CRISPR/Cas9-mediated knockout of the PFK-2 and allosteric activator binding domain of PFK-1 as a novel therapeutic approach for cancer

Abstract

Cancer is a significant health issue causing deaths worldwide, necessitating novel and efficient treatments. Modern cancer therapies primarily target the altered energy metabolism of cancer cells, which enables their accelerated proliferation. A key feature of this metabolic shift is aerobic glycolysis, also called the Warburg effect, where cancer cells convert glucose to lactate despite the presence of oxygen. This metabolic reprogramming supports the rapid proliferation and survival of cancer cells. Phosphofructokinase-1 (PFK-1) plays a crucial role in this process, which catalyses the rate-limiting step of glycolysis, and is allosterically activated by fructose- 2, 6-bisphosphate (F2,6BP), a metabolite produced by the PFK-2. This study presents two complementary CRISPR/Cas9-based approaches to reduce the rate of glycolysis and thereby attenuating cancer cell proliferation by targeting allosteric activation of PFK-1. The first approach involves disruption of the PFKFB3 by targeting exon 2 using a specifically designed gRNA (guide RNA) to knockout PFK-2 and thereby inhibit the synthesis of F2,6BP. In the second approach, the regulatory domain of the PFK-1 will be selectively knocked out by targeting exon 13 using a specific gRNA, disrupting F2,6BP binding while potentially preserving basal catalytic activity. This partial knock-out may significantly impair glycolysis in cancer cells while minimising adverse effects on normal cells. Suitable donor templates were designed using bioinformatic tools to repair the double stranded breaks (DSBs) via Homology Directed Repair (HDR) pathway, by incorporating an EcoRI restriction site, 6× His-tag, and a stop codon facilitate the detection of truncated protein using anti-His Western blot. The pSpCas9(BB)-2Apuro- plasmid harboring PFKFB3-gRNA, along with the corresponding donor template, were transfected into MCF-7 breast cancer cells using PEI followed by puromycin selection. The truncated PFK-2 protein with a His-tag (˜5 kDa) was detected in a Western blot analysis using anti-His antibodies indicating successful K/O of PFKFB3. Silencing PFKFB3 is expected to significantly reduce F2,6BP production, consequently lowering PFK-1 activity and glycolytic flux in cancer cells. Together, these independent approaches offer a promising strategy to disrupt cancer cell metabolism by limiting glycolytic flux, enabling safer and more selective therapies that minimise adverse effects on healthy cells.