Proteome-wide target engagement and selectivity profiling of CDK2 inhibitors using CETSA®

Mitosis and the cell cycle is a pivotal part of cancers and other neoplastic diseases. The cell cycle is tightly regulated, yet frequently deregulated in tumors, making its core regulators attractive targets for anti-tumor therapy. Cyclin-dependent kinases (CDKs) form complexes with cyclins to phosphorylate key proteins that drive cell-cycle progression. Dual CDK4/6 inhibition is an established treatment strategy in subsets of breast cancer, but resistance is common. Targeting CDK2 has therefore emerged as a strategy to overcome resistance to CDK4/6 inhibition. While CDK4/6 primarily controls early cell-cycle progression by regulating phosphorylation of the retinoblastoma-associated protein (RB1), CDK2 participates in this phase and regulates later cell-cycle transitions. CDK2 inhibitors may thus offer additional therapeutic benefits compared with CDK4/6 inhibitors. Several CDK2-selective inhibitors have recently been developed, including Tagtociclib (PF-07104091), Cirtociclib (BLU-222), and INX-315. Here, we investigated the proteome-wide target engagement and selectivity profiles of these CDK2-selective kinase inhibitors and compared them with those of approved CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) and pan-CDK probes. We utilized the cellular thermal shift assay (CETSA®) coupled with mass spectrometry (MS). CETSA monitors drug-induced changes in protein thermal stability and can be applied in intact cells or cell lysates without modifying proteins, ligands, or cells. An MS-based readout enables unbiased, proteome-wide detection of small-molecule-protein interactions, including non-kinase targets and downstream pathway components. All tested CDK2-selective inhibitors engaged CDK2 and additional kinases at high concentrations of 30 µM. Compared with CDK4/6 inhibitors, they showed weaker engagement of CDK4/6 but, like CDK4/6 inhibitors, induced thermal destabilization of RB1, indicating a shared impact on RB1-dependent pathways. Target engagement of CDK1 was restricted to CDK2-selective and pan-CDK inhibitors and was only observed at higher compound concentrations. Together, these data illustrate the utility of CETSA MS for mapping target engagement and selectivity across the proteome, as well as for characterizing the evolution of CDK inhibitors from early pan-CDK compounds to modern CDK2-selective agents.