Abstract 2810: Investigating the oncogenic function of the long noncoding RNA KCNQ1OT1 in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents, with survival for high-risk cases stagnant at less than 30%. Contemporary molecular classification of RMS is into fusion-positive (FP) and fusion-negative (FN) groups, based on expression of fusion oncogenes. Although the majority of FN-RMS tumors exhibit mutations in protein coding genes, especially in the RAS pathway, about 25% instead show only copy number alterations and LOH at chromosome 11p15.5, which harbors tumor suppressor genes (TSGs) CDKN1C, PHLDA2, and SLC22A18. The 90kb long noncoding RNA KCNQ1OT1 (OT1) is a known cis silencer of this locus. We hypothesize that OT1 is upregulated in RMS, contributes to RMS tumorigenesis by repressing critical TSGs, and that targeting OT1 could provide a novel therapeutic approach. Using qPCR and epigenomic interrogation of available RMS tissue, we find OT1 highly expressed in FN-RMS cell lines and banked tumor samples. Complementary genomic methylome data of RMS tumors shows silencing and activation of the CDKN1C and OT1 promoters, respectively. Suppression of OT1 using shRNA against various sites along the transcript, or a dual excision CRISPR knockout (DECKO) of the promoter, inhibits RMS cell proliferation, induces apoptosis, and recovers TSG expression. In a candidate approach, using both gain and loss of function approaches, the Hippo pathway YAP1 transcriptional co-activator was found to modulate OT1 expression. Unbiased genomic screens are planned. Finally, using in silico binding prediction models, we designed OT1-directed anti-sense oligonucleotides (ASOs), demonstrating they can be delivered to RMS cell lines. These ASOs, designed to degrade or inhibit OT1 function, will be tested in RMS cell-based and xenograft models to determine their efficacy and specificity. To generate additional information about regions of OT1 that might be therapeutically tractable, antisense DNA capture probes will be applied to OT1 in RMS cells to generate higher nucleotide resolution maps of OT1 structure and protein binding. This study not only deepens our understanding of OT1's role in RMS but also advances the development of innovative RNA-based treatments, addressing a critical need for effective therapies in this aggressive childhood cancer. Citation Format: Ara Jo, Alex Kovach, Christian Stackhouse, Samantha Weitzel, Brian Masters, Justina McEvoy, Wenyue Sun, Frederic G. Barr, Mauro Calabrese, Xiang Chen, Corinne M. Linardic. Investigating the oncogenic function of the long noncoding RNA KCNQ1OT1 in rhabdomyosarcoma [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 2810.