2014 “Towards an HIV Cure” symposium Melbourne Synergistic activation of HIV-1 expression by compounds targeting the positive transcription elongation factor b (P-TEFb) and by inducers of the NF-B signaling pathway Gilles Darcis Carine Van Lint lab
Jan 15, 2016
2014 “Towards an HIV Cure” symposiumMelbourne
Synergistic activation of HIV-1 expression by compounds targeting the positive
transcription elongation factor b (P-TEFb) and by inducers of the NF-B signaling
pathway
Gilles Darcis
Carine Van Lint lab
Inducers of HIV-1 gene expression
?
Strategies aimed at reducing the size of the persistent reservoirs of latent HIV-1 by forcing viral gene expression
cART + „purging strategy”
nuc-15’
3’
TF (such as NF-KB)ProstratinBryostatinIngenol
Cyto
plas
mNu
cleus
NF-kB sites
TAR
Brd4
P-TEFb
P-TEFb
Cellular genes
JQ1I-BETI-BET151
Tat
P-TEFb
Postintegration latency is a multifactorial phenomenon
HMBA
Chromatin structure and epigenetic modifications
• histone posttranslational modifications
• DNA methylation
Absence of cellular inducible transcription factor
Sequestration of P-TEFb
BETi Cellular modelBanerjee C, et al.
J Leukoc Biol 2012
JQ1 Ach2, U1, J-Lat 10.6resting CD4+ Tcells isolated from ART treated patients
JQ1 reactivates HIV-1 in several cellularsystems and in 1 out of 3 patients.
Li Z, et al. Nucleic Acids Res 2013
JQ1JQ1+Pro
J-Lat A2, 2D10, Jurkats 1G5Hela
JQ1 activates HIV transcription in latently infected Jurkat Tcells.JQ1 dissasociates BRD4 from chromatin and increases Tat binding to HIV-1 LTR
Bartholomeeusen K, et al. J Biol
Chem 2012
JQ1JQ1+SAHA
JΔK Jurkat cell line (lack both NFkB binding sites)
JQ1 activates HIV transcription in JΔK cells JQ1 activates HIV-1 transcription via the release of free P-TEFb from the 7SK snRNP
Zhu J, et al. Cell Rep 2012
JQ1JQ1+ProJQ1+PHA
HeLa, U1, J-Lats, J-Lat A2, 293TCD8+-depleted PBMCs and resting CD4+ Tcells isolated from HIV-1 negative or from ART-treated patients
Enhanced viral reactivation in NL4-3-GFP -HIV-1 infected PBMCs isolated from 6 negative donors.Enhanced viral replication when JQ1 + Prostratin or JQ1+ PHA in 7 out of 19 ART-treated patients.
Boehm D, et al. Cell Cycle 2012
JQ1I-BET
I-BET151JQ1+Pro
JQ1+SAHA
Jurkats, J-LatA2, J-LatA72 Primary T- cell models (Bcl-2 transduced resting Cd4+ T cells isolated from healthy donors)
BETi reactivate HIV from latency in Jurkat cells and in T-cell model of HIV latency. This activation is depenedent on P-TEFb but independent from Tat.
Function
Five recent publications show that BET inhibitors reactivate latent HIV-1
PKC agonists
-Prostratine-Bryostatin-1-Ingenol
Compounds targeting P-TEFb
-JQ1-IBET-IBET151-HMBA
Co-treatment with P-TEFb and NF- B inducers leads to strong synergistic activation of HIV-1 production (I)
Similar results obtained for prostratin
Co-treatment with P-TEFb and NF- B inducers leads to strong synergistic activation of HIV-1 production (II)
Similar results obtained for prostratin
The combination NF- B inducers + PTEFb inducer activates HIV-1 expression in a greater proportion of cells than each
compound alone (I)
Mo
ck
JQ
1
I-B
ET
I-B
ET
151
HM
BA
Bry
os
tati
n-1
JQ
1
I-B
ET
I-B
ET
151
HM
BA
Pro
str
ati
n
JQ
1
I-B
ET
I-B
ET
151
HM
BA
TN
F
0
1
2
3
4
5
6
7
8
synergy JQ1 I-BET I-BET151 HMBA Bryostatin-1 2.6 1.8 2.3 3.3
Prostratin 2.1 1.4 1.9 3.9
J-Lat 9.2 cells
10
15
20
25
Bryostatin-1 Prostratin
0.4 0.4
0.3
0.3 0.3
1.3
3.5
2.1 2
.95
6.0
1 6.3
17
.2
11
.6 15
.0
21
.45
17
.25
% o
f G
FP
(+
) ce
lls
tat
rev5’ 3’
vpr
5’ LTR gag
pol
vif
env GFPGFP
3’ LTR
vpu
Microglial cellsThe combination NF- B inducers + PTEFb inducer activates HIV-1 expression in a greater proportion of cells than each
compound alone (II)
Mo
ck
JQ
1
I-B
ET
I-B
ET
15
1
HM
BA
Bry
os
tati
n-1
JQ
1
I-B
ET
I-B
ET
15
1
HM
BA
Pro
str
ati
n
JQ
1
I-B
ET
I-B
ET
15
1
HM
BA
TN
F
0
2
4
6
8
10
12
14
16
18
20
22
24
synergism (fold)
JQ1 I-BET I-BET151 HMBA
Bryostatin-1 1.6** 1.1 1.2 0.8Prostratin 1.4** 1.0 1.1 0.9
HIV-latently infected microglial cells
Bryostatin-1 Prostratin
1.55
4.65
2.85
3.1 3.4 4.15
12.3
5
6.95 7.8
6.8
4.55
14.8
8.35 9.1
6.65
31.5
% o
f G
FP
(+
) ce
lls
** p<0.005
Evaluation of HIV-1 recovery in CD8(+)-depleted PBMCs from virally suppressed patients (I)
Patients HIV DNA
copies/ 106 PBMCs
Mock JQ1 I-BET I-BET151 HMBA Pro Bryo
Prostratin Bryostatin-1
C+JQ1 I-BET I-BET151 HMBA JQ1 I-BET I-BET151 HMBA
P1 680 - 422 - 584 1016 669 449 3591 141 4200 282 318 - - - -
P2 1217 - - - 237 - - - - - - - - - - - -
P3 70 - - - 584 717 5359 657 694 585 435 - 691 830 - 191 1017
P4 350 - 729 // 1707 944 394 - 271 531 316 187 670 - 14741 308 7027
P5 1782 - - 812 - - 3870 2381 6857 3328 5876 5563 6307 2496 10158 3142 20524
P6 669 - 2418 5579 2505 1582 308 1336 1918 774 1666 472 3936 5741 936 8798 39773
P7 670 - 25034 - - 586 459 296 536 - 797 // 566 - 274 2274 36562
P8 782 - 576 964 650 - 2377 - - 665 - 512 253 - 851 329 526
P9 847 - - 490 257 - - 265 - 273 247 - 261 - - - 6468
P10 345 - - 278 619 - - 2153 644 - - 334 264 - - - -
P11 1435 - 604 784 2380 - - 1304 1699 - 24068 485 4099 962 3274 1007 16036
P12 947 - 357 - - 325 627 - 967 5251 2636 - - - - - 273634
P13 253 - - 381 181 - - - - 185 - - - 224 3194 - -
P14 1823 - 185 - - 789 - 766 193 206 - - 380 - 634 382 -
P15 390 - 167 - - - 1594 377 - 370 - - - - - 790 172
P16 340 - 901 - - - - - - - - 608 6835 - 403 - 4506
P17 263 - - - - - 849 459 3920 - - - 898 9752 - - 4119
P18 333 - 1064 1579 953 451 - 883 2834 1002 517 1502 4161 3171 1190 2056 -
P19 187 - - - - - - 1330 - - - 520 4880 - 428 - 79344
P20 1236 481 914 - - - 1949 2669 3393 783 6281 2597 655 - 212 916 19029
P21 600 693 954 846 - - - - 1039 - 918 - 814 - - - 2717
P22 737 - - - 319 - - 869 1190 14170 - - 965 1855 1395 4230 7879
P23 380 - 1239 647 - 2106 - 1702 109 1032 1870 6074 3833 - - 1092 7267
P24 203 - - 815 430 662 - - 1956 813 - - - - - 402 464
% of activated patients (>150
cop/ml) 8 58 48 54 42 46 67 67 63 54 52 79 33 54 58 75
Evaluation of HIV-1 recovery in CD8(+)-depleted PBMCs from virally suppressed patients (II)
Patients HIV DNA
copies/ 106 PBMCs
Mock JQ1 Ingenol Ingenol +
JQ1 C+
P21 600 693 954 713 // 2717P22 737 0 0 3468 // 7879P23 380 0 1239 // // 7267P24 203 0 0 768 // 464P25 11 0 902 453 2313 373P26 548 0 181 888 // 10407P27 4486 2433 3417 9412 13199 31132P28 2552 1911 920 834 6751 2348
% of activated patients (>150 cop/ml)
38 75 100 100 100
JQ1 combinatory treatments with PKC activators synergistically activate latent HIV ex-vivo in resting CD4 T
cells from virally suppressed patients
Patients HIV DNA
cop/106 resting CD4+ T cells
AgeCD4+ T
cell count
Last treatmentAviremic for
(years)Mock JQ1 Pro Bryo Ing
JQ1
C+Pro Bryo Ing
P1 1200 65 869 TRU EFV 8 - 483 - - 764 - - - 1002
P2 1179 48 670 CBV NVP 15 - - - 4982 648 - - - 460
P3 112 41 848 TRU KLT 4 - 649 5022 676 180 - 1007 4131 1337
P4 327 38 367 TRU NVP 6 - - - - - - - - -
P5 4414 48 418 ATR 7 - 503 - - 545 741 540 7687 -
P6 1777 44 401 TRU NVP 15 - 403 - - - 860 - - 1077
P7 5220 48 670 CBV NVP 2 - - 497 642 643 - 1399 // 7868
P8 911 41 736 KVX NVP 3 - 522 268 - - - - - -
P9 3841 58 818 RTV DRV ETV MVC 9 607 - 369 624 - 538 8858 // -
P10 946 49 663 ATR 8 - 405 - 333 392 - 4155 860 453
P11 525 46 928 TZV 10 - - 400 - - - - - 7880
P12 1523 66 817 KVX NVP 0.5 - - 423 - - 401 - - 235
P13 9248 63 1091 ATR 4 - - 994 3148 261 453 - 2677 2018
P14 4849 45 686 KVX RTV ATV 1 377 - - 609 521 412 - 3839 5497
P15 1177 47 845 TRU NVP 10 221 - 210 218 - - 543 2452 525
P16 758 39 561 ATR 8 - - 218 - - 533 1259 - -
% of activated patients (>150 cop/ml) 19 38 56 50 50 44 50 43 69
% of patients with combinatory beneficial effect (out of activated patients)
71 75 100
• We have identified combinations of compounds exhibiting real potential of reactivation in several different post-integration latency cellular models.
• Some of these combinations might be clinically relevant for reducing/eliminating the cellular reservoirs of latent HIV-1.
Conclusion and perspectives
Laboratory of Molecular Virology, University of Brussels, Belgium• Carine Van Lint
• Anna Kula
• Arsène Burny• Sophie Bouchat• Christelle Cardona• Jean-Stéphane Gatot• Nadège Delacourt•Caroline Vanhulle
St Pierre Hospital, Belgium
• Nathan Clumeck • Stéphane De Wit• Kabamba Kabeya
Necker Hospital,Paris, France
• Christine Rouzioux•Adeline Melard
University of Strasbourg, France
• Olivier Rohr
University of Franche-Comté,France
• Georges Herbein
University of Liège• Michel Moutschen• Dolores Vaira
Acknowledegments
Thanks to L.Gama and to Amazônia Fitomedicamentos, Brazil for providing Ingenol.
P-TEFb inducers increase HIV-1 expression in a dose-dependent manner without cytotoxicity
0
2
4
6
8
10
12
14
16
18
20
mo
ck
mo
ck
mo
ck
JQ1 I-BET I-BET151 HMBA
J-Lat 9.2 cellsm
oc
k
p2
4 a
nti
ge
n le
ve
l (a
rbit
rary
un
its
)
0
25
50
75
100
125
150
mo
ck
mo
ck
mo
ck
mo
ck
JQ1 I-BET I-BET151 HMBA
J-Lat 9.2 cells
% o
f c
ellu
lar
via
bili
ty
0
5
10
15
20
25
30
35
mo
ck
mo
ck
mo
ck
mo
ck
JQ1 I-BET I-BET151 HMBA
U1 cells
p2
4 a
nti
ge
n le
ve
l (a
rbit
rary
un
its
)
0
25
50
75
100
125
150
mo
ck
mo
ck
mo
ck
mo
ck
JQ1 I-BET I-BET151 HMBA
U1 cells
% o
f c
ellu
lar
via
bili
ty
A
DC
B
NF-B inducers increase HIV-1 expression in a dose-dependent manner without cytotoxicity
0
2
4
6
8
10
12
prostratin
20
70
120
170
220
Bryostatin-1
J-Lat 9.2 cells
p24
an
tig
en l
evel
(ar
bit
rary
un
its)
0
25
50
75
100
125
150
175
200
prostratinBryostatin-1
J-Lat 9.2 cells
% o
f ce
llula
r v
iab
ility
0
5
10
15
20
25
30
U1 cells
prostratinBryostatin-1
30
70
110
150
p2
4 a
ntig
en
lev
el (
arb
itra
ry u
nits
)
0
20
40
60
80
100
120
140
160
180
prostratinBryostatin-1
U1 cells
% o
f ce
llula
r v
iab
ility
mock mock mock mock
mock mock mock mock
The combination Bryostatin-1/Prostratin + PTEFb inducer activates HIV-1 transcription
mo
ck
JQ1
I-B
ET
I-B
ET
151
HM
BA
Pro
stra
tin
JQ1
I-B
ET
I-B
ET
151
HM
BA
0.0
2.5
5.0
7.5
10.0
12.5
15.0
tat/actinTAR/actin
20
220
420
620
820
synergyJQ1 I-BET I-BET151 HMBA
TAR 9.2 5.0 3.7 11.4
tat 9.6 5.0 5.2 19.2
Prostratin
Prostratin
J-Lat 9.2
Fo
ld i
nd
uct
ion
mo
ck
JQ1
I-B
ET
I-B
ET
151
HM
BA
Bry
ost
atin
JQ1
I-B
ET
I-B
ET
151
HM
BA
Pro
stra
tin
JQ1
I-B
ET
I-B
ET
151
HM
BA
TN
Fal
ph
a
05
10
15
20
2530
tat/actinTAR/actin
50100150200250300500
1500
2500
JQ1 I-BET I-BET151 HMBA JQ1 I-BET I-BET151 HMBA TAR 5.7 4.5 6.1 5.9 4.3 3.1 3.5 6.6
env 7.3 5.2 6.7 7.5 4.4 3.4 3.5 6.3
synergyBryostatin-1 Prostratin
U1 cells
Bryostatin-1 Prostratin
Fo
ld i
nd
uct
ion
Down-regulation of CD4 receptor expressed on resting CD4+ T cells (I)
resting CD4+ T cells
mock
Day
6JQ
1
Bryost
atin
-1J+
B
Prost
ratin J+
P
Ingen
olJ+
I
CD3/CD28
0
500
1000
1500
2000
2500
3000
3500
4000
4500%
CD
4+ C
D8-
(M
FI)
Global T cell activation (II)
resting CD4+ T cells
mock
Day
6JQ
1
Bryost
atin
-1J+
B
Prost
ratin J+
P
Ingen
olJ+
I
CD3/CD28
05
10152025303540455050
75
100
% C
D38
+ C
D4+
resting CD4+ T cells
mock
Day
6JQ
1
Bryost
atin
-1J+
B
Prost
ratin J+
P
Ingen
olJ+
I
CD3/CD28
05
10152025303540455050
75
100
% C
D69
+ C
D4+
resting CD4+ T cells
mock
Day
6JQ
1
Bryost
atin
-1J+
B
Prost
ratin J+
P
Ingen
olJ+
I
CD3/CD28
0
1
2
3
4
58090
100
% C
D25
+ C
D4+
resting CD4+ T cells
mock
Day
6JQ
1
Bryost
atin
-1J+
B
Prost
ratin J+
P
Ingen
olJ+
I
CD3/CD28
0
25
50
75
100
% H
LA
-DR
+ C
D4+
Global T cell activation (I)
CD8(+)- depleted PBMCs
mock
Day
0
mock
Day
6JQ
1
Bryost
atin
-1J+
B
Prost
ratin J+
P
Ingen
olJ+
I
CD3/CD28
05
10152025303540455050
75
100
% C
D38
+ C
D4+
CD8(+)- depleted PBMCs
mock
Day
0
mock
Day
6JQ
1
Bryost
atin
-1J+
B
Prost
ratin J+
P
Ingen
olJ+
I
CD3/CD28
05
10152025303540455050
75
100
% C
D69
+ C
D4+
CD8(+)- depleted PBMCs
mock
Day
0
mock
Day
6JQ
1
Bryost
atin
-1J+
B
Prost
ratin J+
P
Ingen
olJ+
I
CD3/CD28
0
10
20
30
40
508090
100
% H
LA
-DR
+ C
D4+
CD8(+)- depleted PBMCs
mock
Day
0
mock
Day
6JQ
1
Bryost
atin
-1J+
B
Prost
ratin J+
P
Ingen
olJ+
I
CD3/CD28
05
101520253035404550556065
% H
LA
-DR
+ C
D4+
BET
• BET proteins (BRD2, BRD3, BRD4 and BRDT in human) contain two conserved N-terminal bromodomains (BRDs), small helical modules that specifically recognize acetylated lysine sites in proteins (Muller et al., 2011), an extra terminal domain (ET) and a more divergent C-terminal recruitment domain (CT motif or CTM). They bind to P-TEFb via their CT motif, tethering the complex to acetylated histone tails via their two N-terminal BRDs, resulting in assembly of the transcriptional machinery.
NF-kB
• Inducible transcription factor made up of homo- and heterodimers of P50, P65, P52, re1B, et c-rel subunits that interact with a family of inhibitory IkB proteins, of which IkB alpha is the best characterized. Phosphorylation of IkBalpha at serines 32 and 36 is a key step involved in the activation of NF-kB complexes. This event is mediated par IKK, activated by several upstream kinases including some members of the PKC family.
Evaluation of cellular viability in CD8(+)-depleted PBMCs treated with P-TEFb and NF-B inducers
Prostratin
0
50
100
150
200
250
P1
P2
P3
P4
P5
Bryostatin-1 JQ1 I-BET I-BET151 HMBAmock
81 60 35 31100
% c
ellu
lar
via
bil
ity
CD8(+)-depleted PBMCs isolated from 5 healthy donors
108119 100 101 103 124 111 110 107** * ** *
Evaluation of cellular viability of the combination P-TEFb + NF-B inducer in CD8(+)-depleted PBMCs
Mock JQ1 I-BET I-BET151 HMBA - JQ1 I-BET I-BET151 HMBA
Bryostatin - - - - - + + + + +
0
50
100
150
200
250
P1P2P3P4P5
% c
ell
ula
r v
iab
ilit
y
Mock JQ1 I-BET I-BET151 HMBA - JQ1 I-BET I-BET151 HMBA
Prostratin - - - - - + + + + +
0
20
40
60
80
100
120
140
160
180
200
P1P2P3P4P5
% c
ell
ula
r v
iab
ilit
y
103 117 120 64100 67 125 69 71119
100 103 111 107100 83 84 75 12260
Pla
sma
vira
l R
NA
(co
pie
s/m
l)
50 RNA copies/ml of plasma
Blips
Viral rebound
Limit of detection
+ cART
cART
Persistent residual low-level viremia(1 to 5 viral RNA copies /ml)
Combination antiretroviral therapy (cART) is potent and life-prolonging but does not eradicate HIV infection
Latently-infected resting CD4+ T cells (and/or other cellular reservoirs)
Latently-infected cells, HIV-1 reservoirrare event (0.1-1 per 106 resting CD4+ T cells)long duration (months)
Productively infected cellsmost common - cell death (days)
InfectedUninfected
CD4+ T lymphocytes
Viral cytopathic effectsHost immune response
HIV-1 latent reservoirs are not eliminated by cART
Cellular stimuli (antigens, phorbol esters, mitogens, cytokines,…)
cART