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Koskela, Hanna, Samuli Eldfors, Henrikki Almusa, Emma Andersson, Pekka Ellonen, Henrik Edgren, Pirjo Koistinen et al. "Recurrent Missense Mutations in the STAT3 Gene in LGL Leukemia Provide Insights to Pathogenetic Mechanisms and Suggest Potential Diagnostic and Therapeutic Applications". Blood 118, n. 21 (18 novembre 2011): 936. http://dx.doi.org/10.1182/blood.v118.21.936.936.
Abstract (sommario):
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.
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Chomczynski, Peter W., Michael J. Clemente, Srinivasa Reddy Sanikommu, Alek d. Nielsen, Cassandra M. Hirsch, Hanna Rajala, Mikkael A. Sekeres et al. "Next-Generation Sequencing Analysis of Clonal Hierarchy and Dynamics in T-Large Granular Lymphocyte Leukemia Suggests Emergence of STAT3 Clones within Pre-Existing Dominant T-Cell Repertoire Responses Otherwise Silenced in Normal Individuals". Blood 128, n. 22 (2 dicembre 2016): 2731. http://dx.doi.org/10.1182/blood.v128.22.2731.2731.
Abstract (sommario):
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.
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Lesmana, Harry, Marcela Popescu, Sara Lewis, Sushree Sangita Sahoo, Charnise Goodings-Harris, Mihaela Onciu, John Kim Choi, Clifford Takemoto, Kim E. Nichols e Marcin Wlodarski. "Germline Gain-of-Function JAK3 Mutation in Familial Chronic Lymphoproliferative Disorder of NK Cells". Blood 136, Supplement 1 (5 novembre 2020): 9–10. http://dx.doi.org/10.1182/blood-2020-142078.
Abstract (sommario):
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 > 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>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.
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Rajala, Hanna L. M., Thomas Olson, Sonja Lagström, Pekka Ellonen, Syed Arshi Uz Zaman, Emma I. Andersson, Michael J. Clemente et al. "Multiple STAT3 Mutations In Different Lymphocyte Clones Of Large Granular Lymphocytic Leukemia Patients". Blood 122, n. 21 (15 novembre 2013): 2559. http://dx.doi.org/10.1182/blood.v122.21.2559.2559.
Abstract (sommario):
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.
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Nabhani, Schafiq, Hagit Miskin, Cyrill Schipp, Dan Harlev, Shoshana Revel-Vilk, Michael Gombert, Sebastian Ginzel, Arndt Borkhardt, Polina Stepensky e Ute Fischer. "Activating Mutation of STAT3 Protects Lymphocytes from Apoptosis and Leads to a Clinical Phenotype Resembling the Autoimmune Lymphoproliferative Syndrome". Blood 126, n. 23 (3 dicembre 2015): 2218. http://dx.doi.org/10.1182/blood.v126.23.2218.2218.
Abstract (sommario):
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.
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Wang, T. Tiffany, Jun Yang, Shubha Dighe, Matthew W. Schmachtenberg, Nathan T. Leigh, Emily Farber, Suna Onengut-Gumuscu et al. "Whole Genome Sequencing of Spontaneously Occurring Rat Natural Killer Large Granular Lymphocyte Leukemia Identifies JAK1 Somatic Activating Mutation". Cancers 12, n. 1 (3 gennaio 2020): 126. http://dx.doi.org/10.3390/cancers12010126.
Abstract (sommario):
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.
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Kim, Daehong, Mikko Myllymäki, Matti Kankainen, Timo Jarvinen, Giljun Park, Roberta Bruhn, Edward L. Murphy e Satu Mustjoki. "Somatic STAT3 Mutations in CD8+ T Cells of HTLV-2 Positive Blood Donors". Blood 138, Supplement 1 (5 novembre 2021): 3133. http://dx.doi.org/10.1182/blood-2021-146326.
Abstract (sommario):
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.
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Kunter, Ghada M., Fulu Liu, Maxwell Krem e Daniel Link. "G-CSF Receptor Mutations Found in Patients with Severe Congenital Neutropenia Confer a Strong Competitive Growth Advantage at the Hematopoietic Stem Cell Level That Is Mediated by STAT5 Activation." Blood 108, n. 11 (16 novembre 2006): 632. http://dx.doi.org/10.1182/blood.v108.11.632.632.
Abstract (sommario):
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 < .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.
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Mailloux, Adam W., Jeff Painter, Eric Padron, Thomas P. Loughran e Pearlie K. Epling-Burnette. "Common γ Chain (γc) Cytokine Hypersensitivity Leads To T-Cell Homeostatic Deregulation Independent Of STAT3 Mutation In Large Granular Lymphocyte Leukemia". Blood 122, n. 21 (15 novembre 2013): 1628. http://dx.doi.org/10.1182/blood.v122.21.1628.1628.
Abstract (sommario):
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.
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Pastore, Friederike, Aishwarya Krishnan, Henrik M. Hammarén, Olli Silvennoinen, Benedict Yan e Ross L. Levine. "JAK2S523L, a novel gain-of-function mutation in a critical autoregulatory residue in JAK2V617F− MPNs". Blood Advances 4, n. 18 (21 settembre 2020): 4554–59. http://dx.doi.org/10.1182/bloodadvances.2019001283.
Abstract (sommario):
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.
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Masle-Farquhar, Etienne, Kathryn Payne, Mandeep Singh, Geetha Rao, Ghamdan Al-Eryani, Christopher Jara, Katherine Jackson et al. "The effects of germline STAT3-activating mutations from autoimmunity and lymphoid malignancy on mouse and human T cells". Journal of Immunology 204, n. 1_Supplement (1 maggio 2020): 142.35. http://dx.doi.org/10.4049/jimmunol.204.supp.142.35.
Abstract (sommario):
Abstract Signal transducer and activator of transcription 3 (STAT3) regulates gene expression downstream of cell surface cytokine and hormone receptors. Heterozygous germline loss of function STAT3 mutations lead to the primary immunodeficiency hyper-IgE syndrome while somatic activating STAT3 mutations recur in human solid organ and immune malignancies. Germline heterozygous activating STAT3 mutations result in early-onset autoimmunity with aspects of immunodeficiency. Affected individuals share characteristics with autoimmune lymphoproliferative syndrome and immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, including reduced T regulatory cell numbers. They present with variable autoimmune symptoms including type 1 diabetes, rheumatoid arthritis, gut enteropathies and autoimmune cytopenias. Whilst many effects of STAT3 loss of function on immune cells have been described, the mechanisms behind autoimmunity and immunodeficiency in patients with activating STAT3 mutations remain unclear. Here, we present a detailed characterisation of T cell development and maturation in young and old mice on two different backgrounds with Crispr-engineered germline activating mutations in two domains of STAT3. We use mixed chimeras, flow cytometry, T cell receptor deep sequencing and high throughput single-cell transcriptomics to reveal cell-extrinsic and -autonomous roles of overactive STAT3 in T cells in autoimmunity and immune malignancy. To our knowledge, this is the first report of mice with Stat3 germline activating mutations identical to those in autoimmunity or malignancy. We validate our key findings in humans with gain of function germline STAT3 mutations and childhood-onset autoimmunity.
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Milosevic Feenstra, Jelena D., Harini Nivarthi, Heinz Gisslinger, Emilie Leroy, Elisa Rumi, Ilyas Chachoua, Klaudia Bagienski et al. "Whole Exome Sequencing Identifies Novel MPL and JAK2 M utations in Triple Negative Myeloproliferative Neoplasms". Blood 126, n. 23 (3 dicembre 2015): 606. http://dx.doi.org/10.1182/blood.v126.23.606.606.
Abstract (sommario):
Abstract Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic myeloproliferative neoplasms (MPN) characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. Most of the cases are sporadic and driven by somatic mutations, although familial clustering is observed. The most common mutation affecting 50-60% of the cases is JAK2-V617F, while 25-30% of the patients carry somatic mutations in exon 9 of CALR. MPL exon 10 mutations affect ~5% of the cases. JAK2, CALR and MPL mutations are mutually exclusive and account for >90% of ET and PMF cases. In 12% of ET and 5% of PMF cases the disease drivers remain unknown. These patients are termed as triple negative. The mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR. The aim of this study was to identify disease causing mutation in triple negative cases of ET and PMF. To identify the somatic mutations that are potential disease drivers in triple negative MPN we performed whole exome sequencing (WES) on paired samples from the tumor and control tissue of 4 patients with ET and 4 patients with PMF. We identified somatic mutations in 3/8 analyzed cases. In two PMF cases we identified somatic mutations in genes relevant for MPN- TET2, ASXL1, CBL, SRSF2 and a mutation in MPL-S204P. We did not identify a novel recurrent mutation. In the 5 cases without somatic mutations, we looked for germline mutations in genes relevant for MPN. We identified germline mutations MPL-V285E and JAK2-G571S in one PMF case and one case of ET, respectively. SNP microarray analysis for presence of chromosomal aberrations revealed a uniparental disomy of chromosome 6p in the case with MPL -V285E mutation, suggesting clonal hematopoiesis. To determine the frequency of MPL and JAK2 mutations outside exons 10 and 14 in triple negative MPN, we performed Sanger sequencing of all coding exons of MPL in 62 patients and of JAK2 in 49 patients. We detected variants outside exon 10 of MPL in 6/62 cases (9.7%). MPL-T119I, MPL-S204F, MPL-E230G and MPL-Y591D were somatic mutations, while MPL-R321W was germline. We identified an additional patient with MPL-S204P mutation, however the control tissue was not available. JAK2 variants were found in 4/49 cases (8.1%). JAK2-G335D and JAK2-V625F were germline mutations, while for the patients with JAK2-F556V and JAK2-G571S the control tissue was unavailable. In total, we identified non-canonical MPL mutations in 8/70 (11.4%) and JAK2 mutations in 5/57 (8.8%) triple negative cases of ET and PMF. All mutations were heterozygous. The mutations in MPL and JAK2 were mutually exclusive in our patient cohort. The expression of identified MPL mutants did not induce cytokine independent growth of Ba/F3 cells, but the MPL-Y591D expressing cells showed marked hypersensitivity to TPO compared to the wild type. Using a luciferase reporter assay in JAK2-deficient gamma 2A cells, where we transiently expressed the wild type or mutant MPL cDNAs, JAK2, STAT5, STAT5 reporter Spi-Luc, and pRL-TK for transfection control, we could demonstrate that all identified MPL mutations lead to constitutive activation of JAK/STAT signaling. As the detection of activity required longer times (48h) than for the MPL-W515K (24h), we concluded that the identified mutations have a milder effect of the function of MPL. By Western immunodetection we could demonstrate that expression of JAK2-F556V and JAK2-V625F in Ba/F3-MPL cells, lead to the increased phosphorylation of STAT5 in the absence of cytokines. We also observed increased sensitivity to TPO in the Ba/F3 MPL cell lines expressing JAK2-F556V and JAK2-V625F. JAK2-V625F and JAK2-F556V are mild gain-of-function mutations, while JAK2-G335D and JAK2-G571S do not seem to alter the function on the JAK2 protein. The results of our study suggest that sequencing of all coding exons of MPL and JAK2 is recommended for the diagnostic work-up of the ET and PMF patients who do not carry other more common mutations. The lack of evidence for clonal disease in 50% of the triple negative cases and presence of germline mutations suggests that a proportion of cases are likely to be hereditary MPN-like disorders. Application of whole genome sequencing or RNA sequencing for fusion oncogene detection will likely fill in the gap of the remaining triple negative MPN cases with clonal hematopoiesis in which we did not identify a recurrent driving mutation using WES. Disclosures Gisslinger: AOP ORPHAN: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Consultancy; Geron: Consultancy; Janssen Cilag: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.
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Lukes, Julius, Eliska Potuckova, Julia Starkova, Jan Stary, Jan Zuna, Jan Trka e Marketa Zaliova. "Chromosome 21 Gain Is Dispensable for Transient Myeloproliferative Disorder (TMD) Development". Blood 132, Supplement 1 (29 novembre 2018): 2764. http://dx.doi.org/10.1182/blood-2018-99-112078.
Abstract (sommario):
Abstract Transient myeloproliferative disorder (TMD) is a hematopoietic disease, characterized by a clonal proliferation of immature megakaryoblasts in the neonatal period occurring in approximately 10% of newborns with Down syndrome (DS). Rarely, TMD occurs in non-DS newborns but then it is associated with somatic trisomy 21 (tri21). Tri21 together with in-utero gained mutations in the GATA1 gene encoding a myeloid transcription factor are thus considered essential in TMD. Recently, we have identified a TMD with a typical manifestation and course in a newborn without DS/somatic tri21, which admits that tri21 is dispensable for TMD development. To elucidate the alternative TMD pathogenesis, we performed comprehensive genomic/transcriptomic profiling of this TMD case. We utilized high-density SNP array and whole exome and transcriptome sequencing (WES/RNAseq) to detect copy number changes, mutations and fusion genes. We did not find any aberrations on chromosome 21 and any fusion genes. Two focal intronic losses, likely representing benign germline variants, were found on chromosome X. In addition to 6 missense mutations affecting genes without established roles in hematopoietic disorders, we found in-frame deletions in the GATA1 and JAK1 genes. Both mutations are novel. The GATA1 D65_C228del mutation is predicted to result in an internally truncated protein - GATA1aber. Unlike GATA1s (resulting from GATA1 mutations in DS-TMD) which lacks the transactivation domain (TAD) but retains both Zinc fingers (ZF), GATA1aber lacks part of TAD and the N-terminal ZF. Nevertheless, we hypothesize that GATA1aber substitutes the pathogenetic role of GATA1s. The JAK1 gene encodes a non-receptor tyrosine-kinase engaged in the JAK/STAT signaling pathway. The identified mutation results in the loss of phenylalanine 636 (F636del), which is located in the pseudokinase domain and belongs to a conserved amino acid triad (F636-F575-V658) that is believed to mediate a structural switch controlling the JAK1 catalytic activity (Toms, Nat Struct Mol Biol, 2013). JAK1 mutations are implicated in various hematological malignancies including acute megakaryocytic leukemia, and we hypothesize that JAK1 F636del co-operates with GATA1aber on TMD pathogenesis via deregulation of cytokine/growth factor signaling. We cloned the coding sequences of GATA1aber and JAK1 F636del and transfected them into a model cell line in which we confirmed the expression of both in-silico predicted proteins. Their subcellular trafficking was analogous to that of their wild type counterparts; GATA1aber was found in the nucleus and JAK1 F636del in both the nucleus and cytoplasm. Next, we assessed the kinase activity of JAK1 F636del. To distinguish auto- from trans-phosphorylation, we utilized the JAK1 F636del construct harboring an inactivating mutation of an ATP-binding site (K908G). The JAK1 F636del (but not JAK1 F636del + K908G) was autophosphorylated on Y1034/Y1035 and induced STATs phosphorylation both under steady-state conditions and following non-specific stimulation with PMA. However, at all studied time points all phosphorylation levels were lower compared to wild-type JAK1. Moreover, unlike constitutively active JAK1 V658I, JAK1 F636del did not confer IL3-independent growth to the murine B-cell progenitor cell line BAF3. Interestingly, the transforming potential of double-mutated JAK1 (JAK1 V658I + F636del) was enforced compared to JAK1 V658I. These data show that F636del does not lead to constitutive activation, but in the same time it is not functionally neutral. As the impact of F636del on JAK1 function may vary depending on upstream signaling, we are currently assessing JAK1 F636 kinase activity/transforming potential in BAF3 cells stably expressing the IL6 receptor, which (unlike the IL3 receptor) directly activates JAK1 upon ligand binding. In the future, we plan to study the impact of JAK1 F636del on GATA1s induced deregulation of erythroid/megakaryocytic lineage development and to demonstrate "GATA1s-like" function of GATA1aber. To conclude, we identified two novel mutations affecting GATA1 and JAK1 as likely drivers in an alternative tri21-independent TMD pathogenesis. As the pathogenetic role of tri21 has been poorly understood so far, we believe that by clarifying an alternative mechanism of TMD development, we could improve our understanding of this intriguing disease in general. Support: GAUK 86218 Disclosures No relevant conflicts of interest to declare.
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Viganò, Elena, Gerben Duns, Daisuke Ennishi, Randy D. Gascoyne, Ryan D. Morin, David W. Scott e Christian Steidl. "Recurrent IL4R Somatic Mutations in Diffuse Large B-Cell Lymphoma Lead to an Altered Gene Expression Profile and Changes in Tumor Microenvironment Composition". Blood 132, Supplement 1 (29 novembre 2018): 669. http://dx.doi.org/10.1182/blood-2018-99-110473.
Abstract (sommario):
Abstract The Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is an important and conserved pathway in lymphocytes which is activated by extracellular stimuli such as cytokines and growth factors. Aberrant activation of the JAK-STAT pathway is a hallmark of a variety of lymphomas which leads to increased proliferation/survival as well as immune evasion. Regarding the latter, it has been previously described that activation of the JAK-STAT signaling pathway can alter the secretome of lymphoma cells and the composition of the tumor microenvironment (TME). Specifically, our group reported PTPN1 loss-of-function as well as IL4R gain-of-function (p.I242N) mutations up-regulate the expression of the immune regulatory chemokine CCL17 through a STATs-dependent mechanism in primary mediastinal B cell lymphoma (PMBCL). Here, we assembled a cohort of 340 diffuse large B cell lymphoma (DLBCL) patients uniformly treated with R-CHOP to investigate JAK-STAT signaling mutations (targeted gene sequencing), copy number alteration (SNP arrays), gene expression (RNAseq) and TME composition (Cibersort, IHC). We confirmed the presence of mutations in SOCS1, STAT6 and 9p24 amplification with a frequency of 13.8%, 2.5%, 11.4%, respectively. Interestingly, we also identified the presence of somatic IL4R mutations in DLBCL, including the hotspot p.I242N mutation previously reported in PMBCL. Similarly to what was reported for other alterations in the JAK-STAT pathway, IL4R mutations were significantly enriched in GCB-DLBCL as compared to the ABC subtype, unclassified or double hit lymphomas with DLBCL morphology (p=0.045). Within the GCB group, patients carrying mutations in IL4R showed inferior disease-specific survival (p=0.029) and time to progression (p=0.023) after R-CHOP therapy. Mutational analysis revealed IL4R mutation being significantly concurrent together with mutations in ACTB, KLML6, MYC, STAT6, NLRC5, TNFAIP3 and mutually exclusive with EZH2 mutations (p<0.05). However, among those gene mutations, only mutations in IL4R risk-stratified patients with GCB DLBCL and showed inferior patient outcomes. Moreover, mutations in the extracellular and transmembrane domains of IL4R resulted in gain-of-function leading to constitutive activation of the JAK-STAT pathway in vitro. Gene expression analysis of primary patient samples carrying IL4R mutations displayed increased CCL17 expression (p=0.027), which positively correlated with the level of the T-regulatory marker FOXP3 by IHC (p=0.005). In addition, in silico TME composition analysis revealed a role of IL4R mutations in inducing changes in macrophage polarization. Specifically, we observed an enrichment of an M2-like macrophage phenotype in primary patient specimens carrying IL4R mutations (p=0.01) and a high M2/M1 ratio was significantly associated with inferior patient outcomes (time to progression, log rank p=0.027). In summary, our data suggest a common mechanism between PMBCL and DLBCL where aberrant JAK-STAT activation mediated by mutations in IL4R plays a significant role in altering chemokine expression profiles and TME changes. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Celgene: Consultancy, Honoraria; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding. Steidl:Roche: Consultancy; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: patent holding; Tioma: Research Funding; Juno Therapeutics: Consultancy.
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Gerbaulet, Alexander, Claudia Wickenhauser, Julia Scholten, Katrin Peschke, Sebastian Drube, Hans-Peter Horny, Thomas Kamradt et al. "Mast cell hyperplasia, B-cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit". Blood 117, n. 6 (10 febbraio 2011): 2012–21. http://dx.doi.org/10.1182/blood-2008-11-189605.
Abstract (sommario):
Abstract Signaling through the receptor tyrosine kinase kit controls proliferation and differentiation of hematopoietic precursor cells and mast cells. Somatic point mutations of the receptor that constitutively activate kit signaling are associated with mastocytosis and various hematopoietic malignancies. We generated a Cre/loxP-based bacterial artificial chromosome transgenic mouse model that allows conditional expression of a kit gene carrying the kitD814V mutation (the murine homolog of the most common mutation in human mastocytosis, kitD816V) driven by the kit promoter. Expression of the mutant kit in cells of adult mice, including hematopoietic precursors, caused severe mastocytosis with 100% penetrance at young age frequently associated with additional hematopoietic (mostly B lineage–derived) neoplasms and focal colitis. Restriction of transgene expression to mature mast cells resulted in a similar mast cell disease developing with slower kinetics. Embryonic expression led to a hyperproliferative dysregulation of the erythroid lineage with a high rate of perinatal lethality. In addition, most adult animals developed colitis associated with mucosal mast cell accumulation. Our findings demonstrate that the effects of constitutive kit signaling critically depend on the developmental stage and the state of differentiation of the cell hit by the gain-of-function mutation.
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Manshouri, Taghi, Zeev Estrov, Alfonso Quintas-Cardama, Jorge Cortes, Francis Giles, David Harris, Waldemar Priebe, Hagop Kantarjian e Srdan Verstovsek. "WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation." Blood 108, n. 11 (16 novembre 2006): 4885. http://dx.doi.org/10.1182/blood.v108.11.4885.4885.
Abstract (sommario):
Abstract Myeloproliferative disorders (MPDs) are characterized by proliferation of one or more myeloid cell lineages in bone marrow and peripheral blood, with relatively preserved differentiation. Recent discovery of a dominant gain-of-function mutation in the Janus kinase 2 (JAK2) gene in patients with MPDs, involving the substitution of valine for phenylalanine at position 617 of the JAK2 protein (JAK2 V617F), represents the first acquired somatic mutation in hematopoietic stem cells described in these disorders. This discovery has opened new avenues for the development of targeted therapies for MPDs. WP1066 is a small molecule, a member of a novel class of anticancer agents whose development was based upon the backbone of AG490, a tyrphostin with activity against JAK2 V617F-expressing cell lines but limited in vivo activity. We investigated the inhibitory activity of the WP1066 against the JAK2 V617F-mutant expressing erythroid leukemia HEL cell line and peripheral blood mononuclear cells from patients with polycythemia vera (PV). WP1066 significantly inhibited the phosphorylation of JAK2 and downstream signal transduction proteins STAT3, STAT5, and ERK1/2 in a dose- and time-dependent manner. It induced a time- and dose-dependent antiproliferative and pro-apoptotic effects (activation of caspase 3, release of cytochrome c, and cleavage of PARP) in the JAK2 V617F-bearing HEL cell line in the low micromolar range. Pretreatment of cells with pan-caspase inhibitor Z-VAD abolished WP1066-induced apoptosis. The expression of apoptosis related proteins bcl-2, bax, and XIAP, however, was not changed. More important, WP1066 was effective in inhibiting cell growth in clonogenic assays of mononuclear cells harboring the JAK2 V617F mutation obtained from peripheral blood of patients with PV. We conclude that WP1066 is active both in vitro and ex vivo against cells carrying the JAK2 V617F mutation and represents a solid candidate for the treatment of JAK2 V617V-expressing MPDs.
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Georgescu, Maria-Magdalena. "TAMI-32. TEMPOROSPATIAL INVASION AND GENETIC EVOLUTION FROM INFRATENTORIAL TO SUPRATENTORIAL COMPARTMENT IN DIFFUSE MIDLINE GLIOMA". Neuro-Oncology 22, Supplement_2 (novembre 2020): ii220. http://dx.doi.org/10.1093/neuonc/noaa215.920.
Abstract (sommario):
Abstract Diffuse midline gliomas (DMGs) are very aggressive pediatric brain tumors with dismal prognosis due to therapy-resistant tumor growth and invasion. We performed the first integrated histologic/genomic/proteomic analysis of 21 tumor foci from three pontine DMG cases with supratentorial dissemination. Histone H3.3 K27M was the driver mutation, usually at high variant allele fraction due to recurrent chromosome 1q copy number gain, in combination with germline variants in ATM, FANCM and MYCN genes. Both previously reported and novel recurrent copy number variations and somatic pathogenic mutations in chromatin remodeling, DNA damage response and PI3K/MAPK growth pathways were variably detected, either in multiple or isolated foci. Proteomic analysis showed global upregulation of histone H3, lack of K27 tri-methylation, and further impairment of polycomb repressive complex 2 by ASXL1 downregulation. Activation of oncogenic pathways resulted from combined upregulation of N-Myc, SOX2, p65/p50 NF-kB and STAT3 transcription factors, EGFR, FGFR2, PDGFRa/b and MET receptor tyrosine kinases, and downregulation of PHLPP1/2, PTEN and p16/INK4A tumor suppressors. Upregulation of SMAD4, PAI-1, CD44, and c-Src in multiple foci most likely contributed to invasiveness. This integrated comprehensive analysis allowed spatiotemporal modeling of tumor progression and identified two general pathways of supratentorial invasion, and a multitude of migratory subpopulations within the infratentorial compartment. It also delineated common signaling pathways and potential therapeutic targets, revealing an unsuspected activation of a multitude of oncogenic pathways that may explain the resistance of DMG to current therapies.
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Lee, Jeong Seok, Yohan An, Christopher J. Yoon, Jeong Yeon Kim, Kyung Hwan Kim, Alexandra F. Freeman, Jae-Joon Yim et al. "Germline gain-of-function mutation of STAT1 rescued by somatic mosaicism in immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like disorder". Journal of Allergy and Clinical Immunology 145, n. 3 (marzo 2020): 1017–21. http://dx.doi.org/10.1016/j.jaci.2019.11.028.
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Mikulasova, Aneta, Brian A. Walker, Christopher P. Wardell, Eileen M. Boyle, Alexander Murison, Zuzana Kufova, Ludek Pour, Petr Kuglik, Roman Hajek e Gareth J. Morgan. "Somatic Mutation Spectrum in Monoclonal Gammopathy of Undetermined Significance Compared to Multiple Myeloma". Blood 124, n. 21 (6 dicembre 2014): 3346. http://dx.doi.org/10.1182/blood.v124.21.3346.3346.
Abstract (sommario):
Abstract Introduction: Malignant transformation of normal to tumour cells is a multistep process followed by sequential aggregation of hits at different molecular levels. Genetic events including single nucleotide variants (SNVs), insertion-deletion changes (indels) as well as copy number variants (CNVs) affect the phenotype of the tumour population and consequently patient prognosis. Transformation from a symptomless state, monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) can be used as a unique model for cancer development studies. To date, there is very little data regarding the mechanisms leading to disease progression at molecular level. In our study, we performed exome sequencing together with SNP array analysis on 33 MGUS patients to describe the premalignant phenotype and compared these to advanced tumour cells at the DNA level. We hypothesised that increased genetic instability indicated MGUS patients with a high risk of progression to MM. Methods: 33 MGUS patients (M:F 1.5:3; median age 61, range: 35-86) were included in this study. Plasma cells were isolated from bone marrow by FACSAria (BD Biosciences) system using CD138, CD19 and CD56 markers to obtain a pure abnormal plasma cell population with a purity >90%. Tumour DNA was isolated using Gentra Puregene Kit and amplified using REPLI-g Midi Kit (both Qiagen); control DNA was gained from peripheral white blood cells by MagNA Pure System (Roche Diagnostics). For exome sequencing, NEBNext kit (NEB) and SureSelect Human All Exon V5 (Agilent Technologies) were used and samples were sequenced by HiSeq2000 (Illumina) using 76-bp paired end reads. Unbalanced CNVs were tested by SurePrint G3 CGH+SNP, 4x180K (Agilent Technologies). Results were compared to 463 MM patients. Results: In our analysis, we found acquired SNVs in 100% (33/33) MGUS patients with a median of 89 (range 9-315) SNVs per patient. Non-synonymous SNVs (NS-SNVs) were present in 97% (32/33) cases with a median 19 (range 0–70) NS-SNVs per patient. Overall, 42 genes were recurrently mutated in at least 2 patients and 6 genes were mutated in at least 3 cases including MUC16, IGK, TTN, KLHL6, AKAP9 and NPIPL2. We identified 7 genes which were significantly mutated in MM in our previous study including KRAS (n=2), HIST1H1E (n=2) and NRAS, DIS3, EGR1, LTB, PRKD2 (all n=1). IGH translocations were identified in 27% (9/33) of patients: t(11;14) in 12% (4/33), t(4;14) in 9% (3/33), t(14;16) in 3% (1/33) and t(14;20) in 3% (1/33). We did not find any translocations involving MYC (8q24.21) or the light chain loci IGK (2p12) and IGL (22q11.2). Using SNP arrays, unbalanced CNVs were presented in 67% (22/33) of MGUS patients and detected CNVs showed similarity to MM across the cohort. As previously described in MM, only one type of IGH translocation was found per patient and all 9 cases with IGH translocation did not have additional hyperdiploidy. Furthermore, we identified a patient with two CCND1 (p.K50T, p.E51D) mutations and a t(11;14), a case with a DIS3 (p.D488N) mutation and a 13q loss. Moreover, we noticed a co-segregation of cases t(4;14) and t(14;16) who all had a 13q loss (100%, 4/4). In contrast none of the patients (0/5) with a t(11;14) or a t(14;20) had a 13q loss. Of note 29% (7/24) patients without any IGH translocation had a 13q loss. Sixty seven percent (2/3) of patients with a t(4;14) and the one case with a t(14;16) also had a 1q gain. In comparison, none of patients with a t(11;14) (0%; 0/4) had a 1q gain. Unlike what has previously been described in MM, neither of the 2 MGUS patients with a KRAS (p.Q61L and p.A146T) mutations had a t(11;14). We also identified a patient with both a KRAS (p.Q61L) and an NRAS (p.G13R) mutation which are although not mutually exclusive, negatively correlated in MM. Importantly, we did not find any mutations in TP53, ATM, ATR and ZFHX4 genes involved in DNA repair pathway alterations which were identified as unfavourable factors in survival of MM patients. Summary: We have performed the first comprehensive analysis of 33 MGUS patients using exome sequencing together with SNP arrays and described the main genetic events that are already present in this premalignant state. We found similarities to MM in terms of SNVs, CNVs and their correlations. We identified 6 MGUS cases with NS-SNVs in potential key genes that could indicate a potential high risk to progression. Support: IGA MH CZ NT13492, OPVK CZ.1.07/2.3.00/20.0183. Disclosures Walker: Onyx Pharmaceuticals: Consultancy, Honoraria.
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Nagata, Yasunobu, Terukazu Enami, Kenji Kontani, Keisuke Kataoka, Mamiko Sakata-Yanagimoto, Akira Kitanaka, Aiko Sato et al. "Novel Biological Effects and Distinct Patterns of Rhoa Mutations in Adult T-Cell Leukemia/Lymphoma and Angioimmunoblastic T Cell Lymphoma". Blood 124, n. 21 (6 dicembre 2014): 2215. http://dx.doi.org/10.1182/blood.v124.21.2215.2215.
Abstract (sommario):
Abstract Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma (PTCL). ATL is initiated by immortalization of T-cells by human T-lymphotropic virus type I (HTLV-1) infection during early infancy, followed by an accumulation of multiple genetic hits to develop leukemia. However, little has been known about these genetic hits. Recently, frequent somatic mutations in RHOA, DNMT3A, IDH2 and TET2 have been reported in angioimmunoblastic T cell lymphoma (AITL) and PTCL not otherwise specified (PTCL-NOS), which are characterized by follicular helper T-cell phenotypes. Most of the RHOA mutations in AITL invariably cause an identical amino acid change (Gly17Val), which was shown to inhibit wild-type RHOA function in a dominant-negative manner. In addition, RHOA mutations have been also identified in gastric cancer and Burkitt lymphoma, with mutational hotspots including Tyr42 and Arg5. Interestingly, some of them were reported as working in a gain-of function manner. In the current study, we investigated the mutational status of RHOA, as well as TET2, IDH2, and DNMT3A in a total of 205 patients with ATL in order to reveal their role in ATL pathogenesis. In targeted deep sequencing, RHOA was mutated in 32 (15.6%) cases. In contrast to the exclusive G17V mutation found in AITL and PTCL-NOS, mutations in ATL was widely distributed within the regions responsible for GTP-binding with Cys16Arg being most frequently mutated (N = 11; 5.4%), although Gly17Val mutations were also found in 4 cases (1.9%). Being commonly located in the same amino acid with the GTP binding site, Ala161Pro and Ala161Glu mutations were exclusively found in ATL and AITL/PTCL-NOS, respectively. We found no significant differences in the RHOA mutational status among different ATL subtypes, and no significant prognostic impact of RHOA mutations on survival. TET2 mutations were very common and tightly associated with RHOA mutation in AITL, but much less common (N = 21; 10.2%) and may not associated with RHOA mutations in ATL. Mutations of IDH2 (N = 2; 1%) and DNMT3A (N = 2; 1%) were rare in ATL. RHOA encodes a ras-related GTP-binding protein that functions as a molecular switch in a variety of biological processes through cycling between an active (GTP-bound) state and an inactive (GDP-bound) state. Known biological functions of RHOA are related to actin organization, cell migration and transcriptional activation. Modeling of three-dimensional structures suggested that most of mutations identified in ATL located near nucleotide binding pocket, which affected the GTP-binding ability of RHOA protein. Indeed, we performed luciferase assay to measure transcriptional activities of wild-type and mutant RHOA. As previously reported, dominant-negative Gly17Val RHOA mutant suppressed transcriptional activity. In contrast, ATL specific RHOA mutant such as Cys16Arg or Ala161Pro enhanced transcriptional activity, similar to or even more than wild-type RHOA. In addition, when we assessed actin stress fiber formation in cells transduced with wild-type or mutant RHOA, Gly17Val or Ala161Glu RHOA attenuated actin stress fiber formations, whereas Cys16Arg or Ala161Pro RHOA induced actin stress fiber formation, similar to known gain-of-function Gly14Val RHOA mutant. These functional assays implicated Cys16Arg and Ala161Pro RHOA mutant showed different biological behaviors from Gly17Val and Ala161Glu RHOA mutant, indicating gain-of-function mechanisms of Cys16Arg or Ala161Pro mutants. In summary, RHOA mutations were also common in ATL as in AITL and PTCL-NOS. Nevertheless, mutational patterns and functional consequences of RHOA mutations identified in ATL were distinct from those in other PTCLs, suggesting differential driver roles of RHOA mutations in ATL compared to other PTCLs. Disclosures No relevant conflicts of interest to declare.
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Nothiger, R., M. Jonglez, M. Leuthold, P. Meier-Gerschwiler e T. Weber. "Sex determination in the germ line of Drosophila depends on genetic signals and inductive somatic factors". Development 107, n. 3 (1 novembre 1989): 505–18. http://dx.doi.org/10.1242/dev.107.3.505.
Abstract (sommario):
We have analyzed the mechanism of sex determination in the germ line of Drosophila by manipulating three parameters: (1) the ratio of X-chromosomes to sets of autosomes (X:A); (2) the state of activity of the gene Sex-lethal (Sxl), and (3) the sex of the gonadal soma. To this end, animals with a ratio of 2X:2A and 2X:3A were sexually transformed into pseudomales by mutations at the sex-determining genes Sxl (Sex-lethal), tra (transformer), tra-2 (transformer-2), or dsx (double-sex). Animals with the karyotype 2X;3A were also transformed into pseudofemales by the constitutive mutation SxlM1. The sexual phenotype of the gonads and of the germ cells was assessed by phase-contrast microscopy. Confirming the conclusions of Steinmann-Zwicky et al. (Cell 57, 157, 1989), we found that all three parameters affect sex determination in germ cells. In contrast to the soma in which sex determination is completely cell-autonomous, sex determination in the germ line has a non-autonomous component inasmuch as the sex of the soma can influence the sexual pathway of the germ cells. Somatic induction has a clear effect on 2X;2A germ cells that carry a Sxl+ allele. These cells, which form eggs in an ovary, can enter spermatogenesis in testes. Mutations that cause partial loss of function or gain of function of Sxl thwart somatic induction and, independently of the sex of the soma, dictate spermatogenesis or oogenesis, respectively. Somatic induction has a much weaker effect on 2X;3A germ cells. This ratio is essentially a male signal for germ cells which consistently enter spermatogenesis in testes, even when they carry SxlM1. In a female soma, however, SxlM1 enables the 2X;3A germ cells to form almost normal eggs. Our results show that sex determination in the germ line is more complex than in the soma. They provide further evidence that the state of Sxl, the key gene for sex determination and dosage compensation in the soma, also determines the sex of the germ cells, and that, in the germ line, the state of activity of Sxl is regulated not only by the X:A ratio, but also by somatic inductive stimuli.
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Niemöller, Christoph, Sabine Bleul, Nadja Blagitko-Dorfs, Christine Greil, Kenichi Yoshida, Rainer Claus, Dietmar Pfeifer, Seishi Ogawa, Michael Lübbert e Heiko Becker. "Single Cell Genotyping of Inv(16) AML in CBL Mutated Clonal Hematopoiesis Characterizes Clonal Architecture and Evolution of Exome Sequencing-Identified Mutations in the Protein Tyrosine Phosphatase Ptprt and Other Genes". Blood 126, n. 23 (3 dicembre 2015): 3834. http://dx.doi.org/10.1182/blood.v126.23.3834.3834.
Abstract (sommario):
Abstract INTRODUCTION: We recently described the first case of the evolution of inv(16) AML on the background of a clonal hematopoiesis due to a germline CBL mutation (defining the CBL syndrome), and we identified possibly cooperating mutations by exome sequencing (Becker et al. Blood 2014;123:1883-6). Among the mutated genes was PTPRT, encoding a protein tyrosine phosphatase that inhibits STAT3 activity and is commonly mutated in cancer (reviewed by Zhao et al. Oncogene 2015;34:3885-94). Here, we investigated the co-occurrence of mutated PTPRT with other mutated genes by single cell genotyping in order gain insights into the clonal architecture and sequence of mutation acquisition. METHODS: Exome sequencing of the bulk specimens was previously described; germline or somatic origin of mutations was verified in skin fibroblasts (Becker et al. Blood 2014;123:1883-6). For single cell genotyping, Ficoll-enriched bone marrow aspirates were DAPI stained, and single cells were placed into each well of a PCR plate using a MoFlo high speed cell sorter (Beckman Coulter). Genomic DNA was amplified by whole genome amplification (WGA) using the REPLI-g Mini Kit (Qiagen) according to a modified protocol, and subjected to PCR and Sanger sequencing of the respective mutation loci. As WGA can lead to allele dropout (ADO), we also sequenced single nucleotide polymorphisms (SNPs), that were identified by CytoScan HD array (Affymetrix) to be heterozygous in the sample and that were located nearby the respective mutation loci. RESULTS: Exome sequencing allows prediction of a possible clonal architecture based on the variant allele frequencies (VAFs). VAFs of the mutations identified in the AML were as follows: KIF14 p.V341I (VAF 51%), TMEM125 p.D113N (51%), MIOX p.W225R (46%), CAND1 p.E584* (39%), NID2 p.D319N (38%), ARF3 p.N101S (36%), PRSS16 p.R491C (36%), PTPRT p.T844M (33%), DOCK6 p.R1872_K1873insP (33%), ADAM12 p.A222V (21%), CMIP p.T323M (15%) and MYOCD p.D283N (7%); due to its germline nature, all leukemic cells harbored the CBL p.D390V mutation. In order to verify the co-occurrence of mutations in a clone and thus the clonal architecture, we performed single cell genotyping of the mutations in PTPRT as well as CAND1 and DOCK6. CAND1 and DOCK6 were selected in addition to PTPRT since their comparable VAFs did not allow identifying the sequence of acquisition. Moreover, CAND1 and DOCK6 were affected by likely deleterious mutations and were previously found mutated in AML. To control for ADO, we included the heterozygous SNPs rs2867061 (PTPRT), rs1252402 (C AND1), and rs12980863 (DOCK6) in our analyses. We analyzed 19 single cells for the 6 mutations and SNPs. This resulted in 102 successful sequencing reactions, and yielded informative results for at least 2 mutations in 12 cells and for all 3 mutations in 5 cells. Based on the concurrent presence of the wild-type allele at the mutation locus and ADO at the SNP site, 18 mutation analyses were judged to be inconclusive. Overall, our analyses confirmed that the mutations in CAND1, PTPRT and DOCK6 occurred together in the same clone. Moreover, based on the identification of cells with the presence of both CAND1 and DOCK6 mutations but presence or absence of PTPRT mutations, respectively, we concluded that PTPRT mutations were acquired after the mutations in DOCK6 and CAND1. CONCLUSION: Single cell genotyping verified the co-occurrence of PTPRT, CAND1 and DOCK6 mutations in the same AML clone and revealed a clonal hierarchy, as the PTPRT mutation was acquired after the mutations in CAND1 and DOCK6. These insights into the clonal architecture and evolution had not been possible solely based on exome sequencing and suggest that the sequential expression of mutated PTPRT may cooperate with mutated CBL and inv(16) at a late stage of AML development. Disclosures No relevant conflicts of interest to declare.
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Singh, Manu, Raymond Louie, Claire Milthopre, Thiruni Adikari, Melinda Hardy, Megan Faulks, Matt Field et al. "Multi-omic profiling in coeliac disease reveals somatic driver mutations in rogue T cell clones". Journal of Immunology 210, n. 1_Supplement (1 maggio 2023): 234.12. http://dx.doi.org/10.4049/jimmunol.210.supp.234.12.
Abstract (sommario):
Abstract It has long been proposed that the pathogenesis of human autoimmune diseases may share common origins with lymphoid cancer. However, a major hurdle in understanding the immune pathogenesis of autoimmune diseases is distinguishing self-reactive “rogue” lymphocytes from normal immune cells. In Coeliac disease, both the environmental trigger (gluten) and major autoantigen (transglutaminase 2) are well characterised, but the underlying mechanisms by which rogue lymphocytes initiate and drive disease are unknown. Here, we apply multi-omic technologies that enable detailed DNA, RNA and protein measurements at the single-cell level to profile tens of thousands of immune cells isolated from small intestine duodenum biopsies from individuals with Coeliac disease. We utilise the T-cell receptor as a natural barcode across multiple single-cell experiments to identify expanded rogue T cell clones and their gene and cell-surface protein expression profiles, along with any somatic DNA driver mutations. Strikingly, we identify in multiple patients expanded T cell clones with mRNA and cell-surface protein expression profiles of cytotoxic effector cells, that harboured missense somatic mutations in T cell lymphoma driver genes STAT3, STAT5Band DDX3X.These mutations have been described as strong gain-of-function mutations in multiple T cell lymphomas, highlighting a novel mechanism by which T cell clones escape immune tolerance and adopt a rogue phenotype in Coeliac disease. Overall, our results highlight the power of single-cell multi-omics in identifying and characterising rare pathogenic clones in a common human autoimmune disease and provide direct evidence for a shared pathogenesis of autoimmunity and lymphoid malignancy.
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Materna-Kiryluk, Anna, Agnieszka Pollak, Karol Gawalski, Aleksandra Szczawinska-Poplonyk, Zuzanna Rydzynska, Anna Sosnowska, Bożena Cukrowska et al. "Mosaic IL6ST variant inducing constitutive GP130 cytokine receptor signaling as a cause of neonatal onset immunodeficiency with autoinflammation and dysmorphy". Human Molecular Genetics 30, n. 3-4 (30 gennaio 2021): 226–33. http://dx.doi.org/10.1093/hmg/ddab035.
Abstract (sommario):
Abstract Interleukin-6 signal transducer (IL6ST) encodes the GP130 protein which transduces the proinflammatory signaling of the IL6 cytokine family through Janus kinase signal transducers and activators of transcription pathway (JAK/STAT) activation. Biallelic loss-of-function IL6ST variants cause autosomal recessive hyper-IgE syndrome or a variant of the Stuve–Wiedemann syndrome. Somatic gain-of-function IL6ST mutations, in particular, small monoallelic in-frame deletions of which the most prevalent is the IL6ST Ser187_Tyr190del, are an established cause of inflammatory hepatocellular tumors, but so far, no disease caused by such mutations present constitutively has been described. Herein, we report a pediatric proband with a novel syndrome of neonatal onset immunodeficiency with autoinflammation and dysmorphy associated with the IL6ST Tyr186_Tyr190del variant present constitutively. Tyr186_Tyr190del was found by exome sequencing and was shown to be de novo (absent in proband’s parents and siblings) and mosaic (present in approximately 15–40% of cells depending on the tissue studied—blood, urine sediment, hair bulbs and buccal swab). Functional studies were performed in the Epstein–Barr virus-immortalized patient’s B cell lymphoblastoid cell line, which carried the variant in approximately 95% of the cells. Western blot showed that the patient’s cells exhibited constitutive hyperphosphorylation of Tyr705 in STAT3, which is indicative of IL6-independent activation of GP130. Interestingly, the STAT3 phosphorylation could be inhibited with ruxolitinib as well as tofacitinib, which are clinically approved JAK1 and JAK3 (to lesser extent JAK2 and JAK1) inhibitors, respectively. Given our results and the recent reports of ruxolitinib and tofacitinib use for the treatment of diseases caused by direct activation of STAT3 or STAT1, we speculate that these drugs may be effective in the treatment of our patient’s condition.
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Makishima, Kenichi, Kashima Yukie, Yuta Kuze, Sakurako Suma, Yoshiaki Abe, Yasuhito Suehara, Tatsuhiro Sakamoto et al. "Investigation of Clonal Hematopoiesis in New Japanese Cohort". Blood 142, Supplement 1 (28 novembre 2023): 5608. http://dx.doi.org/10.1182/blood-2023-181324.
Abstract (sommario):
Introduction: Clonal hematopoiesis (CH) is an age-related change in which blood cells with somatic mutations are clonally expanded. CH is known to be a predisposing factor for various age-related diseases. However, the characteristics of blood cells with somatic mutations derived from CH at the single-cell level is not fully understood. Objective: We performed this study to explore the comprehensive properties of mutant cells derived from CH. Methods: We enrolled 51 healthy elderly individuals (male, 13; female, 38) from the Kashiwanoha cohort for this study. To investigate somatic mutations frequently mutated in CH, we designed a custom panel targeting 49 genes. Targeted deep sequencing (TDS) was performed on mononuclear cells, and T, B, and monocyte fractions of peripheral blood (PB). Error-correction process was performed on TDS data. The error-correction process to the TDS data was as follows: Firstly, read families which have more than 5 identical unique molecular identifier (UMI) amplicon were included. Second, at each position, nucleotides were compared and a consensus nucleotide was called if at least 90% the nucleotides were identical. If the agreement was below 90%, the nucleotides were changed to “N ”at that position. Third, if the “N” constituted less than 10% of a read family, it was recognized as a consensus read. Single-cell multiome analysis (sc Multiome) was performed on PB samples of 43 individuals by 10xGenomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression. Sc Multiome analysis was conducted using Seurat and Signac for quality control, integration, and clustering. Additionally, we employed long-read sequencing with the PromethION platform to identify CH mutations in the sc Multiome libraries. Results: The median age of this cohort was 72 years old (range, 50 - 85). After error correction was applied to the TDS data, a total of 56 mutations were detected in 34 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 16; TET2, 10; GNAS, 4; STAT3, 3; KRAS, 1; MYD88, 1; others, 20). The median VAF was 0.0091 (range, 0.003 to 0.164). The number of mutated genes in each individual increased with age, and individuals of under the age of 69 had significantly fewer mutations compared to those over 70 years old. Regarding the TDS data of each fraction, DNMT3A mutations were detected in both monocyte and B-cell fractions in 5 cases, while they were restricted to T-cell fraction in one case. In 3 cases, mutations were detected in all fractions. TET2 mutations were present in both monocyte and B-cell fractions in all 5 cases. On the other hand, KRAS, STAT3 and MYD88 mutations were restricted to T-, T-, and B-cell fractions, respectively. In sc Multiome data, the median number of cells included was 7931 (range, 2998 to 13413). By the sc Multiome long read sequencing, 39 mutations were detected in 22 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 5; TET2, 6; GNAS, 1; STAT3, 3; KRAS, 1; others, 22). The median rate of DNMT3A mutated cells was 0.007(range, 0.003 to 0.014). Similarly, the median TET2 mutated cell population rate was 0.007(range, 0.004 to 0.044). The average cell number of B cells, T cells, monocytes, and NK cells in sc Muliome data were 955 (range, 305 to 2275), 3875 (range, 955 to 7726), 1812 (range, 352 to 4750), and 868 (range, 289 to 6022) respectively. Among B cells, 16 mutations were detected in 13 individuals ( DNMT3A nonR882, 2; TET2, 2; STAT3, 3; others, 9). For T cells, 27 mutations were detected in 20 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 4; TET2, 4; GNAS, 1; STAT3, 3;others, 12). In Monocytes cells 24, mutations were detected in 16 individuals ( DNMT3A R882, 1; DNMT3A nonR882, 4; TET2, 5; GNAS, 1; STAT3, 3;others, 10). Lastly, among NK cells, 23 mutations were detected in 17 individuals ( DNMT3A nonR882, 5; TET2, 4; GNAS, 1; STAT3, 3;others, 10). The mutated cell populations in each cell fraction were 0.037(range, 0.014 to 0.208), 0.013 (range, 0.002 to 0.180), 0.013(range, 0.003 to 0.178), and 0.043(range, 0.009 to 0.284). Conclusion: This study clarified the detailed distribution of CH-derived mutant cells in PB of elderly individuals. We identified 17 mutations that expanded in myeloid fraction and 12 mutations expanded in lymphoid fraction. Sc Multiome analysis combined with long-read sequencing allowed us to gain a deeper understanding of CH mutated cell populations. These findings provide the valuable insights into properties of CH mutant cells.
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Al-Dewik, Nader I., Bruno Cassinat, Jean-Jacques Kiladjian, Alexander Knuth e Mohamed A. Yassin. "Targeted Exome Sequencing Identifies Novel Mutations in Familial Myeloproliferative Neoplasms Patients in the State of Qatar". Blood 124, n. 21 (6 dicembre 2014): 5570. http://dx.doi.org/10.1182/blood.v124.21.5570.5570.
Abstract (sommario):
Abstract Background: Myeloproliferative Neoplasms (MPNs) are clonal hematopoietic disorders characterized by excessive proliferation of one or more myeloid cell lineages. Philadelphia negative MPNs include Polycythemia Vera (PV), Essential Thrombocytosis (ET) & Primary Myelofibrosis (PMF). MPNs are associated with the presence JAK2 V617F mutation in 95% of PV & 50% of ET & PMF patients. Several molecular techniques such as RQ-PCR, HRM & Sequencing are currently used to detect common mutations. However, there are still significant numbers of MPNs that are negative to the most common genetic anomalies & many mutations are still unknown. The advent of Next Generation Sequencing (NGS) gives the opportunity to study relevant mutations in several genes. Aim: Utilizing NGS to identify potential genetic anomalies causing familial MPNs patients in Qatar. Methods: 6 MPNs patients from consanguineous families & 5 healthy individuals were consented into the study & peripheral blood samples were collected. gDNA was extracted & used for multiplex PCR amplification of amplicons targeting cancer associated mutations in 28 key genes (JAK2, MPL, THPO, CBL, LNK, SH2B3, NF1, SOCS1/2/3, TP53, NRAS/KRAS, NF1, IDH1/2, EZH2, ASXL1, TET2, ATM, KIT, RB, TP53, IKZF1, RUNX1, PDGFRB, TERT & CALR) using the Ion AmpliSeq Kit. NGS was performed via the Ion Torrent using the 318 chip & data was analyzed with the Torrent Suite Software. Mutation details were obtained from COSMIC database. A hg 19 sequence was used as reference. The confirmation of NGS data was performed using RQ-PCR or Sequencing. Results: 11 samples were successfully sequenced, with a mean depth of 1500 reads & the FASTQC plugin indicated good quality sequencing metrics. JAK2 V617F, JAK2 exon 12-15 & MPL (S505N, W515 L/K) negative samples tested before via RQ-PCR, HRM & sequencing were called negative by NGS. NGS identified novel deleterious mutations in MPNs patients. Out of 6 familial cases, 5 patients (P1- P5) were ET & 1 patient (P6) was PV. P1 had JAK2 V617F, ASXL1 T600P, CBFB G180S, THPO S184R &ITGA2R76Q, P2 had JAK2 V617F, MPL A554G & ATM F582L, the other three Patients (P3, P4 & P5) had CLAR K385fs*47 & one PV patient (P6) had TYK2 E1163G, ASXL1 P808H, PDGFRB P4L & TERT G300fs. Among the patients & healthy individuals, mutations/SNVs such as MPL P106L, K553N, SH2B3 L476F, ATM F1036F KIT N564S & TET2 T730R were also found Discussion & conclusion: Initial screening of known common genes (JAK2 V617F, JAK2 exon 12-15 & MPL W515 L/K) mutations did not reveal the causative mutations in 3% of 180 PV patients, 52% of 200 ET patients & 77% of 20 PMF patients. In this study, several deleterious somatic & germ-line mutations & SNVs were identified using Targeted Exome Sequencing approach. A complex combination of mutations in JAK2, THPO, ITGA2 & MPL genes occurred in ET patients & coexistence of several oncogenic events in TYK2, ASXL1, PDGFRB & TERT occurred in PV patient. This finding may also suggest that the MPNs phenotype may depend on presence of other mutations. It is worth mentioning that the presence of ATM variant in P2 is associated with increased risk of CLL. Somatic CALR type-2 mutation was identified in 3 ET (nonmutated JAK2 or MPL) patients. This mutation is 5-bp TTGTC insertion in exon 9 that generates a mutant protein with a novel C-terminal (p.K385fs*47). In patients & healthy individuals, a heterozygous germ-line mutation in exon 3 of the MPL gene (MPL P106L) has been observed. it has previously been described as a rare autosomal-dominant disorder. However, this mutation is considered to be frequent in Arabic populations, leading to severe thrombocytosis in homozygotes & occasionally to mild thrombocytosis in heterozygotes. In addition, several unreported variants of uncertain significance were identified. Our preliminary results suggested that MPNs patients in Qatar have several potential disease- associated variants & mutations. Evidences show that there exists a possibility of the disease arising out of the accumulation of genetic alterations & not as the consequence of a single genetic-hit event. This could possibly be due to the high rate of consanguineous marriages in Qatar i.e. the "Founder Effect". Our results recommended carrying out WES to explore & identify mutations which will be crucial to characterize many cases of MPNs with unknown molecular causes, gain a deep understanding of genotype-phenotype correlations & MPNs pathogenesis. Disclosures Al-Dewik: Qatar National Research Fund: Patents & Royalties, Research Funding. Yassin:Qatar National research fund: Patents & Royalties, Research Funding.
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Stockklausner, Clemens, Christin Maria Duffert, Ziwei Zhou, Anne Christine Klotter, Isabelle Nadine Kuhlee e Andreas E. Kulozik. "Mpl Gain-of-Function Mutations Can be Classified By Differential Subcellular Processing, Molecular Mechanisms, Mode of Inheritance and Clinical Impact". Blood 126, n. 23 (3 dicembre 2015): 1634. http://dx.doi.org/10.1182/blood.v126.23.1634.1634.
Abstract (sommario):
Abstract The interaction between the c-Mpl receptor and its ligand thrombopoietin (TPO) on the cell surface is crucial for the regulation of thrombopoiesis. Several mutations in the c-Mpl receptor gene have been linked to a gain-of-function resulting in thrombocytosis. We have analyzed the known gain-of-function mutations in the extracellular part of the Mpl receptor, K39N and P106L, as well as the S505N, W515K and W515L mutations in the transmembrane and juxtamembrane region, respectively. Interestingly, the latter mutations can occur as autosomal dominant and/or as somatic mutations and are known to be associated with myeloproliferative malignancies and AML, whereas the abundant K39N and the P106L mutations are the cause of autosomal recessive hereditary thrombocytosis without a known predisposition to hematologic malignancies. To date, these differences in clinical impact and mode of inheritance are poorly understood. Starting from these clinical observations, we have performed functional analyses of the described gain-of-function mutations to address the key functional properties that might explain the observed clinical differences. Three crucial stages of the c-Mpl receptor life cycle were addressed: (1) post-translational processing of the immature receptor protein and its subcellular distribution, (2) membranous expression of the mature receptor and (3) receptor internalization upon stimulation with its ligand TPO. We first analyzed the post-translational processing of the normal, the K39N and the P106L mutated receptor in comparison with receptors carrying the S505N, the W515K and W515L mutations in a HeLa cell culture model. The normal, the K39N, S505N, W515K and W515L mutated c-Mpl receptors were properly glycosylated during their transport through the Golgi apparatus, whereas the P106L mutated receptor did not enter the Golgi and was not fully glycosylated. The K39N mutant was fully glycosylated but did show different running behavior on the SDS Gel, most likely caused by post-translational modifications different from glycosylation. The S505N, the W515K and the W515L mutated receptors displayed stable surface expression in confocal microscopy and FACS analysis, whereas the P106L mutated receptor was not detectable on the cell surface. After stimulation with TPO, a decrease in mean receptor surface protein could be observed for the wild type and all mutants that were expressed on the surface, namely S505N, W515K and W515L, however not significant (p>0.05). Interestingly, our functional analyses of the TPO/c-Mpl signaling pathways in TPO stimulated c-Mpl transfected BA/F3 cells showed activation of the ERK1/2 pathway in all mutants but only weaker activation of the PI3K/m-TOR and Stat3/5 signaling pathways for the P106L mutant. By contrast, cells transfected with the wild type, the S505N, W515K and W515L c-Mpl mutants showed predominant up-regulation of the PI3K/m-TOR and Stat3/5 pathways. These results show that first, both impaired and regular receptor glycosylation and correlating subcellular distribution may occur in c-Mpl gain-of function mutants. Second, the c-Mpl gain-of-function mutants differ substantially in surface expression levels. Third, our results suggest differences in the maintenance of the TPO negative feedback loop across c-Mpl gain-of-function mutants. Indeed, in contrast to P106L, it seems likely that the TPO negative feedback-loop is preserved in the S505N, the W515K and the W515L mutants. In line with this, highly elevated TPO serum levels have only been described for P106L, but not for the other gain-of-function mutations. We hypothesize that maintenance of the TPO negative feedback-loop is sufficient to prevent dysregulation of TPO levels but not transmission of a harmful c-Mpl gain-of-function effect. Instead, the predominant activation of the PI3K/m-TOR and Stat3/5 pathways might explain the different propensity to induce hematopoietic malignancy. In summary, our findings suggest the existence of different disease causing molecular mechanisms behind the mutations' respective clinical correlates and provide the basis for an important extension to the current classification of c-Mpl mutations that is primordially based on clinical observations. Disclosures No relevant conflicts of interest to declare.
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Sanders, Mathijs A., Annelieke Zeilemakers, Jasper Koenders, Remco Hoogenboezem, François Kavelaars, Rob Henderson, Bob Lowenberg e Peter J. M. Valk. "The Gene Encoding Nuclear Erythroid Factor 2 (NFE2) Is Recurrently Mutated in Acute Myeloid Leukemia". Blood 120, n. 21 (16 novembre 2012): 1392. http://dx.doi.org/10.1182/blood.v120.21.1392.1392.
Abstract (sommario):
Abstract Abstract 1392 Background: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of various acquired (cyto)genetic aberrations in the leukemic blasts. Novel state-of-the-art sequencing technologies enable sequencing of complete disease genomes. Methods: We have used Complete Genomics (CG) next-generation sequencing to identify novel recurrent mutations in AML. We have selected a single AML case, WHO: AML with maturation, FAB: M2, karyotype 45X, -Y and a NPM1 mutation. Mutations in FLT3, CEBPA, ASXL1, IDH1, IDH2, NRAS, KRAS and DNMT3A were absent. By whole genome sequencing of the AML and its corresponding remission sample, we identified acquired mutations in the protein coding regions of 31 genes (CG somatic score >0.1), including NPM1 and PTPN11. Results: Interestingly, a frame-shift mutation in the protein coding region of the Nuclear Erythroid Factor 2 (NFE2) transcription factor gene was identified and acquisition of this mutation was confirmed by Sanger sequencing of both AML and remission samples. The complete NFE2 gene of the index AML patient was sequenced but no additional mutation was present, nor was the remaining allele affected by deletions and/or amplifications. The index mutation introduces a premature stop codon (PTC) in the NFE2 gene, upstream of the region encoding the bZIP domain. To investigate if NFE2 would be recurrently mutated in AML, we screened a cohort of 1139 AML cases by denaturing high performance liquid chromatography (dHPLC) analyses for mutations in a 350bp region surrounding the index mutation in the NFE2 gene. We identified NFE2 mutations in 5 additional cases of AML. Subsequently, we screened the complete NFE2 gene in 254 randomly selected AML cases by Roche 454 sequencing. This analysis revealed 8 NFE2 mutant cases in total. These results indicate that approximately 3.5% (8/254) of unselected primary AML cases carry NFE2 mutations. The acquisition of the NFE2 mutations was confirmed by Sanger sequencing of all NFE2mutant AML cases and their corresponding derived T cells. Frame shift mutations upstream of the NFE2 bZIP domain introducing PTCs were present in 6 out of all identified NFE2 mutant cases (n=13). The remaining cases carried non-synonymous NFE2 mutations and a single case an in-frame insertion/deletion. In our cohort of molecularly and clinically well-characterized cohort of AML patients the NFE2 mutations were not associated with any clinical characteristic or any other (cyto)genetic aberration. Discussion: It is currently unclear if the NFE2 mutations would lead to a gain-of-function or a loss-of-function. Nfe2-deficient mice lack circulating platelets and die of hemorrhage; their megakaryocytes showed no cytoplasmic platelet formation. In addition, NFE2 transgenic mice show MPNs, including thrombocytosis, and spontaneous transformation to acute myeloid leukemia. However, the NFE2 mutations did not associate with abnormal platelet counts in the affected AML cases, nor did the AML cases have consistent megakaryocyte abnormalities. Mutant NFE2 is currently functionally studied by introduction into various cell line models and mouse primary bone marrow. Conclusion: In conclusion, we have identified recurrent mutation in the transcription factor gene NFE2 in a subset of AML cases. The exact role of mutant NFE2 is currently being investigated. Disclosures: No relevant conflicts of interest to declare.
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Przychodzen, Bartlomiej, e Sandra Paulina Smieszek. "Preclinical evaluation of JAK2 specific investigational oligonucleotide for the treatment of MDS/PV." JCO Global Oncology 9, Supplement_1 (agosto 2023): 122. http://dx.doi.org/10.1200/go.2023.9.supplement_1.122.
Abstract (sommario):
122 Background: Myeloproliferative disorders (MPD) are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Polycythemia vera (PV), an acquired MPD characterized by increased blood cell mass and hematocrit and leukocyte count, is associated with incidental myelofibrosis (MF). MDS is characterized by cytopenia and the presence of morphological dysplasia of precursor and mature bone marrow blood cells. PV and MDS leave patients at risk for progression to acute myeloid leukemia. Abnormal cytokine signaling due to an aberrant JAK2-STAT pathway has a vital role in PV and MDS pathogenesis. JAK2 mutations can result in hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway promotes tumor cell proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is observed in hematologic malignancies; somatic JAK2 V617F gain-of-function mutations are found in at least 95% of PV patients and is implicated in MDS cases. We hypothesize that preventing JAK2 transcription by antisense oligonucleotide (ASO) mediated exon masking of the JAK2 intron-exon junction will result in reduced JAK2 mRNA, and thus JAK2 protein, by providing nonsense mediated decay (NMD) in the reading frame. This may alleviate disease manifestations of hematologic malignancies like PV and MDS. Methods: This study utilized a HEL cell line harboring the V617F JAK2 gain-of-function mutation. We designed a series 19mer ASO targeting JAK2 exon-intron junctions ( JAK2 intron-exon junction providing NMD in the reading frame, steric, non-RNAse H1) and tested these at a range of concentrations. The ASOs were designed with a phosphorothioate 2’-O-methoxyethyl backbone and prioritized based on in silico binding affinity and limited off-target binding. HEL cells underwent ASO treatment (1µM) and 72-hour incubation. Results: We observed significant JAK2 protein decrease (~50%) in ASO-treated samples compared to untreated samples. JAK2 qPCR results confirmed 40-60% of target transcript. STAT5 phosphorylation status further confirmed this effect, and we report a 35% pSTAT5 reduction. Furthermore, this ASO showed limited off-target effects in silico. siRNA and CRISPR knockout lysate were used as controls. Our preclinical data support this ASO as a highly specific JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. Conclusions: Around 50-60% of primary MF patients harbor the JAK2 V617F gain-of-function mutation. ASOs offer the capability to directly target JAK2 mutations with high precision and effectively reduce JAK2 protein production, without off-target kinase effects. The ability to reduce JAK2 protein may alleviate the disease burden that patients with hematologic malignancies face, resulting in a higher quality of life through prevention and treatment.
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Coppe, Alessandro, Emma I. Andersson, Andrea Binatti, Vanessa R. Gasparini, Sabrina Bortoluzzi, Michael J. Clemente, Marco Herling, Jaroslaw P. Maciejewski, Satu Mustjoki e Stefania Bortoluzzi. "Subset-Specific Recurrence of Mutations and Identification of Functional Modules Provides New Clues about the Pathogenesis of Large Granular Lymphocyte Leukemia". Blood 128, n. 22 (2 dicembre 2016): 4117. http://dx.doi.org/10.1182/blood.v128.22.4117.4117.
Abstract (sommario):
Abstract Background: Large granular lymphocyte (LGL) leukemia is a rare disease characterized by a clonal persistence of cytotoxic T cells or natural killer (NK) cells. Patients usually suffer from cytopenias and other organ-related autoimmune phenomena. These are putatively mediated by the cytotoxic LGL cells constitutively activated following an antigen-driven immune response. In addition to gain-of-function mutations in the STAT3 gene, which occur in 40-50% of patients, recurrent alterations only in the STAT5b and TNFAIP3 tumor suppressor genes have been described thus far. However, based on gene expression analyses, JAK/STAT pathway activation and deregulation of several pro-apoptotic (sphingolipid and FAS/FAS ligand) and pro-survival signaling pathways (PI3K/AKT and RAS) are common features of LGL leukemia. In this project, we aimed to characterize the genomic landscape of LGL leukemia using exome sequencing and systems genetics approaches in a patient cohort including both T- and NK-LGL cases and patients without known STAT mutations. Methods: The study cohort included 19 patients diagnosed with LGL leukemia that underwent exome sequencing analysis with matched germline controls. 13 patients had CD8+ T LGL and 3 patients CD4+ T LGL phenotype and 3 patients were NK LGL cases. From the T LGL leukemia cases CD8+ or CD4+ T cells were sorted (according to the dominant phenotype) and used as the tumor sample. In NK LGL leukemias, sorted CD3neg,CD16/56+ NK cells constituted the tumor fraction that underwent exome sequencing. Polyclonal blood lymphocytes depleted from LGL cells were used as germline controls. The exome was captured with the Nimblegen SeqCap EZ Exome Library v2.0 and the sequencing was performed with the Illumina HiSeq2000 sequencing platform. All bioinformatics steps were carried out using a custom bioinformatics pipeline. Putative somatic variants were identified by subtracting, for each patient, the ones called in the normal samples from those found in the tumor sample. After filtering by call quality and allele frequency in ExAC database, somatic variants were prioritized according to the predicted impact from the SnpEff software. Genes hit by variants putatively altering their function were finally mapped to Kegg and Reactome to generate pathway-derived meta gene networks for the identification of affected functional components. Results: 4 patients had STAT3 mutations and 4 additional cases had STAT5B mutations. In addition to STAT mutations, a number of novel somatic variants, which were recurrently mutated were discovered. These included the tumor suppressor gene FAT4, the epigenetic regulator KMT2D, as well as genes involved in the control of cell proliferation (CDC27 and ARL13B). With the systems genetics approach based on integration of pathway-derived mutated gene network topologies for identification of connected components we were able to discover affected functional modules. The main network component included key genes, which either directly interact (such as the FLT3 tyrosine kinase) or are functionally connected (such as ADCY3, ANGPT2, CD40LG, PRKCD, PTK2, KRAS, and RAB12 genes) with STAT proteins. Additional modules with putative pathogenetic relevance in LGL leukemia and mutated in the absence of STAT mutations were cell cycle control (CDC27, PLK1, CDC25B, RAD21), Notch signaling (NOTCH2, NOTCH3 and MAML3) and epigenetic regulation through histone-lysine methyltransferase activity (KMT2D and ASH1L). The comparison of various LGL leukemia subtypes revealed that the mutation burden was especially high among the CD4+ T LGL leukemia cases. Part of the genes and modules affected were shared between the different subtypes of LGL leukemia, but for example KIR2DL1 mutations were only found in CD8+ and NK LGL leukemia cases. Conclusions: With the exome sequencing and systems genetic approach we were able to discover specific gene networks, which are recurrently mutated in LGL leukemia and particularly in patients without STAT mutations. As several mutated genes are directly or indirectly connected with the STAT pathway, the data strengthen the key role of JAK/STAT signaling in LGL leukemia. The novel identified pathway modules beyond STAT networks provide intriguing insights into the pathobiology of LGL leukemia. Disclosures Maciejewski: Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau. Mustjoki:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding.
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Takeda, Yusuke, Chiaki Nakaseko, Hiroaki Tanaka, Masahiro Takeuchi, Makiko Yui, Atsunori Saraya, Satoru Miyagi et al. "Direct Activation of STAT5 by TEL-Lyn Fusion Protein Promotes Induction of Myeloproliferative Neoplasms with Myelofibrosis". Blood 116, n. 21 (19 novembre 2010): 4114. http://dx.doi.org/10.1182/blood.v116.21.4114.4114.
Abstract (sommario):
Abstract Abstract 4114 Background Myeloproliferative neoplasms (MPN), a group of hematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. The V617F somatic mutation in the Janus kinase 2 (JAK2) gene has recently been found in the majority of patients with polycythemia vera (PV) and more than half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The expression of JAK2 V617F causes a PV-like disease with myelofibrosis in a murine bone marrow (BM) transplant model. In addition, a gain-of-function c-MPL W515 mutation was described in nearly 10% of patients with JAK2 V617F-negative IMF. However, the mechanism responsible for MPD and the formation of myelofibrosis in patients without JAK2 or c-MPL mutations is still unclear. We previously identified the fusion of the TEL gene to the Lyn gene (TEL-Lyn) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of TEL-Lyn into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged Lyn kinase in the pathogenesis of MPN with myelofibrosis. However, the signaling molecules directly downstream from and activated by TEL-Lyn remain unknown. Design and Methods We examined the signaling pathways activated by TEL-Lyn by Western blotting, immunoprecipitation, and in vitro kinase assay using a TEL-Lyn kinase-dead mutant as a control. We further characterized the functional properties of Stat5-deficient HSCs transduced with TEL-Lyn by colony-forming assay and bone marrow transplantation to evaluate the role of STAT5 in TEL-Lyn-induced MPN. Results TEL-Lyn was demonstrated to be constitutively active as a kinase through autophosphorylation. In TEL-Lyn-expressing cells, STAT5, STAT3, and Akt were constitutively activated. Among these signaling molecules, STAT5 was activated most prominently and this occurred without the activation of Jak2, the major kinase for STAT5. TEL-Lyn was co-immunoprecipitated with STAT5, and STAT5 was phosphorylated when incubated with TEL-Lyn, but not with TEL-Lyn kinase-dead mutant. These results indicate that TEL-Lyn interacts with STAT5 and directly activates STAT5 both in vitro and in vivo. Of note, the capacity of TEL-Lyn to support the formation of hematopoietic colonies under cytokine-free conditions in vitro and to induce MPN with myelofibrosis in vivo was profoundly attenuated in a Stat5-null background. Conclusions In this study, we clearly showed that TEL-Lyn directly activates STAT5 and the capacity of TEL-Lyn to induce MPN with myelofibrosis was profoundly attenuated in the absence of STAT5. Our findings of TEL-Lyn in this study support the role of the Src family kinases in the regulation of STAT pathways and implicate active Lyn in the alternative pathway for STAT activation in pathological cytokine signaling. Our mouse model of MPD with myelofibrosis would be beneficial for the analysis of therapeutic approaches for myelofibrosis. Disclosures: No relevant conflicts of interest to declare.
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Mambet, Cristina, Jean-Philippe Defour, Olga Babosova, Emilie Leroy, Laura Necula, Oana Stanca, Aurelia Tatic et al. "JAK2 R1063H Variant Enhances V617F Constitutive Signaling and Favors Development of Essential Thrombocythemia with Increased Hemoglobin and Neutrophils". Blood 132, Supplement 1 (29 novembre 2018): 3066. http://dx.doi.org/10.1182/blood-2018-99-117019.
Abstract (sommario):
Abstract The clinical consequences of the highly prevalent JAK2 V617F mutation in Ph-negative myeloproliferative neoplasms (MPNs) are well studied. However, the impact on the MPN phenotype of co-occurring JAK2 variants is less well characterized. In this study, we focused on JAK2 R1063H, a variant previously reported in a pediatric case of hereditary erythrocytosis (Kapralova et al, Blood 2016) and in 3 out of 93 polycythemia vera patients that were JAK2 V617F-positive (Levine et al, Cancer Cell 2005). It has been described as a very rare polymorphism in population (frequency 0.004377). In order to gain insight into the clinical and functional relevance of coexisting JAK2 mutations, we tested a cohort of 390 JAK2 V617F-positive MPN patients for JAK2 R1063H and identified 14 carriers of both mutations. From the clinical perspective, the double-positive patients exhibited predominantly an essential thrombocythemia phenotype that was accompanied by significantly higher neutrophil granulocyte counts and hemoglobin values, when compared to those harboring only JAK2 V617F mutation. By employing targeted NGS for genes known to be involved in myeloid malignancies we found that the mutational profile and the number of additional somatic mutations in double-mutant patients seemed to be comparable to previous studies of JAK2 V617F-positive MPNs. Next, we used digital droplet PCR (ddPCR) for JAK2 R1063H and JAK2 V617F allele burden assessment. Quantification of JAK2 R1063H allele indicated that in 8 patients the variant was heterozygous, likely inherited (the percentage of the mutant allele being around 50%). In 3 patients with a high JAK2 V617F allelic burden a nearly homozygous status for R1063H was identified (a fractional abundance >80%) suggesting that one R1063H allele was inherited and the second one was acquired by uniparental disomy, as a result of mitotic recombination. In 3 other patients, the JAK2 R1063H mutation was most likely acquired due to the low percentage of the mutant allele (between 20.7% and 31.5%). For cis/trans configuration analysis of JAK2 V617F and R1063H mutations, we performed single-colony sequencing of subcloned JAK2 cDNA from 9 out of 14 patients. A cis configuration of the mutations was detected in 7 and a trans configuration in 2 cases. In one patient, the cis configuration of JAK2 mutations combined with results obtained by ddPCR (the allele burdens for R1063H and V617F were 31.5% and 52.8%, respectively) suggested that R1063H was acquired, following V617F acquisition. To assess the effect on JAK2 signaling of coexisting JAK2 V617F and R1063H mutations in cis or trans, we created human cDNA JAK2 mutants (V617F, R1063H and V617F/R1063H). STAT5 transcriptional activity of the JAK2 WT and JAK2 mutants in the presence of myeloid dimeric cytokine receptors (EPOR, given the higher hemoglobin levels, G-CSFR, given the observed neutrophilia in patients carrying JAK2 V617F and R1063H, and TPOR) measured in JAK2-deficient γ-2A cells by dual luciferase assay revealed a significantly higher constitutive activity of JAK2 V617F/R1063H (cis mutant) compared to that of JAK2 V617F, in both homozygous and heterozygous configurations and with either of the dimeric myeloid cytokine receptors. In the trans configuration of JAK2 V617F and JAK2 R1063H, we did not observe a significant difference compared to combination of JAK2 V617F and JAK2 WT. Western blot analysis assessing activated phosphorylated forms of JAK2, STAT5 and ERK1/2 demonstrated a higher level of constitutive activation of JAK2 and STAT5 generated by the double V617F/R1063H mutant versus JAK2 V617F and similar effect was shown also for ERK1/2 signaling. Our results suggest that either an acquisition of V617F in cis on a germline R1063H allele or a gain of additional JAK2 R1063H on JAK2 V617F will lead to an increase in downstream signaling by the driver JAK2 V617F. The oncogenic signaling of JAK2 V617F enhanced by the R1063H mutation has a clinical effect, leading to a significantly increased number of neutrophilic granulocytes and hemoglobin values, in agreement with higher levels of signaling via EPOR and G-CSFR. The work was supported by projects COP A1.1.4. ID: P_37_798, Contract 149/26.10.2016, (MySMIS2014+: 106774), MyeloAL, WELBIO F 44/8/5-MCF/UIG-10955, ARC: N° 16/21-073, by Ludwig Institute for Cancer Research, Salus Sanguinis Foundation, Fondation Les avions de Sébastien, Belgium and by GACR 17-05988S and MSMT LTAUSA17142. Disclosures No relevant conflicts of interest to declare.
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Przychodzen, Bart, Sandra P. Smieszek, Christos M. Polymeropoulos, Mihael Polymeropoulos e Gunther Birznieks. "Preclinical Evaluation of JAK2 Specific Investigational Oligonucleotide for the Treatment of MPNs". Blood 142, Supplement 1 (28 novembre 2023): 7138. http://dx.doi.org/10.1182/blood-2023-183035.
Abstract (sommario):
Background: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Polycythemia vera (PV), an acquired MPN characterized by increased blood cell mass and hematocrit and leukocyte count, is associated with incidental myelofibrosis (MF). Myelofibrosis (MF) is a chronic and progressive disorder characterized by bone marrow fibrosis, extramedullary hematopoiesis, leukoerythroblastosis and splenomegaly. Essential Thrombocythemia (ET) is another type of MPN associated with increased number and size of circulating platelets. All MPNs leave patients at risk for progression to leukemia. Abnormal cytokine signaling due to an aberrant JAK2-STAT pathway has a vital role in pathogenesis of various MPNs. JAK2 mutations can result in hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway promotes tumor cell proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is observed in hematologic malignancies; somatic JAK2 V617F gain-of-function mutations are found in large numbers between different subtypes of MPNs patients (40-95%) and are also implicated in MDS-MPN cases. We hypothesize that preventing JAK2 transcriptionby antisense oligonucleotide (ASO) mediated exon masking of the JAK2 intron-exon junction will result in reduced JAK2 mRNA, and thus JAK2 protein, by providing nonsense mediated decay (NMD) in the reading frame. This may alleviate disease manifestations of many patients suffering from different forms of MPNs. Methods: This study utilized a HEL92.1.7 cell line harboring the V617F JAK2 gain-of-function mutation. Antisense Oligonucleotides (ASOs) are small fragments of DNA/RNA that have the ability to reduce production of target protein via interaction with RNA. We designed a series ASOs targeting JAK2 sequence and tested these at a range of concentrations. The ASOs were designed and prioritized based on in silico binding affinity and limited off-target activity. HEL92.1.7 cells underwent treatment with several ASO-prototypes and functional readout at the end of 72h of incubation. Results: We observed significant JAK2 protein decrease (~50%) in ASO-treated samples compared to untreated samples. JAK2 qPCR results confirmed reduction of target transcript in 40-60% range. STAT5 phosphorylation status further confirmed this effect, and we report a ~35% pSTAT5 reduction. Furthermore, this ASO showed limited off-target effects in silico. siRNA and CRISPR knockout protein lysates were used as internal controls. Our preclinical data support our ASO as a highly specific JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. Conclusions: Around 50-60% of primary MF patients harbor the JAK2 V617F gain-of-function mutation. ASOs offer the capability to directly target JAK2-driven neoplasms with highest precision and effectively reduce JAK2 protein production, without off-target effects linked with small molecules targeting various kinase families. The ability to reduce JAK2 protein may alleviate the disease burden that patients with hematologic malignancies face, resulting in a higher quality of life through prevention and treatment.
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Lukes, Julius, Petr Danek, Oriol Alejo, Eliska Potuckova, Ondrej Gahura, Dirk Heckl, Julia Starkova et al. "Characterization of a Novel JAK1 Pseudokinase Mutation in the First Case of Trisomy 21-Independent GATA1-Mutated Transient Abnormal Myelopoiesis". Blood 134, Supplement_1 (13 novembre 2019): 4208. http://dx.doi.org/10.1182/blood-2019-122168.
Abstract (sommario):
Clonal proliferation of megakaryoblasts, called transient abnormal myelopoiesis (TAM), is a rare disease of newborns triggered by trisomy 21 (constitutional or somatic) together with acquired mutations of GATA1 resulting in the exclusive production of its short variant - GATA1s. No other TAM drivers have been described so far. We have diagnosed a unique TAM case with a typical clinical and laboratory manifestation but without the gain (or any other aberration) of chromosome 21. Thorough genomic profiling revealed 4 somatic mutations: GATA1 D65_C228del, JAK1 F636del, FN1 R2420C and SPIRE2 R471W. With respect to the generally accepted 2-hit theory, we hypothesized that this TAM arose from a collaboration of the atypical GATA1 mutation (not inducing GATA1s) with (at least) one of the other identified mutations. Unlike SPIRE2 and FN1 aberrations, various mutations of the JAK1 kinase have been previously described as leukemia drivers, suggesting JAK1 F636del as a top candidate for the second hit. Moreover, JAK1 mutations have been associated with the transformation of TAM into acute megakaryoblastic leukemia (Nikolaev et al., Blood, 2013). The aim of our project was to functionally characterize this novel JAK1 mutation. Phenylalanine 636 belongs to a phylogenetically conserved triad of amino acids suggested to control catalytic activity of JAK1 via mediating a switch between the supposedly active and inactive conformations (Toms et al., Nat Struct Mol Biol, 2013). Hence, F636 seems to be essential for JAK1 function. Surprisingly, homology modeling showed that loss of F636 is compatible with both functionally opposite conformations. Indeed, Western blot analysis of JAK/STAT signaling in transiently transformed HEK293T cells showed that catalytic activity is preserved in JAK1 F636del. However, we observed lower levels of auto- and STATs- phosphorylation compared to wild-type (wt) JAK1 suggesting decreased kinase activity of JAK1 F636del. Subsequently, we tested the oncogenic potential of JAK1 F636del in the Ba/F3 cell assay; unlike the known oncogenic JAK1 variant (JAK1 V658I), JAK1 F636del did not induce IL3-independent growth. To further assess phenotypic impact of F636del, we introduced JAK1 F636del into murine bone marrow and fetal liver hematopoietic stem and progenitor cells (HSPCs) using lentiviruses and performed colony forming assays. The number and morphology of colonies did not differ in JAK1 F636del compared to wt JAK1. Furthermore, we assessed the impact of JAK1 F636del in the context of mutated GATA1. We utilized the in-vitro model recently described by Labuhn et al. (Cancer Cell, 2019), in which the CRISPR/Cas9-mediated induction of Gata1s expression leads to the expansion and sustained proliferation of fetal liver HSPCs from embryonic day 13.5 ROSA26:Cas9-EGFPki/wt mice. Similar to wt JAK1, lentivirally introduced JAK1 F636del had no impact on the proliferation and maturation status of such Gata1-edited HPSCs irrespective of the timing of its introduction (simultaneously with Gata1 editing versus into fully established Gata1-edited culture) or of culturing conditions (fully cytokine-supplemented growth-supportive versus cytokine-depleted growth-restrictive medium). Altogether, we show that unlike known oncogenic variants, F636del identified in the first case of trisomy 21-independent TAM attenuates JAK1 kinase activity. The results of our phenotypic studies question the potential contribution of this mutation to TAM development. Interestingly, Labuhn et al. (2019) recently showed that non-activating JAK mutations occur at higher than random frequency in trisomy 21-dependent TAM. This tempts us to speculate that JAK1 mutations may still play a role in TAM. Yet, this role may significantly differ from that of known oncogenic mutations; it may result from attenuation/modulation instead of activation of downstream signaling and it may remain unrevealed utilizing the currently available sophisticated, yet still imperfect experimental models. Support: GAUK 86218, EHA Research Mobility Grant Disclosures No relevant conflicts of interest to declare.
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Hartert, Keenan, Saber Tadros, Alyssa Bouska, Dalia Moore, Christine Pak, Tayla Heavican, Chih Long Liu et al. "DNA Copy Number Gains of TCF4 (E2-2) Are Associated with Poor Outcome in Diffuse Large B-Cell Lymphoma". Blood 128, n. 22 (2 dicembre 2016): 2686. http://dx.doi.org/10.1182/blood.v128.22.2686.2686.
Abstract (sommario):
Abstract The development of B-cells is a complex process that proceeds through multiple stages and is regulated by a hierarchy of transcription factors and other physiologic signals. Each unique B-cell malignancy can be aligned with a 'normal counterpart' at one or more of these discrete developmental stages. However, with the exception of translocations of transcription factor genes, the genetic basis for this is not well defined. We performed an analysis of high-resolution single nucleotide polymorphism (SNP) microarrays from 694 diffuse large B-cell (DLBCL) tumors to identify significant somatic copy number alterations (SCNA). Through integrative analysis of 249 tumors with matched gene expression profiling (GEP) data, we identified the likely targets of these alterations and found that genes that were targeted by DNA copy number gain were significantly enriched for DNA binding activity and transcription factor function. We extended upon this observation by analyzing SNP microarray data of a further 2,716 tumors from 7 additional subtypes of B-cell malignancy. Through this analysis, we identified patterns of transcription factor alterations that aligned with the differentiation state of the 'normal B-cell counterpart' of each malignancy. This provides evidence that SCNA of B-cell transcription factors may underlie the differentiation state of B-cell malignancies. DLBCL can be divided into two subtypes based upon gene expression profiles that align with either the germinal center B-cell differentiation state (GCB-like) or a post-GCB activated B-cell state (ABC-like). Having observed an enrichment for transcription factor SCNAs in DLBCL, and an alignment between transcription factor alterations and differentiation states in other B-cell malignancies, we hypothesized that SCNAs of transcription factors may also underlie the etiology of these molecular subtypes. By testing for associations between SCNAs and cell of origin subtype, we identified three co-segregating DNA copy number gains that were significantly enriched in the ABC-like subtype. These included gains of the BCL6 and SPIB genes that have been previously observed to be associated with the ABC-like subtype. In addition, we found gains of the TCF4 (E2-2) gene to be significantly enriched in ABC-like tumors. In line with this, TCF4 alterations were significantly associated with reduced overall survival in cohorts of patients treated with either CHOP (n=232, P=0.009) or R-CHOP (n=197, P=0.041). B-cell receptor (BCR) signaling is a key survival pathway in ABC-like DLBCL, and the TCF4 gene has a defined role in promoting the expression of immunoglobulin (Ig) genes that encode the B-cell receptor (BCR). The analysis of paired SCNA and GEP data revealed a significantly higher expression of Ig genes in tumors with TCF4 DNA copy number gain compared to those without, suggesting that normal BCR expression may be deregulated by this genetic alteration. In addition, chromatin-immunoprecipitation sequencing (ChIP-seq) for TCF4 in ABC-like DLBCL cell lines also revealed binding of TCF4 to an Ig gene enhancer region. As BCR signaling can be altered by somatic mutations in the CARD11, CD79B and MYD88 genes, we evaluated the relative representation of these mutations and TCF4 DNA copy number gains using targeted deep sequencing of 124 DLBCL tumors. This revealed that TCF4 DNA copy number gains largely mutually excluded CARD11 mutations, but significantly co-segregated with both MYD88 (FDR=0.005) and CD79B (FDR=0.053) mutations. In addition, we observed significant co-segregation between CD79B and MYD88 mutations (FDR<0.001). This is particularly notable due to the preliminary associations between combined CD79B and MYD88 mutation status and response to an inhibitor of BCR signaling, Ibrutinib. Together these data highlight an association between SCNA of B-cell transcription factors and the differentiation state of the 'normal counterpart' of the respective malignant B-cell. In line with this, we show that DNA copy number gains of the TCF4 transcription factor are associated with the ABC-like subtype of DLBCL, significantly worse overall survival, and increased Ig expression. These characteristics, in addition to the co-association between TCF4 DNA copy number gains and somatic mutations of CD79B and MYD88, suggest that TCF4 may be an important modifier of BCR signaling and contribute to the etiology of ABC-like DLBCL. Disclosures Rosenquist: Gilead Sciences: Speakers Bureau. Lunning:TG Therapeutics: Consultancy; AbbVie: Consultancy; Gilead: Consultancy; Bristol-Myer-Squibb: Consultancy; Juno: Consultancy; Genentech: Consultancy; Spectrum: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.
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Chapuy, Bjoern, Chip Stewart, Andrew Dunford, Jaegil Kim, Kirsty Wienand, Atanas Kamburov, Gabriel Kenneth Griffin et al. "Comprehensive Genomic Analysis of Primary Mediastinal B-Cell Lymphoma". Blood 132, Supplement 1 (29 novembre 2018): 1564. http://dx.doi.org/10.1182/blood-2018-99-118135.
Abstract (sommario):
Abstract Primary mediastinal large B-cell lymphomas (PMBL) typically occur in young women who present with localized, large mediastinal masses. These tumors share certain clinical, pathomorphological and transcriptional features with classical Hodgkin lymphoma (cHL). To date, PMBL genetic analyses focused on limited sets of genes and recurrent somatic copy number alterations (SCNAs). Previously, we identified frequent 9p24.1/PD-L1/PD-L2 copy gains and increased expression of the PD-1 ligands as a genetically-defined immune escape mechanism in PMBL. The demonstrated efficacy of PD-1 blockade in relapsed/refractory PMBL led to recent FDA approval and underscored the importance of characterizing targetable genetic vulnerabilities in this disease. For these reasons, we obtained diagnostic biopsy specimens from 37 patients with PMBL (median age 34; female 70%) and performed whole exome sequencing (WES) with an expanded bait set to capture structural variants (SVs). Somatic alterations (mutations, SCNAs and SVs) were determined using established analytical pipelines including our algorithm for evaluating tumors without paired normal samples. Genes more frequently mutated than by chance, Candidate Cancer Genes (CCGs), were identified with MutSig2CV and recurrent SCNAs were defined with GISTIC2.0. SVs were characterized with a recently described 4-algorithm pipeline (Nature Medicine, 2018;24(5):679-690). First, we identified 15 CCGs (q-value <0.1) including genes with known roles in PMBL, such as IL4R and TNFAIP3 and mutational drivers in additional B-cell lymphomas (B2M, GNA13, STAT6, IKZF3, XPO1, TP53, PAX5) and other cancers (TP53, ZNF217 and XPO1). Overlaying the predicted protein changes onto available 3D protein structures highlighted the likely biological functions of specific alterations, such as mutational clustering in the STAT6 DNA-binding domain. We next analyzed the PMBL mutational signatures and identified 3 cases as hypermutators with MSI signatures, including 2 with MLH1 frameshift mutations and 1 with a nonsense PMS2 mutation. Despite the young age of the PMBL patient cohort, the majority of remaining mutations were caused by spontaneous deamination at CpGs, a genetic signature usually associated with aging. The next most prevalent mutational signatures were APOBEC and, infrequently, AID. We observed a higher median mutational density in PMBL (7.56 mutations/MB), compared to diffuse large B-cell lymphoma (DLBCL) and most solid cancers, providing a potential basis for increased neoantigen production and responsiveness to PD-1 blockade. Next, we identified 18 recurrent SCNAs, including 10 copy gains (2 focal and 8 arm level) and 8 copy losses (7 focal and 1 arm level). Copy gains of 9p24.1/PD-L1/PD-L2 were detected in 70% of cases. SVs were defined at base-pair resolution and included infrequent (2/37) tandem duplications of both PD-1 ligands and inactivating CTIIA SVs (deletions and inversions) in 10% (4/37) of cases. Although PMBL had a higher mutational density than DLBCL, the PMBL alterations involved a smaller number of median genetic drivers (9 [PMBL] vs 17 [DLBCL], respectively). Combined analyses of recurrent CCGs, SCNAs and SVs revealed that certain candidate driver genes were perturbed by multiple mechanisms. Examples include: TNFAIP3 (59% overall, 41% mutations, 24% copy loss, 6% biallelic); and B2M (51% overall, 30% mutations, 27% copy loss, 6% biallelic). Concurrent analyses of the 3 types of genetic alterations also revealed multiple bases of perturbing specific signaling pathways. In this PMBL series, 73% (27/33) of tumors exhibited one or more alterations of JAK/STAT pathway components: IL4R mutations (32%), JAK2 (9p24.1 focal copy gain [70%]) and STAT6 mutations (43%). Additionally, 59% of PMBLs had alterations of antigen presentation pathway components including B2M copy loss or mutations, copy loss of 6q21.33 (which includes the HLA class I/II loci) and SVs of CTIIA. These findings provide a genetic framework for analyzing the precise mechanism of action of PD-1 blockade in PMBL. Taken together, these findings underscore the importance of a comprehensive genomic analysis in PMBL and define additional candidate treatment targets and pathogenetic mechanisms in this disease. ____ BC, CS and AD contributed equally. GG and MAS contributed equally. Disclosures Rodig: Merck: Research Funding; KITE: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding. Shipp:Merck: Research Funding; AstraZeneca: Honoraria; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.
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Papayannidis, Cristina, Anna Ferrari, Stefania Paolini, Carmen Baldazzi, Chiara Sartor, Abbenante maria Chiara, Giovanni Marconi et al. "Very Poor Outcome and Chemoresistance of Acute Myeloid Leukemia Patients with TP53 Mutations: Correlation with Complex Karyotype and Clinical Outcome". Blood 124, n. 21 (6 dicembre 2014): 484. http://dx.doi.org/10.1182/blood.v124.21.484.484.
Abstract (sommario):
Abstract Background: AML is a heterogeneous disease. The karyotype provides important prognostic information that influences therapy and outcome. Identification of AML patients (pts) with poor prognosis such as those with complex karyotype (CK) has great interest and impact on therapeutic strategies. TP53 is the most frequently mutated gene in human tumours. TP53 mutation rate in AML was reported to be low (2.1%), but the incidence of TP53 mutations in AML with a complex aberrant karyotype is still debated. Aims: To investigate the frequency of TP53 mutations in adult AML pts, the types of mutations, the associations with recurrent cytogenetic abnormalities and their relationship with response to therapy, clinical outcome and finally their prognostic role. To this aim, we focused on a subgroup of TOT/886 AML pts treated at the Serˆgnoli Institute of Bologna between 2002 and 2013. Patients and Methods: 886 AML patients were analysed for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM1, DNMT3A, IDH1, IDH2 mutations, WT-1 expression, CBF fusion transcripts). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation Deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform. 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Numberª v7.5 (BioDiscovery). Results: Of the 886 AML patients, 172 pts were screened for TP53 mutations. Sanger sequencing analysis detected TP53 mutations in 29/172 AML patients with 36 different types of mutations; seven pts (4%) had 2 mutations. At diagnosis, the median age of TP53 mutated and wild type patients was 68 years (range 42-86), and 65 years (range 22-97) respectively. Median WBC count was 8955/mmc (range 580-74360/mmc) and 1240/mmc (range 400-238000/mmc). Conventional cytogenetics showed that: a) 52 pts (30,2%) had 3 or more chromosome abnormalities, i.e. complex karyotype; b) 71 (41,3%) presented with one or two cytogenetic abnormalities (other-AML); c) 34 pts (19,8%) had normal karyotype. Most of the TP53 mutated pts (23/29, 79.3%) had complex karyiotype, whereas only 6/29 mutated pts had “no complex Karyotype” (21% and 3% of the entire screened population, respectively). Overall, TP53 frequency was 44.2% in the complex karyotype group, suggesting a pathogenetic role of TP53 mutations in this subgroup of leukemias. As far as the types of TP53 alterations regards, the majority of mutations (32) were deleterious.. Copy Number Alterations (CNAs) analysis performed on 40 cases by Affymetrix SNP arrays showed the presence of several CNAs in all cases: they ranged from loss or gain of the full chromosome (chr) arm to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). In addition to the trisomy of the chr 8, others CNAs, located on chromosomes 5q, 3, 12, 17 are significantly associated (p = 0.05) with TP53 mutations. WES analysis was performed in 37 pts: 32 TP53 were wt while 5 pts were TP53 mutated. Interestingly, TP53 mutated patients had more incidence of complex karyotype, more aneuploidy state, more number of somatic mutations (median mutation rate 30/case vs 10/case, respectively). Regarding the clinical outcome, as previously reported (Grossmann V. et Al. Blood 2013), alterations of TP53 were significantly associated with poor outcome in terms of both overall survival (median survival: 4 and 31 months in TP53 mutated and wild type patients, respectively; p<0.0001) and relapse free-survival (RFS) (p < 0.0001). (Figure 1) Figure 1: Overall Survival curve of 172 AML patients with (red) or without (blue) TP53 mutations (p< 0.0001). Conclusions: Our data demonstrated that TP53 mutations are more frequent at diagnosis in the subgroup of complex karyotype AML (16.86%) (p< 0.0001–Fisher's exact test). They are mostly deleterious mutations and are significantly correlated with worst prognosis, fail to respond to therapy and rapidly progress. We recommend TP53 mutation screening at least in AML pts carrying either complex karyotype or chr. 8 gain. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Universitˆ 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures No relevant conflicts of interest to declare.
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Gopisetty, Apurva, Aniello Federico, Didier Surdez, Yasmine Iddir, Sakina Zaidi, Alexandra Saint-Charles, Joshua Waterfall et al. "Abstract 234: ITCC-P4: Genomic profiling and analyses of pediatric patient tumor and patient-derived xenograft (PDX) models for high throughput in vivo testing". Cancer Research 83, n. 7_Supplement (4 aprile 2023): 234. http://dx.doi.org/10.1158/1538-7445.am2023-234.
Abstract (sommario):
Abstract Advancements in state-of-the-art molecular profiling techniques have resulted in better understanding of pediatric cancers and driver events. It has become apparent that pediatric cancers are significantly more heterogeneous than previously thought as evidenced by the number of novel entities and subtypes that have been identified with distinct molecular and clinical characteristics. For most of these newly recognized entities there are extremely limited treatment options available. The ITCC-P4 consortium is an international collaboration between several European academic centers and pharmaceutical companies, with the overall aim to establish a sustainable platform of >400 molecularly well-characterized PDX models of high-risk pediatric cancers, their tumors and matching controls and to use the PDX models for in vivo testing of novel mechanism-of-action based treatments. Currently, 251 models are fully characterized, including 182 brain and 69 non-brain PDX models, representing 112 primary models, 92 relapse, 42 metastasis and 4 progressions under treatment models. Using low coverage whole-genome and whole exome sequencing, somatic mutation calling, DNA copy number and methylation analysis we aim to define genetic features in our PDX models and estimate the molecular fidelity of PDX models compared to their patient tumor. Based on DNA methylation profiling we identified 43 different tumor subgroups within 18 cancer entities. Mutational landscape analysis identified key somatic and germline oncogenic drivers. Ependymoma PDX models displayed the C11orf95-RELA fusion event, YAP1, C11orf95 and RELA structural variants. Medulloblastoma models were driven by MYCN, TP53, GLI2, SUFU and PTEN. High-grade glioma samples showed TP53, ATRX, MYCN and PIK3CA somatic SNVs, along with focal deletions in CDKN2A in chromosome 9. Neuroblastoma models were enriched for ALK SNVs and/or MYCN focal amplification, ATRX SNVs and CDKN2A/B deletions. Tumor mutational burden across entities and copy number analysis was performed to identify allele-specific copy number detection in tumor-normal pairs. Large chromosomal aberrations (deletions, duplications) detected in the PDX models were concurrent with molecular alterations frequently observed in each tumor type -isochromosome 17 was detected in 5 medulloblastoma models, while deletion of chromosome arm 1p or gain of parts of 17q in neuroblastomas which correlate with tumor progression. We observe clonal evolution of somatic variants not only in certain PDX-tumor pairs but also between disease states. The multi-omics approach in this study provides insight into the mutational landscape and patterns of the PDX models thus providing an overview of molecular mechanisms facilitating the identification and prioritization of oncogenic drivers and potential biomarkers for optimal treatment therapies. Citation Format: Apurva Gopisetty, Aniello Federico, Didier Surdez, Yasmine Iddir, Sakina Zaidi, Alexandra Saint-Charles, Joshua Waterfall, Elnaz Saberi-Ansari, Justyna Wierzbinska, Andreas Schlicker, Norman Mack, Benjamin Schwalm, Christopher Previti, Lena Weiser, Ivo Buchhalter, Anna-Lisa Böttcher, Martin Sill, Robert Autry, Frank Estermann, David Jones, Richard Volckmann, Danny Zwijnenburg, Angelika Eggert, Olaf Heidenreich, Fatima Iradier, Irmela Jeremias, Heinrich Kovar, Jan-Henning Klusmann, Klaus-Michael Debatin, Simon Bomken, Petra Hamerlik, Maureen Hattersley, Olaf Witt, Louis Chesler, Alan Mackay, Johannes Gojo, Stefano Cairo, Julia Schueler, Johannes Schulte, Birgit Geoerger, Jan J. Molenaar, David J. Shields, Hubert N. Caron, Gilles Vassal, Louis F. Stancato, Stefan M. Pfister, Natalie Jaeger, Jan Koster, Marcel Kool, Gudrun Schleiermacher. ITCC-P4: Genomic profiling and analyses of pediatric patient tumor and patient-derived xenograft (PDX) models for high throughput in vivo testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 234.
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Casolari, Debora A., Diana G. Iarossi, Carolyn M. Butcher, Sarah C. Bray, Wendy T. Parker, Chris N. Hahn, Susan Branford et al. "Aberrant Activation of Epidermal Growth Factor Receptor in MPN May Respond to the Kinase Inhibitor Gefitinib". Blood 124, n. 21 (6 dicembre 2014): 1882. http://dx.doi.org/10.1182/blood.v124.21.1882.1882.
Abstract (sommario):
Abstract Epidermal growth factor receptor (EGFR) expression is elevated in peripheral blood (PB) cells of polycythemia vera (PV) patients (Skov et al, Eur J Haematol 2011;87:54-60) and EGFR inhibitors (AEE788, erlotinib) inhibit erythroid burst-forming units (BFUE) from PV patients but not normal donors. The mechanism underlying the effect of EGFR inhibitors on MPN progenitor growth has not been established but could be due to an off-target effect on JAK2 activity (Li et al, J Biol Chem 2007;282:3428-32; Gaikwad et al, Exp Hematol 2007;35:1647-56). Therefore, we investigated the growth of BFUE in the presence and absence of the EGFR inhibitor gefitinib (10µM), which does not inhibit JAK2, and observed inhibition of growth of both erythropoietin (Epo)-dependent and -independent colonies from PB mononuclear cells (PBMNC) from PV patients but not from normal individuals. These results suggest a potential role for EGFR signalling in supporting growth and/or survival of PV progenitors. Therefore, to evaluate the possibility of somatic genetic abnormalities leading to EGFR hypersensitivity in MPN, we performed targeted exon capture and massively parallel sequencing, and sensitive mass array screening of 155 MPN patient samples. We identified a low-frequency, recurrent somatic variant of EGFR (p. C329R) in 3/155 MPN patients. The human EGFR C329 residue is homologous to the residue C359 of the C. elegans gene let-23, target of a known gain-of-function mutation (Katz et al, Mol Cell Biol 1996;16(2):529-37); it also aligns with the cysteine residue affected in the highly-transforming mutant ErbB2 C334S found in lung cancer (Greulich et al, PNAS 2012; 109:14476–14481); and it lies within the extracellular cysteine-rich region of EGFR that is the target of frequent somatic mutations in glioma. To confirm the hypothesis that the EGFR C329R mutant leads to altered cytokine response, we transduced Ba/F3 cells with empty vector, EGFR wild type (WT) or mutant constructs (BaF3/MIG, BaF3/EGFR and BaF3/EGFRC329R, respectively). Both WT and mutant receptors showed constitutive activation and transforming ability when expressed at high levels. However, BaF3/EGFRC329R cells display increased levels of STAT activation associated with a slight proliferation advantage when compared to BaF3/EGFR. Given that gefitinib inhibited the growth of both BaF3/EGFR and BaF3/EGFRC329R but did not affect BaF3/MIG cells grown in IL-3, we next compared the effect of gefitinib (10µM) on the growth of BFUE from PV patient samples with and without the EGFR C329R mutation. We observed significant inhibition of Epo-independent BFUE from all PV samples but not of Epo-dependent BFUE from normal controls (Figure A). Furthermore, genotyping of JAK2 and EGFR from the individual colonies obtained in BFUE assay (treated or not with gefitinib, 10µM) for a PV patient that is positive for EGFR C329R showed that drug treatment significantly reduced the proportion of JAK2 V617F heterozygous BFUE compared to the vehicle-treated control (chi-squared test = 0.0002, Figure B). This suggests that signalling from EGFR contributes to proliferation and/or survival in JAK2 V617F heterozygous BFUE from this patient. The results presented here are consistent with an EGFR signalling role in supporting growth of PV progenitors, particularly in the context of a heterozygous JAK2V617F mutation. STAT5 signalling is essential for PV (Walz et al, Blood 2012;119:3550-3560; Yan et al, Blood 2012;199:3539-3549) and JAK2-independent activation of STAT5 through EGFR (Quesnelle et al, J. Cell. Biochem. 2007; 102:311–319) via various mechanisms may contribute to the level of STAT5 activation required for the PV phenotype. A recent study demonstrating a role for EGFR in hematopoietic stem cells (Doan et al, Nat Med 2013;19:295-304) also highlights the potential of aberrant EGFR signalling to contribute to altered properties of MPN stem cells. Finally, given that gefitinib is currently in clinical use for treatment of solid tumors, these findings raise the possibility that gefitinib may have clinical utility in the context of MPN. Figure 1 Figure 1. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding.
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Palomero, Teresa, Lucile Couronne, Hossein Khiabanian, Mi-Yeon Kim, Alberto Ambesi, Zachary Carpenter, Francesco Abate et al. "Recurrent Rhoa Mutations In Peripheral T-Cell Lymphoma". Blood 122, n. 21 (15 novembre 2013): 846. http://dx.doi.org/10.1182/blood.v122.21.846.846.
Abstract (sommario):
Abstract Peripheral T-cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of aggressive non Hodgkin lymphomas with poor prognosis. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed whole exome sequencing of matched tumor and normal DNA samples from 12 PTCL patients including 6 PTCL not otherwise specified (PTCL-NOS) tumors, 3 angioimunoablastic (AITL) T-cell lymphomas, 2 nasal type NK-/T-cell lymphomas and one enteropathy-associated T-cell lymphoma (EATL). This analysis identified 288 candidate coding somatic mutations in 268 genes and a mean mutation load of 24 non synonymous mutations per sample (range 4 – 57). Among these we noted the presence of a recurrent heterozygous mutation in the RHOA small GTPase gene (RHOA G17V) present in two independent AITL samples and one PTCL NOS biopsy. Analysis of a broad and diverse panel of 126 PTCL samples identified the presence of the RHOA G17V allele in 32 samples with a high prevalence in AITL (24/36, 67%, P < 0.001) and PTCL NOS cases (8/44, 18%, P < 0.002). The RHOA protein belongs to the Rho family of small GTPases, a group of Ras-like proteins responsible for linking a variety of cell-surface receptors to different intracellular signaling proteins. As is the case for RAS and most other small GTPases, RHOA activation is mediated by guanine exchange factors (GEFs), which catalyze the switch of RHOA from an inactive GDP-bound to an active GTP-bound state. Thus, and to test the functional significance of the RHOA G17V mutation we analyzed the capacity of this mutant to load GTP. This analysis revealed that RHOA G17V fails to incorporate GTP in response to an activated GEF in vitro. Moreover, and consistent with its inability to bind GTP, RHOA G17V failed to interact with rhotekin, a RHOA effector protein that selectively interacts with the GTP-bound active form of RHOA. However and most notably, the lack of RHOA G17V activation is not the result of a defect in RHOA-GEF interaction as RHOA G17V pull down assays demonstrated effective binding of this mutant protein to activated GEF proteins in T-cells. Based on these results we proposed an inhibitory role for RHOA G17V via sequestration of active GEF proteins. Consistently, while forced activation of RHOA signaling by GFP-RHOA overexpression induced loss of adhesion and round cell morphology in HEK293T cells, transfection of GFP-RHOA-G17V induced increased elongation and cellular protrusions as result of RHOA inactivation. In addition, immunoflourescence analysis of actin stress fiber formation by RHOA demonstrated effective abrogation of RHOA mediated cytoskeleton remodeling in cells expressing RHOA G17V. Overall these results show novel insight on the genetic basis of PTCLs and demonstrate a prominent role for RHOA G17V in the pathogenesis of AITL via disruption of RHOA signaling. Disclosures: No relevant conflicts of interest to declare.
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Yen, Katharine, Fang Wang, Jeremy Travins, Yue Chen, Hua Yang, Kim Straley, Sung Choe et al. "AG-221 Offers a Survival Advantage In a Primary Human IDH2 Mutant AML Xenograft Model". Blood 122, n. 21 (15 novembre 2013): 240. http://dx.doi.org/10.1182/blood.v122.21.240.240.
Abstract (sommario):
Abstract Somatic point mutations in isocitrate dehydrogenase 1/2 (IDH1/2) confer a gain-of-function in cancer cells resulting in the accumulation and secretion of an onco-metabolite, R (-)-2-hydroxyglutarate (2HG). High levels of 2HG have been shown to inhibit aKG dependent dioxygenases including histone and DNA demethylases, which play a key role in regulating the epigenetic state of cells. Recently, ex vivo treatment with AGI-6780, a potent IDH2 R140Q inhibitor induced cellular differentiation of leukemic blast cells isolated from primary human AML patient samples harboring an IDH2 R140Q mutation. These data provided the first evidence that inhibition of mutant IDH2 can reverse the block in cellular differentiation conferred by high levels of 2HG and could provide a therapeutic benefit to patients. AG-221 is a potent and selective inhibitor of the IDH2 mutant enzyme and is currently being evaluated in a first-in-human study entitled: A Phase 1, Multicenter, Open-Label, Dose-Escalation, Safety, Pharmacokinetic, Pharmacodynamic, and Clinical Activity Study of Orally Administered AG-221 in Subjects with Advanced Hematologic Malignancies with an IDH2 Mutation. The compound has been demonstrated to reduce 2-HG levels by >90% and reverse histone and deoxyribonucleic acid (DNA) hypermethylation in vitro, and to induce differentiation in leukemia cell models. We evaluated the efficacy of AG-221 in a primary human AML xenograft model carrying the IDH2 R140Q mutation. This is an aggressive model with mortality from AML consistently occurring by day 80, following tail vein engraftment. Results show that AG-221 is able to potently reduce 2HG found in the bone marrow, plasma and urine of engrafted mice. Treatment also induced a dose dependent, statistically significant, survival benefit where all mice in the high dose treatment group survived to the end of study. We also saw a dose dependent proliferative burst of the human specific CD45+ blast cells followed by cellular differentiation as measured by the expression of CD11b, CD14 and CD15 and cell morphology. Furthermore, the onset of differentiation correlated with survival, whereas mice that died in the low dose groups failed to show signs of cellular differentiation. These data provide strong preclinical in vivo evidence that AG-221 may have clinical benefit for IDH2 mutant patients through the reduction of 2HG and the induction of blast differentiation. Disclosures: Yen: Agios Pharmaceuticals: Employment, Equity Ownership. Wang:Agios Pharmaceuticals: Employment, Equity Ownership. Travins:Agios Pharmaceuticals: Employment, Equity Ownership. Chen:agios Pharmaceuticals: Employment, Equity Ownership. Yang:Agios Pharmaceuticals: Employment, Equity Ownership. Straley:Agios Pharmaceuticals: Employment, Equity Ownership. Choe:agios Pharmaceuticals: Employment, Equity Ownership. Dorsch:agios Pharmaceuticals: Employment, Equity Ownership. Schenkein:agios Pharmaceuticals: Employment, Equity Ownership. Agresta:agios Pharmaceuticals: Employment, Equity Ownership. Biller:agios Pharmaceuticals: Employment, Equity Ownership. Su:agios Pharmaceuticals: Employment, Equity Ownership.
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Biancon, Giulia, Poorval Joshi, Torben Hunck, Yimeng Gao, Valentina Botti, Ashley Qin, Mukhtar Sadykov et al. "U2AF1 Driver Mutations in Hematopoietic Disorders Alter but Do Not Abrogate RNA Binding and Enlighten Structural Dependencies of the U2AF-RNA Complex". Blood 134, Supplement_1 (13 novembre 2019): 1230. http://dx.doi.org/10.1182/blood-2019-130759.
Abstract (sommario):
Among genetic aberrations responsible for ineffective hematopoiesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia, somatic mutations in splicing factors such as U2AF1 are of significant interest as they are recurrent, mutually exclusive and early occurring. U2AF1 participates in mRNA splicing through the recognition of the intronic 3' splice site, forming the U2AF complex as a heterodimer with U2AF2. Heterozygous hotspot mutations at S34 or Q157, in the two U2AF1 zinc fingers respectively, result in sequence dependent aberrant splicing, suggestive of altered RNA binding. The mechanism by which these mutations alter U2AF1-U2AF2-RNA interactions has to date not been elucidated, yet understanding the structure-function relationship is critical to devise novel therapeutic strategies that either aim to correct or exploit RNA binding and splicing defects. To address this issue, we profiled the transcriptome of HEL erythroleukemic cell lines expressing wild-type (WT) and mutant U2AF1. U2AF1 S34F and Q157R mutants induced widespread alterations in splicing patterns of 3250 and 1791 genes respectively, with an overlap of 23.8% genes. On the other hand, we observed only minor alterations in gene expression levels. Meta-analysis and comparison with published RNA sequencing datasets on U2AF1 mutants revealed both conserved and unique splicing changes, with a strong enrichment for genes involved in cell cycle (P=6.7E-15) and DNA repair (P=2.6E-5). Confirming previous literature, U2AF1 S34F preferentially leads to the exclusion of exons preceded by 3' splice sites bearing an intronic UAG motif, while U2AF1 Q157R preferentially excludes exons starting with the AGA motif (Figure 1A). Collectively, the S34F mutation has a stronger effect on splicing, ultimately decreasing the global translation state of cells. To understand how mutations eventually result in the observed splicing alterations, we also profiled with unprecedented resolution the RNA interactome of the physiological and pathological U2AF heterodimer. We first performed enhanced crosslinking immunoprecipitation (eCLIP) on U2AF1 WT, U2AF1 mutants and U2AF2. Comparison of U2AF1 and U2AF2 binding profiles revealed a high degree of similarity, suggesting that they mostly bind to RNA as a tight dimer. Only by performing fractionated eCLIP on U2AF1 we were able to isolate, at the molecular level, the individual contributions of the U2AF components in the recognition of the 3' splice site. In particular, we deconvolved the U2AF2 signal, insisting on the polypyrimidine region, and the U2AF1 signal, peaking on the AG dinucleotide at the intronic end (Figure 1B). Importantly, the S34F mutant displays an aberrant binding profile, with a specific peak on the nucleotide in position -3, matching the sequence specificity previously observed in aberrant splicing events (Figure 1A-B). Systematic analysis of bound junctions suggests a complex model where the S34F mutation does not simply abrogate the ability of U2AF1 to bind splicing junctions ending with the UAG sequence, but rather alters the conformation of the U2AF complex bound to RNA, resulting in a differential ability to effectively recruit the U2 complex. To confirm this model, we identified and validated a set of gain-of-function splice junctions in genes contributing to hemopoiesis and cell cycle, characterized by increased binding of U2AF1 S34F mutant and parallel decreased binding of U2AF2. In summary, we identified novel RNA sequence and structure determinants of U2AF complex conformation, uncovered by the binding alterations induced by the U2AF1 S34F mutation. Our data further dissect the complexities of post-transcriptional regulation and provide the basis for development of U2AF directed cancer therapies. Disclosures Hunck: B**hringer-Ingelheim Foundation.: Other: During my stay in the Halene Lab I was founded by an MD fellowship.
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Andersson, Emma I., Samuli Eldfors, Hanna L. M. Koskela, Pekka Ellonen, Thomas Olson, Andres Jerez, Michael J. Clemente et al. "Somatic PTPRT and ANGPT2 Mutations in Large Granulocyte Leukemia". Blood 120, n. 21 (16 novembre 2012): 1302. http://dx.doi.org/10.1182/blood.v120.21.1302.1302.
Abstract (sommario):
Abstract Abstract 1302 Introduction: T-cell large granular lymphocyte (T-LGL) leukemia is a rare, clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T-cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene (Koskela et al, NEJM, 2012). In the remaining T-LGL patients, the pathogenetic mutations are not known. Methods: To identify additional somatic mutations, we chose two STAT3 mutation negative T-LGL leukemia patients for exome sequencing. CD8+ T-cells were used as test cells and matched CD4+ T-cells as control. The exome was captured with the Nimblegen SeqCap EZ Exome Library v2.0 and the sequencing was performed with the Illumina HiSeq2000 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. Results: Index patient 1 was diagnosed with T-LGL leukemia at the age of 70 and a TCR repertoire assay revealed one minor T- cell clone in the leukemic sample (Vβ7.1: 28 %). Exome sequencing revealed 10 nonsynonymous nucleotide variants with p-values lower than 0.01, of which the tumor suppressor gene Protein tyrosine phosphatase (PTP) receptor T (PTPRT) had a variant frequency of 14%. PTPRT has previously been found to reverse Tyr705 phosphorylation on STAT3, a modification associated with STAT3 deactivation. In this novel mutation, a highly conserved hydrophobic valine residue is converted into methionine (V995M). The mutation occurs in the cytoplasmic part of the protein, within the tyrosine-protein phosphatase 1 domain. The PTPRT V995M mutation may thereby affect STAT3 activity by reducing dephosphorylation of Tyr705, thus increasing the expression of STAT3 target genes. Index patient 2 was a 40 year-old male with untreated T-LGL leukemia. A TCR repertoire assay showed one predominant T-cell clone in the leukemic T-cells (Vβ13.2: 70%). Exome sequencing revealed 8 nonsynonymous nucleotide variants with p-values lower than 0.01. The missense mutation K436E in Angiopoietin-2 (ANGPT2), presenting with the lowest somatic p-value (1,06−09) and highest variant frequency (34%), was the most relevant candidate involved in the pathogenesis of leukemia. The mutation occurs on the surface of ANGPT2 within the well-conserved fibrinogen C-terminal domain. This domain binds the receptor TIE2 and the change in the polarity induced by K436E mutation is likely to affect the binding of TIE2 by ANGPT2. Overexpression of ANGPT2 has previously been shown to confer an adverse prognostic factor in other forms of leukemia. While these mutations appear biologically relevant and exciting, we have not yet seen them in other LGL patients screened so far (n=80). Conclusions: Somatic mutations in the PTPRT and ANGPT2 genes may represent rare genetic causes for T-LGL leukemia. Screening for these mutations in a larger cohort of patients is warranted. The mutation in the PTPRT gene is particularly exciting as it may directly impact the STAT3 pathway, which is a common pathogenetic event in T-LGL leukemia. Inactivating mutations of the PTPRT gene may have the same functional consequence as activating mutations of STAT3 in LGL patients. 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.
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Soekojo, Cinnie Yentia, Tae-Hoon Chung, Muhammad Shaheryar Furqan e Wee-Joo Chng. "Identifying the Genomic Profile of Functional High-Risk Multiple Myeloma Patients". Blood 136, Supplement 1 (5 novembre 2020): 51–52. http://dx.doi.org/10.1182/blood-2020-136553.
Abstract (sommario):
Background: Multiple myeloma (MM) patients with suboptimal response to induction therapy or early relapse, classified as the functional high-risk (FHR) patients, have been shown to have poor outcomes. However, the current risk stratification at diagnosis has not been able to accurately identify these patients. In clinical practice, we saw patients who were not identified as being high-risk at diagnosis having refractory disease or early relapse. The aim of our study is to evaluate the genomic profile of FHR MM patients at diagnosis. Method: We evaluated newly-diagnosed MM patients in the CoMMpass dataset and divided them into 3 groups: genomic high-risk (GHR) group for patients with t(4;14) or t(14;16) or del17p13 and TP53 mutation or 1q gain and International Staging System (ISS) stage 3; FHR group for patients who were refractory to induction therapy or had early relapse within 12 months and without the markers of GHR group; and standard-risk (SR) group for patients who did not fulfil both criteria. We evaluated the genomic profile based on the differentially expressed genes (DEG), copy number aberrations (CNA), mutational signatures (MS), and gene set enrichment analysis (GSEA). Results: Of 512 evaluable patients, there were 345 patients in the SR group, 106 patients in the GHR group, and 61 patients in the FHR group. On the survival analysis, both FHR and GHR groups had significantly poorer outcomes as compared with the SR group, with FHR group being the worst (FHR: HR=5.19, p=3.42x10-11;GHR: HR=3.55, p=3.5x10-8) (Fig A) The DEGs in FHR and GHR groups were distinct. FHR patients were enriched for genes linked to centromeres, mitosis, DNA repair, C2H2-type zinc finger proteins, and proteasome complex. On the other hand, GHR patients were enriched for genes linked to ribosomal proteins, immunoglobulin proteins, and cell-cell junctions. As FHR patients could not be identified at diagnosis by clinical and genetic parameters, we applied established gene expression signatures of high-risk disease, including proliferation (PI), chromosomal instability (CIN70, CINSARC, CINGEC), centrosome (CI), cell death (HZDCD), and others (EMC92, HMCL7, IFM15, UAMS70, and UAMS80), to see if they could be used. Interestingly, none of these could identify all FHR patients. The best amongst these signatures was able to identify one-third of these patients. Indeed, about one-third of patients were not classified as high-risk by any of these signatures (Fig B). We next explored the use of machine learning methods on the 453 DEGs to identify a predictive model for the FHR group. Our predictive model based on random forest technique resulted in 19 significant genes with accuracy of 0.88, specificity of 0.94, sensitivity of 0.47, AUC - ROC of 0.70, F1 score of 0.48, and Matthews correlation coefficient of 0.42 (Fig C). In terms of CNA, FHR group was predominantly hyperdiploid. On the other hand, GHR group was mostly non-hyperdiploid with fewer gains of odd-numbered chromosomes, more pronounced 13q deletion, and increased 1q gain, as compared with FHR (p=1.45×10-10) and SR groups (p<2.2×10-16). Mutation analysis revealed that IL6-JAK-STAT3 pathway had more mutated genes per patient in FHR group, while estrogen response, KRAS, and WNT β catenin signaling pathways had more mutated genes per patient in GHR group. GHR also had higher mutational load (p=0.00331), and FGFR3 (p=1.63×10-11), PRKD2 (p=2.82×10-7), and TP53 (p=8.7×10-6) were predominantly mutated in GHR group as compared with others. MS analysis using SigProfiler with Catalogue Of Somatic Mutations in Cancer (COSMIC) reference catalogue (version 3.1) showed that as compared with SR group, FHR group had increased activity in the defective homologous recombination-based DNA damage repair signature (SBS3), while GHR group had increased activity in the AID/APOBEC family of cytidine deaminase signatures (SBS2, SBS13). Interestingly, GSEA showed enrichment in cell cycle related gene sets (G2M, E2F) in FHR group and estrogen response gene sets in GHR group (Fig D). Conclusion: Identifying FHR MM patients at diagnosis represents an unmet clinical need and in this study, we report preliminary results in developing a high-specificity classifier. Our study also shows that FHR patients have specific MS and deregulate genes in unique pathways and biological processes as compared with GHR patients. This may provide insights into the biology and potential therapeutics of this group. Figure 1 Disclosures Chng: Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Abbvie: Honoraria.
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Willekens, Christophe, Lucie Laplane, Tracy Dagher, Camélia Benlabiod, Catherine Lacout, Philippe Rameau, Cyril Catelain et al. "SRSF2-P95Hdelays Myelofibrosis Development through Altered JAK/STAT Signaling in JAK2-V617F Megakaryocytes". Blood 138, Supplement 1 (5 novembre 2021): 2544. http://dx.doi.org/10.1182/blood-2021-149757.
Abstract (sommario):
Abstract Background: The gain-of-function JAK2 V617F mutant is the most common driver mutation identified in myeloproliferative neoplasms (MPNs). Additional somatic variants, also found in other malignant hemopathies, are detected in primary myelofibrosis (MF) and supposed to contribute to fibrosis or leukemia development. One of these mutations affects SRSF2, a gene encoding a component of the splicing machinery. SRSF2 heterozygous mutation mainly affects the proline 95 residue of the protein. Its association with JAK2 V617F correlates with a reduced leukemia free survival. Whether and how SRSF2 P95 variants could favor fibrosis development in JAK2 V617F cells remained unknown. Methods & Results: To investigate how homozygous Jak2 V617F and heterozygous Srsf2 P95H could interact in the hematopoietic tissue, we generated conditional knock-in mice in which the CreERt recombinase expression was driven by the HSC-Scl promoter leading to Jak2 V617F and/or Srsf2 P95H hematopoietic-specific expression upon tamoxifen induction. Srsf2 P95H mutation initially exhibited limited effect on Jak2 V617F-induced polycythemia vera (PV) only slightly reducing erythrocytosis and leukocytosis (through a previously described decrease in B220 + B cell number). The expansion of hematopoietic stem cells (SLAM), multipotent progenitors (MPP) and megakaryocyte progenitors (MKP) observed in Jak2 V617F mice was not affected by Srsf2 status. However, while platelet count was decreasing in Jak2 V617F alone mice at later time point due to fibrosis development, Srsf2 P95H/Jak2 V617F combination further increased platelet counts correlating with a significant delay in the development of myelofibrosis. Bone marrow cells (BM) were transplanted into lethally irradiated recipient mice together with GFP-positive wild-type competitor cells and tamoxifen was administrated after transplantation. Double mutant cells initially demonstrated a limited competitive advantage over wild-type cells as compared to Jak2 V617F-only cells. However, serial transplantation revealed a rapid exhaustion of Jak2 V617F single mutant cells leading to lethal pancytopenia, which was not observed in animals transplanted with Jak2 V617F/Srsf2 P95H double mutant cells. As both monocytes and megakaryocytes (MK) were involved in fibrosis development, we further explored the role of these two cell populations. Spectral flow analysis of monocyte subsets in peripheral blood and BM failed to detect any significant change in double compared to single mutant animals. In contrast, double-mutant mice presented a significant delay in MK maturation with normalized expression of c-Mpl and ploidy. Using mass cytometry, we found ex vivo a higher proportion of MKP and MK expressing high levels of P-Stat5 in Jak2 V617F mice, which the addition of Srsf2 P95H tend to reduce, suggesting an altered Mpl/Jak2 signaling pathway. To validate the hypothesis that Srsf2 P95H negatively interfere with Jak2-mediated signaling in MK, we injected high dose of the thrombopoietin-mimetic romiplostim in mice transplanted with wild-type or Srsf2 P95H BM. Both thrombocytosis and myelofibrosis were significantly reduced in Srsf2 P95H transplanted animals. To further decipher the mechanism by which Srsf2 P95H could alter cell signaling, we performed bulk RNA sequencing on sorted MK. Pathway analysis using gene set enrichment analysis identified mostly a down-regulation of signaling pathways, including JAK/STAT signaling, in Jak2 V617F/Srsf2 P95H compared to Jak2 V617F single mutant cells. Further analysis of splicing events in Srsf2 P95H mutant cells identified an increased exon 14 skipping in Jak2, which was validated by RT-qPCR. Summary: Contrary to EZH2 mutation that promotes JAK2 V617F-induced myelofibrosis in mouse models, heterozygous Srsf2 P95H delays myelofibrosis development in Jak2 V617F-transgenic mice. Srsf2 P95H co-mutation prevents the clonal exhaustion induced by serial transplantation of JAK2 V617F BM cells. This effect is associated with a reduced signaling in MK, which may involve abnormal splicing of signaling components including Jak2 exon 14 skipping. Disclosures Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.
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Song, Jihyun, e Josef T. Prchal. "Ropeginterferon Alfa-2b Blunts Hyperactive JAK2 Activity in Polycythemia Vera and Essential Thrombocythemia". Blood 142, Supplement 1 (28 novembre 2023): 3162. http://dx.doi.org/10.1182/blood-2023-190172.
Abstract (sommario):
Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPNs) characterized by phenotype defining somatic mutations of JAK2, cMPL, and CALR genes. These mutations activate the JAK-STAT signaling pathway, leading to augmented cellular proliferation. A major cause of morbidity and mortality in PV and ET is thrombosis. Current therapy options include hydroxyurea (HU), interferon-α (IFN-α), and JAK2 inhibitors. IFN-α treatment normalizes blood cell counts, and reduces thrombotic complications in PV and ET and has been associated with a decrease in JAK2V617F variant allele frequency (VAF), indicating its selective targeting of JAK2V617F-mutated cells. IFN-α treatment also rescues the normal dormant HSCs, resulting in conversion of clonal to polyclonal myelopoiesis (Tashi, Leukemia, 2018). Ropeginterferon alfa (ropeg-IFNα)-2b is mono-pegylated IFNα-2b with greater tolerability and an extended half-life compared to Peg-IFNα-2α and is FDA approved for PV therapy. We recently discovered a patient with a novel germline JAK2R715T mutation, which causes erythrocytosis with PV-like features (Kim, ASH, 2022, Song under review in Blood). This patient had poor tolerance to HU and a JAK2 inhibitor therapy; although the latter normalized her blood cell counts, it was poorly tolerated. She has been on ropeg-IFNα-2b since Mar. 2023. Given that ropeg-IFNα-2b's known function is to reduce mutated cells (Verger, Blood Cancer J. 2018) and all her hematopoietic cells were bearing a JAK2 gain-of-function germline mutation, we did not expect a complete hematological response, but her blood counts completely normalized. This observation led us to hypothesize that ropeg-IFNα-2b induces remission by reduction of JAK2 activity. To measure JAK2 activity, we quantitated phosphorylated STAT5 (p-STAT5) levels in her erythroid progenitors (CD71 +/CD235a +) and observed a 20% reduction in these levels after ropeg-IFNα-2b treatment. This prompted us to investigate PV and ET patients with the typical JAK2V617F mutation treated with ropeg-IFNα-2b. We measured p-STAT5 in erythroid progenitors and platelets (CD61 +) of 8 PV and 5 ET patients treated with ropeg-IFNα-2b and 1 PV and 6 ET patients treated with HU. Levels of p-STAT5 were higher in patients treated with HU compared to those treated with Ropeg-IFNα-2b (Figure A and B). In healthy controls, levels of p-STAT5 were lower than those in HU treated patients but comparable to those in ropeg-IFNα-2b treated patients (Figure A and B). p-STAT5 levels showed a positive correlation with platelet counts (r=0.5235, p=0.0397) and neutrophil counts (r=0.6088, p=0.0235). p-STAT5 levels were then measured before and after ropeg-IFNα-2b treatment in 1 ET patient and showed a 20% reduction, which coincided with a decrease in platelet count from 746 x 10 9/L to 207 x 10 9/L. Peg-IFN-2α downregulates upregulated oxidative stress genes and upregulates downregulated antioxidative defense genes in MPNs (Skov, PLOS One, 2022), suggesting its potential role in reducing oxidative stress.We measured reactive oxygen species (ROS) in erythroid progenitors and found that ROS levels were significantly higher in PV and ET patients treated with HU compared to those treated with ropeg-IFNα-2b and to healthy controls (Figure C). JAK2V617F mutated cells accumulate more ROS compared to unmutated cells (Marty, Leukemia, 2013). We found a positive correlation between ROS levels and p-STAT5 levels (r=0.4778, p=0.0449), suggesting an interaction between JAK2 activity and oxidative stress in PV and ET. Our study demonstrated the impact of ropeg-IFNα-2b treatment on JAK2 activity in PV and ET. Ropeg-IFNα-2b effectively reduced JAK2 activity, leading to suppression of dysregulated cell proliferation, and decreased ROS accumulation. To minimize the influence of confounding factors that could independently affect JAK2 activity, we are now comparing samples from the same patients before and after ropeg-IFNα-2b treatment. These results suggest a novel molecular benefit of ropeg-IFNα-2b therapy in PV and ET.
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Magee, Jeffrey A., Shaina Porter e Andrew Cluster. "FLT3-ITD Has Developmental Context Specific Effects on Hematopoiesis and Myeloid Leukemogenesis". Blood 126, n. 23 (3 dicembre 2015): 843. http://dx.doi.org/10.1182/blood.v126.23.843.843.
Abstract (sommario):
Abstract Much progress has been made toward identifying the mutations that cause human acute myeloid leukemia (AML), and these studies have shown that pediatric and adult AML are often caused by different mutations. Genetic differences between pediatric and adult AML may underlie differences in outcomes and necessitate different treatment strategies, yet we have few insights into why these differences occur. One possibility is that the mechanisms that regulate normal hematopoiesis change with age, and mutations therefore have age-specific phenotypes in pre-leukemic progenitors. To fully understand how AML evolves in children and adults, and how targeting individual pathways might impact cell physiology at different ages, it is important to understand how somatic mutations interface with the normal, temporally dynamic programs that regulate hematopoiesis. The FLT3-Internal Tandem Duplication (FLT3-ITD) mutation is common in adult AML but rare in early childhood AML (30-40% of adult AML, 5-10% of AML in children <10 years old, <1% of infant AML). FLT3-ITD mutations occur late in the clonal evolution of AML cells, and they are thought to drive cell proliferation and survival. In mice, FLT3-ITD has been shown to deplete adult hematopoietic stem cells (HSCs) by promoting myeloid differentiation. This may explain why the mutation occurs late in clonal evolution - HSCs must first acquire mutations that enhance self-renewal - but it also raises the question of why infant HSCs, which have an inherently higher self-renewal capacity, do not give rise to FLT3-ITD positive AML more often than is observed. We used FLT3-ITD knock-in mice to test whether FLT3-ITD has developmental context specific effects on hematopoiesis. In adult mice, FLT3-ITD depleted the HSC pool and expanded multipotent progenitor (MPP) and myeloid progenitor populations consistent with prior studies. In fetal mice, FLT3-ITD had no effect on HSC or MPP numbers, HSC function (as determined by limit dilution transplants) or myelopoiesis. FLT3-ITD did not affect hematopoiesis until shortly after birth. These temporal differences were evident even in the presence of cooperating Runx1 mutations. To understand why fetal and adult progenitors responded differently to FLT3-ITD, we characterized signal transduction and gene expression in fetal, neonatal and adult progenitors. We found that STAT5 was activated by FLT3-ITD at all stages of development, but MAPK was activated only in post-natal progenitors concordant with the onset of HSC and myeloid phenotypes. To our surprise, conditional Stat5a/b deletion exacerbated the HSC depletion and myeloid expansion phenotypes of adult FLT3-ITD mice rather than rescuing them. This suggests that STAT5 helps to maintain adult, FLT3-ITD mutant progenitors in an undifferentiated state even as other effectors promote myeloid differentiation. We next used microarrays to test whether FLT3-ITD has age-specific effects on gene expression in HSCs and MPPs, and to identify normal temporal changes in gene expression that may modulate the FLT3-ITD phenotypes. These studies made several key points: 1) In wild type HSCs, most fetal-specific genes were inactivated and most adult-specific genes were activated between birth and P14. This transition was earlier than prior studies have suggested, and it correlated with the age at which FLT3-ITD induced HSC depletion and myeloid expansion. 2) FLT3-ITD did not alter gene expression until after birth, coincident with onset of the HSC depletion and myeloid expansion phenotypes. 3) FLT3-ITD target genes were more differentially expressed in MPPs than in HSCs, consistent with recent data suggesting that MPPs are a cell of origin for FLT3-ITD driven AML. 4) Most, but not all, FLT3-ITD target genes were STAT5 dependent. Our analyses have identified novel, adult-specific candidate effectors of FLT3-ITD. Moreover, our findings raise the question of whether fetal genetic programs can suppress FLT3-ITD driven leukemogenesis, and we have begun to address this question with gain of function models. AML cells may exhibit "context addiction" (i.e.a sustained requirement for normal adult gene products and a toxic response to fetal gene products), that could be exploited therapeutically. Disclosures No relevant conflicts of interest to declare.
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Cline, Thomas W. "A FEMALE-SPECIFIC LETHAL LESION IN AN X-LINKED POSITIVE REGULATOR OF THE DROSOPHILA SEX DETERMINATION GENE, SEX-LETHAL". Genetics 113, n. 3 (1 luglio 1986): 641–63. http://dx.doi.org/10.1093/genetics/113.3.641.
Abstract (sommario):
ABSTRACT Characterization of a partial-loss-of-function, female-specific lethal mutation has identified an X-linked genetic element (1-34.3; 10B4) that functions as a positive regulator of Sxl, a central gene controlling sex determination in Drosophila melanogaster. The name, sisterless-a, was chosen both to suggest functional similarities that exist between this gene and another positive regulator of Sxl, the maternally acting gene daughterless (da), and also to highlight an important difference; namely, that in contrast to da, it is the zygotic rather than maternal functioning of sis-a that is involved in its interaction with Sxl. As with da, the female-specific lethal phenotype of sis-a is suppressed both by SxlM #1, a gain-of-function mutant allele of the target gene, and, to a lesser extent, by a duplication of Sxl +. Mutations at sis-a, da and Sxl display female-specific dominant synergism, each enhancing the others' lethal effects. The allele specificity with respect to Sxl of these dominant interactions indicates that sis-a and da affect the same aspect of Sxl regulation. As with previous studies of da and Sxl, the masculinizing effects of loss of sis-a function are generally obscured by lethal effects, presumably related to upsets in dosage compensation. The masculinizing effects can be dissociated from lethal effects by analysis of triploid intersexes (XX AAA) or by analysis of diploid females who are also mutant for autosomal genes known to be required for the transcriptional hyperactivation associated with dosage compensation in males. Analysis of foreleg development shows that intersexuality generated by sis-a is of the mosaic type: At the level of individual cells, only male or female development is observed, never an intermediate sexual phenotype characteristic of true intersexes. Sexual development of diplo-X germline and somatic clones of sis-a tissue generated by mitotic recombination during larval stages is normal, as is the sexual phenotype of homozygous sis-a escapers. Considered in their totality, these results indicate that sis-a functions early in development to help establish the activity state of Sxl and thereby initiate the sexual pathway commitment, rather than functioning later in the processes by which Sxl maintains and expresses the sex determination decision.
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Wang, Linghua, Sabina Swierczek, Kimberly Hickman, Soo-Jin Kim, David A. Wheeler e Josef Prchal. "Molecular Characterization Of Polycythemia Vera Based On The Relationship Of JAK2V617F and 9pUPD". Blood 122, n. 21 (15 novembre 2013): 1607. http://dx.doi.org/10.1182/blood.v122.21.1607.1607.
Abstract (sommario):
Abstract The gain-of-function mutation at codon 617 of JAK2 (JAK2V617F) is the most common somatic event observed in patients with polycythemia vera (PV), occurring in over 95% of PV patients. JAK2V617F confers cytokine hypersensitivity and cytokine-independent growth of erythroid progenitors, which are characteristic features of PV. Homozygous JAK2V617F is observed in about half of PV patients, whereas it is rarely seen in essential thrombocythemia (2-4%) and other myeloproliferative neoplasms. Homozygous JAK2V617F has been assumed to result from homozygous recombination, leading to uniparental disomy on 9p (9pUPD). It has been reported that the JAK2 46/1 (GGCC) haplotype may predispose carriers to the JAK2V617F mutation, and the JAK2V617F mutation facilities the acquisition of homozygous JAK2V617F. Challenging this view is a single study reporting 9pUPD in two PV subjects with wild-type JAK2, suggesting that in these two individuals, 9pUPD might have preceded the JAK2V617F mutation (Blood. 2011;118(24):6468-6470). However, the relationship between JAK2V617F and 9pUPD, the frequency of this new PV molecular subtype, its clinical relevance, and the stability of this genotype need to be systematically defined in a larger sample cohort. To address this, we combined whole-exome sequencing (WXS) of DNA from 31 consecutive PV patients with high-resolution SNP arrays, and further validated our findings in two additional cohorts comprising 59 PV consecutive patients collected from a single institution. In addition, we investigated the stability of each molecular subtype by using serial samples collected from 25 PV patients. We obtained an average of 125x coverage on JAK2 locus by WXS (Illumina Hiseq2000) and 2225x coverage by targeted deep sequencing using Ion PGM sequencer. Analysis of these data shows that the relationship between the JAK2 locus and 9pUPD is more complex than originally assumed. We defined 4 subgroups: 41% of patients had JAK2V617F in a heterozygous state without detectable 9pUPD (Subgroup I); 43% of patients had JAK2V617F with an allelic fraction in direct proportion to the level of 9pUPD (Subgroup II; homozygous JAK2V617F); 10% of patients harbored 9pUPD at approximately twice the level of the JAK2V617F allelic burden (Subgroup III; UPD with heterozygous JAK2V617F); and a small subset (6%) of patients exhibited trisomy of 9p, generating 3 copies of the JAK2 allele by chromosome duplication (Subgroup IV). No difference in the frequency of the JAK2 46/1 (GGCC) haplotype was found among these 4 subgroups. We found that this subtype classification was stable over time in over 60% of patients, whereas it transformed among the 9pUPD-positive subtypes in the remaining patients, indicating the outgrowth of a new PV subclone. While 2 PV patients with 9pUPD and wild-type JAK2 were previously reported (Blood. 2011;118(24):6468-6470), we now show a relative high proportion of PV patients having the novel, previously not recognized JAK2 genotype; i.e. JAK2 9pUPD with heterozygous JAK2V617F mutation. Our study will provide novel perspectives on the molecular basis of the evolution of PV and a better understanding of the roles of JAK2V617F and 9pUPD in this disease. Disclosures: No relevant conflicts of interest to declare.
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Morgan, Elizabeth A., Mark N. Lee, Daniel J. DeAngelo, David P. Steensma, Richard M. Stone, Frank C. Kuo, Jon C. Aster, Christopher J. Gibson e R. Coleman Lindsley. "Systematic STAT3 Mutation Testing Identifies Patients with Unsuspected T-Cell Large Granular Lymphocytic Leukemia". Blood 128, n. 22 (2 dicembre 2016): 919. http://dx.doi.org/10.1182/blood.v128.22.919.919.
Abstract (sommario):
Abstract The initial clinical presentation of T-cell large granular lymphocytic leukemia (T-LGL) and myelodysplastic syndromes (MDS) can be similar, each characterized by unexplained peripheral cytopenias. However, these diseases are pathobiologically distinct and associated with stark differences in prognosis and therapy. T-LGL is a clonal lymphoid disorder defined by phenotypically abnormal cytotoxic T cells and an indolent clinical course, while MDS is a clonal disorder of hematopoietic stem cells defined by ineffective hematopoiesis, morphologic dysplasia, and an elevated risk of acute leukemia. Despite these differences, distinction between T-LGL and MDS can be challenging and misdiagnosis can significantly delay initiation of appropriate therapy. The recent identification of STAT3 mutations in LGL may facilitate this distinction: activating STAT3 mutations occur in 40-70% of T-LGL cases, primarily within the SH2 domain, but have not been reported in patients with MDS without concomitant T-LGL. STAT3 is included within our clinical next generation sequencing (NGS) panel, which is used to evaluate patients with known or suspected hematologic malignancies, primarily acute myeloid leukemia, MDS and myeloproliferative neoplasms, as well as various lymphocytic leukemias. We report the frequency and type of STAT3 mutations within our patient population and assess the impact of this information on diagnosis. Between 1/1/2015 and 6/30/2016, 3414 samples (primarily peripheral blood (PB) or bone marrow (BM)) from 2530 unique patients evaluated at Brigham and Women's Hospital and/or Dana-Farber Cancer Institute underwent clinical NGS with a custom, 95-gene, amplicon-based panel (PMID: 27339098). Exons 2-17 and 21-23 of the STAT3 gene were analyzed in each sample using reference transcript 1 (NM_139276). We identified 40 patients with 40 candidate STAT3 mutations (Figure 1). Based on domain localization, variant allele fraction, and population allele frequency, we classified these sequence variants as somatic SH2 domain mutations (n = 21), somatic non-SH2 domain mutations of unknown significance (n = 5) or germline variants (n = 14). Of the 21 patients with somatic SH2 domain mutations, 9 carried a prior diagnosis of T-LGL and 8 were concurrently diagnosed with T-LGL by conventional diagnostic criteria (clonal aberrant T cells in the setting of neutropenia, anemia, or lymphocytosis). The final 4 patients with STAT3 SH2 domain mutations were unexpected diagnoses of T-LGL. These 4 patients were initially referred from outside institutions for MDS based on the reported presence of unilineage erythroid dysplasia (n=2), unquantified ring sideroblasts (n=1), or pancytopenia with unspecified marrow findings (n=1). In 3 of these cases, the STAT3 mutation discovery prompted T-cell flow cytometric analysis of peripheral blood, which revealed an aberrant immunophenotype, and T-cell receptor gamma gene rearrangement studies, which were clonal; these tests are pending in the 4th case. BM evaluation was performed in 12 of 21 patients, including the 4 with suspected MDS; in all cases, the findings did not meet diagnostic criteria for MDS by expert hematopathology review and all showed a normal karyotype. Five additional cases demonstrated somatic non-SH2 domain STAT3 mutations of unknown pathobiologic significance: 3 myeloid neoplasms, 1 chronic lymphocytic leukemia, and 1 autoimmune hemolytic anemia. Additional non-STAT3 mutations were also frequently identified in tumors other than T-LGL. Our experience demonstrates that STAT3 sequencing is a critical component of the evaluation of unexplained cytopenias, and identification of a mutation can clarify ambiguous phenotypes thus averting the consequences of misdiagnosis or diagnostic delay. Notably, several series have reported that MDS and T-LGL can infrequently occur concurrently and thus identification of a STAT3 mutation and a clonal T-LGL population does not exclude the possibility of concomitant MDS. In our cohort, however, no T-LGL patient with a STAT3 mutation demonstrated pathologic evidence of MDS and the majority (19 of 21) showed no other myeloid-associated somatic mutations. In addition, 5 cases in our cohort had likely somatic, non-SH2 domain STAT3 mutations in the context of disparate clinical scenarios, suggesting that these mutations may have a pathogenic role in other hematologic malignancies, a subject of future study. Disclosures DeAngelo: Ariad: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Incyte: Consultancy; Celgene: Consultancy. Stone:Celator: Consultancy; Jansen: Consultancy; ONO: Consultancy; Agios: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Merck: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy.