Abstract 1412: Translational regulation of <i>MYC</i> in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most frequently diagnosed childhood cancer, with a prevalence of almost 25%. ALL cells are auxotrophic for asparagine and have been targeted therapeutically by treatment with bacterial-derived asparaginase. Although asparaginase has been a cornerstone of treatment, ALL can relapse in 10-15% of cases. We previously published that asparagine synthetase (ASNS) induction in response to asparagine depletion is a primary resistance mechanism to asparaginase therapy. Using auxotrophic DND-41 cells, we also showed that asparagine starvation suppresses the expression of c-MYC (MYC) protein, a critical oncogenic driver in ALL cells. We did not observe a change in MYC transcript levels or protein stability, suggesting the acute decrease in protein level was due to a decrease in translation of MYC mRNA. We performed a polysome profiling in DND-41 cells to measure the translation of MYC mRNA. Compared to asparagine-replete control, asparagine starvation caused a significant decrease in the amount of heavier polysomes associated with MYC mRNA. We further performed a Ribo-seq experiment, where cell lysates were treated with RNAse I, leaving ribosome-protected fragments (RPFs) intact for high throughput sequencing. Our analysis showed that RPFs were distributed throughout the MYC coding sequence (CDS) for the control samples but enriched in the N-terminal of the MYC CDS when asparagine was limiting. Of note, these RPFs were around closely spaced asparagine codons (+4, +9, +11) near the translation initiating AUG codon, suggesting this is a critical cis-element regulating the translation of MYC mRNA. We designed a MYC-translation reporter with the MYC N-terminal region fused to an eYFP-DD construct. The fused protein rapidly degrades upon translation due to the destabilization domain (DD), leading to reduced fluorescence. Treating cells with a small molecule, trimethoprim (TMP), stabilizes the fusion protein, enhancing fluorescence. We defined the translation rate by calculating the ratio of fluorescence intensities with and without TMP. Our results showed that fusing the MYC N-terminal region to the reporter significantly reduced translation compared to the eYFP-DD construct upon asparagine starvation. We also found that deleting the asparagine codons partially rescued the reporter translation, when asparagine is limiting. We will use this system to identify trans-factors regulating MYC translation using a dedicated CRISPR library targeting mRNA regulators. Citation Format: Sankalp Srivastava, Jagannath Misra, Rodney Claude, Michael Holmes, Gang Peng, Kirk Staschke, Sandeep Batra, Utpal Dave, Chi Zhang, Ronald Wek, Jing Fan, Ji Zhang. Translational regulation of MYC in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1412.