Phosphatidylserine-Targeting Monoclonal Antibodies Exhibit Distinct Biochemical and Cellular Effects on Anti-CD3/CD28-Stimulated T Cell IFN-γ and TNF-α Production.

Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via ß2-gp1 (ß2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-ɑ production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4+ and CD8+ cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells.

[Correlation of age, body mass index and obesity-related biochemical indexes with semen quality in males intending for a second child].

OBJECTIVE: To explore the influence of age, body mass index (BMI) and obesity-related biochemical indexes on semen quality in adult males intending to have a second child in Guangzhou. METHODS: We conducted a questionnaire investigation among 632 adult males seeking medical advice on their intention for a second child at Guangzhou Women and Children's Medical Center from August 2017 to July 2018. We obtained their lipid metabolism indicators and semen parameters, and analyzed the correlation of semen quality with age, BMI, obesity-related biochemical indexes, living environment and occupation. RESULTS: Age, BMI, season of sperm extraction, living environment and occupation all influenced the semen quality of the males. Age was correlated negatively with the percentage of progressively motile sperm (PMS) (r = －0.109, P < 0.05), BMI positively with the semen volume (r = 0.103, P < 0.05) but negatively with the percentage of morphologically normal sperm (MNS) (r = －0.138, P < 0.05), high-density lipoprotein (HDL) negatively with PMS (r = －0.168, P < 0.01) and the percentage of immotile sperm (IM) (r = －0.135, P < 0.05), low-density lipoprotein (LDL) negatively with the semen volume (r = －0.124, P < 0.01), PMS (r = －0.127, P < 0.05), sperm concentration (r = －0.121, P < 0.05) and total sperm count (r = －0.210, P < 0.01) but positively with IM (r = 0.140, P < 0.01). Multivariate regression analysis showed BMI and LDL to be independent factors influencing the semen volume, uric acid to be an independent factor influencing semen liquefaction time, age, HDL and LDL to be independent factors influencing PMS, age, and HDL to be independent factors influencing IM, LDL to be independent factors influencing total sperm count, while BMI and TG to be independent factors influencing MNS. CONCLUSIONS: Age, BMI, season of sperm extraction, living environment and occupation may affect the semen quality of the males in Guangzhou.

Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase.

BACKGROUND: Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. METHODS: In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. RESULTS: Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. CONCLUSION: These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

KMT2D deficiency drives lung squamous cell carcinoma and hypersensitivity to RTK-RAS inhibition.

Lung squamous cell carcinoma (LUSC) represents a major subtype of lung cancer with limited treatment options. KMT2D is one of the most frequently mutated genes in LUSC (>20%), and yet its role in LUSC oncogenesis remains unknown. Here, we identify KMT2D as a key regulator of LUSC tumorigenesis wherein Kmt2d deletion transforms lung basal cell organoids to LUSC. Kmt2d loss increases activation of receptor tyrosine kinases (RTKs), EGFR and ERBB2, partly through reprogramming the chromatin landscape to repress the expression of protein tyrosine phosphatases. These events provoke a robust elevation in the oncogenic RTK-RAS signaling. Combining SHP2 inhibitor SHP099 and pan-ERBB inhibitor afatinib inhibits lung tumor growth in Kmt2d-deficient LUSC murine models and in patient-derived xenografts (PDXs) harboring KMT2D mutations. Our study identifies KMT2D as a pivotal epigenetic modulator for LUSC oncogenesis and suggests that KMT2D loss renders LUSC therapeutically vulnerable to RTK-RAS inhibition.

Use of proton pump inhibitors for the risk of gastric cancer.

BACKGROUND: This study aimed to systematically analyze the association between long-term use of proton pump inhibitors (PPIs) and the risk of gastric cancer (GC). METHODS: We performed a systematic search of articles on the relationship between long-term use of PPIs and the risk of GC from PubMed and EMBASE. We calculated the pooled odds ratio of GC in PPI users compared to non-PPI users using random-effects models. RESULTS: This meta-analysis included 18 studies from 20 different databases with 4348,905 patients enrolled. In the random effects model, we found that an increased risk of GC among PPI users (OR = 1.94; 95% CI [1.43, 2.64]). The long-term use of PPIs compared with histamine-2 receptor antagonist users did not increase the risk of GC (OR = 1.65; 95% CI [0.92, 2.97]). Stratified analysis showed that PPI users had a significantly increased risk of noncardia GC (OR = 2.53; 95% CI [2.03, 3.15]), but had a relatively small relationship with the risk of gastric cardia cancer. (OR = 1.79; 95% CI [1.06, 3.03]). With the extension of PPI use time, the estimated risk value decreases (<1 year: OR = 6.33, 95% CI [3.76, 10.65]; 1-3 years: OR = 1.82, 95% CI [1.30, 2.55]; >3 years: OR = 1.25, 95% CI [1.00, 1.56]). Despite Helicobacter pylori eradication, the long-term use of PPIs did not alter the increased risk of GC (OR = 2.29; 95% CI [1.57, 3.33]). CONCLUSION: Our meta-analysis found that PPI use may be associated with an increased risk of GC. Further research on the causal relationship between these factors is necessary.

Enrichment and speciation of chromium during basalt weathering: Insights from variably weathered profiles in the Leizhou Peninsula, South China.

Basalt-derived soils are widespread worldwide. Such soils contain high levels of heavy metals like chromium (Cr), which is a serious environmental concern. However, little is known regarding the enrichment and speciation of Cr during the basalt weathering process. Therefore, two basalt-derived soil profiles (Nitisol and Ferralsol) in the Leizhou Peninsula, south tropical China, were investigated to explore the redistribution and transformation of Cr during basalt weathering. All profiles could be divided into three layers: rocks, saprolites, and soils. The Nitisol and Ferralsol profiles exhibited strong (kaolinization) and extreme (laterization) degrees of weathering, respectively. Results showed that Cr concentrations in the saprolites (234 to 315 mg·kg-1) were higher than those in basalt rocks (139 to 159 mg·kg-1), indicating that Cr was enriched with the continuous loss of Si and other mobile macro-elements. While high levels of Cr were also enriched in the soils (178 to 430 mg·kg-1) accompanied with Fe. However, in the upper soils of the Ferralsol profile, the acidity and organic matter could promote the leaching of Cr. Geochemical fractions and EPMA mapping showed that chromite and olivine were the main Cr-bearing minerals in basalt, but Fe-oxides (e.g., goethite and hematite) contained the highest portion of Cr in weathered saprolites and soils. The availability of Cr in the soil was extremely low due to the high stability of Cr bound to Fe-oxides. However, the decreasing contents of Cr bound to Fe-oxides in the upper soils of the Ferralsol profile indicated that Cr could also be released during Fe leaching. In conclusion, the weathering of basalt can lead to the enrichment of Cr in Fe-(hydro)oxides, which are the main controlling minerals for Cr mobility in basalt-derived soils. Further research is needed to evaluate the effect of Fe-(hydro)oxide formation and dissolution on the release of soil Cr.

Loss of TSC1/TSC2 sensitizes immune checkpoint blockade in non-small cell lung cancer.

Tuberous sclerosis complex subunit 1 (TSC1) and 2 (TSC2) are frequently mutated in non-small cell lung cancer (NSCLC), however, their effects on antitumor immunity remained unexplored. A CRISPR screening in murine KrasG12D/Trp53-/- (KP) model identified Tsc1 and Tsc2 as potent regulators of programmed cell death ligand 1 (Pd-l1) expression in vitro and sensitivity to anti-programmed cell death receptor 1 (PD-1) treatment in vivo. TSC1 or TSC2 knockout (KO) promoted the transcriptional and membrane expression of PD-L1 in cell lines. TSC2-deficient tumors manifested an inflamed microenvironment in patient samples and The Cancer Genome Atlas dataset. In syngeneic murine models, KP-Tsc2-KO tumors showed notable response to anti-PD-1 antibody treatment, but Tsc2-wild-type tumors did not. Patients with TSC1/TSC2-mutant NSCLC receiving immune checkpoint blockade (ICB) had increased durable clinical benefit and survival. Collectively, TSC1/TSC2 loss defines a distinct subtype of NSCLC characterized as inflamed tumor microenvironment and superior sensitivity to ICB.

Nemvaleukin alfa, a novel engineered IL-2 fusion protein, drives antitumor immunity and inhibits tumor growth in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is a deadly disease with a 5-year survival of less than 7%. The addition of immunotherapy to chemotherapy was recently approved as first-line treatment; however, the improved clinical benefit is modest, highlighting an urgent need for new treatment strategies. Nemvaleukin alfa, a novel engineered interleukin-2 fusion protein currently in phase I-III studies, is designed to selectively expand cytotoxic natural killer (NK) cells and CD8+ T cells. Here, using a novel SCLC murine model, we investigated the effects of a mouse version of nemvaleukin (mNemvaleukin) on tumor growth and antitumor immunity. METHODS: A novel Rb1 -/- p53 -/- p130 -/- SCLC model that mimics human disease was generated. After confirming tumor burden by MRI, mice were randomized into four treatment groups: vehicle, mNemvaleukin alone, chemotherapy (cisplatin+etoposide) alone, or the combination of mNemvaleukin and chemotherapy. Tumor growth was measured by MRI and survival was recorded. Tumor-infiltrating lymphocytes and peripheral blood immune cells were analyzed by flow cytometry. Cytokine and chemokine secretion were quantified and transcriptomic analysis was performed to characterize the immune gene signatures. RESULTS: mNemvaleukin significantly inhibited SCLC tumor growth, which was further enhanced by the addition of chemotherapy. Combining mNemvaleukin with chemotherapy provided the most significant survival benefit. Profiling of tumor-infiltrating lymphocytes revealed mNemvaleukin expanded the total number of tumor-infiltrating NK and CD8+ T cells. Furthermore, mNemvaleukin increased the frequencies of activated and proliferating NK and CD8+ T cells in tumors. Similar immune alterations were observed in the peripheral blood of mNemvaleukin-treated mice. Of note, combining mNemvaleukin with chemotherapy had the strongest effects in activating effector and cytotoxic CD8+ T cells. mNemvaleukin alone, and in combination with chemotherapy, promoted proinflammatory cytokine and chemokine production, which was further confirmed by transcriptomic analysis. CONCLUSIONS: mNemvaleukin, a novel cytokine-based immunotherapy, significantly inhibited murine SCLC tumor growth and prolonged survival, which was further enhanced by the addition of chemotherapy. mNemvaleukin alone, and in combination with chemotherapy, drove a strong antitumor immune program elicited by cytotoxic immune cells. Our findings support the evaluation of nemvaleukin alone or in combination with chemotherapy in clinical trials for the treatment of SCLC.

Quantification of nickel and cobalt mobility and accumulation via the phloem in the hyperaccumulator Noccaea caerulescens (Brassicaceae).

Hyperaccumulators have exceptional phloem translocation capability for heavy metals. This study aims at quantifying the mobility and accumulation of Ni and Co via the phloem in the model hyperaccumulator Noccaea caerulescens. "Phloem loading capability (PLC)," which is calculated by the "Metal content in phloem sap/Metal content in leaves," was introduced to evaluate the metal phloem mobility, while "Phloem mobility value (PMV)" was used for the normalization of PLC, which sets the PLC of Sr as PMV 0 and that of Rb as 100. The results showed that the PMVs of Ni and Co were 63 and 47, respectively. And the phloem mobility of Rb, Ni, Co, and Sr could be graded as highly mobile, mobile, intermediate, and immobile accordingly. The phloem stream can supply up to 19.1% and 16.0% of the total Ni and Co accumulated in the young leaves, respectively, while for Rb and Sr, the phloem contributes to 29% and 1.4% of the total Rb or Sr, indicating phloem contribution of certain metal is directly linked with its mobility. The results of this study raise the importance of phloem translocation on metal accumulation in shoots and provide insights on the metal cycling process in hyperaccumulators.

Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

Post-translational modifications of the ligands: Requirement for TAM receptor activation.

The Tyro3, Axl, and MerTK (TAM) receptors are three homologous Type I Receptor Tyrosine Kinases that have important homeostatic functions in multicellular organisms by regulating the clearance of apoptotic cells (efferocytosis). Pathologically, TAM receptors are overexpressed in a wide array of human cancers, and often associated with aggressive tumor grade and poor overall survival. In addition to their expression on tumor cells, TAMs are also expressed on infiltrating myeloid-derived cells in the tumor microenvironment, where they appear to act akin to negative immune checkpoints that impair host anti-tumor immunity. The ligands for TAMs are two endogenous proteins, Growth Arrest-Specific 6 (Gas6) and Protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PtdSer) on apoptotic cells and the TAM ectodomains. One interesting feature of TAMs biology is that their ligand proteins require specific post-translational modifications to acquire activities. This chapter summarized these important modifications and explained the molecular mechanisms behind such phenomenon. Current evidences suggest that these modifications help Gas6/Pros1 to achieve optimal PtdSer-binding capacities. In addition, this chapter included recent discovery of regulating machineries of PtdSer dynamic across the plasma membrane, as well as their potential impacts in the tumor microenvironment. Taken together, this review highlights the importance of the upstream PtdSer and Gas6 in regulating TAMs' function and hope to provide researchers with new perspectives to inspire future studies of TAM receptors in human disease models.

Efficacy of Targeting SARS-CoV-2 by CAR-NK Cells.

SARS-CoV-2, which causes COVID-19 disease, is one of greatest global pandemics in history. No effective treatment is currently available for severe COVID-19 disease. One strategy for implementing cell-based immunity involves the use of chimeric antigen receptor (CAR) technology. Unlike CAR T cells, which need to be developed using primary T cells derived from COVID-19 patients with lymphopenia, clinical success of CAR NK cell immunotherapy is possible through the development of allogeneic, universal, and 'off-the-shelf' CAR-NK cells from a third party, which will significantly broaden the application and reduce costs. Here, we develop a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2. CAR-NK cells were generated using the scFv domain of CR3022 (henceforth, CR3022-CAR-NK), a broadly neutralizing antibody for SARS-CoV-1 and SARS-CoV-2. CR3022-CAR-NK cells can specifically bind to RBD of SARS-CoV-2 and pseudotyped SARS-CoV-2 S protein, and can be activated by pseudotyped SARS-CoV-2-S viral particles in vitro. Further, CR3022-CAR-NK cells can specifically kill pseudo-SARS-CoV-2 infected target cells. Thus, 'off-the-shelf' CR3022-CAR-NK cells may have the potential to treat patients with severe COVID-19 disease.

Spraying rhubarb powder solution under gastroscope in the treatment of acute non-varicose upper gastrointestinal bleeding: A systematic review and meta-analysis of randomized controlled trials.

OBJECTIVE: Systematically assessing the safety and effectiveness of spraying rhubarb powder solution under gastroscope for the treatment of acute non-varicose upper gastrointestinal bleeding, and confirmation for further clinical research and application. METHODS: We searched the following databases up till November 2019: PubMed, the Cochrane Library, EMBASE, CNKI, WanFang Data, VIP and SinoMed. Randomized controlled trials (RCTs) were used to compare the curative effect of spraying rhubarb powder solution with other drugs under gastroscope for the treatment of acute non-varicose upper gastrointestinal bleeding. RESULTS: Out of 171 articles, 14 RCTs involving 1493 patients were included. All control groups included in the RCTs were treated with norepinephrine solution. The hemostatic effect of spraying rhubarb powder solution under gastroscope was examined for 24 h at high concentration (0.1 g/mL). The hemostatic effect at higher conc. (0.1 g/mL) found far more better than low conc.(RR = 1.48;95 %CI:1.25,1.75;P﹤0.00001) (0.03 g/mL)as homeostatic effect at low conc.is same that of norepinephrine solution (RR = 1.02;95 %CI:0.94,1.10;P = 0.62). Moreover within 48 h, rhubarb powder solution with 0.1 g/mL or 0.15 g/mL conc. have of significantly higher hemostatic effects than norepinephrine solution (RR = 1.18;95 % CI: 1.08, 1.30;P = 0.0003). Occurrence of rebleeding event within 48 h after successful hemostasis (RR = 0.42;95 %CI:0.24,0.74;P = 0.003) reduced exceptionally. After that the hemostatic effect of rhubarb powder solution with 0.1 g/mL conc.examined within 72 h again exhibited significant improvement than norepinephrine solution (RR = 1.19;95 %CI:1.12,1.26;P < 0.00001). On par with immediate hemostasis time, rhubarb powder solution took unprecedented less time than norepinephrine solution;(MD=-5.56S;95 %CI:-6.16, -4.95;P﹤0.00001). Additionally, the adverse reaction produced by rhubarb powder solution is much lower than norepinephrine solution (RR = 0.22;95 %CI:0.11,0.42;P < 0.00001). CONCLUSIONS: According to meta-analysis, Spraying rhubarb powder solution under gastroscope in the treatment of acute non-varicose upper gastrointestinal bleeding is superior to norepinephrine solution in improving hemostasis effect. Shortening immediate hemostasis time and reducing rebleeding,and is safe to use. Based on the results of this study, physicians can treat patients with acute non-varicose upper gastrointestinal bleeding by spraying rhubarb powder solution under gastroscope according to the patients' condition.However, the sample size included in this study is small and of substandard quality qu, and a large sample size clinical trial with strict design and normative report is needed to verify the safety and efficacy of rhubarb powder solution under gastroscope for acute non-varicose upper gastrointestinal bleeding.

Cyclophilin A Inhibitor Debio-025 Targets Crk, Reduces Metastasis, and Induces Tumor Immunogenicity in Breast Cancer.

The Crk adaptor protein, a critical modifier of multiple signaling pathways, is overexpressed in many cancers where it contributes to tumor progression and metastasis. Recently, we have shown that Crk interacts with the peptidyl prolyl cis-trans isomerase, Cyclophilin A (CypA; PP1A) via a G219P220Y221 (GPY) motif in the carboxyl-terminal linker region of Crk, thereby delaying pY221 phosphorylation and preventing downregulation of Crk signaling. Here, we investigate the physiologic significance of the CypA/Crk interaction and query whether CypA inhibition affects Crk signaling in vitro and in vivo. We show that CypA, when induced under conditions of hypoxia, regulates Crk pY221 phosphorylation and signaling in cancer cell lines. Using nuclear magnetic resonance spectroscopy, we show that CypA binds to the Crk GPY motif via the catalytic PPII domain of CypA, and small-molecule nonimmunosuppressive inhibitors of CypA (Debio-025) disrupt the CypA-CrkII interaction and restores phosphorylation of Crk Y221. In cultured cell lines, Debio-025 suppresses cell migration, and when administered in vivo in an orthotopic model of triple-negative breast cancer, Debio-025 showed antitumor efficacy either alone or in combination with anti-PD-1 mAb, reducing both tumor volume and metastatic lung dispersion. Furthermore, when analyzed by NanoString immune profiling, treatment of Debio-025 with anti-PD-1 mAb increased both T-cell signaling and innate immune signaling in tumor microenvironment. IMPLICATIONS: These data suggest that pharmacologic inhibition of CypA may provide a promising and unanticipated consequence in cancer biology, in part by targeting the CypA/CrkII axis that regulates cell migration, tumor metastasis, and host antitumor immune evasion.

Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances Anti-PD-1 mAb Efficacy in a Murine Model of Triple-Negative Breast Cancer.

Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PS→TAM receptor→PD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 alone or in combination. Tyro3, Axl, and Mertk were differentially expressed on multiple cell subtypes in the tumor microenvironment. Although monotherapeutic administration of either pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 mAb therapy showed partial antitumor activity, combined treatment of BMS-777607 with anti-PD-1 significantly decreased tumor growth and incidence of lung metastasis. Moreover, combined treatment with BMS-777607 and anti-PD-1 showed increased infiltration of immune stimulatory T cells versus either monotherapy treatment alone. RNA NanoString profiling showed enhanced infiltration of antitumor effector T cells and a skewed immunogenic immune profile. Proinflammatory cytokines increased with combinational treatment. Together, these studies indicate that pan-TAM inhibitor BMS-777607 cooperates with anti-PD-1 in a syngeneic mouse model for triple-negative breast cancer and highlights the clinical potential for this combined therapy. SIGNIFICANCE: These findings show that pan-inhibition of TAM receptors in combination with anti-PD-1 may have clinical value as cancer therapeutics to promote an inflammatory tumor microenvironment and improve host antitumor immunity.

Sirt3 deficiency impairs neurovascular recovery in ischemic stroke.

AIMS: Sirt3 is one member of the NAD+ -dependent protein deacetylase family and plays crucial roles in diverse aspects of mammalian biological function. Then the role of Sirt3 on ischemia stroke is unknown. METHODS: To examine the effect of Sirt3 on ischemic stroke, we performed transient middle cerebral artery occlusion (tMCAO) in adult male Sirt3 knockout (KO) and wild-type (WT) mice. RESULTS: The level of Sirt3 in infarct region is decreased after ischemic stroke. In addition, we found that Sirt3 KO mice showed worse neurobehavioral outcome compared with WT mice, accompanied by decreased neurogenesis and angiogenesis as shown by the reduction in number of DCX+ /BrdU+ cells, NeuN+ /BrdU+ cells, and CD31+ /BrdU+ cells in the perifocal region during recovery phase after ischemic stroke. Furthermore, Sirt3 deficiency reduced the activation of vascular endothelial growth factor (VEGF), AKT, and extracellular signal-regulated kinases (ERK) signaling pathways. CONCLUSION: Our results indicated that Sirt3 is beneficial to neurovascular and functional recovery following chronic ischemic stroke.

Requirement of Gamma-Carboxyglutamic Acid Modification and Phosphatidylserine Binding for the Activation of Tyro3, Axl, and Mertk Receptors by Growth Arrest-Specific 6.

The Tyro3, Axl, and Mertk (TAM) receptors are homologous type I receptor tyrosine kinases that have critical functions in the clearance of apoptotic cells in multicellular organisms. TAMs are activated by their endogenous ligands, growth arrest-specific 6 (Gas6), and protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PS) on apoptotic cells and the TAM ectodomains. However, the molecular mechanisms by which Gas6/Pros1 promote TAM activation remains elusive. Using TAM/IFNγR1 reporter cell lines to monitor functional TAM activity, we found that Gas6 activity was exquisitely dependent on vitamin K-mediated γ-carboxylation, whereby replacing vitamin K with anticoagulant warfarin, or by substituting glutamic acid residues involved in PS binding, completely abrogated Gas6 activity as a TAM ligand. Furthermore, using domain and point mutagenesis, Gas6 activity also required both an intact Gla domain and intact EGF-like domains, suggesting these domains function cooperatively in order to achieve TAM activation. Despite the requirement of γ-carboxylation and the functional Gla domain, non-γ-carboxylated Gas6 and Gla deletion/EGF-like domain deletion mutants still retained their ability to bind TAMs and acted as blocking decoy ligands. Finally, we found that distinct sources of PS-positive cells/vesicles (including apoptotic cells, calcium-induced stressed cells, and exosomes) bound Gas6 and acted as cell-derived or exosome-derived ligands to activate TAMs. Taken together, our findings indicate that PS is indispensable for TAM activation by Gas6, and by inference, provides new perspectives on how PS, regulates TAM receptors and efferocytosis.

Crk adaptor protein promotes PD-L1 expression, EMT and immune evasion in a murine model of triple-negative breast cancer.

The tumor infiltration of immune cells in solid cancers can profoundly influence host antitumor responses. In recent years, immunotherapeutic regimens, that target immune checkpoints, demonstrated significant antitumor response by increasing intra-tumoral immune cell populations, including CD8+ effector T cells. However, administration of such immune checkpoint inhibitors is largely inefficacious in inducing immunogenicity and treating breast cancer. Currently, there is a great need to better understand cell autonomous mechanisms of immune evasion in breast cancer to identify upstream therapeutic targets that increase the efficacy of immunotherapy. Here we show that Crk, an SH2 and SH3 domain-containing adaptor protein implicated in focal adhesion signaling, cell migration, and invasion, and frequently up-regulated in human cancers, has an important role in regulating the tumor immune microenvironment. Using a murine 4T1 breast adenocarcinoma model of spontaneous metastasis in immune-competent BALB/C mice, we show that genetic ablation of Crk by CRISPR-Cas9 leads to enhanced anti-tumor immune cell populations, cytotoxic effector and immune surveillance cytokines in primary tumor. Pathologically, this leads to a significant reduction in tumor growth and lung metastasis. Mechanistically, Crk KO suppresses EMT and PD-L1 expression on tumor cells and acts additively with anti-PD1 therapy to suppress tumor growth and metastasis outcomes. Taken together, these data reveal a previously un-described function of Crk adaptor protein expression in tumor cells for cell autonomous regulation of tumor immune microenvironment.

Phosphatidylserine Sensing by TAM Receptors Regulates AKT-Dependent Chemoresistance and PD-L1 Expression.

Tyro3, Axl, and Mertk (collectively TAM receptors) are three homologous receptor tyrosine kinases that bind vitamin K-dependent endogenous ligands, Protein S (ProS), and growth arrest-specific factor 6 (Gas6), and act as bridging molecules to promote phosphatidylserine (PS)-mediated clearance of apoptotic cells (efferocytosis). TAM receptors are overexpressed in a vast array of tumor types, whereby the level of expression correlates with the tumor grade and the emergence of chemo- and radioresistance to targeted therapeutics, but also have been implicated as inhibitory receptors on infiltrating myeloid-derived cells in the tumor microenvironment that can suppress host antitumor immunity. In the present study, we utilized TAM-IFNγR1 reporter lines and expressed TAM receptors in a variety of epithelial cell model systems to show that each TAM receptor has a unique pattern of activation by Gas6 or ProS, as well as unique dependency for PS on apoptotic cells and PS liposomes for activity. In addition, we leveraged this system to engineer epithelial cells that express wild-type TAM receptors and show that although each receptor can promote PS-mediated efferocytosis, AKT-mediated chemoresistance, as well as upregulate the immune checkpoint molecule PD-L1 on tumor cells, Mertk is most dominant in the aforementioned pathways. Functionally, TAM receptor-mediated efferocytosis could be partially blocked by PS-targeting antibody 11.31 and Annexin V, demonstrating the existence of a PS/PS receptor (i.e., TAM receptor)/PD-L1 axis that operates in epithelial cells to foster immune escape. These data provide a rationale that PS-targeting, anti-TAM receptor, and anti-PD-L1-based therapeutics will have merit as combinatorial checkpoint inhibitors.Implications: Many tumor cells are known to upregulate the immune checkpoint inhibitor PD-L1. This study demonstrates a role for PS and TAM receptors in the regulation of PD-L1 on cancer cells. Mol Cancer Res; 15(6); 753-64. ©2017 AACR.