CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

Conclusions: While general expected trends in SAHA-induced histone modifications were consistent with detected HIV activation, this preliminary data suggests that induced histone modifications alone are not sufficient to reactivate HIV from latency. Future work will examine whether SAHA affects other essential components for HIV reactivation, such as key transcription factors (SP-1) or P-TEFb activity (via CDK9 phosphorylation), that contribute to HIV reactivation in latently infected cells. 408 Donor-to-Donor Variation in the Host Gene Expression Response to SAHA Bastiaan Moesker 1 ; Brian Reardon 2 ; Nadejda Beliakova-Bethell 2 ; Akul Singhania 1 ; Michael S. Breen 1 ; Christopher H.Woelk 1 1 University of Southampton, Southampton, United Kingdom; 2 University of California San Diego, La Jolla, CA, US Background: The “shock and kill” approach to eliminating the latent HIV reservoir depends on efficient reactivation of HIV provirus. Multiple studies have observed variability in the levels of HIV reactivation after treatment of latently infected CD4 + T cells with the histone deacetylase inhibitor SAHA. In this study, we hypothesized that the heterogeneity in HIV reactivation is partly related to donor-to-donor differences that are reflected in host gene expression. Therefore, we sought to identify genes displaying a high level of donor- to-donor variability upon treatment with different doses of SAHA. Methods: Primary CD4 + T cells from six healthy seronegative volunteers were incubated with 0.34, 1.0, 3.0 or 10.0 m M SAHA for 24 hours, or left untreated, after which RNA was extracted and gene expression was measured using Illumina HT-12 v4 BeadChips. To identify donor-to-donor variation specific to the SAHA treatment, we initially selected only those genes that were expressed at the same level in the untreated condition for each donor, and then ranked genes according to highest variation between donors upon SAHA treatment. Results: The 100 most variable genes between donors showed higher donor-to-donor variation at the higher SAHA concentrations, 1, 3 and 10 m M compared to 340 nM as determined by Pearson’s correlation analysis. Protein interaction network analysis identified STAT1 (interferon-responsive transcriptional activator) and AKT2 (serine/threonine kinase) as “hub” genes within the 100 most variable genes, and gene ontology analysis revealed a cluster of genes whose functions are relevant to HIV latency. Interesting genes identified were JMJD1A, a lysine demethylase involved in hormone-dependent transcriptional activation; EGR2, a zinc-finger protein shown to interact with HIV Tat; SIN3B, a transcriptional repressor of MYC; L3MBTL3 a putative Polycomb group (PcG) protein and chromatin-interacting transcriptional repressor; and NAE1, which has been shown to interact with HIV-1 Vpr. A separate study of 4 donors treated with 1.0 m M SAHA confirmed similar inter-individual variability at the protein level. Conclusions: It appears that SAHA has different effects in different donors at the level of gene and protein expression. Future work is required to determine if such differences lead to variation in the ability of SAHA to activate HIV in different donors. Future clinical trials with SAHA may need to be tailored to HIV infected individuals likely to respond favorably to this activating compound. 409 Off-Target Effects of SAHA May Inhibit HIV Activation Cory H. White 1 ; Harvey E. Johnston 2 ; Antigoni Manousopoulou 2 ; Celsa A. Spina 1 ; Douglas D. Richman 1 ; Spiros D. Garbis 2 ; Christopher H.Woelk 2 ; Nadejda Beliakova-Bethell 1 1 University of California San Diego, La Jolla, CA, US; 2 University of Southampton, Southampton, United Kingdom Background: The latent HIV reservoir is the obstacle to a cure. HIV reactivation with histone deacetylase inhibitors (HDACis) such as SAHA (Vorinostat) in the presence of Highly Active Anti-Retroviral Therapy (HAART) is a candidate eradication strategy. However, the ability of SAHA to activate HIV in clinical trials appears limited. The primary action of SAHA thought to lead to HIV activation is the hyperacetylation of histones, but the off-target effects of this compound have not been well characterized. We have used an integrated systems biology approach (transcriptomics and proteomics) to reveal previously uncharacterized off-target effects of SAHA. Methods: Primary CD4 T cells were isolated from 10 HIV seronegative donors and either treated with 1 m M of SAHA for 24 hours or DMSO (Dimethyl Sulfoxide). Protein extracts from 4 donors were proteolyzed, labeled with iTRAQ 8-plex and characterized by two-dimensional liquid chromatography-mass spectrometry quantitative proteomics. Total RNA was isolated from the samples of 6 donors and subjected to transcriptomic analysis (Illumina HT12 v4 microarrays). Differentially expressed genes (DEGs) and proteins (DEPs), as well as differentially expressed phosphorylated (DPPs) and acetylated (DAPs) proteins were identified using Limma . Data integration was facilitated by merging data types and mapping the non-redundant set onto biological pathways, gene ontology terms, and protein interaction networks to characterize the off-target effects of SAHA. Results: A total of 368 DEGs, 185 DEPs, 18 DPPs, and 4 DAPs ( p -value < 0.05) were modulated by SAHA. Data integration suggested that SAHA modulates a number of off-target molecules that may enhance (e.g. AES, HSPA1A, KDM1A) or inhibit (e.g. BRD2, ETS1, HMGA1, INBKB, Lef-1) HIV reactivation. For example, HMGA1, which competes with Tat for TAR binding, was upregulated at the transcription and protein levels and acetylated as the result of SAHA treatment.

Poster Abstracts

293

CROI 2015