Distinct p21 dynamics drive alternative routes to whole-genome duplication through a common CDK4/6-dependent polyploid G0 state.

Whole-genome duplication (WGD) fuels tumor evolution and therapy resistance, yet the molecular mechanisms governing the switch from the canonical mitotic cell cycle to the endoreplication cycle remain unclear. Here, we combine single-cell proteomics, manifold learning, and live-cell imaging to map the intersection of the mitotic and endoreplication cycles in breast cancer cells exposed to genotoxic agents. We identify two distinct routes to WGD driven by distinct p21 dynamics. High p21 induction induces G2 exit and endocycling, whereas insufficient p21 permits mitotic entry followed by slippage and endomitosis. This therapy-induced switch acts as a facultative stress response, generating drug-resistant polyploid populations that propagate genomic instability through replication stress and the generation of replication-competent micronuclei. Both paths to WGD converge on a common polyploid G0 state dependent on cyclin D1:CDK4/6 activity to complete the transition to the endoreplication cycle, revealing a shared vulnerability. Sequential treatment with genotoxic agents followed by CDK4/6 inhibitors preserves the cytotoxic efficacy of DNA-damaging drugs while simultaneously blocking entry into the endoreplication cycle and WGD-driven evolutionary rescue. These findings reveal the molecular rules governing the switch from the mitotic to endoreplication cycle and highlight the potential of WGD-blocking drugs as adjuvant therapies to inhibit drug resistance and suppress tumor evolution.