LNK Gene Research Articles

The circadian clock and thermal regulation in plants: novel insights into the role of positive circadian clock regulators in temperature responses.

The impact of rising global temperatures on crop yields is a serious concern, and the development of heat-resistant crop varieties is crucial for mitigating the effects of climate change on agriculture. To achieve this, a better understanding of the molecular basis of the thermal responses of plants is necessary. The circadian clock plays a central role in modulating plant biology in synchrony with environmental changes, including temperature fluctuations. Recent studies have uncovered the role of transcriptional activators of the core circadian network in plant temperature responses. This expert view highlights key novel findings regarding the role of the RVE and LNK gene families in controlling gene expression patterns and plant growth under different temperature conditions, ranging from regular diurnal oscillations to extreme stress temperatures. These findings reinforce the essential role of the circadian clock in plant adaptation to changing temperatures and provide a basis for future studies on crop improvement.

Effect of Silencing and Overexpression of LNK Gene on STAT3 Expression in THP-1 Cells

To investigate the effect of LNK gene silencing and overexpression on the expression of STAT3 gene in human monocytic leukemia cells (THP-1). THP-1 cells were cultured, and the lentivirus was used as a vector to silence and overexpres the LNK gene stably. After transfection for 72 hours, the GFP expression levels were observed by inverted fluorescence microscopy. The lentiviral transfection efficiencies were detected by flow cytometry. The effects of LNK silencing and overexpression were confirmed, and the expression of STAT3 mRNA was detected by RT-PCR. The protein levels of LNK and STAT3 were detected by Western blotting. The GFP expression level of THP-1 cells reached more than 85% after transfection with lentivirus for 72 hours, and the transfection efficiency of cells was above 99%. mRNA expressions levels of LNK and STAT3 in LNK silencing group were signifycantly lower than those in control group, while LNK and STAT3 mRNA levels in the LNK overexpression group was significantly higher than those in control group (P＜0.05). The protein expression levels of LNK and STAT3 in LNK silencing group were significantly lower than those in control group, while that in LNK overexpression group was significantly higher than that in control group (P＜0.05). The THP-1 cell line with LNK gene silencing and overexpression has been successfully established. The LNK gene silencing resulted in decrease of STAT3 expression; LNK gene overexpression and leads to inereases of STAT3 expression indicating that LNK participates in the regulation of STAT3.

Effect of Silencing LNK Gene on the Expression of EPO and EPOR in THP-1 Cells

To investigate the effect of silencing LNK gene on the expression of EPO and EPOR in acute myeloid leukemia cells (THP-1). THP-1 cells were cultured. The lentivirus was used as a vector to silence the LNK gene stably. After 72 hours of infection, GFP expression level was detected by the fluorescent inverted microscopy. The lentiviral Infection efficiencies were monitored by flow cytometry. The LNK silencing effect was confirmed. The mRNA expressions of EPO and EPOR were detected by RT-PCR. The protein levels of LNK, EPO and EPOR were detected by Western blot. At the time-point of 72 hours after lentivirus infection, the expression level of GFP was above 85% detected by fluorescent inverted microscopy. The infection efficiency was above 99% by flow cytometry. mRNA expressions of LNK, EPO and EPOR in LNK silencing group were signifycantly lower than those in control group (P＜0.05). The protein levels of LNK, EPO and EPOR in LNK silencing group were significantly lower than those in the control group (P＜0.05). THP-1 cell line of LNK gene silencing has been successfully established,the LNK gene has been silenced, the expression of EPO and EPOR decrease, indicating that LNK may participate in the regulation of EPO and EPOR.

Expression of LNK Genes in Patients with Acute Leukemia and Its Clinical Significance

To investigate the expression of LNK gene in patients with acute leukemia (AL) and its correlation with the clinical characteristics. Real-time quantitative RT-PCR was used to detect the level of LNK mRNA in bone marrow samples from 80 patients diagnosed as AL(42 cases of ALL, and 38 cases of AML), and its relevance with clinical indicators was statistically analyzed. Western blot was used to detect the expression of LNK protein. The bone marrow samples of 16 healthy volunteers were used as the controls. The LNK mRNA levels in ALL and AML groups were significantly higher than that in control group (P=0.007, P=0.021) and there was no statistical difference between ALL and AML groups. The LNK levels in ALL and AML groups possitively correlated with the risk of patients (P=0.000, P=0.04, r=0.5, r=0.386), And the LNK levels in high risk ALL and AML groups were significantly higher than that in control group (P=0.035, P=0.032), the LNK levels in intermediate risk of AML and ALL groups (P=0.239,P=0.609) and the LNK level in standard risk (P=0.974, P=1) were all higher than that in control group, there was no statistianl significance. but the risks of different groups showed no statistical significance. The LNK protein level in patients with acute leukemia was higher than that in control group. The expression level of LNK gene in AL patients is higher than that in healthy people, and the expression level of LNK gene positively correlates with the risk of patients.

Variation and Clinical Significance of LNK Gene in Essential Thrombocytosis

To explore the mutation and single nucleotide polymorphism(SNP) of LNK gene in the patients with essential thrombocytosis (ET), and to analyze the relationship between LNK gene variation and the occurrence of ET. JAK2V617F mutation was identified by allele-specific PCR. The whole exon of LNK gene was amplified by PCR. The amplified sequences included the Rs3184504 (C/T) and Rs78894077 (A/C/G/T) affecting the expression of amino acids in LNK gene, and the Rs7973120 (A/T) unaffecting the expression of amino acids. The mutation and SNP of LNK gene were analyzed by DNA sequencing. Six cases of ET had LNK mutation, including four types: A300V, R425C, V402L and R426Q. T allele distribution of SNP Rs78894077 Ser in ET group was statistically significantly higher than that in the control group (P<0.05). T allele frequency of SNP Rs3184504 Ser in ET group was higher than that in the control group(P<0.05). LNK mutations exist in ET patients, and the T allele gene carrying LNK SNP Rs78894077 Ser and Rs3184504 Ser in persons may increase the risk of ET.

Variation of LNK Gene in Chronic Myeloid Leukemia

To compare the mutation and single nucleotide polymorphism (SNP) of LNK gene between chronic myeloid leukemia(CML) and control groups, and to explore the relationship between LNK gene variation and the occurrence of CML. A total of 36 patients with CML were selected, 46 healthy persons were used as normal controls. DNA was extracted from bone marrow and peripheral blood, BCR/ABL1 fusion gene was detected by Q-PCR. The whole exon of LNK gene was amplified by PCR. The amplified sequences included the Rs3184504 (C/T) and Rs78894077 (A/C/G/T) affecting the expression of amino acids in LNK gene, and the Rs7973120 (A/T) unaffecting the expression of the amino acids. The mutations and SNP of LNK gene were analyzed by DNA sequencing. Thirty-six cases of CML had BCR/ABL1 mutation, while no mutation was found in the control group. One case of CML had LNK heterozygous mutation (A300V), and the mutation rate was 2.8%, no mutation was seen in normal control group. Rs3184504: C/T allele frequency was 50%/50% in the control group, 94.4%/5.6% in the CML group, and the C allele in CML group was significantly higher than that in the control group; CC genotype accounted for 94.4% (P<0.01). Rs78894077: C/T allele in the control group was 9.8%/90.2%, in CML group was 16.7%/83.3%, the difference was not statistically significant(P>0.05); but CC genotype in CML group was very statistically significant higher than that in control group(P<0.01). Rs7973120: A/T allele frequency was 10.9%/89.1% in the control group, 25%/75% in the CML group, the LNK A allele in CML group was very significantly higher than that in control group (P<0.01). CML patients have been confirmed to have LNK mutation; the SNPs of LNK are related with the development of CML, and the most CML patients carry the LNK Rs3184504 C allele and the Rs7973120 A allele.

LNK Gene Single Nucleotide Polymorphisms and Acute Leukemia Susceptibility

The bone marrow and peripheral blood samples from 31 cases of acute lymphoblastic leukemia, 70 cases of acute myeloid leukemia and 130 healthy persons as the controls were collected. Genotype of LNK SNP Rs3184504(c.784T>C) and Rs78894077(c.724C>T) were determined by PCR-RFLP, and were confirmed by gel electrophoresis and sequencing. The NB4, THP-1 and Raji leukemia cell line models were cultured, the leukemia cell line LNK Rs3184504 and Rs78894077 polymorphism were detected by using direct sequencing. The CC genotype frequencies of Rs3184504 SNP were higher in ALL and AML patients than those in control (P<0.01), but there was no different between the groups in AML and ALL. The frequency of LNK gene Rs3184504 C allele was higher in AL as compared with control (P<0.01). The LNK gene Rs78894077 locus genotype distribution was not significantly different between the AL and the normal control group (P>0.05). Both Rs3184504 and Rs78894077 sites were detected as CC genotype in NB4, THP-1 and Raji cells. The persons carrying C allele of LNK gene Rs3184504 are more prone to develop acute leukemia.

The Polymorphisms in LNK Gene Correlated to the Clinical Type of Myeloproliferative Neoplasms.

ObjectiveLNK is an adapter protein negatively regulating the JAK/STAT cell signaling pathway. In this study, we observed the correlation between variation in LNK gene and the clinical type of myeloproliferative neoplasms (MPN).MethodsA total of 285 MPN cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, primary myelofibrosis (PMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was identified by allele-specific PCR method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in coding exons and their flanking sequences of LNK gene were examined by PCR-sequencing.ResultsMissense mutations of A300V, V402M, and R415H in LNK were found in 8 patients including ET (4 cases, all combined with JAK2-V617F mutation), PV (2 cases, one combined with JAK2-V617F mutation), PMF (one case, combined with JAK2-V617F mutation) and CML (one case, combined with BCR/ABL1 fusion gene). The genotype and allele frequencies of the three SNPs (rs3184504, rs111340708 and rs78894077) in LNK were significantly different between MPN patients and controls. For rs3184504 (T/C, in exon2), the T allele (p.262W) and TT genotype were frequently seen in ET, PV and PMF (P<0.01), and C allele (p.262R) and CC genotype were frequently seen in CML (P<0.01). For rs78894077 (T/C, in exon1), the T allele (p.242S) was frequently found in ET (P<0.05). For rs111340708 (TGGGGx5/TGGGGx4, in intron 5), the TGGGG x4 allele was infrequently found in ET, PMF and CML(P<0.01).ConclusionMutations in LNK could be found in some of MPN patients in the presence or absence of JAK2-V617F mutation. Several polymorphisms in LNK gene may affect the clinical type or the genetic predisposition of MPN.

Lnk prevents inflammatory CD8+ T‐cell proliferation and contributes to intestinal homeostasis

The intracellular adaptor Lnk (also known as SH2B3) regulates cytokine signals that control lymphohematopoiesis, and Lnk(-/-) mice have expanded B-cell, megakaryocyte, and hematopoietic stem-cell populations. Moreover, mutations in the LNK gene are found in patients with myeloproliferative disease, whereas LNK polymorphisms have recently been associated with inflammatory and autoimmune diseases, including celiac disease. Here, we describe a previously unrecognized function of Lnk in the control of inflammatory CD8(+) T-cell proliferation and in intestinal homeostasis. Mature T cells from newly generated Lnk-Venus reporter mice had low but substantial expression of Lnk, whereas Lnk expression was downregulated during homeostatic T-cell proliferation under lymphopenic conditions. The numbers of CD44(hi) IFN-γ(+) CD8(+) effector or memory T cells were found to be increased in Lnk(-/-) mice, which also exhibited shortening of villi in the small intestine. Lnk(-/-) CD8(+) T cells survived longer in response to stimulation with IL-15 and proliferated even in nonlymphopenic hosts. Transfer of Lnk(-/-) CD8(+) T cells together with WT CD4(+) T cells into Rag2-deficient mice recapitulated a sign of villous abnormality. Our results reveal a link between Lnk and immune cell-mediated intestinal tissue destruction.

Lnk adaptor suppresses radiation resistance and radiation-induced B-cell malignancies by inhibiting IL-11 signaling

The Lnk (Sh2b3) adaptor protein dampens the response of hematopoietic stem cells and progenitors (HSPCs) to a variety of cytokines by inhibiting JAK2 signaling. As a consequence, Lnk(-/-) mice develop hematopoietic hyperplasia, which progresses to a phenotype resembling the nonacute phase of myeloproliferative neoplasm. In addition, Lnk mutations have been identified in human myeloproliferative neoplasms and acute leukemia. We find that Lnk suppresses the development of radiation-induced acute B-cell malignancies in mice. Lnk-deficient HSPCs recover more effectively from irradiation than their wild-type counterparts, and this resistance of Lnk(-/-) HSPCs to radiation underlies the subsequent emergence of leukemia. A search for the mechanism responsible for radiation resistance identified the cytokine IL-11 as being critical for the ability of Lnk(-/-) HSPCs to recover from irradiation and subsequently become leukemic. In IL-11 signaling, wild-type Lnk suppresses tyrosine phosphorylation of the Src homology region 2 domain-containing phosphatase-2/protein tyrosine phosphatase nonreceptor type 11 and its association with the growth factor receptor-bound protein 2, as well as activation of the Erk MAP kinase pathway. Indeed, Src homology region 2 domain-containing phosphatase-2 has a binding motif for the Lnk Src Homology 2 domain that is phosphorylated in response to IL-11 stimulation. IL-11 therefore drives a pathway that enhances HSPC radioresistance and radiation-induced B-cell malignancies, but is normally attenuated by the inhibitory adaptor Lnk.

The Polymorphisms In LNK Gene Correlated To The Clinical Type Of Myeloproliferative Disorders

ObjectiveLNK (lymphocyte adaptor protein) is an adapter protein negatively regulating JAK/STAT cell signaling pathway. In this study, we observed the effects of variations in LNK gene on the clinical type of myeloproliferative disorders (MPD). MethodsA total of 285 MPD cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, idiopathic myelofibrosis (IMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was searched by allele-specific primers method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in exons and their flanking sequences of LNK gene were examined by PCR-sequencing. Count data were analyzed by χ2 or Fisher exact test. ResultsThe genotypes of 4 SNPs (rs3184504, rs111340708, rs78894077and rs7973120) could be identified. Missense mutations of A300V, V402M, and R415H in LNK were found in 8 patients, of whom 3 ET patients had homozygous A300V mutation combined with JAK2V617F mutation. The genotypes and allele types of the 3 SNPs (rs3184504, rs111340708 and rs78894077) correlated to the clinical type of MPD. For rs3184504, the allele frequencies of C/T were 0.5/0.5 in normal controls, 0.117/0.883 in ET, 0.132/0.868 in PV, 0.211/0.789 in IMF, and 0.944/0.056 in CML; T allele (p.262Trp) was significantly higher in ET, IMF and PV (P<0.01), and C allele (p.262Arg) was significantly higher in CML (P<0.01), as compared with the controls. For rs78894077, the allele frequencies of C/T were 0.097/0.903 in normal controls, 0.045/0.955 in ET, 0.086/0.914 in PV, 0.053/0.947 in IMF, and 0.167/0.833 in CML; T allele (p.242Ser) was higher in ET (P<0.05) than in normal controls. For rs111340708 which has 2 alleles of repeated sequences (TGGGG x5/TGGGG x4), the allele frequencies of TGGGG x5/TGGGG x4 were 0.462/0.538 in normal controls, 0.719/0.281 in ET, 0.533/0.467 in PV, 0.789/0.211 in IMF, and 0.639/0.361 in CML; TGGGG x4 allele was significantly lower in ET, IMF and CML than in normal controls (P<0.01). However, the allele frequencies of rs3184504 and rs111340708 were unrelated to the presence of JAK2V617F mutation and BCR/ABL1 fusion gene in MPD patients. ConclusionThe polymorphisms of SNPs in LNK gene correlated to the clinical type of MPD. Disclosures:No relevant conflicts of interest to declare.

RUNX1 and NF-E2 Transcriptional Upregulation Is Not Specific For Myeloproliferative Neoplasms But Is Seen In Those Polycythemic Disorders With Augmented HIFs Signaling

▪RUNX1 and NF-E2 are transcription factors that regulate hematopoietic stem cell homeostasis. It has been reported that increased RUNX1 expression in the granulocytes is present in all three classical myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis (Wang et al, Blood 2010), and that elevated NF-E2 promotes erythropoietin (EPO)-independent erythroid maturation of hematopoietic stem cells in vitro (Bogeska et al, Stem Cells Transl Med 2013). A mouse model overexpressing the NF-E2 transgene in hematopoietic cells was reported to be a new model of myeloproliferative neoplasms (Kaufmann et al, J Exp Med 2012).Polycythemic states can be divided into primary polycythemias, characterized by intrinsically hyperproliferative erythroid progenitors that are hypersensitive to EPO, and secondary polycythemias, wherein erythroid progenitors respond normally to EPO but circulating EPO is elevated or inappropriately normal for the level of increased red cell mass. Some congenital disorders including those with mutations in the hypoxia sensing pathway may share features of both primary and secondary polycythemias. We considered the possibility that increased transcripts of RUNX1 and NF-E2 might be the feature of other primary polycythemic states as well.We report a study of 19 polycythemic patients with primary or secondary polycythemia with diverse etiologies including mutations in positive and negative regulators of hypoxia sensing pathway. RUNX1 and NF-E2 transcripts were quantitated in granulocytes and BFU-E colonies by qPCR. All primary polycythemic patients had erythroid progenitors hypersensitive to or independent to EPO; all secondary polycythemic subjects had normal erythroid progenitor response to EPO.RUNX1 and NF-E2 gene transcripts were increased in granulocytes and BFU-E colonies in all PV patients, two unrelated subjects with the VHLR200W homozygous mutation (Chuvash polycythemia), one polycythemic patient homozygous for the VHLP138L exon 2 mutation, and a patient with the HIF2αM535V gain-of-function mutation. We also found upregulated expression of RUNX1 and NF-E2 in granulocytes and BFU-Es from a polycythemic patient (with no detectable EPOR, JAK2V617F or JAK2 exon 12 mutations and low level of EPO < 1 mU/mL) who was heterozygous for a SNP in exon 3 (rs147341899) in the LNK gene. We examined transcripts of RUNX1 and NF-E2 genes in granulocytes from two Croatian polycythemic patients with a homozygous VHLH191D exon 3 mutation whose erythroid progenitors were not hypersensitive to EPO and whose RUNX1, but not NF-E2, transcript was increased. We found similar results in two compound heterozygotes for VHLT124A exon 2 and VHLL188V exon 3 mutations. These two polycythemic siblings had hypersensitive erythroid colonies, increased RUNX1 transcripts and decreased NF-E2 transcripts in granulocytes. RUNX1 and NF-E2 transcripts were normal in two subjects with primary polycythemia due to the EPOR gain-of-function EPORQ434Xmutation, and in five unrelated subjects with secondary polycythemia.We next examined granulocyte transcripts of HIF-regulated genes: TFRC, SLC2A1, VEGF, BNIP3 and HK1, and found them to be increased in all PV patients and all studied polycythemic patients with VHL, HIF2α or LNK mutations, but not in polycythemic EPORQ434Xpatients or five patients with secondary polycythemia. Increased transcripts of HIF regulated genes are compatible with the previously unappreciated Warburg effect in PV (see S. Sana's Abstract at this ASH meeting).We propose that increased expression of RUNX1 and NF-E2 is not specific for myeloproliferative neoplasms but also is not universal for primary polycythemic disorders. Therefore, increased expression of RUNX1 and NF-E2 do not seem to be underlying mechanism for MPNs development but rather represent factors associated with diverse primary polycythemia states with augmented HIF signaling.(Note: KK and LL contributed equally to this work.)This work was supported by 1P01CA108671-O1A2 (NCI) Myeloproliferative Disorders (MPD) Consortium (PI Ron Hoffman) project#1 (PI Prchal) and the Leukemia & Lymphoma Society. Work by KK, LL, MH and VD was in part supported by the European Structural Funds (project CZ.1.07/2.3.00/20.0164 and CZ.1.07/2.3.00/30.0041), grant LF_2013_010 and by Czech Science Foundation (Project-P301/12/1503). Disclosures:No relevant conflicts of interest to declare.

Role of LNK gene mutation in pathogenesis of myeloproliferative neoplasms-review

Myeloproliferative neoplasms ( MPN ) is a class of clonal hematopoietic stem cell disease. Studies found that the JAK-STAT signaling pathway is closely related to the pathogenesis of MPN. The lymphocyte-specific adaptor protein (LNK) gene negatively regulates Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling and may play an important role in the pathogenesis of MPN. Especially in JAK2 mutation-negative MPN, LNK gene specific mutations may be the key to cause MPN subtypes. Certain single nucleotide polymorphism of LNK gene regulation of hematopoietic cells in different directions may also be important influence factors of MPN performance for different subtypes. LNK gene functional changes lead to abnormal activation of the JAK-STAT signaling pathway, and may be a new mechanism of MPN. In this review, the role of LNK gene in MPN pathogenesis is briefly summarized.

The Lnk Adaptor Protein: A Key Regulator of Normal and Pathological Hematopoiesis

The development and function of blood cells are regulated by specific growth factors/cytokines and their receptors' signaling pathways. In this way, these factors influence cell survival, proliferation and differentiation of hematopoietic cells. Central to this positive and/or negative control are the adaptor proteins. Since their identification 10years ago, members of the Lnk adaptor protein family have proved to be important activators and/or inhibitors in the hematopoietic, immune and vascular system. In particular, the generation of animal and cellular models for the Lnk and APS proteins has helped establish the physiological role of these molecules through the identification of their specific signaling pathways and the characterization of their binding partners. Moreover, the recent identification of mutations in the LNK gene in myeloproliferative disorders, as well as the correlation of a single nucleotide polymorphism on LNK with hematological, immune and vascular diseases have suggested its involvement in the pathophysiology of these malignancies. The latter findings have thus raised the possibility of addressing Lnk signaling for the treatment of certain human diseases. This review therefore describes the pathophysiological role of this adaptor protein in hematological malignancies and the potential benefits of Lnk therapeutic targeting.

Inhibition of Lnk in Mouse Induced Pluripotent Stem Cells Promotes Hematopoietic Cell Generation

Embryonic stem (ES) cell- and induced pluripotent stem (iPS) cell-derived hematopoietic stem/progenitor cells (HSPCs) are considered as an unlimited source for HSPC transplantation. However, production of immature hematopoietic cells, especially HSPCs, from ES and iPS cells has been challenging. The adaptor protein Lnk has been shown to negatively regulate HSPC function via the inhibition of thrombopoietin (TPO) and stem cell factor signaling, and Lnk-deficient HSPCs show an enhanced self-renewal and repopulation capacity. In this study, we examined the role of Lnk on the hematopoietic differentiation from mouse ES and iPS cells by the inhibition of Lnk using a dominant-negative mutant of the Lnk (DN-Lnk) gene. We generated mouse ES and iPS cells stably expressing a DN-Lnk, and found that enforced expression of a DN-Lnk in ES and iPS cells led to an enhanced generation of Flk-1-positive mesodermal cells, thereby could increase in the expression of hematopoietic transcription factors, including Scl and Runx1. We also showed that the number of both total hematopoietic cells and immature hematopoietic cells with colony-forming potential in DN-Lnk-expressing cells was significantly increased in comparison with that in control cells. Furthermore, Lnk inhibition by the overexpression of the DN-Lnk gene augmented the TPO-induced phosphorylation of Erk1/2 and Akt, indicating the enhanced sensitivity to TPO. Adenovirus vector-mediated transient DN-Lnk gene expression in ES and iPS cells could also increase the hematopoietic cell production. Our data clearly showed that the inhibition of Lnk in ES and iPS cells could result in the efficient generation and expansion of hematopoietic cells.

Modulation of gurken translation by insulin and TOR signaling in Drosophila

Localized Gurken (Grk) translation specifies the anterior–posterior and dorsal–ventral axes of the developing Drosophila oocyte; spindle-class females lay ventralized eggs resulting from inefficient grk translation. This phenotype is thought to result from inhibition of the Vasa RNA helicase. In a screen for modifiers of the eggshell phenotype in spn-B flies, we identified a mutation in the lnk gene. We show that lnk mutations restore Grk expression but do not suppress the persistence of double-strand breaks nor other spn-B phenotypes. This suppression does not affect Egfr directly, but rather overcomes the translational block of grk messages seen in spindle mutants. Lnk was recently identified as a component of the insulin/insulin-like growth factor signaling (IIS) and TOR pathway. Interestingly, direct inhibition of TOR with rapamycin in spn-B or vas mutant mothers can also suppress the ventralized eggshell phenotype. When dietary protein is inadequate, reduced IIS–TOR activity inhibits cap-dependent translation by promoting the activity of the translation inhibitor eIF4E-binding protein (4EBP). We hypothesize that reduced TOR activity promotes grk translation independent of the canonical Vasa- and cap-dependent mechanism. This model might explain how flies can maintain the translation of developmentally important transcripts during periods of nutrient limitation when bulk cap-dependent translation is repressed.

Modulation ofgurkenTranslation by Insulin/TOR Signaling in Drosophila

Localized Gurken (Grk) translation specifies the anterior-posterior and dorsal-ventral axes of the developing Drosophila oocyte; spindle-class females lay ventralized eggs resulting from inefficient grk translation. This phenotype is thought to result from inhibition of the Vasa RNA helicase. In a screen for modifiers of the eggshell phenotype in spn-B flies, we identified a mutation in the lnk gene. We show that lnk mutations restore Grk expression but do not suppress the persistence of double-strand breaks nor other spn-B phenotypes. This suppression does not affect Egfr directly, but rather overcomes the translational block of grk messages seen in spindle mutants. Lnk was recently identified as a component of the insulin/insulin-like growth factor signaling (IIS) and TOR pathway. Interestingly, direct inhibition of TOR with rapamycin in spn-B or vas mutant mothers can also suppress the ventralized eggshell phenotype. When dietary protein is inadequate, reduced IIS-TOR activity inhibits cap-dependent translation by promoting the activity of the translation inhibitor eIF4E-binding protein (4EBP). We hypothesize that reduced TOR activity promotes grk translation independent of the canonical Vasa- and cap-dependent mechanism. This model might explain how flies can maintain the translation of developmentally important transcripts during periods of nutrient limitation when bulk cap-dependent translation is repressed.

Identification of Novel LNK Mutations In Patients with Chronic Myeloproliferative Neoplasms and Related Disorders

AbstractAbstract 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. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Characterization of Lnk: AN ADAPTOR PROTEIN EXPRESSED IN LYMPHOCYTES

Stimulation of the T cell antigen receptor (TCR) activates a set of non-receptor protein tyrosine kinases that assist in delivering signals to the cell interior. Among the presumed substrates for these kinases, adaptor proteins, which juxtapose effector enzyme systems with the antigen receptor complex, figure prominently. Previous studies suggested that Lnk, a 38-kDa protein consisting of a single SH2 domain and a region containing potential tyrosine phosphorylation sites, might serve to join Grb2, phospholipase C-gamma1, and phosphatidylinositol 3-kinase to the TCR. To elucidate the physiological roles of Lnk in T cell signal transduction, we isolated the mouse Lnk cDNA, characterized the structure of the mouse Lnk gene, and generated transgenic mice that overproduce Lnk in thymocytes. Here we report that although Lnk becomes phosphorylated during T cell activation, it plays no limiting role in the TCR signaling process. Moreover, we have distinguished p38(Lnk) from the more prominent 36-kDa tyrosine phosphoproteins that appear in activated T cells. Together these studies suggest that Lnk participates in signaling from receptors other than antigen receptors in lymphocytes.