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.
