PBMCs from patients with chronic myeloid leukemia treated

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 Bermejo 1, Juan Ambrosioni 2, Guiomar Bautista 3, Núria Climent 2, Elena Mateos 1, Cristina Rovira 2, Rafael Duarte 3, Francisco Cervantes 2, Montserrat Plana 2, José M. Miró 2, José Alcamí 1, Mayte Coiras 1 1 Instituto de Salud Carlos III, Madrid 2 Hospital Clínic, Barcelona 3 Hospital Puerta de Hierro, Madrid #IAS 2017 | @IAS_Conference

#IAS 2017 | @IAS_conference CD 4+ T cell activation during HIV-1 primary infection SAMHD 1 -phosphorylation Reverse transcription enhancement P SAMHD 1 Resting CD 4+ T cell SAMHD 1 Post-integration latency IL-2/IL-7 -7 /IL 2 L I 7 IL- 2 /IL Tc. R-activation Massive activation of infected CD 4+ T cells during acute HIV-1 infection leads to reservoir seeding and T-cell destruction. Low ongoing replication Homeostatic proliferation Apoptosis Full viral replication

#IAS 2017 | @IAS_conference SAMHD 1 is deactivated during T cell activation Stimuli CD 4+ T cells 5 days Coiras et al. , Cell Rep. 2016 Mar 8; 14(9): 2100 -7 p. SAMHD 1 PHA and anti-CD 3 induce SAMHD 1 phosphorylation at T 592 IL-2 and IL-7 sustain SAMHD 1 phosphorylation

#IAS 2017 | @IAS_conference Tyrosine kinase p 56 Lck is essential for T cell activation HIV-1 replication Wang et al. , Front Immunol. 2012 Jul 11; 3: 197 Bermejo et al. , Biochem Pharmacol. 2016; 106: 30 -45

#IAS 2017 | @IAS_conference Tyrosine kinase inhibitors are used in clinic for treating chronic myeloid leukemia Bosutinib Bcr-Abl: quimeric fusion protein with uncontrolled tyrosine kinase activity

#IAS 2017 | @IAS_conference 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: Nilotinib • Third generation TKIs: Ponatinib 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

#IAS 2017 | @IAS_conference Tyrosine kinase inhibitors interfere with HIV-1 replication in vitro PBMCs from healthy donors Imatinib Nilotinib 8. 2 M 9. 2 M IC 50 (n. M) 8259 CC 50 (n. M) R 2 0. 9813 R 2 SI > 20000 - > 2. 42 16. 3 n. M IC 50 (n. M) 9280 CC 50 (n. M) R 2 0. 9608 R 2 SI Bosutinib > 30000 0. 8962 > 3. 23 SI 145 n. M IC 50 (n. M) 618. 4 CC 50 (n. M) R 2 0. 9531 R 2 1. 66 IC 50 (n. M) 16. 34 CC 50 (n. M) R 2 0. 9532 R 2 > 10000 - > 612 Ponatinib 618 n. M SI Dasatinib 1031 0. 9917 IC 50 (n. M) 145. 6 CC 50 (n. M) R 2 0. 9541 R 2 SI 68. 68 10000 0. 9866 Dasatinib and Ponatinib were the most potent inhibitors of HIV-1 replication (n. M concentration)

#IAS 2017 | @IAS_conference Tyrosine kinase inhibitors interfere with SAMHD 1 phosphorylation in vitro PBMCs from healthy donors Imatinib and nilotinib did not interfere with SAMHD 1 phosphorylation induced by CD 3/CD 28/IL-2 • All ITKs interfere with SAMHD 1 phosphorylation induced by IL-2 • Imatinib did not interfere with SAMHD 1 phosphorylation induced by IL-7

#IAS 2017 | @IAS_conference Effect of TKIs on HIV-1 co-receptors expression PBMCs + CD 3/CD 28/IL-2 3 days Flow cytometry: CXCR 4 and CCR 5

#IAS 2017 | @IAS_conference Correlation of TKIs between IC 50 in vitro and Cmax in vivo IC 50 Cmax Imatinib Nilotinib Dasatinib Bosutinib Ponatinib 8. 2 M (4. 07 g/ml) 9. 2 M (4. 91 µg/ml) 16. 3 n. M (8. 26 ng/ml) 618 n. M (327. 28 ng/ml) 145 n. M (77. 46 ng/ml) 41. 52 ng/ml (50 mg once in healthy adults) 120 -141 ng/ml 54. 7 ng/ml (600 mg once in CML patients (45 mg once in and healthy adults, adults) respectively) 2. 35 µg/ml 1. 59 µg/ml (400 mg once (400 mg twice in healthy in CML adults patients) IC 50/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

#IAS 2017 | @IAS_conference PBMCs from CML patients on treatment with TKIs TKI 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 11 52 2. 5 96 (100) 2108 Bosutinib 4 67 2 250 (200) 1903 Ponatinib 1 70 5 15 (15) 1400 Parameters: • Negative for HIV-1 infection • Hematological remission • Cytogenetic remission • CML chronic stage

#IAS 2017 | @IAS_conference PBMCs from CML patients on treatment with TKIs + CD 3/CD 28/IL-2 48 hours 5 days + NL 4 -3_Renilla Proviral DNA Proviral integration Viral proteins Viral protein expression

#IAS 2017 | @IAS_conference PBMCs from CML patients on treatment with TKIs + CD 3/CD 28/IL-2 48 hours 5 days + NL 4 -3_Renilla Proviral integration Viral proteins 5 hours (RT)

#IAS 2017 | @IAS_conference SAMHD 1 phosphorylation in PBMCs from CML patients on treatment with TKIs PBMCs PHA/IL-2 5 days Analysis of p. SAMHD 1 CD 4+ T cells Dasatinib (n=6); Imatinib (n=5); Nilotinib (n=5), Bosutinib (n=2), Ponatinib (n=1), Healthy (n=6)

#IAS 2017 | @IAS_conference Effect of TKIs on T cell proliferation PBMCs + TKI + CD 3/CD 28/IL-2 3 d CFSE, flow cytometry

#IAS 2017 | @IAS_conference Effect of TKIs on T cell proliferation PBMCs + TKI + stimuli Dasatinib and ponatinib interfered with T cells proliferation induced by any stimuli CFSE

#IAS 2017 | @IAS_conference Mechanisms of inhibition of HIV-1 cycle by TKIs 1. TKIs specifically block SAMHD 1 phosphorylation and inhibits viral reverse transcription 2. Through inhibition of T-cell activation, TKIs inhibit full viral replication 3. 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

#IAS 2017 | @IAS_conference Conclusions • In vivo treatment with TKIs preserves SAMHD 1 -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 IC 50 lower than Cmax for CML treatment. • Treatment with dasatinib or ponatinib interfered with CD 4+ 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.

#IAS 2017 | @IAS_conference Research team AIDS Immunopathology, National Center of Microbiología, Instituto Carlos III. Madrid Pepe Alcamí The patients Mayte Elena Mateos Sara Rodríguez. Mercedes MªRosa López-Mora Bermejo Huertas Coiras