1. 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

2. 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

3. SAMHD1 is deactivated during T cell activation
5 days
Stimuli
pSAMHD1
CD4+ T cells
Coiras et al., Cell Rep Mar 8;14(9):2100-7
PHA and anti-CD3 induce SAMHD1 phosphorylation at T592
IL-2 and IL-7 sustain SAMHD1 phosphorylation

4. 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

5. Tyrosine kinase inhibitors are used in clinic for treating chronic myeloid leukemia
Bosutinib
Bcr-Abl: quimeric fusion protein with uncontrolled tyrosine kinase activity

6. 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

7. 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

8. 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

9. Effect of TKIs on HIV-1 co-receptors expression
PBMCs
Flow cytometry: CXCR4 and CCR5
3 days
+ CD3/CD28/IL-2

10. 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
( 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)
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

11. 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

12. 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

13. 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)

14. 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)

15. Effect of TKIs on T cell proliferation
+ CD3/CD28/IL-2 3d
PBMCs
CFSE, flow cytometry

16. Effect of TKIs on T cell proliferation
+ stimuli
PBMCs
CFSE
Dasatinib and ponatinib interfered with T cells proliferation induced by any stimuli

17. 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 May;16(5):

18. 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.

19. 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