Abstract 1182: Modulating splicing of NELFE as a therapeutic strategy for hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is already one of the deadliest cancers with the death rate only expected to increase in the United States over the next few decades. This is, in part, due to its various risk factors that result in a heterogenous disease arising from the accumulation of genetic and epigenetic alterations. Negative Elongation Factor E (NELFE) is altered in more than 30% of HCCs and elevated gene expression is associated with worse patient outcomes. We discovered a rare (<1% general population) germline intronic heterozygous single nucleotide polymorphism (SNP) (G<A) in ∼3.8% of our HCC cohort which is associated with better patient outcomes. Patients with the SNP had decreased NELFE mRNA expression in their tumors compared to wildtype (WT) patients. Moreover, SNP patients had longer recurrence free and overall survival times. Using a minigene, we found the SNP decreases the binding of an essential splicing protein, U2AF65, which promoted exon 10 skipping. Thus, we hypothesize alternative splicing (AS) of NELFE can be used as a therapeutic strategy for HCC. To investigate the effects of exon 10 loss, we employed a pharmacological approach (antisense oligonucleotides (ASOs)) and a genetic based approach (CRISPR/Cas9) to target exon 10 splice junctions. Loss of exon 10 through ASO induced AS and CRISPR-mediated exon deletion produced a shorter isoform we called d.ex10 and decreased full-length NELFE protein expression. Furthermore, cells treated with ASOs had significantly decreased cell proliferation, migration, invasion, and colony formation in vitro and inhibited tumor growth in vivo. To reduce potential off-target and toxicity effects of ASO in vivo, we will utilize FDA-approved ultrasound contrast agents (microbubbles) for targeted delivery of ASO to tumors. In conclusion, NELFE ASOs could be a potential therapeutic strategy for HCC patients. Citation Format: Laura M. Reynolds, Ryan Lamm, Kai Zhang, Anna Barry, Corinne Wessner, Ji-Bin Liu, Alvaro Lucci, Brittany N. Ruiz, Meghan D. Grim, John Eisenbrey, Hien Dang. Modulating splicing of NELFE as a therapeutic strategy for hepatocellular carcinoma [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 1182.