Lck Kinase Activity Research Articles

Targeting Hyperactivated Lck in PAX5 Rearranged Pediatric B-Cell Precursors Acute Lymphoblastic Leukemia

Despite intensive risk-based treatment protocols, 15% of pediatric patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) experience relapse. There is urgent need of novel strategies to target poor prognosis subgroups. We considered 135 B-other cases consecutively enrolled in Italy in the AIEOP-BFM ALL2000/R2006 protocols without recurrent genetic rearrangements and excluding Down syndrome and High Hyper Diploid cases. By integrating gene expression, copy number analyses and fusion genes discovery by target-capture NGS, we identified 59 patients (43·7%) having a Ph-like signature; in addition to ERG-related (26%), High Hyperdiploid-like (17%), ETV6::RUNX1-like (8·9%), MEF2D-rearranged (2·2%) or KMT2A-like (1·5%). CRLF2 rearrangements were almost exclusively found in the Ph-like group, and also the IKZF1-plus profile was prevalent in Ph-like cases (8/9) and associated with a poor outcome (5-year EFS (SE) 37·5% (17·1) vs. 65·5% (7·0) for non-IKZF1-plus cases). Interestingly, PAX5 gene lesions were mostly associated with the Ph-like signature, with CNAs and translocations in 43/131 B-other patients (32·8%), with 31/57 (54·3%) in Ph-like and 12/74 (16%) in non-Ph-like cases. Notably, all the 7 PAX5t were found in the Ph-like group. EFS (p<0·001) and OS (p=0·009) analyses according to the fusion classes reveal statistical significance in Ph-like cases, classified accordingly to different class fusions, such as ABL/JAK-class, PAX5-class, other fusions and negative cases after NGS analysis. In fact, the PAX5t cases have a poor EFS, similar to ABL/JAK-class patients (53% and 50%, respectively) and lower than Ph-like cases negative for any fusion gene (78%). Moreover, PAX5t OS was inferior compared to ABL/JAK-class and negative cases (about 53% vs. 83·3% and 88·9%). These features of PAX5t cases prompted us to explore the feasibility of targeting this aberration. By applying the NetBID2 data-driven network interference algorithms, we constructed the BCP-ALL interactome and identified the hub drivers' activity (DA) based on network gene interaction on either PAX5 translocated patients versus all BCP-ALL excluding Ph-like or versus Ph-like. We also assessed a Differential Expression (DE) of genes belonging to these pathways. Interestingly, in the top NetBID2 drivers we identified the LCK signaling factor showing a significantly higher activity in PAX5t compared to other BCP-ALL subgroups. Furthermore, analysis of LCK activity in BCP-ALL patients with known PAX5 status revealed that it is an exclusive signature of PAX5t cases. Moreover, 22 genes resulted with a positive driver activity in PAX5t vs. Ph-like (p<0.05) and bioinformatic analysis to identify drug approved interactions found 7 targetable genes, including LCK. As previously showed by us, we demonstrated the efficacy of the LCK-inhibitor Nintedanib, as single agent or in combination with conventional chemotherapy, both ex-vivo and in a patient-derived xenograft model, showing a synergistic effect with dexamethasone. We herewith applied a wider ex-vivo drug screening with 174 FDA-approved drugs, by comparative cellular viability of different sub-groups of leukemia PAX5t samples and control-B-lymphoblastoid cell lines (CTR-B-LCSs) in addition to NALL-1 cell lines (carrying PAX5/ETV6 fusion), measured by ATP-Glo based luminescent based assay after exposure of the depicted drugs, whereas Staurosporin was taken as a positive control. Drug sensitivity scores (DSS) are plotted as a clustered heat map, followed by unsupervised hierarchical clustering. From the drug screening, we selected Dasatinib, Bosutinib and Foretinib, known to be among the top 10 most potent LCK ligands. Here we demonstrated their efficacy in PAX5t cases by evaluating the differential DSS, i.e., by avoiding toxicity in CTR-B-LCSs. This study provides new insights in the pathogenic mechanisms of poor risk Ph-like leukemia and identifies a potential novel therapy for targeting the PAX5t group. Moreover, in general this study opens the scenario to target LCK kinase activity in further BCP-ALL subgroups.

Enhanced cytotoxic CD8+ T cell (CTL) antigen recognition by LCK activation

Abstract The anti-tumor response of cytotoxic T cells requires detection of tumor-derived antigenic peptide-MHC complex (pMHC) by the T cell antigen receptor (TCR). Strategies that boost TCR signaling caused by weak pMHC could augment the recognition of poorly immunogenic tumors by T cells. Because LCK kinase phosphorylates the TCR complex and the recruited ZAP-70 kinase, increased LCK activity has been shown to increase TCR signaling in naïve T cells. Our previous study demonstrated that a small molecule inhibitor of CSK kinase, a negative regulator of LCK, increased LCK kinase activity and sensitized naïve T cells such that they became activated by weak antigens ex vivo. Our goal was to determine whether CD8+ cytotoxic T cells (CTL) could also be sensitized to detect weak antigens, and evaluate whether their capacity to recognize and kill cancer cells could be augmented. Because the Nur77-GFP reporter system responds to TCR-mediated signaling, we used it to determine that LCK activation sensitized OT-1 TCR transgenic CTLs to detect weak (low affinity) antigens displayed by splenocytes. We also observed increased phosphorylation of ZAP-70 and other signaling mediators upon TCR stimulation. We will next evaluate whether CTL-mediated effector functions can be enhanced by monitoring killing of MC38 cancer cells that display a weak antigen. Our findings will inform whether LCK kinase can be targeted to improve the recognition of cancer by T cells.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

Abstract 2564: Integrated transcriptomic, proteomic, and metabolomic analyses of human and mouse T cell leukemia

Abstract Nuclear and mitochondrial crosstalk plays an important role in regulating diverse cellular functions. It is contributed partly by non-canonical signaling in these two key subcellular compartments. Disruption of this signaling network has been shown to associate with oncogenic transformation. Our lab has long-term interests in studying the molecular mechanisms of cellular transformation by the oncogenic lymphocyte-specific protein tyrosine kinase (Lck). Previous reports from our lab show novel roles of Lck in both nucleus and mitochondrion of human and mouse T leukemic cell lines that exhibit metabolic reprogramming. However, it remains largely unclear how dysregulated nuclear and mitochondrial crosstalk leads to altered cellular metabolism in Lck-transformed leukemic cells. The purpose of our study is to combine transcriptomic, proteomic, and metabolomic approaches to establish a link between altered nuclear gene expression, mitochondrial activity, and metabolic shift in Lck-transformed leukemic cells. For human T cell leukemia, we compared Jurkat T cells with its Lck-deficient derivative Jcam cell line. For mouse T cell leukemia, we compared EL4 cells that express normal levels of Lck and LSTRA cells that overexpress active Lck kinase. For additional verification, Jcam cells were reconstituted with wild-type Lck and EL4 cells were transfected with constitutively-active Lck mutant kinase. Total RNAs were subjected to transcriptomic analysis to identify genes significantly upregulated or downregulated in Lck-transformed cells. Tyrosine-phosphorylated proteins were enriched from Lck-transformed cells and identified by LC-MS/MS. Hydrophilic metabolites were extracted from whole cells and subjected to targeted metabolomic analysis using LC-MS/MS. Pathway analysis of our transcriptomic data revealed significant changes of gene expression involved in several pathways, including cell adhesion and glycolysis pathways. We verified several glycolytic genes downregulated in Lck-transformed cells. These targets were further confirmed by reduced levels of corresponding metabolites. Proteomic analysis identified a set of novel target proteins potentially phosphorylated by Lck. Cyclin-dependent kinase 1 (CDK1) is of particular interest because its phosphorylation status correlates with its activity in regulating electron transport chain functions within mitochondrion. Our data suggest a link between tyrosine-phosphorylated CDK1 and reduced mitochondrial respiration. In conclusion, integrated transcriptomic, proteomic, and metabolomic analyses enabled us to identify novel target genes regulated, proteins phosphorylated, and metabolites altered in Lck-transformed leukemic cells. Collectively, our data demonstrate a unique pattern of metabolic reprogramming both similar and distinct from the well-known Warburg effect in cancer cells. Citation Format: Ching-Yu Chu, Szu-Yuan Chen, Fu-Yu Chueh, Mei-Ling Cheng, Chao-Lan Yu. Integrated transcriptomic, proteomic, and metabolomic analyses of human and mouse T cell leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2564.

In Silico and Ex Vivo Drug Screening Identifies Dasatinib as a Potential Targeted Therapy for T-ALL

IntroductionT cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy affecting 10-15% of pediatric ALL patients. Current cure rates of pediatric patients is 80% but increasing understanding of the molecular mechanisms of T-ALL provides possibilities for more effective and targeted therapies. During the last decade targeted therapies with tyrosine kinase inhibitors (TKI) have proven to be effective in BCR-ABL1 fusion positive leukemias. Several studies have suggested TKI dasatinib to be effective also in the treatment of NUP214-ABL1 positive T-ALLs, which comprise approximately 4-10% of the T-ALL cases.Materials and MethodsIn silico drug screening was performed by comparing gene expression profiles of 4769 leukemic samples to a library of 13384 compounds and their known targets from the Drug signature database (DsigDB). These compounds included FDA approved therapeutic molecules and molecules under studies. Findings were validated in an ex vivo drug screen, consisting of 20 T-ALL bone marrow samples and 9 healthy bone marrow controls. Samples were treated for 72 hours with five different concentrations of dasatinib in 10-fold dilutions (0.1-1000nM), cell viability was measured and the data normalized to negative (DMSO) and positive (benzethonium chloride) controls. The effect of dasatinib was further explored in vitro by treating one NUP214-ABL1 fusion positive and six fusion negative T-ALL cell lines with dasatinib (1-1000nM). Gene expression levels of the known dasatinib targets in these cell lines were measured by Global Run On sequencing (GRO-seq) assay and qRT-PCR.ResultsIn order to find novel targeted therapies for T-ALL, we performed an in silico drug target screen. A dasatinib-targetable gene LCK was strongly expressed in a number of T-ALL cases whereas normal T-lymphoid cells had lower expression. Chemical screen data of the target specificity of dasatinib showed high inhibition of LCK with percent of control (POC) value of 1, meaning that 0.1µM concentration of dasatinib decreases the kinase activity of LCK to 1% in comparison to control. In vitro dasatinib decreased cell viability in fusion negative Jurkat and MOLT-16 cells, and also in fusion positive cell line Peer. GRO-seq and qRT-PCR confirmed the expression of LCK and several other known dasatinib targets, including other SRC family kinases, in Jurkat and MOLT-16 cell lines. However, some LCK-expressing T-ALL cell lines were less sensitive to dasatinib. In further validation, ex vivo drug testing of patient samples revealed a marked response in 6/20 patient samples with IC50 values ranging between 1.3 - 8.2nM, while in healthy bone marrow controls IC50 values were >1000nM.ConclusionOur in silico drug screen identified dasatinib as a potential targeted therapy for a subgroup of T-ALL cases, and this finding was further supported by both ex vivo and in vitro studies. The exact mechanism remains to be elucidated but a number of SRC family kinases, which could potentially be targeted by dasatinib, showed expression in T-ALL samples. DisclosuresHeckman:Celgene: Research Funding; Pfizer: Research Funding.

TCR Triggering Induces the Formation of Lck-RACK1-Actinin-1 Multiprotein Network Affecting Lck Redistribution.

The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1–Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 s after TCR–CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck, which co-purifies with high molecular weight cellular fractions. The formation of RACK1–Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1–pY394Lck–α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1–Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4+ T-cells by an adenoviral-mediated knockdown of RACK1. These results demonstrate that in T cells, RACK1, as an essential component of the multiprotein complex which upon TCR engagement, links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck.

The kinase Itk and the adaptor TSAd change the specificity of the kinase Lck in T cells by promoting the phosphorylation of Tyr192.

The substrate specificity of Src family kinases (SFKs) is partly determined by their Src homology 2 (SH2) domains. Thus, transient alterations in the SH2 domain of SFKs might change their binding partners and affect intracellular signaling pathways. Lck is an SFK that is central to the initiation of T cell activation in response to ligand binding to the T cell receptor (TCR) and is also critical for later signaling processes. The kinase activity of Lck requires both the phosphorylation of an activating tyrosine residue and the dephosphorylation of an inhibitory tyrosine residue. We found that a third conserved tyrosine phosphorylation site (Tyr(192)) within the SH2 domain of Lck was required for proper T cell activation and formation of cell-cell conjugates between T cells and antigen-presenting cells. Through phosphopeptide arrays and biochemical assays, we identified several regulators of the actin cytoskeleton that preferentially bound to Lck phosphorylated at Tyr(192) compared to Lck that was not phosphorylated at this site. Two of these phosphorylation-dependent binding partners, the kinase Itk (interleukin-2-inducible Tec kinase) and the adaptor protein TSAd (T cell-specific adaptor), promoted the TCR-dependent phosphorylation of Lck at Tyr(192). Our data suggest that phosphorylation transiently alters SH2 domain specificity and provide a potential mechanism whereby SFKs may be rewired from one signaling program to another to enable appropriate cell activation.

OP0176 S100a4 Regulates the Src-Thyrosine Kinase Dependent Differentiation of TH17 Cells in Rheumatoid Arthritis

BackgroundA calcium-binding protein S100A4 regulates non-muscle myosin assembly and activity and plays important part in cell motility and differentiation. The binding of S100A4 to calcium drives conformation changes and permits...

SAP facilitates recruitment and activation of LCK at NTB-A receptors during restimulation-induced cell death. (IRM10P.734)

Abstract Upon TCR restimulation, activated, cycling T cells can undergo a self-regulatory form of apoptosis known as restimulation-induced cell death (RICD). We previously demonstrated that RICD is impaired in T cells from patients with X-linked lymphoproliferative disease (XLP), which lack SLAM-associated protein (SAP) expression. Both SAP and the specific SLAM receptor NK, T, and B cell antigen (NTB-A) are required for RICD, but the mechanism by which these molecules promote a strong, pro-apoptotic signal through the TCR remains unclear. Here we show that the Src-family kinase LCK, but not FYN, associates with NTB-A in activated human T cells. This association increased after TCR restimulation in a SAP-dependent manner, requiring both immunoreceptor tyrosine-based switch motifs (ITSMs) in the NTB-A cytoplasmic tail. Both NTB-A-associated LCK phosphorylation and kinase activity were enhanced in restimulated T cells, amplifying proximal TCR signaling. In contrast, TCR-induced LCK association with NTB-A, as well as phosphorylation and kinase activity, were reduced in XLP patient T cells or normal T cells transfected with SAP-specific siRNA, consistent with RICD resistance. Collectively, our data reveal how SAP nucleates a previously unknown signaling complex involving NTB-A and LCK to potentiate RICD of activated human T cells.

SAP Facilitates Recruitment and Activation of LCK at NTB-A Receptors during Restimulation-Induced Cell Death

Upon TCR restimulation, activated, cycling T cells can undergo a self-regulatory form of apoptosis known as restimulation-induced cell death (RICD). We previously demonstrated that RICD is impaired in T cells from patients with X-linked lymphoproliferative disease, which lack SLAM-associated protein (SAP) expression. Both SAP and the specific SLAM receptor NK, T, and B cell Ag (NTB-A) are required for RICD, but the mechanism by which these molecules promote a strong, proapoptotic signal through the TCR remains unclear. In this article, we show that the Src-family kinase LCK, but not FYN, associates with NTB-A in activated human T cells. This association increased after TCR restimulation in a SAP-dependent manner, requiring both immunoreceptor tyrosine-based switch motifs in the NTB-A cytoplasmic tail. Both NTB-A-associated LCK phosphorylation and kinase activity were enhanced in restimulated T cells, amplifying proximal TCR signaling. In contrast, TCR-induced LCK association with NTB-A, as well as phosphorylation and kinase activity, was reduced in T cells from patients with X-linked lymphoproliferative disease or normal T cells transfected with SAP-specific small interfering RNA, consistent with RICD resistance. Collectively, our data reveal how SAP nucleates a previously unknown signaling complex involving NTB-A and LCK to potentiate RICD of activated human T cells.

Lymphocyte Cell Kinase Activation Mediates Neuroprotection during Ischemic Preconditioning

The molecular mechanisms underlying preconditioning (PC), a powerful endogenous neuroprotective phenomenon, remain to be fully elucidated. Once identified, these endogenous mechanisms could be manipulated for therapeutic gain. We investigated whether lymphocyte cell kinase (Lck), a member of the Src kinases family, mediates PC. We used both in vitro primary cortical neurons and in vivo mouse cerebral focal ischemia models of preconditioning, cellular injury, and neuroprotection. Genetically engineered mice deficient in Lck, gene silencing using siRNA, and pharmacological approaches were used. Cortical neurons preconditioned with sublethal exposure to NMDA or oxygen glucose deprivation (OGD) exhibited enhanced Lck kinase activity, and were resistant to injury on subsequent exposure to lethal levels of NMDA or OGD. Lck gene silencing using siRNA abolished tolerance against both stimuli. Lck-/- mice or neurons isolated from Lck-/- mice did not exhibit PC-induced tolerance. An Lck antagonist administered to wild-type mice significantly attenuated the neuroprotective effect of PC in the mouse focal ischemia model. Using pharmacological and gene silencing strategies, we also showed that PKCε is an upstream regulator of Lck, and Fyn is a downstream target of Lck. We have discovered that Lck plays an essential role in PC in both cellular and animal models of stroke. Our data also show that the PKCε-Lck-Fyn axis is a key mediator of PC. These findings provide new opportunities for stroke therapy development.

Abstract 1343: Tracking T cell leukemogenesis in mice with green fluorescence protein-labeled LSTRA leukemic cells

Abstract LSTRA leukemia was originally induced in a BALB/c mouse by the Moloney murine leukemia virus. The established LSTRA leukemic T cell line is transplantable in BALB/c mice and serves as an important animal model for leukemogenesis. Numerous studies using LSTRA cells have been reported in the field of cancer therapy, including immunotherapy and chemotherapy. Subsequent identification of a lymphocyte-specific protein tyrosine kinase gene Lck in LSTRA cells further highlights the LSTRA leukemia as a key model in studying hematologic malignancy induced by oncogenic Lck protein tyrosine kinase. While intraperitoneal inoculation of LSTRA cells reproducibly induces leukemic death in syngeneic BALB/c mice, very little is known on the details of its malignant progression. It also is not clear if LSTRA leukemia in mice resembles any specific T cell leukemia in humans. To better understand the process of LSTRA leukemia, we engineered LSTRA cells to stably express green fluorescence protein (GFP) by retroviral transduction. GFP-labeled LSTRA cells are easily distinguishable by flow cytometry, immunofluorescence microscopy, and immunoblotting. Biochemical characterization confirms that, like the parental LSTRA leukemia, GFP-tagged LSTRA cells express high levels of active Lck kinase. Consistent with our previous report (J Immunol 159:5206), GFP-tagged LSTRA cells also exhibit constitutive activation of signal transducer and activator of transcription 3 (STAT3) and STAT5. BALB/c mice receiving 10 million GFP-labeled LSTRA cells intraperitoneally show signs of leukemia, such as acute weight loss, abdominal swelling, hunched posture, and squinting eyes, within 1 week. All mice die within 10 days. Even intraperitoneal inoculation of 50 GFP-labeled LSTRA cells results in 40% lethality within a month. By tracking GFP, we were able to identify multi-organ metastasis of LSTRA leukemia, including bone marrow, spleen, and liver. Immunohistochemistry analysis of tissue sections further revealed a unique concentration of LSTRA cells in the red pulp of spleen. It is important to note that mild to moderate lymphocytic infiltration of the spleen red pulp has been reported in patients with T cell large granular lymphocyte (LGL) leukemia. It suggests that LSTRA leukemia may represent a murine model of distinct human T cell maliganacy, such as LGL leukemia. All together, we have shown here that the GFP-labeled LSTRA cell line is a powerful tool to monitor leukemic progression in immune-competent mice. It has important implications in utilizing LSTRA leukemia for future studies in cancer therapy. Experiments are also in progress to define the molecular mechanisms underlying LSTRA infiltration in the spleen red pulp. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1343. doi:1538-7445.AM2012-1343

Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

Abstract 2206: SOCS1 and SOCS3 attenuate Lck-mediated cellular transformation through distinct mechanisms

Abstract The suppressor of cytokine signaling (SOCS) proteins are negative feedback inhibitors of Janus kinase (JAK) – signal transducer and activator of transcription (STAT) signaling in response to cytokine stimulation. Among eight SOCS family members, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2-containing protein) are best characterized both structurally and functionally. We showed previously that CIS, SOCS1 and SOCS3 were not expressed in Lck-transformed cells (Cell Signal 18:851; Int J Oncol 36:1201). It suggests that defective negative feedback control may contribute to constitutive JAK-STAT5 activation in Lck-mediated oncogenesis (J Immunol 159:5206; Mol Cancer Res 4:39). These findings also raise the possibility that SOCS proteins may function as tumor suppressors. Indeed, by enforced SOCS1 and SOCS3 expression, we were able to reduce Lck-induced cellular transformation (Int J Oncol 36:1201). In this report, we further examine the molecular mechanisms underlying SOCS tumor-suppressing activity. Through comparative studies of SOCS1, SOCS2, SOCS3 and CIS, we demonstrate here two distinct properties of SOCS1 and SOCS3 toward oncogenic Lck kinase. First, SOCS1 has the highest affinity in binding to oncogenic Lck kinase. We identified the positive regulatory phospho-tyrosine 394 residue (Y394) in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These data suggest that SOCS1 may inhibit Lck kinase activity in a manner similar to JAK kinases. Second, only SOCS3 becomes phosphorylated in the presence of oncogenic Lck kinase. We identified the phosphorylation sites as Y204 and Y221 located in the C-terminal SOCS box of SOCS3. We further analyzed the contribution of SOCS3 tyrosine phosphorylation on its tumor suppressor activity. Preliminary studies suggested that SOCS3 phosphorylation on Y204 and Y221 might play an important role in blocking cell proliferation and cell cycle progression of Lck-transformed cells. SOCS3 tyrosine phosphorylation can lead to altered protein stability and crosstalk with other signal transduction pathways, such as MAPK signaling. Experiments are in progress to further define the molecular details of SOCS3 phosphorylation involved in tumor suppression. All together, these results provide important mechanistic insights into the distinct roles of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases. This work is supported in part by NIH grant CA107210. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2206. doi:10.1158/1538-7445.AM2011-2206

Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase

Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

CD48: A co-stimulatory receptor of immunity

The CD48 molecule is a glycosyl-phosphatidyl-inositol (GPI)-anchored cell-surface protein of the CD2 family of molecules. Originally described on virally-induced B cells, CD48 has been found on various hematopoietic cells, and its expression is regulated by viral and bacterial products and immune-associated proteins. CD48 binds CD2 and other molecules, yet its high-affinity ligand in both mouse and human systems is 2B4. Despite its lack of an intracellular domain, stimulation of CD48 induces rearrangement of signaling factors in lipid rafts, Lck-kinase activity, and tyrosine phosphorylation. As an adhesion and co-stimulatory molecule, CD48 induces numerous effects in B and T lymphocytes, natural killer cells, mast cells, and eosinophils. Some of these depend upon cell-cell interactions via 2B4-CD48 binding. The structural and phenotypic characteristics of CD48, and its role in physiological and pathophysiological processes, are reviewed herein. Possible CD48-based applications for immune-impaired and inflammatory disorders are discussed as well.

Update on lymphocyte specific kinase inhibitors: a patent survey

Importance of the field: Lck (p56lck or lymphocyte specific kinase) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase activity is critical for T cell receptor (TCR)-mediated signaling, leading to normal T-cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection.Areas covered in this review: This review covers the patents, patent applications and associated publications for small molecule kinase inhibitors of Lck since 2005 and attempts to place them in context from a structural point of view.What the reader will gain: Readers will gain an overview of the structural classes and binding modes of Lck inhibitors, the major players in this area and an insight into the current state of the field.Take home message: The search for a potent and orally active inhibitor of Lck has been an intense area of research for a number of years. Despite tremendous efforts, the identification of a highly selective and potent Lck inhibitor suitable for use as an immunosuppressive agent remains elusive.

Constitutively Active Lck Kinase in T Cells Drives Antigen Receptor Signal Transduction

SummaryT cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to ∼40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-ζ phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors.

Enforced SOCS1 and SOCS3 expression attenuates Lck-mediated cellular transformation

Lck is an Src family protein tyrosine kinase with predominant T cell expression. Aberrant expression or activation of Lck kinase has been reported in both lymphoid and non-lymphoid malignancies. We showed previously that the signal transduction pathway involving Janus kinase (JAK) and signal transducer and activator of transcription (STAT) is constitutively activated and contributes to Lck-mediated oncogenesis. Under normal physiological conditions, active STAT proteins induce the expression of suppressor of cytokine signaling (SOCS) family proteins to inhibit further JAK/STAT signaling. It is not fully understood whether and how SOCS-mediated negative feedback control is dysregulated in Lck-transformed cells. Here we report that two SOCS family members, SOCS1 and SOCS3, are not expressed in Lck-transformed LSTRA leukemia. While SOCS1 gene is silenced by DNA hypermethylation, loss of SOCS3 expression is through a mechanism independent of epigenetic silencing by DNA methylation. Furthermore, ectopic expression of SOCS1 or SOCS3 leads to reduced cell proliferation and increased apoptosis in Lck-transformed cells. This is consistent with the attenuation of Lck kinase activity by exogenous SOCS1 or SOCS3 expression. Downstream STAT5 activity is also inhibited as shown by reduced STAT5 tyrosine phosphorylation and in vitro DNA binding. All together, our data highlight the importance of silencing multiple SOCS genes in tumorigenesis and support the roles of SOCS1 and SOCS3 as tumor suppressors toward oncogenic Lck kinase.

Nilotinib inhibits the Src‐family kinase LCK and T‐cell function in vitro

Dear Editor: In a recent issue of the Journal of Cellular and Molecular Medicine, it was reported that the tyrosine kinase inhibitor nilotinib (AMN-107, Tasigna®; Norvartis Pharmaceuticals, Basel, Switzerland), used for the treatment of chronic myeloid leukaemia, is able to inhibit the function of normal human T lymphocytes in vitro[1]. In addition, this group demonstrated nilotinib inhibits T-cell receptor (TCR) activation and the phosphorylation of signalling proteins involved in TCR activation. We have also investigated the effects of nilotinib on T cells and have expanded on the findings of Chen et al. by demonstrating that like imatinib [2], nilotinib is able to inhibit LCK, a Src-family kinase that plays a critical role in TCR activation [3]. Experiments were performed using normal human peripheral blood mononuclear cells (PBMCs) isolated by density centrifugation. Experimental use of human material was approved by the Royal Adelaide Hospital Ethics Committee and blood was collected with informed consent. 5’6 carboxyfluorescein diacetate succin-imidyl ester (CFSE) staining of human PBMCs was performed as described previously [4]. In the presence of varying concentrations of nilotinib and imatinib (Novartis Pharmaceuticals), T cells were stimulated with 10 μg/ml phytohaemagglutinin (PHA) or Concanavalin A (ConA) (Sigma, St. Louis, MO, USA) or directly using 75 ng/ml of an anti-CD3 antibody (Mabtech, Stockholm, Sweden). Following a 5-day incubation, the proliferation of T cells determined by analysing CFSE dye dilution in cells stained positive by an anti-CD3 PE antibody (BD Biosciences, San Jose, CA, USA) (Fig. 1A). T-cell data were then analysed by Modfit analysis program (Verity Software, Topsham, ME, USA) and the proliferation index determined for each sample. Graphing these values allowed IC50 values for each drug to be determined. Inhibition of LCK by nilotinib and imatinib was analysed by evaluating the effects of each drug on LCK phosphorylation of a substrate peptide. Active LCK kinase (Cell Signaling, Danvers, MA, USA) was incubated with varying concentrations of both drugs in a standard kinase reaction buffer containing a mix of unlabelled and γ32p labelled ATP and a Src-family kinase peptide substrate (Upstate Biotechnology, Lake Placid, NY, USA). The level of γ32pATP labelled substrate, and hence LCK activity, was determined by blotting reactions on p81 filter paper (Whatman, Kent, UK) and determining radioactivity of each sample using a bench top scintillation β-counter. Nilotinib inhibits T-cell proliferation and LCK activity. Following PHA stimulation, nilotinib was able to strongly inhibit the proliferation of T cells as determined by CFSE tracking (A). Using this CFSE data, the proliferation index at various drug concentrations was determined and used to calculate IC50 values for the inhibition of proliferation (B). Values represent the mean from five donors each analysed in different experiments and significant donor variability was seen as represented by large standard deviations. The effects of varying concentrations of nilotinib and imatinib on LCK kinase activity was determined and normalized to a percentage of maximum kinase activity when no drug was present allowing IC50 values to be determined (C). Data presented represent the mean of three independent experiments. Nilotinib inhibited T-cell proliferation at IC50's of 2–5 μM depending on the stimulus used (Fig. 1B). The IC50 values were roughly half that we observed with imatinib. We also observed a similar effect of nilotinib on T-cell activation marker expression and cytokine production (data not shown). LCK kinase activity was inhibited by nilotinib with an IC50 of 550 nM (Fig. 1C). Imatinib inhibited LCK at a similar concentration as reported in the literature [2] with an IC50 of 1250 nM, approximately twice the IC50 of nilotinib. In contrast to our findings, another group [5] found nilotinib to inhibit LCK weakly with an IC50 of 5200 nM. While this is somewhat less potent inhibition to that we obtained, the different IC50s could be due to technical differences in the kinase assays used. Abl has also been implicated in T-cell function [6–8] and it is possible that Abl inhibition by nilotinib may cause, or add to LCK blockade for the inhibitory effect on T-cell function. Nilotinib has 20-fold increased potency against Abl compared with imatinib [9] and we found it to have twice the potency against LCK. As nilotinib inhibited T cells approximately twice as strongly as imatinib, not 20 times as strongly, our data would suggest LCK inhibition may be the main mechanism by which the drug inhibits T-cell activation. Our findings agree well with those of Chen et al.[1] and expand on their results by demonstrating nilotinib inhibits the activity of the Src-family kinase LCK, and propose inhibition of LCK as the likely mechanism by which nilotinib interrupts TCR signalling and the function of T cells. We would like to thank Novartis for supplying the nilotinib used in these experiments. One of the authors, Professor Tim Hughes, is on the Advisory Board of Novartis Pharmaceuticals and also receives research funding from the company.