The tyrosine kinase inhibitor Dasatinib blocks in-vitro HIV-1 production by primary CD4+ T cells from HIV-1 infected patients
Pogliaghi, Manuelaa,b,c; Papagno, Lauraa,b,d; Lambert, Sidoniee,f,g; Calin, Ruxandrae,f,h; Calvez, Vincente,f,g; Katlama, Christinee,f,h; Autran, Brigittea,b
aUPMC Univ Paris 06, UMR-S945
bInserm, U945, Laboratory of Immunity and Infection, Paris, France
cUniversità Vita-Salute, San Raffaele Scientific Institute, Milan, Italy
dORVACS, Hospital Pitié-Salpêtrière
eUPMC Univ Paris 06, UMR-S943, Epidemiology, Therapeutics Strategies and Clinical Virology in HIV Infection, Paris, France
fInserm, U943, Epidemiology, Therapeutics Strategies and Clinical Virology in HIV Infection
gAP-HP, Hospital Pitié-Salpêtrière Hospital, Department of Virology
hAP-HP, Hospital Pitié-Salpêtrière Hospital, Department of Infectious and Tropical Diseases, Paris, France.
Correspondence to Brigitte Autran, Hôpital Pitie-Salpêtriere, Département d’immunologie, Paris, France. Tel: +33 142177930; fax: +33 142177490; e-mail: firstname.lastname@example.org
HIV reservoirs persistence despite antiretroviral therapy (ART) might be related to persistent immune activation and residual HIV production, requiring further therapeutic strategies. We demonstrated that the tyrosine kinase inhibitor (TKI) Dasatinib, used for chronic myeloid leukaemia, significantly blocks in vitro HIV1 production by 3.4 logs in HIV1-infected primary CD4+ T lymphocytes, by inhibiting cell activation and proliferation, without cell toxicity. This molecule deserves to be investigated further for HIV cure strategies to hinder persistent immune activation and residual viral production.
The persistence of HIV despite fully suppressive ART reflects multiple mechanisms among which a low-level HIV production, facilitated by a residual immune activation, contributes to maintain the HIV reservoirs [1–3]. Innovative therapeutic strategies are required to interrupt these phenomenons. TKIs, such as Imatinib, a specific Abl-kinase inhibitor, are used for treating haematological malignancies characterized by constitutive tyrosine kinase activation [4,5]. In addition, Dasatinib, a 300-fold more potent drug, also inhibits the Src-kinase family, strongly decreasing the phosphorylation levels of kinases belonging to the T cell receptor signalling pathway such as LCK, Zap70 and Erk, and inhibits T-cell proliferation triggered by various stimuli , thereby blocking phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells in S and G2/M phases. The antiproliferative effect of this TKI on T lymphocytes is currently considered for treating inflammatory and autoimmune diseases [7–10]. Furthermore, TKIs have recently been shown to block in-vitro Abl-dependent HIV-1-cell fusion process and to inhibit the Env-mediated cell–cell fusion on cell lines and normal lymphocytes infected in vitro by HIV-1 laboratory strains . In an effort to investigate whether TKIs could block a natural HIV infection, we evaluated their inhibitory effect on autologous HIV-1 production in parallel to T cell activation in primary CD4+ T cells from HIV-1 infected patients.
Peripheral blood CD4+ T cells from 13 untreated chronically HIV-1 infected patients (median viral loads: 45 800 copies/ml; range: 26 400–67 900) were positively selected using CD4 Human-Microbeads (Miltenyi, Biotec SAS, Paris, France) (purity >95%), stimulated by PHA and cultured for 10 days with interleukin-2 and interleukin-7 added at D0, D3, D6 and D8. Various doses of Dasatinib (0.2–1000 nmol/l) or Imatinib (1–10 000 nmol/l) were added daily from D0 or D3 up to D10. As control, CD4+ T cells were cultured without addition of TKIs. Flow-cytometry analysis characterized cell expression of HIV-1 p24 (p24-PE), viability (Fixable-Dead-Cell-Stain Kit; Life Technologies SAS, Saint Aubin, France), survival capacity (Bcl2-V450), activation (CD38-PE-Cy5.5, HLADR-PE-Cy7) and proliferation (Ki67-FITC), in parallel to cell count and viability analysis using trypan blue exclusion over the culture period. The p24 production was measured in supernatants by an enzyme-linked immunosorbent assay. All data were compared using a Wilcoxon matched paired test. The IC50 for Dasatinib was calculated at D6, D8 and D10 using nonlinear regression (curve fit).
The Dasatinib addition to cultures from D0 to D10 induced a significant dose-range inhibition of p24+ cell numbers from D3 until D10, with a maximal effect at 75 nmol/l (p24+ cells less than 0.1%, P = 0.03), down to levels comparable to those obtained from unstimulated cultured CD4+ T cells (Fig. 1c). Measuring soluble p24 production in supernatants confirmed the dose-range inhibition (0.2–1000 nmol/l) from D3 to D10, with a mean 2.8 logarithmic reduction at 75 nmol/l (P = 0.007 at D6) and 3.4 logs at 1000 nmol/l. Thus, Dasatinib at 100 nmol/l reduced HIV production by 72% (Fig. 1a). A 4.8 nmol/l mean IC50 of Dasatinib was estimated on the D6–D10 p24 quantification in supernatants (Fig. 1b). A less marked inhibition was observed when Dasatinib was added at D3 after PHA-stimulation (Fig. 1c), suggesting that Dasatinib could not completely switchoff the activation triggered at D0 by PHA if added 3 days later.
In this culture model of HIV-1 infected primary CD4+ cells, we did not observe any significant toxicity of Dasatinib but instead a better viability of PHA-stimulated cells than without Dasatinib, as shown by the D3–D10 dead cell counts (P < 0.03) and the trend for higher CD38−Bcl2+ cells (Fig. 1d). In addition, the lower proportions of activated activated CD38+HLADR+ cells in D3–D10 cultures along with Dasatinib, lower cell counts at D6 (P = 0.007) and lower proportions of CD38+Ki67+ cells from D3 to D10 suggested that Dasatinib inhibits both cell activation and cell proliferation triggered at D0 by PHA (Fig. 1d). As control, Dasatinib did not affect the CD3, CD4 and CCR5 surface expression. Finally, Dasatinib addition on healthy donor's CD4+ T cell cultures using the same culture assay induced the same inhibition of cell activation and proliferation with increased CD38−Bcl2+ percentages. This HIV-negative cell viability was not affected, thus contrasting with the slightly increased cell viability in HIV-1 infected cells cultured with Dasatinib that most probably reflected inhibition of HIV production.
In contrast, Imatinib showed an important toxicity on primary CD4+ T cells, with a very narrow range (5–7 μmol/l) of p24 inhibition (data not shown). The 100-fold higher dose required for Imatinib than for Dasatinib was consistent with results mentioned above showing that Dasatinib was 300-fold more potent than Imatinib at inhibiting the Abl kinase and blocking the virus-cell fusion process in cell lines .
Therefore, our results showed that Dasatinib, a TKI used in clinical practice with a long track record as the second-line long-term treatment of chronic myeloid leukaemia (CML), strongly inhibits in vitro the autologous HIV-1 production by primary CD4+ T lymphocytes from HIV-1 infected patients. The mechanisms appear to involve also the blockade of cell activation and CD3/T-cell receptor signalling without cell toxicity. Interestingly, the concentrations inhibiting HIV production in vitro are far below the well tolerated Dasatinib concentrations achievable in vivo, which are up to 180 nmol/l [12–15]. This concentration is also lower than the 10–17 nmol/l IC50 shown to inhibit the S and G2/M cell cycle phases of T-cell proliferation . Therefore, we suggest that this agent should be evaluated in vivo in combinatorial strategies to achieve an HIV cure. Indeed, its anti-HIV effect, combining inhibition of HIV entry through interference with a virus-cell fusion process and antiproliferative effect [6,11], could help to diminish the HIV reservoirs, particularly for situations in which persisting residual immune activation favours HIV low-level virus production and a reseeding of HIV reservoirs [1–3]. In addition, this drug could help reduce the massive immune activation observed in primary HIV infection or in paradoxical nonresponders to ART, in order to block the massive CD4+ T cell depletion owing to HIV-induced T cell activation and apoptosis and improved CD4+ T cell reconstitution. Of note, other immunosuppressive compounds (hydroxyurea, cyclosporine, mycophenolate) have been used in HIV infection with some benefits [16–19]. Although the capacity of Dasatinib to limit immune activation and establishment or spreading of the HIV reservoirs has never been investigated so far , our findings provide a solid rationale to envisage using this well tolerated drug in therapeutic strategies aiming at reducing HIV reservoirs.
Manuela Pogliaghi was supported by an EACS scholarship programme, CRIPTS and Vita-Salute San Raffaele University. This work was supported by ORVACS and INSERM.
Conflicts of interest
There are no conflicts of interest.
1. Chun TW, Murray D, Justement JS, Hallahan CW, Moir S, Kovacs C, Fauci AS. Relationship between residual plasma viremia and the size of HIV proviral DNA reservoirs in infected individuals receiving effective antiretroviral therapy
. J Infect Dis
2. Hatano H. Immune activation and HIV persistence: considerations for novel therapeutic interventions
. Curr Opin HIV AIDS
3. Hatano H, Jain V, Hunt PW, Lee TH, Sinclair E, Do TD, et al. Cell-based measures of viral persistence are associated with immune activation and programmed cell death protein 1 (PD-1)-expressing CD4+ T cells
. J Infect Dis
4. Jabbour E, Kantarjian H. Chronic myeloid leukemia: 2012 update on diagnosis, monitoring, and management
. Am J Hematol
5. Lai EC, Lau SH, Lau WY. Current management of gastrointestinal stromal tumors: a comprehensive review
. Int J Surg
6. Blake S, Hughes TP, Mayrhofer G, Lyons AB. The Src/ABL kinase inhibitor dasatinib (BMS-354825) inhibits function of normal human T-lymphocytes in vitro
. Clin Immunol
7. Seggewiss R, Lore K, Greiner E, Magnusson MK, Price DA, Douek DC, et al. Imatinib inhibits T-cell receptor-mediated T-cell proliferation and activation in a dose-dependent manner
8. Lissina A, Ladell K, Skowera A, Clement M, Edwards E, Seggewiss R, et al. Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers
. J Immunol Methods
9. Akashi N, Matsumoto I, Tanaka Y, Inoue A, Yamamoto K, Umeda N, et al. Comparative suppressive effects of tyrosine kinase inhibitors imatinib and nilotinib in models of autoimmune arthritis
. Mod Rheumatol
10. Reber L, Da Silva CA, Frossard N. Stem cell factor and its receptor c-Kit as targets for inflammatory diseases
. Eur J Pharmacol
11. Harmon B, Campbell N, Ratner L, et al. Role of Abl kinase and the Wave2 signaling complex in HIV-1 entry at a posthemifusion step
. PLoS Pathog
12. Tyler T. Once-daily dasatinib for treatment of patients with chronic myeloid leukemia
. Ann Pharmacother
13. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia
. N Engl J Med
14. Jabbour E, Cortes J, Kantarjian H. Long-term outcomes in the second-line treatment of chronic myeloid leukemia: a review of tyrosine kinase inhibitors
15. Fullmer A, Kantarjian H, Cortes J, Jabbour E. Dasatinib for the treatment of chronic myeloid leukemia
. Expert Rev Hematol
16. Rizzardi GP, Harari A, Capiluppi B, Tambussi G, Ellefsen K, Ciuffreda D, et al. Treatment of primary HIV-1 infection with cyclosporin A coupled with highly active antiretroviral therapy
. J Clin Invest
17. Chapuis AG, Paolo Rizzardi G, D’Agostino C, Attinger A, Knabenhans C, Fleury S, et al. Effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo
. Nat Med
18. Sankatsing SU, Jurriaans S, Van Swieten P, Van Leth F, Cornelissen M, Miedema F, et al. Highly active antiretroviral therapy with or without mycophenolate mofetil in treatment-naive HIV-1 patients
19. Garcia F, Plana M, Arnedo M, Brunet M, Castro P, Gil C, et al. Effect of mycophenolate mofetil on immune response and plasma and lymphatic tissue viral load during and after interruption of highly active antiretroviral therapy for patients with chronic HIV infection: a randomized pilot study
. J Acquir Immune Defic Syndr
20. Katlama C, Deeks SG, Autran B, Martinez-Picado J, Van Lunzen J, Rouzioux C, et al. Barriers to a cure for HIV: new ways to target and eradicate HIV-1 reservoirs
© 2014 Lippincott Williams & Wilkins, Inc.