Catchet-MS identifies IKZF1-targeting Thalidomide analogues as novel HIV-1 latency reversal agents

Abstract

A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular correlates of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has not been possible. Here we use dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to isolate the latent and activated HIV-1 5′LTR, followed by MS identification of the differentially locus-bound proteins. Catchet-MS identified known and novel latent 5′LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the drug iberdomide, which targets IKZF1 for degradation, to be a clinically advanced novel LRA that reverses HIV-1 latency in CD4+T-cells isolated from virally suppressed HIV-1 infected participants.

The BAF complex inhibitor pyrimethamine reverses HIV-1 latency in people with HIV-1 on antiretroviral therapy

“…Strategies that use combinatorial approaches of LRAs could result in higher efficacy in reactivating HIV-1 latency (5). We have shown in previous studies that the effect of pyrimethamine on HIV-1 latency ex vivo could be potentiated when combined with other LRAs including HDACi (44,46). We therefore designed the study to include a combinatorial arm to test whether the effect of the individual LRAs could be potentiated in vivo.…”

“…Our preclinical studies identified BRG-Brahma Associated Factors (BAF) complex inhibitors (BAFi) as a novel LRA class which we demonstrated enhanced the activity of other LRA classes (44)(45)(46). The BAF (mammalian SWI/SNF) chromatin remodeling complex is a key repressor of HIV-1 latency that uses ATP hydrolysis to position repressive nucleosome nuc-1 at the HIV-1 long terminal repeat, causing transcriptional silencing of HIV-1 gene expression thereby maintaining HIV-1 latency (47,48).…”

PCID2 dysregulates transcription and viral RNA processing to promote HIV-1 latency

“…In a previous study 16 , we identified putative latent promoter-bound regulators of HIV-1 gene expression by dCas9-targeted chromatin immunoprecipitation coupled to mass spectrometry (Catchet-MS) (Figure 1A). Because of differential enrichment at the latent HIV-1 and inactive LTR, we hypothesized that these factors may contribute to the maintenance of HIV-1 latency as putative repressors of HIV-1 gene expression.…”

“…In latent cells, and consistent with literature, there is a basal level of residual transcription and basal expression of GFP 31 (Figure 3C-D), with about 37.7% (mean) of GFP+ cells also expressing vRNA (Figure 3E). To demonstrate the effect of a release in a transcriptional block specifically, we performed FISH-Flow in J-Lat 11.1 cells shRNA depleted of Ikaros zinc finger 1 (IKZF1), a recently identified HIV-1 transcriptional repressor 16 . Indeed, we observed strong viral reactivation in IKZF1-knockdown cells as shown by a marked increase in vRNA and GFP producing cells (Figure 3D), of which 48.6% (mean) also express vRNA (Figure 3E).…”

“…In a previous study, we performed an HIV-1 LTR locus-specific dCas9 chromatin immunoprecipitation coupled to mass spectrometry (Catchet-MS) in latent and active cells and identified putative LTR-bound transcriptional regulators of HIV-1 latency 16 . Interestingly, while a substantial number of latency-associated factors identified were, as expected, DNA associated, we also identified many putative RNA-binding latent HIV-1 promoter enriched regulators involved in RNA metabolism, including CRNKL1, recently described to posttranscriptionally regulate HIV-1 gene expression 17 , and other known regulators of HIV-1 gene expression such as DDX24 18 .…”