PFKFB3 inhibition dictates the adaptive responses and cell fates in tumor cells.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolysis highly expressed in many tumor cells, has a critical role in promoting glycolysis and tumor progression. Consequently, anti-cancer strategies have sought to inhibit PFKFB3 to exploit this metabolic dependency of tumor cells. In this study, we employed a controlled protein degradation system to manipulate the kinetics of PFKFB3 depletion in investigating its impact on tumor cell growth. We show that, while slow depletion allows metabolic adaptation with a minimal effect on proliferation, rapid depletion or acute inhibition suppresses cell growth despite a similar glycolytic reduction. We show that rapid PFKFB3 loss activates AMPK, which perturbs cell cycle progression through the MAPK/ERK pathway. Importantly, AMPK inhibition was sufficient to rescue the adaptation failure caused by rapid PFKFB3 depletion. Together, our findings document that dynamic targeting of PFKFB3 can drive heterogeneous cancer cell fates, shedding light on cells' potential path toward developing resistance to anti-PFKFB3 treatments.