Figure 6 from Translational Activation of ATF4 through Mitochondrial Anaplerotic Metabolic Pathways Is Required for DLBCL Growth and Survival

https://doi.org/10.1158/2643-3230.30698619

figure

posted on 2025-11-24, 22:01 authored by Meng Li, Matthew R. Teater, Jun Young Hong, Noel R. Park, Cihangir Duy, Hao Shen, Ling Wang, Zhengming Chen, Leandro Cerchietti, Shawn M. Davidson, Hening Lin, Ari M. Melnick

ATF4 translation and protein level respond to nutrient level and are regulated by autophagy. A, FCs of mean fluorescence intensity (MFI) of the GFP reporter expressed from the ATF4-5′UTR-GFP translation reporter in Karpas 422 cells containing control or SIRT3 shRNAs under different culture conditions. Top, experiments were done as in the schema. Briefly, cells were cultured with fresh medium for 48 hours and then replenished with same volume of fresh medium or maintained without replenishment (as a control) for another 16 hours. The y-axis denotes GFP signal intensity relative to control cells without expression of reporter, determined with MFI of GFP signals from flow cytometer. B, FCs of MFI of the GFP reporter expressed from the ATF4-5′UTR-GFP translation reporter in Karpas 422 cells with control or SIRT3 shRNAs under control or glutamine starvation or tunicamycin (10 μg/mL) treatment. Both starvation and tunicamycin treatment were maintained for 15 hours at day 4 after viral transduction. C, Western blot results show the GFP expression from the ATF4-5′UTR-GFP translation reporter in Karpas 422 cells from B. D, The heatmap shows that relative abundances of amino acids were detected by metabolic profiling from Karpas 422 cells transduced with SIRT3 or control shRNAs. The metabolite levels were mean value from five to six replicate samples obtained on day 6 after infection. E, Relative activities of the ATF4-5′UTR-GFP reporter in Karpas 422 were cultured for 48 hours, replenished with fresh media or with the indicated nutrients, and then assessed for ATF4 translation reporter activity 16 hours later. The y-axis denotes GFP signal intensity relative to control cells without expression of reporter, determined with MFI of GFP signals from flow cytometer. NT, not treated; Q, glutamine. F, Western blots show ATF4 protein levels from the indicated cell lines cultured as in A and E, and then replenished with NEAA or not replenished, after which immunoblots were performed for ATF4, with tubulin and actin as loading controls, in control or NEAA-added conditions. G, The heatmap shows the amino acid abundance measured with LC/MS from Karpas 422 cells transduced with SIRT3 or control shRNA and cultured with or without DMKG supplementation. The values show the average logFC from three replicates in amino acid abundance as compared with DSMO-treated control shRNA–transduced cells. H, Western blot results show ATF4 level changes in control or ATG5 knockdown Karpas 422 cells under glutamine starvation condition. Samples were collected at different time points, and protein level changes were quantified with densitometry results. I, Western blots show the ATF4 and autophagy changes in Karpas 422 cells treated with DMSO or YC8-02 (YC; 3 μmol/L) for 40 hours. Cell lysates were subjected to Western blot using the indicated antibodies. Protein levels were quantified with densitometry results. J, The barplot shows the relative cell viability after OCI-LY1 and Karpas 422 cells were treated with DMSO, YC8-02 (OCI-LY1: 6 μmol/L; Karpas 422: 2 μmol/L), GCN2IN6 (OCI-LY1: 7.5 μmol/L; Karpas 422: 10 μmol/L), and combinations for 72 hours. Cells were subject to flow cytometry for viability tests (DAPI staining) and counting. *, P < 0.05; **, P < 0.01. NS, not significant. Error bars represent the mean ± SD of three or more replicates.

Funding

NCI

Falk Medical Research Catalyst and Transformational Award

Tri-Institutional Therapeutics Discovery Institute Award

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DOI - Is supplement to Translational Activation of ATF4 through Mitochondrial Anaplerotic Metabolic Pathways Is Required for DLBCL Growth and Survival

ARTICLE ABSTRACT

Diffuse large B-cell lymphomas (DLBCL) are broadly dependent on anaplerotic metabolism regulated by mitochondrial SIRT3. Herein we find that translational upregulation of ATF4 is coupled with anaplerotic metabolism in DLBCLs due to nutrient deprivation caused by SIRT3 driving rapid flux of glutamine into the tricarboxylic acid (TCA) cycle. SIRT3 depletion led to ATF4 downregulation and cell death, which was rescued by ectopic ATF4 expression. Mechanistically, ATF4 translation is inhibited in SIRT3-deficient cells due to the increased pools of amino acids derived from compensatory autophagy and decreased glutamine consumption by the TCA cycle. Absence of ATF4 further aggravates this state through downregulation of its target genes, including genes for amino acid biosynthesis and import. Collectively, we identify a SIRT3–ATF4 axis required to maintain survival of DLBCL cells by enabling them to optimize amino acid uptake and utilization. Targeting ATF4 translation can potentiate the cytotoxic effect of SIRT3 inhibitor to DLBCL cells. We discovered the link between SIRT3 and ATF4 in DLBCL cells, which connected lymphoma amino acid metabolism with ATF4 translation via metabolic stress signals. SIRT3–ATF4 axis is required in DLBCL cells regardless of subtype, which indicates a common metabolic vulnerability in DLBCLs and can serve as a therapeutic target.This article is highlighted in the In This Issue feature, p. 1