983 Syk promotes macrophage immunosuppressive phenotype to suppress anti-tumor immunity
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Abstract:
Background
Macrophages play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in macrophages that promote tumor immunosuppression will provide therapeutic benefits.Methods
Myeloid Syk KO mice, B cell Syk KO mice, LLC, B16 melanoma, NB9464 neuroblastoma, and murine KPC1245 pancreatic adenocarcinoma cell lines were used to elucidate the effect of myeloid Syk on the tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or in combination with immunotherapy or chemotherapy was also determined in various tumor models.Results
We found that Syk promotes immunosuppressive transcriptional programming in tumor-associated macrophages (TAMs) that promotes tumor growth and metastasis. Syk controls stabilization of hypoxia-inducible factor (HIF1/2α) to promote immunosuppression during tumor growth, while genetic deletion of this kinase or use of FDA-approved Syk inhibitor R788 (aka, fostamatinib), activates NFκB signaling, increases the expression of Il12, Ifng, and Nos2 and skews the macrophages toward an immunostimulatory phenotype in vitro and in vivo.1–3 This reprogramming of macrophages towards an immunostimulatory phenotype in Syk-inhibitor-treated tumors was accompanied by increased CD8+ T cell recruitment, enhanced CD8+ T cell proliferation, increased T cell expression of Ifng, Gzm, and Prf and bolstered CD8+ T cell responses in the solid tumors.1–3Conclusions
Syk inhibitors either alone or together with immunotherapy or chemotherapy demonstrate efficacy in multiple tumor models and represent a novel combinatorial approach to activate antitumor immunity.Acknowledgements
This work was supported by NIH grants K22 CA229594, R01NS122835 to Shweta Joshi.References
Joshi S, Liu KX, Zulcic M, Singh AR, Skola D, Glass CK, et al. Macrophage Syk-PI3Kgamma Inhibits Antitumor Immunity: SRX3207, a Novel Dual Syk-PI3K Inhibitory Chemotype Relieves Tumor Immunosuppression. Mol Cancer Ther 2020;19:755–64 Rohila D PI, Pham T, Jones R, Tapia E, Liu KX, Tamayo P, Yu A, Sharabi A, Joshi S. Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma. Frontiers in Immunology 2023 Rohila D PI, Pham TV, Weitz J, Mendoza T, Madheswaran S, Ishfaq M, Beaman C, Tapia E, Sun S, Patel J, Tamayo P, Lowy AM, Joshi S. Syk inhibition reprograms tumor-associated macrophages and overcomes gemcitabine-induced immunosuppression in pancreatic ductal adenocarcinoma. Cancer Research 2023Ethics Approval
Human tissues from deidentified patient samples were received from UC San Diego Moores Cancer Center Biorepository under IRB-approved protocol #181755.Spleen tyrosine kinase (SYK) is a cytosolic nonreceptor protein tyrosine kinase that mediates key signal transduction pathways following the activation of immune cell receptors. SYK regulates cellular events induced by the B-cell receptor and Fc receptors with high intrinsic activity. Furthermore, SYK has been regarded as an attractive target for the treatment of autoimmune diseases and cancers. Here, we report the crystal structures of SYK in complex with seven newly developed inhibitors (G206, G207, O178, O194, O259, O272, and O282) to provide structural insights into which substituents of the inhibitors and binding regions of SYK are essential for lead compound optimization. Our kinase inhibitors exhibited high inhibitory activities against SYK, with half-maximal inhibitory concentrations (IC50 ) of approximately 0.7-33 nm, but they showed dissimilar inhibitory activities against KDR, RET, JAK2, JAK3, and FLT3. Among the seven SYK inhibitors, O272 and O282 exhibited highly specific inhibitions against SYK, whereas O194 exhibited strong inhibition of both SYK and FLT3. Three inhibitors (G206, G207, and O178) more efficiently inhibited FLT3 while still substantially inhibiting SYK activity. The binding mode analysis suggested that a highly selective SYK inhibitor can be developed by optimizing the functional groups that facilitate direct interactions with Asn499.The atomic coordinates and structure factors for human SYK are in the Protein Data Bank under accession codes 4XG2 (inhibitor-free form), 4XG3 (G206), 4XG4 (G207), 5GHV (O178), 4XG6 (O194), 4XG7 (O259), 4XG8 (O272), and 4XG9 (O282).
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Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y).
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Cancer immunotherapy has shown impressive anti-tumor activity in patients with advanced and early-stage malignant tumors, thus improving long-term survival. However, current cancer immunotherapy is limited by barriers such as low tumor specificity, poor response rate, and systemic toxicities, which result in the development of primary, adaptive, or acquired resistance. Immunotherapy resistance has complex mechanisms that depend on the interaction between tumor cells and the tumor microenvironment (TME). Therefore, targeting TME has recently received attention as a feasibility strategy for re-sensitizing resistant neoplastic niches to existing cancer immunotherapy. With the development of nanotechnology, nanoplatforms possess outstanding features, including high loading capacity, tunable porosity, and specific targeting to the desired locus. Therefore, nanoplatforms can significantly improve the effectiveness of immunotherapy while reducing its toxic and side effects on non-target cells that receive intense attention in cancer immunotherapy. This review explores the mechanisms of tumor microenvironment reprogramming in immunotherapy resistance, including TAMs, CAFs, vasculature, and hypoxia. We also examined whether the application of nano-drugs combined with current regimens is improving immunotherapy clinical outcomes in solid tumors.
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Modulation of the tumor microenvironment is becoming an increasingly popular research topic in the field of immunotherapy, and studies regarding immune checkpoint blockades and cancer immunotherapy have pushed cancer immunotherapy to a climax. Simultaneously, the manipulation of the immune regulatory pathway can create an effective immunotherapy strategy; however, the tumor microenvironment serves an important role in suppressing the antitumor immunity by its significant heterogeneity. A number of patients with cancer do not have a good response to monotherapy approaches; therefore, combination strategies are required to achieve optimal therapeutic benefits. Targeting the tumor microenvironment may provide a novel strategy for immunotherapy, break down the resistance of conventional cancer therapy and produce the foundation for personalized precision medicine. The present review summarized the research regarding cancer immunotherapy from the perspective of how the tumor microenvironment affects the immune response, with the aim of proposing a novel strategy for cancer immunotherapy and combination therapy.
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Immune checkpoint
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Syk is a nonreceptor tyrosine kinase critically involved in signaling by various immunoreceptors including B-cell-receptors and activating Fc-receptors. We have previously shown that Syk also mediates immunoreceptor-like signals required for the in vitro development and function of osteoclasts. However, the perinatal lethality of Syk–/– mice precluded the analysis of the role of Syk in in vivo bone metabolism. To overcome that problem, we generated mice with osteoclast-specific (SykΔOC) or hematopoietic (SykΔHaemo) Syk deficiency by conditional deletion of Syk using Cre recombinase expressed under the control of the Ctsk or Vav1 promoter, respectively. Micro-CT analysis revealed increased bone trabecular density in both SykΔOC and SykΔHaemo mice, although hematopoietic Syk deficiency caused a more severe phenotype than osteoclast-specific Syk deficiency. Osteoclast-specific Syk deficiency reduced, whereas hematopoietic Syk deficiency completely blocked in vitro development of osteoclasts. Both interventions inhibited the resorptive activity of osteoclasts and osteoclast-specific gene expression. Kinetic analysis of Syk protein levels, Cre expression and the genomic deletion of the Sykflox allele revealed complete and early deletion of Syk from SykΔHaemo osteoclasts whereas Syk was incompletely deleted at a later stage of osteoclast development from SykΔOC cultures. Those results provide an explanation for the in vivo and in vitro difference between the SykΔOC and SykΔHaemo mutant strains and suggest late activation of, and incomplete target gene deletion upon, osteoclast-specific Cre expression driven by the Ctsk promoter. Taken together, our results indicate that Syk plays an indispensable role in osteoclast-mediated in vivo bone resorption and suggest that Syk-specific inhibitors may provide therapeutic benefit in inflammatory and other diseases characterized by excessive osteoclast-mediated bone resorption.
RANK Ligand
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Στην παρούσα εργασία μελετήθηκε η κυτταροπλασματική κινάση της τυροσίνης SYK (Spleen Tyrosine Kinase), η έκφραση και ο ρόλος της στον καρκίνο του μαστού. Η μελέτη πραγματοποιήθηκε σε ασθενείς με καρκίνο του μαστού καθώς και σε κυτταρικές σειρές καρκίνου του μαστού. Η Syk απαντάται σε όλα τα κύτταρα του ανοσο-αιμοποιητικού συστήματος και λόγω της ευρείας έκφρασής της εκεί, ο ρόλος της ως ένας σημαντικός ρυθμιστής της μεταγωγής σήματος, ήταν γνωστός μέχρι πρόσφατα, μόνο στο σύστημα αυτό. Η Syk είναι από τις πρώτες κινάσες που, προσδένεται αμέσως μετά την ενεργοποίηση των ανοσο-υποδοχέων στις φωσφορυλιωμένες τυροσίνες τους, μέσω των SH2 περιοχών της και των αλληλεπιδράσεων τους. Η δέσμευση προκαλεί την ενεργοποίησή της καθώς και τη συμμετοχή της στην επαγωγή πληθώρας σημάτων, όπως διαφοροποίησης, ωρίμανσης, κλωνικής επέκτασης, αντίσωμα-εξαρτώμενης κυτταροτοξικότητας, φαγοκυττάρωσης κ.α. ανάλογα με τον κυτταρικό τύπο των κυττάρων του ανοσο-αιμοποιητικού συστήματος. Ο ρόλος της κινάσης Syk, ως πιθανό ογκοκατασταλτικό γονίδιο του καρκίνου του μαστού προτάθηκε το 2000. Η έκφρασή της βρέθηκε να μειώνεται σημαντικά ή σε ιστό διηθητικού καρκινώματος μαστού και σε καρκινικές κυτταρικές σειρές μαστού, υψηλού βαθμού κακοήθειας. Επιπρόσθετα , η syk εμπόδιζε τη δημιουργία μεταστατικών αποικιών σε in vivo πειράματα με αθυμικά ποντίκια όπως και καρκινικά κύτταρα να αναπτύσσονται ανεξαρτήτως προσφύσεων και να δημιουργούν διηθητικές αποικίες. Ακολούθησαν και άλλες μελέτες που επιβεβαίωσαν τη μειωμένη έκφραση της Syk σε διηθητικό καρκίνωμα μαστού και τη δυνατότητά της να εμποδίζει τη μετακίνηση και την διήθηση καρκινικών κυττάρων μαστού. Όλα τα παραπάνω έκαναν τη μείωση της έκφρασής της να θεωρείται ως κακός προγνωστικός δείκτης και να συνδέεται με αυξημένες πιθανότητες ανάπτυξης μεταστάσεων. Η υπερμεθυλίωση του γονιδίου της Syk, βρέθηκε να είναι ένας μηχανισμός που οδηγεί στη μείωση της έκφρασή της. Η παρεμπόδιση της προόδου του κυτταρικού κύκλου και κατ’ επέκταση του κυτταρικού πολλαπλασιασμού, προτάθηκε ως ένας από τους πιθανούς μηχανισμούς δράσης της. Στην παρούσα εργασία η έκφραση της syk μελετήθηκε σε δείγματα ασθενών με καρκίνο του μαστού. Σημαντική μείωση της έκφρασής της παρατηρήθηκε σε διηθητικό, σε σχέση με το φυσιολογικό ιστό. Παράλληλα, η έκφρασή της εξαφανίστηκε στους ασθενείς που παρουσίασαν ενδοαγγειακά έμβολα, μία σημαντική ένδειξη ανάπτυξης μεταστατικής νόσου. Επιπρόσθετα, διερευνήθηκε η σχέση της syk και προγνωστικών δεικτών των καρκινωμάτων των ασθενών. Θετική συσχέτιση βρέθηκε να υπάρχει σε πολλές περιπτώσεις, καθιστώντας την εκτίμηση των επίπεδων της έκφρασής ίσως ως ένα ισχυρό μέσο διαλογής, για προγνωστικούς σκοπούς της μεταστατικής νόσου. Δε βρέθηκε να υπάρχει συσχέτιση της έκφρασής της και ρυθμιστών της προόδου του κυτταρικού κύκλου κατά την ύπαρξη κυτταρικής προσκόλλησης, στα δείγματα που εξετάστηκαν, παραπέμποντας πιθανότατα σε διαφορετικό μηχανισμό δράσης της. Επιπρόσθετα, η syk βρέθηκε να σχετίζεται άμεσα με την έκφραση του υποδοχέα Β της Ιντερλευκίνης 8, ρύθμιση του οποίου μπορεί να ευθύνεται για την παρεμπόδιση της κινητικότητας και κατ’ επέκταση της μετανάστευσης των καρκινικών κυττάρων.
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Abstract Background: The tumor microenvironment (TME) has achieved remarkable results in the research of cancer progression in the past few years. it is crucial to understand the nature and function of TME in tumors because of precise treatment strategies for individual cancers having received widespread attention, including immunotherapy. The immune infiltrative profiles of neuroblastoma (NB) have not yet been completely illustrated. The purpose of this research is to analyses tumor immune cell infiltration (ICI) in the microenvironment of NB. Methods: We applied CIBERSORT and ESTIMATE algorithms to evaluate the ICI status of 438 NB samples. Three ICI models were selected and ICI scores were acquired. Subgroups with high ICI scores based on immune activation signaling pathways have better overall survival. Results: The genes of immunosuppressive glycosaminoglycan biosynthesis heparan sulfate signaling pathway were markedly enriched in the low ICI score subgroup. It was inferred that compared with low ICI NB subtypes, patients with high ICI NB subtypes were more likely to respond to immunotherapy and a better prognosis. Conclusion: Notably, our ICI scores not only provided new clinical and theoretical basis for mining NB prognostic markers related to the microenvironment, but also aided new ideas for the development of new NB precision immunotherapy methods.
Cancer Immunotherapy
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Tumor immunotherapy is considered to be a novel and promising therapy for tumors and it has recently become a hot research topic. The clinical success of tumor immunotherapy has been notable, but it has been less than totally satisfactory because tumor immunotherapy has performed poorly in numerous patients although it has shown appreciable efficacy in some patients. A minority of patients demonstrate durable responses but the majority of patients do not respond to tumor immunotherapy as the tumor immune microenvironment is different in different patients for different tumor types. The success of tumor immunotherapy may be affected by the heterogeneity of the tumor immune microenvironment and its components, as these vary widely during neoplastic progression. The deepening of research and the development of technology have improved our understanding of the complexity and heterogeneity of the tumor immune microenvironment and its components, and their effects on response to tumor immunotherapy. Therefore, investigating the tumor immune microenvironment and its components and elucidating their association with tumor immunotherapy should improve the ability to study, predict and guide immunotherapeutic responsiveness, and uncover new therapeutic targets.
Cancer Immunotherapy
Tumor progression
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