Allosteric inhibition reveals SHP2-mediated tumor immunosuppression in colon cancer by single-cell transcriptomics

Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING-TBK1-IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68+ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy.

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC

Tyrosine phosphatase SHP2 inhibitors in tumor-targeted therapies

Src homology containing protein tyrosine phosphatase 2 (SHP2) represents a noteworthy target for various diseases, serving as a well-known oncogenic phosphatase in cancers. As a result of the low cell permeability and poor bioavailability, the traditional inhibitors targeting the protein tyrosine phosphate catalytic sites are generally suffered from unsatisfactory applied efficacy. Recently, a particularly large number of allosteric inhibitors with striking inhibitory potency on SHP2 have been identified. In particular, few clinical trials conducted have made significant progress on solid tumors by using SHP2 allosteric inhibitors. This review summarizes the development and structure-activity relationship studies of the small-molecule SHP2 inhibitors for tumor therapies, with the purpose of assisting the future development of SHP2 inhibitors with improved selectivity, higher oral bioavailability and better physicochemical properties.

The Role of B and T Lymphocyte Attenuator in Immune Regulation and Immune Related Diseases / 中国生物化学与分子生物学报

B / T lymphuocyte attenuator (BTLA) is one of the important checkpoint molecules in immune regulation. BTLA belongs to the CD28 superfamily, and its protein structure is similar to programmed cell death-1 (PD-1) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4). BTLA is mainly expressed in B and T cells, and has also been found in some innate immune cells such as dendritic cells and monocytes. To date, the herpesvirus entry mediator (HVEM) is the only ligand for BTLA found in human cells. HVEM belongs to the Tumor necrosis factor receptor (TNFR) superfamily, and its interaction with BTLA directly connects CD28 and TNFR families. The binding of BTLA with HVEM often mediates immunosuppressive effects and plays an important role in maintaining immune tolerance. However, recent studies have found that the BTLA / HVEM pathway not only provides negative regulatory signals, but also promotes the survival of T cells. This bidirectional signaling system renders BTLA-mediated immune regulation more sophisticated and complex. Growing evidence has shown that BTLA is involved in T cells, B cells and dendritic cell-mediated immune regulation, and therefore plays an important role in a variety of immune related diseases, such as inflammation, tumor, infectious diseases and autoimmune diseases. Based on the latest research progress at home and abroad, this paper reviews the role of BTLA in immune regulation and immune related diseases.

Expression and clinical significance of B and T lymphocyte attenuator on CD4+ and CD8+ T cells from patients with pulmonary tuberculosis

Background: As an immune checkpoint, upregulation of B and T lymphocyte attenuator (BTLA) contributes to T-cell exhaustion in chronic infection. However, the characteristics of BTLA on T cells of patients with pulmonary tuberculosis (PTB) are still uncovered. Aims: The aim of the study was to elucidate the dynamics and clinical significance of BTLA expression on circulating CD4+ and CD8+ T cells of PTB patients. Materials and Methods: BTLA expression on T cells from PTB patients with smear positivity (n = 86) and healthy controls (HCs) (n = 40) were determined using flow cytometry. Results: The levels of BTLA expression on circulating CD4+ and CD8+ T cells of PTB patients with smear positivity were both upregulated, compared with HC. At the same time, the levels of BTLA expression on CD4+ and CD8+ T cells of patients with retreatment were both higher than that of those with initial treatment and gradually upregulated along with the increase of the bacillary load in sputum. In addition, the patients with lung cavity were discovered to present higher levels of BTLA expression on CD4+ and CD8+ T cells than those without lung cavity. Whereas we noted that there was no correlation between the levels of BTLA expression and the positivity or negativity of anti-Mycobacterium tuberculosis antibody. Conclusions: The levels of BTLA expression were upregulated on CD4+ and CD8+ T cells of PTB patients and associated with disease progression. Thereby, BTLA expression on T cells may be considered as a potential clinical indicator and utilized as a therapeutic target for PTB.

Expression of PD1 and BTLA on the CD8+ T Cell and γδT Cell Subsets in Peripheral Blood of Non-Small Cell Lung Cancer Patients / 中国医学科学杂志(英文版)

Objective To investigate the expression and regulation of programmed cell death protein 1 (PD1), B lymphocyte and T lymphocyte attenuator (BTLA) in peripheral blood of patients with non-small cell lung cancer (NSCLC); to examine the correlation of the mRNA levels between PD and BTLA in NSCLC. Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8+ T cells and γδ+ T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals. We compared the expression of PD1 and BTLA on the surfaces of γδ+ T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid. The correlations of PD1 and BTLA, as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform. Results The frequency of PD1 on the surfaces of CD8+ T cells was significantly higher than that of the γδT cells in both healthy controls (t=2.324, P=0.024) and NSCLC patients（t=2.498, P=0.015). The frequency of PD1 on CD8+ T cells, rather than on γδ+ T cells, was significantly upregulated in advanced NSCLC patients compared with that in healthy controls (t=4.829, P<0.001). The PD1+ BTLA+γδT cells of the healthy controls were significantly lower than that of the NSCLC patients (t=2.422, P=0.0185). No differences in percentage of PD1+γδ+ and BTLA+γδ+ T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment. PD1 was positively correlated with BTLA in both lung adenocarcinoma (r=0.54; P<0.05) and lung squamous cell carcinoma (r=0.78; P<0.05). Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8+ T cells and γδT cells in advanced NSCLC, suggesting that these molecules were involved in regulating the inactivation of CD8+ T cells and γδ+ T cells, immune escape and tumor invasion.

A study based on BTLA for mechanism exploration and correlation analysis of lung function decline of patients with KOA / 中华微生物学和免疫学杂志

Objective To observe the changes of lung parameters,the ratios of B and T lymphocyte attenuator(BTLA) and serum cytokines in patients with knee osteoarthritis (KOA),and explore possible molecular mechanism of them.Methods Forty-seven cases of knee osteoarthritis from the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine from 2011 October to 2012 July were analyzed in this study.Pulmonary parameters were detected by spirometer; B and T lymphocyte attenuator(BTLA) was detected by flow cytometry ; Interleukin (IL)-1β,IL-10,matrix metalloproteinase-9 (MMP-9),tissue inhibitor of matrix metalloprotease-1 (TIMP-1) were detected by ELISA;ESR was determined by Westergren method ; hs-CRP was determined by the automatic biochemistry analyzer.Results (1) Compared with NC group,levels of FVC,FEV1,FEV1/FVC,PEF,MEF25.75,MEF50,MEF25,CD3 + BTLA+ T cell,CD4+ BTLA+ Tcell,IL-10,TIMP1 were significantly decreased,IL-1 β,MMP9 were significantly increased.(2)Correlation analysis showed FVC was negatively correlated to Lequesne MG,symptom classify quantization scores,course,MMP9,while positively related to CD3+ BTLA+T cell,IL-10,TIMP1 ;FEV1 was positively correlated to CD3 + BTLA+T cell,CD4+ BTLA+T cell,TIMP1,while negatively correlated to course ; MEF50 was positively related to CD3+BTLA+T cell,CD4+ BTLA+T cell.Conclusion While articular cartilage lesions occurred in KOA patients,the lung function was also declined.The mechanism may be associated with the declination of expression of BTLA,which can cause up-regulating of IL-1 β,MMP9 and down-regulating of IL-10,TIMP1,thus leading to immune dysfunction and abnormal immune response.Those may induce airway injuries and result in lung function decline finally.

Progress on AmpC hyperproduction in Escherichia coli / 医学研究生学报

The ampC ?-lactamases gene in Escherichia coli(E.coli) is different from other Gram-negative bacteria.E.coli contains a chromosomal ampC gene which has a weak promoter as well as a transcriptional attenuator.The promoter of the ampC gene in E.coli is part of the preceding frd operon,the attenuator of the ampC gene is a transcription terminator for the frd operon.The ampC regulatory gene,ampR,is absent.Strains carrying the wild-type gene produce a low basal amount of AmpC.Studies on the molecular basis of AmpC overproduction in E.coli have shown that some hyperproducers contain mutation in the promoter region and/or attenautor and/or ampC-coding region of ampC,while others contain more than one copy of ampC.Acquisition of a stronger promoter or insertion of an insertion element containing promoter sequences or regulatory gene ampR has also been proposed as the molecular basis of hyperproduction of AmpC in some E.coli strains.Plasmid-mediated AmpC ?-lactamases have been discovered frequently in E.coli strains.This is another reason for hyperproduction of AmpC ?-lactamases.