Antigen Receptor Signaling Research Articles

Preclinical Evaluation of a Novel MALT1 Inhibitor CTX-177 for Relapse/Refractory Lymphomas

Among various subtypes of malignant lymphomas, activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), mantle cell lymphoma (MCL), and adult T-cell leukemia/lymphoma (ATL) are clinically intractable as patients with these lymphomas carry a dismal prognosis, with long-term survival rates of 10-30%. Therefore, a novel therapeutic strategy is required to better manage patients with these malignancies. Recently, we and other investigators performed comprehensive genetic studies and revealed frequent genetic alterations in B and T cell antigen receptor signaling and NF-κB pathway, such as CD79A/B and CARD11 mutations in ABC-DLBCL and PLCG1, PRKCB, and CARD11 mutations in ATL, suggesting the biological relevance of this pathway. To exploit a new treatment strategy in these malignant lymphomas, we focused on the protease mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) which is a key regulator of the antigen receptor signaling and NF-κB pathway and forms a complex with CARD11 and BCL10, and developed a novel compound CTX-177 to inhibit MALT1 with high potency and specificity. CTX-177 was efficacious against ABC-DLBCL and MCL models in vitro and in vivo. Moreover, CTX-177 exhibited combination synergistic effect with BTK inhibitor. In addition, the MALT1 inhibitor showed an anti-tumor effect against CARD11 mutated ABC-DLBCL model, which is resistant to BTK inhibitor. To further explore efficacy of CTX-177 against malignant lymphomas, we generated animal models such as genetically engineered mice and patient-derived xenograft models recapitulating molecular features of these diseases, and examined the response to the MALT1 inhibitor. In these experiments, target engagement of CTX-177 was confirmed by detecting digested substrates of MALT1, and mode of action was evaluated by downregulation of oncogenic transcriptional factor IRF4 which is critical for lymphoma survival. Importantly, the relationship of susceptibility to MALT1 inhibition and gene mutations was analyzed to identity a patient selection biomarker for CTX-177. In summary, the novel, selective, small-molecule MALT1 inhibitor CTX-177 demonstrated preclinical efficacy along with target engagement in several lymphoma models with activated antigen receptor signaling and NF-κB pathway. Our results underscore the preclinical therapeutic potential of CTX-177 as a single-agent or in combination with other inhibitors like BTK inhibitor for the treatment of malignant lymphomas. Disclosures Morishita: Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Mizutani:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Tozaki:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Arikawa:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Kataoka:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Genomics: Current equity holder in private company. Yoda:Chordia Therapeutics Inc.: Research Funding. Izutsu:Symbio: Research Funding; Solasia: Research Funding; Celgene: Research Funding; Chugai: Research Funding; Novartis: Research Funding; Ono Pharmaceutical: Research Funding; Bayer pharmaceuticals: Research Funding; Daiichi Sankyo: Research Funding; AstraZeneca: Research Funding; Eisai: Research Funding; Incyte: Research Funding; Abbvie pharmaceuticals: Research Funding; HUYA Japan: Research Funding; Sanofi: Research Funding; Janssen: Research Funding; Yakult: Research Funding. Minami:Bristol-Myers Squibb Company: Honoraria; Novartis Pharma KK: Honoraria; Pfizer Japan Inc.: Honoraria; Takeda: Honoraria. Shimoda:Otsuka Pharmaceutical: Research Funding; Pfizer Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Merck & Co.: Research Funding; Astellas Pharma: Research Funding; AbbVie Inc.: Research Funding; PharmaEssentia Japan: Research Funding; Perseus Proteomics: Research Funding; Celgene: Honoraria; Shire plc: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company: Honoraria; Novartis: Honoraria, Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding. Miyake:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Ogawa:KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding.

Inhibitory Receptor Trap: A Platform for Discovery of Inhibitory Receptors That Utilize Inositol Lipid and Phosphotyrosine Phosphatase Effectors.

Among the areas of most impactful recent progress in immunology is the discovery of inhibitory receptors and the subsequent translation of this knowledge to the clinic. Although the original and canonical member of this family is FcγRIIB, more recent studies defined PD1 as an inhibitory receptor that constrains T cell immunity to tumors. These studies led to development of “checkpoint blockade” immunotherapies (CBT) for cancers in which PD1 interactions with its ligand are blocked. Unfortunately, although very effective in some patients, only a small proportion respond to this therapy. This suggests that additional as yet undescribed inhibitory receptors exist, which could be exploited. Here, we describe a new platform, termed inhibitory receptor trap (IRT), for discovery of members of this family. The approach takes advantage of the fact that many of the known inhibitory receptors mediate signaling by phospho-immunoreceptor tyrosine-based inhibition motif (ITIM) mediated recruitment of Src Homology 2 (SH2) domain-containing phosphatases including the SH2 domain-containing inositol phosphatase SHIP1 encoded by the INPP5D gene and the SH2 domain-containing phosphotyrosine phosphatases SHP1 and SHP2 encoded by the PTPN6 and PTPN11 genes respectively. Here, we describe the IRT discovery platform in which the SH2 domains of inhibitory phosphatases are used for affinity-based isolation and subsequent identification of candidate effectors via immunoblotting and high sensitivity liquid chromatography–mass spectrometry. These receptors may represent alternative targets that can be exploited for improved CBT. Salient observations from these studies include the following: SH2 domains derived from the respective phosphatases bind distinct sets of candidates from different cell types. Thus, cells of different identity and different activation states express partially distinct repertoires of up and downstream phosphatase effectors. Phosphorylated PD1 binds not only SHP2 but also SHIP1, thus the latter may be important in its inhibitory function. B cell antigen receptor signaling leads predominantly to CD79 mono-phosphorylation as indicated by much greater binding to LynSH2 than Syk(SH2)2. This balance of ITAM mono- versus bi-phosphorylation likely tunes signaling by varying activation of inhibitory (Lyn) and stimulatory (Syk) pathways.

Serum levels of soluble PD1 in pemphigus vulgaris: A useful marker for disease severity.

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease targeting the skin and mucous membranes. Programmed cell death protein 1 (PD1) is an immune checkpoint which binds to two ligands, PDL1 and PDL2 resulting in negative regulation of antigen receptor signaling, thus, play a critical role in the immune regulation of autoimmune diseases. In this work we aimed to assess serum levels of soluble PD1 (sPD1) in patients with active PV and in patients in remission in an attempt to evaluate its effect on disease severity. In this case-control study, 60 pemphigus vulgaris patients (30 clinically active and 30 in a clinical remission) and 30 age matched healthy control subjects were included. Severity of PV was assessed using pemphigus disease area index (PDAI) score. Serum levels of sPD1 were measured by ELISA for both patients and healthy control. Serum levels of sPD1 were significantly lower in PV patients than in controls (P<.001) and significantly lower in patients with active disease than in those in remission (P<.001). Serum sPD1 correlated negatively with the severity of the disease (P<.001, r=-0.4). A defect in PD1 pathway is suggested in PV patients, and this defect plays a substantial role in determining the severity of the disease. Thus, sPD1 could be considered a useful marker for disease severity and targeting PD1 pathway could be a potential aim for future therapies of PV.

Dysregulated CARD11 signaling in the development of diffuse large B cell lymphoma

CARD11 is a crucial scaffold protein that controls antigen-induced activation of lymphocytes. Upon antigen receptor signaling, CARD11 engages several signaling pathways, leading to the activation of NF-κB, mTOR, and JNK. CARD11 mutations are frequently found in patients with non-Hodgkin lymphoma and their ability to induce aberrant lymphocyte proliferation may be enhanced by mutations in regulators of CARD11 signal transduction. Here we describe how dysregulated CARD11 activity can promote lymphomagenesis through branched signaling pathways whose components and intermediates provide targets for novel diagnostic and therapeutic approaches. Statement of novelty: This review discusses how gain-of-function CARD11 mutations promote lymphomagenesis by engaging branching signaling pathways and how these different pathways provide multiple targets for therapies.

Immunopharmacology and Quantitative Analysis of Tyrosine Kinase Signaling.

In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kinases is critical for revealing and therapeutically manipulating mechanisms of immune regulation. Quantitative analysis of the amplitude and kinetics of these effects is becoming ever more accessible experimentally and increasingly important for predicting complex downstream effects of therapeutics and for building computational models. Secondarily, quantitative analysis is increasingly expected by reviewers and journal editors, and statistical analysis of biological replicates can bolster claims of experimental rigor and reproducibility. Here we outline methods for perturbing tyrosine kinase activity in cells and quantifying protein phosphorylation in lysates and immunoprecipitates. The immunoblotting techniques are a guide to probing the dynamics of protein abundance, protein–protein interactions, and changes in post‐translational modification. Immunoprecipitated protein complexes can also be subjected to targeted mass spectrometry to probe novel sites of modification and multiply modified or understudied proteins that cannot be resolved by immunoblotting. Together, these protocols form a framework for identifying the unique contributions of tyrosine kinases to cell activation and elucidating the mechanisms governing immune cell regulation in health and disease. © 2020 The Authors. Basic Protocol 1: Quantifying protein phosphorylation via immunoblotting and near‐infrared imaging Alternate Protocol: Visualizing immunoblots using chemiluminescence Basic Protocol 2: Enriching target proteins and isolation of protein complexes by immunoprecipitation Support Protocol: Covalent conjugation of antibodies to functionalized beads Basic Protocol 3: Quantifying proteins and post‐translational modifications by targeted mass spectrometry

MCL-247: Durable Response of a Patient with a Mantle Cell Lymphoma Central Nervous System (CNS) Relapse to Treatment with a Bruton Tyrosine Kinase Inhibitor (BTKi) Monotherapy

MCL accounts for 5% of all non-Hodgkin lymphomas. CNS involvement occurs in 4% of patients with 4 months of median survival. CNS lymphoma treatment usually involves high-dose chemotherapy + radiotherapy. BTKis block the B-cell antigen receptor signaling with decreased proliferation of malignant B-cells and promoting apoptosis. Monotherapy with BTKis is active in relapsed and refractory MCL with an ORR of 68%. BTK inhibitors may be actively transported across the blood-brain barrier or may rely on simple diffusion. We report here a durable response to BTKi monotherapy of a patient with MCL CNS relapse. Clinical case: A 57-year-old patient with MCL IVA was diagnosed on 6/15/2015. He was treated with bendamustine-rituximab and rituximab maintenance therapy. After five years in complete remission, he was admitted on March 12, 2019, with an acute organic brain syndrome. Brain MRI revealed leptomeningeal disease and CSF + for CNS involvement by MCL. He was started on steroids and ibrutinib 560 mg a day. He experienced significant improvement in Discussion: As far as we know there have been only three other patients reported in Europe with MCL CNS relapse treated with ibrutinib, a BTK inhibitor, and achieving durable responses. In our patient, the switch to a different BTK inhibitor has not affected the quality of his response. The treatment of MCL CNS relapses with a BTK inhibitor monotherapy constitutes a significant advance over high-dose chemotherapy and radiotherapy.

Mechanisms determining a differential threshold for sensing Src family kinase activity by B and T cell antigen receptors

Although signal transduction by immunoreceptors such as the T cell antigen receptor (TCR), B cell antigen receptor (BCR), and Fc receptors uses the same schematic and similar molecules, the threshold and the fine-tuning are set differently for each receptor. One manifestation of these differences is that inhibition of Src family kinases (SFK) blocks TCR but not BCR signaling. SFKs are key kinases phosphorylating immunoreceptor tyrosine-based activation motifs (ITAM) in both these receptors. However, it has been proposed that in B cells, downstream kinase SYK can phosphorylate ITAM sequences independently of SFK, allowing it to compensate for the loss of SFK activity, whereas its T cell paralog ZAP-70 is not capable of this compensation. To test this proposal, we examined signaling in SYK- and ZAP-70-deficient B and T cell lines expressing SYK or ZAP-70. We also analyzed signal transduction in T cells expressing BCR or B cells expressing part of the TCR complex. We show that when compared with ZAP-70, SYK lowered the threshold for SFK activity necessary to initiate antigen receptor signaling in both T and B cells. However, neither SYK nor ZAP-70 were able to initiate signaling independently of SFK. We further found that additional important factors are involved in setting this threshold. These include differences between the antigen receptor complexes themselves and the spatial separation of the key transmembrane adaptor protein LAT from the TCR. Thus, immunoreceptor sensing of SFK activity is a complex process regulated at multiple levels.

Bayesian machine learning to discover Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine kinase (BTK) has a crucial role in multiple cell signaling pathways including B-cell antigen receptor (BCR) and Fc receptor (FcR) signaling cascades, which has attracted much attention to find BTK inhibitors to treat autoimmune diseases. In this work, we constructed a Bayesian classification model for virtually seeking novel BTK inhibitors, which showed good performance in terms of screening efficiency and accuracy. Through searching for several chemical libraries including Chembl_17 (1,317,484 compounds), Chembridge (103,473 compounds), and Chemdiv (700,000 compounds) using this model followed by molecular docking and activity prediction, 52 compounds with novel scaffolds were acknowledged as potential BTK inhibitors, which could be promising starting points for further exploration. This study also provided a guide to construct an efficient and effective protocol for virtual screening by integrating machine learning methods.

Abstract 521: CD45 targeted engineered toxin bodies deplete hematopoietic and malignant cells

Abstract CD45 is highly expressed on the cell surface of all nucleated hematopoietic cells, including malignant cells of B, T and myeloid lineage. CD45, a receptor tyrosine phosphatase that has important roles in antigen receptor signaling, has multiple isoforms that are differentially expressed on immune cell subsets. Targeting of CD45 with antibody-based therapies, tested in conjunction with reduced dose chemotherapy and radiation regimens, has shown promise in preclinical models and clinical trials as a conditioning therapy for bone marrow transplant (BMT), although these approaches are associated with safety limitations. A single agent, targeted conditioning method could increase patient safety and eliminate genotoxic effects associated with the conditioning regimens. Conditioning therapy by depletion of CD45 positive cells specifically has potential applications in oncology as well as non-oncologic, hematopoietic disease settings where engraftment of donor or modified stem cells could provide benefit. Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forcing internalization, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes. Targeted ETBs are being developed to specifically target and destroy CD45 expressing cells. ETBs targeting CD45 specifically bind to, internalize into and then trigger cell death of the target cells in vitro. ETBs have been designed using antibody fragments that recognize all isoforms of CD45. The CD45 targeted ETB has shown high potency in immortalized blood cancer cell lines of T cell, B-cell and eosinophil origin, including leukemia and lymphoma cell lines. Additionally, the ability of the CD45 targeted ETBs to deplete primary cells from healthy donors has been demonstrated. The direct cell kill activity of the CD45 targeted ETBs have the potential to deplete immune cells in the periphery and the bone marrow, as well as to deplete malignant hematological cells expressing CD45. In vivo depletion of human immune cells in murine models by CD45 targeted ETBs will be evaluated. Citation Format: Aimee Iberg, Garrett L. Robinson, Sara LeMar, Joseph D. Dekker, Jay Zhao, Hilario J. Ramos, Melissa M. Singh, Erin K. Willert. CD45 targeted engineered toxin bodies deplete hematopoietic and malignant cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 521.

Noncanonical binding of Lck to CD3ε promotes TCR signaling and CAR function.

Initiation of T cell antigen receptor (TCR) signaling involves phosphorylation of CD3 cytoplasmic tails by the tyrosine kinase Lck. How Lck is recruited to the TCR to initiate signaling is not well known. We report a previously unknown binding motif in the CD3ε cytoplasmic tail that interacts in a noncanonical mode with the Lck SH3 domain: the receptor kinase (RK) motif. The RK motif is accessible only upon TCR ligation, demonstrating how ligand binding leads to Lck recruitment. Binding of the Lck SH3 domain to the exposed RK motif resulted in local augmentation of Lck activity, CD3 phosphorylation, T cell activation and thymocyte development. Introducing the RK motif into a well-characterized 41BB-based chimeric antigen receptor enhanced its antitumor function in vitro and in vivo. Our findings underscore how a better understanding of the functioning of the TCR might promote rational improvement of chimeric antigen receptor design for the treatment of cancer.

Chimeric antigen receptor signaling: Functional consequences and design implications.

Chimeric antigen receptor (CAR) T cell therapy has transformed the care of refractory B cell malignancies and holds tremendous promise for many aggressive tumors. Despite overwhelming scientific, clinical, and public interest in this rapidly expanding field, fundamental inquiries into CAR T cell mechanistic functioning are still in their infancy. Because CAR T cells are manufactured from donor T lymphocytes, and because CARs incorporate well-characterized T cell signaling components, it has largely been assumed that CARs signal analogously to canonical T cell receptors (TCRs). However, recent studies demonstrate that many aspects of CAR signaling are unique, distinct from endogenous TCR signaling, and potentially even distinct among various CAR constructs. Thus, rigorous and comprehensive proteomic investigations are required for rational engineering of improved CARs. Here, we review what is known about proximal CAR signaling in T cells, compare it to conventional TCR signaling, and outline unmet challenges to improving CAR T cell therapy.

Lymphocytes in autoimmune-resistant mice may protect the islets through IL-10 production

Abstract We have identified T cells and B cells in the islets of autoimmune resistant mice, yet the role of these lymphocytes is unknown. In the islets of autoimmune resistant C57BL/6 mice, local antigen receptor signaling was assessed using Nur77-GFP mice. Nur77-GFP expression was increased in islet CD8+ T cells and B cells suggesting that these lymphocyte subsets receive local antigenic stimulus. Islet T cell and B cell populations expressed the regulatory cytokine IL-10, assessed using the B6.TIGER reporter mouse. T cell populations lacked expression of the pro-inflammatory cytokine IFN-γ. These findings indicate that islet T cell populations are likely playing a regulatory role. In addition, cultured whole islets produced IL-10 protein, but not IFN-γ protein. To test the stability of the islet lymphocyte populations under inflammatory conditions, mice were exposed to metabolic stress through a diet-induced obesity (DIO) model. Obesity and type 2 diabetes (T2D) are associated with circulating inflammatory cytokines. In T2D patients, islet lymphocytes have been identified, though the function of these populations is largely unknown. C57BL/6 mice were fed a high fat diet (DIO) or a matched control diet (Control) starting at 5 weeks of age, and then analyzed at 16–25 weeks of age. The number and phenotype of lymphocytes in the islets were similar in DIO and control mice. Notably, under the conditions of obesity-associated systemic inflammation, islet lymphocytes in the DIO mice maintained IL-10 production. From these data, we propose that islet lymphocytes play a regulatory role to protect the islets from systemic inflammation through IL-10 production.

Chimeric antigen receptor signaling confers antitumor activity to human regulatory T cells

Abstract Regulatory T cells (Tregs) are a subset of T cells dedicated to suppressing immune responses to ensure immune self tolerance. Treg therapy offers the opportunity to treat transplant rejection and autoimmunity without the toxicity associated with immunosuppressive regimens. Conferring antigen specificity to Tregs using a chimeric antigen receptor (CAR) dramatically expands what targets can be pursued using Treg therapies. However, the consequences of CAR-mediated signaling for human Treg biology remain poorly understood. To address this, we generated anti-CD19 CAR Tregs with a tandem CD28-TCRζ intracellular domain. Upon in vitro co-incubation with CD19-expressing cells, CAR Tregs upregulated activation markers, proliferated, secreted IL-10, and suppressed T cell proliferation, while maintaining high FOXP3 expression and a demethylated TSDR. In humanized mice harboring CD19+ tumor cells, CAR Tregs suppressed CAR T cell proliferation. Strikingly, CAR Tregs also suppressed tumor growth, even in the absence of CAR T cells. This phenomenon was observed across three tumor cell types (B-cell leukemia, myeloid leukemia, and epithelial carcinoma) and two routes of delivery (intravenous and subcutaneous). Real-time monitoring of tumor cell survival and proliferation in vitro confirmed that CAR Tregs suppress tumor cell growth. Interestingly, single-cell cytokine analysis revealed that CAR-mediated activation of Tregs leads to higher production of IFN-γ, TNF-α, perforin, and granzyme B than anti-CD3/28 stimulation. CRISPR/Cas9-mediated ablation of granzyme B in CAR Tregs diminished their antitumor activity in vivo. These results demonstrate that CAR Treg cytotoxicity is an important concern for safe clinical translation.

Disease-associated dominant negative CARD11 mutations interfere with the CARD11 Opening Step during antigen receptor signaling to NF-κB and JNK2

Abstract CARD11 mediates lymphocyte activation by functioning as a scaffold in antigen receptor signaling to NF-κB, JNK, and mTOR. Antigen-induced CARD11 signaling proceeds through an ordered series of steps, including Opening, Cofactor Recruitment, Enzyme Activation, and Complex Disassembly. Recently, several forms of Primary Immunodeficiency have been described that are due to germline CARD11 mutations. One of these syndromes, Combined Immunodeficiency (CID) with atopy, is inherited in an autosomal dominant manner and caused by loss-of-function (LOF) CARD11 alleles, which display dominant negative activity that poisons signaling from co-expressed wild-type CARD11. To investigate the mechanistic basis for disease-associated dominant negative activity we reconstituted CARD11-deficient T cells with a panel of LOF CARD11 variants in the absence and presence of co-expressed wild-type CARD11. In the absence of wild-type CARD11, the LOF variants were indistinguishable, such that each variant failed to promote the signal-induced 1) recruitment of the cofactors Bcl10, HOIP, and MALT1 to CARD11, 2) Bcl10 polyubiquitinylation, 3) MALT1 protease activation, 4) NF-κB activation, and 5) JNK2 activation. In the presence of co-expressed wild-type CARD11, the LOF variants surprisingly displayed a range of dominant negative activity from strong to intermediate to weak, indicating a specific potency of some mutations to disrupt signaling when heterozygous. Our results suggest that disease-associated dominant negative CARD11 mutations interfere with the Opening Step in the CARD11 signaling cycle and provide a mechanistic basis to explain why some LOF CARD11 mutations can cause disease when heterozygous while others do so when homozygous.

SHP1 tyrosine phosphatase gets involved in host defense against Streptococcus agalactiae infection and BCR signaling pathway in Nile tilapia (Oreochromis niloticus)

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1), a kind of protein tyrosine phosphatases (PTPs), is a critical regulator of antigen receptor signal transduction. Signal transduction of BCR is regulated by phosphatases in teleost as in mammals. In this study, SHP1 from Nile tilapia (Oreochromis niloticus) (OnSHP1) was identified and characterized, including the expression pattern against bacterial infection and regulation function in BCR signaling pathway. The open reading frame of OnSHP1 contains 1749 bp of nucleotide sequence, encoding a protein of 582 amino acids. The OnSHP1 protein was highly conversed compared to that of other species, including two amino-terminal SH2 domains at the N terminus and a PTP catalytic domain. Transcriptional expression analysis revealed that OnSHP1 was detected in all examined tissues and highly expressed in spleen. The up-regulated OnSHP1 expression was observed in peripheral blood, spleen and anterior kidney following challenge with Streptococcus agalactiae or lipopolysaccharide (LPS) in vivo, as well as that displayed in leukocytes stimulated with S. agalactiae or LPS in vitro. Further, after induction with mouse anti-tilapia IgM monoclonal antibody in vitro, OnSHP1 was significantly up-regulated in leukocytes. When spleen leukocytes treated with PTP Inhibitor II in vitro, the phosphorylation level of OnSHP1 at the phosphorylation sites (Y535 and Y557) and the cytoplasmic free Ca2+ concentration were up-regulated significantly. Overall, the findings of this study indicate that SHP1 gets involved in host defense against bacterial infection and BCR signaling pathway in Nile tilapia.

Syk degradation restrains plasma cell formation and promotes zonal transitions in germinal centers.

Germinal centers (GCs) are sites at which B cells proliferate and mutate their antibody-encoding genes in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). B cell antigen receptor (BCR) signals induce Syk activation followed by rapid phosphatase-mediated desensitization; however, how degradation events regulate BCR functions in GCs is unclear. Here, we found that Syk degradation restrains plasma cell (PC) formation in GCs and promotes B cell LZ to DZ transition. Using a mouse model defective in Cbl-mediated Syk degradation, we demonstrate that this machinery attenuates BCR signaling intensity by mitigating the Kras/Erk and PI3K/Foxo1 pathways, and restricting the expression of PC transcription factors in GC B cells. Inhibition of Syk degradation perturbed gene expression, specifically in the LZ, and enhanced the generation of PCs without affecting B cell proliferation. These findings reveal how long-lasting attenuation of signal transduction by degradation events regulates cell fate within specialized microanatomical sites.

CD247 expression is associated with differentiation and classification in ovarian cancer.

Ovarian cancer (OC) is one of the most common malignant tumors in female reproductive system and most OC cases are diagnosed at an advanced stage with the overall 5-year survival rate below 40%. The function of CD247 enhances T-cell antigen receptor (TCR) signaling cascade and it is necessary for assembling of the TCR/CD3 complex on the surface of T lymphocytes. It is well established that defective CD247 function leads to impaired activation of T cells upon engagement of the TCR.Flow cytometry was used to examine the difference of CD247+ T lymphocyte between the OC and ovarian cyst, immunohistochemistry analysis was used to investigate the correlation between CD247 expression and clinicopathologic features of epithelial OC patients.Our study showed that the expression of CD247 in peripheral blood lymphocytes from patients with OC is decreased compared with ovarian cyst patients and the expression of CD247 in tumor infiltrating lymphocytes with cancer tissue is decreased compared with adjacent tissues. We showed that abnormal expression of CD247 was related with differentiation and classification in OC.Our findings suggested that CD247-targeted treatment could be used as a potential therapeutic strategy for OC.

Oncogenic Cooperation between EGR1 and BRD4 in Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) grows rapidly and represents 30% to 40% of newly diagnosed lymphomas, and comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). ABC DLBCL is more aggressive with a current cure rate of only ~40%. The B cell antigen receptor (BCR) signaling pathway is known for its critical role in the survival and proliferation of ABC DLBCL cells. Our recent studies revealed that IL6- and IL10-mediated JAK1/STAT3 activation enhances the survival and proliferation of ABC DLBCL cells. Furthermore, we discovered that EGR1, a transcription factor known to be a downstream effector of BCR signaling, is also a downstream target of JAK1 signaling in DLBCL. While EGR1 has been characterized as a tumor suppressor in leukemia, the role of EGR1 in human lymphoma remains unclear. Initial immunohistochemical analysis of 102 DLBCL cases revealed that not only is the level of EGR1 elevated in DLBCL as compared to normal human tonsils, lymph nodes and mantle cell lymphomas, but its level is higher in ABC DLBCL (non-GCB) than GCB DLBCL, indicating functional significance of EGR1 in DLBCL. In addition, we found that 7 out of 8 ABC DLBCL cell lines are sensitive to EGR1 knockdown, whereas only half of 8 GCB DLBCL cell lines are affected. Our genomics studies discovered dual functions of EGR1 in regulating three signaling pathways. EGR1 mediates transcriptional activation associated with two hallmark oncogenic pathways, JAK1/STAT3 and BCR signaling. Our EGR1 ChIP-seq results revealed a large overlap of EGR1 genomic occupancy with that of BRD4, a highly expressed bromo and extraterminal (BET) protein in DLBCL known to induce MYC expression and activation of MYC target genes. This agrees with the RNA-seq results showing that multiple oncogenic pathways including MYC are activated by EGR1 in ABC DLBCL. The synergistic inhibition of cell growth was observed between EGR1 shRNA and JQ1, an inhibitor of BRD4. Surprisingly, the RNA-seq data and pathway analyses also revealed EGR1-mediated suppression of the type I interferon pathway, which otherwise causes cancer cell death. The majority of the interferon pathway genes are up-regulated upon EGR1 knockdown. EGR1-modulated suppression of the interferon pathway appears to be functionally significant as EGR1 knockdown by shRNA synergizes with the type I interferon inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro and in a xenograft mouse model. EGR1 is known to bind the promoter of p300 and activate p300 expression. P300 catalyzes H3K27 acetylation (H3K27ac), an active histone mark that recruits BRD4 for transcription of MYC and MYC pathway genes, among others. Indeed, immunoblot analysis revealed that EGR1 knockdown reduces H3K27ac expression in ABC DLBCL cells, leading to reduced expression of MYC and MYC target genes based on H3K27ac ChIP qPCR analysis. The results provide a molecular basis for oncogenic cooperation between EGR1 and BRD4 in ABC DLBCL. In summary, our study demonstrates that EGR1 is an oncogenic driver orchestrating multiple important signaling pathways. Notably, EGR1 creates therapeutic vulnerability in ABC DLBCL because both EGR1-mediated transcriptional activation and repression provide growth advantage to the cancer cells. Disclosures No relevant conflicts of interest to declare.

BTK Inhibitors Induce ABC-DLBCL Cell Apoptosis By Inhibiting CYLD Phosphorylation

BTK inhibitors induce ABC-DLBCL cell apoptosis by inhibiting CYLD phosphorylation Xin Xu1, Zhigang Zhu1, Feng Liu1 , Weijie Zhong1,Huabao Xiong2, Tiefeng Feng3, and Qingshan Li3 1Geriatrics Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China. 2Precision Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029, U.S.A. 3Hemotology Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China. Activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL) is a common aggressive diffuse large B cell lymphoma (DLBCL). Even though CD20 antibody (Rituximab) combination therapy improved the total prognosis of DLBCL, there are still many relapsed and refractory ABC-DLBCL cases. The new treatment options are urgently needed. Pathogenesis of ABC-DLBCL is different from germinal center B cell-like diffuse large B cell lymphoma (GCB-DLBCL), which is related to activation of B cell antigen receptor(BCR) signaling. The Bruton tyrosine kinase(BTK) plays a central role in BCR signaling. Inhibition of BTK function will affect BCR signaling activity, which is involved in maintaining the malignant phenotype in B-cell lymphoma. Now in clinic, BTK inhibitors like Ibrutinib or Acalabrutinib are used extensively in chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma(MCL) as first line therapy or second line therapy. But in DLBCL its role is unclear now.Some DLBCL clinic trials got the better PFS and OS in BTK inhibitor combination therapy group(combined with Rituximab or RCHOP) but some got the negative results. Whether BTK inhibitors can definitely improve ABC-DLBCL survival and the mechanism how BTK inhibitors inducing cell death are needed further investigations. CYLD (cylindromatosis) is a tumor suppressor, its activity is inhibited by phosphorylation, which is related to apoptosis regulation pathway NF-kB in many kinds of tumors.In this study, we found that CYLD to be constitutively phosphorylated in ABC-DLBCL cell line models (HBL-1 and OCI-Ly10) as well as primary samples from patients with non-GCB DLBCL. We subsequently examined the effect of BTK inhibitors Ibrutinib (PCI-32765) and Acalabrutinib (ACP-196) on CYLD phosphorylation. We found that BTK inhibitors Ibrutinib and Acalabrutinib both induced ABC-DLBCL apoptosis by down-regulating CYLD phosphorylation in vivo and in vitro, even in the Rituximab resistant ABC-DLBCL cell line.Combination of BTK inhibitors and Rituximab would enhance this effect. Knockdown of CYLD in ABC-DLBCL cells reduced the level of cell death induced by BTK inhibitors, which showed that this cell death induced by BTK inhibitors was CYLD dependent. Through this study, we conclude that BTK inhibitors induce CYLD inactivation by regulating CYLD phosphorylation is clinical beneficial in ABC-DLBCL therapy, especially the relapsed or refractory ABC-DLBCL cases after Rituximab therapy. Disclosures No relevant conflicts of interest to declare.

Skin Associated Staphylococcus Aureus Contributes to Disease Progression in CTCL

It has been proposed that bacteria play a direct role in progression of cutaneous T cell lymphoma (CTCL), although definitive evidence is missing, and the underlying mechanism of how microbes contribute to disease progression remains unknown. The skin of CTCL patients is frequently colonized with Staphylococcus aureus (S. aureus) strains and infections with hospital and community associated strains of S. aureus are a frequent cause of morbidity and mortality among patients with advanced CTCL. Here we provide a comprehensive analysis of the association between CTCL and S. aureus colonization, and use our unique pre-clinical animal model of CTCL to determine the cause-effect relationship between skin-associated S. aureus and CTCL progression. To understand the relationship between bacterial colonization and CTCL we collected skin swabs from active lesions, unaffected skin and nares of CTCL patients to perform S. aureus cultures and 16S rRNA gene sequencing. Skin swabs of psoriasis patients and healthy donors served as controls. The frequency of S. aureus colonization determined by culture based techniques revealed that &amp;gt;65% of advanced stage patients had S. aureus present at lesional/tumor sites, while corresponding sites in patients with psoriasis and in healthy controls rarely had detectable S. aureus. Colonization rates correlated positively with the disease stage. Unbiased, 16s sequencing based analysis of the skin microbiome from advanced CTCL patients revealed that the overall skin microbiome of these patients is distinct from that of healthy individuals and patients with psoriasis. A lower phylogenetic diversity and significantly higher relative abundance of Staphylococcus species was found in CTCL patients. To determine the causal relationship between skin flora and progression of CTCL we used our mouse model of CTCL and assessed disease progression in both conventionally housed specific-pathogen-free (SPF) conditions and in germ free (GF) isolators using a standardized clinical score. The CD4CreSTAT3stopfl/+ mice express a hyper-active STAT3C mutant protein selectively in T lymphocytes and virtually all mutant mice develop T cell infiltration in the epidermis causing skin lesions resembling CTCL, by eight months of age. In contrast to the SPF housed animals, GF mice remained disease free or developed only a mild phenotype (clinical score 1 out of 5) after 11 months of follow-up. Notably, when GF CD4CreSTAT3stopfl/+ mice were transitioned to SPF conditions they all developed advanced disease. Finally, we examined the role of T cell antigen receptor (TCR) signaling in mediating the transformation of T lymphocytes. R26STAT3Cstopfl/+CD4Cre Rag2KO OTII mice express only OVA-specific TCRs. T cells from R26STAT3Cstopfl/+ CD4Cre Stim1fl/fl mice express a normal TCR repertoire, but exhibit defective T cell receptor signaling due to compromised calcium influx. Both strains failed to develop typical skin lesions, suggesting an essential role for TCR interaction with tumor microenvironment and microbial antigens in the pathogenesis of CTCL. In conclusion, we demonstrate a strong correlation between CTCL staging and rates of S. aureus colonization. Our study supports a cause-effect relationship between skin flora and CTCL oncogenesis. We propose that CTCL represents an antigen driven malignancy. Further studies using mono-colonization with single bacterial strains are needed to further interrogate the role of specific bacteria. Disclosures Hymes: Celgene: Consultancy. Odum:Micreos human Health B.V: Consultancy. Geskin:Merck: Other: Supported/Contracted Research; UpToDate: Patents &amp; Royalties: Royalty, Receipt of Intellectual Property Rights / Patent Holder; Mallinckrodt: Consultancy, Honoraria, Other: Supported/Contracted Research; Helsinn: Consultancy, Honoraria, Other: Supported/Contracted Research; Stratpharma: Other: Supported/Contracted Research; Medivir: Consultancy, Honoraria; Medscape: Speakers Bureau; Actelion: Other: Supported/Contracted Research.