CDK4/6 inhibitors are among a new generation of therapeutics. Building upon the striking success of the combination of CDK4/6 inhibitors and the hormone receptor antagonist letrozole in breast cancer, many other combinations have recently entered clinical trials in multiple diseases. To achieve maximal benefit with CDK4/6 inhibitors it will be critical to understand the cellular mechanisms by which they act. Here we highlight the mechanisms by which CDK4/6 inhibitors can exert their anti-tumor activities beyond simply enforcing cytostatic growth arrest, and discuss how this knowledge may inform new combinations, improve outcomes, and modify dosing schedules in the future.

CDK4 inhibitors (CDK4i) earned Breakthrough Therapy Designation from the FDA last year and are entering phase III clinical trials in several cancers. However, not all tumors respond favorably to these drugs. CDK4 activity is critical for progression through G1 phase and into the mitotic cell cycle. Inhibiting this kinase induces Rb-positive cells to exit the cell cycle into either a quiescent or senescent state. In this report, using well-differentiated and dedifferentiated liposarcoma (WD/DDLS) cell lines, we show that the proteolytic turnover of MDM2 is required for CDK4i-induced senescence. Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state. Reducing MDM2 in these cells drives them into the more stable senescent state. CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome. These insights identify MDM2 and ATRX as new regulators controlling geroconversion, the process by which quiescent cells become senescent, and this insight may be exploited to improve the activity of CDK4i in cancer therapy.

Senescence is a state of stable cell cycle exit with important implications for development and disease. Here, we demonstrate that the chromatin remodeling enzyme ATRX is required for therapy-induced senescence. ATRX accumulates in nuclear foci and is required for therapy-induced senescence in multiple types of transformed cells exposed to either DNA damaging agents or CDK4 inhibitors. Mobilization into foci depends on the ability of ATRX to interact with H3K9me3 histone and HP1. Foci form soon after cells exit the cell cycle, before other hallmarks of senescence appear. Eliminating ATRX in senescent cells destabilizes the senescence-associated heterochromatic foci. Additionally, ATRX binds to and suppresses expression from the HRAS locus; repression of HRAS is sufficient to promote the transition of quiescent cells into senescence and preventing repression blocks progression into senescence. Thus ATRX is a critical regulator of therapy-induced senescence and acts in multiple ways to drive cells into this state.

Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma. While the initial treatment strategy is highly effective, relapse occurs in 40% of cases. Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs but their single agent efficacy against relapsed DLBCL has been variable, ranging from few complete/partial responses to some stable disease. However, most patients showed no response to HDACi monotherapy for unknown reasons. Here we show that sensitivity and resistance to the hydroxamate HDACi, PXD101, can be modeled in DLBCL cell lines. Sensitivity is characterized by G2/M arrest and apoptosis and resistance by reversible G1 growth arrest. These responses to PXD101 are independent of several negative prognostic indicators such as DLBCL subtype, BCL2 and MYC co-expression, and p53 mutation, suggesting that HDACi might be used effectively against highly aggressive DLBCL tumors if they are combined with other therapeutics that overcome HDACi resistance. Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation. PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G1 arrest. The results suggest several treatment strategies that might increase the efficacy of HDACi against aggressive DLBCL.

CDK4/6 inhibitors are being used to treat a variety of human malignancies. In well-differentiated and dedifferentiated liposarcoma their clinical promise is associated with their ability to downregulate the MDM2 protein. The downregulation of MDM2 following treatment with CDK4/6 inhibitors also induces many cultured tumor cell lines derived from different types of malignancies to progress from quiescence into senescence. Here we used cultured human cell lines and defined a role for PDLIM7 and CDH18, regulating MDM2 protein in CDK4/6 inhibitor-treated cells. Materials from our previous phase II trials with palbociclib were then used to demonstrate that expression of CDH18 protein was associated with response, measured as both progression-free survival and overall survival. This supports the hypothesis that the biologic transition from quiescence to senescence has clinical relevance for this class of drugs.