PBMCs from patients with chronic myeloid leukemia treated with different tyrosine kinase inhibitors show variable susceptibility to HIV-1 infection: searching for the best therapeutic approach Mercedes Bermejo1, Juan Ambrosioni2, Guiomar Bautista3, Núria Climent2, Elena Mateos1, Cristina Rovira2, Rafael Duarte3, Francisco Cervantes2, Montserrat Plana2, José M. Miró2, José Alcamí1, Mayte Coiras1 1Instituto de Salud Carlos III, Madrid 2Hospital Clínic, Barcelona 3Hospital Puerta de Hierro, Madrid
CD4+ T cell activation during HIV-1 primary infection SAMHD1-phosphorylation Reverse transcription enhancement Post-integration latency P SAMHD1 SAMHD1 Massive activation of infected CD4+ T cells during acute HIV-1 infection leads to reservoir seeding and T-cell destruction. IL-2/IL-7 Resting CD4+ T cell IL-2/IL-7 IL-2/IL-7 TcR-activation Low ongoing replication Homeostatic proliferation Apoptosis Full viral replication
SAMHD1 is deactivated during T cell activation 5 days Stimuli pSAMHD1 CD4+ T cells Coiras et al., Cell Rep. 2016 Mar 8;14(9):2100-7 PHA and anti-CD3 induce SAMHD1 phosphorylation at T592 IL-2 and IL-7 sustain SAMHD1 phosphorylation
Tyrosine kinase p56Lck is essential for T cell activation HIV-1 replication Bermejo et al., Biochem Pharmacol. 2016;106:30-45 Wang et al., Front Immunol. 2012 Jul 11;3:197
Tyrosine kinase inhibitors are used in clinic for treating chronic myeloid leukemia Bosutinib Bcr-Abl: quimeric fusion protein with uncontrolled tyrosine kinase activity
Tyrosine kinase inhibitors are used in clinic for treating chronic myeloid leukemia First TKI against BCR-ABL introduced in clinical practice: Imatinib Second generation TKIs: Third generation TKIs: Ponatinib Nilotinib Dasatinib Bosutinib Dasatinib is a multi-targeted inhibitor of Bcr-Abl and Src family kinases that exclusively binds the active conformation of Abl kinase, contrary to most TKIs
Tyrosine kinase inhibitors interfere with HIV-1 replication in vitro PBMCs from healthy donors Imatinib Nilotinib Dasatinib 8.2 M 9.2 M 16.3 nM IC50 (nM) 8259 CC50 (nM) > 20000 R2 0.9813 - SI > 2.42 IC50 (nM) 9280 CC50 (nM) > 30000 R2 0.9608 0.8962 SI > 3.23 IC50 (nM) 16.34 CC50 (nM) > 10000 R2 0.9532 - SI > 612 Bosutinib Ponatinib Dasatinib and Ponatinib were the most potent inhibitors of HIV-1 replication (nM concentration) 618 nM 145 nM IC50 (nM) 618.4 CC50 (nM) 1031 R2 0.9531 0.9917 SI 1.66 IC50 (nM) 145.6 CC50 (nM) 10000 R2 0.9541 0.9866 SI 68.68
Tyrosine kinase inhibitors interfere with SAMHD1 phosphorylation in vitro PBMCs from healthy donors Imatinib and nilotinib did not interfere with SAMHD1 phosphorylation induced by CD3/CD28/IL-2 All ITKs interfere with SAMHD1 phosphorylation induced by IL-2 Imatinib did not interfere with SAMHD1 phosphorylation induced by IL-7
Effect of TKIs on HIV-1 co-receptors expression PBMCs Flow cytometry: CXCR4 and CCR5 3 days + CD3/CD28/IL-2
between IC50 in vitro and Cmax in vivo Correlation of TKIs between IC50 in vitro and Cmax in vivo Imatinib Nilotinib Dasatinib Bosutinib Ponatinib IC50 8.2 M (4.07 g/ml) 9.2 M (4.91 µg/ml) 16.3 nM (8.26 ng/ml) 618 nM (327.28 ng/ml) 145 nM (77.46 ng/ml) Cmax 2.35 µg/ml (400 mg once in healthy adults 1.59 µg/ml (400 mg twice in CML patients) 41.52 ng/ml (50 mg once in healthy adults) 120-141 ng/ml (600 mg once in CML patients and healthy adults, respectively) 54.7 ng/ml (45 mg once in healthy adults) IC50/Cmax 1.7 3.0 0.2 2.3 1.4 Selectivity index > 2.42 > 3.23 > 612 1.66 68.68 Dasatinib would need lower concentration to interfere with HIV-1 replication than for the treatment of CML Similar concentration of imatinib and ponatinib would be needed
PBMCs from CML patients on treatment with TKIs No samples Age at sampling (years) Time of treatment (years) Dose of TKI (mg/d) Lymphocyte count (106/ml) Imatinib 15 67 4.8 392 (400) 1788 Nilotinib 11 51 4.4 585 (600) 2161 Dasatinib 52 2.5 96 (100) 2108 Bosutinib 4 2 250 (200) 1903 Ponatinib 1 70 5 15 (15) 1400 Parameters: Negative for HIV-1 infection Hematological remission Cytogenetic remission CML chronic stage
PBMCs from CML patients on treatment with TKIs Proviral integration Viral proteins 48 hours 5 days + CD3/CD28/IL-2 + NL4-3_Renilla Proviral DNA Viral protein expression
PBMCs from CML patients on treatment with TKIs Proviral integration Viral proteins 48 hours 5 days + CD3/CD28/IL-2 + NL4-3_Renilla 5 hours (RT)
SAMHD1 phosphorylation in PBMCs from CML patients on treatment with TKIs PBMCs PHA/IL-2 5 days Analysis of pSAMHD1 CD4+ T cells Dasatinib (n=6); Imatinib (n=5); Nilotinib (n=5), Bosutinib (n=2), Ponatinib (n=1), Healthy (n=6)
Effect of TKIs on T cell proliferation + CD3/CD28/IL-2 3d PBMCs CFSE, flow cytometry
Effect of TKIs on T cell proliferation + stimuli PBMCs CFSE Dasatinib and ponatinib interfered with T cells proliferation induced by any stimuli
Mechanisms of inhibition of HIV-1 cycle by TKIs TKIs specifically block SAMHD1 phosphorylation and inhibits viral reverse transcription Through inhibition of T-cell activation, TKIs inhibit full viral replication Reservoir expansion is blocked by interfering with the homeostatic proliferation of latently infected T cells Coiras et al., Opin Drug Saf. 2017 May;16(5):547-559
Conclusions In vivo treatment with TKIs preserves SAMHD1-mediated restriction to HIV-1 infection through the inhibition of its phosphorylation. As a consequence, PBMCs from CML patients were refractory to HIV-1 reverse transcription and, consequently, to proviral integration. Dasatinib is the most potent TKI against HIV-1 replication, closely followed by ponatinib. Dasatinib showed the best therapeutic index, with IC50 lower than Cmax for CML treatment. Treatment with dasatinib or ponatinib interfered with CD4+ T cell activation and proliferation induced by several physiological stimuli. Dasatinib could control the size of the reservoir by interfering with its formation and with the replenishment induced by homeostatic proliferation.
Research team AIDS Immunopathology, National Center of Microbiología, Instituto Carlos III. Madrid Pepe Alcamí The patients Mayte Coiras Elena Mateos Sara Rodríguez-Mora Mercedes Bermejo MªRosa López-Huertas