Cytoplasmic Tyrosine Kinase Research Articles

CD4+ T cells are more sensitive to inhibition of TCR signaling under Th17 polarizing conditions

Abstract Following TCR stimulation, in the presence of polarizing cytokines, naïve CD4+ T cells differentiate into effector cells (e.g. Th1, Th2, and Th17). To study how proximal TCR signaling molecules influence T cell differentiation, we used small molecule inhibitors to reduce signaling through the cytoplasmic tyrosine kinases Zap-70 or Lck. We used PP2 to inhibit Lck signaling and a chemical-genetic system to inhibit Zap-70. In this model system, mouse cells expressing an analog-sensitive mutant of Zap-70 are sensitive to inhibitor while wild-type cells are not. Surprisingly, we found that the generation of IL-17 producing Th17 cells was more sensitive to inhibition than the generation of either IFNg producing Th1 cells or IL-4 producing Th2 cells. These results raised the possibility that cells undergoing Th17 differentiation require TCR signaling of a higher intensity. To test this possibility, we analyzed the TCR-induced expression of IRF4 and Nur77-GFP, which are both inducibly expressed in relative proportion to the strength of TCR signaling. For both IRF4 and Nur77-GFP, the magnitude of expression was decreased under Th17 conditions. Additionally, increasing downstream signaling through NFAT rescued the Th17 sensitivity to inhibition. These results suggest that factors in Th17 cultures suppress TCR signal strength, thus cells stimulated under Th17 conditions experience weaker TCR signals and become more sensitive to further inhibition of TCR signaling.

Association of IGF1R Polymorphisms with Growth Traits and its Expression Profiles in Different Pig Breeds

Insulin-like growth factor 1 receptor (IGF1R) is one component of insulin-like growth factor system, which has biological functions of growth traits. The aim of this study is to investigate the entire exons of IGF1R in the three commercial pig breeds, Duroc, Yorkshire and Landrace, to identify novel single nucleotide polymorphisms and their correlation with growth traits. One novel SNP (c.3678C>T) in the exon 20 was detected. This SNP caused the change of amino acid (Ser1217Phe), a portion of cytoplasmic tyrosine kinase domain of IGF1R. At the c.3678C>T site, three genotypes were significantly associated with average daily gain at different growth stage in Yorkshire and Landrace breeds. Meanwhile, we identified the differently expressed pattern of IGF1R in muscle of Yorkshire and Jinhua pigs at different growth stages. Our results provide useful information on understanding the effect of porcine IGF1R gene on growth. The novel IGF1R polymorphism may be useful as molecular markers in pig selection but future studies are required.

Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (remibrutinib, 25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren's syndrome.

Association between focal adhesion kinase and matrix metalloproteinase-9 expression in prostate adenocarcinoma and their influence on the progression of prostatic adenocarcinoma

Focal adhesion kinase (FAK), a member of the non-receptor cytoplasmic tyrosine kinase family, is associated with the development and progression of cancer. Matrix metalloproteinase-9 (MMP-9) is directly involved in the degradation of the extracellular matrix, and basement membrane components promote cancer cell migration and invasion. There is a functional interaction among FAK, MMP-9 and vascular endothelial growth factor (VEGF), which leads to enhanced cancer angiogenesis, cancer cell invasion and progression of malignancy. FAK, MMP-9, VEGF and CD34-positive microvessel density (MVD) were examined in 100 patients with prostate adenocarcinoma using immunohistochemistry. The relationship among these proteins and their impact on angiogenesis and clinicopathological parameters were also evaluated. The FAK expression was found to be positively correlated with the Gleason score, WHO grade group, tumour stage, extracapsular extension and perineural invasion. The MMP-9 expression was positively correlated with the WHO grade group, tumour stage, extracapsular extension, positive surgical margin and lymphovascular and perineural invasion. The FAK expression was also positively correlated with MMP-9 expression and MVD. However, no correlation between FAK and VEGF expression was identified. The MMP-9 expression was positively correlated with FAK expression and MVD. Strong MMP-9 expression was associated with shorter disease-free survival. These results suggest that strong MMP-9 and FAK expressions play an essential role in the progression of prostate adenocarcinoma. Further investigations should be conducted to determine the importance of these proteins as therapeutic targets for patients with prostate adenocarcinomas.

SERINE-910 Phosphorylated Focal Adhesion Kinase Expression Predicts Better Overall and Disease-free Survival in Melanoma.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that mediates multiple cellular functions such as survival, invasion, and migration. FAK has been found to be over-expressed in various human cancers, including melanoma. FAK molecule has several tyrosine, serine, and threonine phosphorylation sites which have an important regulatory role. Tyrosine phosphorylation of FAK has been extensively studied, however little is known about the role of serine phosphorylation. We sought to examine the frequency and extent of serine-910 phosphorylated FAK (P-FAKSer910) expression in a spectrum of melanocytic proliferations as well as it's correlation with other histopathologic predictors and its effect on patient's survival. Clinicopathologic features and immunohistochemical expression of P-FAKSer910 were evaluated in 147 melanocytic proliferations: 73 primary melanoma (PM), 19 metastatic melanoma (MetM), 2 melanoma in situ, and 53 melanocytic nevi (MN). Higher cytoplasmic intensity predicted better overall survival (OS) in PM (χ=5.69; P=0.034) and was associated with 48% decrease in death risk (HR, 0.52; 95% CI, 0.28-0.95; P=0.036). In contrast, increased nuclear intensity was significantly associated with better disease-free survival (DFS) when stratified by tumor stage Log-rank test, trend of survival (χ=5.83, P=0.015) and independently on multivariate analysis when subcategorized into 3-tier categories (HR, 0.43; 95% CI, 0.18-0.98; P=0.045). Also, Clark's level and tumor-infiltrating lymphocytes (TILS) were independent predictors of DFS. Cytoplasmic intensity correlated inversely with American Joint Commission on Cancer stage in primary melanoma cases as well with vascularity in both primary and metastatic melanoma. Nuclear intensity independently correlated negatively with angioinvasion and TILS when subcategorized to 3 tier system. We found American Joint Commission on Cancer tumor stage, Clark's level, depth, ulceration, TILS, mitosis, angioinvasion, and tumor vascularity predictors of both DFS and OS. There was no significant difference in cytoplasmic or nuclear expression among the major categories of melanocytic proliferation. In this pilot immunohistochemistry-based study, we found P-FAKSer910 expression in melanoma by cytoplasmic intensity to correlate with better OS while nuclear intensity correlated with better DFS.

Pretransplant Short-Term Exposure of Donor Graft Cells to ITK Selective Inhibitor ONO-7790500 Prevents Acute Gvhd in Mouse BMT Model

Introduction and Objectives Graft-versus-host disease (GVHD) triggered by activated donor graft cells still remains a major complication and course of non-relapse mortality after HSCT. Interleukin-2-inducible T-cell kinase (ITK) is a TEC family cytoplasmic tyrosine kinase of crucial importance for T cell development and TCR signaling. Recent studies demonstrated that ITK-deficient CD4+ T cells easily differentiated into regulatory T cells (Tregs) and ITK/BTK inhibitor ibrutinib could improve chronic GVHD symptoms in mouse model and human clinical study, suggesting that ITK may have important roles in post-transplant immune tolerance. In this study, we examined the effects of ITK-selective inhibitor (ITKsi; ONO-7790500) on acute GVHD by using murine BMT model. Methods Allogeneic response of ITKsi-treated CD4+ T cells from B6 was evaluated by in vitro system and murine BMT model. In BMT experiments, lethally irradiated 10-week old B6D2F1 recipient mice were transplanted with 5 × 106 TCD BM together with 1.5 × 106 CD4+ T cells from B6 donors. Survival and GVHD severity were clinically monitored, and T cell subset reconstitution and serum cytokine profile were also examined. Results First, to examine the influence of ITKsi on alloreactive T cell to the corresponding antigens in vitro, CD4+ T cells isolated from B6 mice were exposed to ITKsi for 3 hours and then cultured with irradiated Balb/c stimulators. Proliferation and IFN-gamma production of ITKsi-treated T cells in response to allogeneic stimulators were significantly reduced in comparison to those from controls (P Conclusion Taken together, these data showed that pretreatment of CD4+ T cells with ITKsi efficiently regulated alloreactive T cell and improved the symptoms of acute GVHD. ITKsi inhibited both Th1 and Th2 response but spared the expansion of donor-derived Tregs. Our findings suggest that graft manipulation with short-term culture with ITKsi just before transplant could provide a simple but efficient strategy to modulate alloreactive T cell response and clinical GVHD.

Enhancement of FAK alleviates ventilator-induced alveolar epithelial cell injury

Mechanical ventilation induces lung injury by damaging alveolar epithelial cells (AECs), but the pathogenesis remains unknown. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that is involved in cell growth and intracellular signal transduction pathways. This study explored the potential role of FAK in AECs during lung injury induced by mechanical ventilation. High-volume mechanical ventilation (HMV) was used to create a mouse lung injury model, which was validated by analysis of lung weight, bronchoalveolar lavage fluid and histological investigation. The expression of FAK and Akt in AECs were evaluated. In addition, recombinant FAK was administered to mice via the tail vein, and then the extent of lung injury was assessed. Mouse AECs were cultured in vitro, and FAK expression in cells under stretch was investigated. The effects of FAK on cell proliferation, migration and apoptosis were investigated. The results showed that HMV decreased FAK expression in AECs of mice, while FAK supplementation attenuated lung injury, reduced protein levels/cell counts in the bronchoalveolar lavage fluid and decreased histological lung injury and oedema. The protective effect of FAK promoted AEC proliferation and migration and prevented cells from undergoing apoptosis, which restored the integrity of the alveoli through Akt pathway. Therefore, the decrease in FAK expression by HMV is essential for injury to epithelial cells and the disruption of alveolar integrity. FAK supplementation can reduce AEC injury associated with HMV.

Gastrointestinal and Extra-Gastrointestinal Stromal Tumours: A Clinicopathological and Immunohistochemical (c-KIT) Study

Introduction: Gastrointestinal Stromal Tumour (GIST) is one of the most common mesenchymal tumours of GIT. They have been proved to be arising from the smooth muscle pacemaker interstitial cells of Cajal, these cells are involved in gut motility and peristaltic movements. They can also rarely arise outside the Gastrointestinal Tract (GIT) from retroperitoneum, mesentery and omentum called as Extragastrointestinal Stromal Tumor (EGIST). Single best defining feature of GIST is positivity for Cytoplasmic Tyrosine Kinase (c-KIT). Aim: The aim of the study was to analyse clinicopathological features and c-KIT expression in both gastrointestinal and EGISTs. Materials and Methods: This was a retrospective observational (cohort) study; over a period of two years from January 2018 to December 2019, done at Navodaya Medical College and Hospital, Raichur, Karnataka, which comprised of 12 cases of GIST. All the cases were a resected specimens and thorough evaluation of clinical, imaging and histopathological studies were done and forwarded for immunohistochemistry for c-KIT expression. Results: Age of cases ranged from 04-70 years, mean age being 50.6 years and male to female ratio 2:1. Presenting symptoms of most of GIST were pain abdomen, diarrhea and few with vomiting whereas, rectosigmoid GISTs (2 cases and one with metastasis to liver) were associated with pain abdomen, bleeding per rectum and constipation. One case of retroperitoneal (extraintestinal) GIST was asymptomatic and other presented with pain in the right hip due to secondaries and one benign gastric serosal GIST was associated with gastric adenocarcinoma. The CT scan in two of malignant GISTs, confirmed metastasis and c-KIT study was negative in these 2 malignant GISTs. Conclusion: This study reaffirms importance of CD117 in diagnosis of GIST and EGIST, however, the negativity of CD117 does not rule out GIST, which requires thorough clinico-radiological and pathological correlation.

Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

Targeted therapeutics are needed for triple-negative breast cancer (TNBC). In this study, we investigated the activation of Src family of cytoplasmic tyrosine kinases (SFKs) and two SFK substrates-CUB-domain containing protein 1 (CDCP1) and protein kinase C δ (PKCδ)-in 56 formalin-fixed, paraffin-embedded (FFPE) TNBCs. Expression of SFK phosphorylated at Y416 (SFK_pY416+) in tumor cells was strongly associated with phosphorylation of CDCP1 and PKCδ (CDCP1_ pY743+ and PKCδ_pY311+), as assessed by immunohistochemistry, indicating increased SFK activity in situ. To enable biochemical analysis, protein extraction from FFPE tissue was optimized. Cleaved CDCP1 isoform (70 kDa) was expressed to a varying degree in all samples but only phosphorylated in TNBC tumor cells that expressed SFK_pY416. Interestingly, active SFK was found to be biphosphorylated (SFK_pY416+/pY527+). Biphosphorylated active SFK was observed more frequently in forkhead box protein A1 (FOXA1)- TNBCs. In addition, in SFK_pY416- samples, FOXA1+ TNBC tended to be SFK_pY527+ (classic inactive SFK), and FOXA1- TNBC tended to be SFK_pY527- (SFK poised for activation). Strong SFK_pY416 staining was also observed in tumor-infiltrating lymphocytes in a subset of TNBCs with high tumor-infiltrating lymphocyte content. This report will facilitate protein biochemical analysis of FFPE tumor samples and justifies the development oftherapies targeting the SFK/CDCP1/PKCδ pathway for TNBC treatment.

A Systematic Review and Meta-Analysis of Cardio-Vascular Side Effects with Fostamatinib a Spleen Tyrosine Kinase Inhibitor

Introduction: Spleen tyrosine kinase (Syk), a cytoplasmic tyrosine kinase, is a member of the non-receptor type protein kinase family. Apart from the hematopoietic cells, it is also expressed in the epithelial and endothelial cells. Fostamatinib, a Syk inhibitor, has been proven beneficial in autoimmune disease like rheumatoid arthritis (RA) and immune thrombocytopenia (ITP). Murine models have shown a direct correlation between hypertension and level of R406, the active metabolite of fostamatinib. In this study, we sought to examine the cardiovascular profile of fostamatinib in published and unpublished randomized controlled trials. Methods: A systematic search of Pubmed, Medline and Scopus databases were done from inception till date to identify all phase 2 and 3 clinical trials of fostamatinib in patients with ITP and RA by two independent reviewers. Trials were included if they reported side effects including but not limited to cerebral ischemia or infarction, myocardial ischemia and myocardial infarction, hypertension and cardiac rhythm disorders. 35 trials were retrieved in the initial search which were excluded according to PRISMA (Preferred Reporting Items for Systematic review and Meta-Analyses) guidelines. 11 trials met the eligibility criteria and were included in the final analysis. All statistical analysis was carried out using OpenMetaAnalyst software. Categorical variables from each study are presented as proportions. The proportions from each study were subjected to arcsine transformation and pooled using a random-effects model. This yielded the pooled estimate with 95% confidence intervals. The I2 statistic was used to assess heterogeneity and a value of I2 = 25%-50% was considered mild, 50%-75% as moderate, and > 75% as severe. A P value of <0.05 was considered significant in all cases. Results: 11 trials included in final study had 3941 patients. Cardio-vascular side effects were reported by all trials which ranged from 0.7% to 19.2%. The pooled estimate was 11.7% (8.6%-15.3%, P<0.001 (I2=89.97%)). The most common adverse event reported was hypertension by 10 trials with pooled estimate of 11.1% (8.3%-14.2%, p<0.001(I2=87.27%). The FIT-3 trial reported hypertension as the sole cardiovascular adverse event in 16.7% (n=21) of patients. Other adverse events reported were arrhythmias by 3 trials with incidence of 0.4% (n=11), angina was reported by 5 trials with incidence of 0.8% (n=19). Malignant hypertension was reported in 3 trials with incidence of 0.2%(n=4). 3 trials report heart failure with incidence of 0.3% (n=7). Carotid artery stenosis was observed in only one patient in Oskira 2 trial. Only 2 patients were found to have stroke from all trials, no incidence of aortic stenosis/aneurysm reported. Ventricular fibrillation and peripheral ischemia were observed in one patient respectively and circulatory shock was seen in two patients. Left bundle branch block and pericarditis were also rare side effects observed in only one patient. Conclusion: Our study shows that the use of fostamatinib is significantly associated with cardiovascular side effects, of which hypertension is most significant adverse event. We also recorded a few serious adverse events like hypertensive crises, myocardial infarction, coronary artery disease and congestive heart failure in few patients, but at a very low rate. Larger longitudinal studies are needed to determine the side effect profile of fostamatinib. Disclosures No relevant conflicts of interest to declare.

Bruton's Tyrosine Kinase (BTK) Is a Novel KMT2A Partner Gene

Histone-lysine N-methyltransferase 2A gene (KMT2A) rearrangements are common genetic events in acute leukemia. They more frequent in infants and are found in 30-45% of acute myeloid leukemias (AML). They demonstrate great molecular heterogeneity with more than 90 partner genes and multiple KMT2A breakpoints involved (Meyer et al., 2018). These characteristics challenge both initial molecular diagnostics and MRD monitoring in ALs with KMT2A rearranged. As the accurate detection of all KMT2A-r types is crucial in order to correct patients' therapy and risk groups' definition, we aim to fully characterize KMT2A rearrangements within our cohort of patients using various techniques. Our recent study demonstrates a novel KMT2A partner gene - Bruton's tyrosine kinase (BTK), in a case of childhood AML. The patient is 9 m.o. girl who presented with WBC 69,3*109/L and hepatosplenomegaly. Morphological, immunochemical and immunological examination revealed acute monocytic leukemia. Bone marrow aspirates were analyzed by G-banded karyotyping, FISH with KMT2A breakapart probe. The karyotype was 45,X,der(X?)t(X;11)(q22.1;q23.3),del(11)(q14),-20[7] with 95% KMT2A-rearranged nuclei. Real-time RT-PCR for 8 most common KMT2A rearrangements screening was negative. Targeted RNA-seq with FusionPlex Myeloid kit (ArcherDX, CO, USA) followed by high-throughput sequencing identified novel fusion transcript KMT2A-BTK with exon 9-exon 2 breakpoint junction. Sanger sequencing was used for validation. The achieved data on KMT2A translocation partner and breakpoint location was used for subsequent patient-specific MRD monitoring. The patient was treated according to AML-MRD-2018 local protocol and achieved complete MRD-negative remission after induction course. Then, a severe myelodepression developed after consolidation therapy, and the patient died due to infectious complications. BTK gene is located at Xq22.1 and encodes for Bruton's tyrosine kinase protein (Hashimoto et al., 1996), which is a cytoplasmic non-receptor tyrosine kinase essential for B-cell maturation and development (Shinners et al., 2007). BTK deficiency is strongly associated with X-linked agammaglobulinemia, where 80-90% of patients express either truncated or misfolded or mutated BTK protein (Valiaho, Smith, & Vihinen, 2006). BTK is also implemented in B-cell acute lymphoblastic leukemia, where it often has a deleted or a truncated kinase domain (Feldhahn et al., 2005). The opposite situation was reported for AML in several studies showing increased BTK activity (Gu et al., 2011; Tomasson et al., 2008). The presented t(X;11)(q22.1;q23.3) childhood AML is to our knowledge the first case of BTK translocations in hematologic malignancies, and its value for leukemogenesis is to be further investigated. However, as the breakpoint junction is located in BTK intron 1, upstream of its coding region (the translation starts in exon 2), we can speculate that BTK's structure itself is not committed and its activity is increased due to upregulated expression together with KMT2A. Therefore, the novel fusion KMT2A-BTK was for the first time revealed during the molecular profiling of KMT2A-rearranged AML in Russian Federation. The work was supported by RFBR grant №17-29-06052. Disclosures No relevant conflicts of interest to declare.

Design, Synthesis, and Evaluation of Highly Potent FAK-Targeting PROTACs.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase, exerts kinase-dependent enzymatic functions and kinase-independent scaffolding functions, both of which are crucial in cancer development, early embryonic development, and reproduction. However, previous efforts for FAK blocking mainly focus on kinase inhibitors. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that allow direct post-translational knockdown of proteins via ubiquitination of a target protein by E3 ubiquitin ligase and subsequent proteasomal degradation. Here, we designed and synthesized a FAK PROTAC library with FAK inhibitor (PF562271 or VS6063) and CRBN E3 ligand. A novel FAK-targeting PROTAC, FC-11, showed a rapid and reversible FAK degradation with a picomolar of DC50 in various cell lines in vitro, which imply that FAK-PROTACs could be useful as expand tools for studying functions of FAK in biological system and as potential therapeutic agents.

Uncommon EGFR Mutations in Non-Small Cell Lung Cancer and Their Impact on the Treatment.

Epidermal growth factor receptor (EGFR) mutations play an important role in the pathogenesis of non-small cell lung cancer. Because these alterations are so-called targetable mutations, their identification is important in daily clinical practice. The diagnostic standard of EGFR mutations is currently based on polymerase chain reaction methods, particularly the quantitative real-time polymerase chain reaction. In recent years, new generation sequencing has become increasingly important. In patients with EGFR mutations, a significant improvement in therapeutic outcomes was achieved with the administration of targeted therapy using tyrosine kinase inhibitors. EGFR is composed of four domains: extracellular with a ligand binding site, a transmembrane domain, a cytoplasmic tyrosine kinase catalytic domain, and a C-terminal domain. The key structures of the tyrosine kinase domain responsible for signal activation and transmission are encoded within exons 18-21 on chromosome7. EGFR mutations are highly heterogeneous. About 90% of EGFR mutations are deletions of exon 19 and point mutation L858R in exon 21. These are referred to as classic mutations. Approximately 10% of the total number of EGFR mutations is attributable to less frequent alterations in the EGFR gene. Due to the low incidence of non-small cell lung cancer with less frequent EGFR mutations, information on their predictive significance is still incomplete. Most of the data for the treatment of cases with uncommon mutations were gathered from retrospective analyses and evaluations of small cohorts. The aim of this review is to summarise the current options for diagnosing and treating non-small cell lung cancer patients with uncommon EGFR mutations. This work was supported by the MEYS - NPS I - LO1413 and MH CR - DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 6. 2019 Accepted: 26. 8. 2019.

Effects of BTK signalling in pathogenic microorganism infections.

As a cytoplasmic protein tyrosine kinase, Bruton's tyrosine kinase (Btk) is widely considered as a vital kinase in many aspects of different physiologic processes. It is engaged in many important signalling pathways related to the immune response, such as the B cell receptor pathway, pattern‐recognition receptor pathway, and triggering receptor expressed on myeloid cell pathway. Recent studies have increasingly focused on the important role of Btk in various inflammatory diseases, which are related to Btk expression in myeloid innate immune cells, such as macrophages, dendritic cells and neutrophils. Although some investigations have explored the role of Btk in microbial infections, many aspects remain elusive, and some of the results are opposite and controversial. Considering the complicated and multiple roles of Btk in the immune system, we summarized the engagement of Btk signalling in various pathogenic microorganism infections, the possible mechanisms involved and its therapeutic potential in the control of infectious diseases.

Targeting Upstream Kinases of STAT3 in Human Medulloblastoma Cells.

Medulloblastoma is the most common malignant brain tumor in children. Despite improvement in overall survival rate, it still lacks an effective targeted treatment strategy. The Janus family of cytoplasmic tyrosine kinases (JAKs) and Src kinases, upstream protein kinases of signal transducer and activator of transcription 3 (STAT3), play important roles in medulloblastoma pathogenesis and therefore represent potential therapeutic targets. In this report, we examined the inhibitory efficacy of the JAK1/2 inhibitor, ruxolitinib, the JAK3 inhibitor, tofacitinib and two Src inhibitors, KX2-391 and dasatinib. These small molecule drugs significantly reduce cell viability and inhibit cell migration and colony formation in human medulloblastoma cells in vitro. Src inhibitors have more potent efficacy than JAK inhibitors in inhibiting medulloblastoma cell migration ability. The Src inhibitors can inhibit both phosphorylation of STAT3 and Src while JAK inhibitors reduce JAK/STAT3 phosphorylation. We also investigated the combined effect of the Src inhibitor, dasatinib with cisplatin. The results show that dasatinib exerts synergistic effects with cisplatin in human medulloblastoma cells through the inhibition of STAT3 and Src. Our results suggest that the small molecule inhibitors of STAT3 upstream kinases, ruxolitinib, tofacitinib, KX2-391, and dasatinib could be novel and attractive candidate drugs for the treatment of human medulloblastoma.

Abstract 109: Myeloid Syk-PI3Kg-HIF axis inhibits anti-tumor adaptive immunity: In silico design of a “first in class” novel dual-Syk/PI3K inhibitor, SRX3207, to block the immunosuppressive tumor microenvironment

Abstract Introduction: Macrophages (MΘs) play important roles in the initiation and progression of solid tumors and in establishing an immunosuppressive tumor microenvironment (TME) that dampens effective anti-tumor immune responses in cancer. Hence, targeting signaling pathways in MΘs that promote tumor immunosuppression will provide therapeutic benefit. Syk kinase is a well-established cytoplasmic protein tyrosine kinase that has been extensively studied in adaptive immune responses, but its role in macrophage-mediated inflammatory responses and innate immune responses remains poorly understood. The role of PI3Kγ in tumor growth and immunosuppression has been recently established by our and other groups. Our current study examines whether targeting two crucial signaling entities, which promote macrophage-mediated anti-inflammatory responses viz. Syk kinase and PI3K, would maximally activate the anti-tumor immune response. Methodology: 1) We generated myeloid-specific conditional Syk k/o mice to investigate if the deletion of Syk has any role in a) macrophage polarization in vitro and in vivo; b) tumor growth in various syngeneic mouse models including LLC, B16, CT26, and MC38 carcinoma; c) tumor immunosuppression including polarization of macrophages into immunosuppressive phenotype, infiltration of myeloid-derived suppressor cells, regulatory T cells; and d) infiltration and activation of CD8+T cells in the TME. 2) We used commercially available Syk inhibitor, R788, to study the role of Syk in the control of the aforementioned phenotypes. 3) We used computational chemistry methods to develop a novel chemotype, SRX3207, which inhibits both PI3K and Syk (with a single molecule) as a strategy for combinatorial activation of anti-cancer immunity. Results: Our results suggest that macrophage, Syk, functions upstream of Rac2 GTPase and PI-3K to modulate integrin (αvβ3/αvβ5&α4β1)-mediated polarization of immunosuppressive macrophages and tumor growth in in vivo syngeneic tumor models. Genetic or pharmacological blockade of Syk in MΘs promotes a pro-inflammatory MΘ phenotype, restores CD8+ T cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Moreover, Syk-regulated immune response gene signature predicts survival in cancer patients. Novel dual-Syk/PI3K inhibitor, SRX3207, shows great efficacy in various syngeneic tumor models with no toxicity. Conclusions: Our results validate the concept of combined Syk and PI3K inhibition as an effective approach to treat macrophage-driven, devastating cancers. This study will open new avenues to explore this chemotype in combination with other immuno-oncologic agents as the majority of cancer patients do not respond to single agent anti-PD1 or other checkpoint inhibitors currently in clinical use. Citation Format: Shweta Joshi, Kevin Liu, Muamera Zulcic, Alok Singh, Timothy Pham, Christopher K. Glass, Andrew Sharabi, Guillermo A. Morales, Joseph R. Garlich, Donald Durden. Myeloid Syk-PI3Kg-HIF axis inhibits anti-tumor adaptive immunity: In silico design of a “first in class” novel dual-Syk/PI3K inhibitor, SRX3207, to block the immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 109.

Benefits of camellia sinensis

Tea is one of the most widely consumed beverages worldwide, and is available in various forms. Green tea is richer in antioxidants compared to other forms of tea. Tea is composed of polyphenols, caffeine, minerals, and trace amounts of vitamins, amino acids, and carbohydrates. The composition of the tea varies depending on the fermentation process employed to produce it. The phytochemicals present in green tea are known to stimulate the central nervous system and maintain overall health in humans. Skin aging is a complex process mediated by intrinsic factors such as senescence, along with extrinsic damage induced by external factors such as chronic exposure to ultraviolet (UV) irradiation—A process known as photoaging—Which can lead to erythema, edema, sunburn, hyperplasia, premature aging, and the development of non-melanoma and melanoma skin cancers. UV can cause skin damage either directly, through absorption of energy by biomolecules, or indirectly, by increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Green tea phytochemicals are a potent source of exogenous antioxidant candidates that could nullify excess endogenous ROS and RNS inside the body, and thereby diminish the impact of photoaging. Several in vivo and in vitro studies suggest that green tea supplementation increases the collagen and elastin fiber content, and suppresses collagen degrading enzyme MMP-3 production in the skin, conferring an anti-wrinkle effect. The precise mechanism behind the anti-photoaging effect of green tea has not been explored yet. Studies using the worm model have suggested that green tea mediated lifespan extension depends on the DAF-16 pathway. Apart from this, green tea has been reported to have stress resistance and neuroprotective properties. Its ROS scavenging activity makes it a potent stress mediator, as it can also regulate the stress induced by metal ions. It is known that tea polyphenols can induce the expression of different antioxidant enzymes and hinder the DNA oxidative damage. Growing evidence suggests that green tea can also be used as a potential agent to mediate neurodegenerative diseases, including Alzheimer’s disease. EGCG, an abundant catechin in tea, was found to suppress the neurotoxicity induced by Aβ as it activates glycogen synthase kinase-3β (GSK-3β), along with inhibiting c-Abl/FE65—the cytoplasmic non-receptor tyrosine kinase which is involved in the development of the nervous system and in nuclear translocation. Additionally, green tea polyphenols induce autophagy, thereby revitalizing the overall health of the organism consuming it. Green tea was able to activate autophagy in HL-60 xenographs by increasing the activity of PI3 kinase and BECLIN-1. This manuscript describes the reported anti-photoaging, stress resistance, and neuroprotective and autophagy properties of one of the most widely known functional foods-green tea.

Depletion of Host Cell Focal Adhesion Kinase Increases the Susceptibility to Invasion by Trypanosoma cruzi Metacyclic Forms.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK), is implicated in diverse cellular processes, including the regulation of F-actin dynamics. Host cell F-actin rearrangement is critical for invasion of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. It is unknown whether FAK is involved in the internalization process of metacyclic trypomastigote (MT), the parasite form that is important for vectorial transmission. MT can enter the mammalian host through the ocular mucosa, lesion in the skin, or by the oral route. Oral infection by MT is currently a mode of transmission responsible for outbreaks of acute Chagas disease. Here we addressed the question by generating HeLa cell lines deficient in FAK. Host cell invasion assays showed that, as compared to control wild type (WT) cells, FAK-deficient cells were significantly more susceptible to parasite invasion. Lysosome spreading and a disarranged actin cytoskeleton, two features associated with susceptibility to MT invasion, were detected in FAK-deficient cells, as opposed to WT cells that exhibited a more organized F-actin arrangement, and lysosomes concentrated in the perinuclear area. As compared to WT cells, the capacity of FAK-deficient cells to bind a recombinant protein based on gp82, the MT surface molecule that mediates invasion, was higher. On the other hand, when treated with FAK-specific inhibitor PF573228, WT cells exhibited a dense meshwork of actin filaments, lysosome accumulation around the nucleus, and had increased resistance to MT invasion. In cells treated with PF573228, the phosphorylation levels of FAK were reduced and, as a consequence of FAK inactivation, diminished phosphorylation of extracellular signal-regulated protein kinases (ERK1/2) was observed. Fibronectin, known to impair MT invasion, induced the formation of thick bundles of F-actin and ERK1/2 dephosphorylation.

Up-regulated cathepsin C induces macrophage M1 polarization through FAK-triggered p38 MAPK/NF-κB pathway

Increasing evidence indicates that in response to environmental changes, macrophages can dynamically change into two main functional phenotypes, namely M1 and M2. Depending on these different phenotypes, macrophages can produce either pro-inflammatory or anti-inflammatory factors which may affect the outcome of inflammation. Mastering the switching of M1/M2 phenotypes may provide therapeutic approaches to chronic inflammatory disease, such as atherosclerosis, rheumatoid arthritis, even the metabolic disorders. Cathepsin C (CTSC), as a member of the papain family of cysteine proteases, is a key enzyme in the activation of granule serine proteases thereby involved in modulating the inflammatory responses. Moreover, abundant expression of CTSC has been found in M1 macrophages in plaques of atherosclerosis and related to the progression of disease. However, whether CTSC can regulate macrophage activation status in inflammatory responses has not been fully investigated. In the present study, using peritoneal macrophages (PMs) and mouse macrophage cell line RAW264.7 treated with LPS and active monomer of CTSC, we found that CTSC was not only expressed in macrophages in M1 activation status, but also facilitated macrophages towards M1 phenotype, suggesting a self-activation mechanism involved in this process which may lead to a vicious circle in chronic inflammation. Then we attempted to explore the underlying molecular mechanisms of CTSC resulting in M1 activation. Focal adhesion kinase (FAK) is one of the non-receptor cytoplasmic protein tyrosine kinases, serving as an upstream mediator that leads to transcription of many pro-inflammatory factors. We found FAK expression was up-regulated at both mRNA and protein levels following CTSC stimulation, and FAK phosphorylation level was also significantly increased. The p38MAPK/NF-κB pathway, as the downstream of FAK, were also found activated in CTSC-treated macrophages, suggesting that CTSC may promote macrophage towards M1 activation status through FAK-induced p38MAPK/NF-κB signaling pathway activation. Our study provides a new therapeutic target in the treatment of chronic inflammatory diseases.

LATENT MEMBRANE PROTEIN 2A (LMP2A) MIMICS B‐CELL RECEPTOR SIGNALING AND PROMOTES IMMUNE ESCAPE IN EPSTEIN‐BARR VIRUS (EBV)‐POSITIVE DIFFUSE LARGE B‐CELL LYMPHOMA (DLBCL)

Background: EBV+ DLBCL is an aggressive malignancy that is largely resistant to current therapeutic regimens. LMP2A is expressed during different latency stages of EBV-infected B cells in which it triggers activation of cytoplasmic protein tyrosine kinases. Early studies have revealed that an immunoreceptor tyrosine-based activation motif in the cytoplasmic N-terminus of LMP2A can trigger a transient increase of the cytosolic Ca2+ concentration similar to that observed in activated B cells. Meanwhile, tumor cells often express PD-L1 in EBV+ DLBCL, providing a possible mechanism for immune escape. We thus explored the correlations between LMP2A expression, B-cell receptor (BCR) signaling, and PD-L1 expression in EBV+ DLBCL. Design: Two cohorts of DLBCL cases, respectively EBV-positive (n=28) and EBV-negative (n=32), were selected. LMP2A, PD-L1 and the BCR signaling-related molecule, phosphorylated form of SYK (pSYK), were immunohistochemically evaluated on formalin-fixed, paraffin-embedded tumor tissues. The expression status of LMP2A, pSYK and BCR were further validated by a flow cytometry analysis using fresh tissues from 3 EBV-positive and 5 EBV-negative DLBCL patients. Results: The EBV-positive cases, with a median age of 61yrs, were more frequently associated with a high-risk IPI score and a non-GCB phenotype (P=0.029). Compared with EBV-negative ones, patients with EBV-positive tumors showed a worse response to the RCHOP therapy and a shorter median survival (P = 0.041). Twenty-one EBV-positive cases (75 %) expressed LMP2A, whereas none of the EBV-negative cohort expressed this protein. The expression level of pSYK was significantly lower (P = 0.0313) in EBV+ DLBCL cases than those EBV-negative ones, and the pSYK level correlated negatively with LMP2A level (P = 0.119). The expression level of PD-L1 was significantly higher (P = 0.042) in EBV+ DLBCL, which correlated positively with the LMP2A level (P=0.184). Flow cytometry confirmed the LMP2A expression in EBV+ DLBCL, which correlated with a down-regulated BCR and pSYK expression. Conclusions: EBV+ DLBCL seems to feature an inactive BCR signaling, which may be related to the expression of LMP2A. Besides, LMP2A may function and promote the PD-L1 expression. These findings indicate that targeting immune checkpoints including PD-1/PD-L1 instead of BCR signaling molecules seems to be more reasonable and promising for the treatment of EBV+ DLBCL. Keywords: diffuse large B-cell lymphoma (DLBCL); Epstein-Barr virus (EBV).