Indiana University-Purdue University Indianapolis (IUPUI) / Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) cause constitutive activation of the receptor and confer a poor prognosis in acute myeloid leukemia (AML). We hypothesized that Shp2 interacts with FLT3-ITD via protein complexes at tyrosine (Y) 768, 955, and/or 969 and that Shp2 and PI3K work cooperatively to promote FLT3-ITD-induced leukemogenesis. Consistently, mutation of N51-FLT3 tyrosine 768 to phenylalanine reduced proliferation and levels of phospho-Erk compared to N51-FLT3-expressing cells while having no effect on levels of phospho-STAT5. In transplants, C3H/HeJ mice injected with either WT-FLT3-, N51-FLT3-, or N51-Y768F-expressing cells showed that mutation of Y768 had no effect on overall survival. In addition, pharmacologic inhibition of Shp2 with II-B08 or PI3K with GDC-0941 in N51-FLT3-expressing cells and primary patient samples showed decreased proliferation. A possible mechanistic explanation for reduced proliferation and selective reduction of P-Erk levels in the N51-FLT3-Y768-expressing cells is through decreased recruitment of Grb2, which participates with son of sevenless, SOS, to activate the RAS-Erk signaling pathway. The lack of improvement in overall survival could be due to preserved STAT5 signaling, as observed during in vitro experiments. Collectively, these data suggest that the tyrosine 768 residue plays an important role in phospho-Erk signaling in N51-FLT3-expressing cells, and that pharmacologic therapy with Shp2 or PI3K inhibitors may provide a novel treatment approach for FLT3-ITD positive AML. For future directions, we plan to treat mice with the Shp2 inhibitor, II-B08, the PI3K inhibitor, GDC-0941, or a combination to determine the effect on overall survival.

Les arrestines sont connues pour leurs rôles dans la désensibilisation et l'endocytose des récepteurs couplés aux protéines G (RCPGs). Au fil des ans, plusieurs partenaires de ces protéines adaptatrices ont été identifiés, notamment diverses molécules impliquées dans la signalisation, incluant les kinases ERK, JNK, Akt, Raf et Src. Ainsi, les arrestines interagissent avec plusieurs kinases, mais seulement avec deux phosphatases, PPA2 et MKP7. Récemment, notre laboratoire a démontré une nouvelle interaction entre les arrestines non-visuelles et la phosphatase Shp2. En effet, des essais in vitro et in cellulo ont montré une interaction directe entre les deux protéines peuvent interagir ensembles et ce directement. Or, nous en sommes venus à nous demander si les arrestines peuvent être phosphorylées sur leurs résidus tyrosine et si cette modification pourrait être régulée par des partenaires connus, soient Src et Shp2. Il a déjà été montré que l'arrestine 2 peut être phosphorylée sur un résidu tyrosine qui lui est unique et que cette modification expliquerait peut-être les différences de modulation entre les arrestines non-visuelles. Par contre, il n'existe encore aucune preuve que l'arrestine 3 puisse aussi être phosphorylée. D'abord, nous démontrons pour la première fois que l'arrestine 3, tout comme l'arrestine 2, est phosphorylée sur des résidus tyrosines et que cette modification peut être amenée par l'activité de Src. Ensuite, un double mutant ponctuel de l'arrestine 3 a été construit afin de cibler les tyrosines régulées. Il semble que les tyrosines 380 et 404 de l'arrestine 3, soient d'importants sites de phosphorylation. Ce nouveau mutant de l'arrestine 3 représente un excellent outil afin de déterminer le rôle de la phosphorylation des tyrosines de l'arrestine 3. Aussi, puisque ces deux tyrosines sont absentes de la séquence de l'arrestine 2, leur phosphorylation pourrait être à la base des différences fonctionnelles entre les arrestines 2 et 3. De plus, nous faisons la démonstration, en conditions de surexpression en cellules, que l'activité de Shp2 peut mener à la déphosphorylation des arrestines non-visuelles. Dès lors, nos études montrent d'abord que, tout comme son homologue, l'arrestine 3 peut être phosphorylée sur ses tyrosines. De plus, cette phosphorylation est non seulement régulée par la phosphatase Shp2, mais représente également un nouveau mécanisme potentiel de régulation des multiples fonctions des arrestines.

The response of cells to mechanical inputs is a key determinant of cell behavior. In response to changes in the mechanical environment of epithelial cells, E-cadherin initiates signal transduction cascades that allow the cells to modulate their contractility to withstand the force. Much attention has focused on identifying the E-cadherin signaling pathways that promote contractility, but the negative regulators remain undefined. In this thesis, we identify SHP2 as a force-activated phosphatase that negatively regulates E-cadherin force transmission by dephosphorylating vinculin Y822. To specifically probe a role for SHP2 in E-cadherin mechanotransduction, we innovatively mutated vinculin so that it retains its phosphorylation but cannot be dephosphorylated. Cells expressing the mutant vinculins have increased contractility. This work provides the first mechanism for inactivating E-cadherin mechanotransduction and provides a new method for specifically targeting the action of phosphatases in cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm that lacks effective chemotherapies. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in SHP2, leading to hyperactivation of ERK and AKT and hyperproliferation of cells in response to granulocyte macrophage-colony stimulating factor (GM-CSF). Our lab previously showed that p110δ, the hematopoietic-specific catalytic subunit of phosphoinositide 3-kinase, is a crucial mediator of mutant Shp2-induced GM-CSF hypersensitivity in vitro. We treated oncogenic Shp2-expressing mice with a p110δ inhibitor and showed that the strong effect our lab observed in vitro translated into reduced splenomegaly and prolonged survival in vivo. We investigated molecules potentially cooperating with p110δ signaling and discovered that Bruton’s tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2 myeloid cells. We used specific BTK and p110δ inhibitors to demonstrate that BTK cooperates with p110δ to hyperactivate Akt/Erk and to promote hyperproliferation. GOF Shp2-expressing mice treated in vivo with the drug combination targeting p110δ and BTK have significantly decreased splenomegaly and WBC counts. We also explored the mechanism of BTK signaling and hypothesized that B cell adaptor for PI3K (BCAP) mediated BTK upregulation of PI3K activity. In mutant Shp2 macrophages, we observed BCAP phosphorylation specifically in the larger isoforms needed for PI3K activation, and BTK inhibition led to a dose-dependent reduction in this phosphorylation. We also demonstrated reduced interaction between BCAP and the PI3K regulatory p85α subunit bearing mutated SH2 domains. Finally, we investigated the effects of mutated DNA methyltransferase 3A (Dnmt3a) in conjunction with GOF Shp2. Double mutant mice quickly became moribund with pronounced splenomegaly and leukocytosis. There was an expansion of mature myeloid cells in the periphery and myeloid progenitors in the bone marrow, plus anemia with evidence of compensatory erythropoiesis in the spleen. Our findings show that the myeloproliferative neoplasm caused by GOF Shp2 is due to hyperactive p110δ, and this is further promoted by BTK, which forms a positive feedback loop with PI3K and BCAP, thus leading to more Akt/Erk hyperphosphorylation and more hyperproliferation in response to GM-CSF. The dual inhibition of p110δ and BTK represents a novel effective treatment strategy for JMML and other diseases induced by oncogenic Shp2.

The microtubule-associated protein tau normally functions to bind to and stabilize microtubules. However, evidence now indicates that tau may also play a critical role in signaling pathways linked to neuronal development and neurodegeneration. The tau association with numerous signaling proteins such as tyrosine kinases, adaptor proteins, and scaffold proteins support this hypothesis. Phospho-Y18 tau was previously found in Alzheimer’s disease (AD) brain. Interestingly, this phosphorylation appeared to be regulated during neurodegeneration possibly by a tyrosine phosphatase(s). Identifying a candidate phosphatase, our lab found the association between tau and SHP2 in a neuronal cell line and dephosphorylation of phospho-Y18 by protein tyrosine phosphatase SHP2 in vitro. Since both tau and SHP2 play a critical role in NGF-induced signaling pathway, these findings raised the possibility that the tau-SHP2 association has a role in NGF signaling. The aim of this dissertation research is to characterize the tau-SHP2 association and its role in neuronal signaling. Here, we provide evidence that tau phosphorylation is not required for SHP2 association but significantly enhances the interaction. The SHP2 binding region of tau napped to residues 256-273, which contain the microtubule binding repeat 1 of tau. Using in situ proximity ligation assay (PLA), we also showed the presence of endogenous tau-SHP2 and tau-activated SHP2 complexes in neuronal cells. The number of complexes was increased in the cells in response to NGF. Our PLA data also showed the localization of these complexes to actin ruffles. In NGF signaling, we showed that phosphorylation at T231 of tau was necessary for the increase in tau-SHP2 association. Lastly, we provide evidence that tau-SHP2 complexes are present in mouse primary neuronal cultures and mouse brain sections. Together, these findings show a role for tau phosphorylation in SHP2 binding and a potential role for tau-SHP2 interaction in neuronal signal transduction. Based on our findings, we speculate that there is a role for tau-SHP2 association during early brain development and in neurodegenerative disease.

Mast cells are granulocytes that are a key component of the innate and adaptive immune system, and contribute to allergic disorders. Mast cell activation following clustering of the high affinity IgE receptor (FcepsilonRI) by multivalent antigens requires reversible tyrosine phosphorylation of myriad signaling proteins. Activated mast cells rapidly release granule contents (eg. histamine and serine proteases) that cause vascular permeability, and in a more delayed manner they also synthesize and secrete eicosanoids and numerous cytokines (eg. IL-6 and TNFalpha) that recruit activated leukocytes. FcepsilonRI signaling is initiated by Lyn, a Src Family Kinase (SFK), that phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) found on the FcepsilonRI beta and gamma chains. This allows recruitment of Fyn SFK and Syk kinase that bind ITAMs and phosphorylate numerous downstream targets. Src Homology 2 domain-containing Phosphatase 2 (SHP2, encoded by ptpn11/shp2) is known to be recruited to several phosphorylated proteins following FcepsilonRI aggregation in mast cells, however attempts to define the role of SHP2 have been hampered by its essential role during embryonic development and hematopoiesis in mice. Recently, conditional SHP2 knock-out mice (shp2fl/fl) have been created allowing for shp2 inactivation in a tissue-specific manner by Cre recombinase. Here we describe the use of transgenic mice expressing a modified estrogen receptor-Cre Recombinase (TgCreER*) on a shp2fl/fl genetic background, that allows for maturation of bone marrow-derived mast cells (BMMCs) prior to shp2-inactivation using 4-hydroxytamoxifen (4OH-TM). SHP2-depleted BMMCs display reduced phosphorylation of the FcepsilonRI beta chain, but exhibit extended phosphorylation of Syk kinase. Additionally, SHP2-deficient cells display a defect in the activation of both Erk mitogen-activated protein kinase and Akt, which correlates with an observed defect in the production of TNFalpha and Leukotriene C4. Finally, we show that SHP2-deficient BMMCs display elevated FcepsilonRI-evoked phosphorylation of Csk-Binding Protein (Cbp or PAG) on residue Y317, which recruits C-terminal Src kinase (Csk) that phosphorylates SFKs on an inhibitory tyrosine. This hyperphosphorylation of Cbp correlates with elevated phosphorylation of the C-terminal inhibitory tyrosine on Fyn kinase. This study provides new insights into the role of SHP2 as a positive effector of FcepsilonRI signaling and cytokine production in mast cells. / Thesis (Master, Biochemistry) -- Queen's University, 2008-08-20 13:51:18.751

Le diabète de type 2 (DT2) est une maladie qui affecte de plus en plus de personnes à travers le monde et comporte plusieurs complications. Les moyens thérapeutiques actuels sont assez limités, car même s'ils sont efficaces, ils sont associés à d'importants effets secondaires. Ainsi, il est important de trouver de nouvelles cibles thérapeutiques pour améliorer la sensibilité à l'insuline en situation d'obésité ou de diabète. Nous nous intéressons ici à une nouvelle cible potentielle, appelée SHP2, qui est une protéine tyrosine phosphatase impliquée dans la transduction du signal en régulant plusieurs voies canoniques (MAPK, PI3K). Cette protéine est connue pour ses rôles cruciaux dans le développement ainsi que son implication dans le métabolisme glucidique. Cependant, cette dernière fonction est encore assez peu comprise, car l'effet d'une délétion de SHP2 sur la sensibilité à l'insuline est différent suivant les tissus et son rôle global n'est pas connu. Nous utilisons ici un modèle original pour étudier l'impact de SHP2 sur le métabolisme glucidique au niveau du corps entier, en travaillant sur le syndrome de Noonan (SN). En effet, cette maladie génétique est principalement causée par une mutation hyperactivatrice du gène PTPN11 codant la protéine SHP2. L'étude du métabolisme glucidique dans le contexte du SN a permis de mettre en évidence une intolérance au glucose, qui est dissociée de l'adiposité réduite, à la fois chez les patients et dans le modèle murin de la maladie (SHP2D61G/+). Nous montrons que les souris SN présentent une inflammation caractérisée par une surexpression de marqueurs pro-inflammatoires, ainsi qu'une augmentation de macrophages pro-inflammatoires dans les tissus métaboliques. [...] / Type 2 diabetes (T2D) is a disease that affects more and more people worldwide and has many severe, lifethreatening complications. The current therapies are rather limited, because even if they are effective, they are associated with significant side effects. Thus, it is important to find new therapeutic targets to improve insulin sensitivity in obesity or diabetes. We are interested here in a new potential target called SHP2, a protein tyrosine phosphatase involved in signal transduction by regulating several canonical pathways (MAPK, PI3K). This protein is known for its crucial roles in development as well as its involvement in glucidic metabolism. However, this latter function is still poorly understood because the effect of a deletion of SHP2 on insulin sensitivity is different between tissues and its overall role is not known. Here, to study the impact of SHP2 on whole body glucidic metabolism we used Noonan Syndrome (NS) as an original model system. Indeed, this genetic disease is mainly caused by a hyperactivating mutation of the gene PTPN11 encoding the protein SHP2. The study of glucidic metabolism in the context of SN has revealed glucose intolerance, which is dissociated from reduced adiposity, both in patients and in the murine model of the disease (SHP2D61G/+). We show that NS mice exhibit inflammation characterized by overexpression of pro-inflammatory markers, as well as an increase of pro-inflammatory macrophages in metabolic tissues. Thanks to bone marrow transplantation and clodronate treatment, we show this inflammation comes from macrophage and is the cause of the insulin resistance in SN.[...]

KIT receptor (CD117) is a receptor tyrosine kinase crucial for homeostasis of mast cells (MCs) in tissues and recruitment to sites of inflammation and tumors in response to its ligand Stem cell factor (SCF). Gain of function mutations in KIT (e.g. D816V) are frequently observed in systemic mastocytosis and other cancer types. Src Homology 2 domain containing phosphatase-2 (SHP2 or PTPN11) is a protein tyrosine phosphatase that promotes cell proliferation, survival and motility in multiple pathways and cell types. To study SHP2 function in MCs, we generated novel MC-specific Shp2 knock-out (KO) mice (MC-shp2 KO). These mice had reduced numbers of MCs in skin and peritoneum, and defective contact hypersensitivity responses compared to control mice, consistent with SHP2 promoting MC homeostasis. Using an inducible SHP2 KO bone marrow-derived MC (BMMC) culture model, we found that SHP2 KO cells were prone to apoptosis and had no MC repopulating activity in vivo. Mechanistically, SHP2 enhanced ERK activation and downregulation of pro-apoptotic protein Bim. SHP2 KO BMMCs also had defects in chemotaxis towards SCF, due to impaired activation of a Lyn/Vav/Rac pathway in SHP2 KO BMMCs. This correlated with defects in cell spreading, and F-actin polymerization in response to SCF. Treatment of BMMCs with a SHP2 inhibitor (II-B08) also led to reduced chemotaxis, consistent with SHP2 phosphatase activity being required for KIT-induced chemotaxis. Lastly, we tested whether SHP2 regulates oncogenic KIT signaling using a P815 mouse mastocytoma model. Stable silencing of SHP2 in P815 cells led to reduced cell growth and survival in vitro, and less aggressive systemic mastocytosis development in syngeneic mice. Overall, these studies identify SHP2 as a key node in SCF/KIT and oncogenic KIT pathways, and as a potential therapeutic target in several human diseases. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-06-25 12:03:57.818

The gene PTPN11 was identified in the early 1990s, and encodes the non-transmembrane protein tyrosine phosphatase SHP2. SHP2 is expressed ubiquitously in cells, and plays an important role in cancer. Unlike most phosphatases, SHP2 positively regulates several signaling pathways including the Ras/MAPK and Src signaling pathways and acts as a proto-oncogene. SHP2 is also a cancer essential gene in certain types of carcinomas, and promotes growth, survival, and epithelial to mesenchymal transformation. Gain of function (GOF) SHP2 mutations are known leukemic oncogenes, and have been identified to a smaller extent in solid tumors as well. Currently, the roles of SHP2 in lung carcinoma are not fully understood. While GOF SHP2 mutations have been detected in lung cancer, their contributions to cellular transformation had not been established. In addition, SHP2 is known to promote EGF growth factor receptor (EGFR) signaling. Since GOF EGFR mutations induce transformation of lung epithelial cells, it is possible that SHP2 plays a role in promoting GOF EGFR mutant driven tumorigenesis. The objective of this dissertation is to determine whether SHP2 can act as an oncogene in lung epithelial cells and whether SHP2 inhibition can affect GOF EGFR mutant induced lung cancer. To achieve these aims, we generated two novel doxycycline (Dox) inducible transgenic mouse models which express either the GOF SHP2E76K leukemic oncogene or the dominant negative SHP2CSDA mutant under the control of the Clara cell secretory protein (CCSP) promoter to regulate transgene expression to type II pneumocytes. To determine whether SHP2 plays a role in promoting GOF EGFR mutant signaling, we started by disrupting SHP2 function in vitro. Two non small lung cancer cell lines were used for this project: HCC827 carries the LREA deletion in exon 19, and H1975 co-expresses the EGFRL858R point mutation and the EGFRT790M gatekeeper mutation. After SHP2 PTP inhibition or knock-down by shRNA and siRNA, both cell lines exhibited decreased cell proliferation and reduced levels of pErk1/2 and c-Myc. Based on these results, we acquired a transgenic mouse line which expresses the EGFRL858R mutant under the control of the tet-O promoter and generated bitransgenic CCSP-rtTA/tetO-EGFRL858R and tritransgenic CCSP-rtTA/tetO-EGFRL858R/tetO-SHP2CSDA mice to study the effects of the dominant negative SHP2CDSA mutant on EGFRL858R mediated carcinogenesis in vivo. After 4, 6, and 8 weeks of Dox induction, pErk1/2 and pSrc levels were increased in the lungs of bitransgenic mice compared to wild type controls. Both kinases were suppressed by SHP2CSDA expression in tritransgenic mice. In addition, SHP2CSDA expression delayed tumor onset and prevented progression to a more aggressive phenotype. Tritransgenic mice also developed a smaller tumor burden compared to bitransgenic animals. These results suggest that SHP2 is critical for GOF EGFR mutant mediated lung tumorigenesis and describe a new role of SHP2 as a potential therapeutic target for the development of novel NSCLC drugs. Once we generated our CCSP-rtTA/tetO-SHP2E76K transgenic mouse model, we administered Dox for one month and found that SHP2E76K expression upregulates pErk1/2, pSrc, pGab1, c-Myc and Mdm2 levels in the lungs of bitransgenic mice compared to controls. After six to nine months of Dox induction, SHP2E76K expression caused formation of lung adenomas and adenocarcinoma. We then took advantage of the reversible feature of our mouse model to test whether lung tumors are dependent on sustained SHP2E76K expression for survival. MRI analysis of lung adenocarcinomas showed full regression of the lung tumors after Dox withdrawal. Histological evaluation of lung tissues revealed residual hyperplastic lesions as well as evidence of necrosis, while biochemical analysis showed that pGab1, pErk1/2, pSrc and c-Myc returned to basal levels. These results demonstrate that sustained SHP2E76K expression is required for lung tumor maintenance. Moreover, this data describe a novel function of SHP2E76K as an oncogene in lung carcinoma.

SHP2, a protein-tyrosine phosphatase (PTP), is a key signaling regulator in multiple cell signaling processes including the Ras/MAPK pathway. This has led to SHP2 being identified as a therapeutic target in multiple types of cancers and autoimmune disorders. However, the role that the C-terminal residues play when they interact with other SHP2 domains remains unknown. This study presents data to illustrate that the C-terminal residues interact intramolecularly with other domains to inhibit PTP activity. Additionally, the identification of a specific and potent SHP2 inhibitor has proven difficult because of the high homology and positive nature of the PTP active site. This study presents the data from a high throughput fragment screen identifying several promising HIT compounds that may be further developed into potent and selective SHP2 inhibitors. Furthermore, data supporting the development of a selective SHP2 covalent inhibitor from a nonselective core molecule is presented.