from one RCDII patient into immunodeficient mice overexpressing the human IL-15 transgene in their gut epithelium (Rag-/-Gc-/- IL-15TgE; IRGC) to test the efficacy of JAK/STAT inhibitors in vivo. Treatment of xenografted mice with ruxolitinib, a potent inhibitor of JAK1/JAK2 decreased the frequency, number and cytotoxic potential of human tumoral cells and allowed clinical restoration. These preliminary results are encouraging but need to be confirmed. The second objective of my thesis was to test whether the Stat3 pD661V mutation is sufficient to induce the intraepithelial lymphoma in an IL-15-rich context in IRGC mice. We have successfully generated murine iCD3+ innate IEL in vitro, resembling their human counterparts from common lymphoid precursors by combining NOTCH and IL-15 signals. We then transduced CLP with a retroviral vector containing wild-type or mutated Stat3 pD661V. The transduced cells were injected into IRGC mice that subsequently were followed-up during a period of 8 weeks. In vitro generated iCD3+ innate IEL preferentially homed to the intestine. However, no development of intraepithelial lymphoma was observed suggesting that the Stat3 pD661V variant alone is not sufficient to induce the intraepithelial lymphoma. These preliminary results need to be reproduced and confirmed. The murine model used to test the role of STAT3 will now be used to evaluate the respective contribution of canonical mutations in JAK1 and STAT3 and of other recurrent mutations identified in RCDII

Abstract The study of genetic epidemiology and the statistical tools it uses requires at least a rudimentary understanding of the principles of molecular biology on which the phenomena of transmission of genes are based. This chapter provides a general survey of the structure of DNA, chromosomes, and genes, their reproduction by mitosis in somatic cell division and by meiosis in the creation of the gametes that are transmitted from parents to offspring, their expression in proteins, and the types of polymorphisms that can occur. Readers interested in more detail are encouraged to read one of the excellent textbooks on molecular biology, such as that of Strachan and Read (1999) or Lewin (1997). Research in genetic or molecular epidemiology inevitably involves collaboration with laboratory scientists, and some understanding of the methods they use to obtain, amplify, and store DNA and to detect polymorphisms or mutations is also essential. Since these techniques are in a rapid state of evolution, I do not attempt to describe them here, but rather encourage readers to consult with their molecular science colleagues about how best to gain this experience.

Postzygotic mutations of the PIK3CA gene are associated with a series of clinical phenotypes characterized by segmental overgrowth and recently grouped under the term PIK3CA-related overgrowth spectrum (PROS). This chapter provides an overview of the clinical features shared by the phenotypes in PROS, including both the conditions with isolated features and the ones with syndromal presentation. The somatic overgrowth in cases with PROS is asymmetric, progressive, and “ballooning” in appearance and tends to involve predominantly the limbs, including fingers and toes, although the trunk and face are often affected as well. The tissues affected in the overgrowth can include all or some of these types: fibrous, adipose, vascular, nervous, and skeletal. Somatic gain-of-function mutations of PIK3CA cause activation of the PI3K-AKT pathway, leading to excessive cell growth and proliferation. Timing of PIK3CA mutations, tissue specificity, and type of mutation may play a role in the phenotypic variability of PROS.

The project’s general objectives were to determine specific polymorphisms at the DNA level responsible for observed quantitative trait loci (QTLs) and to estimate their effects, frequencies, and selection potential in the Holstein dairy cattle breed. The specific objectives were to (1) localize the causative polymorphisms to small chromosomal segments based on analysis of 52 U.S. Holstein bulls each with at least 100 sons with high-reliability genetic evaluations using the a posteriori granddaughter design; (2) sequence the complete genomes of at least 40 of those bulls to 20 coverage; (3) determine causative polymorphisms based on concordance between the bulls’ genotypes for specific polymorphisms and their status for a QTL; (4) validate putative quantitative trait variants by genotyping a sample of Israeli Holstein cows; and (5) perform gene expression analysis using statistical methodologies, including determination of signatures of selection, based on somatic cells of cows that are homozygous for contrasting quantitative trait variants; and (6) analyze genes with putative quantitative trait variants using data mining techniques. Current methods for genomic evaluation are based on population-wide linkage disequilibrium between markers and actual alleles that affect traits of interest. Those methods have approximately doubled the rate of genetic gain for most traits in the U.S. Holstein population. With determination of causative polymorphisms, increasing the accuracy of genomic evaluations should be possible by including those genotypes as fixed effects in the analysis models. Determination of causative polymorphisms should also yield useful information on gene function and genetic architecture of complex traits. Concordance between QTL genotype as determined by the a posteriori granddaughter design and marker genotype was determined for 30 trait-by-chromosomal segment effects that are segregating in the U.S. Holstein population; a probability of <10²⁰ was used to accept the null hypothesis that no segregating gene within the chromosomal segment was affecting the trait. Genotypes for 83 grandsires and 17,217 sons were determined by either complete sequence or imputation for 3,148,506 polymorphisms across the entire genome. Variant sites were identified from previous studies (such as the 1000 Bull Genomes Project) and from DNA sequencing of bulls unique to this project, which is one of the largest marker variant surveys conducted for the Holstein breed of cattle. Effects for stature on chromosome 11, daughter pregnancy rate on chromosome 18, and protein percentage on chromosome 20 met 3 criteria: (1) complete or nearly complete concordance, (2) nominal significance of the polymorphism effect after correction for all other polymorphisms, and (3) marker coefficient of determination >40% of total multiple-regression coefficient of determination for the 30 polymorphisms with highest concordance. The missense polymorphism Phe279Tyr in GHR at 31,909,478 base pairs on chromosome 20 was confirmed as the causative mutation for fat and protein concentration. For effect on fat percentage, 12 additional missensepolymorphisms on chromosome 14 were found that had nearly complete concordance with the suggested causative polymorphism (missense mutation Ala232Glu in DGAT1). The markers used in routine U.S. genomic evaluations were increased from 60,000 to 80,000 by adding markers for known QTLs and markers detected in BARD and other research projects. Objectives 1 and 2 were completely accomplished, and objective 3 was partially accomplished. Because no new clear-cut causative polymorphisms were discovered, objectives 4 through 6 were not completed.