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Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor GCN2 represses the expression of the precursor of ribosomal RNA, 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, GCN2 activity supports cell proliferation through the transcription stimulation of 47S rRNA, independently of the canonical ISR axis. However, impairment of GCN2 activity prevents nuclear translocation of the methionyl tRNA synthetase (MetRS) underlying the generation of a nucleolar stress, mTORC1 inhibition and autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA pol I inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls the ribosome biogenesis according the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and the RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically-stressed COAD cell.

Activating transcription factor 4 (ATF4) is involved in muscle atrophy through the overexpression of some atrogenes. However, it also controls the transcription of genes involved in muscle homeostasis maintenance. Here, we explored the effect of ATF4 activation by the pharmacological molecule halofuginone during hindlimb suspension (HS)-induced muscle atrophy. Firstly, we reported that periodic activation of ATF4-regulated atrogenes (Gadd45a, Cdkn1a, and Eif4ebp1) by halofuginone was not associated with muscle atrophy in healthy mice. Secondly, halofuginone-treated mice even showed reduced atrophy during HS, although the induction of the ATF4 pathway was identical to that in untreated HS mice. We further showed that halofuginone inhibited transforming growth factor-β (TGF-β) signalling, while promoting bone morphogenetic protein (BMP) signalling in healthy mice and slightly preserved protein synthesis during HS. Finally, ATF4-regulated atrogenes were also induced in the atrophy-resistant muscles of hibernating brown bears, in which we previously also reported concurrent TGF-β inhibition and BMP activation. Overall, we show that ATF4-induced atrogenes can be uncoupled from muscle atrophy. In addition, our data also indicate that halofuginone can control the TGF-β/BMP balance towards muscle mass maintenance. Whether halofuginone-induced BMP signalling can counteract the effect of ATF4-induced atrogenes needs to be further investigated and may open a new avenue to fight muscle atrophy. Finally, our study opens the way for further studies to identify well-tolerated chemical compounds in humans that are able to fine-tune the TGF-β/BMP balance and could be used to preserve muscle mass during catabolic situations.

International audience; Genetic alterations in non-small cell lung cancers (NSCLC) stimulate the generation of energy and biomass to promote tumor development. However, the efficacy of the translation process is finely regulated by stress sensors, themselves often controlled by nutrient availability and chemotoxic agents. Yet, the crosstalk between therapeutic treatment and glucose availability on cell mass generation remains understudied. Herein, we investigated the impact of pemetrexed (PEM) treatment, a first-line agent for NSCLC, on protein synthesis, depending on high or low glucose availability. PEM treatment drastically repressed cell mass and translation when glucose was abundant. Surprisingly, inhibition of protein synthesis caused by low glucose levels was partially dampened upon co-treatment with PEM. Moreover, PEM counteracted the elevation of the endoplasmic reticulum stress (ERS) signal produced upon low glucose availability, providing a molecular explanation for the differential impact of the drug on translation according to glucose levels. Collectively, these data indicate that the ERS constitutes a molecular crosstalk between microenvironmental stressors, contributing to translation reprogramming and proteostasis plasticity.

International audience; Muscle atrophy arises from a multiplicity of physio-pathological situations and has very detrimental consequences for the whole body. Although knowledge of muscle atrophy mechanisms keeps growing, there is still no proven treatment to date. This study aimed at identifying new drivers for muscle atrophy resistance. We selected an innovative approach that compares muscle transcriptome between an original model of natural resistance to muscle atrophy, the hibernating brown bear, and a classical model of induced atrophy, the unloaded mouse. Using RNA sequencing, we identified 4415 differentially expressed genes, including 1746 up- and 2369 down-regulated genes, in bear muscles between the active versus hibernating period. We focused on the Transforming Growth Factor (TGF)-β and the Bone Morphogenetic Protein (BMP) pathways, respectively, involved in muscle mass loss and maintenance. TGF-β- and BMP-related genes were overall down- and up-regulated in the non-atrophied muscles of the hibernating bear, respectively, and the opposite occurred for the atrophied muscles of the unloaded mouse. This was further substantiated at the protein level. Our data suggest TGF-β/BMP balance is crucial for muscle mass maintenance during long-term physical inactivity in the hibernating bear. Thus, concurrent activation of the BMP pathway may potentiate TGF-β inhibiting therapies already targeted to prevent muscle atrophy.

Increased expression of Hypoxia-Inducible Factor 1α (HIF-1α) in breast tumours is associated with poor survival outcome. HIF-1α promotes phenotypic adaptations including metabolic reorganisation, angiogenesis and increased cell invasiveness. Notably, HIF-1α activity is increased in Triple receptor Negative Breast Cancer (TNBC) compared with other breast cancer subtypes. One reason for this elevated HIF-1α stabilisation in TNBC has been described; intracellular cysteine depletion which leads to impaired prolyl hydroxylase-domain (PHD) enzyme activity. Normally, cystine is acquired in exchange for intracellular glutamate using the xCT antiporter. However, in TNBC the excessive secretion and extracellular accumulation of glutamate impairs cystine exchange. Since the majority of glutamate produced in TNBC is derived from glutaminase-catalysed hydrolysis of glutamine, we hypothesised that glutaminase inhibition using a potent and selective inhibitor, CB-839, would improve glutamate/cystine exchange and reduce HIF-1α levels. We detected normoxic HIF-1α expression in BT549, Hs578T and SUM159PT TNBC cell cultures. In these cases HIF-1α protein was destabilised upon treatment with CB-839 and the expression of some HIF-1α-target genes was significantly decreased (e.g. CA9, BNIP3, PDK1, LDHA) but not others (e.g. ADM and VEGFA). HIF-1α destabilisation corresponded with increased PHD-dependent hydroxylation, supporting the hypothesis that glutaminase inhibition can improve PHD function. Consistently, WT but not a proline hydroxylation-null mutant HIF-1α protein was depleted by CB-839. We confirmed that CB-839 decreased glutamate secretion but, unexpectedly, HIF-1α levels were not restabilised by addition of excess extracellular glutamate. Thus glutamine metabolism drives normoxic HIF-1α stabilisation via an additional mechanism distinct from perturbation of the glutamate/cystine exchange system. To elucidate the underlying process we focused on the glutamine-derived metabolite (S)-2-Hydroxyglutarate [(S)-2HG], a competitive inhibitor of 2-Oxoglutarate (2OG)-dependent enzymes, including the PHD enzymes. Glutaminase inhibition decreased the levels of (S)-2HG. Furthermore, growing CB-839-treated cells in culture medium containing cell permeable (S)-2HG, but not (R)-2HG, could restabilise HIF-1α. In addition, increasing 2OG levels in these cells further stabilised HIF-1α, an effect that was blunted by inhibition of lactate dehydrogenase A (LDHA), a promiscuous source of (S)-2HG generated via reduction of 2OG. Conclusion: CB-839 has demonstrated encouraging clinical responses in patients with heavily pretreated and therapy-resistant metastatic TNBC. Our work demonstrates that high levels of glutamine metabolism provoke HIF-1α stabilisation through the LDHA-catalysed production of (S)-2HG. Assessment of HIF-1α activity may be a useful approach to identify glutamine-addicted tumours and predict responsiveness to glutaminase inhibitor therapy. Citation Format: Laura Cussonneau, Anne-Lise Dechaume, Pamela M. Murray, Henegama Liyanage, Tet-Woo Lee, Frederik Pruijn, Euphemia Y. Leung, Dean C. Singleton. HIF-1α stabilisation in triple receptor negative breast cancer is sensitive to glutaminase inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3561.