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ATF4 REGULATES AMINO ACID BIOSYNTHESIS AND TRNA CHARGING TO COORDINATE NUCLEOTIDE SYNTHESIS AND SELECTIVE PROTEIN TRANSLATION TO DRIVE THE G1/S TRANSITION
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2025-05
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Biomedical Sciences
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https://doi.org/10.34944/fczs-jd97
Abstract
Metabolic programming is a common feature in many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators of metabolic processes that are commandeered in AML remain largely unknown. We previously discovered that the Activating Transcription Factor 4 (ATF4) supports disease progression in experimental models of AML. We also observed that the expression of ATF4 is significantly elevated in numerous genetic subtypes of AML compared to healthy hematopoietic stem and progenitor cells. This suggests that ATF4 is broadly deregulated in AML and interventions targeting this pathway may be broadly applicable. To better understand the molecular role of ATF4 in AML cell biology, we engineered multiple human AML cells lines (NOMO-1, MV4-11, and OCIAML-3) to express control (shNT) or shRNAs targeting ATF4 under the regulation of a tetracycline-inducible promoter. Using these models, we found that ATF4 inhibition promotes AML cell cycle arrest, differentiation, and death suggesting that ATF4 supports the differentiation blockade. To identify downstream gene targets of ATF4 in AML, we carried out RNA-sequencing analysis in NOMO-1 cells at a time point where we observe maximal knockdown of ATF4 protein (24 hours post-doxycycline [DOX] treatment), but prior to the evolution of cellular changes (~60-72 hours post-DOX). An enrichment analysis of genes that were specifically downregulated by ATF4 inhibition uncovered that genes associated with amino acid metabolism and tRNA aminoacylation are regulated by ATF4 – these results were confirmed by qPCR in all of our human cell line models. Additionally, chromatin immunoprecipitation (ChIP) assays showed that ATF4 localized to the promoters of many of these genes, suggesting that they are direct transcriptional targets.
Metabolomic assays were also conducted in NOMO-1 cells to further investigate how ATF4 regulates these pathways. A pathway enrichment analysis of total steady-state polar metabolites indicated that ATF4 inhibition reduces metabolites involved in the synthesis and catabolism of several amino acids, disrupts the de novo synthesis of nucleotides, and diminishes metabolites required for aminoacyl-tRNA biosynthesis. To further understand the impact ATF4 has on these amino acid metabolic pathways we conducted several stable-isotope tracing metabolomic assays. These analyses showed that ATF4 inhibition disrupts serine and cysteine metabolism, synthesis of TCA cycle derived amino acids, and nucleotide synthesis through reduced glucose and glutamine flux.
Quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC) was also utilized to understand the direct effects ATF4 regulation of tRNA aminoacylation has on changes in the proteome. This analysis showed immense proteomic and transcriptomic overlap of many proteins involved in amino acid metabolism and tRNA aminoacylation, however, only 56% of significantly decreased proteins could be explained by decreased RNA expression. Further analysis revealed that ATF4 inhibition affects the translation of selective proteins that are unaffected at the RNA level, fold enrichment and STRING analyses uncovered that these selective proteins are involved in the G1/S transition.
From these data it is apparent that ATF4 acts as an upstream regulator to promote metabolic reprogramming in AML, specifically in amino acid metabolism and subsequent nucleotide synthesis, as well as linking these newly produced amino acids to protein synthesis through tRNA aminoacylation for selective protein translation to drive the G1/S transition. Our results have shown that ATF4 could be a potential broadly applicable therapeutic target in AML. Additionally, our findings will likely extend beyond AML as this pathway has been implicated in the pathogenesis of a variety of human cancers. Further investigation is needed to uncover the potential therapeutic effects of a pharmacological inhibitor that targets ATF4, as well as various components of the pathways ATF4 regulates, in combination with current standard of care therapies.
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