Regulation of Metabolism and Epitranscriptomic Modifications By RNA Binding Protein IGF2BP3
Authors: Sharma Gunjan , Gutierrez Matin , Jones Anthony E. , Neeb Zachary T. , Rios Amy , Jaiswal Amit K. , Thaxton Michelle L. , Lin Tasha L. , Tran Tiffany , Kabbani Lyna E.S. , Ritter Alexander J. , Stiles Linsey , Hoeve Johanna Ten , Sanford Jeremy R. , Divakaruni Ajit S. , Rao Dinesh S. 
Published: December 2024

N6-methyladenosine (m6A) RNA modification has emerged as a crucial regulator of gene expression, with significant implications in various biological processes and diseases, including leukemogenesis. Recently, the RNA binding protein IGF2BP3 has been identified as a non-canonical reader of N6-methyladenosine (m6A) -modified mRNAs. Our prior work and others established IGF2BP3 as a critical regulator of leukemogenesis in KMT2A translocated B-acute lymphoblastic leukemia. This occurs via its function in binding to and stabilizing oncogenic transcripts and regulating many genes involved in metabolism. Yet, the interplay of how m6A modifications impact the function of IGF2BP3 on leukemogenesis remains to be fully elucidated.

Building on prior work showing gene expression changes in key metabolic pathways, we undertook studies to define the effect of IGF2BP3 on cellular metabolism. Respirometry measurements using Seahorse XF Analysis showed a compromised stress response and mitochondrial reserve capacity. Rates of extracellular acidification, a surrogate for glycolysis, were also decreased, suggesting a defect in energy metabolism. Metabolomics analysis by GC/MS showed a decrease in the steady-state levels of pyruvate, lactate, and α-ketoglutarate. LC/MS-based metabolomics also revealed reproducible reductions in serine, glycine, S-adenosylmethionine (SAM), and cystathionine.

By adopting a combined high through-put analysis approach using differential expression data from IGF2BP3 KOs, cross-link immunoprecipitation with RNA sequencing (CLIP-seq) and m6A- sequencing, we identified several direct targets of IGF2BP3 in the central pathways controlling cancer cell metabolism, such as the Glycine-Serine cycle and the Folate cycle. Additionally, polysome profiling revealed that IGF2BP3 impacts the translation of the genes involved in these pathways to regulate the epitranscriptome. Targeted western blotting showed that IGF2BP3 KO cells showed reduced levels of direct target oncogenes such as MYC and BCL2 but also of several metabolic regulators, including MAT2A, the rate-limiting enzyme in the production of SAM.

Given the impact on SAM, the primary methyl donor for cellular methylation reactions, we next queried what may happen to downstream methylation. Interestingly, as previously demonstrated, SAM depletion in the IGF2BP3 KO led to reduced histone methylation as assayed by H3K4me1 and H3K4me3 western blot analysis. While DNA methylation was not similarly altered, m6A modifications showed a reduction in the IGF2BP3 KO. This reduction was not related to a change in RNA m6A-methylase or demethylase activity within the cells, and the key enzymes involved in m6A showed no change in protein level expression. Using an orthogonal method, we employed a novel IGF2BP3-specific inhibitor, I3IN002, in vitro and in vivo to assess its effect on global m6A methylation patterns and metabolism. Our results demonstrated a significant reduction in m6A levels upon IGF2BP3 inhibition and compromised energy production in leukemic cells, similar to the IGF2BP3 KO. Conversely, overexpressing IGF2BP3 back in IGF2BP3-depleted cells rescued growth and the m6A-modified mRNA levels.

These data collectively suggest that m6A reader IGF2BP3 is a key driver of changes in RNA methylation and oncogene expression, primarily through the regulation of cancer cell metabolism. This results in a pervasive shift in gene expression, maintaining a cancerous phenotype. By unraveling the complex interplay between IGF2BP3 and m6A RNA methylation, we can potentially develop innovative strategies to modulate m6A pathways in leukemia therapy, offering a promising outlook for future therapeutic advancements.