Galectin-9 Triggers Neutrophil-Mediated Anticancer Immunity

In earlier studies, galectin-9 (Gal-9) was identified as a multifaceted player in both adaptive and innate immunity. Further, Gal-9 had direct cytotoxic and tumor-selective activity towards cancer cell lines of various origins. In the current study, we identified that treatment with Gal-9 triggered pronounced membrane alterations in cancer cells. Specifically, phosphatidyl serine (PS) was rapidly externalized, and the anti-phagocytic regulator, CD47, was downregulated within minutes. In line with this, treatment of mixed neutrophil/tumor cell cultures with Gal-9 triggered trogocytosis and augmented antibody-dependent cellular phagocytosis of cancer cells. Interestingly, this pro-trogocytic effect was also due to the Gal-9-mediated activation of neutrophils with upregulation of adhesion markers and mobilization of gelatinase, secretory, and specific granules. These activation events were accompanied by a decrease in cancer cell adhesion in mixed cultures of leukocytes and cancer cells. Further, prominent cytotoxicity was detected when leukocytes were mixed with pre-adhered cancer cells, which was abrogated when neutrophils were depleted. Taken together, Gal-9 treatment potently activated neutrophil-mediated anticancer immunity, resulting in the elimination of epithelial cancer cells.

Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the “Kennedy pathway” is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.

Targeting Venetoclax-Resistant CLL By Bcl-XL Degradation

Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in adults and is associated with resistance to apoptosis. Clinical responses are dramatically improved with the use of targeted therapies such as venetoclax, a selective inhibitor of the anti-apoptotic protein Bcl-2, compared to chemoimmunotherapy. However, some patients can ultimately develop resistant CLL. Bcl-xL overexpression has been implicated in multiple mechanisms of venetoclax resistance, representing a switch that confers cell survival despite Bcl-2 inhibition. Mechanisms of venetoclax resistance include mutated Bcl-2 (ex. G101V), switches in survival dependence to alternative BCL2 family members, and microenvironmental interactions (Reviewed in Bose et al., 2017). Moreover, disease progression on venetoclax is accelerated in CLL patients with aberrant p53 , and our laboratory identified Bcl-xL as one of the main contributors to the oncogenic abilities of mutant p53.Thus, Bcl-xL is a promising target for re-sensitizing CLL to apoptosis, but since it is crucial for platelet viability, direct targeting has not been clinically successful. New Bcl-xL PROTAC degraders provide an exciting opportunity to target Bcl-xL in venetoclax-resistant CLL. Due to differences in the expression of VHL E3 ligase in cancer cells versus platelets, these degraders have shown potency and selectivity to target this protein for degradation in multiple Bcl-xL-dependent malignancies (He, Y. et al., 2020). Our results show that exposing primary CLL samples (n=4) from treatment-naïve patients to the Bcl-xL degrader DT2216 is associated with apoptosis at concentrations known to be non-toxic to platelets (EC50 = 162 nM at 18 hr treatment). Furthermore, the dual Bcl-xL/Bcl-2 degrader PZ18753B was synthesized with enhanced potency against Bcl-xL and Bcl-2 co-dependent cancers. Our results confirm enhanced potency in primary cells from treatment naïve patients with CLL (EC50 = 7.602 nM at 18 hr treatment), compared to DT2216. Also, the degraders exhibit a similar kinetic profile at 10-fold concentrations (n=5, t1/2 = 2.3 hr for 1 uM DT2216, and t1/2 = 3.1 hr for 100 nM PZ18753B). For these results, apoptosis via the mitochondrial pathway was studied by flow cytometry, assessing extracellular exposure of phosphatidyl serine and mitochondrial depolarization by Annexin V and TMRM staining, respectively. Consistently, as confirmed by Western Blot, PZ18753B is more potent at degrading Bcl-xL than DT2216, achieving 50% degradation in RS4;11 leukemic cells at 46 hr treatment at a concentration of 0.86 nM, compared to 7.83 nM for DT2216. Comparing dose-response curves showing apoptosis by AnnexinV/TMRM staining and protein degradation by Western Blot, we confirmed that Bcl-xL degradation precedes cell death. Given that Bcl-xL overexpression confers a survival advantage to CLL cells, we hypothesize that venetoclax-resistant CLL cells can be targeted with the new Bcl-xL degraders. We studied the potency of the degraders against RS4;11 leukemic cells which express wildtype (WT) Bcl-2, or also overexpress G101V mutant Bcl-2. This is one of the mutations associated with venetoclax resistance and, combining data from various cohorts, it was identified in 23% venetoclax-resistant patients (Blombery et al., 2019 and 2020; Tausch et al., 2019; Weiss et al., 2019). PZ18753B induced apoptosis of RS4;11 Bcl-2 WT cells within 46 hrs (EC50 = 11.1 nM) as well as in RS4;11 cells expressing Bcl-2 G101V mutation (EC50 = 109.1 nM). PZ18753B degraded both Bcl-xL and Bcl-2 in RS4;11 WT cells effectively. In RS4;11 Bcl-2 G101V cells, it did not affect Bcl-2 but retained potent Bcl-xL degradation (86% vs 75% at 1 uM in WT vs mutant Bcl-2 cell lines, respectively). Interestingly, DT2216 did show ability to degrade G101V mutant Bcl-2 at the highest concentrations tested (Ex. 36% and 33%, respectively, at 1 uM). We also demonstrated that CLL cells from chemo-naïve patients depend primarily on Bcl-2 for survival, as measured by their responses to BH3 mimetic peptides in a BH3 profiling assay. However, venetoclax-resistant CLL cells undergo a shift in dependence to alternative Bcl-2 family proteins, such as Bcl-xL and Mcl-1, as a mechanism for resistance to apoptosis. Thus, resistant CLL represents an excellent setting in which to continue testing the efficacy of these potent Bcl-xL degraders, to overcome resistance to Bcl-2 inhibitors. Disclosures Zheng: Dialectic Therapeutics: Other: Co-founder and stakeholder. Inventor of Bcl-xL PROTACS disclosed in this study.. Zhou: Dialectic Therapeutics: Other: Co-founder and stakeholder. Inventor of Bcl-xL PROTACS disclosed in this study.. Zhang: Dialectic Therapeutics: Other: Co-founder and stakeholder. Co-inventor of Bcl-xL PROTACs disclosed in this study.. Wierda: Oncternal Therapeutics, Inc.: Research Funding; Genentech: Research Funding; Juno Therapeutics: Research Funding; Xencor: Research Funding; KITE Pharma: Research Funding; Sunesis: Research Funding; GSK/Novartis: Research Funding; Janssen: Research Funding; Loxo Oncology, Inc.: Research Funding; Acerta Pharma Inc.: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; Karyopharm: Research Funding; Miragen: Research Funding; Cyclacel: Research Funding; AstraZeneca: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding. Jain: Incyte: Research Funding; Servier: Honoraria, Research Funding; Precision Biosciences: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Beigene: Honoraria; Cellectis: Honoraria, Research Funding; Pfizer: Research Funding; Fate Therapeutics: Research Funding; Aprea Therapeutics: Research Funding; Genentech: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Research Funding; Adaptive Biotechnologies: Honoraria, Research Funding; TG Therapeutics: Honoraria; Janssen: Honoraria; AbbVie: Honoraria, Research Funding; Pharmacyclics: Research Funding. Woyach: AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; Gilead Sciences Inc: Other: Data & Safety; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy.

Anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) in isolated lupus anticoagulant (LA): is their presence linked to dual test positivity?

Objectives Anti phosphatidylserine/prothrombin antibodies (aPS/PT) are often present in patients with antiphospholipid syndrome (APS) and might be relevant in the pathogenesis of this condition. They are major determinant of lupus anticoagulant (LA) in triple-positive antiphospholipid (aPL) profile. Whether they are present and pathogenic in patients with isolated LA [negative anticardiolipin (aCL) and anti β2-Glycoprotein I (aβ2GPI) antibodies] is a matter of debate. Methods We measured aPS/PT in a large number of isolated LA with the aim to ascertain whether there is a link between the way isolated LA is assessed and the presence of these antibodies. APS/PT were measured in 86 patients with isolated LA (aCL- and abeta2GPI-). LA was assessed by two test systems, the dilute Russell Viper Venom Time (dRVVT) and the Silica Clotting Time (SCT). Results Sixty-six (77%) individuals with isolated LA were positive for aPS/PT (IgM 44, IgG and IgM 15, IgG in 7). Diagnosis of LA was made based on positive results in both dRVVT and SCT in 40 patients (Group 1) and based on only one positive test in the remaining 46 patients (Group 2). The rate of positive aPS/PT antibodies was significantly higher in Group 1 (OR=7.2, 95% CI 1.9–27.0, p<0.002). Moreover, the titre of IgM aPS/PT was significantly increased in Group 1 as compared to Group 2 (137 U, IQR 64–179 vs. 43 U, IQR 11–120, p=0.008). Conclusions These data indicate an association between LA based on two positive coagulation tests and the presence of aPS/PT antibodies, especially of IgM isotype.

Cerebrovascular events in patients with isolated anti-phosphatidyl-serine/prothrombin antibodies

The interest of extra-criteria antiphospholipid antibodies is growing, especially in patients negative for conventional antibodies. In this study we aimed to assess the clinical utility of anti-phosphatidyl-serine/prothrombin antibodies (aPS/PT) testing in patients negative for Beta2-Glycoprotein 1(β2GPI)-dependent tests, for identifying antiphospholipid syndrome (APS) patients that developed cerebrovascular events (CVE). When screening APS patients attending our center, out of 119 aPS/PT IgG/IgM-positive patients, thus patients negative for aβ2GPI and aCL, 42 patients (35%) tested negative for β2GPI-dependent tests and were tested with thrombin generation assay (TGA). Ten patients (24%), with isolated aPS/PT IgG/IgM, had a history of CVE. Lupus anticoagulant (LA)-positive test was more frequently observed in patients with CVE (8/22 vs. 2/20; p = 0.045). Out of the 10 patients who experienced CVE, 3 patients were aPS/PT IgG positive (all LA positive), and 8 patients were aPS/PT IgM positive (6/8 LA positive). One patient was positive for both aPS/PT IgG and IgM. LA-positive patients had only high titers of aPS/PT IgG/IgM, all of them being ≥ 80 U/ml, while the 2 LA-negative patients were aPS/PT IgM positive with medium titers [40–60 U/ml]. LA-positive patients had significantly altered TGA profile when compared to those who were LA negative, considering all TGA parameters. LA-positive patients had significantly higher tLag (8.4 ± 3.3 min vs. 6.6 ± 1.8 min; p = 0.046), higher tPeak (14 ± 4.3 min vs. 11 ± 2.7 min; p = 0.015) and lower Peak (207 ± 152 nM vs. 356.3 ± 104.7 nM; p < 0.001) and lower AUC (2109.7 ± 1006.9 nM vs. 2772.5 ± 776.8 nM; p = 0.033). The use of aPS/PT might be of help in identifying patients with CVE and APS, as also confirmed by TGA testing.

Lipidomic profiling of the developing kernel clarifies the lipid metabolism of Paeonia ostii

Lipid components in the developing kernel of Paeonia ostii were determined, and the fatty acid (FA) distributions in triacylglycerol and phospholipids were characterized. The lipids in the kernel were mainly phospholipids (43%), neutral glycerides (24%), fatty acyls (26%), and sphingolipids (4.5%). The dominant neutral glycerides were TAG and diacylglycerol. The PL components included phosphatidic acid, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, and phosphatidyl ethanolamine. As the kernel developed, the profiles of the molecular species comprising TAG and PL changed, especially during the earlier phases of oil accumulation. During rapid oil accumulation, the abundances of sphingosine-1-phosphate, pyruvic acid, stearic acid, and alpha-linolenic acid changed significantly; the sphingolipid metabolism and unsaturated FAs biosynthesis pathways were significantly enriched in these differentially abundant metabolites. Our results improve our understanding of lipid accumulation in tree peony seeds, and provide a framework for the analysis of lipid metabolisms in other oil crops.

Anti-cardiolipin and other anti-phospholipid antibodies in critically ill COVID-19 positive and negative patients

ABSTRACTBackgroundReports of severe COVID-19 being associated with thrombosis, anti-phospholipid antibodies (APLA), anti-phospholipid syndrome (APS) have yielded disparate conclusions. Studies comparing COVID-19 patients with contemporaneous controls of similar severity are lacking.Methods22 COVID+ and 20 COVID− patients with respiratory failure admitted to intensive care were studied longitudinally. Demographic and clinical data were obtained from the day of admission. APLA testing included anti-cardiolipin (aCL), anti-β2glycoprotien 1 (β2GP1), anti-domain 1 beta2 glycoprotein 1 (β2GP1) and anti-phosphatidyl serine/prothrombin complex (PS/PT). Anti-nuclear antibodies (ANA) were detected by immunofluorescence and antibodies to cytokines by a commercially available multiplexed array. ANOVA was used for continuous variables and Fisher’s exact test was used for categorical variables with α=0.05 and the false discovery rate at q=0.05.ResultsAPLA were predominantly IgG aCL (48%) followed by IgM (21%) in all patients, with a tendency toward higher frequency among the COVID+. aCL was not associated with surrogate markers of thrombosis but IgG aCL was strongly associated with worse disease severity and higher ANA titers regardless of COVID-19 status. An association between aCL and anti-cytokine autoantibodies tended to be higher among the COVID+.ConclusionsPositive APLA serology was associated with more severe disease regardless of COVID-19 status.KEY MESSAGESWhat is already known about this subject?COVID-19 is associated with coagulopathy and high morbidity and mortality.COVID-19 shares some of these clinical features with anti-phospholipid syndrome.Reports of an association of anti-phospholipid antibodies with high risk COVID-19 have yielded disparate conclusions, but they lacked longitudinal follow up and control groups of similar severity.What does this study add?Anti-phospholipid syndrome serology assessed longitudinally was predominantly anticardiolipin IgG autoantibodies, in 48% of patients.Anticardiolipin serology was associated with worse disease severity in both COVID-19 positive and negative patients.How might this impact on clinical practice or future developments?The use of anti-phospholipid antibodies tests in the COVID-19 clinical setting needs to be taken in context; whereas they are associated with more serve disease, they do not discriminate between COVID-19 positive and negative patients.

Roles of Lipid Profiles in Human Non-Small Cell Lung Cancer

Aims: This review aims to identify lipid biomarkers of non-small cell lung cancer (NSCLC) in human tissue samples and discuss the roles of lipids in tissue molecular identification, the discovery of potential biomarkers, and surgical margin assessment. Methods: A review of the literature focused on lipid-related research using mass spectrometry (MS) techniques in human NSCLC tissues from January 1, 2015, to November 20, 2020, was conducted. The quality of included studies was assessed using the QUADAS-2 tool. Results: Twelve studies met the inclusion criteria and were included in the review. The risk of bias was unclear in the majority of the studies. The contents of lipids including fatty acids, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, cardiolipin, phosphatidyl serine, phosphatidyl glycerol, ceramide, lysophosphatidylethanolamine, lysophosphatidylcholine, and lysophosphatidylglycerol differed significantly between cancer and healthy tissues. The sensitivity or specificity of the discrimination model was reported in 8 studies, and the sensitivity and specificity varied among the reported methods. The lipid profiles differed between adenocarcinoma and squamous cell carcinoma NSCLC subtypes. Conclusion: In preclinical studies, MS analysis and multiple discrimination models can be combined to distinguish NSCLC tissues from healthy tissues based on lipid profiles, which provides a new opportunity to evaluate the surgical margin and cancer subtype intraoperatively. Future studies should provide guidance for selecting patients and discrimination models to develop an improved method for clinical application.

The Gardos effect drives erythrocyte senescence and leads to Lu/BCAM and CD44 adhesion molecule activation

Senescence of erythrocytes is characterized by a series of changes that precede their removal from the circulation, including loss of red cell hydration, membrane shedding, loss of deformability, phosphatidyl serine exposure, reduced membrane sialic acid content, and adhesion molecule activation. Little is known about the mechanisms that initiate these changes nor is it known whether they are interrelated. In this study, we show that Ca2+-dependent K+ efflux (the Gardos effect) drives erythrocyte senescence. We found that increased intracellular Ca2+ activates the Gardos channel, leading to shedding of glycophorin-C (GPC)–containing vesicles. This results in a loss of erythrocyte deformability but also in a marked loss of membrane sialic acid content. We found that GPC-derived sialic acid residues suppress activity of both Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 by the formation of a complex on the erythrocyte membrane, and Gardos channel–mediated shedding of GPC results in Lu/BCAM and CD44 activation. This phenomenon was observed as erythrocytes aged and on erythrocytes that were otherwise prone to clearance from the circulation, such as sickle erythrocytes, erythrocytes stored for transfusion, or artificially dehydrated erythrocytes. These novel findings provide a unifying concept on erythrocyte senescence in health and disease through initiation of the Gardos effect.

A second PI(4,5)P2 binding site determines PI(4,5)P2 sensitivity of the tubby domain

ABSTRACTPhosphosinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. Such domains are also used widely as fluorescence-coupled biosensors for cellular PIs. For PI(4,5)P2, the dominant PI of the plasma membrane (PM), only two recognition domains have been characterized in detail and used as sensors. One of them, the tubby domain, which is conserved in the tubby-like protein (TULP) family, is essential for targeting proteins into cilia in a process involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic.Here we used coarse-grained molecular dynamics (MD) simulations to explore PI(4,5)P2 binding by the prototypic tubby domain (tubbyCT). While the MD simulations showed a comparatively low PI(4,5)P2 affinity of the previously described canonical binding site, they unexpectedly revealed an adjacent second binding site, consisting of a conserved cationic cluster at the protein-membrane interface. Population of this second site dramatically increased membrane association of tubbyCT. Although less specific than the canonical binding pocket, this second site preferred binding of PI(4,5)P2 over PI(4)P and phosphatidyl serine. Mutations in this site impaired PI(4,5)P2-dependent PM localization in living cells and PI(4,5)P2 interaction in silico.Thus, the second binding site essentially contributes to the effective affinity and hence PM association of the tubby domain. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of TULPs that underlies delivery of ciliary cargo.