Abstract Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a disorder characterized by the loss of fumarate hydratase (FH) activity, leading to the development of cutaneous leiomyomas, uterine leiomyomas, and aggressive early-onset type 2 papillary renal cell carcinoma. In HLRCC, the disease is driven by the somatic loss of the remaining functional FH allele in individuals carrying a germline pathogenic variant in one copy of the FH gene. The complete loss of FH activity disrupts the tricarboxylic acid (TCA) cycle, resulting in the accumulation of fumarate, which disrupts signaling pathways and contributes to the oncogenic phenotype. We recently identified a single nucleotide polymorphism (SNP) in an intronic region of the fumarate hydratase (FH) gene that leads to the loss of FH activity (Crooks et al., 2023). This SNP introduces a novel splice-acceptor site, resulting in the inclusion of an aberrant exon between wild-type exons 9 and 10. This ‘poison-like’ exon encodes a premature STOP codon, producing a nonfunctional truncated FH protein. Previous studies have shown that restoring FH activity can abrogate pathogenic behavior in patient-derived cells, highlighting the potential for therapeutic interventions targeting FH activity restoration. Our goal is to restore FH enzymatic activity by preventing the inclusion of this cryptic exon in carriers of this variant. To achieve this, we are exploring strategies to restore functional FH production by interfering with the inclusion of the pathogenic intron during splicing by removing the splice acceptor using CRISPR-Cas9 or base editing technologies. We tested these approaches by generating a reporter cell line that recapitulates the splicing pattern observed in patients, where expression of GFP depends on correct splicing between exon 9 and 10. We successfully identified antisense oligonucleotide (ASO) and single-guide RNA (sgRNA) sequences that restore GFP expression in our reporter cell lines. We are now evaluating the efficacy of these reagents in patient-derived cells. An alternative approach involves using synthetic RNA to restore FH expression. The expression of proteins from synthetic RNAs is a promising strategy for treating cancer and infectious diseases. We are currently investigating the efficiency of restoring FH activity through in-vitro transcribed (IVT) mRNA. This approach has the advantage of enabling FH function restoration in cells with mutations that are not amenable to antisense oligonucleotide (ASO) or gene editing techniques. This work serves as an avenue to investigate the application of nucleic acid therapies to restoring activity tumor suppressor genes in kidney cancer. Citation Format: Siddhardha S. Maligireddy, Mariana D. Mandler, Jodie C. Lunger, Christina M. Fitzsimmons, Daniel R. Crooks, Geetha M. Cawthon, Christopher J. Ricketts, Cathy D. Vocke, W. Marson Linehan, Pedro J. Batista. Rescuing expression of a tumor suppressor gene – restoring FH in Hereditary leiomyomatosis and renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B006.

Abstract B006: Rescuing expression of a tumor suppressor gene – restoring FH in Hereditary leiomyomatosis and renal cell carcinoma