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Title
HIV proviral reactivation is impaired in effector memory CD4+ T cells by immunometabolic reprogramming with dasatinib
Presenter
Guiomar Casado Fernández
Authors
G. Casado Fernández1,2,3, M. Martínez Velasco4, S. Rodríguez Mora1,3, M. Torres1, C. Sánchez Menéndez1,5, A. Simón Rueda1,6, M. Cervero7, C. Hoffmann8, C. Wyen9, E. San José4, V. Planelles10, M. Coiras1,3
Institutions
1Instituto de Salud Carlos III, Immunopathology Unit-National Center of Microbiology, Madrid, Spain, 2Universidad de Alcalá, Faculty of Sciences, Madrid, Spain, 3Biomedical Research Center Network in Infectious Diseases (CIBERINFEC), Madrid, Spain, 4Universidad Europea de Madrid, Health Sciences Department, Faculty of Biomedical and Health Sciences, Madrid, Spain, 5Hospital Universitario Ramón y Cajal, Hematology and Hemotherapy Service, Madrid, Spain, 6Universidad Complutense de Madrid, Faculty of Sciences, Madrid, Spain, 7Hospital Universitario Severo Ochoa, Internal Medicine Service, Madrid, Spain, 8ICH Study Center, Hamburg, Germany, 9University Hospital of Cologne, Department of Medicine I, Cologne, Germany, 10University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake, United States

BACKGROUND: We evaluated the use of dasatinib as latency promoting agent (LPA) by reducing the metabolic activity of viable CD4 cells to interfere with HIV reservoir replenishment.
METHODS: CD4 from PLWH and healthy donors were isolated and provirus was reactivated ± dasatinib 75nM 72h. Phosphoproteome was analyzed by LC-MS/MS. Mitochondrial ATP was quantified every 24h. Phosphoglumutase-2 (PGM2), pyruvate kinase M1/2 (PKM), aldolase (ALDOA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities were analyzed by ELISA. Fluorescent glucose analog 2-NBDG uptake, GLUT-1 expression, T-cell memory subpopulations distribution, and T-cell viability were determined by flow cytometry.
RESULTS: 1) Dasatinib decreased proviral reactivation 2.47-fold (p=0.0469) in CD4 from PLWH. 2)Dasatinib modified phosphorylation of >130 proteins involved in metabolic pathways: energy metabolism, glycolysis/glyconeogenesis, inositol-phosphate metabolism, fatty-acid synthesis. 3)Dasatinib interfered with phosphorylation of 4 essential enzymes that belong to irreversible steps of glycolysis and tricarboxylic acid (TCA): PKM (-4.63;p=0.002), PGM2 (-1.74;p=0.020), ACSS2 (Acyl-CoA Synthetase Short Chain Family Member 2) (-3.48;p=0.031), and ACACA (Acetyl-CoA Carboxylase Alpha) (-3.06;p=0.017). 4)Three proteins from reversible steps of glycolytic pathway showed increased phosphorylated status after dasatinib treatment: Aldolase (+4.34;p=0.040), GAPDH (+2.76;p=0.033), and Enolase-1 (ENO1) (+1.94;p=0.014). 5)Dasatinib decreased the activity of GAPDH (3.15-fold;p=0.0379) and ALDOA (4.70-fold;p=0.0416); PKM and PGM2 enzymatic activity was unmodified. 6)Dasatinib reduced 2.1-fold (p=0.0112) mitochondrial ATP in viable CD4 cells in correlation with reduced susceptibility to provirus reactivation. 7)Dasatinib interfered with GLUT-1 expression (Figure 1) and glucose uptake in all CD4 memory subpopulations, including TEM and TEMRA. NK remained metabolically active. 8)PLWH on ART and dasatinib showed reduced CD4 TEM and TEMRA subpopulations with decreased metabolic activity.

CONCLUSIONS: Dasatinib acts as LPA by inducing resting state in viable CD4 cells with stalled glycolysis and mitochondrial ATP synthesis, impeding both HIV infection and reservoir reactivation, while NK cells remained unaffected. Treatment with dasatinib and ART may silence the viral reservoir as a block&lock strategy.

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