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

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. Herein, we identified a splicing isoform of citrate synthase (CS), CS-ΔEx4, and unveiled its role in TCA cycle dysregulation in colorectal cancer (CRC). CS-ΔEx4 was distinctly upregulated in CRC tumors compared with the canonical 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 CRC. Functional experiments revealed the active contribution of CS-ΔEx4 to the aggressive phenotype of CRC 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 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 anti-proliferative effects in CRC 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 CRC progression and identified a molecule that holds potential for targeting the CS-ΔEx4 and CS-FL heterocomplex.