Adaptor Protein SLP-76 Research Articles

ProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting

Determining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling. With ProDOL various factors affecting labeling efficiency, including substrate type, incubation time and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how human immunodeficiency virus-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adapter protein SLP-76 in signaling microclusters. ProDOL proved to be a versatile and robust tool for labeling calibration, enabling determination of labeling efficiencies, optimization of strategies and quantification of protein stoichiometry.

Design, synthesis, and biological evaluation of 2,4-diaminopyrimidine derivatives as potent Hematopoietic Progenitor Kinase 1 (HPK1) inhibitors

HPK1 also referred to as MAP4K1, belongs to the category of mammalian STE20-like protein serine/threonine kinases. Its physiological function involves the down-regulation of T cell signals, and it is regarded as a new immune checkpoint of tumor immunology. In this study, we commenced our investigation with the hit compounds, focusing the efforts on structural optimization and SAR exploration to identify a novel class of 2,4-diaminopyrimidine HPK1 inhibitors. Notably, compound 14g exhibited a remarkable inhibitory effect on HPK1 kinase (IC50 = 0.15 nM), significantly suppressed the phosphorylation of the downstream adaptor protein SLP76 (pSLP76 IC50 = 27.92 nM), and effectively stimulated the secretion of the T cell activation marker IL-2 (EC50 = 46.64 nM). In vitro microsomal stability assay, compound 14g showed moderate stability in HLMs with T1/2 = 38.2 min and CLint = 36.4 µL·min−1·mg−1 proteins. In vivo pharmacokinetic studies, compound 14g demonstrated heightened plasma exposure (AUC0-inf = 644 ng·h·mL−1), extended half-life (T1/2 = 9.98 h), and reduced plasma clearance (CL = 52.3 mL·min−1·kg−1) compared to the reference compound after a single intravenous dose of 2 mg/kg in rats. These results indicated that compound 14g emerged as a promising inhibitor of HPK1.

SHP-1 localization to the activating immune synapse promotes NK cell tolerance in MHC class I deficiency.

Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I-low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A+ NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A+ NK cells by the MHC-I molecule H2Dd led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6, which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2Dd-educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance.

Abstract 1821: Discovery of XNW21015, a novel, potent HPK1 inhibitor with excellent immune modulatory activity

Abstract Hematopoietic progenitor kinase 1 (HPK1), also known as MAP4K1, is a member of the STE20 family of serine/threonine kinases, and restrictedly expressed in hematopoietic cells. In T cells, HPK1 negatively regulated TCR signaling by phosphorylating the adaptor protein SLP76 on Ser376, which leads to the dissociation of the SLP-76/LAT signalosomes and downregulation of T cell priming and activation. HPK1 knockout (KO) or kinase-inactive (KI) mice spontaneously reject tumors with an enhanced immune response compared to wild-type ones. Thus, HPK1 is a novel immuno-oncology (I/O) target. In this work, we described the discovery of a potent HPK1 inhibitor starting from comprehensive SAR-based design with guidance from X-ray crystallography and computational modeling. These efforts led to identification of the potent azaindole inhibitor XNW21015, with excellent kinase selectivity and promising PK profile of low clearance and sufficient exposure for in vivo animal studies. XNW21015 dose-dependently inhibited phosphorylation of SLP76(Ser376) in vitro and in vivo in response to anti-CD3 agonist antibody, with concomitant enhancement in T cell activation. Oral administration of XNW21015 in mouse syngeneic tumor models showed favorable PK profile and robust anti-tumor activity as both single agent and in combination with immune checkpoint inhibitors. In a humanized PDX (patient-derived xenograft) model, XNW21015 enhanced tumor-infiltrated lymphocytes (TIL). In summary, we have discovered a novel, highly potent and selective HPK1 inhibitor XNW21015 as promising small molecule I/O agent, as monotherapy or in combination with immune checkpoint inhibitors. Citation Format: Yuchuan Wu, Rui Hao, Xi Chen, Xiao Liu, Yonghua Xie, Shihua Wang, Bingquan Gao, Jichun Wen, Zhenwei Wu, Wengui Wang, Haiyang Wei, Yonghan Hu, Xiaojun Liu, Meijie Le, Jing Qiang. Discovery of XNW21015, a novel, potent HPK1 inhibitor with excellent immune modulatory activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1821.

Abstract 2122: CRD1600 is a potent HPK-I inhibitor with robust immune modulatory properties

Abstract Background: Haematopoietic Progenitor Kinase (HPK-1, MAP4K1) is a member of the STE20 family of serine/threonine kinases, expressed predominantly in hematopoietic cells. The kinase activity of HPK-1 provides an inhibitory signal that dampens immune responses1. Upon activation of T-cell and/or B-cell receptors, activated HPK-1 phosphorylates the adaptor protein SLP76 at Ser376 in T cells or BLNK at Thr152 in B-cells to trigger proteasomal degradation of the adaptors and disrupt downstream T-cell or B-cell activating signalosome complexes. Blocking HPK-1 extends immune action and since HPK-1 expression is primarily restricted to immune cells, it is a compelling drug target for immune modulation. Anti-tumor efficacy has been demonstrated in HPK-1 kinase dead knock-in mice, HPK-1 knockout mice or using small molecule pharmacological inhibitors of HPK-1 either as monotherapy or in combination regimens2. Methods: Curadev used a pharmacophore based approach to discover novel small molecule HPK-1 inhibitors. SAR studies were carried out using a screening funnel that included efficacy assessment by monitoring in vitro enzymatic and cellular activity, anti-target cross-kinase specificity by assessing cytotoxicity in fresh murine splenocytes and effector mobilization by monitoring IL2 production and human T-cell proliferation under anti-CD3/CD28 stimulation. Results: Curadev’s medicinal chemistry campaign yielded several highly potent HPK-1 inhibitors with sub-nanomolar to nanomolar potency in in vitro human HPK-1 enzyme assays which were active in the cellular SLP76 assay. A single dose of the lead compound CRD1600 to mice treated with an anti-CD3 antibody caused systemic enhancement of pro-inflammatory cytokines such as IL2 and reduced pSLP76 levels in the spleen. These pharmacodynamic effects correlated with plasma levels of CRD1600. Conclusions: Inhibition of HPK-1 is a promising therapeutic modality that could augment the effects of existing anti-cancer treatments including immunotherapy. CRD1600 is a potent, selective HPK-1 inhibitor that has been selected from a rich portfolio of active compounds and shows in vivo activity against the target. Further development of the compound is ongoing.

Abstract 3517: Pharmacologic inhibition of HPK1 kinase activity enhances immune cell activation and T cell mediated anti-tumor activity

Abstract Hematopoietic progenitor kinase 1 (HPK1, mitogen-activated kinase kinase kinase kinase 1 [MAP4K1]), a cytosolic STE20 ser/thr kinase from the germinal center family of kinases, regulates the function of diverse immune populations including T cells, B cells, and dendritic cells. In T cells, activated HPK1 regulates T cell receptor (TCR) signaling by phosphorylating the adaptor protein SLP76 on Ser376, which induces the association of SLP76 with 14-3-3 proteins. This SLP76/14-3-3 complex leads to its disassociation from the TCR signalosome, resulting in reduced downstream TCR signaling. The kinase activity of HPK1 is necessary for its regulatory function as mice expressing a mutant kinase-inactive HPK1 demonstrate enhanced T cell activity and superior tumor growth control compared to their WT counterparts. Here we describe a highly potent, selective and orally bioavailable small molecule inhibitor of HPK1 which displays sub-nanomolar HPK1 inhibition, good selectivity against MAP4K family members and oral bioavailability in preclinical animal models. Pharmacological inhibition of HPK1 kinase activity with Gilead’s HPK1 inhibitor compound demonstrates robust inhibition of pSLP76 S376 with concomitant enhancement in T cell activation, proliferation and effector function in in vitro assays. These immune enhancing effects translate into in vivo preclinical tumor models, where treatment with Gilead’s HPK1 inhibitor compound results in heightened T cell activation and effector function and enhanced tumor growth control both as a single agent and in combination with PD-1 blockade. In summary, our data show that Gilead’s HPK1 inhibitor compound is expected to be a selective, orally bioavailable HPK1 inhibitor with potential to combine with checkpoint inhibitors and other immuno-oncology agents. Citation Format: Jacob S. Lee, Pamela Toy, Gundula Min-Oo, Tony Lee, Patrick Salvo, JeanPhilippe Belzile, Sheila Clancy, Jonathan Nazareno, Mengshu Xu, Weimei Xing, Marilyn Giacomini, Rex Santos, Lance Stapleton, Adele Wang, Vladislava Juric, Scott A. Mitchell, Michelle Kuhne. Pharmacologic inhibition of HPK1 kinase activity enhances immune cell activation and T cell mediated anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3517.

Abstract 5541: BGB-15025, a potent and selective HPK1 inhibitor, is efficacious as a single agent or in combination with PD-1 antibody in multiple tumor models

Abstract Hematopoietic progenitor kinase 1 (HPK1), a hematopoietic cell-restricted serine/threonine protein kinase, has been reported to serve as a critical negative feedback regulator of T lymphocytes by phosphorylating the adaptor protein SLP76 in the TCR complex. The negative feedback role of HPK1 in TCR signaling makes it a promising target for immuno-oncology therapy. It has been recently demonstrated that the kinase activity of HPK1 is essential for its antitumor activity, and HPK1 inhibitors can be potentially combined with immune checkpoint inhibitor therapy (such as PD-1 antibody) for effective cancer treatment. Here, we report a potent and selective HPK1 inhibitor BGB-15025 which has been identified through in-house high throughput screening and structure-guided drug design. BGB-15025 potently inhibits HPK1 kinase activity in biochemical assay, with a 50% inhibitory concentration (IC50) of 1.04 nM under Km concentration of ATP. Consistently, BGB-15025 can potently reduce SLP76 phosphorylation and increase downstream ERK phosphorylation in a concentration dependent manner in T cells-based assay. As a consequence, BGB-15025 induces TCR activation and IL-2 production in T cells. Based on in-house profiling, BGB-15025 shows good selectivity profile among MAP4K family and does not affect ZAP70 phosphorylation up to 1 μM. Oral administration of BGB-15025 demonstrates dose-dependent pSLP76 inhibition in splenic T cells and induces serum IL-2 in mouse model. In efficacy studies, BGB-15025 exhibits anti-tumor activity in GL261 tumor model as single agent. Moreover, BGB-15025 demonstrated combination effect with anti-PD-1 antibody in CT26 and EMT-6 syngeneic tumor models. BGB-15025 is currently in phase I clinical trial to treat patients with advanced solid tumor, both as a single agent and in combination with anti PD-1 antibody tislelizumab. Citation Format: Ye Liu, Jing Li, Zhuo Li, Qin Wang, Xing Zhou, Xiaoxin Liu, Bo Zhang, Xitao Wang, Sanjia Xu, Hanzi Sun, Xiaomin Song, Xi Yuan, Zhiwei Wang, Xuesong Liu. BGB-15025, a potent and selective HPK1 inhibitor, is efficacious as a single agent or in combination with PD-1 antibody in multiple tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5541.

The Small GTPase RHOA Links SLP65 Activation to PTEN Function in Pre B Cells and Is Essential for the Generation and Survival of Normal and Malignant B Cells.

The generation, differentiation, survival and activation of B cells are coordinated by signals emerging from the B cell antigen receptor (BCR) or its precursor, the pre-BCR. The adaptor protein SLP65 (also known as BLNK) is an important signaling factor that controls pre-B cell differentiation by down-regulation of PI3K signaling. Here, we investigated the mechanism by which SLP65 interferes with PI3K signaling. We found that SLP65 induces the activity of the small GTPase RHOA, which activates PTEN, a negative regulator of PI3K signaling, by enabling its translocation to the plasma membrane. The essential role of RHOA is confirmed by the complete block in early B cell development in conditional RhoA-deficient mice. The RhoA-deficient progenitor B cells showed defects in activation of immunoglobulin gene rearrangement and fail to survive both in vitro and in vivo. Reconstituting the RhoA-deficient cells with RhoA or Foxo1, a transcription factor repressed by PI3K signaling and activated by PTEN, completely restores the survival defect. However, the defect in differentiation can only be restored by RhoA suggesting a unique role for RHOA in B cell generation and selection. In full agreement, conditional RhoA-deficient mice develop increased amounts of autoreactive antibodies with age. RHOA function is also required at later stage, as inactivation of RhoA in peripheral B cells or in a transformed mature B cell line resulted in cell loss. Together, these data show that RHOA is the key signaling factor for B cell development and function by providing a crucial SLP65-activated link between BCR signaling and activation of PTEN. Moreover, the identified essential role of RHOA for the survival of transformed B cells offers the opportunity for targeting B cell malignancies by blocking RHOA function.

Reciprocal SH2-SH3 Domain Contacts between ITK Molecules Limit T Cell Receptor Signaling in Th2-type CD4+ T Cells

ABSTRACT The nonreceptor tyrosine kinase ITK is a key component of the T cell receptor (TCR) signaling pathway and is required for cytokine production by CD4+ T cells that have differentiated into Th2 cells. Structural and biochemical studies suggest that contacts between the SH2 and SH3 domains of ITK mediate intermolecular self-association, forming a structure that restrains ITK activity by interfering with interactions between ITK and other components of the TCR signaling pathway. Wild-type (WT) ITK and a panel of ITK mutants containing amino acid substitutions in the SH2 and SH3 domains were tested for self-association and for binding to the adaptor protein SLP76, a key ligand for the ITK SH2 domain. WT and ITK mutants were also expressed in Itk-deficient CD4+ T cells via retroviral-mediated gene delivery to analyze their ability to support TCR signaling and cytokine production by Th2 cells. Specific amino acid substitutions in the ITK SH2 or SH3 domains impaired self-association, with the greatest effects being seen when both intermolecular SH2-SH3 domain contacts were disrupted. Two of the SH2 domain substitutions tested reduced ITK self-association but had no effect on binding to SLP-76. When their function was analyzed in Th2 cells, ITK proteins with diminished self-association activity supported greater IL-4 production and calcium flux in response to TCR stimulation compared to WT ITK. Our findings indicate that intermolecular contacts between ITK molecules can restrain the amplitude of TCR signaling, suggesting ITK is a limiting factor for responses by CD4+ T cells.

Abstract P241: A highly potent HPK1 inhibitor augments immune cell activation and anti-tumor immunity in a syngeneic tumor mouse model

Abstract Background: HPK1 (MAP4K1) is a serine/threonine Ste20-related protein kinase that belongs to the mitogen-activated protein kinase (MAPK) family. HPK1 is mainly expressed in hematopoietic cells and serves as a negative regulator of anti-tumor immunity through modulating the activation of lymphocytes and dendritic cells. Upon TCR activation, HPK1 phosphorylates the adaptor protein SLP76 at Ser376 to destabilize the SLP76 microclusters, which leads to the attenuation of the TCR signaling. The reported anti-tumor efficacy data from HPK1 knockout and kinase-dead knock-in mouse models support HPK1 as a novel intracellular I/O target. Methods: Regor CARD platform (Computer Accelerated Rational Discovery) was deployed to identify potent and selective inhibitors of HPK1. Biochemical assays and primary human pan T cell-based cellular assays were utilized to support the structure-activity relationship (SAR) analysis and inhibitor optimization. In vitro and in vivo target engagement studies were conducted in Jurkat T cells and mouse splenocytes, respectively. In vivo efficacy study data were generated using CT-26 syngeneic tumor mouse model. Results: Highly potent HPK1 inhibitors with good selectivity against the liable immune kinases were identified. RGT-197, one of the lead inhibitors demonstrated a significant increase of IL-2 secretion in primary human pan T cells upon TCR activation. It inhibited TCR-induced phosphorylation of SLP76 at Ser-376 in vitro and in vivo. Anti-tumor efficacy was observed in CT-26 tumor-bearing mice by oral dosing of RGT-197 as a single agent and in combination with an anti-PD1-monoclonal antibody. Furthermore, RGT-197 increased the level of cytokines important for anti-tumor immunity in vivo. Conclusion: RGT-197, a potent and selective HPK1 inhibitor, provides a potential opportunity as a small molecule I/O agent to boost anti-tumor immunity either as monotherapy or in combination with immune checkpoint inhibitors. Citation Format: Hao Liu, Lei Wu, Song Feng, Wei Huang, Huijuan Li, Jing Lin, Yangyang Liu, Liufeng Mei, Baoqi Ren, Julie Xie, Lili Yao, Wenge Zhong. A highly potent HPK1 inhibitor augments immune cell activation and anti-tumor immunity in a syngeneic tumor mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P241.

Abstract 1646: Development of small-molecule HPK1 inhibitors to unleash tumor-specific T cell responses

Abstract Hematopoietic progenitor kinase 1 (HPK1) is an intracellular protein kinase that negatively regulates T cell signaling and proliferation. Upon T cell receptor (TCR) activation, active HPK1 phosphorylates the adaptor protein SLP76 in the TCR complex, recruiting the negative regulator 14-3-3 and targeting components of the TCR signaling complex for degradation. HPK1 thus limits the TCR signaling important for mounting an effective immune response against tumor cells. We are employing structure-guided drug design to develop potent small-molecule inhibitors of HPK1. Our compounds potently inhibit HPK1 in biochemical assays, reduce levels of phosphorylated SLP76 and concomitantly increase IL-2 production by Jurkat T cells. Importantly, our HPK1 inhibitors enhance cytokine production by human and mouse primary T cells above that observed with TCR activation alone. Treatment of mice with orally available HPK1 inhibitors results in increased activation of antigen-specific CD8+ T cells in vivo and decreased tumor growth as single agent and in combination with clinically relevant checkpoint inhibitor antibodies. Our work confirms the importance of HPK1 for T cell function and supports HPK1 as a promising next generation immuno-oncology target. Citation Format: George Katibah, Yamini Ohol, Cyril Bucher, Lavanya Adusumilli, Deepika Kaveri, Omar Robles, Michael Sun, Cynthia Cho, Heather Milestone, Rachel Ames, Scott Jacobson, Dan Nebalasca, Justy Gomez- Guagua, Jerick Sanchez, Molly Grandcolas, Steve Wong, Martin Brovarney, Chandru Ramana, Thant Zaw, Lan Nguyen, Parcharee Tivitmahaisoon, Andrew Ng, Anqi Ma, Blanca Gomez, Michelle Ko, Paul Leger, Jeffrey Jackson, Grant Shibuya, Anton Shakhmin, Delia Bradford, Mengshu Xu, Mikhail Zibinsky, Daniel Poon, David Wustrow, Paul Kassner, Dirk Brockstedt. Development of small-molecule HPK1 inhibitors to unleash tumor-specific T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1646.

Targeting SLP76:ITK interaction separates GVHD from GVL in allo-HSCT

Abstract Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for relapsed hematological malignancies due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also primarily mediated by the same donor T cells. Here we assessed the effect of attenuating TCR-mediated SLP76:ITK interaction in T cell-mediated GVL vs. GVHD effects after allo-HSCT. Neither CD8+ T cells nor CD4+ donor T cells from mice expressing a tyrosine to phenylalanine mutation at position 145 (Y145F) of the adapter protein SLP-76 caused GVHD, T cells cells preserved GVL effects after allogeneic transplantation. SLP76Y145FKI CD8+ and CD4+ donor T cells also produce less inflammatory cytokines and show decreased migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). We also report the development of a novel peptide that can specifically inhibit SLP76 and ITK interactions, which results in decreased phosphorylation of PLCγ1 and ERK, and decreased cytokine production in human T cells. This peptide inhibited donor T cell-mediated GVHD while maintaining GVL effects. Altogether, our data suggest that inhibiting SLP76:ITK interaction could be a therapeutic strategy to reduce GVHD while retaining the beneficial GVL effects after allo-HSCT treatment.

Identification of Potent Reverse Indazole Inhibitors for HPK1.

Hematopoietic progenitor kinase (HPK1), a negative regulator of TCR-mediated T-cell activation, has been recognized as a novel antitumor immunotherapy target. Structural optimization of kinase inhibitor 4 through a systematic two-dimensional diversity screen of pyrazolopyridines led to the identification of potent and selective compounds. Crystallographic studies with HPK1 revealed a favorable water-mediated interaction with Asp155 and a salt bridge to Asp101 with optimized heterocyclic solvent fronts that were critical for enhanced potency and selectivity. Computational studies of model systems revealed differences in torsional profiles that allowed for these beneficial protein-ligand interactions. Further optimization of molecular properties led to identification of potent and selective reverse indazole inhibitor 36 that inhibited phosphorylation of adaptor protein SLP76 in human PBMC and exhibited low clearance with notable bioavailability in in vivo rat studies.

Targeting adaptor protein SLP76 of RAGE as a therapeutic approach for lethal sepsis

Accumulating evidence shows that RAGE has an important function in the pathogenesis of sepsis. However, the mechanisms by which RAGE transduces signals to downstream kinase cascades during septic shock are not clear. Here, we identify SLP76 as a binding partner for the cytosolic tail of RAGE both in vitro and in vivo and demonstrate that SLP76 binds RAGE through its sterile α motif (SAM) to mediate downstream signaling. Genetic deficiency of RAGE or SLP76 reduces AGE-induced phosphorylation of p38 MAPK, ERK1/2 and IKKα/β, as well as cytokine release. Delivery of the SAM domain into macrophages via the TAT cell-penetrating peptide blocks proinflammatory cytokine production. Furthermore, administration of TAT-SAM attenuates inflammatory cytokine release and tissue damage in mice subjected to cecal ligation and puncture (CLP) and protects these mice from the lethality of sepsis. These findings reveal an important function for SLP76 in RAGE-mediated pro-inflammatory signaling and shed light on the development of SLP76-targeted therapeutics for sepsis.

CD28 Expression Distinguishes Plasma Cell Fate in Pediatric Patients Suffering from COVID-19/Kawasaki's Disease Vs MIS-C

The COVID-19 pandemic has ravaged the global community and highlighted the importance of antibody-mediated antiviral immune responses. The SARS-CoV-2 virus is highly pathogenic, but is unique from other viral infections in that pediatric patients are largely spared from severe symptoms. However, a small number of pediatric patients present autoimmune-like symptoms after COVID-19 infection, termed Multisystem Inflammatory Syndrome in Children, or MIS-C. Symptomatically, it shares some similarity to that of Kawasaki's Disease (KD), an autoimmune disorder linked to coronavirus infection thought to be driven by autoantibody production. Understanding the immunological mechanisms that facilitate clearance of SARS-CoV-2 or drive the development of life-threatening autoimmune symptoms in MIS-C or KD is critical for our ability to design successful vaccines that do not elicit autoimmunity in children.Antibodies are produced by terminally differentiated B lymphocytes known as Plasma Cells (PC). Because successful immune responses/vaccination strategies against SARS-CoV-2 are dependent upon effective PC production, the dysregulation of which may lead to the development of Kawasaki's Disease or MIS-C, it is critical to understand the immunological mechanisms that define the pediatric PC response leading to these individual outcomes.We therefore sought to characterize the B cell-PC immune response in pediatric patients that have successfully cleared SARS-CoV-2 and compare the B cell immunological landscape to children who develop either KD or MIS-C. We used the 10X Genomics Platform to interrogate at a single cell level the CD19+ B/PC populations in the peripheral blood of pediatric patients at the transcriptional level as well as with VDJ deep sequencing.We began by clustering the CD19+ populations based on transcriptional similarity and found that both COVID-19 and KD exhibited 12 distinct clusters, but that MIS-C only had 6 clusters. Intriguingly, in evaluating clonal diversity, KD presented a broad spectrum of clonotypes while the COVID-19 and MISC patients were much more limited. This suggested that although KD and MIS-C both present with similar autoimmune-like symptoms, the mechanistic basis for their respective etiology may be distinct. We therefore sought to more deeply probe the subset of cells responsible for antibody production by evaluating the PC subsets.High BLIMP1 expression (PC lineage-defining transcription factor) was present in 2 distinct clusters in COVID-19/KD patients, but was broadly distributed at lower levels in MIS-C. We then looked at genes which were significantly upregulated in the most terminally differentiated cells from each patient. 50 genes were significantly higher in the COVID-19 and KD populations, and 54 in MIS-C. Using gene ontology analysis in COVID-19 and KD, we saw increased expression of transcripts involved in protein trafficking, redox responses, and respiratory metabolism. In comparison, the MIS-C patient demonstrated significance for programs involved in immature B cell development and inflammation. Taken together, this suggests that in COVID-19 and KD there is a program of terminal PC differentiation which is absent in MIS-C. To understand the mechanistic basis for the terminal differentiation in COVID-19 and KD, we sought to probe possible regulators of PC fate.We have recently published that CD28, the canonical T cell costimulatory molecule, is expressed by PC, and CD28 signaling through the adaptor protein SLP-76 leads to increased BLIMP1 expression and an IRF4-mediated metabolic program necessary for PC survival. Interestingly, SLP-76 was expressed at high levels in COVID-19 and KD patients, but was low in MIS-C. Similarly, CD28 was expressed in both COVID-19 and KD patients and correlated with higher IRF4 levels and metabolic genes, but was entirely absent in MIS-C.Taken together, these findings suggest that CD28 signaling may facilitate PC fate in COVID-19 pediatric patients and the development of KD arises from broad antibody specificity, possibly explaining how vascular antigens become targets. Furthermore, MIS-C, although similar in symptomatic presentation to KD, has an etiology driven by antigen-independent inflammation arising from immature B cells due to a lack of CD28-mediated PC differentiation and survival, which can be evaluated diagnostically by simple flow cytometry in a vaccination setting.DisclosuresNo relevant conflicts of interest to declare.

Targeting SLP76/ITK Signaling Interaction Separates GBHD from GVL in Allo-HSCT

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for relapsed hematological malignancies, due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also primarily mediated by the same donor T cells. Here we assessed the role of T cells with attenuated TCR-mediated ITK activation in mediating GVL vs GVHD effects after allo-HSCT. T cells from mice with genomic knock-in to express a tyrosine to phenylalanine mutation at position 145 (Y145F) of the adaptor protein SLP-76 display defective TCR-mediated activation of the downstream kinase ITK. Alloreactive T cells from SLP76 Y145F knock-in (KI) mice did not cause GVHD but preserved cytotoxicity, accompanied by upregulation of Eomesodermin (Eomes), which was necessary for GVL function. We developed a novel peptide that can specifically inhibit SLP76 and ITK interactions, resulting in decrease phosphorylation of PLCγ1, ERK and reduce cytokine production in human T cells. This peptide inhibited donor T cell-mediated GVHD while maintaining GVL effects. Altogether, our data suggest that inhibiting SLP-76/ITK interaction could be a therapeutic strategy to reduce GVHD while maintaining the beneficial GVL effects after allo-HSCT treatment.

Abstract 5317: A high throughput screen of the novel immuno-oncology target HPK1 identifies a range of chemical starting points for the development of potent and selective inhibitors

Abstract The concept of harnessing the immune system to target cancer cells has been an active area of research for decades. The advent of antibodies targeting the checkpoint receptors CTLA-4 and PD-1/PDL-1, have now provided definitive clinical validation for this approach. Since these discoveries the search for small molecule immuno-oncology agents have intensified. Here we present data on HPK1 (hematopoietic progenitor kinase 1), a novel immuno-oncology kinase involved in the negative regulation of T-cell receptor (TCR) signalling and describe a high throughput screen to identify novel chemical starting points to develop potent and selective inhibitors of this kinase. HPK1 is a member of the Ste-20 family of Serine/Threonine protein kinases. It is expressed highly in cells of haematopoietic lineage, including T-cells and is activated upon engagement of the TCR with cell surface MHC complexes. Active HPK1 leads to phosphorylation of an adaptor protein SLP76, triggering a signalling cascade that results in the downregulation of TCR signalling and thus downregulation of T-cell function. Recent reports have demonstrated that the kinase activity is essential for the function of HPK1 in T-cells and support the hypothesis that small molecule inhibitors of HPK1 kinase activity will result in sustained activation of T-cells. To identify inhibitors of HPK1 we developed an enzyme activity assay that could be used in a high throughput assay to screen Sygnature Discovery's proprietary screening library. We utilised the intrinsic ATPase activity of HPK1 to develop an ADP product fluorescent polarisation assay suitable for an HTS screen. We validated the assay using a series of known inhibitors with a broad range of potencies versus HPK1 activity. Next, we developed a cell-based assay encompassing anti-CD3 TCR mediated induction of phosphorylation of SLP76 in Jurkat cells. We tested the activity of our panel of HPK1 tool inhibitors in this assay and demonstrated a strong correlation between the biochemical and cell potencies of the compounds, further validating the robustness and cell potency predictivity of the biochemical assay. The biochemical assay was then used to screen our small molecule chemical library to identify potential inhibitors of HPK1 kinase activity. This library is biased toward lead-like chemical space and has undergone significant triage to remove unwanted chemotypes and PAINs moieties. The initial hits were confirmed and IC50 values determined for a select group of chemotypes. These compounds were subsequently tested in the Jurkat pSLP76 cell assay for cellular activity versus HPK1. In summary we present the results of a successful HTS and hit evaluation to identify novel and attractive chemical starting points for the development of inhibitors of the immuno-oncology target HPK1. Citation Format: Denise Swift, Samantha Hitchin, Chris Tomlinson, Anindita Sengupta, Callum Taylor, Alec O'Keeffe, Grace Boden, John Unitt, Stuart Thomson, Allan M. Jordan. A high throughput screen of the novel immuno-oncology target HPK1 identifies a range of chemical starting points for the development of potent and selective inhibitors [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 5317.

Vav family proteins constitute disparate branching points for distinct BCR signaling pathways.

Antigen recognition by B-cell antigen receptors (BCRs) activates distinct intracellular signaling pathways that control the differentiation fate of activated B lymphocytes. BCR-proximal signaling enzymes comprise protein tyrosine kinases, phosphatases, and plasma membrane lipid-modifying enzymes, whose function is furthermore coordinated by catalytically inert adaptor proteins. Here, we show that an additional class of enzymatic activity provided by guanine-nucleotide exchange factors (GEFs) of the Vav family controls BCR-proximal Ca2+ mobilization, cytoskeletal actin reorganization, and activation of the PI3 kinase/Akt pathway. Whereas Vav1 and Vav3 supported all of those signaling processes to different extents in a human B-cell model system, Vav2 facilitated Actin remodeling, and activation of Akt but did not promote Ca2+ signaling. On BCR activation, Vav1 was directly recruited to the phosphorylated BCR and to the central adaptor protein SLP65 via its Src homology 2 domain. Pharmacological inhibition or genetic inactivation of the substrates of Vav GEFs, small G proteins of the Rho/Rac family, impaired BCR-induced Ca2+ mobilization, probably because phospholipase Cγ2 requires activated Rac proteins for optimal activity. Our findings show that Vav family members are key relays of the BCR signalosome that differentially control distinct signaling pathways both in a catalysis-dependent and -independent manner.

Roscovitine enhances All-trans retinoic acid (ATRA)-induced leukemia cell differentiation: Novel effects on signaling molecules for a putative Cdk2 inhibitor

All-trans retinoic acid (ATRA)-based differentiation therapy has been unsuccessful in treating t(15;17) negative acute myeloid leukemia (AML) patients, motivating interest in combination therapies using ATRA plus other agents. Using the t (15, 17) negative HL-60 human myeloblastic leukemia model, we find that the cyclin-dependent kinase (CDK) inhibitor, roscovitine, augments signaling by an ATRA-induced macromolecular signalsome that propels differentiation and enhances ATRA-induced differentiation. Roscovitine co-treatment enhanced ATRA-induced expression of pS259- pS289/296/301- pS621-c-Raf, pS217/221-Mek, Src Family Kinases (SFKs) Lyn and Fgr and SFK Y416 phosphorylation, adaptor proteins c-Cbl and SLP-76, Vav, and acetylated 14–3-3 in the signalsome. Roscovitine enhanced ATRA-induced c-Raf interaction with Lyn, Vav, and c-Cbl. Consistent with signalsome hyper-activation, roscovitine co-treatment enhanced ATRA-induced G1/0 arrest and expression of differentiation markers, CD11b, ROS and p47 Phox. Because roscovitine regulated Lyn expression, activation and partnering, a stably transfected Lyn knockdown was generated from wt-parental cells to investigate its function in ATRA-induced differentiation. Lyn-knockdown enhanced ATRA-induced up-regulation of key signalsome molecules, c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, Vav1, SLP-76, and Fgr, but with essentially total loss of pY416-SFK. Compared to ATRA-treated wt-parental cells, differentiation markers p47 phox, CD11b, G1/G0 arrest and ROS production were enhanced in ATRA-treated Lyn-knockdown stable transfectants, and addition of roscovitine further enhanced these ATRA-inducible markers. The Lyn-knockdown cells expressed slightly higher c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, and SLP-76 than wt-parental cells, and this was associated with enhanced ATRA-induced upregulation of Fgr and cell differentiation, consistent with heightened signaling, suggesting that enhanced Fgr may have compensated for loss of Lyn to enhance differentiation in the Lyn-knockdown cells.

Molecular design and optimization of hepatic cancer SLP76-derived PLCγ1 SH3-binding peptide with the systematic N-substitution of peptide PXXP motif.

The phospholipase Cγ1 (PLCγ1) is essential for T-cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP-containing decapeptide segment (185 QPPVPPQRPM194 , termed as SLP76185-194 peptide) of adaptor protein SLP76 following T-cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76185-194 PXXP motif with nonnatural N-substituted amino acids, as the proline is the only endogenous N-substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate-sized N-substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N-substituted peptides as well as native SLP76185-194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence-based assays. Two N-substituted peptides, SLP76185-194 (N-Leu187/N-Gln190) and SLP76185-194 (N-Thr187/N-Gln190), are designed to have high potency (Kd =0.67±0.18 and 1.7±0.3μM, respectively), with affinity improvement by, respectively, 8.5-fold and 3.4-fold relative to native peptide (Kd =5.7±1.2μM).