heterogeneous hiv-1 reactivation patterns of disulﬁram …

TRANSLATIONAL RESEARCH

Heterogeneous HIV-1 Reactivation Patterns of Disulfiramand Combined Disulfiram+Romidepsin Treatments

Anna Kula, PhD,a,b Nadège Delacourt, MSc,a Sophie Bouchat, PhD,a Gilles Darcis, MD, PhD,a,c,d

Veronique Avettand-Fenoel, PhD,e, f Roxane Verdikt, MSc,a Francis Corazza, MD, PhD,g

Coca Necsoi, MD, PhD,h Caroline Vanhulle, MSc,a Maryam Bendoumou, MSc,a Arsene Burny, PhD,a

Stephane De Wit, MD, PhD,h Christine Rouzioux, PhD,e, f Oliver Rohr, PhD,i, j and Carine Van Lint, PhDa

Objectives: Few single latency-reversing agents (LRAs) have beentested in vivo, and only some of them have demonstrated an effect,albeit weak, on the decrease of latent reservoir. Therefore, otherLRAs and combinations of LRAs need to be assessed. Here, weevaluated the potential of combined treatments of therapeuticallypromising LRAs, disulfiram and romidepsin.

Setting and Methods: We assessed the reactivation potentialof individual disulfiram or simultaneous or sequential com-bined treatments with romidepsin in vitro in latently infected

cell lines of T-lymphoid and myeloid origins and in ex vivocultures of CD8+-depleted peripheral blood mononuclear cellsisolated from 18 HIV-1+ combination antiretroviral therapy–treated individuals.

Results: We demonstrated heterogeneous reactivation effects ofdisulfiram in vitro in various cell lines of myeloid origin and nolatency reversal neither in vitro in T-lymphoid cells nor ex vivo,even if doses corresponding to maximal plasmatic concentration orhigher were tested. Disulfiram+romidepsin combined treatmentsproduced distinct reactivation patterns in vitro. Ex vivo, thecombined treatments showed a modest reactivation effect whenused simultaneously as opposed to no viral reactivation for thecorresponding sequential treatment.

Conclusions: Exclusive reactivation effects of disulfiram inmyeloid latency cell lines suggest that disulfiram could bea potential LRA for this neglected reservoir. Moreover, distinctreactivation profiles pinpoint heterogeneity of the latent reser-voir and confirm that the mechanisms that contribute to HIVlatency are diverse. Importantly, disulfiram+romidepsin treat-ments are not potent ex vivo and most likely do not represent aneffective drug combination to achieve high levels of latencyreversal in vivo.

Key Words: disulfiram, romidepsin, latency-reversing agents,heterogeneity of HIV reservoir

(J Acquir Immune Defic Syndr 2019;80:605–613)

INTRODUCTIONCombination antiretroviral therapy (cART) has pro-

found health benefits for people with HIV, but cART is notcurative and patients must stay on therapy indefinitely.Persistence during cART of latently infected cells containingintegrated, transcriptionally silent but replication-competentproviruses is a major hurdle for HIV-1 eradication.1 Althoughmany cells may contribute to the latent reservoirs, includingmonocytes and monocyte-derived macrophages (reviewedin2,3), the best characterized one is a small population oflong-lived HIV-1–infected resting memory CD4+ T cells,maintained throughout patient’s life by homeostatic pro-liferation.4 The absence of viral gene expression in latentlyinfected cells renders the virus imperceptible to host immuneresponse. However, various cellular stimuli can induce the

Received for publication June 28, 2018; accepted December 3, 2018.From the aService of Molecular Virology, Département de Biologie

Moléculaire (DBM), Université Libre de Bruxelles (ULB), Gosselies,Belgium; bMalopolska Centre of Biotechnology, Laboratory of Virol-ogy, Jagiellonian University, Krakow, Poland; cAcademic MedicalCenter of the University of Amsterdam, Amsterdam, The Netherlands;dInfectious Diseases Department, Liege University Hospital, Liege,Belgium; eService de Virologie, Université Paris-Descartes, AP-HP,Hopital Necker-Enfants Malades, Paris, France; fUniversité ParisDescartes, Sorbonne Paris Cité, Paris, France; gLaboratory of Immu-nology, Brugmann University Hospital, Université Libre de Bruxelles(ULB), Bruxelles, Belgium; hService des Maladies Infectieuses, CHUSt-Pierre, ULB, Bruxelles, Belgium; iUniversité de Strasbourg, labo-ratoire DHPI EA7292, Schiltigheim, France; and jIUT Louis Pasteur,Université de Strasbourg, Schiltigheim, France.

Supported by the European Union’s Horizon 2020 research and innovationprogramme under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015 to C.V.L. and under the National Science Centre(Poland) and Marie Sk1odowska-Curie grant agreement no. 665778,Polonez Grant UMO-2015/19/P/NZ6/02188 to A.K. Work in C.V.L.’slaboratory was supported from the Belgian Fund for Scientific Research(FRS-FNRS, Belgium), the “Fondation Roi Baudouin”, the NEATprogram (Networking to Enhance the Use of Economics in AnimalHealth Education, Research, and Policy Making), the Walloon Region(Fonds de Maturation), “Les Amis des Instituts Pasteur à Bruxelles, asbl,and the University of Brussels (Action de Recherche Concertée (ARC)grant). The laboratory of C.V.L. is part of the ULB-Cancer ResearchCentre (U-CRC). A.K. is a postdoctoral fellow of ”Les Amis des InstitutsPasteur à Bruxelles, asbl. C.V.L. is “Directeur de Recherches” of theFRS-FNRS (Belgium), respectively.

The authors declare no competing financial interests.The authors S.B. and G.D. have equally contributed to the article.Supplemental digital content is available for this article. Direct URL citations

appear in the printed text and are provided in the HTML and PDFversions of this article on the journal’s Web site (www.jaids.com).

Correspondence to: Carine Van Lint, Service of Molecular Virology,Département de Biologie Moléculaire (DBM), Université Libre deBruxelles (ULB), Rue des Professeurs Jeener et Brachet 12, 6041Gosselies, Belgium (e-mail: [email protected]).

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latent reservoirs, allowing for HIV-1 transcription andsubsequent production of replication-competent virus,5,6

which can represent one potential source of rebound ofviremia after cART interruption.7 One proposed strategy foreliminating the latent reservoir is to pharmacologicallystimulate HIV-1 gene expression in latently infected cells,rendering these cells susceptible to cytolytic T lymphocytesor viral cytopathic effects while maintaining cART toprevent new spreading infection.8 Small molecule latency-reversing agents (LRAs) with potential therapeutical appli-cation as shock agents have been identified includingantialcoholism drug disulfiram (DSF)9,10 and histone deace-tylase inhibitors (HDACIs) such as SAHA, panobinostat,and romidepsin.11–13 Such compounds also include proteinkinase C agonists (prostratin, bryostatin, and ingenols),14,15

bromodomain and extraterminal domain inhibitors (JQ1,I-BET, and I-BET151),6 histone methyltransferases inhib-itors (chaetocin and BIX0194),16 DNA methyltransferaseinhibitors or demethylating agents (5-AzadC),5 and toll-likereceptor 7 agonists (GS-9620).17 DSF has been used for thetreatment of chronic alcoholism for more than 60 years.18 Invitro studies showed that DSF reactivates HIV in latentlyinfected cell lines of myeloid but not T-lymphoid origin19

and in primary Bcl-2–transduced CD4+ T-cell model oflatency.20 A pilot clinical study using an FDA-approveddose showed no overall effect on plasma HIV RNA duringDSF administration, but a transient increase in plasma HIVRNA was noted in a post hoc analysis in some patients.9

Importantly, a dose-escalation study showed very goodsafety profile of DSF, a clear dose-dependent effect ofdisulfiram on HIV transcription; however, a small effect onplasma HIV-1 RNA only for the highest dose wasobserved.10 In addition to antilatency clinical trials usingDSF, several trials using HDACIs have been reported in theHIV field.12,13 Romidepsin, an HDACI, was also evaluatedin vivo and was shown to be the most potent latencyactivator tested in clinical trials to date.13 However, despiteits potent effect, no decrease in the reservoir size wasobserved. Altogether, these studies using LRAs are encour-aging but question the efficacy of LRAs used alone to reducethe size of the HIV-1 reservoirs. Combination of differentclasses of LRAs targeting different mechanisms of latencycan exhibit synergistic effect (ie, result in a higher reac-tivation level than the sum of the reactivations produced byeach compound individually) in reactivation of HIV expres-sion in latently infected cells. We and others have demon-strated proof of concepts for the coadministration of 2 differentclasses of LRAs5,6,21 as a therapeutic perspective to decreasethe pool of latent HIV-1 reservoirs in the presence ofefficient cART.

In this study, we turned our attention to 2 therapeu-tically promising LRAs, DSF and romidepsin, since theformer showed excellent safety profile and the latter potentreactivation in vivo. We evaluated the reactivation poten-tial of individual and combined treatments of DSF andromidepsin in multiple in vitro cellular latency models andin ex vivo cultures of CD8+-depleted peripheral bloodmononuclear cells (PBMCs) isolated from HIV+ aviremiccART-treated individuals.

RESULTS

Heterogeneous Effects of DSF on HIVReactivation In Vitro in Latently InfectedCell Lines

We first determined optimal concentration of DSF interms of both HIV-1 reactivation potential and effect on thecellular viability. We measured induction of HIV-1 p24 capsidprotein production and cellular metabolic activity in well-studied HIV-1 latency cellular model, promonocytic U1 cellline. Increasing doses of DSF for 24, 48, and 72 hoursaugmented p24 antigen level in the cell supernatants in a dose-and time-dependent manner from 5 to 20 mM (Fig. 1A). Wealso assessed the effect of the increasing doses of DSF on cellviability as measured by WST-1 assay that reflects the cellmetabolic activity and cell proliferation. We observed dose-and time-dependent effect on cellular viability for DSF dosesranging from 20 to 50 mM (Fig. 1B). We therefore determinedthat 10 mM of DSF for 48 hours was optimal in terms of bothreactivation effect and effect on cellular viability.

We next assessed the reactivation effect of increasingdoses of DSF in 2 other monocytic-derived cell linescontaining latent HIV-1 proviruses, that is, THP89GFP andCHME-5/HIV microglial cells. These cell lines containinga reporter GFP were treated with increasing doses of DSF for24, 48, and 72 hours (Figs. 1C and E). In THP89GFP cells,20 mM of DSF for 24 hours was optimal in terms of bothreactivation effect and effect on cellular viability. Interest-ingly, in the CHME-5/HIV cell line, we observed 2 peaks ofreactivation (at 0.5 and 10 mM), and the reactivation effectwas the most pronounced after 24-hour stimulation withstrong decrease in reactivation after 48 and 72 hours of DSFtreatment (Fig. 1E). Moreover, cytotoxicity was observedfrom 20 mM DSF (Fig. 1F).

Finally, we also assessed the reactivation effect ofincreasing doses of DSF in a T-lymphoid Jurkat cell–basedlatency model, the J-Lat 9.2 cell line treated for 24, 48, and 72hours (Fig. 1G). We observed no induction of HIV-1 geneexpression in this cell line at any DSF concentration used andat any time-point tested as compared to TNFalpha treatment(see Figure 1, Supplemental Digital Content, http://links.lww.com/QAI/B273). DSF presented no negative effect on cellularviability for concentrations of DSF up to 20 mM treated for 24and 48 hours (Fig. 1H). In addition, no HIV reactivationcould be observed in 2 other Jurkat-based latency model celllines, that is, A2 and A72 cells (see Figure 2, SupplementalDigital Content, http://links.lww.com/QAI/B273). Impor-tantly, Doyon et al19 has also reported that DSF reactivatesHIV-1 expression in the U1 but not in Jurkat-based cell linelatency models. They have shown that DSF significantlyreduces phosphatase and tensin homolog (PTEN) proteinlevels in U1 cells. They have observed that Jurkat cell–basedmodels of latency do not express PTEN, explaining the lackof DSF effect on HIV-1 expression in these cell lines.Notably, we could not observe any expression levels ofPTEN protein neither in J-Lat 9.2 nor in monocytic-derivedU1 and THP89GFP cells (see Figure 3, Supplemental DigitalContent, http://links.lww.com/QAI/B273), but we detectedPTEN in CHME-5/HIV microglial cell line.

Kula et al J Acquir Immune Defic Syndr � Volume 80, Number 5, April 15, 2019

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FIGURE 1. Dose- and time-dependent effect of DSF on HIV-1 production and cellular viability in vitro. Cells were mock-treated ortreated with increasing doses of DSF as indicated. At 24, 48, and 72 hours after treatment, either CA-p24 ELISA production in cellsupernatants (A) was measured or viral protein expression was analyzed by FACS (C, E, and G). In addition, cellular viabilitywas assessed by WST-1 assay that reflects cell metabolic activity, proliferation, and cytotoxicity (B, D, F, and H). Results with themock-treated cells were arbitrary set at a value of 1 (A) or 1% of GFP (+) cells (C, E, and G) or 100% (B, D, F, and H). Mean valuesand SEs of the mean values from duplicate samples are indicated. One representative experiment from 3 is represented.

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In conclusion, our results demonstrated that the in vitroreactivation effect of DSF was heterogeneous with distinctreactivation patterns within myeloid-derived latency cell linesand no reactivation was observed in T-lymphocytic–basedlatency cell lines.

Distinct In Vitro Reactivation Patterns AfterDSF+Romidepsin Combined Treatments inMyeloid HIV Latently Infected Cell Lines

We next investigated whether DSF could synergisti-cally reactivate HIV-1 expression when combined withromidepsin in either U1 or THP89GFP or CHME-5/HIV celllines. Two compounds synergize when their combinationproduces higher effect than the sum of effects arising fromseparate treatments. We tested DSF at 0.5, 10, and 20 mM(concentrations we determined to be optimal doses in latencymodels we investigated). In addition, we also investigatedDSF at 2 and 5 mM. Romidepsin was tested at 2 concen-

trations 0.0175 and 0.04 mM that correspond to dosepreviously established by our laboratory5 and to plasmaticconcentration achieved in patients after romidepsinadministration,13 respectively.

Since, in U1 cells, we observed time-dependent effectof DSF with optimal timing at 48 hours, we investigated bothsimultaneous and sequential DSF+romidepsin combinedtreatments. We compared a simultaneous treatment in whichDSF and romidepsin were added together for 48 hours witha sequential treatment in which 24-hour DSF pretreatmentwas followed by a 24-hour romidepsin induction, correspond-ing to a total of 48-hour DSF treatment. By extracellular p24ELISA assays, we observed that simultaneous DSF+romidep-sin treatments exhibited overall only additive effects (Fig. 2A,max. fold synergy = 1.1) as compared to sequential treatmentthat produced synergistic effects (Fig. 2B, max. fold synergy= 3.4). Of note, 48-hour romidepsin treatment was very toxic(90% of reduction in cellular viability) as compared to 24-hour romidepsin treatment (60% of reduction in cellular

FIGURE 2. A, Regarding simultaneous treatment, at 48 hours post-DSF and romidepsin treatments viral production was estimatedby measuring CA-p24 antigen concentration in culture supernatants. The mock-treated value was arbitrarily set at a value of 1.Means and standard errors of the means from triplicate samples are indicated. B, Regarding sequential treatment, at 24 hourspost-romidepsin treatment and 48h post-DSF treatment, viral production was estimated by measuring CA-p24 antigen con-centration in culture supernatants. C and D, Additionally cellular viability was assessed by WST-1 assay that reflects cell metabolicactivity, proliferation and cytotoxicity. For each combinatory treatment, the fold-synergy was calculated by dividing the effectobserved after co-treatments by the sum of the effects obtained after the individual treatments.




viability) (Figs. 2C, D, respectively). Worth mentioning, weobserved very high synergistic reactivation effects of sequen-tial treatments in which DSF was combined with some otherHDACIs (see Figure 4A, Supplemental Digital Content,http://links.lww.com/QAI/B273) and other classes of LRAs(see Figure 4B, Supplemental Digital Content, http://links.lww.com/QAI/B273).

Next, we evaluated whether DSF could synergize withromidepsin in THP89GFP and CHME-5/HIV cell lines. Weinvestigated only the simultaneous combined treatments inthese cell lines, since DSF reactivation effect was only observedafter 24 hours of treatment. Regarding THP89GFP cells, weobserved synergistic increase in the GFP-positive cells aftersimultaneous cotreatments of 20 mM DSF with either romidep-sin at 0.0175 mM or at 0.04 mM (Fig. 3A, fold synergy = 2.1and fold synergy = 2.3, respectively). Marginal effects oncellular viability were observed (Fig. 3B). In case of CHME-5/HIV cells, we observed only modest synergistic increase inthe GFP-positive cells after simultaneous DSF+romidepsincombined treatments as compared to individual treatments

(Fig. 3C, fold synergy ,2). Cytotoxicity was observed for20 mM DSF and the DSF+romidepsin combined treatments(Fig. 3D).

In conclusion, our results demonstrated that DSF+ro-midepsin combined treatments produced various synergisticeffects, and the highest synergistic effects were observed inU1 cells after sequential DSF+romidepsin treatment.

Simultaneous But Not SequentialDSF+Romidepsin Treatments Lead toModerately Higher HIV-1 Recovery Than theIndividual Drug Treatments in CD8+-DepletedPBMCs From cART-Treated HIV+

Aviremic IndividualsTo address the reactivation potential of the simulta-

neous and sequential DSF+romidepsin treatments in ex vivocultures, we firstly evaluated the cytotoxic effects of thosetreatments on cells from healthy donors. We observed that

FIGURE 3. DSF+romidepsin simultaneous treatments moderately induce HIV-1 as compared to drugs alone in THP89GFP (A) andCHME-5/HIV microglial (C) cells. The CHME-5/HIV microglial cells were mock-treated, treated with DSF, or romidepsin alone or incombination as indicated. At 24 hours after treatment, cells were analyzed by flow cytometry to quantify the proportion of cellsexpressing GFP. Mean values and SEs of the mean values from duplicate samples are indicated. One representative experimentfrom 3 is represented. B and D, Additionally cellular viability was assessed by WST-1 assay that reflects cell metabolic activity,proliferation and cytotoxicity. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observedafter co-treatments by the sum of the effects obtained after the individual treatments.




DSF alone was not toxic and the combined treatmentsproduced cytotoxic effects ranging from 20% to 60% asassessed by cellular viability tests (see Figure 5, SupplementalDigital Content, http://links.lww.com/QAI/B273). Then,CD8+-depleted PBMCs isolated from 18 cART-treated avire-mic HIV-1+ patients were mock-treated, treated with anti-CD3+anti-CD28 antibodies as a positive control for globalT-cell activation or with indicated compounds added ina simultaneous or sequential manner alone or in combination(see Fig. 4 and Table S1, Supplemental Digital Content,http://links.lww.com/QAI/B273). HIV-1 genomic RNA con-centrations in culture supernatants were quantified after 6days of drug(s) treatment (Table 1). We observed in mock-treated cultures a recovery in viral genomic RNA (mean of928 HIV-1 RNA copies/mL) that could be explained byactivation of HIV-infected cells during the purification pro-cedure or during the course of experiment. Importantly, weobserved that DSF at 2 mM (that corresponds to plasmaticconcentration reported by Elliot et al10) and DSF at 5 mM(that has been chosen by us in this ex vivo study) showed noadditional increase in mean HIV-1 RNA levels as comparedto mock-treated cells (597 HIV-1 RNA copies/ml and 646HIV-1 RNA copies/mL, respectively). However, thesechanges were not statistically significant (see Table S2,Supplemental Digital Content, http://links.lww.com/QAI/B273). Furthermore, 2 doses of romidepsin were selectedfor the ex vivo study: a dose of 0.0175 mM that wasdetermined in5 and a dose of 0.04 mM that corresponds toplasmatic concentration measured in HIV reactivation clinicaltrial.13 Romidepsin at 0.0175 and 0.04 mM that was added atday 1 exhibited statistically significant increases in HIVrecovery (mean of 1925 HIV-1 RNA copies/mL and 2546HIV-1 RNA copies/mL, respectively, Fig. 4, see Table S2,Supplemental Digital Content, http://links.lww.com/QAI/B273). Romidepsin at 0.0175 and 0.04 mM that was added

at day 3 produced lower HIV recovery (mean of 1275 HIV-1RNA copies/mL and 1617 HIV-1 RNA copies/mL, respec-tively, Fig. 4) that was not statistically relevant (see Table S2,Supplemental Digital Content, http://links.lww.com/QAI/B273). Combined simultaneous DSF+romidepsin treatmentsexhibited increases in extracellular HIV-1 RNA levels thatwere higher than their corresponding individual treatments(Fig. 4). Those increases were statistically significant whencompared either with mock or individual DSF treatments, butno significance could be observed for comparisons withcorresponding individual romidepsin treatment (see Table S2,Supplemental Digital Content, http://links.lww.com/QAI/B273). Of note, simultaneous combination of 5 mM DSFand 0.04 mM romidepsin induced the highest mean HIVRNA levels; however, this recovery was not statisticallyrelevant when compared with romidepsin individual treat-ment (see Fig. 4 and Table S2, Supplemental Digital Content,http://links.lww.com/QAI/B273).

Moreover, sequential DSF+romidepsin treatments didnot exhibit any positive and statistically relevant effects onextracellular HIV-1 RNA levels (see Fig. 4 and Table S2,Supplemental Digital Content, http://links.lww.com/QAI/B273).

In addition to the increases in mean extracellular HIV-1RNA levels, we demonstrated a higher viral production inseveral cell cultures after combined treatments (Table 1, seeitalicized and bold values).

In conclusion, our results demonstrated that individualDSF and sequential DSF+romidepsin treatments did notreactivate HIV from latency ex vivo in CD8+-depletedPBMCs from HIV+ cART-treated aviremic individuals.Importantly, individual romidepsin treatments producedstatistically significant HIV recoveries. Moreover, simulta-neous DSF+romidepsin treatments produced only moderatebeneficial reactivation effects.

FIGURE 4. Simultaneous but notsequential DSF+romidepsin treatmentslead to moderately higher HIV-1 recoverythan the drugs alone in CD8+-depletedPBMCs from cART-treated HIV+ aviremicpatients. Ex vivo cultures of CD8+-depleted PBMCs purified from blood of18 patients were mock-treated, treatedwith anti-CD3+anti-CD28 antibodies(C+), 2 doses of DSF, and 2 doses of ro-midepsin alone or in combination asindicated. Six days after treatment, con-centrations of extracellular viral RNA (ECHIV-1 RNA) in culture supernatants weredetermined. The results were reported asthe actual HIV RNA copy numbers/ml oras an estimated value calculated as 50%of the smallest value when HIV RNA wasnot detected to assign a log value.Dashed line indicates the 300 HIV-1 RNAcopies/ml limit of detection. The meanvalues are represented. The symbolsrepresent different treatment conditionsfor each ex vivo culture from 18 patients.




TABLE1.Sim

ultaneousandSequentialDSF+

RomidepsinTreatm

ents

inCD8+-D

epletedPBMCsFrom

cART-TreatedHIV

+Aviremic

Patients

Patient

Total

HIV

-1DNA

(Cop

ies/10

6CD8+-

DepletedPBMCs)

LOG

Total

HIV

-1DNA

(Cop

ies/10

6

CD8+-D

epleted

PBMCs)

Mock

DSF

2mM

DSF

5mM

Sim

ultan

eous

Sequential

C+

Rom

i0.0175

mM

Rom

i0.01

75mM

+

Rom

i0.04

mM

Rom

i0.04

mM

+

Rom

i0.0175

mM

Rom

i0.0175

mM

+

Rom

i0.04

mM

Rom

i0.04

mM+

DSF

2mM

DSF

5mM

DSF

2mM

DSF

5mM

DSF

2mM

DSF

5mM

DSF

2mM

DSF

5mM

P1

1567

3.20

1601

3884

I16

3351

6049

1030

98NT

NT

II

1173

NT

NT

NT

2231

P2

789

2.90

II

I53

8685

614

7981

7I

II

1141

810

NT

NT

NT

15,320

P3

362

2.56

1337

II

1705

10,690

4302

3798

8320

I32

6258

261

678

0I

1090

16,310

P4

1171

3.07

1235

I70

739

1661

7224

6952

81NT

NT

4301

365

INT

NT

NT

95,290

P5

317

2.50

1247

867

1141

1367

1945

398

1713

NT

NT

735

736

392

NT

NT

NT

86,990

P6

308

2.49

720

1321

II

929

610

685

524

817

I16

15I

II

NT

879

P7

1905

3.28

II

2614

5317

1022

5990

1395

15,670

6863

3010

7035

4007

5889

2185

NT

64,120

P8

580

2.76

3531

I63

546

1933

2328

2531

9054

3770

,057

517

1484

1833

1617

391

I22

,470

P9

437

2.64

936

II

2280

1627

757

4166

380

4013

1353

2699

393

362

505

1187

25,850

P10

184

2.26

740

II

899

477

617

II

I13

1030

2I

I62

1NT

3552

P11

595

2.77

524

553

690

655

II

I58

034

616

3516

89I

I50

60I

NT

P12

451.65

665

II

II

II

II

I32

349

763

454

668

115

71

P13

427

2.63

992

I73

9I

1882

1438

1587

2690

1081

II

INT

NT

NT

57,170

P14

1329

3.12

319

602

775

2282

2725

12,300

5917

2580

3072

3689

2054

730

866

843

392

1,326,000

P15

923

2.97

II

I68

840

415

0566

6397

033

6670

8I

I14

7361

8NT

20,390

P16

941.97

I93

816

51I

I37

251

0NT

NT

I34

369

3NT

NT

NT

I

P17

2788

3.45

772

I14

1030

7854

2984

1752

0661

6712

,390

1478

445

2065

5207

3397

NT

49,790

P18

570

2.76

I82

8I

I15

6712

4614

9517

95NT

I11

3862

9NT

NT

NT

2190

Culturesof

CD8+-depletedPBMCspurified

from

bloodof

18patientsweremock-treatedor

treatedwith

anti-CD3+

anti-CD28

antib

odies(C+),DSF(2

and5mM)andromidepsin(0.0175and0.04

mM)alone,or

incombinatio

nas

indicated.Six

days

aftertreatm

ent,concentrations

ofgenomicviralR

NAincultu

resupernatantsweredeterm

ined,and

thevalues

wereexpressedas

HIV

-1RNAcopies/m

l.TotalHIV

-1DNAwas

measuredandwas

expressedas

HIV

-1DNAcopies/106

cells.V

aluesrepresentin

ghigher

viralproductio

naftercombinatory

treatm

entthan

aftersingledrug

treatm

entareitalicized.V

aluesrepresentin

greactiv

ationof

thevirusobserved

exclusivelyafterdualtreatm

ents

arerepresentedin

bold.“I”indicatesbelow

the300HIV

-1RNA

copies/m

llim

itof

detection.

“NT”indicatesnottested

conditions.




DISCUSSIONA number of chemical compounds have been identified

as HIV LRAs. DSF has also been studied and broughtforward to clinical trials.9,10 In this report, we evaluatedreactivation potential of DSF alone or combined withromidepsin in vitro in various latency cell lines of myeloidand T-lymphocytic origins and ex vivo in CD8+-depletedPBMCs from HIV+ aviremic cART-treated individuals. Weobserved heterogenous DSF reactivation patterns withinmyeloid-derived latency cell lines and no reactivation wasobserved in Jurkat-based T-cell line, highlighting differentreactivation patterns between models from different cellularlineage but also between models belonging to the samecellular lineage. Importantly, several recent studies haveshown that LRAs have limited reactivation spectra, beingeffective only in some cell types. For instance, recent workfrom Baxter et al22 has demonstrated a heterogeneousresponse of CD4+ population to individual LRAs: bryostatininduced the effector memory CD4+ T-cell reservoir but hadlimited effect on the central/transitional memory CD4+ T-cellcompartment, contrasting with similar effects observed withingenol. In addition, it has been recently demonstrated byChen et al23 that different LRAs reactivate different subsets oflatent proviruses. Moreover, we also observed in a previousreport a great heterogeneity between patients in terms ofreactivation capacity of their ex vivo cell cultures.24 Alto-gether, these recent works and our findings strongly suggestthat LRAs may have limited and heterogeneous reactivationspectra in vivo due to cellular and viral heterogeneity of latentreservoirs and that multiple mechanisms may be involved inthe reactivation of latent HIV. Previously, Doyon et al19 haveinvestigated the mechanism of DSF action on latent HIV inU1 and Jurkat-based cell lines and have shown that DSF actsthrough the Akt signaling pathway by decreasing PTEN inU1 cells but not in Jurkat-based cell lines. By contrary, wecould not detect PTEN in U1 cells, questioning the role of thisfactor in DSF-mediated HIV reactivation in this cell line andsuggesting a different mechanism. Interestingly, Lin et al25

have shown that DSF treatment results in demethylation ofgenes hypermethylated in prostate cancer with subsequent re-expression of these genes, suggesting that DSF can act as anepigenetic drug. It remains to be determined whether DSFcould act as epigenetic drug on HIV expression.

Remarkably, we could not observe any reactivationeffect of DSF ex vivo, in CD8+-depleted PBMCs isolatedfrom HIV+ aviremic cART-treated individuals. This could beexplained at least partially by the fact that most of the HIV-infected cells in blood are CD4+ T cells with HIV-infectedmonocytes being extremely low. Although HIV-1 proviralDNA is only present in less than 1% of circulating monocytes(between 0.01% and 1%), these cells are important viralreservoirs and are responsible for the dissemination of HIV-1into sanctuaries such as the brain.26

Importantly, in our ex vivo studies, we tested 2 and5 mM DSF with the former corresponding to max. plasmaticconcentration from the recent dose-escalation clinical trial.10

This study showed that even if all doses (500, 1000, and 2000mg) produced an increase at the level of intracellular HIV

RNA, only the highest dose increased the plasma HIV RNAalbeit with very low effect. This effect was extremely modestand an order of magnitude lower than that noted previouslywith HDACI, romidepsin.13 In line with this observation,Mohammadi et al27 have also previously shown in theirprimary CD4+ T-cell models that DSF treatment successfullyincreases viral transcription, but fails to effectively enhanceviral translation, as the levels of viral-encoded GFP remainedlow, suggesting the importance of post-transcriptionalblock(s) as one mechanism leading to HIV latency that needsto be relieved to purge the viral reservoir. It is therefore verylikely that DSF may not generate a cellular environment thateffectively sustains HIV particle production ex vivo. Similarobservation has been reported for SAHA, that is, weak effectat the level of viral particle production in vivo.11 Notably,a clear change in plasma HIV-1 RNA with subsequentdecrease in the reservoir size has been seen in vivo only forromidepsin (combined with immunotherapy)28 and for checkpoint inhibitor nivolumab.29 Importantly, no study so faraffected time from interruption of cART to viral rebound.These observations and recent studies demonstrating cell-typespecificity of LRAs suggest that to achieve high levels oflatency reversal, combinations of mechanistically distinctLRAs that exhibit broad spectra of reactivation (being activein multiple cell types) most likely combined with immuno-therapy may be required.

In this report, we tested combined treatments of DSFand romidepsin. This LRA combination is of particularclinical interest. DSF has an excellent safety profile giventhe long history of chronic use for alcohol dependence.Romidepsin has also been evaluated in vivo, and themagnitude of HIV-1 induction after romidepsin administra-tion is greater than anything previously reported for any LRAtested in humans.13 We evaluated simultaneous and sequen-tial combined treatments of those 2 promising LRAs in vitroand ex vivo. Moreover, with the purpose to find the optimalconditions for synergistic studies, we evaluated severalconcentrations of both LRAs including doses correspondingto maximal plasmatic concentrations, achieved in clinicaltrials, for both LRAs. Importantly, we observed significantsynergistic effects only in U1 cells, and only modest addictiveeffect was observed ex vivo. Laird et al21 have also observedthat DSF+romidepsin combined treatments did not producesynergistic but only modest additive effect in ex vivo culturesof resting CD4+ T cells from infected individuals; however,lower dose of DSF (0.5 mM) has been tested in this study.Here, for synergistic ex vivo studies, we evaluated higherdoses of DSF, 2 and 5 mM, with the former corresponding tomax. plasmatic concentration from the recent dose-escalationclinical trial.

In summary, cell-type–specific and distinct re-activation patterns of DSF highlight the heterogeneity ofHIV reservoirs and that multiple molecular mechanismscontribute to HIV latency that need to be relieved to reacha cure. Notably, demonstration of weak ex vivo reactivationeffect of combined DSF+romidepsin treatments should beconsidered for designing future clinical trials and suggestthat to achieve high levels of latency reversal, combination




of broad-spectrum LRAs (being active in multiple cell types)may be required.

Finally, DSF selectivity for myeloid cells suggests thatDSF could represent a good LRA candidate to reactivate thisneglected reservoir of myeloid origin including monocytesand microglial cells.

ACKNOWLEDGMENTSThe authors thank the HIV-1+ patients for their

willingness to participate in this study. The authors thankthe nursing team of CHU Saint-Pierre hospital (Brussels,Belgium), Elodie Goudeseune, Joëlle Cailleau, and AnnickCaestecker, who cared for the patients. The authors thankLudivine David and Adeline Melard from Christine Rou-zioux’s laboratory and Hilde Vereertbrugghen from FrancisCorazza’s laboratory for excellent technical assistance. Theauthors thank the transfusion center from Charleroi (Hai-naut, Belgium) and principally Jacqueline Pineau for pro-viding the blood from HIV-negative donors. Material andMethods section is in Supplemental Digital Content (http://links.lww.com/QAI/B273).

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