Immune Checkpoint LAG3 and Its Ligand FGL1 in Cancer

LAG3 is the most promising immune checkpoint next to PD-1 and CTLA-4. High LAG3 and FGL1 expression boosts tumor growth by inhibiting the immune microenvironment. This review comprises four sections presenting the structure/expression, interaction, biological effects, and clinical application of LAG3/FGL1. D1 and D2 of LAG3 and FD of FGL1 are the LAG3-FGL1 interaction domains. LAG3 accumulates on the surface of lymphocytes in various tumors, but is also found in the cytoplasm in non-small cell lung cancer (NSCLC) cells. FGL1 is found in the cytoplasm in NSCLC cells and on the surface of breast cancer cells. The LAG3-FGL1 interaction mechanism remains unclear, and the intracellular signals require elucidation. LAG3/FGL1 activity is associated with immune cell infiltration, proliferation, and secretion. Cytokine production is enhanced when LAG3/FGL1 are co-expressed with PD-1. IMP321 and relatlimab are promising monoclonal antibodies targeting LAG3 in melanoma. The clinical use of anti-FGL1 antibodies has not been reported. Finally, high FGL1 and LAG3 expression induces EGFR-TKI and gefitinib resistance, and anti-PD-1 therapy resistance, respectively. We present a comprehensive overview of the role of LAG3/FGL1 in cancer, suggesting novel anti-tumor therapy strategies.

The p38 MAPK Components and Modulators as Biomarkers and Molecular Targets in Cancer

The mitogen-activated protein kinase (MAPK) family is an important bridge in the transduction of extracellular and intracellular signals in different responses at the cellular level. Within this MAPK family, the p38 kinases can be found altered in various diseases, including cancer, where these kinases play a fundamental role, sometimes with antagonistic mechanisms of action, depending on several factors. In fact, this family has an immense number of functionalities, many of them yet to be discovered in terms of regulation and action in different types of cancer, being directly involved in the response to cancer therapies. To date, three main groups of MAPKs have been identified in mammals: the extracellular signal-regulated kinases (ERK), Jun N-terminal kinase (JNK), and the different isoforms of p38 (α, β, γ, δ). In this review, we highlight the mechanism of action of these kinases, taking into account their extensive regulation at the cellular level through various modifications and modulations, including a wide variety of microRNAs. We also analyze the importance of the different isoforms expressed in the different tissues and their possible role as biomarkers and molecular targets. In addition, we include the latest preclinical and clinical trials with different p38-related drugs that are ongoing with hopeful expectations in the present/future of developing precision medicine in cancer.

The Effect of IL-6-Primed Platelets on ADAMTS13-Mediated Clearance of Platelet-Bearing ULVWF and Its Mechanism

Inflammation is an essential contributing factor in the development of thrombosis. Using a microfluidic flow chamber, we investigated how the proinflammatory cytokine interleukin 6 (IL-6) affects the cleavage of platelet-bearing ultra-large VWF (ULVWF) by plasma ADAMTS13. We found that IL-6-treated platelets perfused at arteriolar shear stress significantly enhanced the ULVWF-platelet complex formation on activated endothelial cells and suppressed their clearance by ADAMTS13 under flow conditions. We also detected the phosphorylation of the serine/threonine kinase Akt and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in platelets treated with IL-6. Treatment of IL-6-primed platelets with either the phosphoinositol-3 kinase (PI3K) inhibitor LY294002 or the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 reduced the ULVWF-platelet complex formation and restored the clearance of the complex by plasma ADAMTS13, compared to IL-6-primed platelets. Furthermore, IL-6 enhanced the phosphorylation of the intracellular adaptor molecule 14-3-3ζ, which regulates VWF binding to the glycoprotein (GP) Ib-IX complex. The 14-3-3 antagonist R18 significantly increased ADAMTS-13 cleavage of ULVWF strings with adherent IL-6-treated platelets. These findings indicate that IL-6 related intracellular signals of platelet is involved in regulating ULVWF-platelet binding and ULVWF cleavage by ADAMTS13.

Establishment and analysis of conditional Rab1 and Rab5 knockout cells by using the auxin-inducible degron system

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. Since they have recently been shown to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, it has been extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed conditional KO (CKO) cells for Rab1 (Rab1A/1B) and Rab5 (Rab5A/5B/5C) by using the auxin-inducible protein degradation system. Rab1-CKO and Rab5-CKO led to eventual cell death >18 h and >48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and ER-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

The Role of Outer-Membrane Vesicles in Intercellular Communication in Pseudomonas Aeruginosa

<p>Gram-negative bacteria produce outer-membrane vesicles (OMVs) that have biological roles ranging from biofilm formation, modulation of host-cell interactions & delivery of virulence factors. Several studies have shown a role for OMVs to act as intracellular signals to co-ordinate the behaviour of bacteria. This study showed OMVs generated at sub-lethal ciprofloxacin concentrations were capable of programming naïve P. aeruginosa cultures resulting in premature entry into stationary-phase and a significantly lower final culture density reached after 14 hrs. Pyoverdine production was also initiated after 6 hrs in cultures treated with OMVs. Heat-inactivation of OMVs failed to impede OMV-mediated growth inhibition & pyoverdine production. Chloroform-disruption of OMVs prevented OMV-mediated growth inhibition but did not inhibit OMV-induced pyoverdine production. It is likely that these effects are mediated by multiple signals as opposed to a single mechanism. This suggests that a protein is not responsible for OMV-mediated growth inhibition and an intact OMV lipid bilayer is required. Induction of pyoverdine production is likely due to a lipid (such as a homo-serine lactone) or small molecule present within OMVs. Preincubation with OMVs for 2-4 hrs resulted in a substantial decrease in the final culture density from cultures that were exposed to OMVs during the course of growth. This suggests that OMV fusion is capable of programming naïve bacteria to set a predetermined division limit on subsequent daughter cells. We coin this as the ‘Dayflick’ limit due to the similarities of the Hayflick limit in eukaryotic cells. This shows that OMVs act as intercellular messaging vehicles between bacteria that communicate and program naïve bacteria to adapt to the environment under which they were generated in, aiding survival in harsh environments. Further study is needed to determine what OMV components are responsible for initiating these responses and to determine how long the programming is stable.</p>

The Role of Outer-Membrane Vesicles in Intercellular Communication in Pseudomonas Aeruginosa

<p>Gram-negative bacteria produce outer-membrane vesicles (OMVs) that have biological roles ranging from biofilm formation, modulation of host-cell interactions & delivery of virulence factors. Several studies have shown a role for OMVs to act as intracellular signals to co-ordinate the behaviour of bacteria. This study showed OMVs generated at sub-lethal ciprofloxacin concentrations were capable of programming naïve P. aeruginosa cultures resulting in premature entry into stationary-phase and a significantly lower final culture density reached after 14 hrs. Pyoverdine production was also initiated after 6 hrs in cultures treated with OMVs. Heat-inactivation of OMVs failed to impede OMV-mediated growth inhibition & pyoverdine production. Chloroform-disruption of OMVs prevented OMV-mediated growth inhibition but did not inhibit OMV-induced pyoverdine production. It is likely that these effects are mediated by multiple signals as opposed to a single mechanism. This suggests that a protein is not responsible for OMV-mediated growth inhibition and an intact OMV lipid bilayer is required. Induction of pyoverdine production is likely due to a lipid (such as a homo-serine lactone) or small molecule present within OMVs. Preincubation with OMVs for 2-4 hrs resulted in a substantial decrease in the final culture density from cultures that were exposed to OMVs during the course of growth. This suggests that OMV fusion is capable of programming naïve bacteria to set a predetermined division limit on subsequent daughter cells. We coin this as the ‘Dayflick’ limit due to the similarities of the Hayflick limit in eukaryotic cells. This shows that OMVs act as intercellular messaging vehicles between bacteria that communicate and program naïve bacteria to adapt to the environment under which they were generated in, aiding survival in harsh environments. Further study is needed to determine what OMV components are responsible for initiating these responses and to determine how long the programming is stable.</p>

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signaling hubs

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell-cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically-dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane region, pseudokinase and SAM domains. Using small-angle X-ray scattering and crosslinking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosines in the juxtamembrane region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signaling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically-targeted to counter oncogenic signalling.

Peroxiredoxins as Potential Targets for Cardiovascular Disease

Increased oxidative stress (OS) is considered a common etiology in the pathogenesis of cardiovascular disease (CVD). Therefore, the precise regulation of reactive oxygen species (ROS) in cardiovascular cells is essential to maintain normal physiological functions. Numerous regulators of cellular homeostasis are reportedly influenced by ROS. Hydrogen peroxide (H2O2), as an endogenous ROS in aerobic cells, is a toxic substance that can induce OS. However, many studies conducted over the past two decades have provided substantial evidence that H2O2 acts as a diffusible intracellular signaling messenger. Antioxidant enzymes, including superoxide dismutases, catalase, glutathione peroxidases, and peroxiredoxins (Prdxs), maintain the balance of ROS levels against augmentation of ROS production during the pathogenesis of CVD. Especially, Prdxs are regulatory sensors of transduced intracellular signals. The intracellular abundance of Prdxs that specifically react with H2O2 act as regulatory proteins. In this review, we focus on the role of Prdxs in the regulation of ROS-induced pathological changes in the development of CVD.

Downregulation of CD45 Signaling in COVID-19 Patients Is Reversed by C24D, a Novel CD45 Targeting Peptide

CD45, the predominant transmembrane tyrosine phosphatase in leukocytes, is required for the efficient induction of T cell receptor signaling and activation. We recently reported that the CD45-intracellular signals in peripheral blood mononuclear cells (PBMCs) of triple negative breast cancer (TNBC) patients are inhibited. We also reported that C24D, an immune modulating therapeutic peptide, binds to CD45 on immune-suppressed cells and resets the functionality of the immune system via the CD45 signaling pathway. Various studies have demonstrated that also viruses can interfere with the functions of CD45 and that patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are immune-suppressed. Given the similarity between the role of CD45 in viral immune suppression and our findings on TNBC, we hypothesized that the C24D peptide may have a similar “immune-resetting” effect on PBMCs from COVID-19 patients as it did on PBMCs from TNBC patients. We tested this hypothesis by comparing the CD45/TCR intracellular signaling in PBMCs from ten COVID-19 patients vs. PBMCs from ten healthy volunteers. Herein, we report our findings, demonstrating the immune reactivating effect of C24D via the phosphorylation of the tyrosine 505 and 394 in Lck, the tyrosine 493 in ZAP-70 and the tyrosine 172 in VAV-1 proteins in the CD45 signaling pathway. Despite the relatively small number of patients in this report, the results demonstrate that C24D rescued CD45 signaling. Given the central role played by CD45 in the immune system, we suggest CD45 as a potential therapeutic target.