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 determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has been challenging. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to probe the differential protein composition of the latent and activated HIV-1 5′LTR. 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 clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs. We demonstrate that, by targeting IKZF1 for degradation, these compounds reverse HIV-1 latency in CD4+ T-cells isolated from virally suppressed people living with HIV-1 and that they are able to synergize with other known LRAs.

HibeRNAtion: HIV-1 RNA Metabolism and Viral Latency

“…Among those, we found that 10% of the proteins identified in our haploid screen are involved in host cell RNA processing and metabolism, such as DDX46 (also known as PRP5), involved in spliceosome assembly and RNA-protein interactions ( Cordin and Beggs, 2013 ) or EIF2B5, involved in translation initiation ( König et al., 2008 ). In another study, we performed locus-specific chromatin pulldown of the HIV-1 5´LTR latent versus active promoter coupled with mass spectrometry in order to identify differentially abundant putative repressors or activators of latency (in press ( Ne et al., 2022 )). We found that 50% of the putative repressor proteins bound to the latent promoter were proteins involved in RNA processing and metabolism, including members of the TREX1 and TREX2 complexes (DDX39A and PCID2) ( Heath et al., 2016 ; Stewart, 2019 ), and RAMAC/FAM103A1, required for mRNA cap methylation ( Gonatopoulos-Pournatzis et al., 2011 ).…”

“…( Figure 2B ). Spliceosome protein CRNKL1 has been recently implicated in promoting nuclear retention of intron-containing US vRNA species and its depletion leads to improved export of US vRNA ( Figure 2B ) ( Xiao et al., 2021 ; Ne et al., 2022 ). On the contrary, other proteins can act as positive regulators of US vRNA export and their absence leads to nuclear retention of such species.…”

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 factor (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 adenosine triphosphate (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

“…The contribution of these RNA processing mechanisms to the overall repression of HIV-1 gene expression has recently been characterized in both in vitro models of HIV-1 latency and CD4+ T cells obtained from people living with HIV-1 (PLWH) 10–14 , where it has been shown that blocks to HIV-1 gene expression occur largely at post-transcriptional levels. More recently, several unbiased and candidate studies, including our own, have successfully identified co– and post-transcriptional regulators of HIV-1 latency that control vRNA metabolism and either promote or inhibit viral gene expression 12,15,16 .…”