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
Strategies aimed at reducing the size of the persistent
reservoirs of latent HIV-1 by forcing viral gene expression
cART + „purging strategy”
Inducers of HIV-1
gene expression
?
Postintegration latency is a multifactorial phenomenon
Chromatin structure and epigenetic modifications
• histone posttranslational modifications
• DNA methylation
Absence of cellular inducible transcription factor
Sequestration of P-TEFb
TF (such as NF-KB)
Prostratin
Bryostatin
Ingenol
HMBA
P-TEFb
P-TEFb
Tat
5’
JQ1
I-BET
I-BET151
3’
NF-kB sites
P-TEFb
Cellular genes
Brd4
TAR
nuc-1
Five recent publications show that BET inhibitors reactivate latent HIV-1
BETi
Banerjee C, et al.
J Leukoc Biol
2012
JQ1
Li Z, et al. Nucleic
Acids Res 2013
JQ1
JQ1+Pro
Bartholomeeuse
n K, et al. J Biol
Chem 2012
Cellular model
Function
Ach2, U1, J-Lat 10.6
resting CD4+ Tcells isolated from ART
treated patients
JQ1 reactivates HIV-1 in several cellular
systems and in 1 out of 3 patients.
J-Lat A2, 2D10, Jurkats 1G5
Hela
JQ1 activates HIV transcription in latently
infected Jurkat Tcells.
JQ1 dissasociates BRD4 from chromatin and
increases Tat binding to HIV-1 LTR
JQ1
JQ1+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
JQ1
JQ1+Pro
JQ1+PHA
HeLa, U1, J-Lats, J-Lat A2, 293T
CD8+-depleted PBMCs and resting
CD4+ Tcells isolated from HIV-1
negative or from ART-treated patients
Enhanced viral reactivation in NL4-3-GFP -HIV1 infected PBMCs isolated from 6 negative
donors.
Enhanced viral replication when JQ1 +
Prostratin or JQ1+ PHA in 7 out of 19 ARTtreated patients.
Boehm D, et al.
Cell Cycle 2012
JQ1
I-BET
I-BET151
JQ1+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.
Compounds targeting
P-TEFb
-
JQ1
IBET
IBET151
HMBA
PKC agonists
- Prostratine
- Bryostatin-1
- Ingenol
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)
vpr
gag
tat
rev
env
vif
pol
3’
3’ LTR
GFP
I-BET
I-BET151
HMBA
2.6
1.8
2.3
3.3
2.1
1.4
1.9
3.9
J-Lat 9.2 cells
11.6
20
17.2
25
21.45
JQ1
15
15.0
synergy
Bryostatin-1
Prostratin
17.25
vpu
7
6.3
6.01
10
8
6
2.1
4
Bryostatin-1
Prostratin
TNF
HMBA
I-BET151
I-BET
JQ1
Prostratin
HMBA
0.3
HMBA
I-BET151
0.3
I-BET151
I-BET
0.3
I-BET
JQ1
0.4
0
JQ1
1
0.4
2
Bryostatin-1
1.3
3
2.95
3.5
5
Mock
5’ LTR
% of GFP (+) cells
5’
Microglial
The combination NF- B
inducerscells
+ PTEFb inducer activates
HIV-1 expression in a greater proportion of cells than each
compound alone (II)
24
22
JQ1
I-BET
1.6**
1.4**
1.1
1.0
I-BET151 HMBA
1.2
1.1
0.8
0.9
31.5
synergism
(fold)
Bryostatin-1
Prostratin
HIV-latently infected microglial cells
12.35
16
14.8
18
14
** p<0.005
9.1
I-BET151
Prostratin
TNF
HMBA
6.65
8.35
I-BET
4.55
JQ1
HMBA
Bryostatin-1
Prostratin
7.8
I-BET151
6.8
6.95
JQ1
3.4
HMBA
Bryostatin-1
3.1
I-BET151
0
I-BET
2
JQ1
4
1.55
6
2.85
4.65
8
4.15
10
I-BET
12
Mock
% of GFP (+) cells
20
Evaluation of HIV-1 recovery in CD8(+)-depleted PBMCs from
virally suppressed patients (I)
Prostratin
Bryostatin-1
Patients
HIV DNA
copies/ 106
PBMCs
Mock
JQ1
I-BET
I-BET151
HMBA
Pro
Bryo
P1
680
-
422
-
584
1016
669
449
P2
1217
-
-
-
237
-
-
-
-
P3
70
-
-
-
584
717
5359
657
694
P4
350
-
729
//
1707
944
394
-
271
531
316
187
P5
1782
-
-
812
-
-
3870
2381
6857
3328
5876
5563
P6
669
-
2418
5579
2505
1582
308
1336
1918
774
1666
472
P7
670
-
25034
-
-
586
459
296
536
-
797
P8
782
-
576
964
650
-
2377
-
-
665
-
C+
JQ1
I-BET
I-BET151
HMBA
JQ1
I-BET
I-BET151
HMBA
3591
141
4200
282
318
-
-
-
-
-
-
-
-
-
-
-
585
435
-
691
830
-
191
1017
670
-
14741
308
7027
6307
2496
10158
3142
20524
3936
5741
936
8798
39773
//
566
-
274
2274
36562
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
8
58
48
54
42
46
67
67
63
54
52
79
33
54
58
75
% of activated patients (>150
cop/ml)
Evaluation of HIV-1 recovery in CD8(+)-depleted PBMCs from
virally suppressed patients (II)
Patients
P21
P22
P23
P24
P25
P26
P27
P28
HIV DNA
copies/ 106
PBMCs
600
737
380
203
11
548
4486
2552
% of activated patients
(>150 cop/ml)
Mock
JQ1
Ingenol
Ingenol +
JQ1
C+
693
0
0
0
0
0
2433
1911
954
0
1239
0
902
181
3417
920
713
3468
//
768
453
888
9412
834
//
//
//
//
2313
//
13199
6751
2717
7879
7267
464
373
10407
31132
2348
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
JQ1
HIV DNA
Patients cop/106 resting
CD4+ T cells
Age
CD4+ T
cell
count
Last treatment
Aviremic for
(years)
Mock
JQ1
Pro
Bryo
Ing
483
-
-
C+
Pro
Bryo
Ing
764
-
-
-
1002
-
-
460
P1
1200
65
869
TRU EFV
8
-
P2
1179
48
670
CBV NVP
15
-
-
-
4982 648
-
P3
112
41
848
TRU KLT
4
-
649
5022
676
-
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
-
-
19
38
56
50
50
44
50
43
69
71
75
100
% of activated patients (>150 cop/ml)
% of patients with combinatory beneficial effect
(out of activated patients)
180
1007 4131 1337
-
Conclusion and perspectives
• We have identified combinations of compounds exhibiting
real potential of reactivation in several different postintegration latency cellular models.
• Some of these combinations might be clinically relevant for
reducing/eliminating the cellular reservoirs of latent HIV-1.
Acknowledegments
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
Thanks to L.Gama and to Amazônia Fitomedicamentos, Brazil for providing
Ingenol.
University of Strasbourg,
France
• Olivier Rohr
University of Franche-Comté,
France
• Georges Herbein
University of Liège
• Michel Moutschen
• Dolores Vaira
P-TEFb inducers increase HIV-1 expression in a dosedependent manner without cytotoxicity
B
150
J-Lat 9.2 cells
20
18
14
12
10
8
6
4
100
75
50
25
2
JQ1
I-BET
I-BET151
JQ1
C
I-BET
mock
I-BET151
HMBA
D
150
U1 cells
30
U1 cells
20
15
10
100
75
50
0
I-BET
I-BET151
mock
mock
JQ1
mock
0
mock
25
mock
5
HMBA
JQ1
I-BET
mock
25
mock
% of cellular viability
125
mock
35
mock
mock
HMBA
mock
0
mock
mock
mock
mock
0
p24 antigen level (arbitrary units)
J-Lat 9.2 cells
125
16
% of cellular viability
p24 antigen level (arbitrary units)
A
I-BET151
HMBA
NF-B inducers increase HIV-1 expression in a dosedependent manner without cytotoxicity
170
150
J-Lat 9.2 cells
p24 antigen level (arbitrary units)
p24 antigen level (arbitrary units)
220
120
70
20
12
10
8
6
4
2
mock
mock
Bryostatin-1
70
30
30
25
20
15
10
5
mock
mock
Bryostatin-1
prostratin
180
J-Lat 9.2 cells
prostratin
U1 cells
160
% of cellular viability
175
% of cellular viability
U1 cells
0
0
200
110
150
125
100
75
50
25
140
120
100
80
60
40
20
0
0
mock
mock
Bryostatin-1
mock
prostratin
mock
Bryostatin-1
prostratin
The combination Bryostatin-1/Prostratin + PTEFb inducer
activates HIV-1 transcription
TAR
env
10.0
7.5
5.0
Prostratin
HMBA
I-BET151
I-BET
JQ1
Prostratin
HMBA
I-BET151
I-BET
0.0
JQ1
2.5
30
25
20
15
10
5
0
3.1
3.5
6.6
4.4
3.4
3.5
6.3
U1 cells
TAR/actin
tat/actin
Bryostatin-1
Prostratin
TNFalpha
12.5
4.3
7.5
HMBA
Fold induction
20
15.0
mock
Fold induction
220
5.9
6.7
I-BET151
TAR/actin
tat/actin
420
2500
1500
500
300
250
200
150
100
50
6.1
5.2
I-BET
620
J-Lat 9.2
4.5
7.3
JQ1
820
5.7
Prostratin
19.2
HMBA
5.2
I-BET151
5.0
I-BET
9.6
JQ1
11.4
Bryostatin
3.7
HMBA
5.0
Prostratin
I-BET I-BET151 HMBA JQ1 I-BET I-BET151 HMBA
I-BET151
9.2
Bryostatin-1
JQ1
I-BET
TAR
tat
synergy
HMBA
JQ1
Prostratin
I-BET
I-BET151
JQ1
mock
synergy
Down-regulation of CD4 receptor expressed on
resting CD4+ T cells (I)
D
28
J+
I
D
3/
C
C
In
ge
no
l
J+
P
B
os
tr
at
in
J+
Pr
1
os
t
at
in
-
1
JQ
ry
B
oc
k
D
ay
6
4500
4000
3500
3000
2500
2000
1500
1000
500
0
m
% CD4+ CD8- (MFI)
resting CD4+ T cells
C
D
28
I
J+
l
P
ge
no
3/
C
D
In
J+
at
in
B
J+
os
tr
resting CD4+ T cells
in
-1
1
JQ
at
st
B
D
C
J+
B
3/
C
D
l
28
J+
I
no
J+
P
In
ge
D
-1
ra
tin
os
t
Pr
6
1
JQ
ay
D
28
ta
tin
os
ry
k
l
J+
I
3/
C
D
no
J+
P
In
ge
oc
C
J+
B
-1
ra
tin
os
t
Pr
6
1
JQ
ay
D
ta
tin
os
k
oc
ry
m
B
m
% CD38+ CD4+
% CD69+ CD4+
resting CD4+ T cells
Pr
ry
o
0
B
6
1
ay
2
D
3
k
4
% HLA-DR+ CD4+
100
90
80
5
oc
% CD25+ CD4+
100
75
50
50
45
40
35
30
25
20
15
10
5
0
m
28
I
l
J+
no
D
3/
C
tin
J+
P
tr
a
B
J+
-1
1
6
JQ
ay
ge
In
os
D
C
D
ta
tin
k
os
Pr
ry
B
oc
m
Global T cell activation (II)
resting CD4+ T cells
100
75
50
50
45
40
35
30
25
20
15
10
5
0
resting CD4+ T cells
100
75
50
25
0
C
D
3/
C
28
J+
I
J+
P
In
ge
no
l
D
Pr
J+
B
os
tr
at
in
-1
ry
os
ta
tin
6
CD8(+)- depleted PBMCs
JQ
1
ay
0
0
D
ay
10
D
20
oc
k
30
% HLA-DR+ CD4+
40
oc
k
28
J+
I
D
3/
C
D
J+
P
In
ge
no
l
100
90
80
50
m
-1
J+
B
os
tr
at
in
Pr
C
6
0
JQ
1
ay
ay
% HLA-DR+ CD4+
B
C
6
0
l
28
J+
I
D
3/
C
D
In
ge
no
J+
P
J+
B
os
tr
at
in
Pr
JQ
ay
D
ay
D
1
ta
tin
-1
os
oc
k
ry
m
28
J+
I
D
3/
C
D
J+
P
In
ge
no
l
oc
k
C
6
0
J+
B
os
tr
at
in
Pr
JQ
ay
D
ay
D
1
ta
tin
-1
os
oc
k
oc
k
ry
m
B
m
m
% CD38+ CD4+
% CD69+ CD4+
CD8(+)- depleted PBMCs
B
D
D
ry
os
ta
tin
oc
k
oc
k
100
75
50
50
45
40
35
30
25
20
15
10
5
0
m
B
m
m
Global T cell activation (I)
CD8(+)- depleted PBMCs
100
75
50
50
45
40
35
30
25
20
15
10
5
0
CD8(+)- depleted PBMCs
65
60
55
50
45
40
35
30
25
20
15
10
5
0
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
% cellular viability
250
200
P1
150
100
81
*
60
35
31
*
119
108
*
100
101
*
103
124
* 110
111
*
107
P2
P3
P4
100
P5
50
0
mock
Prostratin
Bryostatin-1
JQ1
CD8(+)-depleted PBMCs isolated from 5 healthy donors
I-BET
I-BET151
HMBA
Evaluation of cellular viability of the combination P-TEFb +
NF-B inducer in CD8(+)-depleted PBMCs
% cellular viability
250
100
103
117
120
64
119
67
125
69
71
200
P1
P2
P3
P4
P5
150
100
50
0
Mock
Bryostatin -
JQ1
-
I-BET I-BET151 HMBA
-
-
-
JQ1
I-BET I-BET151 HMBA
-
+
+
+
+
+
107
60
83
84
75
122
% cellular viability
200
180
100
100
103
111
160
140
P1
P2
P3
P4
P5
120
100
80
60
40
20
0
Mock
Prostratin -
JQ1
-
I-BET I-BET151 HMBA
-
-
-
-
JQ1
+
+
I-BET I-BET151 HMBA
+
+
+
Plasma viral RNA (copies/ml)
Combination antiretroviral therapy (cART) is potent and lifeprolonging but does not eradicate HIV infection
+ cART
Viral
rebound
Limit of detection
Blips
50 RNA copies/ml
of plasma
Persistent residual
low-level
Latently-infected
resting
CD4+viremia
T cells
(1 to
5
viral
RNA
copies
/ml)
(and/or other cellular reservoirs)
cART
HIV-1 latent reservoirs are not eliminated by cART
cART
Viral cytopathic effects
Host immune response
Productively infected cells
most common - cell death (days)
Cellular stimuli
(antigens, phorbol esters,
mitogens, cytokines,…)
Uninfected
Infected
CD4+ T lymphocytes
Latently-infected cells, HIV-1
reservoir
rare event (0.1-1 per 106 resting
CD4+ T cells)
long duration (months)