Biology subjects

Neill, G.

Publications and source records attributed to Neill, G..

5 recordsLinked to original sources

bioRxiv2024

RAF inhibitors activate the integrated stress response by direct activation of GCN2

Paradoxical RAF activation by chemical RAF inhibitors (RAFi) is a well-understood on-target biological and clinical response. In this study, we show that a range of RAFi drive ERK1/2-independent activation of the Unfolded Protein Response (UPR), including expression of ATF4 and CHOP, that required the translation initiation factor eIF2. RAFi-induced ATF4 and CHOP expression was not reversed by inhibition of PERK, a known upstream activator of the eIF2-dependent Integrated Stress Response (ISR). Rather, we found that RAFi exposure activated GCN2, an alternate eIF2 kinase, leading to eIF2-dependent (and ERK1/2-independent) ATF4 and CHOP expression. The GCN2 kinase inhibitor A-92, GCN2 RNAi, GCN2 knock-out or ISRIB (an eIF2 antagonist) all reversed RAFi-induced expression of ATF4 and CHOP indicating that RAFi require GCN2 to activate the ISR. RAFi also activated full-length recombinant GCN2 in vitro and in cells, generating a characteristic bell-shaped concentration-response curve, reminiscent of RAFi-driven paradoxical activation of WT RAF dimers. Activation of the ISR by RAFi was abolished by GCN2 kinase dead mutations and M802A or M802G gatekeeper mutations, suggesting that RAFi bind directly to the GCN2 kinase domain; this was supported by mechanistic structural models of RAFi interaction with GCN2. Since the ISR is a critical pathway for determining cell survival or death, our observations may be relevant to the clinical use of RAFi, where paradoxical GCN2 activation may be a previously unappreciated off-target effect that may modulate tumour cell responses.

biochemistry↗

bioRxiv2024

Paradoxical Activation of GCN2 by ATP-competitive inhibitors via allosteric activation and autophosphorylation

Recently it has been found that General Control Non-derepressible 2 (GCN2) can be activated by an array of small molecule ATP-competitive inhibitors, including clinically relevant compounds such as Ponatinib, and compounds specifically designed to be GCN2 inhibitors, such as GCN2iB. Furthermore, we recently showed that GCN2 can be activated in cells by clinically approved small molecule RAF inhibitors. GCN2 is a drug target, specifically in cancers such as mesothelioma, and a better understanding of this paradoxical activation is required to develop drugs which truly inhibit the enzyme. Using biochemical assays and structural mass spectrometry, we present a model for how GCN2 is activated by these compounds by promoting an active conformation in the HisRS domain while competitively inhibiting the kinase domain. This conformation promotes activating phosphorylation of GCN2, potentially through phosphorylation of other activated GCN2 molecules which are not bound to compound. Together this model suggests that efforts to inhibit GCN2 would benefit from exploring allosteric routes rather than targeting the ATP-binding pocket of the kinase domain.

biochemistry↗

bioRxiv2024

Heme binding causes structural rearrangements in HRI to inhibit activation via autophosphorylation

Heme-Regulated Inhibitor (HRI) is one of the four mammalian kinases which phosphorylates eIF2 to facilitate a cellular response to stress through the regulation of mRNA translation. Originally identified for its role as a heme sensor in erythroid progenitor cells, it has since materialised as a potential therapeutic target in both cancer and neurodegeneration. Here we characterise two modes of HRI inhibition of using structural mass spectrometry, biochemical and biophysical techniques. We demonstrate that several ATP-mimetic compounds, including BRAF inhibitors and a compound, GCN2iB, thought to be specific to GCN2, are capable of potently inhibiting HRI. We demonstrate that hemin, a haem-like molecule, inactivates HRI structurally using hydrogen-deuterium exchange mass spectrometry (HDX-MS), and this results in wide-spread structural rearrangement of the protein and how that impacts on the kinase domain through a series of allosteric interactions. This inhibition mainly impacts autophosphorylation, which includes tyrosine phosphorylation, not observed before in the eIF2 kinases.

biochemistry↗

bioRxiv2021

The synthetic triterpenoids CDDO-TFEA and CDDO-Me, but not CDDO, are potent BACH1 inhibitors

The transcription factor BACH1 is a potential target against a variety of chronic conditions linked to oxidative stress and inflammation, and formation of cancer metastasis. However, only a few BACH1 degraders/inhibitors have been described. BACH1 is a transcriptional repressor of heme oxygenase 1 (HMOX1), which is positively regulated by transcription factor NRF2 and is highly inducible by derivatives of the synthetic oleanane triterpenoid 2- cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO). Most of the therapeutic activities of these compounds are due to their anti-inflammatory and antioxidant properties, which are widely attributed to their ability to activate NRF2. However, with such a broad range of action, these drugs may have other molecular targets that have not been fully identified and could also be of importance for their therapeutic profile. Herein we identified BACH1 as a target of CDDO-derivatives, but not CDDO. While both CDDO and CDDO-derivatives activate NRF2 similarly, only CDDO-derivatives inhibit BACH1, which explains the much higher potency of CDDO-derivatives as HMOX1 inducers compared with unmodified CDDO. Notably, we demonstrate that CDDO-derivatives inhibit BACH1 via a novel mechanism that reduces BACH1 nuclear levels while accumulating its cytoplasmic form. Altogether, our study identifies CDDO-derivatives as dual KEAP1/BACH1 inhibitors, providing a rationale for further therapeutic uses of these drugs.

cell biology↗

bioRxiv2020

Dynamic chromatin accessibility landscape changes following interleukin-1 stimulation

Genome-wide methods for examining chromatin modification provide detailed information on regulatory regions of the genome. Dynamic modifications of chromatin allow rapid access of the gene regulatory machinery to condensed genomic regions facilitating subsequent gene expression. Inflammatory cytokine stimulation of cells can cause rapid gene expression changes through direct signalling pathway-mediated transcription factor activation and regulatory element binding. Here we used the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to assess regions of the genome that are differentially accessible following treatment of cells with interleukin-1 (IL-1). We identified 126,483 open chromatin regions, with 241 regions significantly differentially accessible following stimulation, with 64 and 177 more or less accessible, respectively. These differentially accessible regions predominantly correspond to regions of the genome marked as enhancers. Motif searching identified an overrepresentation of a number of transcription factors, most notably RelA in the regions becoming more accessible, with analysis of ChIP-seq data confirmed RelA binding to these regions. A significant correlation in differential chromatin accessibility and gene expression was also observed. Functionality in regulating gene expression was confirmed using CRISPR/Cas9 genome-editing to delete regions for that became more accessible following stimulation in the genes MMP13, IKBKE and C1QTNF1. These same regions were also accessible for activation using a dCas9-transcriptional activator and showed enhancer activity in a cellular model. Together, these data describe and functionally validate a number of dynamically accessible chromatin regions involved in inflammatory signalling.

genomics↗