Статті в журналах: "LNK"

1

Takaki, Satoshi, Julian D. Watts, Katherine A. Forbush, Nhan T. Nguyen, Jun Hayashi, Jose Alberola-Ila, Ruedi Aebersold, and Roger M. Perlmutter. "Characterization of Lnk." Journal of Biological Chemistry 272, no. 23 (June 6, 1997): 14562–70. http://dx.doi.org/10.1074/jbc.272.23.14562.

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2

koren-Michowitz, Maya, Sigal Gery, Daniel Nowak, Arnon Nagler, Aiko Matsubara, Takayuki Tabayashi, Seishi Ogawa, and H. Phillip Koeffler. "Adaptor Protein LNK Binds to and Is Phosphorylated by JAK3 and May Serve as a Scaffold for JAK3 Autophosphorylation In the Absence of An Appropriate Cytokine Receptor." Blood 116, no. 21 (November 19, 2010): 2785. http://dx.doi.org/10.1182/blood.v116.21.2785.2785.

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Abstract Abstract 2785 The adaptor protein LNK has an important role during the development and maturation of cells in the hematopoietic system. LNK KO mice have an expansion of hematopoietic stem cells (HSCS) and an increase in circulating neutrophils, platelets, as well as immature B cells. On the other hand, overexpression of WT LNK under the control of a lymphocyte-specific expression vector results in an impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. Recently, LNK PH domain mutations were described in myeloproliferative neoplasms (MPN). The non-receptor tyrosine kinase JAK3 is expressed mainly in hematopoietic cells and together with JAK1, transmits signals from cytokine receptors of the IL-2 family. While JAK3 loss of function causes severe combined immune deficiency (SCID), JAK3 activating mutations were described in AML, particularly in acute megakaryocytic leukemia (AMKL). The importance of both LNK and JAK3 in lymphopoiesis, coupled with the finding that LNK binds to another JAK family member (JAK2), has led us to study whether LNK and JAK3 interact. LNK and JAK3 co- immunoprecipitated (co-IP) in the following leukemic cell lines- HEL, NALM6 (containing both LNK and WT JAK3), CMK (containing both LNK and an activating JAK3 mutant) and not in K562 (no JAK3). WT-JAK3 and WT LNK also co-IP when overexpressed in 293T cells, and this binding was associated with phosphorylation of LNK. To determine which domain of LNK is responsible for JAK3 binding, WT JAK3 was overexpressed in 293T cells with the following LNK mutants: 1. point mutation in the SH2 domain (R392E); 2. Deletion of the SH2 domain (del SH2); 3. Deletion of the PH and SH2 (del SH2/PH) domains; 4. Point mutation in the PH domain, recently found in a patient with a JAK2 V617F negative MPN (E208Q); 5. LNK SNP (rs3184504) described in GWAS to associate with an increased risk of autoimmune disease and which also causes an amino acid change in the LNK PH domain (W262R). LNK binding was retained in the E208Q and W262R mutants, markedly reduced in the R392E and del SH2 mutants and completely abolished with the del SH2/PH mutant, suggesting that LNK SH2 domain is important for the binding of LNK and WT-JAK3, although some binding may occur even in its absence. The effect of the LNK mutants R392E, E208Q and W262R on WT-JAK3 and LNK phosphorylation was also studied in 293T cells. Notably, WT-JAK3 was phosphorylated in the presence of WT-LNK and the E208Q and W262R mutants and not in the presence of the R392E mutant or an empty vector; also, the WT-LNK, LNK E208Q and W262R were phophorylated and the LNK R392E was not. These results suggest that WT-JAK3 phosphorylates LNK and that the LNK SH2 domain is important for this to occur. Interestingly, we found that the LNK PH domain mutant (E208Q) was much more phosphorylated than the WT- LNK when co-expressed with WT-JAK3. A similar result was obtained when LNK E208Q was co-expressed in 293T cells with vectors expressing an activating JAK3 mutant- A572V and the JAK2 V617F mutant but not when it was co-expressed with a BCR-ABL vector. This, again, supports the fact that direct binding underlines LNK phosphorylation by JAK3, as was previously shown for JAK2, while the BCR-ABL kinase does not bind LNK. The LNK PH domain mutation E208Q may cause a conformational change in LNK, allowing better access to tyrosine residues, or a change in LNK localization. The finding in 293T cells that WT-JAK3 is phophorylated only in the presence of LNK that retains a PH domain is intriguing. The other LNK family members- SH2B1 and APS were previously shown to homo- and heterodimerize. LNK contains a dimerization domain and we speculate that LNK homodimerization may serve as a scaffold for WT-JAK3 in the absence of a cytokine receptor, enabling JAK3 autophosphorylation. Since LNK PH domain mutations were recently described in MPN and may involve activation of the JAK2-STAT pathway we hypothesized that similar mutations might be found in lymphoma cases where JAK3 is reported to be activated. We, therefore, sequenced LNK PH domain in 147 lymphoma samples including- mantle cell lymphoma, peripheral T cell lymphoma (PTCL) and adult T cell leukemia/lymphoma cases but could not detect any mutations. In summary- we found that JAK3 binds and phosphorylates both WT-LNK and to a greater extent, the LNK PH domain mutant E208Q and in the absence of a cytokine receptor can lead to JAK3 phosphorylation. Taken together, LNK may likely control JAK3 activity. Disclosures: No relevant conflicts of interest to declare.

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3

Dou, Huijuan, Andriana Kotini, Wenli Liu, Trevor Fidler, Kaori Endo-Umeda, Xiaoli Sun, Malgorzata Olszewska, et al. "Oxidized Phospholipids Promote NETosis and Arterial Thrombosis in LNK(SH2B3) Deficiency." Circulation 144, no. 24 (December 14, 2021): 1940–54. http://dx.doi.org/10.1161/circulationaha.121.056414.

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Background: LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT ) reduces LNK function and that LNK-deficient mice display prominent platelet–neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet–neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency. Methods: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr –/– mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2 V617F ). Results: Lnk -deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk –/– platelets promoted increased NETosis when incubated with Lnk –/– neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk –/– platelets, as well as increased priming and response of Lnk –/– neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk –/– mice. We also showed increased NETosis when human induced pluripotent stem cell–derived LNK(TT ) neutrophils were incubated with LNK(TT ) platelet/megakaryocytes, but not in isogenic LNK(CC ) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2 VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk +/– and Jak2 VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2 VF Lnk +/+ controls. Conclusions: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2 VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.

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4

Tabayashi, Takayuki, Sigal Gery, Maya Koren-Michowitz, and H. Phillip Koeffler. "Adaptor Protein Lnk Negatively Regulates Bcr-Abl-Induced Cell Proliferation through Inhibition of the Stat5 Signaling Pathway." Blood 116, no. 21 (November 19, 2010): 3406. http://dx.doi.org/10.1182/blood.v116.21.3406.3406.

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Abstract Abstract 3406 Adaptor protein Lnk negatively regulates not only several hematopoietic cytokine receptors including MPL, EpoR and c-Kit, but also non-receptor tyrosine kinases such as JAK2 and Src. Our previous studies demonstrated that Lnk, when expressed in hematopoietic cell lines, binds and regulates the mutant proteins, JAK2V617F and MPLW515L. Recent in vivo studies have shown that Lnk has an important role in the development of myeloproliferative neoplasms. These data suggest that Lnk may have the ability to inhibit constitutively activated signaling pathways in hematopoietic malignancies. However, how Lnk can attenuate the activity of Bcr-Abl is unclear. In the present study, we tested the hypothesis that Lnk might play a role in regulating Bcr-Abl function. In order to assess if Lnk can inhibit the proliferation of Bcr-Abl-positive hematopoietic cells, Bcr-Abl-expressing BaF3 cells were stably transfected with either Lnk (BaF3/Bcr-Abl/Lnk) or vector only (BaF3/ Bcr-Abl). Colony-formation assays revealed that Lnk significantly inhibited the proliferation of Bcr-Abl-expressing BaF3 cells. Similarly, overexpression of Lnk inhibited growth in the human CML cell line, K562. To determine the cause of growth inhibition by Lnk, assays for apoptosis were performed. Annexin V staining demonstrated that Lnk overexpression induced apoptosis in Bcr-Abl-expressing BaF3 cells. Western blotting analysis of protein lysates from BaF3/ Bcr-Abl /Lnk cells and BaF3/ Bcr-Abl cells found that Lnk-mediated growth inhibition was associated with downregulation of the Stat5 signaling pathway, but not associated with MAPK and PI3K signaling pathways. In addition, experiments in 293T cells expressing Bcr-Abl and Stat5 with either wild-type Lnk or SH2 mutant Lnk revealed that wild-type Lnk, but not SH2 mutant Lnk, inhibited phosphorylation of Stat5. Interestingly, Lnk inhibited Bcr-Abl-induced Stat5 phosphorylation in a dose-dependent manner. These data suggest that the SH2 domain of Lnk is essential for Lnk–mediated downregulation of the Stat5 signaling pathway in Bcr-Abl-positive cells. Taken together, our data suggest that Lnk inhibits Bcr-Abl-induced cell proliferation by attenuating the Stat5 signal transduction and may become a therapeutic target for Bcr-Abl-positive leukemias such as chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia. Disclosures: No relevant conflicts of interest to declare.

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5

Gery, Sigal, Saskia Gueller, Julia Sohn, Shayne Nabavinouri, Amanda Leiter, and H. Phillip Koeffler. "Adaptor Protein Lnk Negatively Regulates Mutant MPL and JAK2 Alleles Associated with Myeloproliferative Disorders." Blood 110, no. 11 (November 16, 2007): 1531. http://dx.doi.org/10.1182/blood.v110.11.1531.1531.

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Abstract Activating mutations in the cytokine receptor/JAK2 signaling axis are found at high frequency in myeloproliferative disorders (MPD). Lnk, an SH2-containing adaptor protein, is a negative regulator of several hematopoietic cytokine receptors including MPL and EpoR. Here, we assessed whether Lnk can attenuate the activity of mutant MPLW515L, JAK2V617F and JAK2K539L found in MPD patients. Lnk overexpression in Ba/F3-MPLW515L cells inhibited cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhanced proliferation. Lnk-mediated growth inhibition was associated with downregulation of JAK/STAT, MAPK and PI3K signaling pathways. Similarly, Lnk inhibited cytokine-independent growth conferred by JAK2V617F and JAK2K539L in Ba/F3-EpoR cells. Following thrombopoietin stimulation, Lnk became tyrosyl-phosphorylated and associated with activated wild-type (WT) MPL and MPLW515L at the plasma membrane of Ba/F3 cells. An SH2 mutant Lnk (R392E) failed to bind and inhibit WT MPL and MPLW515L, demonstrating that the SH2 domain is essential for Lnk down-modulation of the receptors. The Lnk-MPL interaction was also detected with endogenously expressed proteins from cultured bone marrow cells. A series of C-terminally truncated Lnk constructs were used to determine which Lnk regions are required for Lnk inhibition of JAK2. Experiments in 293T cells indicated that Lnk SH2 domain binds to phosphorylated JAK2. In addition, other Lnk regions associated with non-phosphorylated JAK2, and these interactions were critical for Lnk inhibition of JAK2V617F and JAK2K539L constitutive activation. Our data suggest a model wherein Lnk downregulation of the receptor/JAK2 signaling involves two mechanisms; one is inhibition of the cytokine receptor utilizing JAK2, the second is direct suppression of JAK2 kinase activity. Furthermore, while the receptor mediated inhibition requires the SH2 domain, direct inhibition of JAK2 mutants, V617F and K539L, relies on other Lnk domains. Further elucidating the molecular mechanisms underlying Lnk inhibition of signaling pathways abnormally activated by oncogenic alleles, will provide insight into the pathogenesis of MPD and may have therapeutic value.

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6

Kiladjian, Jean-Jacques, Fanny Baran-Marszak, Christophe Desterke, Bruno Cassinat, Hajer Magdoud, Elisabeth Mazoyer, Pierre Fenaux, et al. "Abnormal Expression and Function of the Lnk Adaptor Protein in Myeloproliferative Neoplasms (MPN)." Blood 114, no. 22 (November 20, 2009): 2897. http://dx.doi.org/10.1182/blood.v114.22.2897.2897.

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Abstract Abstract 2897 Poster Board II-873 Introduction: We have shown in a deficient mouse model that the adaptor protein Lnk had an important role as negative regulator of cytokine signaling during hematopoiesis (Velazquez et al., J. Exp Med 2002). Lnk-/- animals display abnormal megakaryopoiesis sharing many features with that found in MPN patients. This phenotype is due to loss of Lnk inhibition of thrombopoietin (TPO)-mediated JAK2 activation (Tong et al., J Exp Med 2004). Recent studies have shown that Lnk, when expressed in hematopoietic cell lines, could bind and regulate two mutant proteins found in MPNs, JAK2V617F and MPLW515L. However, the role of Lnk in MPN pathogenesis is still unclear. In the present study, we studied in detail both Lnk expression and function in MPNs. Patients and methods: The study included a total of 82 MPN patients (pts), including 41 essential thrombocytemia (ET), 29 polycythemia vera (PV) and 12 primary myelofibrosis (PMF). Lnk expression was assessed by quantitative RT-PCR. Biochemical and cellular analyses of Lnk and JAK2 interaction were carried out using co-immunoprecipitation, GST pull-down and proliferation assays on primary hematopoietic cells and cell lines expressing either wild-type (WT) or mutant forms of both Lnk and JAK2. Results: Lnk mRNA was clearly overexpressed in platelets and CD34+ cells of most MPN pts compared to controls (P=0.005 and P=0.03, respectively). Moreover, this increased Lnk expression strongly correlated with JAK2V617F allele burden (P=0.02). In contrast, Lnk mRNA levels were reduced in the 18 pts treated with interferon-α compared to the 34 pts treated with hydroxyurea (P=0.04). TPO specifically upregulated Lnk expression at both mRNA and protein levels in both primary and UT7/Mpl megakaryocytic (MK) cells. Analysis of TPO-stimulated platelets from ET patients revealed the existence of a new interaction site between Lnk and JAK2 located in the N-terminal region of Lnk, in addition to the previously known interaction mediated by Lnk SH2 domain. This interaction resulted in Lnk phosphorylation. In JAK2V617F expressing platelets or cell lines, we observed both increased phosphorylation of Lnk, and stronger binding of JAK2 to the N-terminal region of Lnk compared to WT-JAK2 cells. Overexpression of Lnk in JAK2V617F cells showed a dose dependent growth inhibition, as seen in JAK2 WT cells. In addition, overexpression of various mutant forms of Lnk showed that this inhibition required a fully functional SH2 domain. Finally, expression of either WT or mutant forms of Lnk also demonstrated the crucial role of Lnk SH2 domain in growth inhibition of myeloid and MK progenitors in Lnk-/- hematopoietic cells. Conclusion: This first study of a large cohort of 82 patients allowed us to investigate the role of Lnk in MPN: (1) Lnk mRNA was found to be significantly overexpressed in MPN derived platelets and CD34+ cells, and correlated with JAK2V617F allele burden. (2) Lnk expression is upregulated by TPO, an effect likely mediated by JAK2 activation. (3) The Lnk SH2 domain plays a major role in the down-regulation of both normal and MPN-derived hematopoiesis. (4) We describe here a novel interaction site between the N-terminal region of Lnk and JAK2. Stronger interaction of the JAK2V617F mutant form with this N-terminal binding site may account for the dysregulated hematopoiesis observed in MPN patients despite Lnk overexpression. Disclosures: No relevant conflicts of interest to declare.

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7

Eto, Koji, Hitoshi Takizawa, Satoshi Takaki, Hidekazu Nishikii, Atsushi Oda та Hiromitsu Nakauchi. "The Cytokine Signal Inhibitor Lnk Promotes αIIbβ3 Integrin Outside-In Signaling through β3 Tyrosine Phosphorylation in Platelets." Blood 110, № 11 (16 листопада 2007): 3651. http://dx.doi.org/10.1182/blood.v110.11.3651.3651.

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Abstract Lnk is an SH2 domain-containing adapter protein that inhibits cytokine signaling. Lnk−/− mice exhibit a marked increase in numbers of hematopoietic stem cells, megakaryocytes and platelets, presumably due to the lack of negative regulation in thrombopoietin-mediated signals by Lnk. We previously reported that Lnk might play an unanticipated role in platelet integrin αIIbβ3 outside-in signaling. Lnk−/− platelets exhibited defects in full spreading on fibrinogen, clot retraction and formation of thrombi on collagen under flow conditions while they showed normal inside-out signaling (Blood, 106 (11):115a, 2005). However the mechanism(s) in which Lnk participates in αIIbβ3 outside-in signaling had not been elucidated. Here we report that in normal platelets Lnk forms a complex with c-Src, Syk, Fyn and adhesion and degranulation promoting adaptor protein (ADAP) but not SLP-76 in a manner dependent on αIIbβ3 ligation and Src kinase activation. c-Src-, but not Syk-, mediated tyrosine phosphorylation of C-terminus in Lnk appeared to be indispensable for the complex formation and Lnk-mediated function. Furthermore we have shown that Lnk is required for the association of Fyn to αIIbβ3 and for β3 subunit tyrosine phosphorylation while activation of non-receptor tyrosine kinases (c-Src and Syk) in proximity to αIIbβ3 is independent of Lnk. Thus, these results provide new insights into Lnk function and the mechanism by which Lnk contributes to integrin signaling in the adhesion responses of platelets.

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8

de Leone, María José, Carlos Esteban Hernando, Andrés Romanowski, Mariano García-Hourquet, Daniel Careno, Joaquín Casal, Matías Rugnone, Santiago Mora-García, and Marcelo Javier Yanovsky. "The LNK Gene Family: At the Crossroad between Light Signaling and the Circadian Clock." Genes 10, no. 1 (December 20, 2018): 2. http://dx.doi.org/10.3390/genes10010002.

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Light signaling pathways interact with the circadian clock to help organisms synchronize physiological and developmental processes to periodic environmental cycles. The plant photoreceptors responsible for clock resetting have been characterized, but signaling components that link the photoreceptors to the clock remain to be identified. Members of the family of NIGHT LIGHT–INDUCIBLE AND CLOCK-REGULATED (LNK) genes play key roles linking light regulation of gene expression to the control of daily and seasonal rhythms in Arabidopsis thaliana. Particularly, LNK1 and LNK2 were shown to control circadian rhythms, photomorphogenic responses, and photoperiod-dependent flowering time. Here we analyze the role of the four members of the LNK family in Arabidopsis in these processes. We found that depletion of the closely related LNK3 and LNK4 in a lnk1;lnk2 mutant background affects circadian rhythms, but not other clock-regulated processes such as flowering time and seedling photomorphogenesis. Nevertheless, plants defective in all LNK genes (lnkQ quadruple mutants) display developmental alterations that lead to increased rosette size, biomass, and enhanced phototropic responses. Our work indicates that members of the LNK family have both distinctive and partially overlapping functions, and are an essential link to orchestrate light-regulated developmental processes.

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9

Tong, Wei, Jing Zhang, and Harvey F. Lodish. "Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways." Blood 105, no. 12 (June 15, 2005): 4604–12. http://dx.doi.org/10.1182/blood-2004-10-4093.

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Abstract Erythropoietin (Epo), along with its receptor EpoR, is the principal regulator of red cell development. Upon Epo addition, the EpoR signaling through the Janus kinase 2 (JAK2) activates multiple pathways including Stat5, phosphoinositide-3 kinase (PI-3K)/Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor signaling. Here, we showed that Lnk-deficient mice have elevated numbers of erythroid progenitors, and that splenic erythroid colony-forming unit (CFU-e) progenitors are hypersensitive to Epo. Lnk-/- mice also exhibit superior recovery after erythropoietic stress. In addition, Lnk deficiency resulted in enhanced Epo-induced signaling pathways in splenic erythroid progenitors. Conversely, Lnk overexpression inhibits Epo-induced cell growth in 32D/EpoR cells. In primary culture of fetal liver cells, Lnk overexpression inhibits Epo-dependent erythroblast differentiation and induces apoptosis. Lnk blocks 3 major signaling pathways, Stat5, Akt, and MAPK, induced by Epo in primary erythroblasts. In addition, the Lnk Src homology 2 (SH2) domain is essential for its inhibitory function, whereas the conserved tyrosine near the C-terminus and the pleckstrin homology (PH) domain of Lnk are not critical. Furthermore, wild-type Lnk, but not the Lnk SH2 mutant, becomes tyrosine-phosphorylated following Epo administration and inhibits EpoR phosphorylation and JAK2 activation. Hence, Lnk, through its SH2 domain, negatively modulates EpoR signaling by attenuating JAK2 activation, and regulates Epo-mediated erythropoiesis. (Blood. 2005; 105:4604-4612)

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10

Balcerek, Joanna, Jing Jiang, Alexey Bersenev, Yiwen Song, Chao Wu, and Wei Tong. "14-3-3 Regulates the Lnk/JAK2 Pathway In Hematopoietic Stem and Progenitor Cells." Blood 116, no. 21 (November 19, 2010): 86. http://dx.doi.org/10.1182/blood.v116.21.86.86.

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Abstract Abstract 86 Hematopoietic stem and progenitor cell (HSPC) homeostasis is regulated by intricate signaling networks. The tyrosine kinase JAK2 plays an essential role in cytokine signaling during hematopoiesis and its dysregulation can lead to hematologic malignancies. Recently activating mutations in JAK2 were found in a large fraction of patients with myeloproliferative neoplasms (MPNs). We previously demonstrated that lymphocyte adaptor protein (Lnk) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion (Bersenev et al., JCI, 2008;118:2832-2844). We further showed that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPNs in mice (Bersenev et al., JCI, 2010;120:2058-2069). Lnk directly inhibits oncogenic JAK2 as well as acting through JAK2- independent pathways to constrain MPN development. Consistently, aged Lnk–/– mice spontaneously developed a CML-like MPN (Bersenev et al., JCI, 2010;120:2058-2069). More importantly, loss-of-function mutations in Lnk are found in human MPN patients (Oh, et. al., Blood, 2010, in press). This work suggests that Lnk plays a pivotal role in regulating both normal and malignant HSPC expansion. However, it remains to be determined how Lnk attenuates JAK2 activity since Lnk itself does not possess any enzymatic activity. Therefore, we began exploring Lnk regulatory mechanisms by identifying novel Lnk partners through protein purification and mass spectrometric analysis. This resulted in the identification of a number of novel Lnk binding proteins, which include the 14-3-3 proteins as the most robust interactors. 14-3-3s are a group of scaffold proteins that regulate many disease-relevant gene products and play important functions in many aspects of cellular processes. We found that Lnk is phosphorylated at two serine residues, which serve as the critical binding sites for 14-3-3. 14-3-3 abrogates the Lnk-JAK2 interaction thereby alleviating Lnk inhibitory function in both JAK2 signaling and cell growth. Furthermore, 14-3-3 binding is necessary and sufficient to maintain Lnk in an inactive state. We also investigated the signals that regulate Lnk phosphorylation, revealing Lnk as a signaling nodal point that integrates multiple signaling pathways in controlling HSPC homeostasis. The physiological significance of the Lnk-14-3-3 interaction in HSPC development will be discussed. Thus, our data implicate previously unappreciated serine phosphorylation events in Lnk-dependent hematopoietic function and regulation of JAK2. In addition, identification of novel signaling molecules that influence hematopoiesis might facilitate stem cell therapies and provide novel therapeutic targets for the treatment of MPNs. Disclosures: No relevant conflicts of interest to declare.

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11

Lin, De-Chen, Tong Yin, Maya Koren-Michowitz, Ling-Wen Ding, Saskia Gueller, Sigal Gery, Takayuki Tabayashi, et al. "Adaptor protein Lnk binds to and inhibits normal and leukemic FLT3." Blood 120, no. 16 (October 18, 2012): 3310–17. http://dx.doi.org/10.1182/blood-2011-10-388611.

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Abstract Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in approximately one-third of AML patients, mostly by internal tandem duplications (ITDs). Adaptor protein Lnk is a negative regulator of hematopoietic cytokine signaling. In the present study, we show that Lnk interacts physically with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through the SH2 domains. We have identified the tyrosine residues 572, 591, and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Lnk itself was tyrosine phosphorylated by both FLT3 ligand (FL)–activated FLT3-WT and constitutively activated FLT3-ITD. Both shRNA-mediated depletion and forced overexpression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Moreover, Lnk inhibited 32D cell proliferation driven by different FLT3 variants. Analysis of primary BM cells from Lnk-knockout mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors. The results of the present study show that through direct binding to FLT3, Lnk suppresses FLT3-WT/ITD–dependent signaling pathways involved in the proliferation of hematopoietic cells. Therefore, modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD–associated hematopoietic disease.

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12

Nobuhisa, Ikuo, Makiko Takizawa, Satoshi Takaki, Hirofumi Inoue, Keisuke Okita, Masaya Ueno, Kiyoshi Takatsu, and Tetsuya Taga. "Regulation of Hematopoietic Development in the Aorta-Gonad-Mesonephros Region Mediated by Lnk Adaptor Protein." Molecular and Cellular Biology 23, no. 23 (December 1, 2003): 8486–94. http://dx.doi.org/10.1128/mcb.23.23.8486-8494.2003.

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ABSTRACT Development of hematopoietic cells in the aorta-gonad-mesonephros (AGM) region in the midgestation mouse embryo involves a multistep process, sequentially changing from endothelial cell-like cells, including hemangioblasts, into hematopoietic stem cells, progenitors, and/or lineage-committed cells. An adaptor molecule, Lnk, is known to negatively control the production of pro- and pre-B cells and hematopoietic progenitor cells in adult bone marrow. Here we show a role of Lnk in hematopoietic development in the AGM region. Lnk was predominantly expressed in the endothelial cells lining the dorsal aorta at embryonic day 11.5 (E11.5). Overexpression of Lnk in the primary culture of the AGM region at E11.5 suppressed the emergence of CD45+ hematopoietic cells. Point mutation in the SH2 domain of Lnk, which abolishes the binding capability of Lnk to c-Kit upon stimulation with stem cell factor (SCF), led to loss of Lnk-dependent inhibition of hematopoietic cell development in AGM cultures, suggesting Lnk-mediated inhibition of the SCF/c-Kit signaling pathway. In cultured AGM cells from Lnk homozygous mutant mouse embryos, the number of emerged CD45+ cells was 2.5-fold larger than that from heterozygous littermates. Furthermore, aorta cells of E11.5 Lnk homozygous mutant mice also showed enhanced hematopoietic colony-forming activity. Thus, Lnk is a negative regulator of hematopoiesis in the AGM region.

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13

Lasho, Terra, Ayalew Tefferi, Alessandro M. Vannucchi, Christy Finke, Paola Guglielmelli, Lisa Pieri, and Animesh Pardanani. "LNK Mutation Studies In Chronic- and Blast-Phase Myeloproliferative Neoplasms and JAK2 Mutation-Negative Erythrocytosis." Blood 116, no. 21 (November 19, 2010): 4105. http://dx.doi.org/10.1182/blood.v116.21.4105.4105.

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Abstract Abstract 4105 Background: JAK2 mutations are found in approximately 99% of patients with polycythemia vera (PV) and 60% of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF). It is currently assumed that other mutations relevant to JAK signaling contribute to the pathogenesis of JAK2 mutation-negative myeloproliferative neoplasms (MPN). The same might hold true for some cases of “idiopathic erythrocytosis” associated with subnormal serum erythropoietin level (sEpo). LNK is a plasma membrane-bound adaptor protein whose function includes inhibition of wild-type and mutant JAK2 phosphorylation. LNK exon 2 mutations were recently described in two patients with JAK2V617F-negative ET or PMF. Both mutations involved the LNK pleckstrin homology (PH) domain; one was a 5 base-pair deletion and missense mutation leading to a premature stop codon (603_607delGCGCT; 613C>G) and the other a missense mutation (622G>C; glutamic acid to glutamine substitution; E208Q). Objectives: i) To estimate the prevalence of LNK mutations in chronic and blast phase MPN ii) To determine if LNK mutations are mutually exclusive of other MPN-associated mutations iii) To test the hypothesis that LNK mutations might contribute to the pathogenesis of JAK2 mutation-negative PV or otherwise unexplained erythrocytosis Methods: LNK mutation analysis was performed on bone marrow or blood cells using modified primers for amplifying the SH2 and PH domains, using previously published methods (Oh et al. Blood First Edition Paper, prepublished online April 19, 2010; DOI 10.1182/blood-2010-02-270108). Results: LNK mutation studies were performed in 172 patients; paired chronic-blast phase samples were analyzed in 26 cases. Diagnoses in the 172 study patients were as follows: 78 chronic-phase MPN (39 JAK2V617F-positive PV, 25 PMF and 14 ET) enriched for TET2, IDH, JAK2V617F, or MPL mutations, 61 blast-phase MPN (41 blast-phase PMF, 11 blast-phase PV and 9 blast-phase ET), 25 JAK2V617F-negative PV and 8 “unexplained erythrocytosis” associated with subnormal sEpo and negative for Epo receptor (EPOR) mutations. Ten novel heterozygous LNK mutations, all but one affecting exon 2 in the PH domain, were identified: 6 missense mutations involving codons 215, 220, 223, 229 and 234, one nonsense mutation involving codon 208 (622G>T leading to glutamic acid to a stop codon substitution; E208X), one synonymous mutation involving codon 208, and 2 deletion mutations involving exon 2 (685-691_delGGCCCCG) or exon 5 (955_delA). LNK mutations were most frequent in blast-phase MPN, occurring in 6 (9.8%) of the 61 informative patients; chronic-phase sample analysis in 4 of these revealed the same mutation in only one case. Mutant LNK was detected in chronic-phase samples only in 2 additional patients with blast-phase MPN. Among the 8 LNK-mutated blast-phase MPN cases, 7 had blast-phase PMF. The clinical phenotype in the remaining 2 LNK-mutated patients was that of an isolated erythrocytosis without overt features of PV. JAK2V617F was documented in 3 and IDHR140Q in 1 LNK-mutated patients. Conclusions: LNK mutations i) target an exon 2 ‘hot spot' in the PH domain spanning residues E208-D234, ii) might be most prevalent in blast-phase PMF iii) are not mutually exclusive of JAK2 or IDH mutations and iv) might be part of the “missing link” in the pathogenesis of JAK2 mutation-negative “idiopathic” erythrocytosis. Disclosures: No relevant conflicts of interest to declare.

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Velazquez, Laura, Alec M. Cheng, Heather E. Fleming, Caren Furlonger, Shirly Vesely, Alan Bernstein, Christopher J. Paige, and Tony Pawson. "Cytokine Signaling and Hematopoietic Homeostasis Are Disrupted in Lnk-deficient Mice." Journal of Experimental Medicine 195, no. 12 (June 17, 2002): 1599–611. http://dx.doi.org/10.1084/jem.20011883.

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The adaptor protein Lnk, and the closely related proteins APS and SH2B, form a subfamily of SH2 domain-containing proteins implicated in growth factor, cytokine, and immunoreceptor signaling. To elucidate the physiological function of Lnk, we derived Lnk-deficient mice. Lnk−/− mice are viable, but display marked changes in the hematopoietic compartment, including splenomegaly and abnormal lymphoid and myeloid homeostasis. The in vitro proliferative capacity and absolute numbers of hematopoietic progenitors from Lnk−/− mice are greatly increased, in part due to hypersensitivity to several cytokines. Moreover, an increased synergy between stem cell factor and either interleukin (IL)-3 or IL-7 was observed in Lnk−/− cells. Furthermore, Lnk inactivation causes abnormal modulation of IL-3 and stem cell factor–mediated signaling pathways. Consistent with these results, we also show that Lnk is highly expressed in multipotent cells and committed precursors in the erythroid, megakaryocyte, and myeloid lineages. These data implicate Lnk as playing an important role in hematopoiesis and in the regulation of growth factor and cytokine receptor–mediated signaling.

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15

Hao, Meihua, Feng Yuan, Chenchen Jin, Zehong Zhou, Qi Cao, Ling Xu, Guanlei Wang, et al. "Overexpression of Lnk in the Ovaries Is Involved in Insulin Resistance in Women With Polycystic Ovary Syndrome." Endocrinology 157, no. 10 (July 26, 2016): 3709–18. http://dx.doi.org/10.1210/en.2016-1234.

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Polycystic ovary syndrome (PCOS) progression involves abnormal insulin signaling. SH2 domain-containing adaptor protein (Lnk) may be an important regulator of the insulin signaling pathway. We investigated whether Lnk was involved in insulin resistance (IR). Thirty-seven women due to receive laparoscopic surgery from June 2011 to February 2012 were included from the gynecologic department of the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. Samples of polycystic and normal ovary tissues were examined by immunohistochemistry. Ovarian cell lines underwent insulin stimulation and Lnk overexpression. Expressed Lnk underwent coimmunoprecipitation tests with green fluorescent protein-labeled insulin receptor and His-tagged insulin receptor substrate 1 (IRS1), and their colocalization in HEK293T cells was examined. Ovarian tissues from PCOS patients with IR exhibited higher expression of Lnk than ovaries from normal control subjects and PCOS patients without IR; mainly in follicular granulosa cells, the follicular fluid and plasma of oocytes in secondary follicles, and atretic follicles. Lnk was coimmunoprecipitated with insulin receptor and IRS1. Lnk and insulin receptor/IRS1 locations overlapped around the nucleus. IR, protein kinase B (Akt), and ERK1/2 activities were inhibited by Lnk overexpression and inhibited further after insulin stimulation, whereas IRS1 serine activity was increased. Insulin receptor (Tyr1150/1151), Akt (Thr308), and ERK1/2 (Thr202/Tyr204) phosphorylation was decreased, whereas IRS1 (Ser307) phosphorylation was increased with Lnk overexpression. In conclusion, Lnk inhibits the phosphatidylinositol 3 kinase-AKT and MAPK-ERK signaling response to insulin. Higher expression of Lnk in PCOS suggests that Lnk probably plays a role in the development of IR.

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16

Simon, Clotilde, Elisabetta Dondi, Amandine Chaix, Paulo de Sepulveda, Terrance J. Kubiseski, Nadine Varin-Blank, and Laura Velazquez. "Lnk adaptor protein down-regulates specific Kit-induced signaling pathways in primary mast cells." Blood 112, no. 10 (November 15, 2008): 4039–47. http://dx.doi.org/10.1182/blood-2008-05-154849.

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Abstract Stem cell factor (SCF) plays critical roles in proliferation, survival, migration, and function of hematopoietic progenitor and mast cells through binding to Kit receptor. Previous studies have implicated the adaptor protein Lnk as an important negative regulator of SCF signaling. However, the molecular mechanism underlying this regulation is unclear. Here, we showed that the Src homology 2 domain (SH2) of Lnk binds directly and preferentially to phosphorylated tyrosine 567 in Kit juxtamembrane domain. Using Lnk−/− bone marrow mast cells (BMMCs) transduced with different Lnk proteins, we demonstrated that Lnk down-regulates SCF-induced proliferation with attenuation of mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase signaling. Furthermore, we showed that Lnk−/− BMMCs displayed increased SCF-dependent migration compared with wild-type cells, revealing a novel Lnk-mediated inhibitory function. This correlated with enhanced Rac and p38 MAPK activation. Finally, we found that Lnk domains and carboxy-terminal tyrosine contribute differently to inhibition of in vitro expansion of hematopoietic progenitors. Altogether, our results demonstrate that Lnk, through its binding to Kit tyrosine 567, negatively modulates specific SCF-dependent signaling pathways involved in the proliferation and migration of primary hematopoietic cells.

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17

Gueller, Saskia, Helen S. Goodridge, Hongtao Xing, Sigal Gery, Hubert Serve, and H. Phillip Koeffler. "Adaptor Protein Lnk Inhibits C-Fms Mediated Macrophage Function." Blood 112, no. 11 (November 16, 2008): 3555. http://dx.doi.org/10.1182/blood.v112.11.3555.3555.

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Abstract The macrophage colony-stimulating factor receptor (c-Fms) plays an important role in proliferation, differentiation and survival of macrophages and is involved in the regulation of distinct macrophage functions. Interaction with the ligand M-CSF results in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. One such protein is the adaptor Lnk that negatively regulates several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain and a pleckstrin homology domain. The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. In this study, we investigated the ability of Lnk to interact and modulate the function of c-Fms. In order to determine if Lnk can bind to c-Fms, immunoprecipitation was performed with lysates from 293T cells co-transfected with the cDNAs for c-Fms and Lnk. Only after exposure to M-CSF, Lnk bound to c-Fms, and binding was dependent on an intact SH2 domain. To elucidate further if Lnk exhibits biological and functional effects on macrophages, we examined both in-vitro differentiated macrophages derived from the bone marrow and also macrophages harvested from peritoneum from Lnk deleted (KO) and wild type (WT) mice. These cells appeared to be at a similar stage of differentiation because expression levels of myeloid and macrophage surface markers such as F4/80, CD11b and CD11c were the same in both bone marrow-derived and peritoneum-derived macrophages from Lnk KO and WT mice. Clonogenic assays demonstrated that the number of M-CFUs in the bone marrow were elevated in Lnk KO as compared to WT mice. Furthermore, the M-CSF-induced phosphorylation of AKT in these Lnk KO macrophages was increased and prolonged compared to WT macrophages. This was associated with prominent up-regulation of c-Fms in macrophages from Lnk KO mice. We found that Lnk additionally had several functional effects on bone marrow-derived macrophages. Production of reactive oxygen species (ROS) was dramatically increased in a M-CSF-dependent manner in Lnk KO macrophages upon stimulation with zymosan. In addition, knock-out of Lnk led to altered cytokine production of macrophages: Stimulation with zymosan caused increased levels of TNFalpha and IL-6 in the KO cells, while bacterial lipoproteins (Pam3CSK4) decreased levels of TNFalpha in KO compared to WT macrophages. Last, Lnk inhibited M-CSF-induced migration of macrophages in the Boyden chamber as Lnk KO macrophages showed a significantly higher migration capacity than WT macrophages. In summary, we show for the first time that Lnk can bind to c-Fms and can blunt the stimulation of M-CSF. Modulation of levels of Lnk in macrophages may provide a unique therapeutic approach to increase innate host defenses.

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18

Gueller, Saskia, Sigal Gery, Verena Nowak, Liqin Liu, Hubert Serve, and H. Phillip Koeffler. "Adaptor protein Lnk associates with Tyr568 in c-Kit." Biochemical Journal 415, no. 2 (September 25, 2008): 241–45. http://dx.doi.org/10.1042/bj20080102.

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The adaptor protein Lnk is expressed in haemopoietic cells and plays a critical role in haemopoiesis. Animal model studies demonstrated that Lnk acts as a broad inhibitor of signalling pathways in haemopoietic lineages. Lnk belongs to a family of proteins sharing several structural motifs, including an SH2 (Src homology 2) domain which binds phosphotyrosine residues in various signal-transducing proteins. The SH2 domain is essential for Lnk-mediated negative regulation of several cytokine receptors [e.g. Mpl, EpoR (erythropoietin receptor), c-Kit]. Therefore inhibition of the binding of Lnk to cytokine receptors might lead to enhanced downstream signalling of the receptor and thereby to improved haemopoiesis in response to exposure to cytokines (e.g. erythropoietin in anaemic patients). This hypothesis led us to define the exact binding site of Lnk to the stem cell factor receptor c-Kit. Pull-down experiments using GST (glutathione transferase)-fusion proteins of the different domains of c-Kit showed that Lnk almost exclusively binds to the phosphorylated juxtamembrane domain. Binding of Lnk to the juxtamembrane domain was abolished by point mutation of Tyr568 and was competed by peptides with a phosphotyrosine residue at position 568. Co-immunoprecipitation with full-length wild-type or Y568F mutant c-Kit and Lnk confirmed these results, thus showing the importance of this phosphorylated tyrosine residue. Lnk bound directly to c-Kit without requiring other interacting partners. The identification of the binding site of Lnk to c-Kit will be useful to discover inhibitory molecules that prevent the binding of these two proteins, thus making haemopoietic cells more sensitive to growth factors.

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19

Eto, Koji, Hitoshi Takizawa, Hidekazu Nishikii, Jun Seita, Atsushi Oda, Noriko Tamura, Shinya Goto, Kiyoshi Takatsu, Satoshi Takaki, and Hiromitsu Nakauchi. "Negative Hematopoietic Scaffold Lnk Upregulates Integrin Outside-In Signaling in Platelets." Blood 106, no. 11 (November 16, 2005): 382. http://dx.doi.org/10.1182/blood.v106.11.382.382.

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Abstract The adaptor molecule Lnk negatively regulates signaling downstream of cytokine receptors in self-renewal of hematopoietic stem cells, proliferation of B precursors and differentiation/maturation of megakaryocytes. The circulating number of platelets in Lnk-deficient mice is increased by 5-folds to control, presumably owing to its negative effects on TPO-elicited signaling. Lnk expression is maintained in platelets, however, the precise functions in platelets remain unknown. Here we report that Lnk deficiency results in the prolonged bleeding time (2-folds, p<0.01) due to, at least in part, defective the outside-in signaling from α IIbβ 3 integrin. Lnk-null platelets exhibited less activation of PAK kinase, a Rac effector in fibrinogen-adherent platelets. These were concomitant with the defect of lamellipodial formation upon adhesion to fibrinogen in the absence of Lnk, compared to control platelets (p<0.01). Defective Lnk-null platelet functions were also demonstrated by the reduced three-dimensional growth (height) of platelet thrombi on the collagen surface under whole blood flow condition with the adjusted same platelet number at high wall shear rate of 1,500 s-1 (8.1±1.4 μ m in Lnk-deficient vs. 12.1±2.2 μ m in control, p<0.001), while the two-dimensional surface coverage was not impaired. On the other hand, there was no difference between Lnk-null and control platelets in aggregation and α IIbβ 3 integrin activation in response to stimulation with thrombin or collagen. Taken together, implication of Lnk to sites of adhesion may explain the phenotype of Lnk-deficient platelet function. The findings indicate that Lnk is a unique “dual-functioning” scaffolding molecule, which upregulates thrombus formation, while suppressing cytokine-mediated signaling.

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20

Gery, Sigal, Saskia Gueller, Katya Chumakova, Norihiko Kawamata, Liqin Liu, and H. Phillip Koeffler. "Adaptor protein Lnk negatively regulates the mutant MPL, MPLW515L associated with myeloproliferative disorders." Blood 110, no. 9 (November 1, 2007): 3360–64. http://dx.doi.org/10.1182/blood-2007-05-089326.

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Abstract Recently, activating myeloproliferative leukemia virus oncogene (MPL) mutations, MPLW515L/K, were described in myeloproliferative disorder (MPD) patients. MPLW515L leads to activation of downstream signaling pathways and cytokine-independent proliferation in hematopoietic cells. The adaptor protein Lnk is a negative regulator of several cytokine receptors, including MPL. We show that overexpression of Lnk in Ba/F3-MPLW515L cells inhibits cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhances proliferation. Lnk blocks the activation of Jak2, Stat3, Erk, and Akt in these cells. Furthermore, MPLW515L-expressing cells are more susceptible to Lnk inhibitory functions than their MPL wild-type (MPLWT)–expressing counterparts. Lnk associates with activated MPLWT and MPLW515L and colocalizes with the receptors at the plasma membrane. The SH2 domain of Lnk is essential for its binding and for its down-regulation of MPLWT and MPLW515L. Lnk itself is tyrosine-phosphorylated following thrombopoietin stimulation. Further elucidating the cellular pathways that attenuate MPLW515L will provide insight into the pathogenesis of MPD and could help develop specific therapeutic approaches.

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21

Takizawa, Hitoshi, Chiyomi Kubo-Akashi, Ikuo Nobuhisa, Sang-Mo Kwon, Masanori Iseki, Tetsuya Taga, Kiyoshi Takatsu, and Satoshi Takaki. "Enhanced engraftment of hematopoietic stem/progenitor cells by the transient inhibition of an adaptor protein, Lnk." Blood 107, no. 7 (April 1, 2006): 2968–75. http://dx.doi.org/10.1182/blood-2005-05-2138.

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AbstractHematopoietic stem cells (HSCs) are the key elements responsible for maintaining blood-cell production throughout life and for lymphohematopoietic reconstitution following bone marrow (BM) transplantation. Enhancement of the engrafting potential and expansion capabilities of HSCs as well as hematopoietic progenitor cells (HPCs) has been a long-time desire as a means of reducing the risks and difficulties that accompany BM transplantation. The ability of HSCs/HPCs to reconstitute the hematopoietic system of irradiated hosts is negatively regulated by an intracellular adaptor protein, Lnk. Here we have identified the functional domains of Lnk and developed a dominant-negative (DN) Lnk mutant that inhibits the functions of Lnk endogenously expressed in the HSCs/HPCs and thereby potentiates the HSCs/HPCs for engraftment. Importantly, even transient expression of DN-Lnk in HSCs/HPCs facilitated their engraftment under nonmyeloablative conditions and fully reconstituted the lymphoid compartments of immunodeficient host animals. HPCs expressing DN-Lnk were efficiently trapped by immobilized vascular cell adhesion molecule-1 (VCAM-1) in a transwell migration assay, suggesting involvement of Lnk in the regulation of cell mobility or cellular interaction in microenvironments. Transient inhibition of Lnk or Lnk-mediated pathways could be a potent approach to augment engraftment of HSCs/HPCs without obvious side effects.

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22

Tong, Wei, and Harvey F. Lodish. "Lnk Inhibits Tpo–mpl Signaling and Tpo-mediated Megakaryocytopoiesis." Journal of Experimental Medicine 200, no. 5 (August 30, 2004): 569–80. http://dx.doi.org/10.1084/jem.20040762.

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Thrombopoietin (Tpo) is the primary cytokine regulating megakaryocyte development and platelet production. Tpo signaling through its receptor, c-mpl, activates multiple pathways including signal transducer and activator of transcription (STAT)3, STAT5, phosphoinositide 3-kinase–Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor and immunoreceptor signaling. Here, we show that Lnk overexpression negatively regulates Tpo-mediated cell proliferation and endomitosis in hematopoietic cell lines and primary hematopoietic cells. Lnk attenuates Tpo-induced S-phase progression in 32D cells expressing mpl, and Lnk decreases Tpo-dependent megakaryocyte growth in bone marrow (BM)–derived megakaryocyte culture. Consistent with this result, we found that in both BM and spleen, Lnk-deficient mice exhibited increased numbers of megakaryocytes with increased ploidy compared with wild-type mice. In addition, Lnk-deficient megakaryocytes derived from BM and spleen showed enhanced sensitivity to Tpo during culture. The absence of Lnk caused enhanced and prolonged Tpo induction of STAT3, STAT5, Akt, and MAPK signaling pathways in CD41+ megakaryocytes. Furthermore, the Src homology 2 domain of Lnk is essential for Lnk's inhibitory function. In contrast, the conserved tyrosine near the COOH terminus is dispensable and the pleckstrin homology domain of Lnk contributes to, but is not essential for, inhibiting Tpo-dependent 32D cell growth or megakaryocyte development. Thus, Lnk negatively modulates mpl signaling pathways and is important for Tpo-mediated megakaryocytopoiesis in vivo.

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23

Pérez-García, Pablo, Yuan Ma, Marcelo J. Yanovsky, and Paloma Mas. "Time-dependent sequestration of RVE8 by LNK proteins shapes the diurnal oscillation of anthocyanin biosynthesis." Proceedings of the National Academy of Sciences 112, no. 16 (April 6, 2015): 5249–53. http://dx.doi.org/10.1073/pnas.1420792112.

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Circadian clocks sustain 24-h rhythms in physiology and metabolism that are synchronized with the day/night cycle. In plants, the regulatory network responsible for the generation of rhythms has been broadly investigated over the past years. However, little is known about the intersecting pathways that link the environmental signals with rhythms in cellular metabolism. Here, we examine the role of the circadian components REVEILLE8/LHY-CCA1-LIKE5 (RVE8/LCL5) and NIGHT LIGHT–INDUCIBLE AND CLOCK-REGULATED genes (LNK) shaping the diurnal oscillation of the anthocyanin metabolic pathway. Around dawn, RVE8 up-regulates anthocyanin gene expression by directly associating to the promoters of a subset of anthocyanin biosynthetic genes. The up-regulation is overcome at midday by the repressing activity of LNK proteins, as inferred by the increased anthocyanin gene expression in lnk1/lnk2 double mutant plants. Chromatin immunoprecipitation assays using LNK and RVE8 misexpressing plants show that RVE8 binding to target promoters is precluded in LNK overexpressing plants and conversely, binding is enhanced in the absence of functional LNKs, which provides a mechanism by which LNKs antagonize RVE8 function in the regulation of anthocyanin accumulation. Based on their previously described transcriptional coactivating function, our study defines a switch in the regulatory activity of RVE8–LNK interaction, from a synergic coactivating role of evening-expressed clock genes to a repressive antagonistic function modulating anthocyanin biosynthesis around midday.

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Yano, Mio, Toshihiko Imamura, Kenichi Sakamoto, Daisuke Asai, Gen Kano, Hajime Hosoi, Takao Deguchi, et al. "Clinical Significance of LNK (SH2B3) Expression in Pediatric B Cell Precursor Acute Lymphoblastic Leukemia." Blood 124, no. 21 (December 6, 2014): 3772. http://dx.doi.org/10.1182/blood.v124.21.3772.3772.

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Abstract Background: In pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), aberrant JAK-STAT signaling is one of the key leukemogenic mechanisms. Although activating mutations of JAK2 are found in high-risk BCP-ALL patients in western countries, such mutations are rare in Japanese cohorts, led to speculation of other factors contributing abnormal activation of JAK-STAT pathway. The adaptor protein LNK (SH2B3) is one of the negative regulator of JAK-STAT signaling, and its loss of function mutations have been identified in myeloproliferative neoplasms and high-risk BCP-ALL. In addition, the loss of function of LNK has been demonstrated to increase proliferation of B cells in vivo. Based on these findings, we conducted genetic analysis to determine the prognostic impact of LNKin BCP-ALL, and functional analysis to investigate its possible mechanisms. Methods: For genetic analysis, we evaluated diagnostic bone marrow or peripheral blood samples of 164 pediatric BCP-ALL patients treated with the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol along with peripheral blood samples from 9 healthy volunteers. The LNK expression level was determined by qRT-PCR. Deletion of IKZF1 was determined by MLPA, and direct sequencing was employed to detect LNK mutations in patients with IKZF1 deletion. For functional analysis, thrombopoietin (TPO)-dependent BaF3-MPL cells expressing LNK (BaF3-MPL-LNK) was established by retroviral transduction to investigate if LNKexpression levels impact on drug sensitivity for prednisolone (PSL), doxorubicin (DOX) or vincristine (VCR). For growth assay, BaF3-MPL cells with or without LNK were cultured for four days in the presence of TPO and the viable cell number was counted. For drug sensitivity test, BaF3-MPL cells with or without LNK were treated with each drug for 48 hours in the presence of TPO, then the IC50 was calculated. Phospho-specific flow cytometory (Phos-Flow) was performed to measure JAK-STAT activation. Results: The LNK expression levels in pediatric BCP-ALL patients’ samples were significantly lower than in samples from healthy donors (P < 0.01). When analyzing all 164 cases, the expression level of LNK was decreased in relapsed patients but there was no statistical significance (P = 0.067). IKZF1 deletion was found in 25 (15% of all) patients, and in these patients, LNK expression level dose not relate to relapse (P = 0.39). Intriguingly, when patients with known high-risk factor (i.e., IKZF1 deletion or poor response to PSL) were excluded, the expression level of LNK was significantly higher in non-relapsed patients (P < 0.05). In functional assay, we observed inhibition of TPO dependent growth of BaF3-MPL cells by expression of LNK (P < 0.01), consistent with previous reports. Phos-Flow analysis revealed that LNK expression suppressed TPO–induced phosphorylation of STAT5 in BaF3-MPL cells. In drug sensitivity test, we found that IC50 of PSL and DOX were substantially lower in BaF3-MPL-LNK cells from in BaF3-MPL-mock cells (0.70 vs 3.93 nM, P < 0.01 and 0.61 vs 1.14 nM, P < 0.05, respectively). Decline in IC50 of VCR by LNK expression was not statistically significant (1.38 vs 2.45 nM, P = 0.056). We next compared the impact of LNK with Ruxolitinib (RUX), a potent synthesized JAK2 inhibitor. The diminution in IC50 of PSL in BaF3-MPL-mock cells treated with RUX (50 nM) was comparable of that in BaF3-MPL-LNK cells (0.31 nM, combination index (CI) = 0.39), consistent with our hypothesis that LNK is working as a JAK2 inhibitor. Since we identified two amino-acid substitutions in N-terminal proline-rich dimerization domain (R139H) and PH domain (P242S), we also examined their function. Transductions of these genes in BaF3-MPL cells, however, did not alter cell growth, suggesting they are single nucleotide variants. Discussions: Our findings that high LNK expression is associated with low relapse rate in intermediate risk (IKZF1 intact, good PSL response) patients indicate potential of LNK to restrain relapse in such patients, presumably by suppressing JAK-STAT signaling. Since we proved the impact of LNK expression to improve sensitivity of PSL in vitro which was comparable to RUX, RUX could compensate lack of internal LNK expression to induce cell death of BCP-ALL cells. Collectively, targeting JAK-STAT pathway will be promising therapeutic option for intermediate risk BCP-ALL patients with low expression level of LNK. Disclosures No relevant conflicts of interest to declare.

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25

Komartin, Raluca Sanda, Brindusa Balanuca, Madalina Ioana Necolau, Anca Cojocaru, and Raluca Stan. "Composite Materials from Renewable Resources as Sustainable Corrosion Protection Coatings." Polymers 13, no. 21 (November 2, 2021): 3792. http://dx.doi.org/10.3390/polym13213792.

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Epoxidized linseed oil (ELO) and kraft lignin (LnK) were used to obtain new sustainable composites as corrosion protection layers through a double-curing procedure involving UV radiation and thermal curing to ensure homogeneous distribution of the filler. The crosslinked structures were confirmed by Fourier-transform infrared spectrometry (FTIR), by comparative monitorization of the absorption band at 825 cm−1, attributed to the stretching vibration of epoxy rings. Thermal degradation behavior under N2 gas indicates that the higher LnK content, the better thermal stability of the composites (over 30 °C of Td10% for ELO + 15% LnK), while for the experiment under air-oxidant atmosphere, the lower LnK content (5%) conducted to the more thermo-stable material. Dynamic-mechanic behavior and water affinity of the new materials were also investigated. The increase of the Tg values with the increase of the LnK content (20 °C for the composite with 15% LnK) denote the reinforcement effect of the LnK, while the surface and bulk water affinity were not dramatically affected. All the obtained composites were tested as carbon steel corrosion protection coatings, resulting in significant increase of corrosion inhibition efficiency (IE) of 140–380%, highlighting the great potential of the bio-based ELO-LnK composites as a future perspective for industrial application.

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26

Baran-Marszak, Fanny, Hajer Magdoud, Christophe Desterke, Anabell Alvarado, Claudine Roger, Stéphanie Harel, Elizabeth Mazoyer, et al. "Expression level and differential JAK2-V617F–binding of the adaptor protein Lnk regulates JAK2-mediated signals in myeloproliferative neoplasms." Blood 116, no. 26 (December 23, 2010): 5961–71. http://dx.doi.org/10.1182/blood-2009-12-256768.

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Abstract Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F–dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F–binding regulate JAK2-mediated signals in MPNs.

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27

Morris, Rhiannon, Liesl Butler, Andrew Perkins, Nadia J. Kershaw, and Jeffrey J. Babon. "The Role of LNK (SH2B3) in the Regulation of JAK-STAT Signalling in Haematopoiesis." Pharmaceuticals 15, no. 1 (December 24, 2021): 24. http://dx.doi.org/10.3390/ph15010024.

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LNK is a member of the SH2B family of adaptor proteins and is a non-redundant regulator of cytokine signalling. Cytokines are secreted intercellular messengers that bind to specific receptors on the surface of target cells to activate the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signalling pathway. Activation of the JAK-STAT pathway leads to proliferative and often inflammatory effects, and so the amplitude and duration of signalling are tightly controlled. LNK binds phosphotyrosine residues to signalling proteins downstream of cytokines and constrains JAK-STAT signalling. Mutations in LNK have been identified in a range of haematological and inflammatory diseases due to increased signalling following the loss of LNK function. Here, we review the regulation of JAK-STAT signalling via the adaptor protein LNK and discuss the role of LNK in haematological diseases.

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Koren-Michowitz, Maya, Sigal Gery, Daniel Nowak, and Phillip H. Koeffler. "Adaptor Protein Lnk Binds to JAK3 and Negatively Regulates a Mutant Activating Allele Associated with Megakaryocytic Leukemia." Blood 114, no. 22 (November 20, 2009): 5044. http://dx.doi.org/10.1182/blood.v114.22.5044.5044.

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Abstract Abstract 5044 The adaptor protein Lnk is known to associate with hematopoietic cytokine receptors such as cKIT, MPL and PDGFR, as well as, non-receptor tyrosine kinases such as JAK2, and is considered to have an inhibitory effect on these signaling pathways. JAK3 is expressed mainly in the hematopoietic system and its absence is associated with autosomal recessive severe combined immunodeficiency (SCID). Recently, activating mutations of JAK3 were described in transient myeloproliferative disorder (TMD) and acute megakaryocytic leukemia (AMKL) in Down syndrome (DS) patients as well as adult non-DS AMKL. JAK3 mutations were also rarely described in solid tumors and B-ALL. The 50% homology between JAK3 and JAK2 has led us to study the association between Lnk and JAK3.293T cells were co-transfected with cDNAs encoding either wild-type (WT) JAK3 or JAK3 harboring an activating A572V mutation (JAK3 A572V), as well as the WT V5-tagged Lnk. Whole cell lysates were used for immunoprecipitation with either V5-tag or JAK3 antibodies. Binding of Lnk and JAK3 was detected by Western blot probed with JAK3 or V5-tag antibodies. To determine which domain of Lnk is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including a mutation in the SH2 domain (R392E), deletion of the SH2 domain (del SH2) and deletion of the PH and SH2 (del SH2/PH) domains. Our results show that WT Lnk binds to WT JAK3, as well as JAK3 A572V. The R392E and del SH2 Lnk mutants retained JAK3 binding capacity while deletion of both SH2 and PH domains of Lnk abolished JAK3 binding. In order to study the biological effect of Lnk binding to JAK3, we infected CMK cells, a megakaryocytic leukemia cell line harboring JAK3 A572V, with a bicistronic retroviral MSCV-IRES-GFP (MIG) WT Lnk vector. Effect on growth was assessed in GFP positive sorted cells by cell count and colony formation in methylcellulose. CMK cells infected with MIG WT Lnk grew slower in liquid culture and had decreased clonogenic growth in soft agar culture compared to cells infected with MIG vector alone. In summary, we show for the first time that Lnk can bind to WT and mutant JAK3 and slow the growth of leukemic cells harboring an activating JAK3 mutation. Developing a small molecule mimetic of Lnk may have a therapeutic role in the treatment of hematopoietic malignancies associated with a variety of activated tyrosine kinase receptors and non-receptor tyrosine kinases including JAK3, as well as secondary signaling proteins. Disclosures No relevant conflicts of interest to declare.

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Lin, Dechen, Tong Yin, Maya koren-Michowitz, Ling-Wen Ding, Saskia Gueller, Sigal Gery, Takayuki Tabayashi, et al. "Adaptor Protein Lnk Binds to and Inhibits Normal and Leukemic FLT3." Blood 120, no. 21 (November 16, 2012): 1312. http://dx.doi.org/10.1182/blood.v120.21.1312.1312.

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Abstract Abstract 1312 Background The production and lineage commitment of hematopoietic cells is controlled by the actions of a complex network of signaling pathways. Mutations and translocations of tyrosine kinases within these pathways lead to constitutive signaling and enhanced proliferation. Classic examples are BCR-ABL in CML, Janus kinase 2 (JAK2) mutations in MPN, Fms-like tyrosine kinase 3 (FLT3) and c-KIT mutations in AML. FLT3 is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in about 1/3 of AML patients, mostly by internal tandem duplications (ITD). Adaptor protein Lnk is expressed in hematopoietic cells and is an important negative regulator in cytokine signaling and hematopoiesis. Previously, we and others have shown that Lnk interacts with the JXM domain of c-KIT, PDGFRA, PDGFRB and FMS, all of which share a similar sequence in this domain. The fact that FLT3 harbors a conserved JXM domain prompted us to investigate whether Lnk interacts with FLT3. Methods and Results Co-immunoprecipitation and GST-pulldown assay showed that Lnk physically interacts with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through its SH2 domain in multiple types of hematopoietic cells. Through affinity fishing assay with immobilized peptides, we identified the tyrosine residues 572, 591 and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Importantly, Lnk itself was tyrosine-phosphorylated by both FLT3 ligand (FL)-activated FLT3-WT and constitutively activated FLT3-ITD. Functionally, both shRNA-mediated depletion and ectopic expression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Consequently, Lnk inhibited 32D cell proliferation driven by different FLT3 oncogenic variants. Moreover, analysis of primary bone marrow cells from Lnk−/−mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors, mainly through inhibiting MAPK-ERK activation by FL. Conclusions This study reveals that through direct binding to FLT3-WT and FLT3-ITD, Lnk constrains FLT3-WT/ITD-dependent signaling pathways involved in the proliferation and expansion of hematopoietic cells as well as related leukemic cells. Modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD-associated hematopoietic diseases. Disclosures: No relevant conflicts of interest to declare.

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Bersenev, Alexey, Chao Wu, Joanna Balcerek, and Wei Tong. "Lnk Constrains Oncogenic JAK2-Induced Myeloproliferative Disease in Mice." Blood 114, no. 22 (November 20, 2009): 1437. http://dx.doi.org/10.1182/blood.v114.22.1437.1437.

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Abstract Abstract 1437 Poster Board I-460 Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2), which in turn triggers a cascade of downstream signal transduction pathways that regulate key aspects of cell development. Mice that lack the inhibitory adaptor protein Lnk harbor a vastly expanded HSC pool with enhanced self-renewal. We previously demonstrated that Lnk controls HSC self-renewal predominantly through the Mpl/JAK2 pathway. Lnk binds directly to phosphorylated tyrosine 813 in JAK2 upon Tpo stimulation. Moreover, Lnk-deficient HSCs display potentiated JAK2 activation. Dysregulation of cytokine receptor signaling pathways frequently lead to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML) in human patients. Moreover, the JAK2 V617F mutation has been observed at high frequency in several myeloproliferative diseases (MPDs). The JAK2V617F retains Lnk binding, suggesting that alterations in Lnk could influence MPD development. Indeed, we found that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPD in mouse transplant models. Specifically, Lnk deficiency enhanced cytokine signaling, thereby augmenting the ability of oncogenic JAK2 to expand myeloid progenitors. To test whether the interaction between Lnk and JAK2V617F directly constrains MPD development in mice, we transplanted wild-type bone marrow cells expressing the JAK2V617F/Y813F double mutant that does not interact with Lnk (WT;JAK2VF/YF). WT;JAK2VF/YF engrafted mice exhibited increased myeloid expansion when compared to WT;JAK2VF mice, and conferred accelerated polycythemia vera development in secondary transplants. In summary, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may constrain leukemic transformation conferred by oncogenic JAK2. Disclosures No relevant conflicts of interest to declare.

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Cheng, Ying, Kudakwashe Chikwava, Chao Wu, Anchit Bhagat, John K. Choi, and Wei Tong. "LNK (SH2B3) Synergizes with TP53 in Suppressing B-Precursor Acute Lymphoblastic Leukemia through IL-7 Receptor Signaling." Blood 124, no. 21 (December 6, 2014): 1074. http://dx.doi.org/10.1182/blood.v124.21.1074.1074.

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Abstract B-precursor acute lymphoblastic leukemia (B-ALL) is the leading cause of cancer-related deaths in children and commonly has a poor outcome in adults. Gene profiling and exome sequencing of high-risk ALLs led to the recent identification of the Philadelphia chromosome (Ph)-like ALL subtype. These leukemias have gene expression profiles similar to BCR-ABL1-positive (Ph+) ALL, but lack a BCR-ABL1 rearrangement, and often result in poor outcomes. Many Ph-like ALL-associated mutations identified to date are known or predicted to activate oncogenic cytokine receptor signaling pathways, particularly those associated with JAK. The lymphocyte adaptor protein LNK (also called SH2B3) has emerged as a powerful negative regulator of cytokine-mediated JAK2 signaling in hematopoietic stem cells (HSCs). Loss-of-function LNK mutations and deletions have recently been described in B-ALL, and germline loss of LNK contributes to pediatric ALL development. However, the mechanisms by which LNK alterations impact leukemogenesis remain poorly understood. Here we show that LNK synergizes with TP53 and INK4a in suppressing B-ALL development in mice. Tp53-/-Lnk-/- mice developed highly aggressive and transplantable B-ALL with 100% penetrance in contrast to T-lymphoma or sarcoma development observed in Tp53-/- mice. Importantly, gene expression profiles of Tp53-/-Lnk-/- B-ALL blasts were similar to those of human Ph-like B-ALLs, validating the relevance of this model for preclinical studies. Tp53-/-Lnk-/- pro-B progenitors initiated B-ALL in the transplanted recipients, and Lnk loss-of-heterozygosity (LOH) was found exclusively in B-ALL cells from Tp53-/-Lnk+/- mice, but not in HSCs, common lymphoid progenitors, or cells of myeloid/T cell lineages, attesting that LNK is a bona fide tumor suppressor in the committed B-cell precursors. Mechanistically, we found that pre-leukemic Lnk-/-Tp53-/- pro-B progenitors were hypersensitive to IL-7 and showed markedly enhanced self-renewal ability in vitro and in vivo. Our genetic studies revealed that LNK controls normal B cell and B-ALL development independently of its effects on HSCs and specifically regulates pro-B cell homeostasis. A novel phosphoflow cytometry approach in freshly isolated BM cells that combines the surface marker B220, intracellular mu-heavy chain detection, and phospho-specific antibodies enabled separation of subpopulations of B progenitors with distinct IL-7 responsiveness. Using this approach, we demonstrated that LNK deficiency potentiated STAT5 activation in response to IL-7 in pre-leukemic pro-B cells. Of note, Lnk-/-p53-/- leukemic blasts with elevated pSTAT5, were less sensitive to JAK inhibitors than pre-leukemic B progenitors, in part due to constitutive activation of MAPK and AKT/mTOR pathways. Our results invoke the targeting of these pathways as novel therapeutic approaches in B-ALL. In sum, we have developed a novel B-ALL mouse model suitable for preclinical studies aimed at further deciphering the pathogenic mechanisms underlying this disease and exploring new therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

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Bersenev, Alexey, Chao Wu, Joanna Balcerek, and Wei Tong. "Lnk Controls Hematopoietic Stem Cell Self-Renewal through Direct Interactions with JAK2 and Contributes to Oncogenic JAK2-Induced Myeloproliferative Diseases in Mice." Blood 112, no. 11 (November 16, 2008): 895. http://dx.doi.org/10.1182/blood.v112.11.895.895.

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Abstract Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2) that triggers a cascade of downstream signal transduction pathways that regulates many aspects of cell development. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. Surprisingly, we found that Lnk−/− HSCs have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeated 5-Florouracil (5-FU) treatments in vivo compared to wild type HSCs. We further provided genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal predominantly through Mpl. Consistent with this observation, Lnk deficiency in HSCs potentiates JAK2 activation in response to TPO. Biochemical experiments reveal that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Dysregulation of cytokine receptor signaling pathways leads to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML). Recently, the JAK2 V617F mutation has been observed at high frequencies in several myeloproliferative diseases (MPDs). The JAK2V617F mutant retains Lnk binding ability, suggesting Lnk status could modify MPD development. Indeed, we found that loss of Lnk accelerates oncogenic JAK2- induced CML/MPD in the mouse transplant models. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may contribute to leukemic transformation conferred by oncogenic JAK2.

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Holdreith, Nicholas, Grace Lee, Vemika Chandra, Carlo Salas Salinas, Peter Nicholas, Timothy S. Olson, and Wei Tong. "LNK (SH2B3) inhibition expands healthy and Fanconi anemia human hematopoietic stem and progenitor cells." Blood Advances 6, no. 3 (January 27, 2022): 731–45. http://dx.doi.org/10.1182/bloodadvances.2021004205.

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Abstract Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for a variety of hematological diseases. Allogenic HSCT requires hematopoietic stem cells (HSCs) from matched donors and comes with cytotoxicity and mortality. Recent advances in genome modification of HSCs have demonstrated the possibility of using autologous HSCT-based gene therapy to alleviate hematologic symptoms in monogenic diseases, such as the inherited bone marrow failure (BMF) syndrome Fanconi anemia (FA). However, for FA and other BMF syndromes, insufficient HSC numbers with functional defects results in delayed hematopoietic recovery and increased risk of graft failure. We and others previously identified the adaptor protein LNK (SH2B3) as a critical negative regulator of murine HSC homeostasis. However, whether LNK controls human HSCs has not been studied. Here, we demonstrate that depletion of LNK via lentiviral expression of miR30-based short hairpin RNAs results in robust expansion of transplantable human HSCs that provided balanced multilineage reconstitution in primary and secondary mouse recipients. Importantly, LNK depletion enhances cytokine-mediated JAK/STAT activation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Moreover, we demonstrate that LNK depletion expands primary HSPCs associated with FA. In xenotransplant, engraftment of FANCD2-depleted FA-like HSCs was markedly improved by LNK inhibition. Finally, targeting LNK in primary bone marrow HSPCs from FA patients enhanced their colony forming potential in vitro. Together, these results demonstrate the potential of targeting LNK to expand HSCs to improve HSCT and HSCT-based gene therapy.

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Matsumoto, Tomoyuki, Masaaki Ii, Hiromi Nishimura, Taro Shoji, Yutaka Mifune, Atsuhiko Kawamoto, Ryosuke Kuroda, et al. "Lnk-dependent axis of SCF–cKit signal for osteogenesis in bone fracture healing." Journal of Experimental Medicine 207, no. 10 (September 20, 2010): 2207–23. http://dx.doi.org/10.1084/jem.20100321.

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The therapeutic potential of hematopoietic stem cells/endothelial progenitor cells (HSCs/EPCs) for fracture healing has been demonstrated with evidence for enhanced vasculogenesis/angiogenesis and osteogenesis at the site of fracture. The adaptor protein Lnk has recently been identified as an essential inhibitor of stem cell factor (SCF)–cKit signaling during stem cell self-renewal, and Lnk-deficient mice demonstrate enhanced hematopoietic reconstitution. In this study, we investigated whether the loss of Lnk signaling enhances the regenerative response during fracture healing. Radiological and histological examination showed accelerated fracture healing and remodeling in Lnk-deficient mice compared with wild-type mice. Molecular, physiological, and morphological approaches showed that vasculogenesis/angiogenesis and osteogenesis were promoted in Lnk-deficient mice by the mobilization and recruitment of HSCs/EPCs via activation of the SCF–cKit signaling pathway in the perifracture zone, which established a favorable environment for bone healing and remodeling. In addition, osteoblasts (OBs) from Lnk-deficient mice had a greater potential for terminal differentiation in response to SCF–cKit signaling in vitro. These findings suggest that inhibition of Lnk may have therapeutic potential by promoting an environment conducive to vasculogenesis/angiogenesis and osteogenesis and by facilitating OB terminal differentiation, leading to enhanced fracture healing.

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Gueller, Saskia, Sigal Gery, and H. Phillip Koeffler. "Adaptor Protein Lnk Binds to PDGFRA, PDGFRB and FIP1L1-PDGFRA, but Not to the TEL-PDGFRB Fusion Protein." Blood 110, no. 11 (November 16, 2007): 2213. http://dx.doi.org/10.1182/blood.v110.11.2213.2213.

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Abstract PDGFRA and PDGFRB (platelet derived growth factor receptors alpha and beta) are frequently expressed on malignant hematopoietic cells and regulate various cellular responses such as development, proliferation, differentiation, cell survival and cellular transformation. Stimulation by either autocrine loops or constitutional activation by chromosomal translocation (i.e. chronic myelomonocytic leukemia [CMML, TEL-PDGFRB] or chronic eosinophilic leukemia [CEL, FIP1L1-PDGFRA]) makes them important factors in development of hematopoietic disorders. Normally, interaction with the ligand PDGF, induces dimerization of two distinct receptor subunits, resulting in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. We hypothesized that one such protein may be the adaptor Lnk, a negative regulator of several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain, a pleckstrin homology domain (PH) and a dimerization domain (DD). The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. We investigated the ability of Lnk to bind to PDGFRA, PDGFRB, FIP1L1-PDGFRA and TEL-PDGFRB. To determine the domain of Lnk that is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including: a mutation in the SH2 domain (R392E); deletion of the SH2 domain; deletion of the PH and SH2 domains and a construct only containing the DD domain. 293T cells were co-transfected with cDNAs encoding either PDGFRA, PDGFRB or one of the translocation products and either wild-type or mutant Lnk. Whole cell lysates were used to perform immunoprecipitation with either V5-tag or PDGFR antibodies. Binding of Lnk and PDGFR was detected by Western blot probed with PDGFR or V5-tag antibodies. NIH3T3 cells were transfected either with empty vector or Lnk cDNA, transfectants were selected for 5 days with G418, serum starved for 16 hours and induced with PDGF for 10 minutes. Phosphorylation of downstream targets of PDGFRA and PDGFRB was detected by Western blot. Our data showed that Lnk bound to PDGFRA and PDGFRB only after exposure of the cells to PDGF and to the FIP1L1-PDGFRA fusion protein independent of PDGF exposure. Mutation or deletion of the Lnk SH2 domain abolished binding completely in PDGFRA and FIP1L1-PDGFRA, but just partly in PDGFRB. Expression of Lnk in NIH3T3 cells inhibited phosphorylation of ERK after treatment with PDGF. In other experiments, we determined that Lnk bound the juxtamembrane region of this class of receptors. Interestingly, the TEL-PDGFRB fusion protein was unable to bind Lnk, although its breakpoint in PDGFRB is distal to the juxtamembrane domain and the whole intracellular region of PDGFRB is included in the fusion protein. Further exploration of the mechanisms by which Lnk affects wild-type or PDGFR fusion product will provide insight into the molecular pathophysiology of myeloid disorders and could help develop new treatments.

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Takaki, Satoshi, Hatsue Morita, Yoshinari Tezuka, and Kiyoshi Takatsu. "Enhanced Hematopoiesis by Hematopoietic Progenitor Cells Lacking Intracellular Adaptor Protein, Lnk." Journal of Experimental Medicine 195, no. 2 (January 14, 2002): 151–60. http://dx.doi.org/10.1084/jem.20011170.

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Hematopoietic stem cells (HSCs) give rise to variety of hematopoietic cells via pluripotential progenitors and lineage-committed progenitors and are responsible for blood production throughout adult life. Amplification of HSCs or progenitors represents a potentially powerful approach to the treatment of various blood disorders and to applying gene therapy by bone marrow transplantation. Lnk is an adaptor protein regulating the production of B cells. Here we show that Lnk is also expressed in hematopoietic progenitors in bone marrow, and that in the absence of Lnk, the number and the hematopoietic ability of progenitors are significantly increased. Augmented growth signals through c-Kit partly contributed to the enhanced hematopoiesis by lnk−/− cells. Lnk was phosphorylated by and associated with c-Kit, and selectively inhibited c-Kit–mediated proliferation by attenuating phosphorylation of Gab2 and activation of mitogen-activated protein kinase cascade. These observations indicate that Lnk plays critical roles in the expansion and function of early hematopoietic progenitors, and provide useful clues for the amplification of hematopoietic progenitor cells.

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Bunting, Kevin D. "Another Lnk to STAT activation." Blood 116, no. 6 (August 12, 2010): 862–64. http://dx.doi.org/10.1182/blood-2010-05-283176.

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McMullin, Mary Frances, and Holger Cario. "LNK mutations and myeloproliferative disorders." American Journal of Hematology 91, no. 2 (January 22, 2016): 248–51. http://dx.doi.org/10.1002/ajh.24259.

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Wang, Wei, Yang Tang, Ying Wang, Joanna Balcerek, Wei Tong, Ross L. Levine, Carrie Welch, Alan Tall, and Nan Wang. "SH2B3/LNK Loss of Function Promotes Atherosclerosis and Thrombosis." Blood 126, no. 23 (December 3, 2015): 3443. http://dx.doi.org/10.1182/blood.v126.23.3443.3443.

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Abstract Human genome-wide association studies (GWAS) have revealed many novel genetic loci that are associated with coronary heart disease (CHD) but do not involve traditional risk factors. However, the relevant genes and mechanisms are largely unknown. One such locus resides in SH2B3/LNK, which is expressed in hematopoietic cells and suppresses thrombopoietin (TPO) signaling via its receptor (MPL). The common risk single nucleotide polymorphism (SNP) of SH2B3/LNK is associated with CHD, increased platelet and myeloid cell counts in peripheral blood and JAK2V617F positive myeloproliferative neoplasms. Analysis of human cord blood revealed that samples with TT risk SNP (R262W) in SH2B3/LNK was associated with expansion of hematopoietic stem cells (HSCs), increased MPL signaling in HSCs, increased megakaryopoiesis, and a paradoxical increased expression of LNK mRNA, indicating reduced LNK function but induction of LNK transcription downstream of increased MPL signaling. Since humans are hypercholesterolemic relative to mice, to assess the role of reduced LNK function in athero-thrombosis, we transplanted WT or Lnk-/- bone marrow (BM) cells into irradiated WT or low-density lipoprotein receptor knock-out (Ldlr-/-) recipient mice and fed the recipients with chow and western type diet (WTD), respectively. Although Lnk-/- BM recipients showed similarly increased platelet counts on both diets, we found that platelet activation and aggregation, formation of platelet/leukocyte aggregates, atherosclerosis and arterial thrombosis were markedly increased by the combination of hyperlipidemia and hematopoietic LNK deficiency. These diet-genotype interactions mirrored an increase in mean platelet volume, protease-activated receptor-4 (PAR4) agonist-induced P-selectin exposure on platelets and platelet protein kinase C (PKC) activity, suggesting increased platelet granule secretion. We also observed an increased number of bone marrow myeloid progenitor cells expressing elevated levels of the common-beta subunit (CBS) of the interleukin-3/granulocyte-macrophage colony-stimulating factor (IL-3/GM-CSF) receptor, which promotes leukocytosis and the formation of platelet/leukocyte aggregates in the bloodstream. The increased P-selectin exposure, platelet aggregation and PKC activity were largely reversed by high-density lipoprotein (HDL) or cyclodextrin, reagents that remove cholesterol from platelets. Consistently, cholesterol loading of platelets from the chow-fed mice led to increased P-selectin exposure and PKC activity upon PAR4 agonist stimulation but the increase was more pronounced in Lnk-/- platelets, indicating a cholesterol-genotype interaction. LNK deficiency also resulted in defective MPL-mediated TPO internalization in platelets, leading to increased plasma TPO levels in mice on both diets. Elevated TPO levels and enhanced MPL signaling likely acted together to increase the pool of HSCs and megakaryopoiesis, leading to marked thrombocytosis. These studies suggest that SH2B3/LNK loss of function variants lead to HSC expansion, overproduction of platelets, and in the setting of hypercholesterolemia, markedly increased myeloid cells, platelet activation and formation of platelet/leukocyte aggregates, leading to accelerated atherosclerosis and arterial thrombosis. Our studies point to the importance of treating dyslipidemia in the setting of platelet and myeloid cell overproduction, as observed in subjects with SH2B3/LNK risk alleles. Disclosures Levine: Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy. Tall:Amgen: Consultancy; Arisaph: Consultancy; CSL: Consultancy.

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Oh, Stephen T., Jacob M. Zahn, Carol D. Jones, Bing Zhang, Mignon L. Loh, Hagop Kantarjian, Erin F. Simonds, et al. "Identification of Novel LNK Mutations In Patients with Chronic Myeloproliferative Neoplasms and Related Disorders." Blood 116, no. 21 (November 19, 2010): 315. http://dx.doi.org/10.1182/blood.v116.21.315.315.

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Abstract Abstract 315 Introduction: Dysregulated JAK-STAT signaling in chronic myeloproliferative neoplasms (MPNs) has primarily been attributed to activating mutations in tyrosine kinases. However, JAK-STAT activation can be demonstrated in some patients lacking JAK2 or MPL mutations, suggesting alteration of other regulatory elements in this pathway. One regulator of JAK-STAT signaling is LNK (SH2B3), an adapter protein that contains a proline-rich N-terminal dimerization domain (Pro/DD), a pleckstrin homology (PH) domain (plasma membrane localization), and an SH2 domain. LNK binds to cytokine receptors (e.g. MPL, EPOR) and JAK2 via its SH2 domain, inhibiting downstream STAT activation and providing critical negative feedback regulation. LNK-/- mice exhibit features consistent with an MPN phenotype. We recently reported the first human disease-related LNK mutations in two JAK2 V617F-negative MPN patients (Oh et al, Blood, Aug 12, 2010). One patient with primary myelofibrosis (PMF) exhibited a 5 base-pair (bp) deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains. A second patient with essential thrombocythemia (ET) was found to have a missense mutation (E208Q) in the PH domain. Both mutations conferred aberrant JAK-STAT signaling in cell lines and primary patient samples, indicating that loss of LNK negative feedback regulation contributes to MPN pathogenesis. We now report the results of a comprehensive screen of a large cohort of MPN, overlap myelodysplastic syndrome (MDS)/MPN, and post-MDS/MPN acute myeloid leukemia (AML) patients for LNK mutations. Methods: A total of 341 samples were sequenced (Table 1; polycythemia vera (PV)=34, erythrocytosis=7, ET=61, PMF=75, post-PV/ET MF=25, MPN-U=7, chronic myelomonocytic leukemia (CMML)=71, juvenile myelomonocytic leukemia=20, MDS/MPN=8, MDS with fibrosis=2, refractory anemia with ring sideroblasts and thrombocytosis=4, idiopathic hypereosinophilic syndrome/chronic eosinophilic leukemia=4, systemic mastocytosis=4, and post MDS/MPN AML=19). A deep sequencing approach (Illumina multiplexing system) was used to evaluate 84 samples, in which all exons of LNK were sequenced. For the remainder of the samples, direct sequencing was performed on exon 2, the region containing the previously reported DEL and E208Q mutations. Results: After excluding variants previously reported in SNP databases, a total of 11/341 (3.2%) patients were found to have non-synonymous mutations, including 3/61 (4.9%) ET, 3/75 (4.0%) PMF, and 5/71 (7.0%) CMML patients (Table 1). Each of the mutations localized to exon 2 of LNK, implicating this region as a possible mutational hotspot. This included the aforementioned patients with the DEL and E208Q mutations, which were confirmed by deep sequencing. In two other patients, sequencing of DNA from cultured skin fibroblasts DNA indicated that the mutations were germline. For the remaining seven patients, germline analysis is currently ongoing. In one patient with CMML, a 1 bp deletion leading to a frameshift and premature stop codon was identified (Q72fs). This mutation localized to the Pro/DD, likely resulting in a complete loss of LNK function. Interestingly, this patient who is wild type for the JAK2 and RAS genes, also carries a heterozygous CBL mutation (C396Y), suggesting that LNK and CBL mutations may have cooperative effects. Four patients (one with PMF, three with CMML) were found to have a missense mutation (S186I) at a highly conserved residue in the Pro/DD. The previously reported E208Q mutation was also found in one patient with ET and one patient with CMML. None of the 81 patients known to be JAK2 V617F-positive exhibited somatic LNK mutations, suggesting that LNK mutations may provide an alternative basis for JAK-STAT activation in the absence of JAK2 V617F. Conclusion: Missense and deletion mutations of the LNK gene occur at a low frequency in MPNs and MDS/MPNs and segregate predominantly in exon 2. Further analysis of post-MPN AML samples (represented at a low frequency in the current cohort) and other subtypes of acute and chronic myeloid malignancies is warranted to better characterize the disease spectrum of LNK mutations and whether they are mutually exclusive of JAK2 V617F. We are currently investigating whether loss of negative feedback regulation of JAK-STAT signaling is related to haploinsufficiency of LNK or dominant negative effects of the mutant protein. Disclosures: No relevant conflicts of interest to declare.

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41

Rumi, Elisa, Ashot S. Harutyunyan, Daniela Pietra, Jelena D. Milosevic Feenstra, Chiara Cavalloni, Elisa Roncoroni, Ilaria Casetti, et al. "LNK mutations in familial myeloproliferative neoplasms." Blood 128, no. 1 (July 7, 2016): 144–45. http://dx.doi.org/10.1182/blood-2016-04-711150.

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42

Bersenev, Alexey, Chao Wu, Joanna Balcerek, Jiang Jing, Mondira Kundu, Gerd A. Blobel, Kudakwashe R. Chikwava, and Wei Tong. "Lnk constrains myeloproliferative diseases in mice." Journal of Clinical Investigation 120, no. 6 (June 1, 2010): 2058–69. http://dx.doi.org/10.1172/jci42032.

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43

Norddahl, Gudmundur L., Martin Wahlestedt, Santiago Gisler, Mikael Sigvardsson, and David Bryder. "Enhanced Cytokine Responsiveness Counteracts Age-Induced Decline in Hematopoietic Stem Cell Function." Blood 118, no. 21 (November 18, 2011): 2342. http://dx.doi.org/10.1182/blood.v118.21.2342.2342.

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Abstract Abstract 2342 In recent years, it has become increasingly clear that physiologic aging has several profound cell intrinsic effects on hematopoietic stem cell (HSC) aging. This includes an altered output of mature progeny and an expansion of the HSC pool, although the latter is accompanied with several functional shortcomings. Because of the hierarchical structure of hematopoiesis, improving HSC function should present a promising avenue to restore aberrant hematopoietic function. The signal adaptor protein Lnk, a relay of cytokine signaling, has been shown to negatively regulate hematopoiesis at several cellular stages. As a consequence of Lnk deficiency, mice display increased B lymphopoiesis and increased HSC numbers. The increase in HSC numbers has been suggested to arise by an increased ability of HSCs to self-renew. However, it has remained unclear how the enhanced sensitivity to cytokine signaling would affect hematopoiesis upon physiological age. In the present study, we investigated the effects of physiological aging on hematopoiesis in the Lnk−/− mice. Aged wild-type mice showed a number of age-related alterations in the hematopoietic system including; increased HSC numbers, decreased lymphopoiesis and decreased reconstitution potential upon competitive transplantation. Most of these parameters were dramatically altered as a consequence of Lnk deficiency. When competitively transplanted, aged Lnk−/− HSCs displayed a dramatically increased overall reconstitution potential compared to WT mice. Increases in reconstitution potential were observed in both primary and serially reconstituted recipient animals. Upon physiologic age, the lineage distribution of transplanted aged wild-type HSCs is skewed towards the myeloid lineage. By contrast, transplanted aged Lnk−/− HSCs gave rise to a hematopoietic system with a balanced lineage distribution resembling that generated by young wild-type HSCs. The enhanced HSC potential of aged Lnk−/− mice was uncoupled from effects on telomere length maintenance but displayed a distinct molecular profile in whole genome expression analysis. In summary, we demonstrate that deficiency of the signal adaptor molecule Lnk appears to functionally protect HSCs from many of the consequences of physiologic age, which at a broader level perhaps suggest a potential of cytokines to counteract age-related HSC decline. Disclosures: No relevant conflicts of interest to declare.

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44

Oh, Stephen T., Erin F. Simonds, Carol Jones, Matthew B. Hale, Yury Goltsev, Kenneth D. Gibbs, Jason D. Merker, James L. Zehnder, Garry P. Nolan, and Jason Gotlib. "Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms." Blood 116, no. 6 (August 12, 2010): 988–92. http://dx.doi.org/10.1182/blood-2010-02-270108.

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Abstract Dysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis.

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45

Ema, Hideo, Jun Seita, Jun Ooehara, Akiko Iseki, Hina Takano, and Hiromitsu Nakauchi. "Adaptor Protein Lnk Negatively Controls the Likelihood of Self-Renewal in Hematopoietic Stem Cells." Blood 108, no. 11 (November 16, 2006): 1316. http://dx.doi.org/10.1182/blood.v108.11.1316.1316.

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Abstract Great progresses are promised for the development of stem cell-based regenerative medicine if we can manipulate stem cell self-renewal. Thus, one of the central tasks in stem cell biology is to understand how stem cell fate is determined. Hematopoietic stem cells (HSCs) are the best studied stem cells. Their in vivo self-renewal has been extensively studied, but its in vitro recapitulation remains so difficult. We previously reported that HSCs undergo asymmetrical self-renewal division in culture with stem cell factor (SCF) and thrombopoietin (TPO). Since then, we have sought any condition in which HSCs can symmetrically self-renew. We now report in vitro symmetrical self-renewal division of HSCs in the absence of Lnk. Lnk is an adaptor protein containing praline-rich domain, pleckstrin homology domain, and Src homology domain. Lnk-deficient mice have over 10-fold HSCs due to increased self-renewal capacity. CD34−Kit+Sca-1+Lin− cells were purified from bone marrow of wild-type or Lnk-deficient B6 mice, and were subjected to serum-free single cell cultures in the presence of a variety of cyokines. We found that Lnk-deficient CD34−Kit+Sca-1+Lin− cells are hypersensitive to TPO. Repopulating activity in 40 CD34−Kit+Sca-1+Lin− cells from Lnk-deficient mice increased after 3 day-culture with TPO or with SCF and TPO, but not after 3 day-culture with SCF. In contrast, repopulating activity in 40 CD34−Kit+Sca-1+Lin− cells from wild type mice did not significantly change after 3 day-culture with SCF, TPO, or SCF and TPO. Moreover, paired daughter cell-experiments clearly showed that Lnk-deficient, but not wild-type long-term repopulating cells are able to undergo symmetrical self-renewal division at least once in the presence of SCF and TPO. These data suggest that Lnk acts just like a tuner in the regulation of HSC self-renewal downstream of TPO/Mpl signaling. We further investigated TPO-mediated signal transduction pathways in CD34−Kit+Sca-1+Lin− cells. To this end, we developed a novel assay which allowed us to analyze signal transduction in a very limited number of cells. We detected enhanced up-regulation of STAT5 and Akt pathways, and inversely enhanced down-regulation of p38 MAPK pathway in Lnk-deficient CD34−Kit+Sca-1+Lin− cells, as compared with normal ones. These data suggest that these combinational changes in signal transduction lead to initiation of self-renewal in HSCs. We propose that stem cell self-renewal is determined by a balance of positive and negative signals in multiple signal transduction pathways rather than by any particular self-renewal signals.

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46

Vila González, Patricia Elizabet, and María Noel Pereyra. "Interpretación a partir del análisis cinético de los resultados del ensayo acelerado (IRAM 1674) para la reacción álcali-sílice pavimentos." Métodos y Materiales 7 (May 22, 2018): 1–10. http://dx.doi.org/10.15517/mym.v7i1.29716.

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En este trabajo se presentan los resultados de expansión según el ensayo acelerado de la barra de mortero (IRAM 1674) hasta los 14 días de inmersión en solución agresiva y el análisis cinético según las recomendaciones RILEM AAR-2. El objetivo principal del trabajo es contribuir a la interpretación de los resultados del ensayo acelerado a partir del análisis del crecimiento de la expansión con la edad del ensayo (análisis cinético), otorgándole así mayor confiabilidad al ensayo. El análisis cinético consiste en ajustar por mínimos cuadrados los datos experimentales de expansión a un modelo de comportamiento conocido (modelo KAMJ), de dónde surgen los parámetros Lnk y M. El cálculo de los parámetros cinéticos Lnk y M es un buen indicador para analizar el comportamiento de la reacción. Si bien en la bibliografía se propone un valor límite para Lnk de -6 (valores menores, agregados no reactivos y valores mayores agregados deletéreos), al analizar los datos experimentales, se observó que valores bajos de este parámetro podrían no estar asociados a un crecimiento asintótico de la expansión, y por lo tanto, presentar un comportamiento deletéreo. Se considera que el coeficiente Lnk/M es un buen indicador para evaluar el comportamiento del agregado. Sin embargo - para poder definir un valor límite para el coeficiente Lnk/M - resulta necesario establecer una correlación con los ensayos en primas de hormigón de larga duración (1año o más) o de la experiencia en campo de comportamiento del agregado.

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47

Balcerek, Joanna, Jing Jiang, Qinqin Jiang, Kaosheng Lyu, Roger Greenberg, and Wei Tong. "LNK (SH2B3) Deficiency Ameliorates Hematopoietic Stem Cell Defects in Fanconi Anemi." Blood 128, no. 22 (December 2, 2016): 2670. http://dx.doi.org/10.1182/blood.v128.22.2670.2670.

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Abstract Fanconi Anemia (FA) is one of the most common inherited bone marrow failure (iBMF) syndromes. Although initially identified over 85 years ago, FA remains a fatal genetic disease. Nineteen FA genes cooperate in a genome stability pathway that is essential for repair of DNA damage and tolerance of replication stress. Cells derived from FA patients are hypersensitive to DNA interstrand crosslink (ICL)-inducing agents, such as Mitomycin C (MMC), and exhibit DNA damage checkpoint and mitosis defects. Mutations in FA genes result in hematopoietic stem cell (HSC) defects, bone marrow failure and cancer predisposition. Importantly, interventions to mitigate HSC defects do not exist, aside from allogeneic bone marrow transplantation (BMT). HSCs deficient for FancD2, a central component of the FA signaling pathway, are markedly compromised in reconstituting the hematopoietic system in murine BMT models. Remarkably, we found that loss of the adaptor protein Lnk (also called Sh2b3) restores HSC function in FancD2 knockout mice without accelerating neoplastic transformation. LNK negatively regulates HSC self-renewal, in part by attenuating cytokine signaling-activated JAK2 signaling in HSCs (J Clin Invest. 2008;118(8):2832-2844). Fancd2-/-;Lnk-/- (DKO) mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals, and DKOHSC function is also largely restored to WT levels in serial transplantation assays. Primary DKOHSC and progenitors (HSPCs) are still sensitive to MMC, indicating that LNK does not play an overt role in ICL repair. Instead, Lnk deficiency notably reduces spontaneous DNA damage and genome instability. This is in agreement with recent studies that reveal a requirement for FA proteins in replication stress, which is a separable function from their role in DNA repair (Cell. 2011;145(4):529-542; Cancer Cell. 2012;22(1):106-116). Strikingly, we demonstrated that Lnk deficiency mitigates replication stress by stabilizing stalled replication forks, and that this effect is dependent upon cytokine signaling. Together, our data demonstrate that Lnk deficiency ameliorates FA HSPC defects by alleviating replication stress associated DNA damage and genome instability. To our knowledge, this is one of the first examples of in vivo genetic suppression of FA-associated HSPC defects. Our work sheds light on mechanisms underlying the origin of bone marrow failure in FA patients and has implications for new therapeutic strategies to treat FA associated bone marrow failure. Disclosures No relevant conflicts of interest to declare.

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Chen, Jingbo, Jiawen Xu, Yan Sun, Yuhuan Xue, Yang Zhao, Dongzi Yang, Shuijie Li, and Xiaomiao Zhao. "Gut Microbiota Dysbiosis Ameliorates in LNK-Deficient Mouse Models with Obesity-Induced Insulin Resistance Improvement." Journal of Clinical Medicine 12, no. 5 (February 22, 2023): 1767. http://dx.doi.org/10.3390/jcm12051767.

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Purpose: To investigate the potential role of gut microbiota in obesity-induced insulin resistance (IR). Methods: Four-week-old male C57BL/6 wild-type mice (n = 6) and whole-body SH2 domain-containing adaptor protein (LNK)-deficient in C57BL/6 genetic backgrounds mice (n = 7) were fed with a high-fat diet (HFD, 60% calories from fat) for 16 weeks. The gut microbiota of 13 mice feces samples was analyzed by using a 16 s rRNA sequencing analysis. Results: The structure and composition of the gut microbiota community of WT mice were significantly different from those in the LNK-/- group. The abundance of the lipopolysaccharide (LPS)-producing genus Proteobacteria was increased in WT mice, while some short-chain fatty acid (SCFA)-producing genera in WT groups were significantly lower than in LNK-/- groups (p < 0.05). Conclusions: The structure and composition of the intestinal microbiota community of obese WT mice were significantly different from those in the LNK-/- group. The abnormality of the gut microbial structure and composition might interfere with glucolipid metabolism and exacerbate obesity-induced IR by increasing LPS-producing genera while reducing SCFA-producing probiotics.

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Dale, Bethany L., and Meena S. Madhur. "Linking inflammation and hypertension via LNK/SH2B3." Current Opinion in Nephrology and Hypertension 25, no. 2 (March 2016): 87–93. http://dx.doi.org/10.1097/mnh.0000000000000196.

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50

Ding, L.-W., Q.-Y. Sun, D.-C. Lin, W. Chien, N. Hattori, X.-M. Dong, S. Gery, et al. "LNK (SH2B3): paradoxical effects in ovarian cancer." Oncogene 34, no. 11 (April 7, 2014): 1463–74. http://dx.doi.org/10.1038/onc.2014.34.

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