Early WEE1 inhibition prevents senescence-like epigenetic reprogramming of pioneer TMZ-resistant cells and synergizes with temozolomide in MYC-high glioblastoma.

In proliferative glioblastoma, a subset of tumor cells survives early temozolomide (TMZ) exposure and drives eventual-resistance, yet the underlying mechanism remains unclear. We found that MYC target activation was associated with attenuated TMZ survival benefit in CGGA cohorts. In patient-derived MYC-high glioma cells, TMZ increased apoptosis but left a larger residual viable population due to enhanced proliferation; we defined these surviving cells as pioneer resistant (P-R) cells, which exhibited reduced TMZ sensitivity and a competitive survival advantage. Mechanistically, P-R cells adopted a senescence-like state via two coordinated arms of chromatin remodeling. On one hand, H3K27ac-dependent super-enhancers were gained at senescence-associated secretory genes; the CBP/p300 inhibitor A485 reverted the senescence-like state and restored TMZ cytotoxicity; On another hand, chromatin closed, reducing MYC occupancy at cell-cycle genes despite unchanged MYC protein, and suppressed nascent transcription and m6A modification, accompanied by cell-cycle arrest in some P-R cells. Furthermore, time-course analysis revealed that chromatin closure at cell-cycle genes is likely a temporally ordered epigenetic remodeling process, initiated by DNA damage-associated G2/M arrest mediated by WEE1 activation, followed by γH2AX-mediated recruitment of SUV39H1, which deposits H3K9me3 at MYC-bound loci to close chromatin. Functionally, early, but not delayed, inhibition of WEE1 with the novel brain-penetrant inhibitor TSL2304 blocked senescence-like epigenetic reprogramming, synergized with TMZ in vitro, and significantly prolonged survival in an orthotopic glioblastoma model. Together, these findings reveal a temporally ordered epigenetic remodeling in MYC-high glioblastoma that promotes survival after early TMZ exposure, and identify early WEE1 inhibition as a strategy to synergize with TMZ.