Identification of ATF4 As a Key Upstream Regulator of Acute Myeloid Leukemia Cell Metabolism

Abstract

Jacklyn M. Huhn: Jacklyn Huhn, B.S., Fox Chase Cancer Center, 333 Cottman Ave, Reimann Building R354, Philadelphia, PA 19111, USA. 215-214-1600.jmh@temple.edu Metabolic reprogramming 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 (NOMO1, MV4-11, and OCI-AML3) 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 NOMO1 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 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 NOMO1 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. Lastly, to assess the therapeutic potential of targeting the ATF4 pathway, we assessed how depletion of amino acids from growth media with and without ATF4 inhibition affected AML cell growth. From this analysis, we observed that the depletion of amino acids from growth media suppressed AML cell growth, which was further attenuated by the inhibition of ATF4. From these data it is apparent that ATF4 acts as an upstream regulator to promote metabolic reprogramming in AML, specifically in amino acid synthesis and catabolism, as well as linking these newly produced amino acids to protein synthesis through tRNA aminoacylation. Our results thus far have shown that ATF4 could be a potential broadly applicable therapeutic target. Further investigation is needed through stable-isotope tracing metabolomics to understand the impact ATF4 has on these amino acid metabolic pathways, which are currently ongoing; as well as to understand the direct effects ATF4 regulation of tRNA aminoacylation has on changes in the proteome, which we are currently investigating through quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC).