Abstract 3711: Molecular and cancer-related roles of ATP-citrate lyase exon 14 (2023)

Abstract Introduction: ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism as well as provides acetyl-CoA necessary for histone acetylation, linking cell nutrient status and epigenetic regulation. Several studies have shown that ACLY is upregulated in multiple cancer types. ACLY has two major splice isoforms: the full-length canonical “long” isoform and an uncharacterized “short” isoform where exon 14 is spliced out. RNAseq data from The Cancer Genome Atlas (TCGA) shows that the Percent Spliced In (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers compared to normal tissues. We therefore hypothesize that ACLY exon 14 inclusion can support tumor growth. Experimental Procedures: Extensive statistical analysis was performed on TCGA data to explore the connection between ACLY PSI and clinical parameters including tumor type, stage, and survival. We also noted a serine within exon 14 that is dynamically phosphorylated according to multiple phosphoproteomics studies. We used an in vitro activity assay and differential scanning fluorimetry to biochemically characterize purified ACLY isoforms and phosphomutants. We also expressed these isoforms and mutants in cells lacking endogenous ACLY to evaluated rescue of known ACLY functions including proliferation, de novo lipogenesis, and histone acetylation. Since exon 14 is located near a putative nuclear localization sequence, we also used confocal microscopy to study subcellular localization of these constructs. Ultimately, we developed a new C57BL6/J mouse model with ACLY exon 14 deleted by CRISPR to study the role of exon 14 in metabolic physiology and tumor growth in vivo. Results: In vitro assays showed no differences in activity and stability between ACLY isoforms and phosphomutants. Similarly, both isoforms and mutants were able to rescue known ACLY functions in cells, and deletion of exon 14 in mice does not impact normal metabolic physiology. Analysis of TCGA data offers compelling evidence that the long ACLY isoform is upregulated early in tumorigenesis and is associated with deadlier tumors and certain immune cell signatures, suggesting that exon 14 is particularly relevant in this disease context. Our mouse model was cross-bred to ApcMin/+ mice, a genetic model of colon cancer, and studies are underway to determine the impact of exon 14 on colon tumors and immune infiltration. Conclusions: ACLY exon 14 does not have obvious roles in enzymatic activity and known ACLY functions. However, alternative splicing could impact pathophysiological phenotypes such as cancer or other yet undescribed functions of ACLY. Citation Format: Julianna G. Supplee, Hayley C. Affronti, Richard Duan, Rebekah C. Brooks, Phuong Nguyen, Kollin Schultz, Ronen Marmorstein, Kathryn E. Wellen. Molecular and cancer-related roles of ATP-citrate lyase exon 14. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3711.