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Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response

ABSTRACT: Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl-prolyl-tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild type cells and cells lacking GCN2 and those incapable of phosphorylating eIF2. Proline supplementation rescues the observed HF-induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2-to-eIF2 pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF-treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated, and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic.

SUBMITTER: Dr. Aleksandra, P. Pitera

PROVIDER: S-SCDT-EMBOJ-2021-109985 | biostudies-other |

REPOSITORIES: biostudies-other

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Targeting the Integrated Stress Response Kinase GCN2 to Modulate Retroviral Integration.

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Microarray profiling reveals the integrated stress response is activated by halofuginone in mammary epithelial cells.

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Transcriptional responses of wild-type and Gcn2-/- Th17 cells to halofuginone and rapamycin

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2014-05-29 | E-GEOD-47478 | biostudies-arrayexpress

Transcriptional responses of wild-type and Gcn2-/- Th17 cells to halofuginone and rapamycin

2014-05-29 | GSE47478 | GEO

Microarray profiling reveals Integrated Stress Response is activated by Halofuginone in mammary epithelial cells

Project description:The small molecule Halofuginone (HF) is a potent regulator of extracellular matrix (ECM ) gene expression and is unique in its therapeutic potential. While the basis for HF effects is unknown, inhibition of TGFb signaling and activation of the AAR have been linked to HF transcriptional control of a number of ECM components and amelioration of fibrosis and alleviation of autoimmune disease by regulation of Th17 cell differentiation, respectively. The aim of this study was to generate a global expression profile of HF targets in epithelial cells to identify potential mediators of HF function in this cell type. We report that HF modulation of the ECM remodeling protein Mmp13 in epithelial cells is separable from previously reported effects of HF on TGFß signal inhibition, and use microarray expression analysis to correlate this with transcriptional responses characteristic of the Integrated Stress Response (ISR). Our findings suggest a common mechanism underlying HF anti-fibrotic and anti-angiogenic effects in parenchymal cells and HF effects on Th17 differentiation. Moreover, our results point away from a central role of TGFb signaling in the HF mechanism of action and suggest a new approach to small molecule based regulation of the ECM transcriptional program in vivo. NMuMG mammary epithelial cells were treated with 10 nM Halofuginone or 10 nM MAZ1310 (a non-functional analog of Halofuginone used as a control) for 8 hours. Each treatment was performed in biological triplicate. Following RNA extraction, we used Phalanx Mouse Whole Genome OneArray to measure mRNA abundance of Halofuginone-treated and MAZ1310 control samples. Each array was performed in triplicate. Expression of transcripts in Halofuginone versus MAZ1310 treated cells was examined.

2011-10-17 | E-GEOD-30667 | biostudies-arrayexpress

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Project description:Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58(IPK), the cellular inhibitor of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2alpha phosphorylation and unphosphorylated eIF2alpha is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2alpha phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2alpha kinases and phosphorylatable eIF2alpha. When eIF2alpha is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10- to 100-fold. eIF2alpha phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2alpha phosphorylation facilitates reovirus replication in two ways-first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.

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Microarray profiling reveals Integrated Stress Response is activated by Halofuginone in mammary epithelial cells

2011-10-18 | GSE30667 | GEO

Integrated stress response modulates cellular redox state via induction of cystathionine ?-lyase: cross-talk between integrated stress response and thiol metabolism.

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Signaling through alternative Integrated Stress Response pathways compensates for GCN2 loss in a mouse model of soft tissue sarcoma.

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Project description:The integrated stress response (ISR), a vital homeostatic pathway, is an emerging therapeutic target for a broad range of clinical indications. Halofuginone (HF) is a phase 2 clinical compound that induces the ISR by inhibiting the glutamyl-prolyl-tRNA synthetase (EPRS). Here we report that although HF induces the predicted canonical ISR adaptations consisting of attenuation of protein synthesis and gene expression reprogramming, the former surprisingly occurs independently of GCN2 and eIF2 phosphorylation. Proline supplementation rescues the observed HF-induced changes indicating that they result from an on-target effect due to inhibition of EPRS. We find that attenuation of translation initiation through GCN2-to-eIF2 signaling is not robust enough to prevent translation elongation defects caused by HF. Exploiting this vulnerability, we show that cancer cells presenting an increased proline dependency are sensitized to HF. This work provides novel insights on ISR signaling and a molecular framework to guide the targeted development of HF.

2022-04-27 | PXD028744 | Pride

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