p4E-BP1 protein levels upon MET inhibition: Whole-cell lysates were subjected to Western blotting using a specific antibody against p4E-BP1 following the treatment either with vehicle or 50nM tepotinib (EMD1214063, shortly EMD) for either 4 or 24h as indicated. Western blots representative of N=3 independent experiments are shown.

Differential abundance analysis and ion annotation matches for non-targeted metabolomics measurements.

Supplementations with nucleosides and hypoxanthine: (A) Proliferation of GTL-16 and EBC-1 cells upon supplementation with nucleosides or hypoxanthine. (B) Apoptosis (caspase-3 activation) in GTL-16 and EBC-1 upon supplementation with nucleosides, hypoxanthine or their combination.

Differential expression analysis of the GTL-16 and EBC-1 transcriptomics data.

Summary of the significance analysis for the survival analyses.

GART protein expression upon MAPK and PI3K targeting: GART protein levels in untreated GTL-16 and EBC-1 cells and after MAPK (AZD6244) and PI3K (LY294002) pathways inhibition. ß Actin was used as a loading control.

GART and E2F1 mRNA levels following METi combined with IR: mRNA levels of GART (left) and E2F1 (right) following METi (50nM, 24 hr), IR (10 Gy, 1 hr) and their combination in GTL-16 and EBC-1 cells.

Supplementations with glutamine, serine, and folic acid: (A) Proliferation of GTL-16 and EBC-1 cells upon supplementation with glutamine, serine or folic acid. (B) Apoptosis (caspase-3 activation) in GTL-16 and EBC-1 cells upon supplementation with glutamine, serine or folic acid.

Basic statistics of the downloaded TCGA data.

IntroductionWe sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States.MethodsSerum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires.ResultsSerum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range.DiscussionIn the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.

Survival analysis focused on the MET-E2F1-purine synthesis axis in clinical samples of TCGA cancer cohorts. Samples are differentiated by the under- and overexpression of individual transcripts. A, Impact of E2F1 and MET mRNA co-expression on survival of patients with lung, head and neck, stomach, and colorectal cancers. MET low/high percentiles 33/67. B, Impact of the mRNA co-expression of MET and GART/purine synthesis pathway-related enzymes on survival of patients with lung cancer. MET low/high percentiles 25/75. C, Impact of MET and RNR subunit (RRM1 and RRM2) mRNA co-expression on survival of patients with lung cancer. MET low/high percentiles 25/75.

Therapeutic implications of the MET-E2F1-purine synthesis pathway as a component of the MET-DDR crosstalk. A, Proliferation of EBC-1 and HCC827 cells upon treatments with the indicated doses of tepotinib, cisplatin, and their combination assessed by crystal violet assay 72 hours posttreatment. Data points are represented as mean ± SD of technical replicates (dots). Statistical analyses were done using one-way ANOVA. B, Schematic representation of the proposed MET-E2F1-purine synthesis pathway and its interplay with the DDR in MET-driven cancer cells. MET inhibition by the small-molecule inhibitor tepotinib downregulates E2F1 via the MAPK pathway, consequently downregulating purine synthesis enzymes (e.g., GART) and RNR. This leads to the depletion of dNTPs, compromising the ability of cells to repair DNA damage. Created with BioRender.com.

METi-dependent DNA damage induction mediated by E2F1 downregulation. A, Whole-cell lysates of MET-driven (GTL-16 and EBC-1) and non-MET-driven (HCC827 and H1648) cells were subjected to Western blotting using specific antibodies against pMET, pERK1/2, RAD51, GART, and E2F1 following treatment with vehicle or 50 nmol/L tepotinib for 24 (pMET, pERK1/2, and RAD51) or 48 (GART and E2F1) hours. Lysates for pMET and pERK1/2 detection were prepared by using the NP-40 lysis buffer, and lysates for detection of the other proteins were prepared by using the urea lysis buffer. ß-Actin was used as a loading control. Representative images of N = 3 independent experiments are shown. B, Whole-cell lysates were subjected to Western blotting using specific antibody against E2F1 in untreated empty vector and E2F1-overexpessing GTL-16 cells (72 hours post transfection). ß-Actin was used as a loading control. Representative images of N = 3 independent experiments are shown. C, mRNA levels of RRM1, RRM2, and GART following E2F1 overexpression in GTL-16 (72 hours post transfection and 48 hours post tepotinib treatment) as assessed by quantitative real-time PCR (N = 3, unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). D, Whole-cell lysates were subjected to Western blotting using specific antibodies against pMET, GART, and E2F1 following indicated treatments in E2F1-overexpessing cell line GTL-16 (72 hours post transfection and 48 hours post tepotinib treatment). ß-Actin was used as a loading control. Representative images of N = 3 independent experiments are shown. E, Representative images of nuclear γH2AX foci in perturbed (tepotinib 50 nmol/L, 8 hours) and unperturbed E2F1-overexpressing (16 hours post transfection) GTL-16 cells and in empty vector control cells (top). Bottom, γH2AX foci count per cell (blue: DAPI; red: γH2AX; green: E2F1-overexpressing cells), N = 3, unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. F, dNTP levels in MET-driven E2F1-overexpressing GTL-16 cells in response to METi (50 nmol/L, 8 hours). Cells were collected 16 hours post transfection [N = 3, unpaired Student t test, (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001)].

Human Ebola virus (EBOV) outbreaks caused by persistent EBOV infection raises questions on the role of zoonotic spillover in filovirus epidemiology. To characterise filovirus zoonotic exposure, we collected cross-sectional serum samples from bushmeat hunters (n=498) in Macenta Prefecture Guinea, adjacent to the index site of the 2013 EBOV-Makona spillover event. We identified distinct immune signatures (20/498, 4.0%) to multiple EBOV antigens (GP, NP, VP40) using stepwise ELISA and Western blot analysis and, live EBOV neutralisation (5/20; 25%). Using comparative serological data from PCR-confirmed survivors of the 2013-2016 EBOV outbreak, we demonstrated that most signatures (15/20) were not plausibly explained by prior EBOV-Makona exposure. Subsequent data-driven modelling of EBOV immunological outcomes to remote-sensing environmental data also revealed consistent associations with intact closed canopy forest. Together our findings suggest exposure to other closely related filoviruses prior to the 2013-2016 West Africa epidemic and highlight future surveillance priorities.

Impact of MET inhibition on the purine synthesis pathway. A, Transcript levels of purine synthesis enzymes are downregulated upon 24 hours of METi for EBC-1 and GTL-16 cells (N = 2). B, mRNA levels of PRPS1, PPAT, and GART upon METi in MET-driven (EBC-1 and GTL-16) and non-MET-driven (HCC821 and H820) cell lines assessed by quantitative real-time PCR (N = 3, unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). C, Whole-cell lysates were subjected to Western blotting using specific antibodies against GART following treatment either with vehicle or 50 nmol/L tepotinib for 24, 48, and 72 hours in MET-driven (EBC-1 and GTL-16) and non-MET-driven (HCC821 and H820) cell lines. Representative images of N = 3 independent experiments are shown. D, GART expression levels in EBC-1 (top) and HCC827 (bottom) tumor xenografts treated by vehicle or METi. Representative images (top) and quantification of GART-positive area (bottom) are provided [(EBC-1: N = 5 (control), N = 5 (tepotinib); HCC827: N = 3 (control), N = 2 (tepotinib); unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001)]. E, MET, PRPS1, PPAT, GART, PFAS, PAICS, ADSL, and ATIC transcript distribution in normal tissue vs. primary tumor in lung, colorectal, stomach, and head and neck cancer TCGA datasets.

Metabolic changes in MET-driven and non-MET-driven cancer cells and xenografts. A, Pathway enrichment analysis upon MET inhibition (N = 3, |log2FC| > 0.5; adj. P value < 0.01) revealed alterations observed in 12 MET-expressing cell lines upon 24 hours of METi. B, Changes in the abundance of the three metabolite ions [5′-phosphoribosyl-N-formylglycinamide (FGAR), phosphoribosyl pyrophosphate (PRPP), and deoxythymidine diphosphate (dTDP)] that change most upon METi in MET-driven cell lines. Symbol sizes represent the adjusted P values from 1 (smallest) to 10−8 (largest) using a log scale. No significant changes in these ions occurred in the non-MET-driven cell lines H1648, H820, HCC827, and Y1248H. C, Left, Differences in metabolite ion (symbols) abundance in METi- and vehicle-treated tumor xenografts. Symbols corresponding to statistically significant differences (|log2FC| > 0.5; P value < 0.01) are indicated in dark blue, and FGAR is marked in red. Right, Changes in the FGAR metabolite ion abundance in HCC827 and EBC-1 tumor xenografts upon METi.

Abstract Various lines of investigation support a signaling interphase shared by receptor tyrosine kinases and the DNA damage response. However, the underlying network nodes and their contribution to the maintenance of DNA integrity remain unknown. We explored MET-related metabolic pathways in which interruption compromises proper resolution of DNA damage. Discovery metabolomics combined with transcriptomics identified changes in pathways relevant to DNA repair following MET inhibition (METi). METi by tepotinib was associated with the formation of γH2AX foci and with significant alterations in major metabolic circuits such as glycolysis, gluconeogenesis, and purine, pyrimidine, amino acid, and lipid metabolism. 5′-Phosphoribosyl-N-formylglycinamide, a de novo purine synthesis pathway metabolite, was consistently decreased in in vitro and in vivo MET-dependent models, and METi-related depletion of dNTPs was observed. METi instigated the downregulation of critical purine synthesis enzymes including phosphoribosylglycinamide formyltransferase, which catalyzes 5′-phosphoribosyl-N-formylglycinamide synthesis. Genes encoding these enzymes are regulated through E2F1, whose levels decrease upon METi in MET-driven cells and xenografts. Transient E2F1 overexpression prevented dNTP depletion and the concomitant METi-associated DNA damage in MET-driven cells. We conclude that DNA damage following METi results from dNTP reduction via downregulation of E2F1 and a consequent decline of de novo purine synthesis. Significance: Maintenance of genome stability prevents disease and affiliates with growth factor receptor tyrosine kinases. We identified de novo purine synthesis as a pathway in which key enzymatic players are regulated through MET receptor and whose depletion via MET targeting explains MET inhibition-associated formation of DNA double-strand breaks. The mechanistic importance of MET inhibition-dependent E2F1 downregulation for interference with DNA integrity has translational implications for MET-targeting-based treatment of malignancies.

Responses of a panel of MET-positive MET-driven and non-MET-driven human cancer cell models to selective MET targeting. A, Whole-cell lysates were subjected to Western blotting using specific antibodies against MET, pMET, and pS6 following the treatment either with vehicle or 50 nmol/L tepotinib (EMD1214063, shortly EMD) for 24 hours. Western blots representative of N = 3 independent experiments are shown. B, Whole-cell lysates were subjected to Western blotting using a specific antibody against p4E-BP1 following the treatment either with vehicle or 50 nmol/L tepotinib (EMD1214063, shortly EMD) for 24 hours. Western blots representative of N = 3 independent experiments are shown. C, EBC-1 (top) and HCC827 (bottom) xenograft tumor volume upon vehicle or METi treatment (N = 10). D, pMET Tyr1234/5 expression in EBC-1 (left) and HCC827 (right) tumor xenografts treated by vehicle or METi (sampling was performed 2 hours posttreatment). Representative images (top) and quantification of pMET-positive area (bottom) are provided [EBC-1: N = 10 (control), N = 10 (tepotinib); HCC827: N = 6 (control), N = 4 (tepotinib); unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001]. E, Representative images (left) of nuclear foci of γH2AX in MET-driven GTL-16 and EBC-1 and non-MET-driven HCC827 and H820 cells. Untreated or METi-treated (50 nmol/L) cells were fixed; γH2AX-positive foci were visualized by confocal microscopy and counted (right), N = 3, unpaired Student t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Abstract Background Natural killer (NK) cells can both amplify and diminish immune responses to vaccination. Studies in humans and animals have observed NK cell activation within days after mRNA vaccination. In this study, we sought to determine if baseline NK cell frequencies, phenotype, or function correlate with antibody responses or inflammatory side effects induced by the Pfizer-BioNTech COVID-19 vaccine (BNT162b2). Methods We analyzed serum and peripheral blood mononuclear cells (PBMCs) from 188 participants in the Prospective Assessment of SARS-CoV-2 Seroconversion study, an observational study evaluating immune responses in healthcare workers. Baseline serum samples and PBMCs were collected from all participants prior to any SARS-CoV-2 infection or vaccination. Spike-specific IgG antibodies were quantified at one and six months post-vaccination by microsphere-based multiplex immunoassay. NK cell frequencies and phenotypes were assessed on pre-vaccination PBMCs from all participants by multi-color flow cytometry, and on a subset of participants at time points after the 1st and 2nd doses of BNT162b2. Inflammatory side effects were assessed by structured symptom questionnaires, and baseline NK cell functionality was quantified by an in vitro killing assay on participants that reported high or low post-vaccination symptom scores. Results Key observations include: 1) circulating NK cells exhibit an increase in CD56dim CD16- NK cells compared to baseline levels, providing evidence of NK cell activation in the week following vaccination, 2) individuals with high symptom scores after 1st vaccination had higher pre-vaccination NK cytotoxicity indices compared to individuals with low symptoms scores (195.7 [SD 170.1] vs 118.5 [SD 77.5], p=0.04), and 3) pre-vaccination NK cell numbers were negatively correlated with spike-specific IgG levels six months after two BNT162b2 doses (Rho= -0.14, p=0.043). Conclusion These results suggest that NK cell activation by BNT162b2 vaccination may contribute to vaccine-induced inflammatory symptoms and reduce durability of vaccine-induced antibody responses. Disclosures David Tribble, MD, DrPH, AstraZeneca: The IDCRP and HJF were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial as part of US Govt COVID response Timothy Burgess, MD, MPH, AstraZeneca: The IDCRP and the Henry M. Jackson Foundation (HJF) were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial Simon Pollett, MBBS, AstraZeneca: The IDCRP and the Henry M. Jackson Foundation (HJF) were funded to conduct an unrelated phase III COVID-19 monoclonal antibody immunoprophylaxis trial

IntroductionNatural killer (NK) cells can both amplify and regulate immune responses to vaccination. Studies in humans and animals have observed NK cell activation within days after mRNA vaccination. In this study, we sought to determine if baseline NK cell frequencies, phenotype, or function correlate with antibody responses or inflammatory side effects induced by the Pfizer-BioNTech COVID-19 vaccine (BNT162b2).MethodsWe analyzed serum and peripheral blood mononuclear cells (PBMCs) from 188 participants in the Prospective Assessment of SARS-CoV-2 Seroconversion study, an observational study evaluating immune responses in healthcare workers. Baseline serum samples and PBMCs were collected from all participants prior to any SARS-CoV-2 infection or vaccination. Spike-specific IgG antibodies were quantified at one and six months post-vaccination by microsphere-based multiplex immunoassay. NK cell frequencies and phenotypes were assessed on pre-vaccination PBMCs from all participants by multi-color flow cytometry, and on a subset of participants at time points after the 1st and 2nd doses of BNT162b2. Inflammatory side effects were assessed by structured symptom questionnaires, and baseline NK cell functionality was quantified by an in vitro killing assay on participants that reported high or low post-vaccination symptom scores.ResultsKey observations include: 1) circulating NK cells exhibit evidence of activation in the week following vaccination, 2) individuals with high symptom scores after 1st vaccination had higher pre-vaccination NK cytotoxicity indices, 3) high pre-vaccination NK cell numbers were associated with lower spike-specific IgG levels six months after two BNT162b2 doses, and 4) expression of the inhibitory marker NKG2A on immature NK cells was associated with higher antibody responses 1 and 6 months post-vaccination.DiscussionThese results suggest that NK cell activation by BNT162b2 vaccination may contribute to vaccine-induced inflammatory symptoms and reduce durability of vaccine-induced antibody responses.