Kinase Lck Research Articles

Abstract B177: Exploration of the signaling pathways triggered by exogenous galectin-9 in human T and B cells

Abstract Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin which is frequently released in the extra-cellular medium where it can modulate various biological activities (cell adhesion, migration), but it acts mainly as a negative regulator of the immune response. Our team has shown an intense and inappropriate production of gal-9 by malignant epithelial cells in Epstein-Barr virus-associated nasopharyngeal carcinoma. Other groups and ourselves have shown that gal-9 is very abundant in plasma samples from patients chronically infected by hepatitis C or B virus, and particularly in those with hepatocellular carcinomas. The most prominent immunosuppressive effects reported for gal-9 are the induction of apoptosis of CD4+ T-helper 1 (Th1) cells, exhaustion of CD8+ T cells, and stimulation of regulatory T cell activity. However, a recent study (and our own data) demonstrates that gal-9 can also activate and expand a subset of IFNγ; producing Th1 cells and central memory T cells in the surviving population. Despite its critical role in regulating the immune response, our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. Our studies reveal that several proximal components of the TCR-CD3 complex including the Lck kinase were required for the calcium (Ca2+) mobilization induced by gal-9 in Jurkat cells. In contrast, these components were not necessary for the apoptosis pathway triggered by gal-9. These data were confirmed in human CD4+ blood T cells: Lck inhibition with a specific inhibitor abrogates Ca2+ mobilization, but not apoptosis induction in CD4+ T cells. Moreover, this cytosolic Ca2+ release leads to the production of Th1 type cytokines, i.e. IL-2 and IFNγ; also dependent on Lck. These findings support the notion that gal-9 acts on T cells by two distinct pathways: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis which is independent of this complex. More recent data suggest that the CD45 molecule could be a putative receptor for gal-9 on human T cells. Indeed, the J45.01 cell line (CD45-deficient) was not susceptible to gal-9-induced apoptosis. In parallel to this work, we investigated the same cellular events (apoptosis and Ca2+ mobilization) in human B cells. In fact, there are currently very few publications dealing with the effects of gal-9 on B cells. Our preliminary results show that some Burkitt's lymphoma cell lines (BL2, BL41 and Ramos) and CD20+ B cells from blood, were both sensitive to gal-9-induced apoptosis. Besides, gal-9 induced a cytosolic Ca2+ mobilization in BL2 and BL41 cells which is efficiently abrogated by the chemical Lck inhibitor. In contrast, we didn't observe any Ca2+ release in the Ramos cell line, in which the Lck kinase is not functional, as well as in non-lymphoid cells. These preliminary results suggest that gal-9 activates in B cells a signaling pathway somehow similar to the TCR pathway and previously unexplored in B cells. Citation Format: Claire Lhuillier, Clement Barjon, Toshiro Niki, Aurore Gelin, Nadira Delhem, Mitsuomi Hirashima, Ming Wei, Olivier Dellis, Pierre Busson. Exploration of the signaling pathways triggered by exogenous galectin-9 in human T and B cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B177.

Construction of an immunorelated protein–protein interaction network for clarifying the mechanism of burn

Background and aimSevere burn is known to induce a series of pathological responses resulting in increased susceptibility to systemic inflammatory response and multiple organ failure, but the underlying molecular mechanism remains unclear at present. The main aim of this study was to expand our understanding of the events leading to circulating leukocyte response after burn by subjecting the gene expression profiles to a bioinformatic analysis. Materials and methodsComprehensive gene expression analysis was performed to identify differentially expressed genes (DEGs) using the expression profile GSE7404 (Mus musculus, circulating leukocyte, 25% of total body surface area (TBSA), full thickness) downloaded from the Gene Expression Omnibus, followed by the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In addition, a postburn protein–protein interaction (PPI) network was constructed to identify potential biomarkers. ResultsMaximum changes in the gene expression profile were detected 1 day post burn. Separate Gene Ontology (GO) functional enrichment analysis for upregulated and downregulated DEGs revealed significant alterations of genes related to biological process such as “response to stimuli,” “metabolic,” “cellular and immune system processes,” “biological regulation,” and “death” in the leukocyte transcriptome after the burn. The KEGG pathway enrichment analysis showed that the upregulated DEGs were significantly enriched in the nodes of immunorelated and signal transduction-related pathways, and the downregulated genes were significantly enriched for the immunorelated pathways. The PPI network and module analysis revealed that, 1 day after the burn, lymphocyte-specific protein tyrosine kinase (Lck) (downregulated), Jun (upregulated), Cd19 (downregulated), Stat1 (downregulated), and Cdk1 (upregulated) were located centrally in both the PPI network and modules. ConclusionsBased on an integrated bioinformatic analysis, we concluded that Lck, Jun, Cd19, Stat1, and Cdk1 may be critical 1 day after the burn. These findings expand our understanding of the molecular mechanisms of this important pathological process. Further studies are needed to support our work, focused on identifying candidate biomarkers with sufficient predictive power to act as prognostic and therapeutic biomarkers for burn injury.

Oncogenic Role for the Lck/ZAP70/PLCG2 Signaling Pathway in Pre-B-ALL Pathogenesis

Although the treatment and prognosis of patients with pediatric acute lymphoblastic leukemia (ALL) have improved during the last decades, there is still a clinical need for more effective/selective and less toxic therapies.To address this, we have interrogated various signaling pathways in human ALL cells and mouse strains that express E2A-PBX1, which is present in 5-7% of pediatric ALL. Phospho-flow analysis revealed basal hyper-phosphorylation levels of PLCγ2 in mouse E2A-PBX1 leukemias, consistent with hyper-activation of upstream signaling pathways. Efficient shRNA-mediated depletion of PLCγ2 reduced colony formation of mouse E2A-PBX1+ leukemias in vitro and increased disease-free survival after secondary bone marrow transplantation in vivo. Furthermore, PLCγ2-depleted human ALL cell lines including E2A-PBX1+ cells, showed reduced proliferation. These data suggest a pathogenic role of hyperactivated PLCγ2 in pre-B-ALL.Bioinformatics analysis of E2A-PBX1 target genes in human ALLs revealed an enrichment of B- and T-cell activation pathways, which include the SRC-family kinase LCK and the cytoplasmic kinase ZAP70, upstream of PLCγ2. Comparative analyses of global transcriptional profiles in human primary and mouse leukemias and preleukemias induced by the E2A-PBX1 oncogene identified the signaling kinase ZAP70 as one of the earliest and most consistently up-regulated genes in E2A-PBX1 leukemias. Using a candidate gene approach, we identified LCK with increased expression levels in E2A-PBX1 leukemia cells compared to normal B-cell progenitors.Mouse and human E2A-PBX1 leukemia cells were dependent on the E2A-PBX1 target genes ZAP70 and LCK for proliferation and survival as confirmed by shRNA knock-down experiments. Hence, efficient depletion of these genes resulted in a decrease of phosphorylated PLCγ2, suggesting therapeutic targets in E2A-PBX1 leukemias. Combined suppression of ZAP70 and LCK using double-shRNA experiments showed an additive effect on inhibition of cell proliferation and decrease of phosphorylated PLCγ2. These results provide a rationale for combination therapy to block this hyper-activated signaling pathway at different levels.Several small molecule inhibitors were evaluated for their effects on PLCγ2 upstream pathways in E2A-PBX1 leukemia cells. SRC-family kinase inhibitors including dasatinib were most effective in reducing phosphorylation of PLCγ2 and inhibiting cell proliferation. Furthermore, dasatinib showed promising preclinical efficacy in vitro in colony forming assays and in vivo after secondary bone marrow transplantation of leukemias.In summary, our studies demonstrate that the proliferation and survival of E2A-PBX1 leukemias are dependent on PLCγ2 and upstream signaling pathways, which are suitable for pharmacological inhibition. DisclosuresNo relevant conflicts of interest to declare.

SLP76 Is Ectopically Expressed in Chronic Lymphocytic Leukemia Cells and Involves in B-Cell Receptor Signaling

In the last decade, the B-cell receptor (BCR) has emerged as a pivotal stimulus in CLL pathogenesis. The BCR responsiveness in CLL cells is heterogeneous among patients and correlates with disease aggressiveness. Here we show for the first time, that SLP76 a key scaffold protein in T-cell receptor (TCR) signaling, is ectopically expressed in CD19+ purified CLL cells (purity >95%) in the majority of patients, and is co-expressed with other components of the TCR pathway, including LCK and ZAP70. SLP76 mRNA levels correlated with its protein expression and SLP76 protein levels were higher in unmutated IGHV and ZAP70+ CLL cells. SLP76 was found to be functionally active in CLL cells, as it becomes phosphorylated in response to BCR engagement in a time dependent manner. Activation with anti-IgM antibody results in phosphorylation of SLP76 on the positive regulatory tyrosine residue Y128 residue with increased physical association with Btk, peaking after 15 minutes. The negative regulatory residue S376 became phosphorylated only after 45', concomitantly with downregulation of the tyrosine phosphorylation. SLP76 phosphorylation in response to BCR engagement did not correlate with total ZAP70 expression. Pre-incubation of CLL cells with the LCK kinase inhibitor LCKi and with the SYK inhibitor R406, inhibited SLP76 phosphorylation in response to BCR activation, while the BTK inhibitor-ibrutinib had no effect. These suggest that LCK and SYK, but not ZAP70, play a central role in the upstream signaling involved in SLP76 activation in CLL cells. Knockdown of SLP76 in CLL cells resulted in decreased induction of BTK and PLCγ2 phosphorylation after BCR activation with anti-IgM. Consistent with our findings that SLP76 in involved in BCR signaling in CLL cells, we found that high total SLP76 expression was associated with a shorter time to first progression or need for treatment. In conclusion, SLP76 is ectopically expressed in CLL cells and plays a role in BCR signaling in those cells. DisclosuresNo relevant conflicts of interest to declare.

Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells

The protein tyrosine kinase LCK plays a key role in TCR signaling, and its activity is dynamically controlled by the tyrosine kinase C-terminal Src kinase (CSK) and the tyrosine phosphatase CD45. CSK is brought in contiguity to LCK via binding to a transmembrane adaptor known as phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG). The lack of a blatant phenotype in PAG-deficient mice has impeded our understanding of the mechanisms through which PAG exerts its negative-regulatory role in TCR signaling. We used quantitative mass spectrometry and both thymocytes and CD4(+) T cells from mice in which a tag for affinity purification was knocked in the gene coding for PAG to determine the composition and dynamics of the multiprotein complexes that are found around PAG over 5 min of activation. Most of the high-confidence interactions that we observed were previously unknown. Using phosphoproteomic analysis, PAG showed low levels of tyrosine phosphorylation in resting primary mouse CD4(+) T cells; the levels of tyrosine phosphorylation increased and reached a maximum 2 min after stimulation. Analysis of the dynamics of association of the protein tyrosine phosphatase PTPN22 and lipid phosphatase SHIP-1 with PAG following T cell activation suggests that both cooperate with CSK to terminate T cell activation. Our findings provide a model of the role for PAG in mouse primary CD4(+) T cells that is consistent with recent phosphoproteomic studies of the Jurkat T cell line but difficult to reconcile with former biochemical studies indicating that PAG is constitutively phosphorylated in resting T cells and rapidly dephosphorylated once the TCR is engaged.

Pyrazolopyrimidine Derivatives as Antineoplastic Agents: with a Special Focus on Thyroid Cancer.

Tyrosine kinase inhibitors (TKIs) are molecules that compete with ATP on tyrosine kinase receptors (TKRs), blocking tyrosine kinase (TK) activation and then oncogenic pathways; they have been studied, and some of them are right approved for the treatment of many types of cancer. Among TKIs, one of the most explored chemical template is the pyrazolo[3,4-d]pyrimidine (PP) heterocyclic core, which proved to be a useful scaffold for the obtainment of effective compounds. Actually, derivatives belonging to this structural class show a large spectrum of activity, thus standing out as multi-target agents. Different PP compounds have been shown to act as: a) ABL inhibitors and antiproliferative agents against human leukemia cell lines; b) Src kinase inhibitors in neuroblastoma, medulloblastoma and osteosarcoma; c) Phospholipase D inhibitors in different neoplasias; d) Urokinase plasminogen activator inhibitors, in breast cancer. In thyroid cancer (TC), PP1 and PP2 (inhibitors of RET, Hck, lck, and fynT kinases, and a good inhibitor of c-Src and platelet-derived growth factor receptor) showed antineoplastic actvity in human papillary TC cell lines that carry spontaneous RET/PTC1 rearrangements. More recently, new derivatives, (R)-1-phenethyl-N-(1-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4- amine, namely, CLM3 and CLM29, have been demonstrated to exert a multiple signal transduction inhibition (including the RET-TK, BRAF, EGFR, and with antiangiogenic activity), showing antineoplastic activity, in vitro and in vivo, in papillary dedifferentiated, medullary and anaplastic TC. These data have shown the antineoplastic activity of PP in different neoplasias, opening the way to a future clinical evaluation in human cancers.

The phosphoproteome of human Jurkat T cell clones upon costimulation with anti-CD3/anti-CD28 antibodies

Phosphorylation is a reversible post-translational modification, playing a vital role in protein function. In T cells, protein phosphorylation is the key mechanism regulating T cell receptor-driven signaling pathways. In order to gain insights into the phosphoproteome evolution of T cell activation, we performed a large-scale quantitative phosphoproteomics study of Jurkat E6.1 (wild type) and Jurkat gamma1 (Phospholipase gamma1 null) cell clones upon costimulation with anti-CD3 and anti-CD28 antibodies at times ranging from 15min to as long as 120min. In total, we identified 5585 phosphopeptides belonging to 2008 phosphoproteins from both cell clones. We detected 130 and 114 novel phosphopeptides in Jurkat E6.1 and Jurkat gamma1 clones, respectively. A significantly lower number of proteins containing regulated phosphorylation sites were identified in Jurkat gamma1 in comparison to Jurkat E6.1, reflecting the vital role of Phospholipase gamma1 in T cell signaling. Several new phosphorylation sites from lymphocyte-specific protein tyrosine kinase (Lck) were identified. Of these, serine-121 showed significant changes in JE6.1 while only small changes in the Jgamma1 clone. Our data may contribute to the current human T cell phosphoproteome and provide a better understanding on T cell receptor signaling. Data are available via ProteomeXchange with identifier PXD002871.

TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

Lymphocyte phosphatase‐associated phosphoprotein proteoforms analyzed using monoclonal antibodies

Phosphatase CD45 regulates the activation of lymphocytes by controlling the level of receptor and signal molecule phosphorylation. However, it remains unknown which molecules mediate the phosphatase activity of CD45. A candidate for such a molecule is a small transmembrane adapter protein called lymphocyte phosphatase-associated phosphoprotein (LPAP). LPAP forms a supramolecular complex that consists of not only CD45 molecule but also CD4 and Lck kinase. The function of LPAP has not been defined clearly. In our study, we determined the pattern of LPAP expression in various cell types and characterized its proteoforms using new monoclonal antibodies generated against the intracellular portion of the protein. We show that LPAP is a pan-lymphocyte marker, and its expression in cells correlates with the expression of CD45. The majority of T, B and NK cells express high levels of LPAP, whereas monocytes, granulocytes, monocyte-derived dendritic cells, platelets and red blood cells are negative for LPAP. Using one- and two-dimensional protein gel electrophoresis, we demonstrate that LPAP has at least four sites of phosphorylation. The resting cells express at least six different LPAP phosphoforms representing mono-, di- and tri-phosphorylated LPAP. T and B cells differ in the distribution of the protein between phosphoforms. The activation of lymphocytes with PMA reduces the diversity of phosphorylated forms. Our experiments on Lck-deficient Jurkat cells show that Lck kinase is not involved in LPAP phosphorylation. Thus, LPAP is a dynamically phosphorylated protein, the function of which can be understood, when all phosphosites and kinases involved in its phosphorylation will be identified.

Conserved Motifs within Hepatitis C Virus Envelope (E2) RNA and Protein Independently Inhibit T Cell Activation.

T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp’s contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence.

Rapid and transient palmitoylation of the tyrosine kinase Lck mediates Fas signaling

Palmitoylation is the posttranslational modification of proteins with a 16-carbon fatty acid chain through a labile thioester bond. The reversibility of protein palmitoylation and its profound effect on protein function suggest that this modification could play an important role as an intracellular signaling mechanism. Evidence that palmitoylation of proteins occurs with the kinetics required for signal transduction is not clear, however. Here we show that engagement of the Fas receptor by its ligand leads to an extremely rapid and transient increase in palmitoylation levels of the tyrosine kinase Lck. Lck palmitoylation kinetics are consistent with the activation of downstream signaling proteins, such as Zap70 and PLC-γ1. Inhibiting Lck palmitoylation not only disrupts proximal Fas signaling events, but also renders cells resistant to Fas-mediated apoptosis. Knockdown of the palmitoyl acyl transferase DHHC21 eliminates activation of Lck and downstream signaling after Fas receptor stimulation. Our findings demonstrate highly dynamic Lck palmitoylation kinetics that are essential for signaling downstream of the Fas receptor.

T cell receptor dwell times control the kinase activity of Zap70.

Kinase recruitment to membrane receptors is essential for signal transduction. However, the underlying regulatory mechanisms are poorly understood. We investigated how conformational changes control T cell receptor (TCR) association and activity of the kinase Zap70. Structural analysis showed that TCR binding or phosphorylation of Zap70 triggers a transition from a closed, autoinhibited conformation to an open conformation. Using Zap70 mutants with defined conformations, we found that TCR dwell times controlled Zap70 activity. The closed conformation minimized TCR dwell times and thereby prevented activation by membrane-associated kinases. Parallel recruitment of coreceptor-associated Lck kinase to the TCR ensured Zap70 phosphorylation and stabilized Zap70 TCR binding. Our study suggests that the dynamics of cytosolic enzyme recruitment to the plasma membrane regulate the activity and function of receptors lacking intrinsic catalytic activity.

Abstract 3041: Lymphocyte-specific protein tyrosine kinase (Lck) interacts with CR6-interacting factor 1 (CRIF1) in mitochondria to repress oxidative phosphorylation

Abstract The purpose of our study is to determine whether and how lymphocyte-specific protein tyrosine kinase (Lck) may exhibit novel functions in cancer cell mitochondria. Mitochondria are responsible for producing cellular energy via oxidative phosphorylation (OXPHOS) of glucose and fatty acids. Compared to normal differentiated cells, cancer cells rely more on aerobic glycosis than mitochondrial OXPHOS. This shift in energy metabolism allows more resources to be converted into biomass for cancer cell's uncontrolled growth and proliferation. Numerous mechanisms contribute to altered mitochondrial activity, including phosphorylation of mitochondrial proteins. Recent studies showed that cytoplasmic protein tyrosine kinases (PTK) also translocate to the mitochondria and regulate mitochondrial functions. It represents an important non-canonical pathway of how PTK modulates mitochondrial activity. As a Src-family PTK, Lck is essential for T cell activation by transmitting signals downstream of cell surface receptors. Overexpression and aberrant activity of Lck have been reported in both blood malignancies and solid tumors. We showed previously that Lck exhibited additional oncogenic property when translocated to the nucleus (BBRC 417:1058). To explore the possibility that Lck may also translocate to mitochondria, we examined human Jurkat T cell leukemia and its Lck-deficient derivative Jcam cell line. Subcellular fractionation and microscopy both confirmed the presence of Lck in Jurkat mitochondria. Compared to Jcam, Jurkat cells exhibit high levels of mitochondrial protein tyrosine phosphorylation, disrupted mitochondrial morphology, and reduced OXPHOS. Subsequent mass spectrometry and in situ proximity ligation assay identified CR6-interacting factor 1 (CRIF1) as a novel Lck-interacting partner in mitochondria. CRIF1 interacts with mitoribosome and Tid1 to facilitate translation and membrane insertion of mitochondrion-encoded proteins, respectively. Compared to Jcam, Jurkat cells express less mitochondrial proteins and show reduced CRIF1-Tid1 interaction. Knocking down CRIF1 in Jcam also results in decreased mitochondrial protein level and mitochondrial dysfunction. Interestingly, CRIF1 is not phosphorylated on tyrosine by Lck, suggesting the presence of other Lck substrates in mitochondria. All together, these results support the role of mitochondrial Lck as a negative regulator of CRIF1 through competitive binding and independent of its kinase activity. Disrupted mitochondrial protein translation and membrane insertion then lead to reduced OXPHOS and abnormal morphology. These findings raise the possibility of targeting mitochondrial Lck to redirect the metabolic shift in cancer cells. Citation Format: Shahrooz Vahedi, Fu-Yu Chueh, Chao-Lan Yu. Lymphocyte-specific protein tyrosine kinase (Lck) interacts with CR6-interacting factor 1 (CRIF1) in mitochondria to repress oxidative phosphorylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3041. doi:10.1158/1538-7445.AM2015-3041

Abstract 698: CPI-based Crizotinib systemic study on discovery of phenotypic off-target

Abstract Crizotinib, a first ever ALK/MET multi-targeted receptor tyrosine kinase inhibitor for the treatment of ALK-rearranged NSCLC patients. We hypothesize that crizotinib acts on other molecular targets in addition to tyrosine kinases ALK/MET and may play critical role given that there is a complex network of kinases that work together to regulate a number of important cellular processes and different disease signature. Approaching with a comprehensive docking method with our established chemical-protein interactome (CPI) and crizotinib, we have discovered 301 PDB-deposited proteins corresponding to 353 ligand binding pockets among a total of 1,780 PDB-deposited human protein entries. Systemic pharmacology approach also applied including validation the molecular target(s) of TKIs in vitro. Especially multiple human cellular models (including NSCLC and melanoma) and signaling study, luc-reporter, autophagy/apoptosis, proteomics study and NGS analysis applied. Interestingly, crizotinib besides had a high CPI binding score (ZZ_score) of -2.2778 against tyrosine kinase Lck, also had -1.4672, -1.1242, -0.7033 and -1.5384 against MAPK3, S6K, GSK-3β and HDAC 7A, respectively, which indicated crizotinib had potential broad range interaction with both TK and ser/thr kinases. Our data have showed that the crizotinib induced autophagy by activated LC3 in vitro and inhibited oncogenic Akt/mTOR signaling in human multiple melanoma cells resulting in G2/M cell cycle arrest. Furthermore, crizotinib increased p21Waf1/cip1 and Foxo3A expression and tumor suppressor miR-146a in human melanoma cells. Taken together, crizotinib induces miR-146a, autophagy and p21waf1/cip1 may potentially treat other type of human cancer by concurrently targeting tyrosine kinase, Akt/mTOR/Foxo axis and nuclear receptor, shedding a light for future both anti-cancer and anti-metabolic disorder drug discovery and development. This is the first CPI-based systemic study on leading discovery of crizotinib off-target phenotypic importance. These findings suggest that non-tyrosine kinase target of crizotinib may play critical role(s) for drug repositioning may represent a new and effective approach to expanding the application of existing drugs. Citation Format: Jiazhi Sun, Lun Yang, Minghua Li, Shimin Zhang, Ian E. Heller, Johnathan Samuelson, Steven Cindric, Kunal Mishra, Eslam Mohamed, Feng Cheng, Kevin B. Sneed, Lin He, Shu-Feng Zhou. CPI-based Crizotinib systemic study on discovery of phenotypic off-target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 698. doi:10.1158/1538-7445.AM2015-698

Impact of Exogenous Galectin-9 on Human T Cells: CONTRIBUTION OF THE T CELL RECEPTOR COMPLEX TO ANTIGEN-INDEPENDENT ACTIVATION BUT NOT TO APOPTOSIS INDUCTION

Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca(2+)) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca(2+) mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca(2+) mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4(+) T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca(2+) mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4(+) T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex.

Vav1 Regulates T-Cell Activation through a Feedback Mechanism and Crosstalk between the T-Cell Receptor and CD28.

Vav1, a Rac/Rho guanine nucleotide exchange factor and a critical component of the T-cell receptor (TCR) signaling cascade is tyrosine phosphorylated rapidly in response to T-cell activation. Vav1 has established roles in proliferation, cytokine secretion, Ca(2+) responses, and actin cytoskeleton regulation; however, its function in the regulation of phosphorylation of TCR components, including the ζ chain, the CD3 δ, ε, γ chains, and the associated kinases Lck and ZAP-70, is not well established. To obtain a more comprehensive picture of the role of Vav1 in receptor proximal signaling, we performed a wide-scale characterization of Vav1-dependent tyrosine phosphorylation events using quantitative phosphoproteomic analysis of Vav1-deficient T cells across a time course of TCR stimulation. Importantly, this study revealed a new function for Vav1 in the negative feedback regulation of the phosphorylation of immunoreceptor tyrosine-based activation motifs within the ζ chains, CD3 δ, ε, γ chains, as well as activation sites on the critical T cell tyrosine kinases Itk, Lck, and ZAP-70. Our study also uncovered a previously unappreciated role for Vav1 in crosstalk between the CD28 and TCR signaling pathways.

Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells.

Overexpression and hyperactivation of lymphocyte-specific protein tyrosine kinase (Lck) have been associated with leukemia development. We previously showed that, other than its known function as a cytoplasmic signal transducer, Lck also acts as a nuclear transcription factor in mouse leukemic cells. In the present study, we demonstrated the presence of nuclear Lck in human leukemic T cells and in primary cells. We further established a positive correlation between Lck nuclear localization and its kinase activity. Proteomic analysis identified CR6-interacting factor 1 (CRIF1) as one of the Lck-interacting proteins. CRIF1 and Lck association in the nucleus was confirmed both by immunofluorescence microscopy and co-immunoprecipitation in human leukemic T cells. Close-range interaction between Lck and CRIF1 was validated by in situ proximity ligation assay (PLA). Consistent with the role of nuclear CRIF1 as a tumor suppressor, CRIF1 silencing promotes leukemic T cell survival in the absence of growth factors. This protective effect can be recapitulated by endogenous Lck or reconstituted Lck in leukemic T cells. All together, our results support a novel function of nuclear Lck in promoting human leukemic T cell survival through interaction with a tumor suppressor. It has important implications in defining a paradigm shift of non-canonical protein tyrosine kinase signaling.

The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway.

T cell activation by antigens binding to the T cell receptor (TCR) must be properly regulated to ensure normal T cell development and effective immune responses to pathogens and transformed cells while avoiding autoimmunity. The Src family kinase Lck and the Syk family kinase ZAP-70 (ζ chain-associated protein kinase of 70 kD) are sequentially activated in response to TCR engagement and serve as critical components of the TCR signaling machinery that leads to T cell activation. We performed a mass spectrometry-based phosphoproteomic study comparing the quantitative differences in the temporal dynamics of phosphorylation in stimulated and unstimulated T cells with or without inhibition of ZAP-70 catalytic activity. The data indicated that the kinase activity of ZAP-70 stimulates negative feedback pathways that target Lck and thereby modulate the phosphorylation patterns of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ chain components of the TCR and of signaling molecules downstream of Lck, including ZAP-70. We developed a computational model that provides a mechanistic explanation for the experimental findings on ITAM phosphorylation in wild-type cells, ZAP-70-deficient cells, and cells with inhibited ZAP-70 catalytic activity. This model incorporated negative feedback regulation of Lck activity by the kinase activity of ZAP-70 and predicted the order in which tyrosines in the ITAMs of TCR ζ chains must be phosphorylated to be consistent with the experimental data.

PTPROt maintains T cell immunity in the microenvironment of hepatocellular carcinom (TUM9P.1002)

Abstract Background & Aims: Intratumoral T cells play a central role in anti-tumor immunity, and the balance between T effector cells (Teff) and regulatory T cells (Treg) affects the prognosis of cancer patients. However, educated by tumor microenvironment, T cells frequently fail in their responsibility. In this study, we aimed to investigate the role of truncated isoform of protein tyrosine phosphatase receptor - type O (PTPROt) in T cell mediated anti-tumor immunity. Methods: We recruited 70 hepatocellular carcinoma (HCC) patients and 30 healthy volunteers for clinical investigation, and analyzed cellular tumor immunity by using ptpro-/- C57BL/6 mice and NOD/SCID mice. Results: PTPROt expression was significantly down-regulated in human HCC infiltrating T cells due to the hypoxia microenvironment (***P<0.001); PTPROt expression highly correlated with the intratumoral Teff/Treg ratio (**P<0.01) and clinicopathologic characteristics (*P<0.05). Moreover, PTPROt deficiency attenuated T cell mediated anti-tumor immunity and remarkably promoted mouse HCC growth (***P<0.001). Mechanistically, deletion of PTPROt decreased Teff quantity and quality through phosphorylation of lymphocyte specific tyrosine kinase (Lck), but increased Treg differentiation through phosphorylation of signal transducer and activator of transcription (STAT) 5. Conclusions: In support of the Teff/Treg homeostasis, PTPROt serves as an important tumor suppressor in HCC microenvironment.

Reconstitution of TCR/CD3 expression in B cell lymphoma to dissect the contribution of CD3 conformational change from downstream signaling events in the process of TCR/CD3 downregulation. (IRM7P.714)

Abstract T cell receptor (TCR) stimulation induces conformational change in CD3 (CD3Δc), followed by phosphorylation of ITAMs that leads to receptor downregulation among other T cell responses. To begin to dissect the contribution of CD3Δc from downstream signal transduction mechanisms associated with these events, we have transduced the mouse B cell lymphoma X63.Ag8.653, which lacks expression of the src-family kinase Lck required for proper ITAM phosphorylation, with the OT-I TCR chains Vα2 and Vβ5, together with all CD3 subunits and the coreceptor CD8αβ, generating the cell line X63.OT1.CD8. Stimulation of the OT-I TCR with anti-CD3 antibody or the cognate antigen SIINFEKL-H2Kb induces CD3Δc in this cell line. As expected by the absence of Lck, CD3 ITAM phosphorylation was undetectable in our cell line and no calcium mobilization was found upon antibody stimulation. Unexpectedly, stimulation with either SIINFEKL-H2Kb or anti-CD3 did induce TCR/CD3 downregulation in our cells. These data suggest CD3ε PRS exposure via CD3Δc might be sufficient to transduce some signals that contribute to TCR downregulation in the absence of CD3 ITAM phosphorylation.