Immunization of MERS-CoV-2-infected mice with a sublethal dose of MERS-CoV or VRP-MERS-S.

Here, we detail the immunization of mice with a sublethal dose of MERS-CoV or two doses of replication-incompetent alphavirus replicon particles expressing MERS-CoV spike protein. We then describe steps to determine the outcome of immunization by challenging immunized mice with a lethal dose of MERS-CoV, as well as by detecting virus-specific neutralizing antibody and virus-specific T cell response via neutralization assay and flow cytometry, respectively. This protocol can be used to evaluate other CoV infections or vaccine-induced immune responses. For complete details on the use and execution of this protocol, please refer to Zheng et al. (2021).1.

S-08-04 High-dimensional approaches for immune profiling of 2D materials: towards safe-by-design

Understanding the impact of nanomaterials on the immune system is of paramount importance. We recently proposed the "nano-immunity-by-design" approach according to which the characterization of 2D nanomaterials is based not only on their physicochemical parameters but also on immune profiling. Immune profiling can be performed using high-dimensional approaches such as single-cell mass cytometry on graphene and other 2D materials including transition metal carbides/carbonitrides (MXenes). Our studies revealed that amino functionalization of graphene oxide (GO) increased the immune compatibility of this material (Orecchioni et al. Nat Commun. 2017). Moreover, by functionalizing GO with AgInS2 nanocrystals, detection by single-cell mass cytometry was enabled in a large variety of primary immune cells (Orecchioni et al. Small. 2020). Recently, we reported on the immune modulation by Mxenes, including Ti3C2Tx, in combination with their antiviral properties against SARS-CoV-2 by using single-cell mass cytometry and other high-dimensional approaches (Unal et al. Nano Today. 2021). The present talk will also cover recent (unpublished) findings from the EU-funded projects, G-IMMUNOMICS and CARBO-IMmap, on a variety of 2D materials, including Mxenes, MoS2, WS2, and other materials, on human immune cells as well as in murine and porcine models. Our results illustrate that the chemical and immunological design of 2D materials offers new strategies for their safe exploitation in medicine. Copyright © 2022 Elsevier B.V.

A sex-biased imbalance between Tfr, Tph, and atypical B cells determines antibody responses in COVID-19 patients.

Sex-biased humoral immune responses to COVID-19 patients have been observed, but the cellular basis for this is not understood. Using single-cell proteomics by mass cytometry, we find disrupted regulation of humoral immunity in COVID-19 patients, with a sex-biased loss of circulating follicular regulatory T cells (cTfr) at a significantly greater rate in male patients. In addition, a male sex-associated cellular network of T-peripheral helper, plasma blasts, proliferating and extrafollicular/atypical CD11c+ memory B cells was strongly positively correlated with neutralizing antibody concentrations and negatively correlated with cTfr frequency. These results suggest that sex-specific differences to the balance of cTfr and a network of extrafollicular antibody production-associated cell types may be a key factor in the altered humoral immune responses between male and female COVID-19 patients.

A combined fine needle aspiration and spectral flow cytometry approach to assess human germinal center responses to SARS-CoV-2 vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines drive the generation of affinity-matured B cell responses through germinal center (GC) reactions in vaccine draining lymph nodes. Herein, we describe a procedure for the acquisition of human lymph node samples via an ultrasound-guided fine needle aspiration-based approach. Additionally, we outline a suggested approach for the analysis of CD4 T helper cell subsets as well as antigen-specific GC B cells, memory B cells, and plasmablasts by high-parameter spectral flow cytometry. For complete details on the use and execution of this protocol, please refer to Lederer et al. (2022).1.

Flow cytometric protocol to characterize human memory B cells directed against SARS-CoV-2 spike protein antigens.

Memory B cells (MBCs), part of the immune response elicited by infection or vaccination, can persist in lymphoid organs and peripheral blood and are capable of rapid reactivation upon secondary antigen exposure. Here, we describe a flow cytometric assay to identify antigen-specific MBCs from peripheral blood mononuclear cells and characterize their isotypes and activation status. We detail steps to use fluorescently labeled antigen probes derived from the SARS-CoV-2 spike protein. These can be adapted to detect MBCs against other antigens. For complete details on the use and execution of this protocol, please refer to Weskamm et al. (2022).1.

Navigating the cellular landscape in tissue: Recent advances in defining the pathogenesis of human disease.

Over the past decade, our understanding of human diseases has rapidly grown from the rise of single-cell spatial biology. While conventional tissue imaging has focused on visualizing morphological features, the development of multiplex tissue imaging from fluorescence-based methods to DNA- and mass cytometry-based methods has allowed visualization of over 60 markers on a single tissue section. The advancement of spatial biology with a single-cell resolution has enabled the visualization of cell-cell interactions and the tissue microenvironment, a crucial part to understanding the mechanisms underlying pathogenesis. Alongside the development of extensive marker panels which can distinguish distinct cell phenotypes, multiplex tissue imaging has facilitated the analysis of high dimensional data to identify novel biomarkers and therapeutic targets, while considering the spatial context of the cellular environment. This mini-review provides an overview of the recent advancements in multiplex imaging technologies and examines how these methods have been used in exploring pathogenesis and biomarker discovery in cancer, autoimmune and infectious diseases.

Protocol to quantify and phenotype SARS-CoV-2-specific T cell response using a rapid flow-cytometry-based whole blood assay.

Monitoring antigen-specific T cell frequency, function, and phenotype is essential to assess the host immune response to pathogens or novel vaccines. Here, we describe a rapid and simple ex vivo whole blood assay to detect and phenotype the SARS-CoV-2-specific T cell response. We detail steps for whole blood stimulation with SARS-CoV-2 spike peptide and subsequent cell fixation and cryopreservation. We further describe thawing and cell staining steps for flow cytometry analysis. This approach minimizes sample manipulation and has a quick turnaround time. For complete details on the use and execution of this protocol, please refer to Riou et al. (2021).

Rapid increase of myeloid-derived suppressor cells and prolonged innate immune dysfunctions in patients with COVID-19

Background: PMN and monocytic myeloid-derived suppressor cells (PMN-MDSCs, M-MDSCs) are immunosuppressive cells rising during infections. Aim: To characterize the dynamic of MDSCs in relation with immune parameters in COVID-19 patients followed for 3 months. Methods: 56 SARS-CoV-2 infected adult patients hospitalized at CHUV were included. Blood was obtained at inclusion and 3 months later in 21 patients, and from 10 healthy controls. Blood was stimulated with TLR ligands. Leukocyte populations and cytokines were analyzed by flow cytometry, mass cytometry, multiplex bead assay and ELISA. Results: At hospital admission, PMN-MDSCs and M-MDSCs were increased 2-4-fold in COVID-19 patients (P <0.05). PMN-MDSCs and M-MDSCs counts were higher in severe than in moderate COVID-19 patients (P <0.005). PMN-MDSCs and M-MDSCs correlated positively with EGF and HGF (P <0.05). M-MDSCs correlated positively with IL-1β, IL-7, PDGF and VEGF (P <0.05). In whole blood stimulated with TLR ligands, the proportion of TNF and IL-6- producing monocytes and DCs were reduced in patients. After 32 months, MDSCs were back to normal levels, while the production of cytokines by blood, monocytes and DCs was still largely affected. Conclusions: PMN-MDSCs and M-MDSCs were elevated and correlated with disease severity in patients analyzed at hospitalization. Innate immune blood responses were impaired in patients, which persisted for up to 3 months. Our results suggest that COVID-19 induces rapid and long-standing innate immune dysregulation.

Protocol for isolation and characterization of lung tissue resident memory T cells and airway trained innate immunity after intranasal vaccination in mice.

Vaccination route dictates the quality and localization of immune responses within tissues. Intranasal vaccination seeds tissue-resident adaptive immunity, alongside trained innate responses within the lung/airways, critical for superior protection against SARS-CoV-2. This protocol encompasses intranasal vaccination in mice, step-by-step bronchoalveolar lavage for both cellular and acellular airway components, lung mononuclear cell isolation, and detailed flow cytometric characterization of lung tissue-resident memory T cell responses, and airway macrophage-trained innate immunity. For complete details on the use and execution of this protocol, please refer to Afkhami et al. (2022).

Integrated plasma proteomic and single-cell immune signaling network signatures demarcate mild, moderate, and severe COVID-19.

The biological determinants underlying the range of coronavirus 2019 (COVID-19) clinical manifestations are not fully understood. Here, over 1,400 plasma proteins and 2,600 single-cell immune features comprising cell phenotype, endogenous signaling activity, and signaling responses to inflammatory ligands are cross-sectionally assessed in peripheral blood from 97 patients with mild, moderate, and severe COVID-19 and 40 uninfected patients. Using an integrated computational approach to analyze the combined plasma and single-cell proteomic data, we identify and independently validate a multi-variate model classifying COVID-19 severity (multi-class area under the curve [AUC]training = 0.799, p = 4.2e-6; multi-class AUCvalidation = 0.773, p = 7.7e-6). Examination of informative model features reveals biological signatures of COVID-19 severity, including the dysregulation of JAK/STAT, MAPK/mTOR, and nuclear factor κB (NF-κB) immune signaling networks in addition to recapitulating known hallmarks of COVID-19. These results provide a set of early determinants of COVID-19 severity that may point to therapeutic targets for prevention and/or treatment of COVID-19 progression.

High-dimensional immune phenotyping of blood cells by mass cytometry in patients infected with hepatitis C virus.

OBJECTIVES: Chronic hepatitis C virus (HCV) infection affects the immune system. Whether elimination of HCV with direct-acting antivirals (DAA) restores immunity is unclear. We used mass cytometry to get a broad and in-depth assessment of blood cell populations of patients with chronic HCV before and after DAA therapy. METHODS: Before and 12 weeks after sustained virological response (SVR12) to DAA therapy, 22 cell populations were analysed by mass cytometry in blood collected from ten healthy control individuals and 20 HCV-infected patients with (ten patients) or without (ten patients) human immunodeficiency virus (HIV) infection. RESULTS: HCV infection altered the frequency of 14/22 (64%) blood cell populations. At baseline, the frequencies (median, interquartile range (IQR); control, HCV, HCV/HIV) of intermediate monocytes (1.2, IQR 0.47-1.46; 1.76, IQR 0.83-2.66; 0.78, IQR 0.28-1.77), non-classical monocytes (1.11, IQR 0.49-1.26; 0.9, IQR 0.18-0.99; 0.54, IQR 0.28-1.77), conventional dendritic cells type 2 (0.55, IQR 0.35-0.59; 0.31, IQR 0.16-0.38; 0.19, IQR 0.11-0.36) and CD56dim natural killer cells (8.08, IQR 5.34-9.79; 4.72, IQR 2.59-6.05) 3.61, IQR 2.98-5.07) were reduced by 35% to 65%, particularly in HCV/HIV co-infected patients. In contrast, activated double-negative T cells (0.07, IQR 0.06-0.10; 0.10, IQR 0.09-0.19; 0.19, IQR 0.12-0.25), activated CD4 T cells (0.28, IQR 0.21-0.36; 0.56, IQR 0.33-0.77; 0.40, IQR 0.22-0.53) and activated CD8 T cells (0.23, IQR 0.14-0.42; 0.74, IQR 0.30-1.65; 0.80, IQR 0.58-1.16) were increased 1.4 to 3.5 times. Upon stimulation with Toll-like receptor ligands, the expression of cytokines was up-regulated in 7/9 (78%) and 17/19 (89%) of the conditions in HCV- and HCV/HIV-infected patients, respectively. Most alterations persisted at SVR12. CONCLUSIONS: Chronic HCV and HCV/HIV infections induce profound and durable perturbations of innate and adaptive immune homeostasis.

Immune-mediated Hepatitis associated with SARS-CoV-2 mRNA vaccination

In a fraction of SARS-CoV-2 vaccinees, hepatitis compatible with features of autoimmune hepatitis (AIH) have been observed. However, it remains unclear whether the association is coincidental, reflects drug-induced liver injury, or involves vaccine-induced antigen-specific immune activation. Here, we report a case of a 52-year-old male developing a transient hepatitis after the first mRNA vaccination and severe AIH-compatible hepatitis after the second. The intrahepatic immune cell infiltrate was analysed by highly multiplexed imaging mass cytometry broadly covering key immune cell populations. Liver and longitudinal blood samples were analysed for the presence and phenotype of SARS-CoV-2 Spike-specific CD8 T cells using MHC class I tetramer technology. Additionally, Serum titers against SARS-Cov2-Spike antibodies were assessed. We identified a panlobular CD8 T cell dominant immune cell infiltrate in the liver without significant plasma cell components. Spike-specific CD8 T cells were highly enriched within the intrahepatic CD8 T cell population expressing activation markers and a tissue-resident phenotype. The activation phenotype correlated with the circulating Spike-specific CD8 T cell profile and longitudinal analysis revealed a rapid decline of T cell activation after the initiation of budesonid therapy. However, the patient experienced a mild relapse under therapy that was paralleled by the peripheral activation of Spike-specific CD8 T cells and was controlled under systemic steroid therapy. Collectively, our results indicate that an immune-mediated hepatitis after COVID19 vaccination can present with typical clinical features of an AIH but can be pathophysiologically separated from a classical AIH. Whether a long-term immunosuppressive regimen will be required remains to be determined.

A cell-based system combined with flow cytometry to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in patients with COVID-19.

This protocol describes a flow cytometry approach to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in COVID-19-positive patient sera samples without the need of specific laboratory facilities for viral infection. We developed a human-cell-based system using spike-expressing HEK293T cells that mimics membrane insertion and N-glycosylation of viral integral membrane proteins in host cells. This assay represents a powerful tool to test antibody responses against SARS-CoV-2 variants and vaccine effectiveness. For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).

TITAN: An end-to-end data analysis environment for the Hyperion™ imaging system.

Imaging Mass Cytometry (IMC) is a powerful high-throughput technique enabling resolution of up to 37 markers in a single fixed tissue section while also preserving in situ spatial relationships. Currently, IMC processing and analysis necessitates the use of multiple different software, labour-intensive pipeline development, different operating systems and knowledge of bioinformatics, all of which are a barrier to many potential users. Here we present TITAN - an open-source, single environment, end-to-end pipeline that can be utilized for image visualization, segmentation, analysis and export of IMC data. TITAN is implemented as an extension within the publicly available 3D Slicer software. We demonstrate the utility, application, reliability and comparability of TITAN using publicly available IMC data from recently-published breast cancer and COVID-19 lung injury studies. Compared with current IMC analysis methods, TITAN provides a user-friendly, efficient single environment to accurately visualize, segment, and analyze IMC data for all users.

Facile isolation of high-affinity nanobodies from synthetic libraries using CDR-swapping mutagenesis.

The generation of high-affinity nanobodies for diverse biomedical applications typically requires immunization or affinity maturation. Here, we report a simple protocol using complementarity-determining region (CDR)-swapping mutagenesis to isolate high-affinity nanobodies from common framework libraries. This approach involves shuffling the CDRs of low-affinity variants during the sorting of yeast-displayed libraries to directly isolate high-affinity nanobodies without the need for lead isolation and optimization. We expect this approach, which we demonstrate for SARS-CoV-2 neutralizing nanobodies, will simplify the generation of high-affinity nanobodies. For complete details on the use and execution of this profile, please refer to Zupancic et al. (2021).

FIRST HIGH DIMENSIONAL EXAMINATION OF INTESTINAL BIOPSIES IN PATIENTS WITH COVID-19

Introduction: SARS-CoV-2, the causative pathogen for COVID-19, engages host ACE2 receptor for cellular entry. The brush border of the small intestines express high levels of ACE2. Gastrointestinal (GI) manifestations are common among COVID-19 patients. However, to date, there is limited information regarding intestinal response to SARS-CoV-2 infection. Methods: Intestinal biopsies were obtained from 17 COVID-19 patients (17.3 ± positive nasal swab) for cellular and transcriptomic analyses using mass cytometry and RNA-sequencing, respectively. Ten uninfected individuals served as compartment (EC) and lamina propria (LP) were analyzed separately. Results: The cellular profiles from LP of COVID-19 patients showed reduced frequencies of CD206+ conventional dendritic cells (CDC2s) and plasmacytoid (CD123+) dendritic cells Effector T cell (PD1+CD38+) frequency was increased in the LP and Intraepithelial lymphocytes (IEL) were increased in the EC of COVID-19 patients, with a concomitant decrease in CD206+ CDC2s. RNA sequencing active downregulation of genes involved in inflammatory pathways including IBD-associated pathways, while an upregulation of intestinal barrier function Gene expression of Neuropilin-1 (NRP-1), a putative SARS-CoV-2 receptor as well as key inflammatory cytokines (IL-1b, IFN-g, CCL24 and CXCL8) were significantly reduced in controls. A low intensity antiviral host response signature was observed predominantly in EC as opposed to LP suggesting viral localization to epithelium. Conclusions: Epithelial, myeloid and lymphoid cell alterations characterize intestinal response to SARS-CoV-2 infection with an unanticipated downregulation of key inflammatory pathways that have been implicated in adverse outcomes associated with These data stand in contrast to the inflammatory response reported in the systemic compartments and identify a potential mitigating role of the GI

Prolonged enhancement of cytotoxic T lymphocytes in the post-recovery state of severe COVID-19.

We evaluated the peripheral blood immune responses of lymphocytes in severe Coronavirus disease 2019 (COVID-19) patients in different stages of recovery using single-cell mass cytometry. The patients with prolonged hospitalization did not show recovery of B lymphocyte counts and CD4-positive T lymphocyte counts but did show abundant CD8-positive T lymphocytes. CD4 and CD8 T cells expressing high levels of T-bet and Granzyme B were more abundant in post-recovery patients. This study showed that cytotoxic Th1 and CD8 T cells are recruited to the peripheral blood long after recovery from COVID-19.

Manufacture and Immunological Characterization of GMP-Compliant Functional Sars-COV-2 Cytotoxic T Lymphocytes (CTLs) Utilizing the Clinimacs ® Cytokine Capture System

Background: The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pandemic that has taken millions of lives around the globe. Treatment of patients with moderate and severe COVID-19 disease has included dexamethasone, tocilizumab, Remdesivir, convalescent plasma, and targeted antibodies, however, currently, there are no FDA approved targeted cellular therapies in the treatment of mild or moderate SARS-CoV-2 disease. Virus-specific cytotoxic T cell lymphocytes (vCTLs) have shown therapeutic efficacy in immunocompromised patients with viral infections. We developed a multicenter and multidisciplinary Viral Cytotoxic T-Cell Consortium (VIRCTLC) to investigate the use of vCTLs manufactured by direct enrichment using the Cytokine Capture System (CCS) on the CliniMACS® Prodigy device. SARS-CoV-2 specific PepTivator Peptides consist of overlapping peptides that span the entire sequence of the protein (Protein N and M), or the length of its immunodominant domain (Protein S). The peptides can bind to either MHC class I or MHC class II molecules and are therefore able to target both CD4 and CD8 T cells. Objective: To screen, manufacture, and characterize SARS-CoV-2 vCTLs generated from convalescent COVID-19 donors using the CliniMACS® Cytokine Capture System on the CliniMACS® Prodigy device. Methods: Donor screening was done utilizing PBMNCs from 15 convalescent COVID-19 donors after informed consent. PBMNCs were stimulated with a mix of PepTivator peptides (Miltenyi Biotech®) contained in the S, M and N proteins. IFN-γ levels were examined in CD3, CD4, and CD8 T cells by flow cytometry analysis. After informed consent, PBMNCs from three convalescent COVID-19 donors who screened positively to the PepTivator® peptide pools of SARS-CoV-2 Proteins M, N and S were collected by apheresis using the SPECTRA Optia® apheresis instrument. PBMNCs were incubated with the PepTivator® peptide pools for 4 hours. After incubation, the SARS-CoV-2 vCTLs were enriched using the CliniMACS Cytokine Capture System as we have previously described (Flower/Cairo, et al, ASTCT, 2020). Samples were taken from the enriched vCTLs and tested in gram stains, sterility cultures, cell counts, viability and IFN-γ cytokine staining (CD3/CD4/CD8/IFN-γ marker panel) by flow cytometry. Amplification and sequencing of TCRβ CDR3 regions of pre-stimulated PBMNC, stimulated PBMNCs samples taken from the QC bag (QC samples) and the enriched SARS-CoV-2 vCTLs were performed on the ImmunoSEQ platform using ImmunoSEQ® TCRB Assay kit (Adaptive Biotechnologies, Seattle, WA, USA). Characterization of immune subsets was done by mass cytometry analysis with 41 Immunophenotypic markers. Transcriptome of the immune landscape of QC samples, and enriched vCTLs was compared with the pre samples using the human nCounter PanCancer Immune Profiling Panel on the nCounter system. Results: We demonstrate that 93.3% of convalescent donor blood samples passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that consisted of 79% + 21% (mean + SEM) IFNγ + T cells (Fig.1). TCRβ sequencing showed that convalescent COVID-19 donors have a highly diverse TCR repertoire and we identified TCRβ CDR3 clones that are known to be associated with SARS-CoV-2 T cell responses. Immunophenotyping analysis demonstrated more CD4 T cells than CD8 T cells in the SARS CoV-2 vCTLs, an increase in memory CD8 and CD4 cells, especially CD8 T EM, CD4 T cm and CD4 T EMRA cells (Fig.2) and an increase DC cells in the SARS CoV-2 vCTL products as compared to pre-stimulated PBMNCs. Expression of the exhaustion markers was not enhanced in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Transcriptome analysis showed increased gene expression in T-cell function, interleukin, pathogen defense, and TNF superfamily pathway genes in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Conclusion: Our study demonstrates that highly functional SARS-CoV-2 vCTLs can be rapidly generat d by direct cytokine enrichment from convalescent donor peripheral blood mononuclear cells. These data serve as pre-clinical validation for an ongoing clinical trial utilizing related HLA-matched and haplo-identical SARS CoV-2 vCTLs for the treatment of patients with mild and moderate SARS-CoV-2 disease (IND #27260, NCT# 04896606). [Formula presented] Disclosures: Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding;Courier Therapeutics: Current holder of individual stocks in a privately-held company. Johnson: Miltenyi Biotec: Research Funding. Cairo: Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Amgen: Speakers Bureau;Sanofi: Speakers Bureau;Servier: Speakers Bureau;Sobi: Speakers Bureau;Omeros: Membership on an entity's Board of Directors or advisory committees;Nektar: Membership on an entity's Board of Directors or advisory committees.

Inhibition of De Novo Pyrimidine Synthesis Depletes Acute Myleogenous Leukemia Stem Cells (LSCs) Burden and Triggers Apoptosis and Differentiation Associated with Oxidative Stress

Background: De novo nucleotide synthesis is necessary to meet the enormous demand for nucleotides, other macromolecules associated with acute myeloid leukemia (AML) progression 1, 2, 34. Hence, we hypothesized that targeting de novo nucleotide synthesis would lead to the depletion of the nucleotide pool, pyrimidine starvation and increase oxidative stress preferentially in leukemic cells compared to their non-malignant counterparts, impacting proliferative and differentiation pathways. Emvododstat (PTC299) is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis that is currently in a clinical trial for the treatment of AML. Objectives: The goals of these studies were to understand the emvododstat-mediated effects on leukemia growth, differentiation and impact on Leukemia Stem Cells(LSCs). Comprehensive analyses of mitochondrial function, metabolic signaling in PI3K/AKT pathways, apoptotic signatures, and DNA damage responses were carried out. The rationale for clinical testing emvododstat was confirmed in an AML-PDX model. Results: Emvododstat treatment in cytarabine-resistant AML cells and primary AML blasts induced apoptosis, differentiation, and reduced proliferation, with corresponding decreased in cell number and increases in annexin V- and CD14-positive cells. Indeed, the inhibition of de novo nucleotide synthesis compromises the dynamic metabolic landscape and mitochondrial function, as indicated by alterations in the oxygen consumption rate (OCR) and mitochondrial ROS/membrane potential and corresponding differentiation, apoptosis, and/or inhibition of proliferation of LSCs. These effects can be reversed by the addition of exogenous uridine and orotate. Further immunoblotting and mass cytometry (CyTOF) analyses demonstrated changes in apoptotic and cell signaling proteins (cleaved PARP, cleaved caspase-3) and DNA damage responses (TP53, γH2AX) and PI3/AKT pathway downregulation in response to emvododstat. Importantly, emvododstat treatment reduced leukemic cell burden in a mouse model of AML PDX ( Complex karyotype, mutation in ASXL1, IDH2, NRAS), decreased levels of leukemia stem cells frequency (1 in 522,460 Vs 1 in 3,623,599 in vehicle vs emvododstat treated mice), and improved survival. The median survival 40 days vs. 30 days, P=0.0002 in primary transplantation and 36 days vs 53.5 days, P=0.005 in secondary transpantation in a PDX mouse model of human AML. This corresponded with a reduction in the bone marrow burden of leukemia and increased expression of differentiation markers in mice treated with emvododstat (Fig. 1). These data demonstrate effect of emvododstat on mitochondrial functions. Conclusion: Inhibition of de novo pyrimidine synthesis triggers differentiation, apoptosis, and depletes LSCs in AML models. Emvododstat is a novel dihydroorotate dehydrogenase inhibitor being tested in a clinical trial for the treatment of myeloid malignancies and COVID-19. Keywords: AML, emvododstat, DHODH, apoptosis, differentiation References: 1 Thomas, D. & Majeti, R. Biology and relevance of human acute myeloid leukemia stem cells. Blood 129, 1577-1585, doi:10.1182/blood-2016-10-696054 (2017). 2 Quek, L. et al. Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage. The Journal of experimental medicine 213, 1513-1535, doi:10.1084/jem.20151775 (2016). 3 Villa, E., Ali, E. S., Sahu, U. & Ben-Sahra, I. Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides. Cancers (Basel) 11, doi:10.3390/cancers11050688 (2019). 4 DeBerardinis, R. J. & Chandel, N. S. Fundamentals of cancer metabolism. Sci Adv 2, e1600200, doi:10.1126/sciadv.1600200 (2016). [Formula presented] Disclosures: Weetall: PTC therapeutics: Current Employment. Sheedy: PTC therapeutics: Current Employment. Ray: PTC therapeutics: Current Employment. Andreeff: Karyopharm: Research Funding;AstraZeneca: Research Funding;Oxford Biomedica UK: Research Funding;Aptose: Consultancy;Daiich -Sankyo: Consultancy, Research Funding;Syndax: Consultancy;Breast Cancer Research Foundation: Research Funding;Reata, Aptose, Eutropics, SentiBio;Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company;Novartis, Cancer UK;Leukemia & Lymphoma Society (LLS), German Research Council;NCI-RDCRN (Rare Disease Clin Network), CLL Foundation;Novartis: Membership on an entity's Board of Directors or advisory committees;Senti-Bio: Consultancy;Medicxi: Consultancy;ONO Pharmaceuticals: Research Funding;Amgen: Research Funding;Glycomimetics: Consultancy. Borthakur: ArgenX: Membership on an entity's Board of Directors or advisory committees;Protagonist: Consultancy;Astex: Research Funding;University of Texas MD Anderson Cancer Center: Current Employment;Ryvu: Research Funding;Takeda: Membership on an entity's Board of Directors or advisory committees;Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees;GSK: Consultancy.

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