Inhibition of the TRIM24 bromodomain reactivates latent HIV-1

Abstract

Expression of the HIV-1 genome by RNA Polymerase II is regulated at multiple steps, as are most cellular genes, including recruitment of general transcription factors and control of transcriptional elongation from the core promoter. We discovered that tripartite motif protein TRIM24 is recruited to the HIV-1 Long Terminal Repeat (LTR) by interaction with TFII-I and causes transcriptional elongation by stimulating association of PTEF-b/ CDK9. Because TRIM24 is required for stimulation of transcription from the HIV-1 LTR, we were surprised to find that IACS-9571, a specific inhibitor of the TRIM24 C-terminal bromodomain, induces HIV-1 provirus expression in otherwise untreated cells. IACS-9571 reactivates HIV-1 in T cell lines bearing multiple different provirus models of HIV-1 latency. Additionally, treatment with this TRIM24 bromodomain inhibitor encourages productive HIV-1 expression in newly infected cells and inhibits formation of immediate latent repressed provirus. IACS-9571 synergizes with PMA, ionomycin, TNF-α, PEP005, and JQ1 to activate HIV-1 expression. Furthermore, co-treatment of CD4+ T cells from individuals with HIV-1 on antiretroviral therapy (ART) with PEP005 and IACS-9571 caused robust provirus expression. Notably, IACS-9571 did not cause global activation of T cells; rather, it inhibited induction of IL2 and CD69 expression in human PBMCs and Jurkat T cells treated with PEP005 or PMA. These observations indicate the TRIM24 bromodomain inhibitor IACS-9571 represents a novel HIV-1 latency reversing agent (LRA), and unlike other compounds with this activity, causes partial suppression of T cell activation while inducing expression of latent provirus.

CDK8 inhibitors antagonize HIV-1 reactivation and promote provirus latency in T cells

“…10C, PMA, PEP005). In contrast we found that treatment of RBH infected CDK8 KO cells with the histone deacetylase inhibitor SAHA, or the bromodomain inhibitors JQ1 and IACS-957 18 failed to recapitulate the level of productive infections observed for wildtype cells (Fig. 10C, SAHA, JQ1, IACS-9571).…”

Upstream Stimulatory Factors regulate HIV-1 latency and are required for robust T cell activation

“…To examine specific effects of USF1 and USF2 on HIV-1 expression we produced USF1 and USF2 gene knockouts (KO) (Fig. 1A, 1B) in the Jurkat Tat mHIV-Luc T cell line bearing an integrated HIV-1 mini-virus where luciferase expression is driven by the 5' LTR (Bernhard et al 2011) (Horvath, Brumme, and Sadowski 2023). Surprisingly, we noted that USF1 protein abundance was reduced in all USF2 knockout lines (Fig.…”

“…Next, we examined effect of the USFs on reactivation in response to the bromodomain inhibitors JQ1 and IACS-9571. JQ1 is a well-studied LRA that inhibits the BRD4 bromodomain causing increased Tat transactivation (Li et al 2013), while IACS-9571 inhibits the TRIM24 bromodomain causing increased LTR occupancy of this factor and stimulation of transcriptional elongation Horvath, Brumme, and Sadowski 2023) .…”

“…ChIP-qPCR was performed as in ) and Horvath et al, 2023 (Horvath, Brumme, and. Antibodies used are as follows: Myc (10 μg) -Santa Cruz Biotechnology sc-40, USF2 (10 μg) -Abcam ab264330.…”

“…Mutations of the RBE1 and RBE3 elements abrogate binding of RBF-2 (USF1/2, TFII-I) in vivo, and render proviruses incapable of transcriptional induction in response to T cell signaling (Chen et al 2005) (Malcolm et al 2007) (Malcolm et al 2008) (Matthew S. Dahabieh et al 2011). This effect is mediated at least partially by recruitment of the co-factor TRIM24 to the HIV-1 LTR resulting in stimulated transcriptional elongation (Horvath, Brumme, and Sadowski 2023) . However, despite that USF1 and USF2 were among the first cellular factors shown to bind the HIV-1 LTR (Roy 1997) (Du, Roy, and Roeder 1993), the functional significance of these factors for regulation of provirus transcription and latency are unknown.…”