The search for CDK4/6 inhibitor biomarkers has been hampered by inappropriate proliferation assays

CDK4/6 inhibitors are effective at treating advanced HR+ /HER2- breast cancer, however biomarkers that can predict response are urgently needed. We demonstrate here that previous large-scale screens designed to identify which tumour types or genotypes are most sensitive to CDK4/6 inhibitors have misrepresented the responsive cell lines because of a reliance on metabolic proliferation assays. CDK4/6-inhibited cells arrest in G1 but continue to grow in size, thereby producing more mitochondria. We show that this growth obscures the arrest using ATP-based proliferation assays but not if DNA-based assays are used instead. Furthermore, lymphoma lines, previously identified as the most sensitive, simply appear to respond the best using ATP-based assays because they fail to overgrow during the G1 arrest. Similarly, the CDK4/6 inhibitor abemaciclib appears to inhibit proliferation better than palbociclib because it also restricts cellular overgrowth through off-target effects. DepMap analysis of screening data using reliable assay types, demonstrates that palbociclib-sensitive cell types are also sensitive to Cyclin D1, CDK4 and CDK6 knockout/knockdown, whereas the palbociclib-resistant lines are sensitive to Cyclin E1, CDK2 and SKP2 knockout/knockdown. Potential biomarkers of palbociclib-sensitive cells are increased expression of CCND1 and RB1, and reduced expression of CCNE1 and CDKN2A. Probing DepMap with similar data from metabolic assays fails to reveal these associations. Together, this demonstrates why CDK4/6 inhibitors, and any other anti-cancer drugs that arrest the cell cycle but permit continued cell growth, must now be re-screened against a wide-range of cell types using an appropriate proliferation assay. This would help to better inform clinical trials and to identify much needed biomarkers of response.</p

CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal

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

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence

Summary. Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2− breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients

Oncogenic signals prime cancer cells for toxic cell overgrowth during a G1 cell cycle arrest

CDK4/6 inhibitors are remarkable anti-cancer drugs that can arrest tumor cells in G1 and induce their senescence while causing only relatively mild toxicities in healthy tissues. How they achieve this mechanistically is unclear. We show here that tumor 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 progression from G1 or inducing 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 the cells to CDK4/6 inhibition. Together, this demonstrates that cell cycle arrest and oncogenic cell growth is a synthetic lethal combination that is exploited by CDK4/6 inhibitors to induce tumor-specific toxicity

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2− breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients