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1
Xiang, Zhifu, Yu Zhao, Vesselin Mitaksov, Daved H. Fremont, Yumi Kasai, AnnaLynn Molitoris, Rhonda E. Ries, et al. "Identification of somatic JAK1 mutations in patients with acute myeloid leukemia." Blood 111, no. 9 (May 1, 2008): 4809–12. http://dx.doi.org/10.1182/blood-2007-05-090308.
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Abstract Somatic mutations in JAK2 are frequently found in myeloproliferative diseases, and gain-of-function JAK3 alleles have been identified in M7 acute myeloid leukemia (AML), but a role for JAK1 in AML has not been described. We screened the entire coding region of JAK1 by total exonic resequencing of bone marrow DNA samples from 94 patients with de novo AML. We identified 2 novel somatic mutations in highly conserved residues of the JAK1 gene (T478S, V623A), in 2 separate patients and confirmed these by resequencing germ line DNA samples from the same patients. Overexpression of mutant JAK1 did not transform primary murine cells in standard assays, but compared with wild-type JAK1, JAK1T478S, and JAK1V623A expression was associated with increased STAT1 activation in response to type I interferon and activation of multiple downstream signaling pathways. This is the first report to demonstrate somatic JAK1 mutations in AML and suggests that JAK1 mutations may function as disease-modifying mutations in AML pathogenesis.
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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, no. 3-4 (January 30, 2021): 226–33. http://dx.doi.org/10.1093/hmg/ddab035.
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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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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, no. 1 (January 3, 2020): 126. http://dx.doi.org/10.3390/cancers12010126.
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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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Lesmana, Harry, Marcela Popescu, Sara Lewis, Sushree Sangita Sahoo, Charnise Goodings-Harris, Mihaela Onciu, John Kim Choi, Clifford Takemoto, Kim E. Nichols, and Marcin Wlodarski. "Germline Gain-of-Function JAK3 Mutation in Familial Chronic Lymphoproliferative Disorder of NK Cells." Blood 136, Supplement 1 (November 5, 2020): 9–10. http://dx.doi.org/10.1182/blood-2020-142078.
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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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Lukes, Julius, Eliska Potuckova, Julia Starkova, Jan Stary, Jan Zuna, Jan Trka, and Marketa Zaliova. "Chromosome 21 Gain Is Dispensable for Transient Myeloproliferative Disorder (TMD) Development." Blood 132, Supplement 1 (November 29, 2018): 2764. http://dx.doi.org/10.1182/blood-2018-99-112078.
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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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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 (November 13, 2019): 4208. http://dx.doi.org/10.1182/blood-2019-122168.
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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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Kim, Daehong, Mikko Myllymäki, Matti Kankainen, Timo Jarvinen, Giljun Park, Roberta Bruhn, Edward L. Murphy, and Satu Mustjoki. "Somatic STAT3 Mutations in CD8+ T Cells of HTLV-2 Positive Blood Donors." Blood 138, Supplement 1 (November 5, 2021): 3133. http://dx.doi.org/10.1182/blood-2021-146326.
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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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Wahnschaffe, Linus, Till Braun, Sanna Timonen, Anil K. Giri, Alexandra Schrader, Prerana Wagle, Henrikki Almusa, et al. "JAK/STAT-Activating Genomic Alterations Are a Hallmark of T-PLL." Cancers 11, no. 12 (November 21, 2019): 1833. http://dx.doi.org/10.3390/cancers11121833.
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T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell leukemia. Recent studies detected genomic aberrations affecting JAK and STAT genes in T-PLL. Due to the limited number of primary patient samples available, genomic analyses of the JAK/STAT pathway have been performed in rather small cohorts. Therefore, we conducted—via a primary-data based pipeline—a meta-analysis that re-evaluated the genomic landscape of T-PLL. It included all available data sets with sequence information on JAK or STAT gene loci in 275 T-PLL. We eliminated overlapping cases and determined a cumulative rate of 62.1% of cases with mutated JAK or STAT genes. Most frequently, JAK1 (6.3%), JAK3 (36.4%), and STAT5B (18.8%) carried somatic single-nucleotide variants (SNVs), with missense mutations in the SH2 or pseudokinase domains as most prevalent. Importantly, these lesions were predominantly subclonal. We did not detect any strong association between mutations of a JAK or STAT gene with clinical characteristics. Irrespective of the presence of gain-of-function (GOF) SNVs, basal phosphorylation of STAT5B was elevated in all analyzed T-PLL. Fittingly, a significant proportion of genes encoding for potential negative regulators of STAT5B showed genomic losses (in 71.4% of T-PLL in total, in 68.4% of T-PLL without any JAK or STAT mutations). They included DUSP4, CD45, TCPTP, SHP1, SOCS1, SOCS3, and HDAC9. Overall, considering such losses of negative regulators and the GOF mutations in JAK and STAT genes, a total of 89.8% of T-PLL revealed a genomic aberration potentially explaining enhanced STAT5B activity. In essence, we present a comprehensive meta-analysis on the highly prevalent genomic lesions that affect genes encoding JAK/STAT signaling components. This provides an overview of possible modes of activation of this pathway in a large cohort of T-PLL. In light of new advances in JAK/STAT inhibitor development, we also outline translational contexts for harnessing active JAK/STAT signaling, which has emerged as a ‘secondary’ hallmark of T-PLL.
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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, and 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, no. 22 (December 2, 2016): 4117. http://dx.doi.org/10.1182/blood.v128.22.4117.4117.
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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, no. 21 (November 19, 2010): 4114. http://dx.doi.org/10.1182/blood.v116.21.4114.4114.
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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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Chen, Jing, Yong Zhang, Michael N. Petrus, Wenming Xiao, Alina Nicolae, Mark Raffeld, Stefania Pittaluga, et al. "Cytokine receptor signaling is required for the survival of ALK− anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations." Proceedings of the National Academy of Sciences 114, no. 15 (March 29, 2017): 3975–80. http://dx.doi.org/10.1073/pnas.1700682114.
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Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)− anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK− ALCL, we treated ALK− cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK− ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK− ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK− ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK− ALCL who are phosphorylated STAT3+.
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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, no. 23 (December 3, 2015): 606. http://dx.doi.org/10.1182/blood.v126.23.606.606.
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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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Pastore, Friederike, Aishwarya Krishnan, Henrik M. Hammarén, Olli Silvennoinen, Benedict Yan, and Ross L. Levine. "JAK2S523L, a novel gain-of-function mutation in a critical autoregulatory residue in JAK2V617F− MPNs." Blood Advances 4, no. 18 (September 21, 2020): 4554–59. http://dx.doi.org/10.1182/bloodadvances.2019001283.
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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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Hangse, Tiffany, Shalini Pullarkat, Aaron Boothby, Olga Sala Torra, Lan W. Beppu, Sophie H. R. Storz, Miranda P. G. Zalusky, et al. "Expanding the Role of SH2B3: T Cell Large Granular Lymphocytosis (LGL) and Common Variable Immune Deficiency in the Setting of Biallelic SH2B3 Mutations." Blood 144, Supplement 1 (November 5, 2024): 3919. https://doi.org/10.1182/blood-2024-204188.
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SH2B3 encodes a lymphocyte adaptor protein (LNK) involved in the negative regulation of multiple signaling pathways including hematopoiesis, with mutations reported in myeloproliferative neoplasms and B cell acute lymphoblastic lymphoma. Variants are also associated with coronary artery disease and autoimmune conditions. We report a 41-year-old male of European descent with history of immune thrombocytopenia (ITP), common variable immunodeficiency (CVID), and coronary artery disease who was found to have T cell Large Granular Lymphocytosis (LGL) in the setting of biallelic SH2B3 mutations. The patient presented with thrombocytopenia in adulthood and was diagnosed with ITP given improvement with corticosteroids. Following this diagnosis he suffered several ITP relapses which responded to corticosteroids, but subsequently developed recurrent upper respiratory infections. Additional workup revealed hypogammaglobulinemia, poor vaccine responses, and imaging concerning for granulomatous lymphocytic interstitial lung disease (GLILD) and he was diagnosed with CVID. He was a healthy child, apart from mild short stature and brachydactyly, but suffered vitiligo and frequent cutaneous fungal infections as an adult. Family history revealed early onset coronary artery disease in both parents, rheumatoid arthritis (mother), and splenectomy for unknown reasons (sister). Several years later he developed diffuse lymphadenopathy and splenomegaly with lymph node biopsy showing follicular hyperplasia. Worsening abdominal pain prompted splenectomy. Splenic and bone marrow pathology, B cell immunophenotyping, lymphocyte subsets, molecular testing including mutation panels and B and T cell clonality were processed as routine clinical specimens. Fibroblasts from patient skin biopsy were cultured and sequenced. Long read sequencing (LRS) on Oxford Nanopore (ONT) PromethION was done using R10.4.1 flow cells and LSK114 ligation kit and on a ONT P2 solo in duplicate library preps and combined for data analysis. Targeted sequencing of the parents and high resolution immunophenotyping by flow cytometry are planned. Splenic pathology showed white pulp expansion though workup for lymphoproliferative disorders was negative. Sustained post splenectomy thrombocytosis prompted a bone marrow biopsy, which showed myeloid and megakaryocyte hyperplasia, mild atypia in erythroids and megakaryocytes, and an atypical lymphoid infiltrate. Heme gene panel revealed two missense variants in SH2B3, one pathogenic (c.1183G>A, p.E395K, VAF 48%) and one of uncertain significance (VUS) (c.232G>A, p.E78K, VAF 52%). Peripheral blood flow revealed a clonal population of CD8+ large granular lymphocytes (T-LGL). STAT3 and STAT5B somatic genetic testing was negative. Total B and T cells were increased (total B+T 20.3 K/mm3). CD4/CD8 ratio was reduced (0.85). B cell immunophenotyping was consistent with CVID, including increased immature B cells, CD21-low anergic activated B cells, and virtual absence of class-switched memory B cells. Both SH2B3 variants were identified in fibroblast-derived DNA, supporting germline inheritance. Analysis of phased LRS data from fibroblast derived-DNA confirmed the variants are in trans configuration. This case represents several novel associations between SH2B3 variants and disease states (CVID, and T-LGL). The c.1183G>A pathogenic variant is associated with primary familial polycythemia, thrombocythemia, and erythrocytosis. The c.232G>A VUS has been reported in the setting of idiopathic erythrocytosis and we suspect together they explain his MPN-like phenotype and are related to his significant coronary disease. History of CVID may also be related, as monogenic drivers including STAT3 gain of function are increasingly recognized and loss of LNK likely results in increased JAK/STAT signaling which may create an analogous immune milieu. Similarly, T-LGL is postulated to arise from dysregulated apoptosis, with increased JAK-STAT signaling as an initial step. Further research is needed to examine the functional implications of these variants on the development of CVID and T-LGL.
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Viganò, Elena, Gerben Duns, Daisuke Ennishi, Randy D. Gascoyne, Ryan D. Morin, David W. Scott, and 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 (November 29, 2018): 669. http://dx.doi.org/10.1182/blood-2018-99-110473.
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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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Flex, Elisabetta, Valentina Petrangeli, Lorenzo Stella, Sabina Chiaretti, Tekla Hornakova, Laurent Knoops, Cristina Ariola, et al. "Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia." Journal of Experimental Medicine 205, no. 4 (March 24, 2008): 751–58. http://dx.doi.org/10.1084/jem.20072182.
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Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3–independent growth in Ba/F3 cells and/or IL-9–independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.
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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, no. 1_Supplement (May 1, 2020): 142.35. http://dx.doi.org/10.4049/jimmunol.204.supp.142.35.
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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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Mottok, Anja, Christoph Renné, Klaus Willenbrock, Martin-Leo Hansmann, and Andreas Bräuninger. "Somatic hypermutation of SOCS1 in lymphocyte-predominant Hodgkin lymphoma is accompanied by high JAK2 expression and activation of STAT6." Blood 110, no. 9 (November 1, 2007): 3387–90. http://dx.doi.org/10.1182/blood-2007-03-082511.
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Abstract Aberrant activities of JAK/STAT signaling pathways have been observed in several hematologic malignancies. Here, we show high expression of JAK2 in the tumor cells of lymphocyte-predominant Hodgkin lymphoma in 85% of cases and activation of JAK2 in 39% of cases. STAT6, which is a target of JAK2, was activated in 50% of the cases. SOCS1 controls JAK2 activity and degradation. Mutations in SOCS1 of either somatic or germ-line origin were observed in micromanipulated tumor cells of 50% of cases. Most mutations truncated SOCS1 or caused replacement of amino acids in functional important regions. Activating mutations in exon 12 of JAK2, which are frequent in myeloproliferative diseases, were not observed. In lymphocyte-predominant Hodgkin lymphoma SOCS1 function may thus be frequently impaired by mutations, and this may contribute to high JAK2 expression and activation of the JAK2/STAT6 pathway.
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Przychodzen, Bartlomiej, and Sandra Paulina Smieszek. "Preclinical evaluation of JAK2 specific investigational oligonucleotide for the treatment of MDS/PV." JCO Global Oncology 9, Supplement_1 (August 2023): 122. http://dx.doi.org/10.1200/go.2023.9.supplement_1.122.
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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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Przychodzen, Bart, Sandra P. Smieszek, Christos M. Polymeropoulos, Mihael Polymeropoulos, and Gunther Birznieks. "Preclinical Evaluation of JAK2 Specific Investigational Oligonucleotide for the Treatment of MPNs." Blood 142, Supplement 1 (November 28, 2023): 7138. http://dx.doi.org/10.1182/blood-2023-183035.
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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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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 (November 29, 2018): 3066. http://dx.doi.org/10.1182/blood-2018-99-117019.
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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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Nabhani, Schafiq, Hagit Miskin, Cyrill Schipp, Dan Harlev, Shoshana Revel-Vilk, Michael Gombert, Sebastian Ginzel, Arndt Borkhardt, Polina Stepensky, and Ute Fischer. "Activating Mutation of STAT3 Protects Lymphocytes from Apoptosis and Leads to a Clinical Phenotype Resembling the Autoimmune Lymphoproliferative Syndrome." Blood 126, no. 23 (December 3, 2015): 2218. http://dx.doi.org/10.1182/blood.v126.23.2218.2218.
Full textAbstract:
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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Al-Dewik, Nader I., Bruno Cassinat, Jean-Jacques Kiladjian, Alexander Knuth, and Mohamed A. Yassin. "Targeted Exome Sequencing Identifies Novel Mutations in Familial Myeloproliferative Neoplasms Patients in the State of Qatar." Blood 124, no. 21 (December 6, 2014): 5570. http://dx.doi.org/10.1182/blood.v124.21.5570.5570.
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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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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 (November 5, 2021): 2544. http://dx.doi.org/10.1182/blood-2021-149757.
Full textAbstract:
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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Awan-Toor, Sarah, Jimena Castorena, Denis Clay, Juliette Soret-Dulphy, Rafael Daltro De Oliveira, Ludovic Drouet, Hélène Pasquer, et al. "SH2B3 Loss of Function Variants Are Potential Drivers of Severe Thrombocytosis." Blood 144, Supplement 1 (November 5, 2024): 1755. https://doi.org/10.1182/blood-2024-210339.
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Introduction:LNK/SH2B3 adaptor protein is an important negative regulator of cytokine signaling pathways in the hematopoietic and vascular system. The identification of acquired and germline SH2B3 mutations alone or in co-existence with known driver mutations in myeloid and lymphoid malignancies, autoimmune and cardiovascular disorders confirmed LNK implication in these neoplastic and chronic inflammatory diseases (Maslah, N., et al., Leukemia, 2017). Platelet dysfunction and megakaryocyte hyperplasia are two biological features of Sh2b3 KO mice; the latter phenotype being recently observed in two SH2B3 TN MPN cases (Zhang, X., et al., AJH, 2024). However, association of SH2B3 loss of function (LoF) variants with thrombocytosis and thrombotic clinical traits, in the absence of a driver mutation, has not yet been reported. Our aim was to identify LoF SH2B3 variants associated with TN thrombocytosis and to examine their mechanism of action by characterizing affected patients, murine and human cell line models. Methods:A comprehensiveNGS panel of 36 genes involved in myeloproliferative neoplasms (MPN) pathogenesis wasperformed to screen a cohort of 117 patients with TN thrombocytosis followed in our institution. Clinical and biological data, as well as blood samples were obtained from patients carrying an SH2B3 variant. Nail DNA was extracted to confirm or not germline origin. Platelets isolated from patients and human cell line transduced to express SH2B3 variants were examined by flow cytometry and western blots. Results: We identified an unreported subset of five patients with isolated SH2B3 variants of germline and somatic origin in our cohort of TN thrombocytosis patients. All variants were within the pleckstrin (PH) domain, with the exception of one located at the beginning of the SH2 domain, and were characterized by frameshift (fs) mutations, all heterozygous. The analysis of nail DNA from these patients highlighted three germline and two somatic cases. Most important was the clinical features of patients with SH2B3 fs variants. Indeed, all five probands presented severe thrombotic events with portal vein thrombosis, myocardial infarction, several stroke episodes, and cerebral venous thrombosis. We identified two different mutations, the novel mutation c.1093_1094ins31 p.(F365Wfs*30) in one proband and the mutation c.685_691dup p.(D231Gfs*39) in 4 distinct patients. To confirm the latter result, we cross-examined an external independent cohort of TN MPN patients, out of which two probands from the same family (mother and daughter) were identified with that mutation. This result strongly suggests that SH2B3 D231Gfs*39 variant is involved in the thrombocytosis phenotype and severe thrombotic events affecting these patients. To assess the mechanism of action of our recurrent SH2B3 fs mutation, we analyzed LNK expression level in platelets isolated from p.(D231Gfs*39) mutated patients by western blot. No truncated LNK form was detected; instead, a significant reduction of the WT LNK protein was observed compared to healthy donors. Human HEK cells expressing this SH2B3 variant confirmed this result, indicating that this fs mutation leads to a defect in total LNK protein. Indeed, in silico modeling of this SH2B3 variant showed a disruption of the LNK PH domain, causing an unstable WT LNK protein unable to effectively regulate signaling complexes at the membrane. Flow cytometry analysis of TPO signaling of these platelets showed a significant increase in STAT5 activation compared to healthy controls, confirming MPL/JAK2/STAT5 hyper-signaling as a functional consequence of these LoF SH2B3 mutations. Conclusions: We identified frameshift heterozygous SH2B3 germline variants which provide for the first-time evidence of SH2B3 association with the thrombocytosis phenotype displayed in TN patients. A defect on LNK expression can cause abnormal function and JAK/STAT upregulation, possibly in a driver-like fashion. Notably, D231Gfs*39 heterozygous variant displayed different clinical traits from those reported at the homozygous state (Perez-Garcia, A., et al, Blood, 2013; Arfeuille, C., et al, Haematologica, 2023), suggesting that SH2B3 genetic status may determine the severity of the outcome phenotype observed in patients. Ongoing studies on KO and heterozygous Sh2b3 mice expressing this SH2B3 variant will shed light on the scope of the genetic condition.
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Skoda, Radek C. "Predisposition to Myeloproliferative Neoplasms." Blood 124, no. 21 (December 6, 2014): SCI—33—SCI—33. http://dx.doi.org/10.1182/blood.v124.21.sci-33.sci-33.
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Familial forms of myeloproliferative neoplasms (MPN) and genetic contribution to sporadic cases of MPN have long been recognized. In the majority of cases, familial MPN is inherited as an autosomal dominant trait. The penetrance varies from around 20% to up to 100% in some pedigrees. We can distinguish two types of familial MPN. Type 1 has high penetrance, polyclonal hematopoiesis and hyperproliferation of a single hematopoietic lineage, caused by mutations in a single gene and usually manifesting at birth or early childhood. Examples are mutations in the genes for the erythropoietin receptor, thrombopoietin or its receptor, MPL. The type 2 familial MPNs are characterized by clonal hematopoiesis, low penetrance and manifestation beginning in most cases later in adult life. These type 2 familial MPNs are classical examples of inherited predisposition to a clonal malignant disease, in which acquired somatic mutations in hematopoietic cells are required for disease manifestation. Affected family members typically display the same acquired driver mutations in the genes for JAK2 (JAK2-V617F or JAK2-exon12), MPL (MPL-W515), or calreticulin (CALR) as patients with sporadic MPN. The mutated genes and the mechanism of how these inherited germline mutations predispose to MPN have not yet been elucidated. The search for these germline mutations has been hampered by the low penetrance of MPN manifestation and the rare occurrence of pedigrees that are large enough for genetic studies. Furthermore, the few candidate gene mutations that have been identified to date do not map to one gene locus and the function of the candidate genes does not fall into a common category. Two models of how the germline predisposition interacts with acquired driver mutations can be considered. First, the germline mutation may increase the mutation rate for gene mutations in JAK2, MPL, and CALR. Second, the germline mutation functionally synergizes with mutations in JAK2, MPL, and CALR and promotes disease initiation. The current state of our studies and studies in other laboratories will be discussed. Disclosures Skoda: Novartis: Consultancy; Sanofi: Consultancy.
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Manshouri, Taghi, Zeev Estrov, Alfonso Quintas-Cardama, Jorge Cortes, Francis Giles, David Harris, Waldemar Priebe, Hagop Kantarjian, and Srdan Verstovsek. "WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation." Blood 108, no. 11 (November 16, 2006): 4885. http://dx.doi.org/10.1182/blood.v108.11.4885.4885.
Full textAbstract:
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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Song, Jihyun, and Josef T. Prchal. "Ropeginterferon Alfa-2b Blunts Hyperactive JAK2 Activity in Polycythemia Vera and Essential Thrombocythemia." Blood 142, Supplement 1 (November 28, 2023): 3162. http://dx.doi.org/10.1182/blood-2023-190172.
Full textAbstract:
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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Hou, Yujuan, Hans Peter Gratz, Guillermo Ureña-Bailén, Paul G. Gratz, Karin Schilbach-Stückle, Tina Renno, Derya Güngör, et al. "Somatic Reversion of a Novel IL2RG Mutation Resulting in Atypical X-Linked Combined Immunodeficiency." Genes 13, no. 1 (December 23, 2021): 35. http://dx.doi.org/10.3390/genes13010035.
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Mutations of the IL2RG gene, which encodes for the interleukin-2 receptor common gamma chain (γC, CD132), can lead to X-linked severe combined immunodeficiency (X-SCID) associated with a T−B+NK− phenotype as a result of dysfunctional γC-JAK3-STAT5 signaling. Lately, hypomorphic mutations of the IL2RG gene have been described causing atypical SCID with a milder phenotype. Here, we report three brothers with low-normal lymphocyte counts and susceptibility to recurrent respiratory infections and cutaneous warts. The clinical presentation combined with dysgammaglobulinemia suspected an inherited immunity disorder, which has been proven by Next Generation Sequencing as a novel c.458T > C; p.Ile153Thr IL2RG missense-mutation. Subsequent functional characterization revealed impaired T-cell proliferation, low TREC levels and a skewed TCR Vβ repertoire in all three patients. Interestingly, investigation of various subpopulations showed normal expression of CD132 but with partially impaired STAT5 phosphorylation compared to healthy controls. Additionally, we performed precise genetic analysis of subpopulations revealing spontaneous somatic reversion, predominately in lymphoid derived CD3+, CD4+ and CD8+ T cells. Our data demonstrate that the atypical SCID phenotype noticed in these three brothers is due to the combination of hypomorphic IL-2RG function and somatic reversion.
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Wienand, Kirsty, Bjoern Chapuy, Chip Stewart, Andrew Dunford, David Wu, Jaegil Kim, Atanas Kamburov, et al. "Comprehensive Genomic Analysis of Flow-Sorted Hodgkin Reed Sternberg Cells Reveals Additional Genetic Bases of Immune Evasion." Blood 132, Supplement 1 (November 29, 2018): 1559. http://dx.doi.org/10.1182/blood-2018-99-118453.
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Abstract Classical Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin Reed Sternberg (HRS) cells within an extensive, but ineffective, inflammatory/immune cell infiltrate. Emerging data suggests that cHLs use multiple genetic mechanisms to evade immune recognition. We previously found that HRS cells exhibit near-universal somatic copy number alterations (SCNAs) involving chromosome 9p24.1/PD-1-L1/PD-L2 and rare chromosomal rearrangements of PD-L1 or PD-L2. The 9p24.1 amplicon also includes JAK2, which increases JAK2 copy numbers, augments JAK2/STAT signaling and further induces PD-1 ligand expression. However, HRS cells also have inactivating mutations of B2M and decreased or absent MHC class I expression. In cHL, clinical responses to PD-1 blockade are unrelated to HRS cell expression of MHC class I but closely associated with HRS cell expression of MHC class II, highlighting the potential role of CD4+ T-cell effectors (J Clin Oncol 2018;36:942-50). To define genetic bases of response and resistance to PD-1 blockade and identify complementary treatment targets, we performed whole exome sequencing (WES) of HRS cells. We first used a previously described multi-color flow cytometric sorting protocol (Methods 2012; 57:368-75) to obtain highly purified CD30+ HRS cells and normal B cells from the excisional biopsies of 25 newly diagnosed cHLs. The isolated HRS cells and paired normal B cells were then subjected to WES using an optimized workflow for low input samples and an expanded bait set to capture structural variants (SVs). We used established analytical pipelines to identify significantly mutated genes (candidate cancer genes [CCGs], MutSig2CV), SCNAs (GISTIC2.0) and SVs (4 algorithms). With improved methodology and purity (median of 80%) of the isolated HRS cells, we defined 15 significantly mutated CCGs, 21 recurrent SCNAs, including 6 CN gains (4 focal and 2 arm level) and 15 CN losses (14 focal and 1 arm level), and low frequency SVs. We identified 2 cHLs as hypermutators with MSI signatures due to splice site mutations in MSH2 or missense mutations in POLE. Excluding the 2 hypermutators, the analyzed cHLs had a median mutational density of 6.4 mutations/Mb, that falls within the top quartile of reported cancer mutational frequencies (Nature 2013 499:214). We also identified a previously unappreciated high incidence of ARID1A mutations (24%) in cHL. This is noteworthy because ARID1A deficiency increases mutational load and augments the efficacy of PD-1 blockade in murine models (Nature Med 2018;24:556). Together, the observed MSI signatures, relatively high mutational burden and newly identified ARID1A mutations in cHL represent additional potential genetic bases for the efficacy of PD-1 blockade. Notably, these cHLs also exhibited recurrent 9p24.1 copy gain (80%) and multiple genetic bases of enhanced JAK/STAT signaling including JAK2 copy gain (80%), STAT6 mutations (32%) involving known hotspots (D419 and N421) in the DNA-binding domain and frequent inactivating SOCS1 mutations (68%). We also identified multiple genetic bases for immune evasion, including B2M inactivating mutations (36%), HLA-B mutations (16%) and 6p21.32/HLA-B copy loss (28%), copy loss of the larger 6p21.32 region and inactivating CIITA SVs (8%). Additional signaling pathways were perturbed by multiple genetic mechanisms in these cHLs. For example, NF-κB pathway alterations included: TNFAIP3 mutations (24%) and 6q23.2/TNFAIP3 copy loss (56%), 12% biallelic; NFKBIE mutations (24%) and 6q21.32/NFKBIE copy loss (12%); and NFKBIA mutations (16%). The gene encoding the nuclear export protein, XPO1, was perturbed by E571K mutations (24%) and frequent 2p15/XPO1 copy gain (72%). Additionally, GNA13, an activator of RHOA and modifier of PI3K signaling, was mutated in 24% of cases. Of interest, cHL recurrent alterations including B2M, TNFAIP3, STAT6, and GNA13 mutations and 6q23.2 and 9p24.1 SCNAs were also identified in > 20% of examined primary mediastinal B-cell lymphomas, highlighting shared pathogenetic mechanisms in these diseases. In summary, comprehensive genomic analyses of purified HRS cells reveal new genetic bases of immune evasion, potential mechanisms of response and resistance to PD-1 blockade and additional targetable alterations. KW, BC, CS, AD and DW contributed equally. JF, GG and MS contributed equally. Disclosures Rodig: Affimed: Research Funding; KITE: Research Funding; Merck: Research Funding; Bristol Myers Squibb: Research Funding. Shipp:Merck: Research Funding; Bayer: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria.
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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, no. 21 (November 15, 2013): 2559. http://dx.doi.org/10.1182/blood.v122.21.2559.2559.
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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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Stockklausner, Clemens, Christin Maria Duffert, Ziwei Zhou, Anne Christine Klotter, Isabelle Nadine Kuhlee, and 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, no. 23 (December 3, 2015): 1634. http://dx.doi.org/10.1182/blood.v126.23.1634.1634.
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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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AL Assaf, Carla, Petros Papadopoulos, Laura Guttierez, Sanne Smits, Carlos Graux, Jan Emmerechts, Els Lierman, Timothy Devos, Lucienne Michaux, and Peter Vandenberghe. "MPL p.S204P Is a Recurrent Mutation in Essential Thrombocythemia." Blood 126, no. 23 (December 3, 2015): 2837. http://dx.doi.org/10.1182/blood.v126.23.2837.2837.
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Abstract Introduction: The JAK2 p.V617F, MPL p.W515K/L and CALR indels occur in a mutually exclusive pattern in 80-90% of cases with Essential Thrombocythemia (ET), but the driver mutations are unknown in the remaining 10-20%. In this study we aimed to identify driver mutations in the latter group of triple negative (TN) ET by exome sequencing of 10 such cases. Results: We found 27 somatic variants, including indels, in 6 out of 10 TN ET patients (range: 1-10 mutations/case; mean: 2,7 mutations/case), none of which were recurrent. In one case, we found a MPL c.610T>C (p.S204P) mutation, which is located in the extracellular domain of the MPLreceptor. By Sanger sequencing of MPL exon 4 in 20 additional TN ET cases, an additional patient with the MPL S204P mutation was identified. Moreover, this mutation was previously reported in one case with idiopathic myelofibrosis1. In order to study the effect of this mutation on the function of MPL, we produced stable Ba/F3 cell lines expressing MPL S204P, MPL W515K or MPL WT, and assessed the dependence of their growth on exogenous thrombopoietin (TPO). Only MPL W515K transduced Ba/F3 cells proliferated in the absence of TPO, but growth of MPL S204P Ba/F3 and of MPL WT Ba/F3 could be rescued by exogenous TPO, indicating the proper surface expression and the functionality of the transduced receptors. The levels of phospho-JAK2 and phospho-STAT5 were low in cytokine-deprived MPL S204P cells but increased upon TPO stimulation. In contrast, phospho-JAK2 and phospho-STAT5 were detectable in MPL W515K transduced Ba/F3 in the absence of cytokines as assessed by Western blotting. Culture of MPL S204P transduced Ba/F3 in the presence of TPO over a range of concentrations (0,01-10 ng/ml) yielded growth curves comparable with MPL WT transduced Ba/F3. Using flow cytometry, we also explored cell surface marker expression on peripheral blood platelets from the two MPL S204P ET patients. Data were compared with healthy donors or ET patients with JAK2 or CALR mutations. MPL S204P ET platelets displayed higher expression of CD61 than platelets from healthy donors or from JAK2 or CALR mutated ET (p<0,01). In addition, there was a trend for higher expression of KIT, CD36 and CD42b on platelets from the MPL S204P ET cases. Moreover, following platelet activation through the protease activated receptor 1, the degranulation response of platelets from MPL S204P ET was decreased in comparison with JAK2 or CALR mutated ET. Conclusion: The MPL S204P mutation is a recurrent mutation in TN ET, with a frequency of 7% (2/30) in this series, but this mutation does not induce TPO-independent growth nor increased TPO-sensitivity in Ba/F3 cells. However, preliminary phenotypic and functional evidence supports the notion that MPL S204P platelets display specific characteristics as compared with JAK2 or CALR mutated ET. The mechanisms by which the MPL S204P mutation influences megakaryopoiesis and platelet function remain to be elucidated. 1. Williams DM, et al. Phenotypic variations and new mutations in JAK2 V617F-negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis. Exp Hematol 2007; 35: 1641. Disclosures Graux: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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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 (November 29, 2018): 1564. http://dx.doi.org/10.1182/blood-2018-99-118135.
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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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Zara, Greta, Sonia Rodriguez-Rodriguez, and Nadia Carlesso. "MyD88L265P Mutation Impairs Bone Marrow Hematopoietic Stem Cell Function in a Cell-Autonomous Way Resulting in an MPN-like Phenotype." Blood 144, Supplement 1 (November 5, 2024): 2653. https://doi.org/10.1182/blood-2024-201808.
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Background:The cytosolic adapter proteinMyeloid Differentiation 88 (MYD88) plays a pivotal role in the innate and adaptive immune response by initiating the Toll-Like and Interleukin-1 Receptor (TLR and IL1R) signaling cascades. In humans, the somatic gain of function L265P mutation within the C-terminal Toll-IL1 receptor (TIR)-interaction domain of MYD88 has been associated to the development and progression of B-cell neoplasms. In the context of such malignancies, MyD88L265P leads to the constitutive activation of the Nuclear Factor Kappa-light-chain-enhancer of activated B-cells (NF-κB) and Janus Kinase2/Signal Transducer and Activator of Transcription (JAK2/STAT) pathways, fostering the secretion of inflammatory cytokines. Similarly, the spontaneous activation of NF-κB and JAK2/STAT pathways driven by the JAK2V617F gain of function mutation, and the subsequent overproduction of inflammatory cytokines, are central to the pathogenesis of myeloproliferative neoplasms (MPN). Nonetheless, a compelling link between MyD88L265P and MPN remains elusive. Using a model of severe inflammation induced by bacterial sepsis or lipopolysaccharide (LPS), our lab previously reported that “inflamed” hematopoietic stem cells (HSC) underwent dysfunctional expansion associated with a transient inability to differentiate into the downstream myeloid progenitors. Loss of MyD88 was able to abrogate myeloid dysregulation, suggesting that akin to JAK2, abnormal expression of MyD88 may significantly contribute to the breakdown of myeloid homeostasis. Therefore, we propose that MyD88L265P could emulate the inflammation-driven effects of JAK2V617F mutation, potentially resulting in a disease resembling MPN. Objective: Investigate the impact of MyD88L265P-induced inflammation on bone marrow (BM) HSC cycling, differentiation, and reconstitution potential and explore the hypothesis that such gain of function mutation in mice results in a phenotype that mimics MPN in patients. Results: Mutant MyD88L265P HSC displayed abnormal cycling and expansion, resulting in impaired differentiation, hyperproliferation and accumulation of granulocyte-monocyte progenitors (GMP) in the BM. As a consequence, the BM neutrophil compartment was exhausted while white blood cells massively mobilized into the peripheral blood. The latter also exhibited significantly increased immature neutrophils, along with anemia and profound thrombocytopenia. Concurrently, extramedullary hematopoiesis was observed in the liver and spleen (SP) of MyD88 mutant mice, leading to splenomegaly and hepatomegaly, which are hallmark clinical manifestations of MPN with primary myelofibrosis (PMF) in humans. A similar phenotype was observed after transplantation of mutant MyD88 HSC into healthy recipient mice, suggesting that MyD88L265P impairs the functionality of BM HSC in a cell-autonomous fashion. The expression of specific cytokines such as CXCL9, CXCL10, and CXCL12 associated with fibrosis and migration of clonal hematopoietic cells from the BM to the SP and liver are found altered in MPN patients with PMF. Notably, secretomics analysis identified this same abnormal cytokine profile in our MyD88 gain of function mouse model. Immunohistochemistry revealed a severe increase of STAT3 in the BM, SP, liver, and lymph nodes of mutant MyD88 animals. Moreover, while CD68+ monocytes/macrophages were barely detected, CD163+ monocytes/macrophages were expanded in all the tissues of the MyD88L265P mice. Similarly, increased CD163+ cells are found in PMF MPN patients. In addition, the BM of mutant mice showed hypercellularity with proliferation of pleomorphic megakaryocytes, while their SP, liver, and lymph nodes displayed disrupted tissue architecture and inflammatory infiltrates, again reflecting traits found in PMF MPN patients. Conclusion: Our data show that MyD88L265P mutant mice exhibit hallmarks of human PMF MPN induced by the hyperactivation of the JAK2/STAT pathway. Significance: Our MyD88L265P mouse model recapitulating the human PMF MPN phenotype could provide insight to elucidate the mutational landscape of patients with MPN, yielding further potential therapeutic avenues. 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, no. 1_Supplement (May 1, 2023): 234.12. http://dx.doi.org/10.4049/jimmunol.210.supp.234.12.
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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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Al-Naqeeb, Ghadah, Aaron Cypess, Anna Zenno, and Sanjay Jumani. "OR08-5 Associations Between Signal Transducer and Activator of Transcription (STAT) Mutations and Galactorrhea." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A588—A589. http://dx.doi.org/10.1210/jendso/bvac150.1219.
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Abstract Background Prolactin (PRL) is responsible for the development of mammary glands and milk production. STAT1 and STAT3 are genes that express transcription factors that mediate PRL's intracellular signaling, but an association between their function and galactorrhea has not yet been identified. Here we describe three patients with somatic STAT mutations and galactorrhea. A 27-year-old woman with hyper-IgE syndrome (Job's Syndrome) due to a STAT3 deletion mutation (p.Val463del) associated with recurrent infections reported thin, white, bilateral discharge with breast manipulation beginning in adolescence. She did not have spontaneous leakage or pain. At presentation, PRL was slightly elevated to 27.9 (ref. 2.0-25.0 mcg/L) with otherwise normal levels of anterior pituitary hormones. She was not on any drugs known to cause hyperprolactinemia. MRI showed a 5×4 mm microadenoma without any mass effect. A 32-year-old woman also with Job's syndrome due to a heterozygous loss-of-function STAT3 mutation (p.R382Q) associated with recurrent infections and papillary thyroid cancer described unilateral, chronic, thin, white nipple discharge with light touch of her right breast for "as long as she could remember." She had a history of breast abscesses, but at presentation there was no evidence of abscess or infection. PRL was 15.1 mcg/L, and other anterior pituitary hormones were normal. MRI showed a structurally normal pituitary. A 22-year-old woman with a STAT1 activating mutation (p.E235A) associated with recurrent infections, autoimmune hypothyroidism, T1DM, and hypogonadotropic hypogonadism requiring pubertal induction at age 12y reported bilateral galactorrhea starting at age 18y. A white, milky discharge could be expressed with light manipulation, but no discharge was produced spontaneously. PRL was 16.2 mcg/L with serial dilution and has consistently been in the reference range. Anterior pituitary evaluation was normal except for elevated TSH of 12.5 (ref. 0.27-4.2) consistent with autoimmune hypothyroidism. MRI revealed a diffusely enlarged pituitary gland (12mm in the cranio-caudal dimension) without focal hypo-enhancement. Conclusions It is known that PRL signaling involves the JAK/STAT pathway, and an analysis of human milk cells showed upregulated STAT3 and downregulated STAT1 during lactation (PMID 26909879). These three cases provide clinical evidence for a mechanistic connection between STAT signaling and galactorrhea and suggest that STAT3 and STAT1 function in opposite directions to regulate lactation. In our cases, increased STAT1 or decreased STAT3 activity are associated with normal-PRL, inducible galactorrhea. Beyond understanding the specific intracellular signaling pathways leading to galactorrhea, these observations may be utilized for developing therapies for those with impaired lactation. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.
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Kunter, Ghada M., Fulu Liu, Maxwell Krem, and 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, no. 11 (November 16, 2006): 632. http://dx.doi.org/10.1182/blood.v108.11.632.632.
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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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Küppers, Ralf. "Genomic Analysis of Hodgkin Lymphoma." Blood 134, Supplement_1 (November 13, 2019): SCI—7—SCI—7. http://dx.doi.org/10.1182/blood-2019-121073.
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The genetic analysis of the Hodgkin and Reed/Sternberg (HRS) tumor cells in Hodgkin lymphoma (HL) is very much hampered by the rarity of these cells, typically accounting for only about 1% of cells in the lymphoma tissue. Moreover, only very few cell lines were established from HL, partly with an uncertain origin. Thus, for the identification of somatic mutations in HRS cells, methods had to be established to isolate the rare HRS cells by microdissection from tissue sections or by cell sorting from cell suspensions. Initial studies were focussed on candidate gene approaches, often inspired by results from the prior analysis of HL cell lines. These early studies revealed numerous recurrent mutations in members of the NF-κB and JAK/STAT pathways, causing or at least contributing to the constitutive activation of these signaling pathways.1 The pathobiological relevance of the constitutive activation of NF-κB and JAK/STAT factors was validated by functional studies with HL cell lines. A first whole exome sequencing analysis of flow-sorted HRS cells was published by Reichel and colleagues.2 B2M was found to be inactivated by somatic mutations in 7/10 cases, and caused loss of major histocompatibility complex 1 expression. Although later studies with larger case series identified such mutations in only about 20-30% of cases,3,4 B2M inactivation is nevertheless an important factor for immune evasion of HRS cells and a frequently mutated gene in HL. A second exome sequencing study of HRS cells used microdissected tumor cells and revealed as a major novel finding frequent somatic mutations in the STAT6 gene (ca. 30% of cases).3 The genetic lesions in STAT6 are gain-of-function mutations. Overall nearly 90% of HL cases show genetic alterations in members of the JAK/STAT pathway, further demonstrating the major role of this pathway in HL pathogenesis. Our group is currently performing studies of microdissected HRS cells by whole genome sequencing, aiming to identify further genetic lesions in HRS cells beyond those affecting exons. An alternative attractive approach to uncover mutations in HRS cells is the genetic analysis of circulating DNA in plasma, because HRS cell-derived DNA fragments can be identified in most patients at the time of diagnosis. A targeted sequencing study of circulating tumor DNA showed the feasibility of this approach and validated, for example, the frequent occurrence of STAT6 mutations in HL. In rare instances, a HL can co-occur with another lymphoma in the same patient. Such composite lymphomas frequently show a common clonal origin of both lymphomas from a mature B cell.5 Composite lymphomas are therefore elegant models to study the multi-step transformation process in lymphomagenesis. We are currently performing a whole exome sequencing analysis of composite classical HL and B-cell Non-Hodgkin lymphomas, to reveal the pattern of early shared mutations and later distinct mutations. In each of the cases under investigations, such shared and distinct mutations were indeed identified. Küppers R, Engert A, Hansmann ML. Hodgkin lymphoma. J Clin Invest. 2012; 122: 3439. Reichel J, Chadburn A, Rubinstein PG, et al. Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells. Blood. 2015; 125: 1061. Tiacci E, Ladewig E, Schiavoni G, et al. Pervasive mutations of JAK-STAT pathway genes in classical Hodgkin lymphoma. Blood. 2018; 131: 2454. Spina V, Bruscaggin A, Cuccaro A, et al. Circulating tumor DNA reveals genetics, clonal evolution, and residual disease in classical Hodgkinlymphoma. Blood. 2018; 131: 2413. Küppers R, Dührsen U, Hansmann ML. Pathogenesis, diagnosis, and treatment of composite lymphomas. Lancet Oncol. 2014; 15: e435. Disclosures No relevant conflicts of interest to declare.
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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, no. 21 (December 6, 2014): 1882. http://dx.doi.org/10.1182/blood.v124.21.1882.1882.
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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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Perez-Garcia, Arianne, Charles A. LeDuc, Kara A. Kelly, Chaim Jalas, Wendy K. Chung, and Adolfo A. Ferrando. "Familial and Acquired SH2B3 mutations in ALL." Blood 120, no. 21 (November 16, 2012): 1326. http://dx.doi.org/10.1182/blood.v120.21.1326.1326.
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Abstract Abstract 1326 Acute lymphoblastic leukemia (ALL) is a heterogeneous disease in which multiple genetic alterations contribute to the malignant transformation of lymphoid progenitor cells. Although the pathogenesis of ALL is considered to be multifactorial, few environmental factors have been conclusively associated with increased risk. In addition, although recent genome-wide association studies of childhood ALL have suggested that common variation at some genetic loci may predispose to ALL, the risk associated with these variants seems to be modest. Here we hypothesized that leukemia development in the context of rare familial inherited disorders may indicate a tumor suppressor activity for the underlying genetic defect present in these kindreds. In this context, we studied a pediatric B precursor ALL female patient of Eastern European Ashkenazi Jewish background with a history of remote consanguinity, short stature and mild developmental delay. Her family history is significant for a brother with similar growth retardation and mild developmental defects, neonatal anemia, thrombocytopenia and elevated white cell count, who was diagnosed with chronic hepatitis of probable autoimmune origin and Hashimoto's autoimmune thyroiditis. A second male sibling was healthy. We proposed that an underlying autosomal recessive genetic defect resulting in dysregulation of the development and function of the immune system could contribute to the concurrence of a lymphoid neoplasia and an autoimmune disorder in two siblings in this family. We performed homozygosity mapping and linkage analysis in the family. Whole exome sequencing of each of the two affected siblings identified a homozygous frameshift c.671insGGCCCCG p.Asp231fs mutation in the SH2B3 gene. Dideoxynucleotide sequencing of SH2B3 confirmed that both affected siblings were homozygous for this mutation, while the unaffected brother and both parents were carriers. Analysis of 2,000 normal Ashkenazi Jewish adults identified 1 SH2B3 c.671insGGCCCCG p.Asp231fs carrier and no homozygous individuals. The SH2B adaptor protein gene 3 (SH2B3) encodes an adaptor protein involved in the negative regulation of the JAK STAT signaling pathway. Western Blot analysis of immortalized lymphoblastoid cell lines from the two affected siblings and an unrelated normal control showed complete loss of SH2B3 protein expression in SH2B3 mutant cells. Consistently, analysis of JAK STAT signaling in these cells showed increased JAK2 and STAT3 phosphorylation compared with controls. Moreover, SH2B3 mutant lymphoblastoid cells showed increased proliferation and accelerated cell cycle kinetics, which was particularly pronounced in low serum conditions. Consistently, expression of wild type SH2B3 in these cells impaired cell growth and decreased JAK-STAT signaling. Analysis of a cohort of 167 additional ALLs including 119 B-precursor ALLs and 48 T-ALLs identified a homozygous c.1279insCTGTTGCCGTGTGC p.Val426fs SH2B3 mutation in a B-precursor ALL sample and a homozygous nonsense c.908C>A p.Ser303* SH2B3 mutation in a pediatric T-cell ALL. Notably, analysis of normal (remission) DNA demonstrated the somatic origin of the c.908C>A p.Ser303* SH2B3 mutation. In addition, our sequence analysis of SH2B3 in ALL identified two additional heterozygous germline single nucleotide variants resulting in amino acid substitutions (c.557G>T p.Ser186Ile and c.232G>A p.Glu78Lys). Expression of each of these variants in SH2B3 null cells resulted in partial correction of the increased cell growth phenotype and JAK-STAT signaling associated with SH2B3 loss compared with wild type controls. Notably, heterozygous somatic mutations in SH2B3 have been observed in patients with myeloproliferative neoplasms and heterozygous germline SH2B3 alleles have been linked to essential erythrocytosis, suggesting that the hypomorphic SH2B3 p.Ser186Ile and p.Glu78Lys variants found in our analysis may represent rare leukemia predisposing SH2B3 alleles. Overall these results link the loss of SH2B3 with the pathogenesis of a familial developmental delay, autoimmunity and leukemia predisposition syndrome and demonstrate a tumor suppressor role for SH2B3 in the pathogenesis of B-precursor and T-cell ALL. Disclosures: No relevant conflicts of interest to declare.
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Ricci, Kiersten, Erika Huber, Ashok Raj, Mohammad Azam, and Theodosia A. Kalfa. "Compound Heterozygosity of Two Novel JAK2 Mutations in Hereditary Essential Thrombocythemia Implicates Important Monomer-Monomer Interactions in Thrombopoiesis Signaling." Blood 128, no. 22 (December 2, 2016): 3137. http://dx.doi.org/10.1182/blood.v128.22.3137.3137.
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Abstract Essential thrombocythemia (ET) is a myeloproliferative neoplasm that is characterized by pathological overproduction of platelets with an increased risk of thromboembolic events, progression to myelofibrosis and leukemic transformation. Mutations of the Janus Kinase 2 (JAK2) gene, most commonly JAK2V617F, are known driving factors of ET pathogenesis. However, the molecular processes involved in JAK2 activation and regulation and the specific mechanism(s) by which JAK2 mutations disrupt hematopoiesis remain unclear. Most mutations identified in hereditary ET involve the thrombopoietin or thrombopoietin receptor gene; only 4 germline JAK2 mutations have been reported, all of which are inherited in an autosomal dominant pattern. We report the first case in which 2 novel germline JAK2 mutations, JAK2L815P and JAK2V1123G, were found in the compound heterozygous state in 2 siblings with ET. Family members who carry either one of these variants have normal blood counts. The purpose of this study was to demonstrate causality and to explore the mechanism(s) by which these JAK2 mutations, in compound heterozygous state, result in thrombopoietic dysregulation. To study signaling in the JAK2-signal transducer and activator of transcription (STAT) pathway, wild-type (WT) and mutated forms of murine JAK2 were transduced into murine pro-B cells (Ba/F3) stably expressing the thrombopoietin receptor (MPL), Ba/F3-MPL cells. Immunoblotting analysis was performed on Ba/F3-MPL cells that were starved of cytokines overnight and then treated with 10 ng/mL thrombopoietin (TPO) or vehicle for 5 minutes. At baseline (under starved conditions), JAK2L815P + JAK2V1123G Ba/F3-MPL cells demonstrated increased STAT3 and STAT5 phosphorylation compared to WT JAK2 Ba/F3-MPL cells. However, JAK2 double mutant Ba/F3-MPL cells had less JAK2, STAT1 and STAT3 phosphorylation in comparison to JAK2V617F Ba/F3-MPL cells at baseline. After TPO stimulation, both JAK2 double mutant and JAK2V617F Ba/F3-MPL cells showed increased phosphorylated JAK2, STAT1 and STAT3 compared to WT JAK2 Ba/F3-MPL cells. To investigate JAK2-STAT signaling in the patients' cells, immunoblotting analyses of platelets and megakaryocytes grown in vitro from patients' CD34 cells were performed. Our patients' platelets demonstrated increased JAK2, STAT1, STAT3 and STAT5 phosphorylation compared to platelets from a normal control but less JAK2, STAT1 and STAT3 phosphorylation compared to the platelets from a pediatric ET patient with a known JAK2V617F mutation, a similar signaling pattern to that seen in JAK2 double mutant Ba/F3-MPL cells. Similarly, in vitro megakaryocytes from the patients' CD34 cells showed increased STAT5 activation compared to those from normal volunteers in the unstimulated state. To assess cell proliferative capability, IL-3 dependent Ba/F3 cells were starved of cytokines and viable cells were counted every 24 hours for a total of 72 hours. The JAK2V617F and JAK2 double mutant Ba/F3-MPL cells easily proliferated without cytokines and had a large fold-increase at 24, 48 and 72 hours compared to 0 hours. The JAK2L815P + JAK2V1123G Ba/F3-MPL cells had a significantly higher proliferation rate compared to JAK2V617F and WT JAK2 Ba/F3-MPL cells. As expected, the number of viable parental Ba/F3 cells declined over time in the absence of cytokines. To evaluate response to ruxolitinib, a JAK1 and JAK2 inhibitor, IL-3 independent Ba/F3-MPL cells were grown under starved conditions in the presence of 0 to 1 mM ruxolitinib. After 48 hours, the number of viable Ba/F3-MPL cells was measured as a percentage of the dimethylsulfoxide (DMSO) control. The 50% inhibitory concentration (IC50) value was higher in JAK2L815P + JAK2V1123G Ba/F3-MPL cells (147+/-20 nM) compared to JAK2V617F Ba/F3-MPL cells (93+/-1 nM). In the compound heterozygous state, the novel germline JAK2 mutations, JAK2L815P and JAK2V1123G, appear to function cooperatively to increase cell proliferation through activation of the JAK2-STAT pathway. This finding implicates that within the JAK2 dimer in the MPL-JAK2-STAT pathway, the monomer-monomer interactions are important in JAK2 activation and regulation. Additional in vitro and in vivo studies are currently underway to elucidate the exact mechanism(s) of thrombopoietic dysregulation resulting in the siblings' ET and to gain further insight into JAK2 activation and regulation. Disclosures Raj: Novartis: Speakers Bureau. Kalfa:Baxter/Baxalta/Shire: Research Funding.
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Wang, Linghua, Sabina Swierczek, Kimberly Hickman, Soo-Jin Kim, David A. Wheeler, and Josef Prchal. "Molecular Characterization Of Polycythemia Vera Based On The Relationship Of JAK2V617F and 9pUPD." Blood 122, no. 21 (November 15, 2013): 1607. http://dx.doi.org/10.1182/blood.v122.21.1607.1607.
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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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Oh, Stephen T., Jeffrey I. Zwicker, Kamal Patel, Erin L. Crowgey, Cynthia Timmers, Patricia Feldman, Justine Carl, et al. "Molecular Predictors of Disease Progression to Myelofibrosis (MF) in Patients (Pts) with Polycythemia Vera (PV) Enrolled in Reveal." Blood 144, Supplement 1 (November 5, 2024): 3145. https://doi.org/10.1182/blood-2024-199195.
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Introduction: Progression of PV to MF represents a major cause of morbidity and mortality; however, predictors of progression remain unclear. The Prospective Observational Study of Patients With Polycythemia Vera in US Clinical Practices Trial (REVEAL; NCT02252159) followed pts for a median of ~4 years, with optional biospecimen collection every 12 months. This analysis investigated the clonal architecture and molecular mechanisms that predict transformation to MF in pts with PV. Methods: Of 2510 pts enrolled in REVEAL, 1880 had an available biospecimen and a confirmed JAK2 mutation by ddPCR. Of these pts, 114 had MF transformation during the study period based on modified World Health Organization criteria. Whole exome sequencing was performed on enrollment biospecimens (pretransformation) from the 114 pts, as well as a control cohort of 340 pts without transformation, matched (1:3) by propensity score based on age, sex, duration of PV, body mass index, and history of thrombosis. Sequencing was performed using the Illumina (paired-end) platform at an average depth of 300× and processed using Genome Analysis Toolkit best practices. Variants were filtered (VarSeq) according to the following criteria: ≥60 reads coverage, ≥3 reads support, >1% variant allele frequency (VAF), and <1% (or missing) minor allele frequency in the Genome Aggregation Database. Variants were annotated via tiers based on predicted pathogenicity using multiple algorithms. Data were analyzed using fast gene set enrichment analysis (FGSEA) and cell type-specific enrichment analysis (CSEA). Results: There were no significant differences in clinical characteristics at enrollment between the transformed and nontransformed groups. Median (range) enrollment duration was 18.2 (0-30.0) and 19.1 months (0-32.5) in the transformed and nontransformed groups. A wide JAK2 V617F VAF distribution was observed in both cohorts; however, median (range) VAF was significantly higher in the transformed vs nontransformed group (84.21 [0.02-97.99] vs 57.45 [0.05-98.65]; P<0.0001). For both transformed and nontransformed pts, prevalence of commonly mutated genes was consistent with observations in other myeloid malignancies. A higher number of combined Tier I/II mutations in myeloid-related genes was observed per pt in the transformed vs nontransformed group (mean [range], 5.6 [2-14] vs 4.8 [1-12]; P<0.05). In particular, SF3B1, IDH1/2, EZH2, and TP53 mutations were enriched in the transformed group, with VAFs that trended higher compared with the nontransformed group. In addition, the presence of multiple clones was suggested by a difference in VAF between JAK2 V617F and co-occurring somatic variants. To identify novel genes potentially contributing to PV transformation, genes with Tier I variants significantly associated with transformation (P<0.05) and an odds ratio >2 were prioritized. 83 genes were identified, including the tumor suppressor gene PTPN2, which has a known role in regulation of inflammation. Copy number variant (CNV) analysis identified 25 genes with gains and 28 with losses in the transformed vs nontransformed group. More pts in the transformed group had a CNV gain in TYRO3, which has been linked to myeloid leukemia cell growth. Pathways with the highest enrichment scores via FGSEA comparison of the transformed vs nontransformed group included Wnt/β-catenin signaling, inflammatory response, and DNA repair, whereas CSEA indicated a strong signal in bone marrow granulocytes and blood monocytes. Analysis of Tier II variants further identified markers of upregulated interferon-α response, notch signaling, and IL-6 and JAK-STAT3 signaling. Together, these data suggest a potential signature of inflammation in PV transformation to MF. Conclusions: Although clinical characteristics were similar at enrollment for all pts in this analysis, JAK2 V617F VAF was significantly higher in pts with PV who transformed to MF. In addition, the VAF of nondriver somatic mutations trended higher in multiple genes with a known role in myeloid malignancy. Ongoing analyses of novel genes suggest a role of inflammatory pathways in PV transformation and may uncover additional pathways that contribute to this process. Further investigation will include analysis of clonal evolution via intra-pt longitudinal biospecimens and development of a machine learning approach to predict pts at increased risk of transformation.
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Constantinescu, Stefan N. "Immunotherapy targeting mutant calreticulins in myeloid blood cancer." South East European Journal of Immunology 8, CITIM (March 25, 2025): 033. https://doi.org/10.3889/seejim.2025.6094.
Full textAbstract:
Myeloproliferative neoplasms (MPNs) comprise of group of hematopoietic stem cell diseases where acquired mutations lead to excessive formation of myeloid cells, red blood cells, platelets and granulocytes. Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary and Secondary myelofibrosis (MF) are the most prevalent MPNs. The phenotypic driving mutations are the acquired unique somatic JAK2 V617 mutation, MPL/TpoR (thrombopoietin receptor mutations) and calreticulin (CALR) mutations. The latter are +1 frameshift mutations in exon 9 which lead to a new positively charged sequence in the place of the negatively charged C-terminus of wild type CALR, and lacking the ER retention KDEL sequence. Our group established that these mutants are inducing MPNs (15-20% of ET and 27% of MF) via binding in the ER and dimerizing the TpoR, which is then transported in a partially immature state to the cell surface by the rogue chaperone function of mutant CALR. Exposure at the cell-surface is obligatory for oncogenic signal of TpoR in the absence of Tpo ligand. Since the novel sequence is constituted of 36 novel amino-acids that should function as a tumor antigen we examined the possibility of using immunotherapy against this mutant sequence. In addition to the cell-surface exposed tumor antigen, we report that structure of the N-terminus non-mutated region of CALR slightly differs between wild type and mutant CALR. We used vaccination and showed that naive mice C57-B6 mice respond with antibody and T cell responses to immunization with CALR mutant peptide. In contrast, Calr del52 knock-in animals or transplanted animals expressing both wild type Calr and Calr del52 knock-in mutant do not respond. We discuss alternate vaccination methods that might be able to break this tolerance. Furthermore, we found that the mutant CALR is also secreted from cells of the clone that do not express TpoR. At levels equivalent to those in patients the circulating mutant CALR acts as a rogue cytokine, forming complexes with endogenous cell-surface mutant CALR and TpoR, but not with TpoR alone, explaining why the mutant CALR only stimulates clonal expansion and not polyclonal thrombocytosis. The circulating mutant CALR will interact with antibodies that recognize the tumor antigen and may exert a sink effect at low antibody doses. Based on our structural studies examining by deuterium exchange mass spectrometry we propose several precise sequences to be targeted by immunotherapy and discuss recent efforts using anti mutant CALR antibodies or bispsecific approaches.
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Stubbs, Matthew C., Hamza Celik, Yanran Ai, Xin He, Hong Chang, Angela Lei, Mark Rupar, et al. "Preclinical Evaluation of INCB160058 - a Novel and Potentially Disease-Modifying Therapy for JAK2V617F Mutant Myeloproliferative Neoplasms." Blood 142, Supplement 1 (November 28, 2023): 860. http://dx.doi.org/10.1182/blood-2023-179369.
Full textAbstract:
The Janus kinase 2 ( JAK2) mutation, JAK2V617F, is the most common oncogenic driver in myeloproliferative neoplasms (MPNs), with nearly all cases of polycythemia vera (PV) and over half of primary myelofibrosis (MF) and essential thrombocythemia (ET) patients positive for the somatic mutation. Approved therapies for MPNs such as ruxolitinib, which act by directly inhibiting activity of the kinase domain (JH1) of JAK2, have demonstrated impressive clinical efficacy and safety in patients with MPNs; however, they do not address JAK2V617F allelic burden or achieve molecular remission of disease. A targeted JAK2V617F selective agent sparing wild-type (WT) JAK2 activity has potential to eliminate mutant cells, induce molecular remission, and theoretically lead to functional cure of MPNs. We report herein the preclinical development of pseudokinase (JH2)-targeting INCB160058, a first-in-class, orally bioavailable small molecule with the ability to selectively target JAK2V617F + cell populations derived from patients with JAK-mutant MPNs. Using structure- and function-guided molecular design, INCB160058 was designed to bind with picomolar affinity to the JH2 domain of JAK2V617F at the canonical ATP-binding site, with high specificity (>2500-fold) relative to binding at the active kinase domain (JH1) targeted by currently approved JAK inhibitors. Live cell single-molecule fluorescence microscopy showed that INCB160058 binding to JAK2V617F blocked ligand-independent thrombopoietin receptor dimerization induced by the mutation, and consequently led to loss of JH1 domain kinase activity. X-ray crystallography analysis indicates that the observed inhibition is likely driven by conformational disruption of the ⍺C helix motif at Phe 594 and Phe 595 in conjunction with a shift of the upstream region from Leu 583 to Asn 589 upon INCB160058 binding to the JH2 domain of JAK2V617F. We utilized both CD34 + human multipotent hematopoietic stem cells derived from patients with JAK2-mutant MF, engineered JAK2-mutant human hematopoietic cancer cell lines (eg, SET2 and UKE-1), and murine BA/F3 cell lines to explore the selective effects of INCB160058 on JAK2V617F compared with WT JAK2. INCB160058 treatment selectively reduced pathogenic phospho-STAT5 levels, decreased abnormal megakaryopoiesis, and suppressed colony formation only in JAK2V617F + CD34 + cells but not in CD34 + cells from healthy volunteers. Importantly, continuous exposure of mutant and WT JAK2 cells to INCB160058 in co-cultures at concentrations below IC 50 resulted in progressive elimination of JAK2V617F + cells without affecting WT cells. At the end of the testing period, the JAK2V617F-harboring population was no longer detectable in the co-culture assay. In NSG mice subcutaneously inoculated with JAK2V617F-expressing SET2 cells, INCB160058 was tolerated and exhibited significant antitumor activity. In addition, following INCB160058 treatment, a significant reduction in the engraftment of total human cells, particularly human erythroid progenitors (hCD45 − mCD45 − Ter119 − hCD71 + hCD235a +), was observed in NSGS mice xenotransplanted with JAK2V617F + CD34 + cells. Moreover, INCB160058 treatment also led to the normalization of various pathogenic cytokines, such as interleukin (IL)-6 and IL-8. Importantly, these observations were absent in NSGS mice engrafted with CD34 + cells from healthy volunteers following INCB160058 treatment, further demonstrating the selectivity of INCB160058 for JAK2V617F. In summary, our results indicate a novel mechanism of action of INCB160058, a high-affinity pseudokinase (JH2) binding inhibitor of JAK2V617F that blocks cytokine-independent activity of JAK2V617F while preserving cytokine-dependent signaling. Extended treatment with INCB160058 at low therapeutic doses results in the specific elimination of mutant JAK2V617F-harboring cells in mouse models and human cancer cells with minimal impact on WT counterparts. Clinical testing of INCB160058 may allow patients with MPNs to achieve molecular remission by eliminating cells with the main genetic aberration and afford an opportunity to overcome the disease.
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Campanelli, Rita, Gabriela Fois, Carlotta Abbà, Mara De Amici, Laura Villani, Elisa Bonetti, Paolo Catarsi, et al. "Possible Role of Impaired Erk1,2 Phosphorilation and Increased sIL2r Alpha Plasma Levels in the Reduced Frequency of Circulating T Regulatory Cells of Patients with Primary Myelofibrosis." Blood 126, no. 23 (December 3, 2015): 1639. http://dx.doi.org/10.1182/blood.v126.23.1639.1639.
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Abstract Background. Primary myelofibrosis (PMF) is a clonal, neoplastic disorder of the hematopoietic stem cells, characterized by an increased number of circulating CD34+ cells. Available evidence indicates that enhanced activation of JAK-STAT pathway is the main molecular mechanism of myeloproliferation due to somatic mutations in JAK2, CALR or MPL genes. A subset of patients display immune-related abnormalities that suggest an (auto)immune pathogenesis. In addition, it has been reported a significant association between personal history of a broad span of autoimmune diseases and subsequent risk of myeloproliferative neoplasm (Kristinsson SY Haematologica 2010; 95:1216-1220). CD4+ T cells maintain cancer surveillance and immune tolerance. Chronic inflammation has been proposed as a driver of clonal evolution in myeloproliferative neoplasms (MPN) (Hasselbalch HC et al. Leuk Res 2013;37:214-20), suggesting that T cells play an important role in their pathogenesis. A recent paper by Keohane C et al (BJH 2015; doi 10.1111/bjh 13519) shows that peripheral blood (PB) T regulatory cells (Tregs) are reduced in MPN patients compared to healthy subjects (CTRLs) and that this decrease is even more pronounced following JAKi therapy (Massa M et al Leukemia 2014;28:449-51). Unpublished data from our group further indicate a significant (p<0.001) decrease of PB Tregs in PMF (n=193, CD4+CD25highCD127low/- FOXP3+ cells expressed as % of CD4+ cells, median 0.87, 0-10.2) versus CTRLs (n=16, median 2.09, 0.54-6.5). Based on the distribution of Tregs in CTRLs, we established a cut-off point of 1.0% to define an actual reduction of Tregs. Study design. To investigate the possible factors favouring a decrease of Tregs, we examined in patients with PMF and <1% Tregs (n=15), and in CTRLs (n=10): 1) the percentage of PB CD4+, CD4+CD25+, and CD4+CD25++CD127low/- cells expressing membrane Annexin V at time of sampling as a measure of spontaneous apoptosis 2) the phosphorylation of Stat1, Stat3, Stat5, Stat6, Erk1,2 and p38-MAPK determined by using flow-based intracellular staining (BD Biosciences) at baseline and following stimulation with the appropriate cytokine in CD4+, CD4+CD25-, CD4+CD25+, and CD4+CD25++FOXP3+ cells 3) the levels of circulating soluble IL2 receptor alpha (sIL2rα) in 34 patients with PMF and Tregs <1% and 11 CTRLs by ELISA (R&D), due to the relevance of IL2 in the generation, function, and survival of Tregs and data reported by Tefferi A et al (J Clin Onc 2011; 29:1356-63). Results. 1) The percentages of PB CD4+, CD4+CD25+, or CD4+CD25++CD127low/- cells expressing membrane Annexin V were comparable in patients with PMF and CTRLs (data not shown). 2) The phosphorilation of Stat1, Stat3, Stat5, Stat6, and p38-MAPK (expressed as mean fluorescence intensity: MFI) on CD4+, CD4+CD25-, CD4+CD25+, and CD4+CD25++FOXP3+ cells before and after stimulation with IFNα, IL6, IL4, or PMA was comparable in patients and CTRLs. The Erk1,2 phosphorilation was comparable in patients and CTRLs at baseline condition, while in patients with PMF Erk1,2 phosphorilation following stimulation with PMA was significantly lower in both CD4+CD25+ cells (p=0.039; median MFI 5.4, range 0.3-40) and CD4+CD25++FOXP3+ cells (p=0.01; median MFI 4.3, range 1.4-9.3) than in CTRLs (CD4+CD25+ cells median MFI 8.1, range 4.9-20; CD4+CD25++FOXP3+ cells median MFI 7.3, 3.7-10.8). 3) sIL2rα plasma levels were higher (p=0.01) in patients with PMF (1658 pg/ml, range 476-8968) than in CTRLs (1040 pg/ml, 482-1719); the sIL2rα plasma levels were significantly (R=-0.35, p=0.039) inversely correlated with the percentages of circulating Tregs. No significant correlation was found with the presence (n=13)/absence (n=14) of JAK2V617F mutation. Conclusions. These results indicate that in patients with PMF Tregs present an untuned Erk signaling; moreover, sIL2rα plasma levels are higher in patients than in CTRLs, and significantly inversely correlated with Treg percentages. These findings may partly explain the reduced frequency of Tregs in PMF since Erk signaling is pivotal in T cell differentiation, regulation of T cell homeostasis, and defects in Erk expression have been associated with autoimmunity (Chiung-Fang Chang J Immunol 2012; 189:721-731). Moreover, increased levels of sIL2rα may impact on IL-2-mediated functions and alterate the optimal activation of Erk1/2 that is IL-2 dependent (Lequn Li, Blood. 2005;106:3068-3073), Disclosures No relevant conflicts of interest to declare.
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Leeman-Neill, Rebecca J., Devang Thakkar, Sarah L. Ondrejka, Eric D. Hsi, Amy Chadburn, Mateo Mejia Saldarriaga, Sarah C. Rutherford, et al. "Genomic and Transcriptional Characterization of Primary Mediastinal Large B Cell Lymphoma." Blood 138, Supplement 1 (November 5, 2021): 2398. http://dx.doi.org/10.1182/blood-2021-149745.
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Abstract Introduction: Primary mediastinal large B-cell lymphoma (PMBL) is a rare non-Hodgkin lymphoma subtype that occurs predominantly in young adults, with an overall favorable prognosis. The cell of origin is presumed to be thymic medullary B-cells and the gene expression profile of PMBL is similar to classic Hodgkin lymphoma. Recent studies have begun unravelling the genomic alterations underlying PMBL. Frequent, recurrent mutations (e.g. B2M, TNFAIP3, SOCS1, STAT6, GNA13) have been reported, but most of the studies have analyzed a small number of cases. To gain further insights into disease biology, we recruited 63 cases of PMBL as part of the Atlas of Blood Cancer Genomes (ABC-G) initiative, a consortium consisting of 25 institutions. Methods: Formalin-fixed paraffin-embedded (FFPE) biopsies and clinical data were collected. All cases were subjected to centralized review by an experienced panel of hematopathologists to ensure accurate diagnosis. Whole-exome DNA and RNA sequencing was performed using the Illumina platform and the DNA and RNA reads aligned to the GRCh38 genome and transcriptome respectively. Exonic variants were filtered using a set of paired normal samples and population-based databases to identify putative driver mutations, which were then aggregated at the gene level. Mutational analysis was performed on 56 samples that passed quality filtering and expression analysis on 45 samples. RNAseq data was normalized using DESeq2. Results: The cohort included samples from 16 males and 24 females, with a median age of 33 years (range 16 - 72) at the time of diagnosis. The majority of patients were treated with R-CHOP (47%) or R-EPOCH (43%), with 93% of patients surviving through the end of follow-up (median follow-up: 60.1 months). Besides the known recurrent mutations involving the JAK-STAT (STAT6 -21%, SOCS1 - 26%), NFKB (TNFAIP3 - 27%, NFKB1A - 7%), immune escape (B2M - 20%, LTB - 11%, IRF8 - 9%, IRF4 -9%), and chromatin modification (ZNF217 - 16%, CREBBP - 11%, KMT2D -11%) pathways , we discovered recurrent somatic variants in novel candidate driver genes in this disease, including NOTCH4 (7%), DICER1 (11%), MCL1 (7%), amongst others. EZH2, EP300, and XPO1 mutations were not detected. CIITA mutations and fusions were observed in 14% and 11% of cases, respectively, with novel partner genes (IGHA2, IGHG1, CDC6) detected in 67% of the fusion positive cases. Copy number alterations included gains at 2p16.1 (REL - 20%) and 9p24.2 (JAK2/PDL1/PDL2 - 24%), as well as loci not previously implicated in PMBL, 8q24.3 and 9q34.3 (each in 20%). Of note, CIITA alterations and 9p24 gains were virtually mutually exclusive, highlighting diverse mechanisms of immune escape in this entity. The transcriptomes of cases harboring CIITA alterations demonstrated differential enrichment of genes involved in protein glycosylation. The PMBLs in our series showed significant enrichment of the reported PMBL genetic classifier score, compared to nodal diffuse large B cell lymphoma (DLBCL) (p=0.0003). Finally, the gene expression profile of thymic B cells was more similar to that of PMBL than nodal DLBCL (p=0.0144). Conclusions: Our study, representing one of the largest comprehensive genomic and transcriptomic analyses of PMBL, expands the mutational landscape of PMBL, provides evidence for biologically distinct disease subsets and suggests an origin of PMBLs from thymic B-cells. Disclosures Hsi: AbbVie: Research Funding; Eli Lilly: Research Funding; Cytomx: Honoraria; Seattle Genetics: Honoraria. McKinney: BTG: Consultancy; Celgene: Consultancy, Research Funding; Epizyme: Consultancy; Genetech: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria, Speakers Bureau; Molecular Templates: Consultancy, Research Funding; Nordic Nanovector: Research Funding; Novartis: Research Funding; Pharmacyclics: Consultancy; Verastem: Consultancy; Beigene: Research Funding; ADC Therapeutics: Consultancy, Speakers Bureau. Jaye: Stemline Therapeutics: Honoraria. Cohen: Genentech, Takeda, BMS/Celgene, BioInvent, LAM, Astra Zeneca, Novartis, Loxo/Lilly: Research Funding; Janssen, Adaptive, Aptitude Health, BeiGene, Cellectar, Adicet, Loxo/Lilly, AStra ZenecaKite/Gilead: Consultancy. Behdad: Lilly: Speakers Bureau; Roche/Foundation Medicine: Speakers Bureau; Thermo Fisher: Speakers Bureau. Dave: Data Driven Bioscience: Current equity holder in publicly-traded company.
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Minakawa, Keiji, Koki Ueda, Osamu Nakajima, Tetsuro Yokokawa, Yusuke Kinishima, Tomofumi Misaka, Kazuei Ogawa, Takayuki Ikezoe, Yasuchika Takeishi, and Kazuhiko Ikeda. "Knock-Ins of Type-2 Calr Mutants Cause Myeloproliferative Neoplasm (MPN)-like Hematopoiesis in Mice." Blood 134, Supplement_1 (November 13, 2019): 2964. http://dx.doi.org/10.1182/blood-2019-124575.
Full textAbstract:
MPNs, including polycythemia vera, essential thrombocythemia (ET) and myelofibrosis (MF), are characterized by proliferation of mature myeloid cells. A somatic mutation in a hematopoietic stem cell (HSC) that activates JAK/STAT pathway drives MPN. Following the JAK2V617F, the CALR insertion/deletion mutations (indels) are the second most frequent driver and present in 20-30% ET and primary MF. Two major indels, a 52-bp deletion (type 1, p.L367fs*46) and a 5-bp insertion (type 2, p.K385fs*47), account for 80% of the CALR mutations. In addition, there have been more than 100 other indels, which can be classified as type 1- and type 2-like mutations based on the length of negatively-charged amino acid (AA) stretch at the C-terminal side of mutated CALR. Patients with type 1/type 1-like CALR mutations exhibit more incidence of MF while type 2/type 2-like mutations are associated with higher platelet counts in ET (Petra et al, Leukemia, 2016). In previous studies, mice carrying type 1/type 1-like mutations, including knock-in (KI) models, showed mild ET- or MF-like hematopoiesis. Although wild-type (WT) CALR AA sequences are highly conserved between human and mouse, there have been no KI models of type 2/type 2-like CALR mutations. Here, we generated 2 lines of KI mice carrying type 2-like Calr mutations, 2-bp insertion (CR2i, p.K378fs*53) and 10-bp deletion (CR10d, p.K375fs*52), using the CRISPR/Cas9 method. Both KIs removed KDEL, altered AA charges and increased values for isoelectric point, which are similar to type 2/type 2-like mutations in MPN patients. Compared with WT mice, peripheral platelets (1277 ± 228 vs 1560 ± 344 x 109/L, p = 0.004) and leukocytes (14.4 ± 3.7 vs 18.7 ± 4.9 x 109/L, p = 0.006) were increased in CR10d mice, whereas blood cell counts were not different between CR2i and WT mice. In FACS, both CR10d (p = 0.04) and CR2i (p = 0.04) mice exhibited an increased myeloid-cell ratio in bone marrow (BM). Splenomegaly was not present, but histopathological study showed a significant increase and accumulation of large megakaryocytes in BM and spleen of both KI mouse lines. BM fibrosis was not present in any sample. Therefore, CR10d and CR2i mice mimicked ET-like and unclassifiable MPN-like hematopoiesis, respectively. Next, we studied the basis of MPN-like hematopoiesis in CR10d and CR2i mice. Colony forming-cell assay in the presence of cytokines showed reduced growth of CFU-E, especially in CR2i mice (p = 0.01) compared with WT mice, while there was no difference in growth of CFU-Mk between CR10d or CR2i mice and WT mice. TPO-independent colony growth was not observed in both KI mice. Correspondingly, FACS showed comparable expression of phospho-STAT3 (pSTAT3) in BM cells between CR10d or CR2i mice and WT mice in the absence of TPO. However, pSTAT3 was significantly upregulated both in CR10d and CR2i mice compared with WT mice in the presence of TPO, suggesting that high sensitivity of HSCs or progenitor cells to TPO contributes to MPN phenotype in these mice. Thus, we investigated HSC function by a competitive repopulation assay, in which we transplanted a mixture of BM cells from KI mice (Ly5.2) and Ly5.1 mice at a 1:1 ratio into lethally irradiated Ly5.1 mice, showed reduced repopulating capacity, especially in CR2i mice. In the second transplant recipients, cells derived from either CR2i or CR10d mice were markedly diminished, suggesting the reduced self-renewal capacity of an HSC carrying a type 2/type 2-like Calr mutation. Finally, we performed RNAseq for FACS-sorted HSC-enriched lineage-Sca1+Kit+ (LSK) cells, which revealed that approximately 70% of genes among differentially expressed genes were commonly upregulated or downregulated in CR2i and CR10d mice, suggesting a similarity in gene expression profile of LSK cells of these KI mouse lines. As a result, there were several pathways commonly affected in both CR2i and CR10d mice in gene set enrichment analysis, including upregulation of JAK/STAT pathway (FDRq = 0.060 in CR2i and 0.111 in CR10d). On the other hand, targets of polycomb recessive complex 2, which are important for HSC functions in MPNs (Ueda et al, Blood Adv, 2017), were downregulated in both KI mouse lines (FDRq = 0 in both CR2i and CR10d), possibly explaining the reduced repopulating capacities of CR2i and CR10d HSCs. In conclusion, our data indicate that type 2/type 2-like Calr mutation can cause MPN-like hematopoiesis. For disease progression, further mechanism may be required. Disclosures Yokokawa: Actelion Pharmaceuticals Ltd: Other: Donated Fund Laboratory. Ikeda:Kyokuto Pharmaceutical: Research Funding; Hokuyo Denki: Research Funding; Novartis Pharma: Honoraria; Takeda Pharmaceutical: Honoraria, Research Funding.
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Salehi, Matin, Maria Cristina Pirosa, Alessio Bruscaggin, Lodovico Terzi Di Bergamo, Federico Jauk, Gabriela Forestieri, Simone Bocchetta, et al. "A Comprehensive Genetic Study of Classical Hodgkin Lymphoma Using Ctdna." Blood 144, Supplement 1 (November 5, 2024): 854. https://doi.org/10.1182/blood-2024-207234.
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Introduction. We leveraged advanced analytical methods focusing on ctDNA to provide an in-depth overview of the genetic landscape of classical Hodgkin lymphoma (cHL) and its connection to disease pathophysiology and clinical course. Methods. Our study investigated somatic mutations, somatic copy number abnormalities (SCNA), fusions, and whole genome duplication (WGD) in ctDNA from 297 patients enrolled in the IOSI-EMA003-NCT03280394 (treatment-naïve - TN: 215; relapsed/refractory - RR treated with checkpoint inhibitors: 37) and FIL-RougeBIO-NCT05066555 studies (N=45, all TN) using LyV4.0 CAPP-seq. Results. The genes most frequently affected by non-synonymous mutations were SOCS1, TNFAIP3, STAT6, B2M, GNA13, and ITPKB. When considering SCNA, we identified gains in 2p16.1 (REL, XPO1), 3q29 (BCL6), 7q36.3 (EZH2), 9p24.2 (JAK2, CD274, PDCD1LG2), 8q24.23 (MYC), 15q26.3 (IGF1R), and deletions in 1p36.13 (TNFRSF14), 1p12 (CD58), 3q27.1 (KLHL6), 6q23.3 (TNFAIP3), 13q13.3 (FOXO1), 14q12 (NFKBIA), 15q25.3 (B2M), 16p13.3 (CREBBP), 20q13.13 (PTPN1). Recurrent fusions were observed in 32% of cases. No mutations or SCNA were found to correlate with reduced progression-free survival (PFS) in TN cHL. WGD was detected in 24% of patients, which is higher than the frequency observed in other mature B-cell malignancies. cHL with WGD showed a higher incidence of CCNE1 gain, which affects the checkpoint associated with polyploidization. In contrast, multi-hit TP53 lesions or RB1 abnormalities were infrequent and did not correlate with WGD. WGD was significantly linked to a shorter PFS in TN cHL of the IOSI-EMA003 training cohort by univariate and multivariate analyses (HR: 2.4; p=0.02) and further confirmed as a prognostic factor for PFS in the FILRougeBIO validation cohort (HR: 8.9, p=0.01). EBV infection was detected in 18% of cases. EBV+ cases were mostly mutated in SOCS1, infrequently mutated in STAT6, and lacked the loss of PTPN1 and NFKBIA. EBV+ cHL exhibited a significantly lower burden of mutations and SCNA. Two major classes of microenvironments emerged through digital cytometry deconvolution and tissue microarray analysis of cHL biopsies. One class was marked by macrophages (52% of cases) and the other by T-cells and immune checkpoints (48% of cases). Mutations, SCNA, and WGD were evenly distributed across both microenvironmental classes. Conversely, cHL cases with a T-cell-enriched microenvironment had fewer subclonal MHC-I neoantigens, indicating selective pressure exerted from T-cells. Consistently, RR cHL treated with checkpoint inhibitors had longer PFS if they lacked subclonal MHC-I neoantigens. LyV4.0 CAPP-seq together with NMF clustering, reproduced the classification of the C1-C5 genetic subtypes in 235 untreated DLBCL patients. In cHL, two clusters were identified using the same approach. Although the two clusters had similar baseline characteristics, EBV infection rates, ctDNA load, and outcomes, the two clusters differed in their underlying mechanisms of genetic instability. One cluster was defined by aneuploidy and a higher number of SCNA (36% of cases), while the other was characterized by mutations in genes targeted by AID-hypermutation (64% of cases) and had a higher fraction of mutations associated with AID activity signatures (SBS39, SBS84, SBS85). Both clusters shared hallmark lesions of cHL, including STAT6 mutations, and gains in 2p/2p16.1 and 9p/9p24.2. In multivariate regression analysis, MTV and WGD were the most important variables influencing pre-treatment ctDNA quantity. Conversely, the fraction of tumor cells and proliferating cells in the biopsy, and levels of DNASE1L3 showed minimal importance in explaining ctDNA concentration. While we could confirm pre-treatment ctDNA levels above 2.5 Log10 hGE/ml of plasma as a prognostic factor for DLBCL patients, they did not impact PFS in either the IOSI-EMA003 or in the FILRougeBIO cohorts. Conclusions. WGD is the only confirmed genetic factor for predicting the prognosis of TN cHL. MHC-I neoantigen load correlates with the microenvironment characteristics and the response to checkpoint inhibitors rather than specific genetic mutations. The genetic subtypes of cHL are mainly defined by the mechanisms driving genetic instability rather than by individual genetic lesions or EBV infection. The ctDNA load has no prognostic significance in cHL.