Src Family Tyrosine Kinases Research Articles

The SH3 domains of the protein kinases ITK and LCK compete for adjacent sites on T cell–specific adapter protein

T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2-inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239-274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239-256 and aa 257-274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242-268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.

Mutated MYD88 regulates transcription of the pro-survival kinase HCK in MYD88 driven B-cell lymphomas.

Activating mutations that lead to constitutive growth and survival signals are one of the major drivers for the development of cancer. By whole genome sequencing, we discovered the highly recurring MYD88 L265P (MYD88L265P) mutation in >90% of WM patients.[Treon et al, N Engl J Med., 2012] Hematopoietic cell kinase (HCK) is a member of the SRC family tyrosine kinases (SFKs), and is normally expressed in cells of myeloid and B-lymphocyte lineages. In B-lymphocyte lineages, HCK is commonly expressed in earlier B-cell progenitors and is downregulated in mature B-cells.

Loss of tau and Fyn reduces compensatory effects of MAP2 for tau and reveals a Fyn-independent effect of tau on calcium.

Microtubule-associated protein tau associates with Src family tyrosine kinase Fyn and is tyrosine phosphorylated by Fyn. The presence of tyrosine phosphorylated tau in AD and the involvement of Fyn in AD has drawn attention to the tau-Fyn complex. In this study, a tau-Fyn double knockout (DKO) mouse was generated to investigate the role of the complex. DKO mice resembled Fyn KO in novel object recognition and contextual fear conditioning tasks and resembled tau KO mice in the pole test and protection from pentylenetetrazole-induced seizures. In glutamate-induced Ca2+ response, Fyn KO was decreased relative to WT and DKO had a greater reduction relative to Fyn KO, suggesting that tau may have a Fyn-independent role. Since tau KO resembled WT in its Ca2+ response, we investigated whether microtubule-associated protein 2 (MAP2) served to compensate for tau, since the MAP2 level was increased in tau KO but decreased in DKO mice. We found that like tau, MAP2 increased Fyn activity. Moreover, tau KO neurons had increased density of dendritic MAP2-Fyn complexes relative to WT neurons. Therefore, we hypothesize that in the tau KO, the absence of tau would be compensated by MAP2, especially in the dendrites, where tau-Fyn complexes are of critical importance. In the DKO, decreased levels of MAP2 made compensation more difficult, thus revealing the effect of tau in the Ca2+ response.

A reevaluation of the spleen tyrosine kinase (SYK) activation mechanism

Spleen tyrosine kinase (SYK) is a signaling node in many immune pathways and comprises two tandem Src homology (SH) 2 domains, an SH2-kinase linker, and a C-terminal tyrosine kinase domain. Two prevalent models of SYK activation exist. The "OR-gate" model contends that SYK can be fully activated by phosphorylation or binding of its SH2 domains to a dual-phosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model proposes that SYK activation requires ppITAM binding and phosphorylation of the SH2-kinase linker by a SRC family kinase such as LYN proto-oncogene, SRC family tyrosine kinase (LYN). To evaluate these two models, we generated directly comparable unphosphorylated (upSYK) and phosphorylated (pSYK) proteins with or without an N-terminal glutathione S-transferase (GST) tag, resulting in monomeric or obligatory dimeric SYK, respectively. We assessed the ability of a ppITAM peptide and LYN to activate these SYK proteins. The ppITAM peptide strongly activated GST-SYK but was less effective in activating upSYK untagged with GST. LYN alone activated untagged upSYK to a greater extent than did ppITAM, and inclusion of both proteins rapidly and fully activated upSYK. Using immunoblot and phosphoproteomic approaches, we correlated the kinetics and order of site-specific SYK phosphorylation. Our results are consistent with the alternative model, indicating that ppITAM binding primes SYK for rapid LYN-mediated phosphorylation of Tyr-352 and then Tyr-348 of the SH2-kinase linker, which facilitates activation loop phosphorylation and full SYK activation. This gradual activation mechanism may also explain how SYK maintains ligand-independent tonic signaling, important for B-cell development and survival.

Conformational insights into the inhibitory mechanism of phyto-compounds against Src kinase family members implicated in psoriasis

Psoriasis is a chronic immune mediated disorder of the skin. There is growing evidence that the Src family tyrosine kinases (SFK) are highly upregulated in psoriasis. The SFK are the key components of the signaling pathways triggering cell growth and differentiation in addition to the immune cascades. In the current work, the interactions between SFK and selective phyto-compounds were studied using molecular docking approach. Based on the results of docking and binding energy calculations quercetin was identified as potential lead compound. To get a deeper insight into the binding of quercetin with the SFK, a combined molecular dynamics and binding free energy calculations were performed. The binding of quercetin disrupted the intra-molecular contacts making the SFK compact except Src kinase. The MM/PBSA free energy decomposition analysis highlighted the significance of hydrophobic and polar residues which are involved in the binding of quercetin. An experimental validation was carried out against the activated forms of Fyn, Lyn and Src kinases, the top three proteins which showed high preference for quercetin. The flow cytometry analysis showed that the expression levels of Fyn, Lyn and Src kinases were dramatically increased in HaCaT cells. However, the treatment of quercetin at the concentration of 51.65 µM for 24 h markedly decreased their expression in HaCaT cells. Besides, similar results were also observed when the HaCaT cells were treated with the kinase inhibitor Ponitinib (1.43 µM) for 24 h.Communicated by Ramaswamy H. Sarma

Antidepressant amitriptyline-induced matrix metalloproteinase-9 activation is mediated by Src family tyrosine kinase, which leads to glial cell line-derived neurotrophic factor mRNA expression in rat astroglial cells.

BackgroundAstrocytes have been implicated in the pathophysiology of mood disorders and in the mechanism of the pharmacological effects of antidepressant drugs by the production of neurotrophic/growth factors. Previous studies have identified astrocyte‐expressed Gαi/o‐coupled lysophosphatidic acid receptor 1 (LPAR1), as being involved in antidepressant‐induced production of glial cell line‐derived neurotrophic factor (GDNF) and matrix metalloproteinase‐9 (MMP‐9) activation, an important step in the production of GNDF. However, the precise mechanism of MMP‐9 activation by antidepressants has yet to be identified, in particular the intracellular signaling pathway between LPAR1/Gαi/o and MMP‐9.Methods and ResultsTreatment of rat C6 astroglial cells (C6 cells) with amitriptyline increased Src family tyrosine kinase phosphorylation in a time and concentration‐dependent manner. Amitriptyline‐induced GDNF mRNA expression was blocked by Src family tyrosine kinase inhibitors. In addition, inhibiting Src family tyrosine kinase blocked amitriptyline‐induced zymographic MMP‐9 activation in C6 cells. The amitriptyline‐induced zymographic MMP‐9 activity was completely blocked by selective inhibition of Gαi/o protein and LPAR1. Furthermore, the amitriptyline‐induced Src family tyrosine kinase phosphorylation was blocked by LPAR1, but not MMP‐9 inhibition, indicating that Src family tyrosine kinase involvement is downstream of LPAR1.ConclusionsThe current findings suggest that the pharmacological effect of antidepressant such as amitriptyline is mediated through an intracellular signaling pathway via the LPAR1/Gαi/o/Src family tyrosine kinase, which leads to MMP‐9 activation and GDNF production.

Role of MSK1 in the Induction of NF-κB by the Chemokine CX3CL1 in Microglial Cells.

Microglial cells are essential mediators of neuroinflammatory processes involved in several pathologies. Moreover, the chemokine fractalkine (CX3CL1) is essential in the crosstalk between neurons and microglia. However, the exact roles of CX3CL1, CX3CL1 receptor (CX3CR1) and microglia signalling are not fully understood in neuroinflammation. In addition, the findings reported on this subject are controversial. In this work, we investigated whether CX3CL1 induced pro-inflammatory signalling activation through NF-κB pathway. We were able to show that CX3CL1 activates the pro-inflammatory pathway mediated by the transcription factor NF-κB as an early response in microglial cells. On the other side, CX3CR1-deficient microglia showed impaired NF-κB axis. Phospho-kinase assay proteome profiles indicated that CX3CL1 induced several kinases such as MAPK's (ERK and JNK), SRC-family tyrosine kinases (YES, FGR, LCK and LYN) and most interesting and also related to NF-κB, the mitogen- and stress-activated kinase-1 (MSK1). Knockdown of MSK1 with short interfering RNAs decreased partially MSK1 protein levels (about 50%), enough to decrease the mRNA levels of Il-1β, Tnf-α and iNos triggered by stimulation with CX3CL1. These results indicate the relevance of CX3CL1 in the activation of the pro-inflammatory NF-κB signalling pathway through MSK1 in microglial cells.

Prognostic Significance of Phosphorylated Fyn in Patients with Lung Adenocarcinoma after Lung Resection.

Purpose: Src family tyrosine kinases, including Fyn, are non-receptor tyrosine kinases that drive malignancy in various kinds of cancers. Fyn has also been suggested to be an effector of epidermal growth factor receptor (EGFR) signaling, and is recognized as a potential therapeutic target. However, little is known about the clinical importance of phosphorylated Fyn (pFyn) in lung adenocarcinoma. The purpose of this study is to examine the prognostic significance of pFyn in this disease.Methods: A total of 282 lung adenocarcinoma specimens were collected from patients who underwent surgery at our institute. A tissue microarray was assembled from paraffin-embedded tumor blocks. pFyn expression was analyzed through immunostaining of the tissue microarray and each case was classified as positive or negative. The association of clinical information with pFyn expression was analyzed statistically.Results: pFyn was positive in 107 cases. A pFyn-positive status was significantly associated with male gender, p53 mutant, pathological stage, tumor size, plural invasion, lymphatic invasion, vascular invasion, and differentiation. pFyn positivity was associated with poor relapse-free survival (RFS; hazard ratio [HR]: 2.11, 95% confidence interval [CI]: 1.32–3.42, p <0.01) and poor overall survival (OS; HR: 1.95, 95% CI: 1.17–3.33, p = 0.01).Conclusion: pFyn expression may affect the prognosis of patients with lung adenocarcinoma after lung resection.

A dynamic transcriptomic atlas of cytokine-induced killer cells

Several clinical immunotherapy trials with cytokine-induced killer (CIK) cells have been reported. However, molecular evidence of cell expansion, acquisition of tumor cytotoxicity, and safety of CIK cells is required before putting them to clinical use. Here, we performed dynamic transcriptomic analyses of CIKs generated from primary peripheral blood mononuclear cells exposed to interferon-γ, OKT3, and interleukin-2. CIK mRNAs were extracted and sequenced at days 0, 1, 7, and 14 and subjected to bioinformatics analyses. Using weighted correlation network analysis (WGCNA), we identified two major gene modules that mediate immune cell activation and mitosis. We found that activation and cytotoxicity of CIK cells likely rely on cluster of differentiation 8 (CD8) and its protein partner LCK proto-oncogene, Src family tyrosine kinase (LCK). A time-course series analysis revealed that CIK cells have relatively low immunogenicity because of decreased expression of some self-antigens. Importantly, we identified several crucial activating receptors and auxiliary adhesion receptors expressed on CIK cells that may function as tumor sensors. Interestingly, cytotoxicity-associated genes, including those encoding PRF1, GZMB, FASL, and several cytokines, were up-regulated in mature CIK cells. Most immune-checkpoint molecules and inflammatory tumor-promoting factors were down-regulated in the CIK cells, suggesting efficacy and safety in future clinical trials. Notably, insulin-like growth factor 1 (IGF-1) was highly expressed in CIK cells and may promote cytotoxicity, although it also could facilitate tumorigenesis. The transcriptomic atlas of CIK cells presented here may inform efforts to improve CIK-associated tumor cytotoxicity and safety in clinical trials.

Deregulation of the SRC Family Tyrosine Kinases in Gastric Carcinogenesis in Non-human Primates.

The evolution of gastric carcinogenesis remains largely unknown, as the regulatory mechanisms involved in the aggressiveness of gastric cancer are still poorly understood. Kinases are downstream modulators and effectors of various cell signaling cascades and play key roles in the development of neoplastic diseases. The objective of this study was to evaluate the expression of genes and proteins of the SRC family, including FYN, YES, BLK, FGR, LYN and SRC, in a model of intestinal gastric carcinogenesis generated by treating Cebus apella, a New World non-human primate, with N-methyl nitrosourea (MNU). mRNA expression of genes was measured by real-time reverse transcription quantitative PCR (RT-qPCR) and protein expression was measured by western blotting in six Cebus apella treated with N-methyl-nitrosourea (MNU) for about 2.5 years. Elevated mRNA and protein expression mainly of the SRC and LYN kinases was observed. Their expression was gradually increasing as non-atrophic gastritis was evolving to gastric cancer. SRC family kinases play a key role in tumor progression and metastasis and may be a promising target for the treatment of gastric cancer.

The Two Isoforms of Lyn Display Different Intramolecular Fuzzy Complexes with the SH3 Domain.

The function of the intrinsically disordered Unique domain of the Src family of tyrosine kinases (SFK), where the largest differences between family members are concentrated, remains poorly understood. Recent studies in c-Src have demonstrated that the Unique region forms transient interactions, described as an intramolecular fuzzy complex, with the SH3 domain and suggested that similar complexes could be formed by other SFKs. Src and Lyn are members of a distinct subfamily of SFKs. Lyn is a key player in the immunologic response and exists in two isoforms originating from alternative splicing in the Unique domain. We have used NMR to compare the intramolecular interactions in the two isoforms and found that the alternatively spliced segment interacts specifically with the so-called RT-loop in the SH3 domain and that this interaction is abolished when a polyproline ligand binds to the SH3 domain. These results support the generality of the fuzzy complex formation in distinct subfamilies of SFKs and its physiological role, as the naturally occurring alternative splicing modulates the interactions in this complex.

Protein Conformational Transitions from All-Atom Adaptively Biased Path Optimization.

Simulation methods are valuable for elucidating protein conformational transitions between functionally diverse states given that transition pathways are difficult to capture experimentally. Nonetheless, specific computational algorithms are required because of the high free energy barriers between these different protein conformational states. Adaptively biased path optimization (ABPO) is an unrestrained, transition-path optimization method that works in a reduced-variable space to construct an adaptive biasing potential to aid convergence. ABPO was previously applied using a coarse-grained Go̅-model to study conformational activation of Lyn, a Src family tyrosine kinase. How effectively ABPO can be applied at the higher resolution of an all-atom model to explore protein conformational transitions is not yet known. Here, we report the all-atom conformational transition paths of three protein systems constructed using the ABPO methodology. Two systems, triose phosphate isomerase and dihydrofolate reductase, undergo local flipping of a short loop that promotes ligand binding. The third system, estrogen receptor α ligand binding domain, has a helix that adopts different conformations when the protein is bound to an agonist or an antagonist. For each protein, distance-based or torsion-angle reduced variables were identified from unbiased trajectories. ABPO was computed in this reduced variable space to obtain the transition path between the two states. The all-atom ABPO is shown to successfully converge an optimal transition path for each of the three systems.

Inhibition of hematopoietic cell kinase dysregulates microglial function and accelerates early stage Alzheimer's disease-like neuropathology.

Emerging evidence have posited that dysregulated microglia impair clearance and containment of amyloid-β (Aβ) species in the brain, resulting in aberrant buildup of Aβ and onset of Alzheimer's disease (AD). Hematopoietic cell kinase (Hck) is one of the key regulators of phagocytosis among the Src family tyrosine kinases (SFKs) in myeloid cells, and its expression is found to be significantly altered in AD brains. However, the role of Hck signaling in AD pathogenesis is unknown. We employed pharmacological inhibition and genetic ablation of Hck in BV2 microglial cells and J20 mouse model of AD, respectively, to evaluate the impact of Hck deficiency on Aβ-stimulated microglial phagocytosis, Aβ clearance, and resultant AD-like neuropathology. Our in vitro data reveal that pharmacological inhibition of SFKs/Hck in BV2 cells and genetic ablation of their downstream kinase, spleen tyrosine kinase (Syk), in primary microglia significantly attenuate Aβ oligomers-stimulated microglial phagocytosis. Whereas in Hck-deficient J20 mice, we observed exacerbated Aβ plaque burden, reduced microglial coverage, containment, and phagocytosis of Aβ plaques, and induced iNOS expression in plaque-associated microglial clusters. These multifactorial changes in microglial activities led to attenuated PSD95 levels in hippocampal DG and CA3 regions, but did not alter the postsynaptic dendritic spine morphology at the CA1 region nor cognitive function of the mice. Hck inhibition thus accelerates early stage AD-like neuropathology by dysregulating microglial function and inducing neuroinflammation. Our data implicate that Hck pathway plays a prominent role in regulating microglial neuroprotective function during the early stage of AD development.

Cutting Edge: Deletion of Ezrin in B Cells of Lyn-Deficient Mice Downregulates Lupus Pathology.

Genetic deletion of the Src family tyrosine kinase Lyn in mice recapitulates human systemic lupus erythematosus, characterized by hyperactive BCR signaling, splenomegaly, autoantibody generation, and glomerulonephritis. However, the molecular regulators of autoimmunity in Lyn-deficient mice and in human lupus remain poorly characterized. In this study, we report that conditional deletion of the membrane-cytoskeleton linker protein ezrin in B cells of Lyn-deficient mice (double knockout [DKO] mice) ameliorates B cell activation and lupus pathogenesis. B cells from DKO mice respond poorly to BCR stimulation, with severe downregulation of major signaling pathways. DKO mice exhibit reduced splenomegaly as well as significantly lower levels of autoantibodies against a variety of autoantigens, including dsDNA, histone, and chromatin. Leukocyte infiltration and deposition of IgG and complement component C3 in the kidney glomeruli of DKO mice are markedly reduced. Our data demonstrate that ezrin is a novel molecular regulator of B cell-associated lupus pathology.

Mechanisms underlying modulation of podocyte TRPC6 channels by suPAR: Role of NADPH oxidases and Src family tyrosine kinases

The soluble urokinase receptor (suPAR) has been implicated in the pathogenesis of chronic kidney diseases (CKD) and may function as a circulating “permeability factor” driving primary focal and segmental glomerulosclerosis (FSGS). Here we examined the mechanisms whereby suPAR causes mobilization and increased activation of Ca2+-permeable TRPC6 channels, which are also implicated in FSGS. Treatment of immortalized mouse podocytes with recombinant suPAR for 24 h caused a marked increase in cytosolic reactive oxygen species (ROS) that required signaling through integrins. This effect was associated with increased assembly of active cell surface NADPH oxidase 2 (Nox2) complexes and was blocked by the Nox2 inhibitor apoycynin. Treatment with suPAR also evoked a functionally measurable increase in TRPC6 channels that was blocked by concurrent treatment with the ROS-quencher TEMPOL as well as by inhibition of Rac1, an essential component of active Nox2 complexes. Elevated ROS evoked by exposing cells to suPAR or H2O2 caused a marked increase in the abundance of tyrosine-phosphorylated proteins including Src, and suPAR-evoked Src activation was blocked by TEMPOL. Moreover, mobilization and increased activation of TRPC6 by suPAR or H2O2 was blocked by concurrent exposure to PP2, an inhibitor of Src family tyrosine kinases. These data suggest that suPAR induces oxidative stress in podocytes that in turn drives signaling through Src family kinases to upregulate TRPC6 channels. The combination of oxidative stress and altered Ca2+ signaling may contribute to loss of podocytes and progression of various forms of CKD.

Abstract 437: Tyrosine Phosphorylation: Regulator of Cardiac Contraction

Myocardial contractile function is depressed in heart failure. Contractile function of the normal and failing heart is in part determined by kinase mediated serine/threonine phosphorylation. While non-receptor tyrosine specific kinases are expressed in the heart and their activity is increased in disease, the role of tyrosine kinase signaling to modulate cardiac contraction is unknown. Previous work demonstrated the cardiac contractile regulatory protein troponin I (TnI) is phosphorylated at tyrosine 26 (Tyr26). Subsequently, we demonstrated TnI Tyr26 phosphorylation alters myofilament function thereby identifying the first tyrosine phosphorylation to directly modulate cardiac contraction. The role of TnI Tyr26 phosphorylation in the normal or diseased heart and the signaling pathways responsible for its phosphorylation are unknown. To identify the kinase responsible for TnI Tyr26 phosphorylation and its effect on in vivo heart function, we generated a novel tyrosine kinase activator and a TnI Tyr26 phosphorylation (Tg Y26E) mouse. Biochemical kinase assays and kinase activation in ventricular myocytes identify Src family kinases as sufficient to induce TnI Tyr26 phosphorylation. Tg Y26E mice exhibit decreased myofilament calcium sensitivity and accelerated relaxation. Finally, we demonstrate TnI Tyr26 phosphorylation is increased during in vivo myocardial ischemia when Src family tyrosine kinases are also activated. Together these data support targeting Src family tyrosine kinase and TnI Tyr26 phosphorylation to improve heart function in myocardial ischemia and the failing heart.

Positive and Negative Regulatory Roles of C-Terminal Src Kinase (CSK) in FcεRI-Mediated Mast Cell Activation, Independent of the Transmembrane Adaptor PAG/CSK-Binding Protein.

C-terminal Src kinase (CSK) is a major negative regulator of Src family tyrosine kinases (SFKs) that play critical roles in immunoreceptor signaling. CSK is brought in contiguity to the plasma membrane-bound SFKs via binding to transmembrane adaptor PAG, also known as CSK-binding protein. The recent finding that PAG can function as a positive regulator of the high-affinity IgE receptor (FcεRI)-mediated mast cell signaling suggested that PAG and CSK have some non-overlapping regulatory functions in mast cell activation. To determine the regulatory roles of CSK in FcεRI signaling, we derived bone marrow-derived mast cells (BMMCs) with reduced or enhanced expression of CSK from wild-type (WT) or PAG knockout (KO) mice and analyzed their FcεRI-mediated activation events. We found that in contrast to PAG-KO cells, antigen-activated BMMCs with CSK knockdown (KD) exhibited significantly higher degranulation, calcium response, and tyrosine phosphorylation of FcεRI, SYK, and phospholipase C. Interestingly, FcεRI-mediated events in BMMCs with PAG-KO were restored upon CSK silencing. BMMCs with CSK-KD/PAG-KO resembled BMMCs with CSK-KD alone. Unexpectedly, cells with CSK-KD showed reduced kinase activity of LYN and decreased phosphorylation of transcription factor STAT5. This was accompanied by impaired production of proinflammatory cytokines and chemokines in antigen-activated cells. In line with this, BMMCs with CSK-KD exhibited enhanced phosphorylation of protein phosphatase SHP-1, which provides a negative feedback loop for regulating phosphorylation of STAT5 and LYN kinase activity. Furthermore, we found that in WT BMMCs SHP-1 forms complexes containing LYN, CSK, and STAT5. Altogether, our data demonstrate that in FcεRI-activated mast cells CSK is a negative regulator of degranulation and chemotaxis, but a positive regulator of adhesion to fibronectin and production of proinflammatory cytokines. Some of these pathways are not dependent on the presence of PAG.

Integrative analysis of promising molecular biomarkers and pathways for coronary artery disease using WGCNA and MetaDE methods.

The present study aimed to examine the molecular mechanisms of coronary artery disease (CAD). A total of four microarray datasets (training dataset no. GSE12288; validation dataset nos. GSE20680, GSE20681 and GSE42148) were downloaded from the Gene Expression Omnibus database, which included CAD and healthy samples. Weighted gene co-expression network analysis was applied to identify highly preserved modules across the four datasets. Differentially expressed genes (DEGs) with significant consistency in the four datasets were selected using the MetaDE method. The overlapping genes amongst the DEGs with significant consistency and in the preserved modules were used to construct a protein-protein interaction (PPI) network, followed by functional enrichment analysis. A total of 11 modules were established in the training dataset, and five of them were highly preserved across all four datasets, including 873 genes. There was a total of 836 DEGs with significant consistency in the four datasets. A total of 177 overlapping genes were selected, with which a PPI network was constructed. The top five genes of the PPI network were identified based on their degrees: LCK proto-oncogene, Src family tyrosine kinase (LCK), euchromatic histone lysine methyltransferase 2 (EHMT2), inosine monophosphate dehydrogenase 2 (IMPDH2), protein phosphatase 4 catalytic subunit (PPP4C) and ζ-chain of T-cell receptor associated protein kinase 70 (ZAP70). Genes in the PPI network were significantly involved in a number of Kyoto Encyclopedia Genes and Genomes pathways, including the ‘natural killer cell mediated cytotoxicity’, ‘primary immunodeficiency’ and ‘Fc gamma R-mediated phagocytosis’ pathways. LCK, EHMT2, IMPDH2, PPP4C and ZAP70 are suggested as promising molecular biomarkers for CAD. The ‘natural killer cell mediated cytotoxicity’, ‘primary immunodeficiency’ and ‘Fc gamma R-mediated phagocytosis’ pathways may serve important roles in CAD.

Abstract 1820: Overcoming drug resistance in NSCLC with SRC inhibitor

Abstract Lung cancer accounts for about 27 percent of all cancer deaths in the United States. Approximately 85 percent of lung cancers are non-small cell type (NSCLC) and, when treated with both chemotherapy and targeted therapies, have a five-year overall survival rate of approximately 16 percent. These statistics clearly emphasize the need for developing treatment methodologies more effective than the current standard of care. Here, we evaluated the efficacy of combined inhibition of SRC and AXL in NSCLC.Dasatinib, a BCR/abl and src family tyrosine kinase inhibitor (TKI) is a drug that has a potential to be used against both EGFR and KRAS mutated lung cancer patients, alone (phase II clinical trial) and in combination with cetuximab (in phase III clinical trial), osimertinib and erlotinib (both in phase II clinical trials) by exploiting its SRC inhibition capabilities. However, NSCLC patients treated with dasatinib often develop resistance and is the major reason for the low median survival in stage IV patients. Recently, AXL pathway has been shown to be upregulated in Dasatinib resistant cells causing activation of EMT, AKT and survival pathways. It has been suggested that auto-phosphorylation of DDR2 and subsequent activation of SHP2/RAS/AKT pathway as a result of AXL upregulation is one of the major reasons for failure to induce apoptosis in cells during dasatinib resistance. Therefore, we examined whether inhibition of AXL resensitized TKI resistant NSCLC to dasatinib. Our results show that AXL knockdown can resensitize cells to dasatinib. IC50 values decreased significantly following AXL inhibition. We have also demonstrated that nanoparticle-mediated AXL knockdown also produces similar resensitization. The details of the study will be presented. Citation Format: Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. Overcoming drug resistance in NSCLC with SRC inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1820.

Tumor suppressor CD99 is downregulated in plasma cell neoplasms lacking CCND1 translocation and distinguishes neoplastic from normal plasma cells and B-cell lymphomas with plasmacytic differentiation from primary plasma cell neoplasms

AbstractCD99(MIC2) is a widely expressed cell surface glycoprotein and functions as a tumor suppressor involved in downregulation of SRC family of tyrosine kinase. CD99 expression is tightly regulated through B-cell development. The principal aims of this study were to investigate the clinical utility of CD99 expression (i) in distinguishing normal plasma cells from primary plasma cell neoplasms; (ii) in detection of minimal residual disease in primary plasma cell neoplasms; and (iii) in distinguishing plasma cell component of B-cell lymphomas from primary plasma cell neoplasms. We analyzed expression of CD99 by flow cytometry and immunohistochemistry in lymph nodes, peripheral blood, and bone marrow samples. CD99 showed stage-specific expression with highest expression seen in precursor B and plasma cells. In contrast to the uniform bright expression on normal plasma cells, CD99 expression on neoplastic plasma cells was lost in 39 out of 56 (69.6%) cases. Furthermore, 8 out of 56 samples (14%) showed visibly (>10-fold) reduced CD99 expression. Overall, CD99 expression was informative (absent or visibly dimmer than normal) in 84% of primary plasma cell neoplasm. In the context of minimal residual disease detection, CD99 showed superior utility in separating normal and abnormal plasma cells over currently established antigens CD117, CD81, and CD27 by principal component analysis. Preservation of CD99 expression was strongly associated with cyclin D1 translocation in myeloma (p < 0.05). B-cell lymphomas with plasma cell component could be distinguished from myeloma by CD99 expression. In summary, we established that tumor suppressor CD99 is markedly downregulated in multiple myeloma. The loss is highly specific for identification of abnormal cells in primary plasma cell neoplasms, and can be exploited for diagnostic purposes. The role of CD99 in myeloma pathogenesis requires further investigation.