Combined Inhibition of Wee1 and Checkpoint Kinases Synergistically Provokes S-Phase Progression and Mitotic Entry, and Promotes Cell Death Under Heat Stress.

Hyperthermia, induced by heat stress (HS), inhibits cancer cell proliferation and induces cell death. We have previously demonstrated that the inhibition of checkpoint kinase 1/2 (Chk1/2) abrogated G2/M arrest and promoted cell death under HS conditions. Here, we investigated whether Wee1 inhibition promoted mitotic entry and cell death in cells exposed to HS in combination with a Chk1/2 inhibitor. MK-1775, a selective Wee1 inhibitor, promoted HS-induced loss of cell viability and increased the SubG1 population in HeLa S3 cells, accompanied by enhanced recovery of EdU incorporation and reduced G2-phase accumulation following HS. Either MK-1775 or AZD-7762, a Chk1/2 inhibitor, alone counteracted HS-induced suppression of EdU incorporation and G2 arrest, whereas their combination further promoted EdU incorporation, mitotic entry, and loss of cell viability under HS. These findings suggest that Wee1 and Chk1/2 cooperatively regulate both S-phase progression and mitotic entry under HS, thereby contributing to cell survival. Moreover, the combination of MK-1775 and AZD-7762 abrogated G2 arrest and enhanced HS-induced cell death in MG-63 and HSC-3 cells. These findings indicate that simultaneous inhibition of Wee1 and Chk1/2 could be a promising strategy for augmenting the therapeutic efficacy of hyperthermia.