Chidamide, a histone deacetylase inhibitor-based anticancer drug, effectively reactivates latent HIV-1 provirus

Accepted Manuscript Chidamide, a histone deacetylase inhibitor-based anticancer drug, effectively reactivates latent HIV-1 provirus Wenqian Yang, Zhiwu Sun, Chen Hua, Qian Wang, Wei Xu, Qiwen Deng, Yanbin Pan, Lu Lu, Shibo Jiang PII:

S1286-4579(17)30178-8

DOI:

10.1016/j.micinf.2017.10.003

Reference:

MICINF 4516

To appear in:

Microbes and Infection

Received Date: 19 September 2017 Revised Date:

18 October 2017

Accepted Date: 20 October 2017

Please cite this article as: W. Yang, Z. Sun, C. Hua, Q. Wang, W. Xu, Q. Deng, Y. Pan, L. Lu, S. Jiang, Chidamide, a histone deacetylase inhibitor-based anticancer drug, effectively reactivates latent HIV-1 provirus, Microbes and Infection (2017), doi: 10.1016/j.micinf.2017.10.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Chidamide, a histone deacetylase inhibitor-based anticancer drug,

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effectively reactivates latent HIV-1 provirus

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Wenqian Yang a,#, Zhiwu Sun a,#, Chen Hua a, Qian Wang a, Wei Xu a, Qiwen Deng b,

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Yanbin Pan c, Lu Lu a,*, Shibo Jiang a,b,d,*

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Key Lab of Medical Molecular Virology of Ministries of Education and Health, School of

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Basic Medical Sciences & Shanghai Public Health Clinical Center, Fudan University,

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Shanghai, China b

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Shenzhen Nanshan People's Hospital of Shenzhen University, Shenzhen 518052, China c

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Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065, USA

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These authors have equal contribution.

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Aris Pharmaceuticals Inc., Bristol, PA19007, USA

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*Address for correspondence: Shibo Jiang: [email protected]; Lu Lu:

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[email protected].

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Abstract Although combination antiretroviral therapy (cART) is highly effective in suppressing

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human immunodeficiency virus type 1 (HIV-1) replication, it fails to eradicate the virus from

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HIV-1-infected individuals because HIV-1 integrates into the resting CD4+ T cells,

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establishing latently infected reservoirs. Histone deacetylation is a key element in regulating

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HIV-1 latent infection. Chidamide, a new anticancer drug, is a novel type of selective histone

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deacetylase inhibitor. Here we showed that chidamide effectively reactivated HIV-1 latent

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provirus in different latently infected cell lines in a dose- and time-dependent manner.

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Chidamide had relatively low cytotoxicity to peripheral blood mononuclear cells (PBMCs)

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and other latent cell lines. We have demonstrated that chidamide reactivates HIV-1 latent

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provirus through the NF-κB signaling pathway. The replication of the newly reactivated

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HIV-1 could then be effectively inhibited by the anti-HIV drugs Zidovudine, Nevirapine, and

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Indinavir. Therefore, chidamide might be used in combination with cART for functional

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HIV-1 cure.

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Key Words: Histone deacetylase inhibitor; Chidamide; HIV-1 latent infection

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1. Introduction Combination antiretroviral therapy (cART) has succeeded in reducing human

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immunodeficiency virus type 1 (HIV-1) load to undetectable levels in most patients [1], but

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the therapy must be maintained throughout life. Once cART is withdrawn, viral loads could

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return to even pretreatment levels within weeks. One of the principal obstacles to a thorough

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cleaning of the virus is the integration of HIV-1 genome into that of the resting CD4+ T cells

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during acute infection, thereby establishing the latently infected reservoir that harbors

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transcriptionally silent proviruses refractory to current cART [2-4]. Therefore, the

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development of HIV-1 therapeutic strategies to eliminate this latent reservoir is urgently

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needed.

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Some host cell-mediated molecular mechanisms maintain the quiescence of HIV-1 gene

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expression, including epigenetic silencing of the proviral promoter, transcription complex

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formation, transcript initiation, and transcription complex processivity, all of which might act

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as therapeutic targets to disrupt HIV-1 latency [5-8]. Among these mechanisms, histone

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deacetylase (HDAC)-mediated chromosomal suppression of HIV-1 long terminal repeat (LTR)

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is a key regulator leading to the establishment of HIV-1 latency [7, 9]. It was reported the host

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transcription factors comprised of NF-κB family members are involved in HDAC1

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recruitment [10]. HDAC inhibitors have been shown to increase cell-associated HIV-1 RNA

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production and/or plasma viremia in vivo [6, 11, 12], suggesting that they could play a role in

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reactivating latent HIV-1 provirus.

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A strategy known as ‘shock and kill’, or ‘kick and kill’, has been developed for HIV-1

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functional cure [13, 14]. To explain, the latent HIV-1 provirus was first reactivated with some

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small-molecule HIV-1 latency-reversing agents (LRAs) [15] and then killed by the patient’s

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immune system or suppressed by cART. Clinical trials have demonstrated that administration

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of HDAC inhibitors, such as vorinostat [16, 17] or panobinostat [18], to the patients in whom

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viremia was fully suppressed by cART, resulted in a significant increase in HIV-1 RNA

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production in resting CD4+ cells. Another clinical trial had shown that the HDAC inhibitor

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romidepsin was effective in disrupting HIV-1 latency in vivo [19]. However, some LRAs may

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be toxic and mutagenic or harmful to host brain during prolonged treatment [17, 20, 21].

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Therefore, identification of new HDAC inhibitors with improved efficacy and safety is fully

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essential.

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Chidamide is a novel benzamide chemical class of HDAC inhibitor that selectively inhibits

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the histone deacetylation activities of HDAC1, 2, 3, as well as 10 [22]. It has been approved

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by the Chinese Food and Drug Administration for the treatment of patients with recurrent or

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refractory peripheral T-cell lymphoma (PTCL) [23]. Recently, a study screening active LRAs

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in a primary cell model indicated that the HDAC inhibitors containing a benzamide functional

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group and a pyridyl cap are the most effective, as exemplified by chidamide [24]. However,

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unlike vorinostat, which belongs to the class of hydroxamic acids [6], little is known about

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chidamide’s potential use and mechanism to reactivate latent HIV-1 provirus in latently

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infected cells. Therefore, in this study, we dissected the effect of chidamide on reactivation of

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HIV-1 provirus in latently infected cell models, as well as its possible mechanism of action.

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We found chidamide to be much safer with more long-lasting effect than vorinostat. Our

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results suggest that chidamide has the potential to be further developed as an effective and

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safe LRA for HIV-1 functional cure.

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2. Materials and Methods

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2.1. Cell Culture and Chemical Treatment

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J-Lat A2 cells [25] (obtained from NIH AIDS Reagent Program), J-Lat C11 cells [26],

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PBMCs (Allcells), ACH-2 cells [27] and U1 cells [28] were cultured in RPMI 1640 medium

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with 10% fetal bovine serum (FBS) and 1% Pen/strep in a 37 °C incubator containing 5%

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CO2. HEK 293 cells and TZM-Bl cells [29] were grown in Dulbecco’s modified Eagle’s

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medium (DMEM) with 10% fetal bovine serum and 1% Pen/strep at 37 °C under 5% CO2.

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Chidamide

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Pyrrolidinedithiocarbamic acid (PDTC) (Sigma), vorinostat (Sigma) and chidamide were

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dissolved in anhydrous dimethyl sulfoxide (DMSO) and were stored at -20 °C. Recombinant

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Human TNF-alpha Protein (R&D Systems) was dissolved in sterile PBS.

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2.2. Flow Cytometry

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Aspirin

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C11 and A2 cells were incubated with the indicated concentrations of chidamide or

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vorinostat at different time points, or pretreated with various concentrations of Aspirin for 3 h

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and subsequently treated with chidamide, TNF-α or control DMSO for indicated hours. Cells

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were washed and resuspended in PBS 3 times. GFP expression was measured by a BD Accuri

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C6 Flow Cytometer (BD Biosciences) as previously described [30, 31]. All experiments were

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performed independently at least three times in triplicate per experimental point.

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2.3. HIV-1 antigen p24 assay

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ACH-2 and U1 cells (4×104 cells) were seeded into a 96-well plate and then incubated with

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various concentrations of chidamide and vorinostat at different time points. Viral release in

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the supernatant was quantified by p24 ELISA assay as previously described [32]. All

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experiments were performed independently at least three times in triplicate per experimental

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point.

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2.4. Cytotoxicity Assay

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Cell viability was measured following the instructions in the protocol provided in the cell

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counting kit-8 (CCK-8; Dojindo Molecular Technologies, Gaithersburg, MD, USA) as

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described [33]. J-Lat cells, HEK 293 T cells and PBMCs were seeded into a 96-well plate,

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approximately 4×104 cells per well; 20 µL of CCK-8 solution were added to each well of the

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plate, and cells were treated with or without chidamide for 48 h. After 2 to 3 h of incubation

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at 37 °C, the absorbance at 450 nm was taken by using a microplate reader (Infinite F200

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PRO, Tecan, Switzerland). In this assay, the 50% cytotoxic concentration (CC50) was

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calculated by use of the CalcuSyn software program (Biosoft, Ferguson, MO) [34] as we

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previously described [35].

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2.5. Western Blot

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Western blot analysis was performed as described [36]. Cells were harvested, washed once

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with ice-cold PBS, and lysed in ice-cold RIPA buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1%

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NP-40, and 2 mM EDTA)] containing PMSF and phosphatase inhibitor cocktails

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(Thermofisher Scientific, Omaha, NE) for 15 minutes. Protein samples were separated by

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SDS-PAGE and transferred onto nitrocellulose membrane (Pall Corporation, Port Washington,

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NY). After blocking with 5% non-fat dry milk in PBS containing 0.1% Tween 20, proteins of

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interest were probed with the corresponding primary antibodies [pp65 (Ser536) and IκBα

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antibodies obtained from Cell Signaling (Danvers, MA)], followed by appropriate

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infrared-conjugated secondary antibodies, Alexa Fluor 800 or 680 (Carlsbad, CA). The

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immunoblotting was carried out using the LiCOR (Lincoln, NE) Odyssey scanner.

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2.6. Luciferase Assay

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1×104 ACH-2 cells per well were treated with chidamide or vorinostat and incubated with

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the anti-HIV-1 drugs for 4 days at 37 °C, followed by collection of 100 µl culture

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supernatants from each well on the fourth day. Then all supernatants were added into the

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96-well polystyrene plate coated with TZM-Bl cells. After 48 h, TZM-Bl cells were lysed in

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200µl of luciferase reporter lysis buffer. Luciferase activity was measured using luciferase

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assay regents (Promega, Madison, WI, USA) and a luminescence counter (Infinite M200 Pro)

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according to the manufacturer’s instructions as previously described [37].

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2.7. Statistical Analysis

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Means and standard errors (SE) were calculated for all data points from at least 3

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independent experiments in triplicate. Statistical significance was determined using the

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Student’s unpaired two-tailed t-test. All statistical analyses were carried out using GraphPad

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Prism 6.0 (GraphPad Software). P < 0.05 was considered statistically significant.

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3. Results

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3.1. Activation of HIV-1 expression in latently infected cells by chidamide in a

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dose-dependent manner

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To investigate if chidamide has the potential to induce latent HIV-1 expression, we

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performed an experiment on the J-Lat C11 cell line, which is a latently infected Jurkat T cell

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line with a single provirus integrated into intron of RNPS1 and an EGFP gene under control

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of the HIV-1 LTR [26]. We treated C11 cells with increasing concentrations of chidamide,

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while using vorinostat as a positive control, for 48 h and measured the percentage of

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GFP-positive cells by flow cytometry. We found a 4- to 25-fold increase in the percentage of

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GFP-positive cells from the C11 cells treated with chidamide subjected to background levels

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(Fig. 1A).

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GFP-expressing cells reached approximately 82.6%. Similarly, the percentage of

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GFP-positive cells rose from 3.2% to 87.7% after treatment of 0 to 2 µM vorinostat on C11

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cells, but these percentages soon decreased after higher concentration of vorinostat was

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introduced (Fig. 1A). The A2 cell line is another well-established HIV-1 J-Lat clone [25].

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Therefore, as a check, we detected the effect of chidamide on the J-Lat A2 cell line to test

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whether the same result could be achieved. Consistently, as shown in Figure 1B, the

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percentage of GFP-positive cells was positively associated with the 0 to 4 µM concentration

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of chidamide after 2 days of incubation in the culture medium, and the maximal percentage

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increased to about 80.6% at 4 µM of chidamide over mock treatment. In addition, we found

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When the concentration of chidamide was increased to 4 µM, the percentage of

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that vorinostat was toxic to C11 cells and A2 cells when its concentration exceeded 3 µM

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based on the obvious reduction of the percentage of GFP-positive cells. Activity of chidamide as an HIV-1 reactivation agent was further confirmed in latent

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HIV-1 chronically infected ACH-2 cells [27] and U1 cells [28], according to the production

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of HIV-1 antigen p24 in the supernatant of cells cultured in the presence of LRAs. As shown

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in Figure 1C and D, when the concentration of chidamide increased from 0 µM to 4 µM, p24

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antigen production gradually rose. In ACH-2 cells, HIV-1 reactivation induced by chidamide

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was positively correlated with 0 to 4 µM concentration, and its effect was frequently higher

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than that of vorinostat (Fig. 1C). We also observed that the decreased activation of HIV-1

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with vorinostat at 4 µM, as measured by p24 antigen production, was likely caused by

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excessive cytotoxicity of vorinostat during continual 48 hours incubation in U1 cells (Fig. 1D).

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These results demonstrated that chidamide potently induced HIV-1 LTR reactivation,

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indicating its effect on HIV-1 production in a dose-dependent manner.

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3.2. Chidamide activates HIV-1 expression in latent HIV-1 infected cells in a time-dependent

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manner

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To assess the kinetics of HIV-1 LTR expression induced by chidamide, we performed a

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kinetics experiment in which J-Lat A2 cells and ACH-2 cells were grown for 1 to 4 days with

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or without chidamide for different concentrations. As shown in Figure 2A, after A2 cells were

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treated with 1 µM chidamide, the percentage of GFP-positive cells increased over time and

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rose to maximum (approximately 55%) on the fourth day. The kinetics of HIV-1 LTR

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expression induction by vorinostat showed an apparent rise for the first 2 days, but then

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tended toward constancy, or even reduction, by day 4 (Fig. 2A). We then tested the effect on

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ACH-2 cells (Fig. 2B). From the first 3 days, we found that the effect of activation, as

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measured by p24 antigen production, was almost the same between the treatment of

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chidamide and vorinostat (Fig. 2B). In particular, p24 antigen production of the chidamide

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group continuously increased after 3 days, while treatment with vorinostat resulted in only

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negligible difference in variation. On the fourth day, we noticed that the effect of chidamide

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on activation was significantly much higher than that of vorinostat in both cell lines (Fig. 2C

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and D). These results indicated a time-dependent effect of chidamide on HIV-1 expression,

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and as an HDAC inhibitor, chidamide could sustain reactivation over a period of time longer

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than that shown by vorinostat.

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3.3. Chidamide has minimal cytotoxicity in vitro

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To test the cytotoxicity of chidamide on different cells, we measured cell viability in A2,

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C11, HEK 293 and PBMC cells treated with different concentrations of chidamide. After

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treatment for 48h, cells were analyzed by CCK-8 assay. In the PBMC cells derived from

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healthy HIV-negative donors, we did not find a large reduction in cell viability when the

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concentration increased from 1 µM to 100 µM, while cell viability was clearly reduced at

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concentrations > 25 µM, following treatment with vorinostat (Fig. 3A). In HEK293 T cells,

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the CC50 was 37.3 µM (chidamide) and 12.5 µM (vorinostat) (Fig. 3B), respectively. This

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result indicated that chidamide has relatively low toxicity compared to vorinostat on normal

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human cells. The lower toxicity of chidamide compared with vorinostat was also observed in

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J-Lat cells, including A2 cells and C11 cells, following incubation with chidamide at the same

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concentrations as vorinostat based on the curve shown in Figure 3C and 3D, respectively.

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CC50 of chidamide in J-Lat A2 cells and C11 cells was 22.3 µM and 18.6 µM, respectively.

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CC50 of vorinostat in J-Lat A2 and C11 cells was 14 µM and 7.38 µM, respectively. Because

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chidamide is an anticancer drug, it could be somewhat toxic to A2 and C11 cells from acute T

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cell leukemia Jurkat cells. However, considering the lower toxicity in normal cells, such as

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PBMCs and 293 T cells, our results illustrated that chidamide was safe at its active

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concentration.

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3.4. Chidamide reactivates HIV-1 latent provirus through NF-κB signaling

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Binding sites for several inducible transcription factors, such as NF-κB, AP-1, and Sp1, can

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be found in the HIV-1 LTR region [5, 10, 38]. Among these, the host transcription factor

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NF-κB is critical for HIV-1 replication and has been most studied by LRAs [20, 39-44]. To

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determine whether chidamide-mediated activation of HIV-1 is related to the NF-κB pathway,

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we first examined protein expression of two components of the NF-κB pathway in J-Lat A2

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cells by western blot analysis using antibodies specific for two NF-κB families, including

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phospho-p65 (pp65) and IκBα. Our data showed a significant increase in the expression of

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pp65 following treatment with chidamide as early as 2.5h (Fig. 4A and B), indicating that

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NF-κB p65 was induced to be phosphorylated at Ser536 and transformed to pp65. The

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activation of NF-κB usually involves degradation of the IκBα subunit bound to the NF-κB

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dimer. Therefore, we next examined IκBα protein levels in J-Lat A2 cells stimulated with

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chidamide (Fig. 4A). We observed an increased rate of IκB degradation following 2 hours of

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treatment with chidamide (Fig. 4C). To dissect the involvement of the NF-κB pathway in viral

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reactivation mediated by chidamide, the effect of some agents inhibiting the NF-κB pathway,

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such as Aspirin and PDTC, was determined when used prior to chidamide treatment. J-Lat A2

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cells were pretreated with different concentrations of aspirin, which could inhibit

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TNF-α-induced activation of NF-κB [44, 45], and subsequently treated with chidamide or

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TNF-α. We observed that the pretreatment of aspirin not only significantly inhibited

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TNF-α-induced GFP expression in a dose-dependent manner, but also inhibited GFP

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expression induced by chidamide at the concentrations tested (Fig. 4D). PDTC, a NF-κB

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selective inhibitor [46], was also evaluated for HIV-1 p24 production in ACH-2 cells. The

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increasing addition of PDTC to ACH-2 cells contributed to the significant inhibition of

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chidamide-mediated reactivation from latency (Fig. 4E). Our findings collectively indicated

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that chidamide-induced reactivation of latent HIV-1 might be mediated through the NF-κB

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pathway.

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3.5. Combination of chidamide and anti-HIV-1 drugs inhibits viral infection To investigate whether chidamide could be combined with several anti-HIV-1 drugs to

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inhibit active HIV-1 infection, we used TZM-Bl cells that contain a stably-integrated HIV-1

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LTR linked to the luciferase reporter gene in their genome to examine the effect. ACH-2 cells

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were treated with chidamide or anti-HIV-1 drugs, including AZT (Zidovudine), NVP

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(Nevirapine), and IDV (Indinavir), for 4 days. Then we collected the supernatant of ACH-2

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cells and added it to TZM-Bl cells to detect HIV-1 infection through luciferase assay. As

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shown in Figure 5, in the absence of stimulation by chidamide, TZM-Bl cells did not appear

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to express HIV-1 LTR with the treatment of anti-HIV drugs. When we performed chidamide

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treatment alone, a 10-fold increase of HIV-1 expression could be evaluated by detecting the

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expression of luciferase reporter gene, which demonstrated that 1) the latently infected HIV-1

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was activated by chidamide, and 2) the reactivated HIV-1 could effectively infect and

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replicate in the TZM-Bl cells that express CD4, CCR5 and CXCR4. By combining chidamide

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with anti-HIV-1 drugs, the expression of HIV-1 LTR showed a rapid decrease, illustrating

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that replication of the chidamide-reactivated HIV-1 in TZM-Bl cells was significantly

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suppressed by the antivirus drugs tested. These results suggest that chidamide has a potential

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to be used for HIV-1 functional cure based on the “shock and kill” strategy.

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4. Discussion

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Since HIV-1 remains integrated in the DNA of memory CD4+ T cells, cART for HIV-1

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infection limits HIV-1 replication, but it still does not eliminate the virus [3]. The emergence

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of the ‘‘shock and kill’’ strategy seems to be a harbinger of some new phase of HIV-1

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therapeutics. This promising strategy means that the latent virus can be forced out of its

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sanctuary, resulting in the eradication of the HIV-1 reservoir with targeted immunotherapy or

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cART [5, 14, 47]. Therefore, identification of a highly effective LRA is critical for

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reactivation of most, if not all, of the latently infected provirus, in order to eliminate the latent

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HIV-1 reservoir [48]. It was reported that some reactivators have been applied in clinical trials,

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such as the HDAC inhibitors romidepsin [19], panobinostat [49], and vorinostat [16]. It is

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well known that histone deacetylases (HDACs) belong to a family of enzymes equipped to

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remove the acetyl group from histone lysine residues, inducing transcriptional repression

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through chromatin condensation [50]. Thus, it can be seen that HDAC blocking is an

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attractive means of inducing broad reactivation of latent HIV-1.

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Chidamide was a newly identified orally active benzamide class of HDAC inhibitor. In

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recent years, researchers have made great effort in studying the HIV latency reversing effect

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of the HDAC inhibitors belonging to the class of hydroxamic acids (e.g., vorinostat,

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panobinostat and belinostat) or the class of cyclic peptide (e.g., apicidin and romidepsin).

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However, few of the benzamide class of HDAC inhibitors have ever been reported to

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reactivate the latent HIV provirus. Therefore, it is necessary to determine the ability of

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chidamide to reverse HIV-1 latency. In this study, we explored chidamide’s potency in

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reactivating latent HIV-1 expression on different latently infected cell models, including J-Lat

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A2 cells, C11 cells, ACH-2 cells and U1 cells [28, 51]. Among these latent models, both C11

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and J-Lat clone A2 cells are latently infected Jurkat T cells encoding GFP gene under the

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control of HIV-1 LTR. The expression of GFP is used as a marker of latent HIV-1 expression,

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and it can be detected by fluorescence microscopy and flow cytometry.

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Jurkat T cells infected with pNL4-3-EGFP, which was reconstructed from pNL4-3 through the

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introduction of gene-encoded enhancement of green fluorescent protein and mutation in Vpr

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and Env [26]. Consequently, the same stimulation by chidamide or vorinostat could cause a

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slight difference between these two cell lines. Moreover, as a marker of reactivation efficacy,

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HIV-1 p24 antigen production levels were measured with chidamide treatment in ACH-2 and

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U1 cells. These cell lines exhibit low levels of basal transcription, despite mutations in Tat

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C11 cells were

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(U1 cell) and TAR (ACH-2 cell) [27, 51]. In spite of the different integration, or mutation, site

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in the cell lines from various origins, our results powerfully demonstrated that chidamide

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could broadly and effectively activate HIV-1 production in a manner that was both

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concentration- and time-dependent and do so in vitro at micromolar levels in these cells. It

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was worth noting that chidamide induces HIV-1 LTR with lower toxicity in various cell lines

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than vorinostat. Consistently, the benzamide class of HDAC inhibitors showed the least

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pronounced toxicity in primary resting CD4+ T cells among most of the HDAC inhibitors [24].

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Furthermore, in contrast to vorinostat, chidamide could retain its ability to reactivate HIV-1

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much longer. Therefore, when carrying out the “shock and kill” strategy in clinical trials, we

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recommend reducing the frequency of chidamide dosing in order to lessen its toxicity.

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After integration of HIV-1 into the host chromatin, proviral expression is largely controlled

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by host cellular factors. Host transcription factors, including nuclear factor-κB (NF-κB),

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nuclear factor of activated T cells (NFAT), AP1 and SP1, are sequestered in the cytoplasm in

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resting cells and thus do not promote HIV-1 transcription until an appropriate cellular

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activation signal is transmitted [38]. Among these transcription factors, NF-κB plays a central

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role in the activation pathway of the HIV-1 provirus [10]. Therefore, the identification of

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NF-κB activities has evoked a new direction in strategies to combat the effects of HIV-1

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latency [31]. Chidamide could induce increasing levels of phospho-p65 expression and lead to

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the degradation of IκBα which is an inhibited subunit in latently infected cells. It has been

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reported that Aspirin can inhibit NF-κB activation induced by TNF-α by preventing the

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phosphorylation and degradation of IκBα and nuclear translocation of NF-κB [45]. Therefore,

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we examined the effect through Aspirin-inhibition experiments, following chidamide

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treatment with TNF-α as a positive control, in J-Lat A2 cells. PDTC has been studied as a

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NF-κB inhibitor, as well as an antioxidant agent, and we also showed that pretreatment of

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ACH-2 cells with PDTC could remarkably prevent chidamide from inducing HIV-1

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reactivation through expression of p24. The main activity of NF-κB on chidamide effects was

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confirmed by using both inhibitors in viral reactivation experiments, which showed that

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chidamide-mediated HIV-1 reactivation was abrogated. An increasing number of LRAs, such

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as Oxaliplatin [30], M344 [44], Bryostatin [52], and PEP005 [40], have been demonstrated to

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be involved in the NF-κB signaling pathway. However, besides M344, it has not be

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demonstrated that other HDAC inhibitor, which contains a benzamide functional group, such

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as entinostat and pimelic diphenylamide 106 [24], is involved in this pathway. Therefore, we

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wanted to know if chidamide and other HDACs containing a benzamide functional group use

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the NF-κB signal pathway or other pathways to activate latent HIV-1.

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To achieve the “shock and kill” strategy for an HIV-1 cure, the role of anti-HIV-1 drugs on

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viral clearance cannot be ignored. Therefore, we combined chidamide with anti-HIV-1 drugs

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in order to determine if the active virus could be inhibited by antivirus drugs after activation

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of HIV-1 from latently infected cells by chidamide. Among a variety of anti-HIV drugs, we

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chose some drugs appearing to have optimal effect on eliminating HIV-1, such as nucleoside

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reverse transcriptase inhibitors (NRTI), AZT , nonnucleoside reverse transcriptase inhibitors

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(NNRTI), NVP, or the protease inhibitor IDV [53]. Our results showed that infection of

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TZM-Bl cells by HIV-1 was significantly lower after treatment of AZT, NVP or IDV

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cotreatment with chidamide, suggesting that treatments of such highly active antiretroviral

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therapy could protect uninfected cells from becoming reinfected when using a combination of

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HIV activators. Moreover, when combined with chidamide, we found that such anti-HIV-1

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drugs as AZT, NVP and IDV did not lose their anti-HIV-1 potency, suggesting that these

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anti-HIV-1 drugs can be used in combination therapy aimed at eliminating latent HIV-1

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reservoirs.

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To sum up, our results have demonstrated that chidamide plays an important role in histone

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modification via the NF-κB pathway to regulate HIV-1 LTR gene expression and to reactivate

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latent HIV-1, thus having good potential to be used in combination with anti-HIV drugs for

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HIV-1 functional cure.

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Conflict of Interests

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The authors declare no conflict of interests regarding the publication of this paper.

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Acknowledgments

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This work was supported by grants from the National Natural Science Foundation of China

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(81630090 to S.J.; 81373456, 81672019 and 81661128041 to L.L.), the Sanming Project of

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Medicine in Shenzhen, the National 863 Program of China (2015AA020930 to LL) and the

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Shanghai Rising-Star Program (16QA1400300).

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Figure legends

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Fig. 1. Reactivation of latent HIV-1 in different latently infected cells by chidamide. (A) J-Lat

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clone C11 cells were treated with chidamide and vorinostat for 48 h at the indicated

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concentrations. Results are expressed as a percentage of GFP-positive cells within the entire

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population. (B) J-Lat clone A2 cells treated with chidamide and vorinostat for 48 h at the

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indicated concentrations and analyzed as in (A). ACH-2 cells (C) or U1 (D) cells were

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stimulated with increasing concentrations of chidamide and vorinostat for 48 h. Viral

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replication was measured in the supernatant of the cells using p24 ELISA. Data show

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dose-dependent effects of chidamide on HIV-1 production in different latently infected cells

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and represent the means ± standard deviations of three independent experiments.

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Fig. 2. Time-dependent effects of chidamide on HIV-1 production. J-Lat A2 cells (A) or

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ACH-2 cells (B) were treated with DMSO or with 1µM chidamide or vorinostat for 0, 24, 48,

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72, 96 h. A time-dependent curve on HIV-1 transcription is represented by two latent cell

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models. (C) and (D) represented the comparison of the effect on activating latently infected

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cells between chidamide and vorinostat at the third day and the fourth day. Data show

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time-dependent effects of chidamide on activating latently infected cell lines and represent the

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means ± standard deviations of three independent experiments. (**P < 0.01 and***P < 0.001).

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Fig. 3. Viability of cells treated with chidamide or vorinostat. PBMCs from HIV-negative

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donors (A), HEK293 T cells (B), J-Lat A2 cells (C) and C11 cells (D) were treated with

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chidamide and vorinostat from 0 to 100 µM for 48 h, and cytotoxicity was measured by

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CCK-8 kit. The absorbance at 450 nm (OD450) of each well was determined as the readout of

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cell viability. Data represent the means ± standard deviations of three independent

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experiments.

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Fig. 4. Reactivation of HIV-1 latent provirus by chidamide through NF-κB pathway. (A) J-Lat

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A2 cells were treated with 2 µM of chidamide for up to 3.5 h hours. Western blot analysis was

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performed to detect the expression of p-p65 and IκBα. (B) Quantitation of phosphorylation of

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p65 in J-Lat A2 cells after 3.5 hours treatment with chidamide in panel (A). Relative band

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intensities from three independent experiments in J-Lat A2 cells, as determined using Image J

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(NIH), are shown in the bar graph. (C) Quantitation of IκBα in J-Lat A2 cells after 3.5 hours

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treatment with chidamide in panel (A). Relative band intensities from three independent

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experiments in J-Lat A2 cells, as determined using ImageJ (NIH), are shown in the bar graph.

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(D) J-Lat A2 cells were pretreated with various concentrations of Aspirin for 3 h and

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subsequently treated with chidamide (1, 2 and 5 µM), TNF-a (10 ng/mL) or DMSO as control

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for 48 h. The percentage of GFP-positive cells in drug-treated cells in the absence or presence

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of the inhibitor Aspirin was measured by flow cytometry. (E) ACH-2 cells were pretreated

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with increasing concentrations of PDTC for 2 h and subsequently treated with chidamide (2

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µM); p24 production from latently infected cells was measured by p24 ELISA. Data represent

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the means ± standard deviations of three independent experiments. (*P < 0.05, P < **0.01 and

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***P < 0.001).

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Fig. 5. Combination of chidamide with anti-HIV-1 drugs to suppress infection of reactivated

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HIV-1. (a) 1×104 ACH-2 cells were treated with chidamide, vorinostat or PBS in the presence

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or absence of the anti-HIV drugs AZT, NVP, or IDV for 4 days. The supernatants of ACH-2

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cells containing the residual reactivated HIV-1 were cultured with TZM-Bl cells for 48 hours

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before testing residual HIV-1 infectivity using the luciferase assay. Data represent the means ±

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standard deviations of three independent experiments (***P < 0.001).

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Fig. 1. C11 cells

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A2 cells A A

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ACCEPTED MANUSCRIPT Elsevier Editorial System(tm) for Microbes and Infection Manuscript Draft Manuscript Number: MICINF-D-17-00218R1

Article Type: Special Issue

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Title: Chidamide, a histone deacetylase inhibitor-based anticancer drug, effectively reactivates latent HIV-1 provirus

Keywords: Key Words: Histone deacetylase inhibitor; Chidamide; HIV-1 latent infection Corresponding Author: Professor Shibo Jiang,

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Corresponding Author's Institution: Fudan University First Author: Wenqian Yang

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Order of Authors: Wenqian Yang; Zhiwu Sun; Chen Hua; Qian Wang; Wei Xu; Qiwen Deng; Yanbin Pan; Lu Lu; Shibo Jiang

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Abstract: Although combination antiretroviral therapy (cART) is highly effective in suppressing human immunodeficiency virus type 1 (HIV-1) replication, it fails to eradicate the virus from HIV-1-infected individuals because HIV-1 integrates into the resting CD4+ T cells, establishing latently infected reservoirs. Histone deacetylation is a key element in regulating HIV-1 latent infection. Chidamide, a new anticancer drug, is a novel type of selective histone deacetylase inhibitor. Here we showed that chidamide effectively reactivated HIV-1 latent provirus in different latently infected cell lines in a dose- and time-dependent manner. Chidamide had relatively low cytotoxicity to peripheral blood mononuclear cells (PBMCs) and other latent cell lines. We have demonstrated that chidamide reactivates HIV-1 latent provirus through the NF-κB signaling pathway. The replication of the newly reactivated HIV-1 could then be effectively inhibited by the anti-HIV drugs Zidovudine, Nevirapine, and Indinavir. Therefore, chidamide might be used in combination with cART for functional HIV-1 cure.

Cover Letter

ACCEPTED MANUSCRIPT Dear Dr. Ojcius: Thank you very much for your further consideration of our manuscript (MICINF-D-17-00218) entitled “Chidamide, a histone deacetylase inhibitor-based anticancer drug, effectively reactivates latent HIV-1 provirus” for publication in the special issue of Microbes and Infection.

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We would like to express our sincere appreciation for your time and efforts in handling the above referenced manuscript and to the reviewers for their constructive and thoughtful comments. We have now revised our manuscript according to these suggestions and tried our best to address all the concerns. The added or modified sentences were highlighted in red for your convenience to review. The point-by-point responses to reviewers’ comments were submitted separately.

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We hope that we have satisfactorily addressed the reviewers’ concerns and that the revised manuscript will now be found acceptable for publication in Microbes and Infection. Sincerely yours,

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Dr. Shibo Jiang Professor and Director, Institute of Medical Microbiology Key Laboratory of Medical Molecular Virology of Ministries of Education and Health School of Basic Medical Sciences, Fudan University 130 Dong An Road, Building #13, Rm 611 Xuhui District, Shanghai 200032, China Phone: +86 21-54237673 Fax: +86 21-54237465 E-mail: [email protected]

*Manuscript Click here to view linked References

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Chidamide, a histone deacetylase inhibitor-based anticancer drug,

1

effectively reactivates latent HIV-1 provirus

2 3

Wenqian Yang a,#, Zhiwu Sun a,#, Chen Hua a, Qian Wang a, Wei Xu a, Qiwen Deng b,

5

Yanbin Pan c, Lu Lu a,*, Shibo Jiang a,b,d,*

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6 a

Key Lab of Medical Molecular Virology of Ministries of Education and Health, School of

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Basic Medical Sciences & Shanghai Public Health Clinical Center, Fudan University,

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Shanghai, China b

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Shenzhen Nanshan People's Hospital of Shenzhen University, Shenzhen 518052, China c

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Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065, USA

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These authors have equal contribution.

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Aris Pharmaceuticals Inc., Bristol, PA19007, USA

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*Address for correspondence: Shibo Jiang: [email protected]; Lu Lu:

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[email protected].

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Abstract Although combination antiretroviral therapy (cART) is highly effective in suppressing

25

human immunodeficiency virus type 1 (HIV-1) replication, it fails to eradicate the virus from

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HIV-1-infected individuals because HIV-1 integrates into the resting CD4+ T cells,

27

establishing latently infected reservoirs. Histone deacetylation is a key element in regulating

28

HIV-1 latent infection. Chidamide, a new anticancer drug, is a novel type of selective histone

29

deacetylase inhibitor. Here we showed that chidamide effectively reactivated HIV-1 latent

30

provirus in different latently infected cell lines in a dose- and time-dependent manner.

31

Chidamide had relatively low cytotoxicity to peripheral blood mononuclear cells (PBMCs)

32

and other latent cell lines. We have demonstrated that chidamide reactivates HIV-1 latent

33

provirus through the NF-κB signaling pathway. The replication of the newly reactivated

34

HIV-1 could then be effectively inhibited by the anti-HIV drugs Zidovudine, Nevirapine, and

35

Indinavir. Therefore, chidamide might be used in combination with cART for functional

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HIV-1 cure.

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Key Words: Histone deacetylase inhibitor; Chidamide; HIV-1 latent infection

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1. Introduction Combination antiretroviral therapy (cART) has succeeded in reducing human

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immunodeficiency virus type 1 (HIV-1) load to undetectable levels in most patients [1], but

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the therapy must be maintained throughout life. Once cART is withdrawn, viral loads could

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return to even pretreatment levels within weeks. One of the principal obstacles to a thorough

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cleaning of the virus is the integration of HIV-1 genome into that of the resting CD4+ T cells

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during acute infection, thereby establishing the latently infected reservoir that harbors

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transcriptionally silent proviruses refractory to current cART [2-4]. Therefore, the

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development of HIV-1 therapeutic strategies to eliminate this latent reservoir is urgently

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needed.

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Some host cell-mediated molecular mechanisms maintain the quiescence of HIV-1 gene

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expression, including epigenetic silencing of the proviral promoter, transcription complex

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formation, transcript initiation, and transcription complex processivity, all of which might act

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as therapeutic targets to disrupt HIV-1 latency [5-8]. Among these mechanisms, histone

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deacetylase (HDAC)-mediated chromosomal suppression of HIV-1 long terminal repeat (LTR)

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is a key regulator leading to the establishment of HIV-1 latency [7, 9]. It was reported the host

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transcription factors comprised of NF-κB family members are involved in HDAC1

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recruitment [10]. HDAC inhibitors have been shown to increase cell-associated HIV-1 RNA

57

production and/or plasma viremia in vivo [6, 11, 12], suggesting that they could play a role in

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reactivating latent HIV-1 provirus.

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A strategy known as ‘shock and kill’, or ‘kick and kill’, has been developed for HIV-1

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functional cure [13, 14]. To explain, the latent HIV-1 provirus was first reactivated with some

61

small-molecule HIV-1 latency-reversing agents (LRAs) [15] and then killed by the patient’s

62

immune system or suppressed by cART. Clinical trials have demonstrated that administration

63

of HDAC inhibitors, such as vorinostat [16, 17] or panobinostat [18], to the patients in whom

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viremia was fully suppressed by cART, resulted in a significant increase in HIV-1 RNA

65

production in resting CD4+ cells. Another clinical trial had shown that the HDAC inhibitor

66

romidepsin was effective in disrupting HIV-1 latency in vivo [19]. However, some LRAs may

67

be toxic and mutagenic or harmful to host brain during prolonged treatment [17, 20, 21].

68

Therefore, identification of new HDAC inhibitors with improved efficacy and safety is fully

69

essential.

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Chidamide is a novel benzamide chemical class of HDAC inhibitor that selectively inhibits

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the histone deacetylation activities of HDAC1, 2, 3, as well as 10 [22]. It has been approved

72

by the Chinese Food and Drug Administration for the treatment of patients with recurrent or

73

refractory peripheral T-cell lymphoma (PTCL) [23]. Recently, a study screening active LRAs

74

in a primary cell model indicated that the HDAC inhibitors containing a benzamide functional

75

group and a pyridyl cap are the most effective, as exemplified by chidamide [24]. However,

76

unlike vorinostat, which belongs to the class of hydroxamic acids [6], little is known about

77

chidamide’s potential use and mechanism to reactivate latent HIV-1 provirus in latently

78

infected cells. Therefore, in this study, we dissected the effect of chidamide on reactivation of

79

HIV-1 provirus in latently infected cell models, as well as its possible mechanism of action.

80

We found chidamide to be much safer with more long-lasting effect than vorinostat. Our

81

results suggest that chidamide has the potential to be further developed as an effective and

82

safe LRA for HIV-1 functional cure.

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2. Materials and Methods

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2.1. Cell Culture and Chemical Treatment

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J-Lat A2 cells [25] (obtained from NIH AIDS Reagent Program), J-Lat C11 cells [26],

87

PBMCs (Allcells), ACH-2 cells [27] and U1 cells [28] were cultured in RPMI 1640 medium

88

with 10% fetal bovine serum (FBS) and 1% Pen/strep in a 37 °C incubator containing 5%

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CO2. HEK 293 cells and TZM-Bl cells [29] were grown in Dulbecco’s modified Eagle’s

90

medium (DMEM) with 10% fetal bovine serum and 1% Pen/strep at 37 °C under 5% CO2.

91

Chidamide

92

Pyrrolidinedithiocarbamic acid (PDTC) (Sigma), vorinostat (Sigma) and chidamide were

93

dissolved in anhydrous dimethyl sulfoxide (DMSO) and were stored at -20 °C. Recombinant

94

Human TNF-alpha Protein (R&D Systems) was dissolved in sterile PBS.

95

2.2. Flow Cytometry

provided

by

Aris

Pharmaceuticals

Inc.

Aspirin

(Sigma),

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C11 and A2 cells were incubated with the indicated concentrations of chidamide or

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vorinostat at different time points, or pretreated with various concentrations of Aspirin for 3 h

98

and subsequently treated with chidamide, TNF-α or control DMSO for indicated hours. Cells

99

were washed and resuspended in PBS 3 times. GFP expression was measured by a BD Accuri

100

C6 Flow Cytometer (BD Biosciences) as previously described [30, 31]. All experiments were

101

performed independently at least three times in triplicate per experimental point.

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2.3. HIV-1 antigen p24 assay

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ACH-2 and U1 cells (4×104 cells) were seeded into a 96-well plate and then incubated with

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various concentrations of chidamide and vorinostat at different time points. Viral release in

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the supernatant was quantified by p24 ELISA assay as previously described [32]. All

106

experiments were performed independently at least three times in triplicate per experimental

107

point.

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2.4. Cytotoxicity Assay

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Cell viability was measured following the instructions in the protocol provided in the cell

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counting kit-8 (CCK-8; Dojindo Molecular Technologies, Gaithersburg, MD, USA) as

111

described [33]. J-Lat cells, HEK 293 T cells and PBMCs were seeded into a 96-well plate,

112

approximately 4×104 cells per well; 20 μL of CCK-8 solution were added to each well of the

113

plate, and cells were treated with or without chidamide for 48 h. After 2 to 3 h of incubation

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at 37 °C, the absorbance at 450 nm was taken by using a microplate reader (Infinite F200

115

PRO, Tecan, Switzerland). In this assay, the 50% cytotoxic concentration (CC50) was

116

calculated by use of the CalcuSyn software program (Biosoft, Ferguson, MO) [34] as we

117

previously described [35].

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2.5. Western Blot

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Western blot analysis was performed as described [36]. Cells were harvested, washed once

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with ice-cold PBS, and lysed in ice-cold RIPA buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1%

121

NP-40, and 2 mM EDTA)] containing PMSF and phosphatase inhibitor cocktails

122

(Thermofisher Scientific, Omaha, NE) for 15 minutes. Protein samples were separated by

123

SDS-PAGE and transferred onto nitrocellulose membrane (Pall Corporation, Port Washington,

124

NY). After blocking with 5% non-fat dry milk in PBS containing 0.1% Tween 20, proteins of

125

interest were probed with the corresponding primary antibodies [pp65 (Ser536) and IκBα

126

antibodies obtained from Cell Signaling (Danvers, MA)], followed by appropriate

127

infrared-conjugated secondary antibodies, Alexa Fluor 800 or 680 (Carlsbad, CA). The

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immunoblotting was carried out using the LiCOR (Lincoln, NE) Odyssey scanner.

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2.6. Luciferase Assay

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1×104 ACH-2 cells per well were treated with chidamide or vorinostat and incubated with

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the anti-HIV-1 drugs for 4 days at 37 °C, followed by collection of 100 μl culture

132

supernatants from each well on the fourth day. Then all supernatants were added into the

133

96-well polystyrene plate coated with TZM-Bl cells. After 48 h, TZM-Bl cells were lysed in

134

200μl of luciferase reporter lysis buffer. Luciferase activity was measured using luciferase

135

assay regents (Promega, Madison, WI, USA) and a luminescence counter (Infinite M200 Pro)

136

according to the manufacturer’s instructions as previously described [37].

137

2.7. Statistical Analysis

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Means and standard errors (SE) were calculated for all data points from at least 3

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independent experiments in triplicate. Statistical significance was determined using the

140

Student’s unpaired two-tailed t-test. All statistical analyses were carried out using GraphPad

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Prism 6.0 (GraphPad Software). P < 0.05 was considered statistically significant.

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3. Results

144

3.1. Activation of HIV-1 expression in latently infected cells by chidamide in a

145

dose-dependent manner

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To investigate if chidamide has the potential to induce latent HIV-1 expression, we

147

performed an experiment on the J-Lat C11 cell line, which is a latently infected Jurkat T cell

148

line with a single provirus integrated into intron of RNPS1 and an EGFP gene under control

149

of the HIV-1 LTR [26]. We treated C11 cells with increasing concentrations of chidamide,

150

while using vorinostat as a positive control, for 48 h and measured the percentage of

151

GFP-positive cells by flow cytometry. We found a 4- to 25-fold increase in the percentage of

152

GFP-positive cells from the C11 cells treated with chidamide subjected to background levels

153

(Fig. 1A). When the concentration of chidamide was increased to 4 μM, the percentage of

154

GFP-expressing cells reached approximately 82.6%. Similarly, the percentage of

155

GFP-positive cells rose from 3.2% to 87.7% after treatment of 0 to 2 μM vorinostat on C11

156

cells, but these percentages soon decreased after higher concentration of vorinostat was

157

introduced (Fig. 1A). The A2 cell line is another well-established HIV-1 J-Lat clone [25].

158

Therefore, as a check, we detected the effect of chidamide on the J-Lat A2 cell line to test

159

whether the same result could be achieved. Consistently, as shown in Figure 1B, the

160

percentage of GFP-positive cells was positively associated with the 0 to 4 μM concentration

161

of chidamide after 2 days of incubation in the culture medium, and the maximal percentage

162

increased to about 80.6% at 4 μM of chidamide over mock treatment. In addition, we found

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that vorinostat was toxic to C11 cells and A2 cells when its concentration exceeded 3 μM

164

based on the obvious reduction of the percentage of GFP-positive cells. Activity of chidamide as an HIV-1 reactivation agent was further confirmed in latent

166

HIV-1 chronically infected ACH-2 cells [27] and U1 cells [28], according to the production

167

of HIV-1 antigen p24 in the supernatant of cells cultured in the presence of LRAs. As shown

168

in Figure 1C and D, when the concentration of chidamide increased from 0 μM to 4 μM, p24

169

antigen production gradually rose. In ACH-2 cells, HIV-1 reactivation induced by chidamide

170

was positively correlated with 0 to 4 μM concentration, and its effect was frequently higher

171

than that of vorinostat (Fig. 1C). We also observed that the decreased activation of HIV-1

172

with vorinostat at 4 μM, as measured by p24 antigen production, was likely caused by

173

excessive cytotoxicity of vorinostat during continual 48 hours incubation in U1 cells (Fig. 1D).

174

These results demonstrated that chidamide potently induced HIV-1 LTR reactivation,

175

indicating its effect on HIV-1 production in a dose-dependent manner.

176

3.2. Chidamide activates HIV-1 expression in latent HIV-1 infected cells in a time-dependent

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manner

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To assess the kinetics of HIV-1 LTR expression induced by chidamide, we performed a

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kinetics experiment in which J-Lat A2 cells and ACH-2 cells were grown for 1 to 4 days with

180

or without chidamide for different concentrations. As shown in Figure 2A, after A2 cells were

181

treated with 1 μM chidamide, the percentage of GFP-positive cells increased over time and

182

rose to maximum (approximately 55%) on the fourth day. The kinetics of HIV-1 LTR

183

expression induction by vorinostat showed an apparent rise for the first 2 days, but then

184

tended toward constancy, or even reduction, by day 4 (Fig. 2A). We then tested the effect on

185

ACH-2 cells (Fig. 2B). From the first 3 days, we found that the effect of activation, as

186

measured by p24 antigen production, was almost the same between the treatment of

187

chidamide and vorinostat (Fig. 2B). In particular, p24 antigen production of the chidamide

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group continuously increased after 3 days, while treatment with vorinostat resulted in only

189

negligible difference in variation. On the fourth day, we noticed that the effect of chidamide

190

on activation was significantly much higher than that of vorinostat in both cell lines (Fig. 2C

191

and D). These results indicated a time-dependent effect of chidamide on HIV-1 expression,

192

and as an HDAC inhibitor, chidamide could sustain reactivation over a period of time longer

193

than that shown by vorinostat.

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3.3. Chidamide has minimal cytotoxicity in vitro

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To test the cytotoxicity of chidamide on different cells, we measured cell viability in A2,

196

C11, HEK 293 and PBMC cells treated with different concentrations of chidamide. After

197

treatment for 48h, cells were analyzed by CCK-8 assay. In the PBMC cells derived from

198

healthy HIV-negative donors, we did not find a large reduction in cell viability when the

199

concentration increased from 1 μM to 100 μM, while cell viability was clearly reduced at

200

concentrations > 25 μM, following treatment with vorinostat (Fig. 3A). In HEK293 T cells,

201

the CC50 was 37.3 μM (chidamide) and 12.5 μM (vorinostat) (Fig. 3B), respectively. This

202

result indicated that chidamide has relatively low toxicity compared to vorinostat on normal

203

human cells. The lower toxicity of chidamide compared with vorinostat was also observed in

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J-Lat cells, including A2 cells and C11 cells, following incubation with chidamide at the same

205

concentrations as vorinostat based on the curve shown in Figure 3C and 3D, respectively.

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CC50 of chidamide in J-Lat A2 cells and C11 cells was 22.3 μM and 18.6 μM, respectively.

207

CC50 of vorinostat in J-Lat A2 and C11 cells was 14 μM and 7.38 μM, respectively. Because

208

chidamide is an anticancer drug, it could be somewhat toxic to A2 and C11 cells from acute T

209

cell leukemia Jurkat cells. However, considering the lower toxicity in normal cells, such as

210

PBMCs and 293 T cells, our results illustrated that chidamide was safe at its active

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concentration.

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3.4. Chidamide reactivates HIV-1 latent provirus through NF-κB signaling

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Binding sites for several inducible transcription factors, such as NF-κB, AP-1, and Sp1, can

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be found in the HIV-1 LTR region [5, 10, 38]. Among these, the host transcription factor

215

NF-κB is critical for HIV-1 replication and has been most studied by LRAs [20, 39-44]. To

216

determine whether chidamide-mediated activation of HIV-1 is related to the NF-κB pathway,

217

we first examined protein expression of two components of the NF-κB pathway in J-Lat A2

218

cells by western blot analysis using antibodies specific for two NF-κB families, including

219

phospho-p65 (pp65) and IκBα. Our data showed a significant increase in the expression of

220

pp65 following treatment with chidamide as early as 2.5h (Fig. 4A and B), indicating that

221

NF-κB p65 was induced to be phosphorylated at Ser536 and transformed to pp65. The

222

activation of NF-κB usually involves degradation of the IκBα subunit bound to the NF-κB

223

dimer. Therefore, we next examined IκBα protein levels in J-Lat A2 cells stimulated with

224

chidamide (Fig. 4A). We observed an increased rate of IκB degradation following 2 hours of

225

treatment with chidamide (Fig. 4C). To dissect the involvement of the NF-κB pathway in viral

226

reactivation mediated by chidamide, the effect of some agents inhibiting the NF-κB pathway,

227

such as Aspirin and PDTC, was determined when used prior to chidamide treatment. J-Lat A2

228

cells were pretreated with different concentrations of aspirin, which could inhibit

229

TNF-α-induced activation of NF-κB [44, 45], and subsequently treated with chidamide or

230

TNF-α. We observed that the pretreatment of aspirin not only significantly inhibited

231

TNF-α-induced GFP expression in a dose-dependent manner, but also inhibited GFP

232

expression induced by chidamide at the concentrations tested (Fig. 4D). PDTC, a NF-κB

233

selective inhibitor [46], was also evaluated for HIV-1 p24 production in ACH-2 cells. The

234

increasing addition of PDTC to ACH-2 cells contributed to the significant inhibition of

235

chidamide-mediated reactivation from latency (Fig. 4E). Our findings collectively indicated

236

that chidamide-induced reactivation of latent HIV-1 might be mediated through the NF-κB

237

pathway.

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3.5. Combination of chidamide and anti-HIV-1 drugs inhibits viral infection To investigate whether chidamide could be combined with several anti-HIV-1 drugs to

240

inhibit active HIV-1 infection, we used TZM-Bl cells that contain a stably-integrated HIV-1

241

LTR linked to the luciferase reporter gene in their genome to examine the effect. ACH-2 cells

242

were treated with chidamide or anti-HIV-1 drugs, including AZT (Zidovudine), NVP

243

(Nevirapine), and IDV (Indinavir), for 4 days. Then we collected the supernatant of ACH-2

244

cells and added it to TZM-Bl cells to detect HIV-1 infection through luciferase assay. As

245

shown in Figure 5, in the absence of stimulation by chidamide, TZM-Bl cells did not appear

246

to express HIV-1 LTR with the treatment of anti-HIV drugs. When we performed chidamide

247

treatment alone, a 10-fold increase of HIV-1 expression could be evaluated by detecting the

248

expression of luciferase reporter gene, which demonstrated that 1) the latently infected HIV-1

249

was activated by chidamide, and 2) the reactivated HIV-1 could effectively infect and

250

replicate in the TZM-Bl cells that express CD4, CCR5 and CXCR4. By combining chidamide

251

with anti-HIV-1 drugs, the expression of HIV-1 LTR showed a rapid decrease, illustrating

252

that replication of the chidamide-reactivated HIV-1 in TZM-Bl cells was significantly

253

suppressed by the antivirus drugs tested. These results suggest that chidamide has a potential

254

to be used for HIV-1 functional cure based on the “shock and kill” strategy.

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4. Discussion

257

Since HIV-1 remains integrated in the DNA of memory CD4+ T cells, cART for HIV-1

258

infection limits HIV-1 replication, but it still does not eliminate the virus [3]. The emergence

259

of the ‘‘shock and kill’’ strategy seems to be a harbinger of some new phase of HIV-1

260

therapeutics. This promising strategy means that the latent virus can be forced out of its

261

sanctuary, resulting in the eradication of the HIV-1 reservoir with targeted immunotherapy or

262

cART [5, 14, 47]. Therefore, identification of a highly effective LRA is critical for

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reactivation of most, if not all, of the latently infected provirus, in order to eliminate the latent

264

HIV-1 reservoir [48]. It was reported that some reactivators have been applied in clinical trials,

265

such as the HDAC inhibitors romidepsin [19], panobinostat [49], and vorinostat [16]. It is

266

well known that histone deacetylases (HDACs) belong to a family of enzymes equipped to

267

remove the acetyl group from histone lysine residues, inducing transcriptional repression

268

through chromatin condensation [50]. Thus, it can be seen that HDAC blocking is an

269

attractive means of inducing broad reactivation of latent HIV-1.

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Chidamide was a newly identified orally active benzamide class of HDAC inhibitor. In

271

recent years, researchers have made great effort in studying the HIV latency reversing effect

272

of the HDAC inhibitors belonging to the class of hydroxamic acids (e.g., vorinostat,

273

panobinostat and belinostat) or the class of cyclic peptide (e.g., apicidin and romidepsin).

274

However, few of the benzamide class of HDAC inhibitors have ever been reported to

275

reactivate the latent HIV provirus. Therefore, it is necessary to determine the ability of

276

chidamide to reverse HIV-1 latency. In this study, we explored chidamide’s potency in

277

reactivating latent HIV-1 expression on different latently infected cell models, including J-Lat

278

A2 cells, C11 cells, ACH-2 cells and U1 cells [28, 51]. Among these latent models, both C11

279

and J-Lat clone A2 cells are latently infected Jurkat T cells encoding GFP gene under the

280

control of HIV-1 LTR. The expression of GFP is used as a marker of latent HIV-1 expression,

281

and it can be detected by fluorescence microscopy and flow cytometry.

282

Jurkat T cells infected with pNL4-3-EGFP, which was reconstructed from pNL4-3 through the

283

introduction of gene-encoded enhancement of green fluorescent protein and mutation in Vpr

284

and Env [26]. Consequently, the same stimulation by chidamide or vorinostat could cause a

285

slight difference between these two cell lines. Moreover, as a marker of reactivation efficacy,

286

HIV-1 p24 antigen production levels were measured with chidamide treatment in ACH-2 and

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U1 cells. These cell lines exhibit low levels of basal transcription, despite mutations in Tat

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C11 cells were

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(U1 cell) and TAR (ACH-2 cell) [27, 51]. In spite of the different integration, or mutation, site

289

in the cell lines from various origins, our results powerfully demonstrated that chidamide

290

could broadly and effectively activate HIV-1 production in a manner that was both

291

concentration- and time-dependent and do so in vitro at micromolar levels in these cells. It

292

was worth noting that chidamide induces HIV-1 LTR with lower toxicity in various cell lines

293

than vorinostat. Consistently, the benzamide class of HDAC inhibitors showed the least

294

pronounced toxicity in primary resting CD4+ T cells among most of the HDAC inhibitors [24].

295

Furthermore, in contrast to vorinostat, chidamide could retain its ability to reactivate HIV-1

296

much longer. Therefore, when carrying out the “shock and kill” strategy in clinical trials, we

297

recommend reducing the frequency of chidamide dosing in order to lessen its toxicity.

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After integration of HIV-1 into the host chromatin, proviral expression is largely controlled

299

by host cellular factors. Host transcription factors, including nuclear factor-κB (NF-κB),

300

nuclear factor of activated T cells (NFAT), AP1 and SP1, are sequestered in the cytoplasm in

301

resting cells and thus do not promote HIV-1 transcription until an appropriate cellular

302

activation signal is transmitted [38]. Among these transcription factors, NF-κB plays a central

303

role in the activation pathway of the HIV-1 provirus [10]. Therefore, the identification of

304

NF-κB activities has evoked a new direction in strategies to combat the effects of HIV-1

305

latency [31]. Chidamide could induce increasing levels of phospho-p65 expression and lead to

306

the degradation of IκBα which is an inhibited subunit in latently infected cells. It has been

307

reported that Aspirin can inhibit NF-κB activation induced by TNF-α by preventing the

308

phosphorylation and degradation of IκBα and nuclear translocation of NF-κB [45]. Therefore,

309

we examined the effect through Aspirin-inhibition experiments, following chidamide

310

treatment with TNF-α as a positive control, in J-Lat A2 cells. PDTC has been studied as a

311

NF-κB inhibitor, as well as an antioxidant agent, and we also showed that pretreatment of

312

ACH-2 cells with PDTC could remarkably prevent chidamide from inducing HIV-1

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reactivation through expression of p24. The main activity of NF-κB on chidamide effects was

314

confirmed by using both inhibitors in viral reactivation experiments, which showed that

315

chidamide-mediated HIV-1 reactivation was abrogated. An increasing number of LRAs, such

316

as Oxaliplatin [30], M344 [44], Bryostatin [52], and PEP005 [40], have been demonstrated to

317

be involved in the NF-κB signaling pathway. However, besides M344, it has not be

318

demonstrated that other HDAC inhibitor, which contains a benzamide functional group, such

319

as entinostat and pimelic diphenylamide 106 [24], is involved in this pathway. Therefore, we

320

wanted to know if chidamide and other HDACs containing a benzamide functional group use

321

the NF-κB signal pathway or other pathways to activate latent HIV-1.

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To achieve the “shock and kill” strategy for an HIV-1 cure, the role of anti-HIV-1 drugs on

323

viral clearance cannot be ignored. Therefore, we combined chidamide with anti-HIV-1 drugs

324

in order to determine if the active virus could be inhibited by antivirus drugs after activation

325

of HIV-1 from latently infected cells by chidamide. Among a variety of anti-HIV drugs, we

326

chose some drugs appearing to have optimal effect on eliminating HIV-1, such as nucleoside

327

reverse transcriptase inhibitors (NRTI), AZT , nonnucleoside reverse transcriptase inhibitors

328

(NNRTI), NVP, or the protease inhibitor IDV [53]. Our results showed that infection of

329

TZM-Bl cells by HIV-1 was significantly lower after treatment of AZT, NVP or IDV

330

cotreatment with chidamide, suggesting that treatments of such highly active antiretroviral

331

therapy could protect uninfected cells from becoming reinfected when using a combination of

332

HIV activators. Moreover, when combined with chidamide, we found that such anti-HIV-1

333

drugs as AZT, NVP and IDV did not lose their anti-HIV-1 potency, suggesting that these

334

anti-HIV-1 drugs can be used in combination therapy aimed at eliminating latent HIV-1

335

reservoirs.

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To sum up, our results have demonstrated that chidamide plays an important role in histone

337

modification via the NF-κB pathway to regulate HIV-1 LTR gene expression and to reactivate

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latent HIV-1, thus having good potential to be used in combination with anti-HIV drugs for

339

HIV-1 functional cure.

340

Conflict of Interests

342

The authors declare no conflict of interests regarding the publication of this paper.

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Acknowledgments

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This work was supported by grants from the National Natural Science Foundation of China

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(81630090 to S.J.; 81373456, 81672019 and 81661128041 to L.L.), the Sanming Project of

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Medicine in Shenzhen, the National 863 Program of China (2015AA020930 to LL) and the

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Shanghai Rising-Star Program (16QA1400300).

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Figure legends

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Fig. 1. Reactivation of latent HIV-1 in different latently infected cells by chidamide. (A) J-Lat

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clone C11 cells were treated with chidamide and vorinostat for 48 h at the indicated

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concentrations. Results are expressed as a percentage of GFP-positive cells within the entire

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population. (B) J-Lat clone A2 cells treated with chidamide and vorinostat for 48 h at the

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indicated concentrations and analyzed as in (A). ACH-2 cells (C) or U1 (D) cells were

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stimulated with increasing concentrations of chidamide and vorinostat for 48 h. Viral

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replication was measured in the supernatant of the cells using p24 ELISA. Data show

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dose-dependent effects of chidamide on HIV-1 production in different latently infected cells

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and represent the means ± standard deviations of three independent experiments.

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Fig. 2. Time-dependent effects of chidamide on HIV-1 production. J-Lat A2 cells (A) or

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ACH-2 cells (B) were treated with DMSO or with 1μM chidamide or vorinostat for 0, 24, 48,

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72, 96 h. A time-dependent curve on HIV-1 transcription is represented by two latent cell

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models. (C) and (D) represented the comparison of the effect on activating latently infected

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cells between chidamide and vorinostat at the third day and the fourth day. Data show

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time-dependent effects of chidamide on activating latently infected cell lines and represent the

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means ± standard deviations of three independent experiments. (**P < 0.01 and***P < 0.001).

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Fig. 3. Viability of cells treated with chidamide or vorinostat. PBMCs from HIV-negative

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donors (A), HEK293 T cells (B), J-Lat A2 cells (C) and C11 cells (D) were treated with

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chidamide and vorinostat from 0 to 100 μM for 48 h, and cytotoxicity was measured by

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CCK-8 kit. The absorbance at 450 nm (OD450) of each well was determined as the readout of

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cell viability. Data represent the means ± standard deviations of three independent

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experiments.

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Fig. 4. Reactivation of HIV-1 latent provirus by chidamide through NF-κB pathway. (A) J-Lat

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A2 cells were treated with 2 μM of chidamide for up to 3.5 h hours. Western blot analysis was

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performed to detect the expression of p-p65 and IκBα. (B) Quantitation of phosphorylation of

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p65 in J-Lat A2 cells after 3.5 hours treatment with chidamide in panel (A). Relative band

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intensities from three independent experiments in J-Lat A2 cells, as determined using Image J

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(NIH), are shown in the bar graph. (C) Quantitation of IκBα in J-Lat A2 cells after 3.5 hours

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treatment with chidamide in panel (A). Relative band intensities from three independent

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experiments in J-Lat A2 cells, as determined using ImageJ (NIH), are shown in the bar graph.

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(D) J-Lat A2 cells were pretreated with various concentrations of Aspirin for 3 h and

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subsequently treated with chidamide (1, 2 and 5 μM), TNF-a (10 ng/mL) or DMSO as control

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for 48 h. The percentage of GFP-positive cells in drug-treated cells in the absence or presence

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of the inhibitor Aspirin was measured by flow cytometry. (E) ACH-2 cells were pretreated

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with increasing concentrations of PDTC for 2 h and subsequently treated with chidamide (2

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μM); p24 production from latently infected cells was measured by p24 ELISA. Data represent

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the means ± standard deviations of three independent experiments. (*P < 0.05, P < **0.01 and

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***P < 0.001).

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Fig. 5. Combination of chidamide with anti-HIV-1 drugs to suppress infection of reactivated

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HIV-1. (a) 1×104 ACH-2 cells were treated with chidamide, vorinostat or PBS in the presence

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or absence of the anti-HIV drugs AZT, NVP, or IDV for 4 days. The supernatants of ACH-2

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cells containing the residual reactivated HIV-1 were cultured with TZM-Bl cells for 48 hours

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before testing residual HIV-1 infectivity using the luciferase assay. Data represent the means ±

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standard deviations of three independent experiments (***P < 0.001).

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Figure 1

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Fig. 1. C11 cells

A2 cells

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U1 cells D

Figure 2

Fig. 2.

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A2 cells A A

ACH-2 cells

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Figure 3

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Fig. 3. PBMCs

HEK 293 T cells

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C11 cells

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Fig. 4.

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B

Chidamide 0h

1h

2h

2.5 h 3 h

3.5 h

p-p65 (S536) β-actin

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IκBα

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β-actin

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Figure 5

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Fig. 5.

*Detailed Response to Reviewers

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Responses to the Reviewers' comments

Reviewer #1: Minor comments

into that of resting…"

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1. Line 44: "integration of HIV-1 into into", change to "integration of HIV-1 genome

2. Line 51-52, "histone deacetylation" should be "histone deacetylase", because that

3. Line 111 "twenty µL" change to "20 µl".

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is what HDAC refers to in the rest part of the manuscript.

Response: We thank the reviewer for the constructive comments, and we have

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corrected these errors accordingly in the revised manuscript.

4. In the cytotoxicity assay (Figure 3), how were the CC50 values determined? If a nonlinear regression model is used for curving fitting, please describe the details in the method.

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Response: We used the Calcusyn software program (Biosoft, Ferguson, MO) to calculate CC50 values and we have added the description of the method and

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references in the Materials and Methods section accordingly.

5. Line 190, "The lower toxicity compared …" change to "The lower toxicity of

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chidamide compared …”

6. Line 236, "latently infected HIV-1" change to "reactivated HIV-1" 7. Line 240-243, "In the presence of anti-HIV-1 drugs …"should be rewritten to, e.g. "These results indicated that anti-HIV-1 drugs could prevent the spread of newly synthesized viruses reactivated by chidamide." Response: Thanks, we have changed these sentences in the revised manuscript according to the reviewer's suggestion.

Reviewer #2:

ACCEPTED MANUSCRIPT 1. In line 247 where the "shock and kill" strategy is described, the following reference (Gallo RC. Shock and kill with caution. Science 2016;354:177-8) should be cited and the related points should be discussed. Response: We appreciate the reviewer's insightful suggestion to improve our

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manuscript. We have cited this reference and added relevant discussion in the revised manuscript.

2. In the Materials and Methods, the method for Western blot should be provided.

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Response: We have added the following paragraph in the revised manuscript:

“Western blot analysis was performed as described [1]. Cells were harvested, washed

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once with ice-cold PBS, and lysed in ice-cold RIPA buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40, and 2 mM EDTA)] containing PMSF and phosphatase inhibitor cocktails (Thermofisher Scientific, Omaha, NE) for 15 minutes. Protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane (Pall Corporation, Port Washington, NY). After blocking with 5% non-fat dry milk in

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PBS containing 0.1% Tween 20, proteins of interest were probed with the corresponding primary antibodies [pp65 (Ser536) and IκBα antibodies obtained from Cell Signaling (Danvers, MA)], followed by appropriate infrared-conjugated

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secondary antibodies, Alexa Fluor 800 or 680 (Carlsbad, CA). The immunoblotting

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was carried out using the LiCOR (Lincoln, NE) Odyssey scanner.”

3. In line 208, the space between "Ser" and "536" should be deleted. Response: We have corrected this error.

4. In the Figure 5, the scale on the Y axis should be changed to scientific notation. 5. Figure 1A and 1B, Figure 2A and 2C, and Figure 4D, the "GFP-Positive cells (%)" in the Y-axis should be "GFP-positive cells (%)". Response: We have made correction accordingly.

Reviewer #3:

ACCEPTED MANUSCRIPT Minor comments 1. The authors could discuss in more detail about the advantages of chidamide as a LRA, compared with other HDACi-based anti-cancer drugs. Response: We appreciate the reviewer's insightful comment and have added the

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chidamide’s advantages in the Discussion section in the revised manuscript.

2. Some repeated sentences about the result in the Discussion section should be removed.

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Response: We have deleted the redundant parts in the revised manuscript.

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3. In the legend of Fig. 5, "ACH-2 cells were treated with chidamide, vorinostat or PBS in the presence or absence of the anti-HIV drugs AZT, NVP, or IDV for 96 h", while in the Results (lines 228-230), " ACH-2 cells were treated with chidamide or anti-HIV-1 drugs, including AZT (Zidovudine), NVP (Nevirapine), and IDV (Indinavir), for 4 days". To keep consistence, 96 h should be changed to 4 days.

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Response: Thanks, we have corrected it accordingly.

4. The manuscripts need some proofreads in English.

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Response: We have thoroughly checked our revised manuscript, paying attention to English grammar, spelling, punctuation, subject/verb agreement, and proper use of

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singular and plural as well as meaning.

References: [1]

Gan J, Wang C, Jin Y, Guo Y, Xu F, Zhu Q, et al. Proteomic profiling identifies the SIM-associated complex of KSHV-encoded LANA. Proteomics 2015;15:2023-37.