Syk Activation Research Articles

AT9283, 1-Cyclopropyl-3-(3-(5-(Morpholinomethyl)-1H-Benzo[d]Imidazole-2-yl)-1H-Pyrazol-4-yl) Urea, Inhibits Syk to Suppress Mast Cell-Mediated Allergic Response.

Mast cells are an effector cell that plays a pivotal role in type I hypersensitive immune responses. Mast cells exist in connective tissues, such as skin and mucosal tissue, and contain granules which contain bioactive substances such as histamine and heparin in cells. The granules of mast cells are secreted by antigen stimulation to cause the type I allergic hypersensitivity. In addition, stimulated by antigen, mast cells synthesize and secrete various eicosanoids and cytokines. While AT9283 is known to have anticancer effects, the therapeutic effect of AT9283 on allergic disorders is completely unknown. In this study, it was found that AT9283 reversibly inhibited antigen-IgE binding-induced degranulation in mast cells (IC50, approx. 0.58 μM) and suppressed the secretion of the inflammatory cytokines IL-4 (IC50, approx. 0.09 μM) and TNF-α (IC50, approx. 0.19 μM). For a mechanism of mast cell inhibition, while not inhibiting Syk phosphorylation, AT9283 suppressed the activation of LAT, a downstream substrate protein of Syk, in a dose-dependent manner. As expected, AT9283 also inhibited the activation of PLCγ1 and Akt, downstream signaling molecules of Syk/LAT, and MAP kinases such as JNK, Erk1/2, and P38. In an in vitro protein tyrosine kinase assay, AT9283 directly inhibited Syk activity. Next, AT9283 dose-dependently inhibited passive cutaneous anaphylaxis (PCA), an IgE-mediated allergic acute response, in mice (ED50, approx. 34 mg/kg, p.o.). These findings suggest that AT9283 has potential to use as a new drug for alleviating the symptoms of IgE-mediated allergic disorders.

Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation.

The function of intracellular trafficking in immune-complex triggered inflammation remains poorly understood. Here, we investigated the role of Insulin-Regulated Amino Peptidase (IRAP)-positive endosomal compartments in Fc receptor (FcR)-induced inflammation. Less severe FcγR-triggered arthritis, active systemic anaphylaxis and FcεRI-triggered passive systemic anaphylaxis were observed in IRAP-deficient versus wild-type mice. In mast cells FcεRI stimulation induced rapid plasma membrane recruitment of IRAP-positive endosomes. IRAP-deficient cells exhibited reduced secretory responses, calcium signaling and activating SykY519/520 phosphorylation albeit receptor tyrosine phosphorylation on β and γ subunits was not different. By contrast, in the absence of IRAP, SHP1-inactivating phosphorylation on Ser591 that controls Syk activity was decreased. Ex-vivo cell profiling after FcγR-triggered anaphylaxis confirmed decreased phosphorylation of both SykY519/520 and SHP-1S591 in IRAP-deficient neutrophils and monocytes. Thus, IRAP-positive endosomal compartments, in promoting inhibition of SHP-1 during FcR signaling, control the extent of phosphorylation events at the plasma membrane and contribute to setting the intensity of immune-complex triggered inflammatory diseases.

Targeting lipid-protein interaction to treat Syk-mediated acute myeloid leukemia.

Membrane lipids control the cellular activity of kinases containing the Src homology 2 (SH2) domain through direct lipid-SH2 domain interactions. Here we report development of new nonlipidic small molecule inhibitors of the lipid-SH2 domain interaction that block the cellular activity of their host proteins. As a pilot study, we evaluated the efficacy of lipid-SH2 domain interaction inhibitors for spleen tyrosine kinase (Syk), which is implicated in hematopoietic malignancies, including acute myeloid leukemia (AML). An optimized inhibitor (WC36) specifically and potently suppressed oncogenic activities of Syk in AML cell lines and patient-derived AML cells. Unlike ATP-competitive Syk inhibitors, WC36 was refractory to de novo and acquired drug resistance due to its ability to block not only the Syk kinase activity, but also its noncatalytic scaffolding function that is linked to drug resistance. Collectively, our study shows that targeting lipid-protein interaction is a powerful approach to developing new small molecule drugs.

Mouse Ocilrp2/Clec2i negatively regulates LPS-mediated IL-6 production by blocking Dap12-Syk interaction in macrophage.

C-type lectin Ocilrp2/Clec2i is widely expressed in dendritic cells, lymphokine-activated killer cells and activated T cells. Previous studies have shown that Ocilrp2 is an important regulator in the activation of T cells and NK cells. However, the role of Ocilrp2 in the inflammatory responses by activated macrophages is currently unknown. This study investigated the expression of inflammatory cytokines in LPS-induced macrophages from primary peritoneal macrophages silenced by specific siRNA target Ocilrp2. Ocilrp2 was significantly downregulated in macrophages via NF-κB and pathways upon LPS stimuli or VSV infection. Silencing Ocilrp2 resulted in the increased expression of IL-6 in LPS-stimulated peritoneal macrophages and mice. Moreover, IL-6 expression was reduced in LPS-induced Ocilrp2 over-expressing iBMDM cells. Furthermore, we found that Ocilrp2-related Syk activation is responsible for expressing inflammatory cytokines in LPS-stimulated macrophages. Silencing Ocilrp2 significantly promotes the binding of Syk to Dap12. Altogether, we identified the Ocilrp2 as a critical role in the TLR4 signaling pathway and inflammatory macrophages’ immune regulation, and added mechanistic insights into the crosstalk between TLR and Syk signaling.

SYK coordinates neuroprotective microglial responses in neurodegenerative disease

SUMMARYRecent studies have begun to reveal critical roles for the brain’s professional phagocytes, microglia, and their receptors in the control of neurotoxic amyloid beta (Aβ) and myelin debris accumulation in neurodegenerative disease. However, the critical intracellular molecules that orchestrate neuroprotective functions of microglia remain poorly understood. In our studies, we find that targeted deletion of SYK in microglia leads to exacerbated Aβ deposition, aggravated neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer’s disease (AD). Disruption of SYK signaling in this AD model was further shown to impede the development of disease-associated microglia (DAM), alter AKT/GSK3β-signaling, and restrict Aβ phagocytosis by microglia. Conversely, receptor-mediated activation of SYK limits Aβ load. We also found that SYK critically regulates microglial phagocytosis and DAM acquisition in demyelinating disease. Collectively, these results broaden our understanding of the key innate immune signaling molecules that instruct beneficial microglial functions in response to neurotoxic material.

Docking of Syk to FcεRI is enhanced by Lyn but limited in duration by SHIP1.

The high-affinity immunoglobulin E (IgE) receptor, FcεRI, is the primary immune receptor found on mast cells and basophils. Signal initiation is classically attributed to phosphorylation of FcεRIβ-and γ-subunits by the Src family kinase (SFK) Lyn, followed by the recruitment and activation of the tyrosine kinase Syk. FcεRI signaling is tuned by the balance between Syk-driven positive signaling and the engagement of inhibitory molecules, including SHIP1. Here, we investigate the mechanistic contributions of Lyn, Syk, and SHIP1 to the formation of the FcεRI signalosome. Using Lyn-deficient RBL-2H3 mast cells, we found that another SFK can weakly monophosphorylate the γ-subunit, yet Syk still binds the incompletely phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs). Once recruited, Syk further enhances γ-phosphorylation to propagate signaling. In contrast, the loss of SHIP1 recruitment indicates that Lyn is required for phosphorylation of the β-subunit. We demonstrate two noncanonical Syk binding modes, trans γ-bridging and direct β-binding, that can support signaling when SHIP1 is absent. Using single particle tracking, we reveal a novel role of SHIP1 in regulating Syk activity, where the presence of SHIP1 in the signaling complex acts to increase the Syk:receptor off-rate. These data suggest that the composition and dynamics of the signalosome modulate immunoreceptor signaling activities.

Spatial requirements for ITAM signaling in an intracellular natural killer cell model membrane

FcγRIIIa-FcεRIγ complexes, upon stimulation by antibodies, cluster to initiate intracellular signaling and activate natural killer (NK) cells. Intracellular signaling involves Lck phosphorylation of ITAMs of each monomer of a FcεRIγ homodimer in a FcγRIIIa-FcεRIγ complex and subsequent binding of two phosphotyrosines (pY) in tandem by a Syk family kinase. However, how FcR clustering triggers ITAM signaling is not resolved. Molecular modeling and dynamics (MD) simulations are applied to generate ensembles of structures of the FcγRIIIa and FcεRIγ homodimeric cytoplasmic tails of FcγRIIIa-FcεRIγ complexes based on the transmembrane helices and cytoplasmic tails spaced 120, 80, and 50 Å apart to model different extents of clustering. Site-identification by ligand competitive saturation method with Monte Carlo sampling (SILCS-MC) is used to model how Lck could phosphorylate a diversity of ITAM conformations. At 80 Å separation between FcγRIIIa-FcεRIγ complexes, Lck can perform multiple phosphorylations on individual and multiple ITAMs across complexes, including potential sequential phosphorylation events. Syk may then potentially bind the two pYs within a single ITAM in tandem in isolated FcγRIIIa-FcεRIγ complexes, as observed in CD3ε and ζ chains of T cell receptors by the Syk family kinase ZAP-70. In addition, at 50 Å separation between complexes, unique to natural killer cells over T cells, Syk could potentially bind in tandem to pYs in different ITAMs across FcγRIIIa-FcεRIγ complexes. Thus, we predict that an ensemble of spatial orientations of the ITAMS of FcγRIIIa-FcεRIγ complexes that occur upon clustering lead to ITAM phosphorylation by Lck and subsequent Syk activity thereby facilitating downstream signaling.

P1267: HSP110 FAVORS BCR SIGNALING THROUGH SYK PHOSPHORYLATION IN DIFFUSE LARGE B CELL LYMPHOMA

Background: Heat-shock proteins (HSPs) are molecular chaperones highly expressed in hematological malignancies. They promote proliferation, immune escape, and resistance to chemotherapies. Our team and others identified HSP110 as a pro-survival factor in diffuse large Bcell lymphomas (DLBCL). In particular, HSP110 sustains aberrant NFkB signaling in activated B-cell DLBCL (ABC-DLBCL) through MyD88 stabilization (Boudesco et al. Blood 2018 Aug 2;132(5):510-520). These findings highlighted HSP110 as a new potential therapeutic target in DLBCL. However, the role of HSP110 in the global signaling pathway of DLBCL has not been studied yet. In addition, the impact of HSP110 inhibition in in vivo xenograft models has been hampered by the absence of specific inhibitors. Aims: We recently identified two foldamers by their capacity to specifically disrupt HSP110 functions in colorectal cancer models (Gozzi et al. Cell Death Differ. 2020 Jan;27(1):117-129). These first HSP110 inhibitors have given us the opportunity to gain more insight into the signaling pathways controlled by HSP110 in DLBCL in vitro and in vivo. Methods: We treated DLBCL cell lines in vitro, and in vivo in xenografted NSG mice. Analyses were done using Western blot, IHC, proximity ligation assay, and in vitro phosphorylation assay. Results: We first validated the two HSP110 inhibitors in DLCBL since they efficiently disrupted the binding of HSP110 to MyD88 in DLBCL cell lines. They impaired cell growth and led to cell death in the Myd88L265P cell lines OCI-Ly10, HBL1, TMD8 and OCI-Ly3 in vitro. Treatment of TMD8-xenografted NSG mice with increasing doses of HSP110 inhibitors limited tumor growth, without tissue toxicity. Analysis of xenografted tumors and in vitro treatments of DLCBL cell lines revealed that the MyD88/NFkB pathway was altered as expected, but also the BCR pathway since we observed a decrease in the activation of BTK, SYK and PLCy2. Using immunoprecipitations and DuolinkTM assays, we identified an in vitro and in cellulo physical interaction between HSP110 and SYK (but not BTK) in several DLBCL cell lines. Furthermore, HSP110 inhibitors disrupted the binding of HSP110 with SYK in vitro, suggesting that the foldamers may act through this mechanism in vivo. We confirmed this assumption using DuolinkTM assays, which showed fewer interactions in xenografted tumors after treatment. Moreover, the phosphorylation of SYK recombinant protein is increased in vitro in the presence of a HSP110 recombinant protein, suggesting that HSP110 acts as a molecular chaperone for optimal activation of SYK through phosphorylation. In addition to the MyD88L265P promotion of the NFkB pathway, the chronic BCR activation in DLBCL has been shown to mediate the activation of both the SYK/BTK and PI3K/Akt pathways, converging to NFkB. Targeting these pathways with ibrutinib and copanlisib, respectively, synergistically suppresses tumor growth in xenograft models (Paul et al. Cancer Cell 31, 64–78, January 9, 2017). We showed that the combination of HSP110 inhibitors with copanlisib also decreases SYK/BTK and AKT phosphorylation synergistically, leading to less NFkB activation in vitro and in vivo. Accordingly, the drug combination stopped the progression of the xenograft. Summary/Conclusion: In conclusion, we identified HSP110 as a regulator of BCR signaling through SYK chaperoning in DLBCL. This finding highlights the major potential of HSP110 as a therapeutic target in DLBCL and suggests a broader role in immune responses.

Egfl6 promotes ovarian cancer progression by inducing the immunosuppressive functions of tumor-infiltrating myeloid cells.

Abstract Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is epidermal growth factor-like 6 (EGFL6), a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. We found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of SYK and ERK signaling, (i) acts as a chemotactic factor for myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Moreover, in an immune ‘hot’ tumor model, EGFL6 completely inhibited response to a-PD-L-1 therapy. Combined our data show that EGFL6 induces the recruitment of myeloid cells into the ovarian TME and subsequently promotes their immunosuppressive functions. This suggest EGFL6 is a potential novel therapeutic target to enhance response to immune therapy in OvCa patients. Supported by Ovarian Cancer Research Alliance

Small molecule inhibition of spleen tyrosine kinase mitigates gut-liver axis inflammation and histopathology in a novel model of IBD-associated liver disease.

Abstract In light of recent studies identifying gain-of-function variants of spleen tyrosine kinase (Syk) as a cause of multi-organ inflammatory disease, Syk inhibition has become an attractive pharmacological target. Therapeutic potential of Syk inhibition may extend beyond that of gain-of-function mutations, since Syk activity initiates downstream responses to several pattern-recognition receptors, which are collectively thought to trigger pathology in several inflammatory diseases, including inflammatory bowel disease (IBD). We previously identified the IBD-linked gene Tnfaip3 (A20) as a suppressor of Syk-dependent inflammatory signals in dendritic cells (DCs) and report here that small molecule inhibition of Syk with FDA-approved drug Fostamatinib mitigates the small intestinal inflammation that develops spontaneously in mice with DC-specific loss of A20 (A20cko mice). In our investigation of IBD in these mice we surprisingly found that A20cko mice also had striking inflammation of the liver that shared many biochemical and histological features characteristic of IBD-associated liver disease, including extensive bridging fibrosis. Importantly, Syk inhibition normalized liver biochemistries and significantly reduced intrahepatic inflammation and fibrosis. Mechanistically, plasma proteomics and liver immunophenotyping pinpointed the key impact of Syk inhibition on restricting chemokine expression linked to recruitment of pro-inflammatory monocytes into in the liver. Collectively this work identifies the interplay of Syk and A20 signaling as drivers of inflammation along the gut-liver axis and suggests Syk is a valid target for pharmacological intervention for IBD and IBD-associated liver disease. Supported by R01AI145930-01A1

Tankyrase represses autoinflammation through the attenuation of TLR2 signaling.

Dysregulation of Toll-like receptor (TLR) signaling contributes to the pathogenesis of autoimmune diseases. Here, we provide genetic evidence that tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, negatively regulates TLR2 signaling. We show that mice lacking tankyrase in myeloid cells developed severe systemic inflammation with high serum inflammatory cytokine levels. We provide mechanistic evidence that tankyrase deficiency resulted in tyrosine phosphorylation and activation of TLR2 and show that phosphorylation of tyrosine 647 within the TIR domain by SRC and SYK kinases was critical for TLR2 stabilization and signaling. Last, we show that the elevated cytokine production and inflammation observed in mice lacking tankyrase in myeloid cells were dependent on the adaptor protein 3BP2, which is required for SRC and SYK activation. These data demonstrate that tankyrase provides a checkpoint on the TLR-mediated innate immune response.

A new role for the SRC family kinase HCK as a driver of SYK activation in MYD88 mutated lymphomas

AbstractThe SRC family kinase (SFK) HCK is transcriptionally upregulated and activated by mutated MYD88 (MYD88Mut), a key adaptor for Toll-receptor signaling. HCK activates BTK, AKT, and ERK in MYD88Mut lymphomas. SYK, a B-cell receptor (BCR) component, is activated in MYD88Mut lymphoma cells. Although the SFK LYN serves as a trigger for SYK activation in MYD88Mut ABC DLBCL cells, LYN activity is muted in MYD88Mut Waldenstrom macroglobulinemia (WM) cells. We therefore investigated a role for HCK in mediating SYK activation. Overexpression of wild-type (WT) (HCKWT) or gatekeeper mutated (HCKThr333Met) HCK in MYD88Mut lymphoma cells triggered SYK activation. Conversely, HCK knockdown reduced p-SYK in MYD88Mut lymphoma cells. Coimmunoprecipitation experiments showed that HCK was complexed with p-SYK in MYD88Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. Rescue experiments in MYD88Mut lymphoma cells expressing HCKThr333Met led to persistent HCK and SYK activation and resistance to the HCK inhibitor A419259. Treatment of primary MYD88Mut WM cells with A419259 reduced p-HCK and p-SYK expression. Taken together, our findings show that SYK is activated by HCK in MYD88Mut B-cell lymphomas cells, broaden the prosurvival signaling generated by aberrant HCK expression in response to MYD88Mut, and help define HCK as an important therapeutic target in MYD88Mut B-cell lymphomas.

Clustering extent‐dependent differential signaling by CLEC‐2 receptors in platelets

BackgroundC‐type lectin receptor family members play a role in many cells including platelets, where they are crucial in the separation of lymphatic and blood vessels during development. The C‐type lectin‐like receptor 2 (CLEC‐2) receptor contains the canonical intracellular hemITAM motif through which it signals to activate Syk. ObjectivesOne proposed hypothesis for signaling cascade is that Syk bridges two receptors through phosphorylated hemITAM motifs. We demonstrated that the phosphorylated hemITAM stimulates PI3 kinase/Btk pathways to activate Syk. To address this controversy, we used a CLEC‐2 selective agonist and studied the role of Btk in platelet activation. Results and ConclusionsPlatelet activation and downstream signaling were abolished in murine and human platelets in the presence of the Btk inhibitors ibrutinib or acalabrutinib when a low concentration of a CLEC‐2 antibody was used to crosslink CLEC‐2 receptors. This inhibition was overcome by increasing concentrations of the CLEC‐2 antibody. Similar results were obtained in X‐linked immunodeficient mouse platelets, with an inactivating mutation in Btk or in Lyn null platelets. We conclude that at low crosslinking conditions of CLEC‐2, Btk plays an important role in the activation of Syk, but at higher crosslinking conditions their role becomes less important and other mechanisms take over to activate Syk. [Display omitted]

Dual BTK/SYK inhibition with CG-806 (luxeptinib) disrupts B-cell receptor and Bcl-2 signaling networks in mantle cell lymphoma

Aberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.

Anti-Inflammatory Functions of Methanol Extract from Malus baccata (L.) Borkh. Leaves and Shoots by Targeting the NF-κB Pathway.

Malus baccata (L.) Borkh. is a widely used medical plant in Asia. Since the anti-inflammatory mechanism of this plant is not fully understood, the aim of this study was to explore the anti-inflammatory function and mechanism of Malus baccata (L.) Borkh. methanol extract (Mb-ME). For in vitro experiments, nitric oxide production assay, PCR, overexpression strategy, immunoblotting, luciferase reporter assay, and immunoprecipitation were employed to explore the molecular mechanism and the target proteins of Mb-ME. For in vivo experiments, an HCl/EtOH-induced gastritis mouse model was used to confirm the anti-inflammatory function. Mb-ME showed a strong ability to inhibit the production of nitric oxide and the expression of inflammatory genes. Mb-ME decreased NF-κB luciferase activity mediated by MyD88 and TRIF. Moreover, Mb-ME blocked the activation of Src, Syk, p85, Akt, p50, p60, IKKα/β, and IκBα in LPS-induced RAW264.7 cells. Overexpression and immunoprecipitation analyses suggested Syk and Src as the target enzymes of Mb-ME. In vitro results showed that Mb-ME could alleviate gastritis and relieve the protein expression of p-Src, p-Syk, and COX-2, as well as the gene expression of COX-2 and TNF-α. In summary, this study implied that Mb-ME performs an anti-inflammatory role by suppressing Syk and Src in the NF-κB signaling pathway, both in vivo and in vitro.

SLAMF3 and SLAMF4 are immune checkpoints that constrain macrophage phagocytosis of hematopoietic tumors.

The interaction of SIRPα with CD47 represents a major mechanism for preventing macrophage phagocytosis. However, CD47-independent mechanisms are poorly defined. Here, we report a critical role of SLAM family receptors (SFRs), ubiquitously expressed on hematopoietic cells and forming homotypic interactions, in constraining macrophage phagocytosis. We found that SFR deficiency triggered macrophage phagocytosis of hematopoietic cells, leading to severe rejection of donor hematopoietic graft in recipient mice. Specific SFR members, mainly SLAMF3 and SLAMF4, were identified as "don't eat me" receptors on macrophages. These receptors inhibited "eat me" signals, such as LRP1-mediated activation of mTOR and Syk, through SH2 domain-containing phosphatases. SFRs combined with, but were independent of, CD47 to mitigate macrophage phagocytosis, and the combined deletion of SFRs and CD47 resulted in hematopoietic cytopenia in mice. This SFR-mediated tolerance was compromised in patients with hemophagocytic lymphohistiocytosis, a syndrome characterized by inappropriate phagocytosis toward hematopoietic cells. Loss of SFRs potently elicited macrophage rejection of hematopoietic tumors. Deletion of SFRs also significantly enhanced the phagocytosis of CD19-positive hematopoietic targets by the macrophages expressing the chimeric CD19 antigen receptor. Therefore, SFR-mediated inhibition of macrophage phagocytosis is critical to hematopoietic homeostasis, and SFRs may represent previously unknown targets for tumor immunotherapy.

Syk-MyD88 Axis Is a Critical Determinant of Inflammatory-Response in Activated Macrophages.

BackgroundInflammation, a vital immune response to infection and injury, is mediated by macrophage activation. While spleen tyrosine kinase (Syk) and myeloid differentiation primary response 88 (MyD88) are reportedly involved in inflammatory responses in macrophages, their roles and underlying mechanisms are largely unknown.MethodsHere, the role of the MyD88-Syk axis and the mechanism by which Syk and MyD88 cooperate during macrophage-mediated inflammatory responses are explored using knockout conditions of these proteins and mutation strategy as well as flowcytometric and immunoblotting analyses.ResultsSyk rapidly activates the nuclear factor-kappa B (NF-κB) signaling pathway in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the activation of the NF-κB signaling pathway is abolished in Syk−/− RAW264.7 cells. MyD88 activates Syk and Syk-induced activation of NF-κB signaling pathway in LPS-stimulated RAW264.7 cells but Syk-induced inflammatory responses are significantly inhibited in MyD88−/− RAW264.7 cells. MyD88 interacts with Syk through the tyrosine 58 residue (Y58) in the hemi-immunoreceptor tyrosine-based activation motif (ITAM) of MyD88, leading to Syk activation and Syk-induced activation of the NF-κB signaling pathway. Src activates MyD88 by phosphorylation at Y58 via the Src kinase domain. In addition, Ras-related C3 botulinum toxin substrate 1 (Rac1) activation and Rac1-induced formation of filamentous actin (F actin) activate Src in LPS-stimulated RAW264.7 cells.ConclusionsThese results suggest that the MyD88-Syk axis is a critical player in macrophage-mediated inflammatory responses, and its function is promoted by an upstream Src kinase activated by Rac1-generated filamentous actin (F-actin).

Role and mechanism of the Dectin-1-mediated Syk/NF-κB signaling pathway in Talaromyces marneffei infection.

Dendritic cell-associated C-type lectin-1 (Dectin-1), a C-type lectin receptor, serves a critical role in host antifungal immunity. However, the molecular mechanism and function of Dectin-1-mediated signaling in response to infection by the pathogenic fungus Talaromyces marneffei remains unclear. To understand the role of Dectin-1 signaling against T. marneffei infection, the phosphorylation of spleen tyrosine kinase (Syk), nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα) and NF-κB were analyzed using western blotting, and the secretion of cytokines was detected using ELISA. Upon sporular or hyphal heat-killed T. marneffei stimulation, Dectin-1 in THP-1 macrophages recognized and induced the activation of Syk, and in turn triggered phosphorylation of downstream molecules IκBα and NF-κB, thus increasing the secretion of TNF-α and IL-8. Conversely, knockdown of Dectin-1 in THP-1 macrophages downregulated the phosphorylation of Syk, IκBα and NF-κB molecules, and significantly decreased the production of TNF-α and IL-8. These results indicated that Dectin-1 may have a crucial role in inducing the inflammatory response via increasing levels of TNF-α and IL-8 induced by T. marneffei, whereas NF-κB may be the key downstream molecule involved in the response to T. marneffei infection. Subsequently, THP-1 macrophages could orchestrate the innate immune system by releasing the cytokines TNF-α and IL-8. Therefore, it was hypothesized that regulation of the Dectin-1 signaling pathway may effectively interfere with the defense ability of the host against T. marneffei infection.

A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

Inhibition of Pre-BCR Signaling Mediates a Metabolic Switch in B-Cell Progenitor Acute Lymphoblastic Leukemia

Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether.To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway.We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism.To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004).Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. DisclosuresMullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.