A Citrate Synthase Splice Variant Rewires the TCA Cycle to Promote Colorectal Cancer Progression

Abstract Metabolic reprogramming, notably alterations in the tricarboxylic acid (TCA) cycle, has emerged as a hallmark of cancer that supports tumor growth and metastasis. Despite the TCA cycle being a classical central metabolic pathway, further exploration is needed to fully elucidate the intricate manifestations and contributory mechanisms of TCA cycle rewiring in colorectal carcinogenesis. In this study, we identified a splicing isoform of citrate synthase (CS), CS-ΔEx4, and unveiled its role in TCA cycle dysregulation in colorectal cancer. CS-ΔEx4 was distinctly upregulated in colorectal cancer tumors compared with the canonical CS full-length (CS-FL) isoform. Clinical analyses established a strong correlation between elevated CS-ΔEx4 expression and cancer recurrence as well as inferior survival outcomes in patients with colorectal cancer. Functional experiments revealed the active contribution of CS-ΔEx4 to the aggressive phenotype of colorectal cancer cells both in vitro and in vivo. Mechanistically, CS-ΔEx4 formed a heterocomplex with CS-FL within the mitochondria that influenced the enzymatic function of canonical CS and accelerated TCA cycle flux, thereby promoting the accumulation of the oncometabolite 2-hydroxyglutarate. The CS-ΔEx4–mediated metabolic alterations engendered epigenomic modulations that drove the upregulation of oncogenic gene signatures. In silico screening identified a small molecule with potent antiproliferative effects in colorectal cancer cell line and organoid models that selectively antagonized the CS-ΔEx4 and CS-FL heterocomplex activity while sparing the CS-FL homodimers. Together, this study discovered the presence of a spliced CS isoform that promotes colorectal cancer progression and identified a molecule that holds potential for targeting the CS-ΔEx4 and CS-FL heterocomplex. Significance: The citrate synthase variant CS-ΔEx4 augments TCA metabolic flux to facilitate epigenetic reprogramming and colorectal carcinogenesis and can be targeted with a small molecule, providing a promising treatment strategy for colorectal cancer. See related commentary by Schatton and Frezza, p. XX .