Preclinical study of nucleic acid therapeutics targeting the Warburg effect

[Background] Cancer cells rely on glycolysis for energy, known as the Warburg effect, which supports survival by promoting nucleotide synthesis, reducing oxidative stress, and inhibiting cell death. Polypyrimidine tract-binding protein 1 (PTBP1) regulates pyruvate kinase muscle (PKM1/PKM2) splicing, maintaining a PKM2-dominant state. Suppressing PTBP1 shifts cellular metabolism to a PKM1-dominant state, reduces glycolysis, alters ATP production, and increases oxidative stress, ultimately resulting in cell death. We developed optimized chemically modified siR-PTBP1 derivatives to evaluate their therapeutic potential. [Methods] Anti-proliferative activity in human colorectal DLD-1 cells was tested by chemically modified siR-PTBP1 derivatives. Cell viability was assessed by CVS assay, and PTBP1, PKM1/PKM2, and cleaved PARP protein levels were analyzed by Western blotting and immunostaining after the transfection. ATP production and oxidative stress assay were performed to examine the alternation from glycolysis-dominant to oxidative phosphorylation via TCA cycle. Metabolome analysis was also examined. [Results] Among 14 derivatives, the derivative siR-2-6 significantly suppressed PTBP1, increased PKM1 expression, and raised the PKM1/PKM2 ratio. Increased cleaved PARP expression indicated apoptosis induction and Warburg effect suppression. Also, siR-2-6 increased ATP production and oxidative stress, resulting in apoptosis. The higher RNase resistance and biological activity under RNase-containing conditions were shown in comparison to those of the SiR-2-1 that is not chemically modified. [Conclusion] Optimized siR-PTBP1 derivatives increased the PKM1/PKM2 ratio and partially shifted cellular metabolism from glycolysis dependence to oxidative phosphorylation, highlighting their potential as nucleic acid therapeutics. The enhanced RNase resistance of siR-2-6 further supports its promise for in vivo application and clinical translation.