2014 “ Towards an HIV Cure ” symposium Melbourne

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


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

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mo

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mo

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J-Lat 9.2 cellsm

oc

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p2

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nti

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n le

ve

l (a

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rary

un

its

)

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25

50

75

100

125

150

mo

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J-Lat 9.2 cells

% o

f c

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lar

via

bili

ty

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5

10

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20

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30

35

mo

ck

mo

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mo

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mo

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U1 cells

p2

4 a

nti

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l (a

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rary

un

its

)

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25

50

75

100

125

150

mo

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mo

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U1 cells

% o

f c

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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


30

70

110

150

p2

4 a

ntig

en

lev

el (

arb

itra

ry u

nits

)

0

20

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60

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180


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


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)


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+


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+


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+


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+


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+


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+


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

Documents

2014 “ Towards an HIV Cure ” symposium Melbourne