CDK4/6 inhibitors arrest the cell cycle in G1-phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a temporary cell cycle arrest in G1 causes long-lasting effects on tumour growth. Here we demonstrate that a prolonged G1-arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. This causes a failure in DNA replication after release from that arrest, resulting in a p53-dependent withdrawal from the cell cycle. If p53 is absent, then cells bypass the G2-checkpoint and undergo a catastrophic mitosis resulting in excessive DNA damage. These data therefore link CDK4/6 inhibition to genotoxic stress; a phenotype that is shared by most other broad-spectrum anti-cancer drugs. This provides a rationale to predict responsive tumour types and effective combination therapies, as demonstrated by the fact that chemotherapeutics that cause replication stress also induce sensitivity to CDK4/6 inhibition.

CDK4/6 inhibitors arrest the cell cycle in G1‐phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long‐lasting effects on tumour growth. Here, we demonstrate that a prolonged G1 arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. Upon release from that arrest, many cells fail to complete DNA replication and exit the cell cycle in a p53‐dependent manner. If cells fail to withdraw from the cell cycle following DNA replication problems, they enter mitosis and missegregate chromosomes causing excessive DNA damage, which further limits their proliferative potential. These effects are observed in a range of tumour types, including breast cancer, implying that genotoxic stress is a common outcome of CDK4/6 inhibition. This unanticipated ability of CDK4/6 inhibitors to induce DNA damage now provides a rationale to better predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

Cell size and the cell cycle are intrinsically coupled and abnormal increases in cell size are associated with senescence. The mechanism by which overgrowth primes cells to exit the cell cycle remains unclear. We investigate this using CDK4/6 inhibitors that arrest cell cycle progression in G0/G1 and are used to treat ER+/HER2- metastatic breast cancer. We demonstrate that long-term CDK4/6 inhibition promotes cellular overgrowth during the G0/G1 arrest, causing widespread proteome remodeling and p38-p53-p21-dependent cell cycle exit. Cell cycle exit is triggered by two waves of p21 induction. First, overgrowth during a G0/G1 arrest induces an osmotic stress response, producing the first wave of p21 induction. Second, when CDK4/6 inhibitors are removed, a fraction of cells escape G0/G1 arrest and enter S-phase where overgrowth-driven replication stress results in a second wave of p21 induction that causes cell cycle withdrawal from G2, or the subsequent G1. This could explain why cellular hypertrophy is associated with senescence and why CDK4/6 inhibitors have long-lasting anti-proliferative effects in patients.

A long-term goal in cancer research has been to inhibit the cell cycle in tumour cells without causing toxicity in proliferative healthy tissues. The best evidence that this is achievable is provided by CDK4/6 inhibitors, which arrest the cell cycle in G1, are well-tolerated in patients, and are effective in treating ER+/HER2- breast cancer. CDK4/6 inhibitors are effective because they arrest tumour cells more efficiently than some healthy cell types and, in addition, they affect the tumour microenvironment to enhance anti-tumour immunity. We demonstrate here another reason to explain their efficacy. Tumour cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing exit from G1 or by inducing replication stress and genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize cells to CDK4/6 inhibition. Together, this demonstrates how oncogenic signals that have evolved to stimulate constitutive tumour growth and proliferation can be driven to cause toxic cell growth and irreversible cell cycle exit when proliferation is halted in G1.

CDK4/6 inhibitors arrest the cell cycle in G1 and are used in combination with hormone therapy to treat advanced HR+/HER- breast cancer. To allow more effective use of these drugs in breast cancer, and to facilitate their use in other tumour types, biomarkers that can predict response are urgently needed. We demonstrate here that previous large-scale screens designed to identify the most sensitive tumour types and genotypes have misrepresented the responsive cell lines because of a reliance on ATP-based proliferation assays. When cells arrest in G1 following CDK4/6 inhibition, they continue to grow in size, producing more mitochondria and ATP. This cellular overgrowth masks an efficient arrest using metabolic ATP-based assays, but not if DNA-based assays are used instead. By comparing tumour cells using different assay types, we demonstrate that the lymphoma lines previously identified as the most responsive cell types, simply appear to respond the best because they fail to overgrow during the G1 arrest. Similarly, the CDK4/6 inhibitor abemaciclib appears to inhibit proliferation better than palbociclib, but this is because it also inhibits cell overgrowth through off-target effects. DepMap analysis of previous screening data using only the reliable assay types, demonstrates that palbociclib-sensitivity is associated with sensitivity to Cyclin D1, CDK4 and CDK6 knockout/knockdown, and resistance is associated with sensitivity to Cyclin E1, CDK2 and SKP2 knockout/knockdown. Furthermore, potential biomarkers of palbociclib-sensitivity are increased expression of Cyclin D1 (CCND1) and RB1, and reduced expression of Cyclin E1 (CCNE1) and CDKN2A. None of these associations are present when analysing DepMap using similar data from metabolic assays. This reinforces the importance of new screens to assess CDK4/6 inhibitors against a wide range of cancer cell types using an appropriate proliferation assay. This would help to better inform clinical trials and to identify much needed biomarkers of response.