MOLECULAR CANCER THERAPEUTICS | REVIEW The Ongoing Search for Biomarkers of CDK4/6 Inhibitor Responsiveness in Breast Cancer Scott F. Schoninger1 and Stacy W. Blain2 ABSTRACT ◥ CDK4 inhibitors (CDK4/6i), such as palbociclib, ribociclib, benefit from these agents, often switching to chemotherapy and abemaciclib, are approved in combination with hormonal within 6 months. Some patients initially benefit from treatment, therapy as a front-line treatment for metastatic HRþ,HER2- but later develop secondary resistance. This highlights the need breast cancer. Their targets, CDK4 and CDK6, are cell-cycle for complementary or companion diagnostics to pinpoint – regulatory proteins governing the G1 S phase transition across patients who would respond. In addition, because CDK4 is a many tissue types. A key challenge remains to uncover biomar- bona fide target in other tumor types where CDK4/6i therapy is kers to identify those patients that may benefit from this class of currently in clinical trials, the lack of target identification may drugs. Although CDK4/6i addition to estrogen modulation ther- obscure benefit to a subset of patients there as well. This review apy essentially doubles the median progression-free survival, summarizes the current status of CDK4/6i biomarker test devel- overall survival is not significantly increased. However, in reality opment, both in clinical trials and at the bench, with particular only a subset of treated patients respond. Many patients exhibit attention paid to those which have a strong biological basis as primary resistance to CDK4/6 inhibition and do not derive any well as supportive clinical data. Introduction well (7). Although these results are promising, the clinical use of CDK4/6 inhibitors is confounded by the high individual variability in Breast cancer is the most common women's cancer worldwide, clinical response. Many patients exhibit primary resistance to CDK4/6 comprising 25% of total new cases diagnosed in 2018, and is the second inhibition and do not derive any benefit from treatment with these leading cause of cancer-related deaths (1). Treatment depends on agents, often switching to chemotherapy within 6 months. Many other hormone receptor status, as well as the expression of the receptor patients derive some benefit from treatment, but invariably become tyrosine kinase HER2/neu. For patients with estrogen receptor (ER) refractory, defined as secondary resistance. and/or progesterone receptor (PR)–positive (HRþ), HER2-negative Most targeted anticancer agents have a companion or comple- metastatic disease, antiestrogen therapy is the backbone of treatment, mentary diagnostic biomarker to determine which patients have the which can be accomplished by ER downregulation (e.g., fulvestrant) or actionable target, and are therefore likely to respond to treatment. A modulation (e.g., tamoxifen), or by aromatase inhibition (e.g., letro- complementary diagnostic is a test which provides information zole; ref. 2). relevant to determining the risk versus benefit of a particular Palbociclib, ribociclib, and abemaciclib are first-generation cyclin- treatment, but is not required by regulatory agencies in order to dependent kinase 4/6 (CKD4/6) inhibitors (CDK4/6i), which have use the treatment (8). Complementary diagnostics often provide demonstrated activity against HRþ, HER2- breast cancer (3–5). information relevant to a specific class of drugs, as opposed to a Beginning with the FDA approval of palbociclib in 2015, the addition specific agent. A companion diagnostic, on the contrary, provides of these agents to antihormonal therapy has become a first-line option information that is required prior to using a drug, and is therefore for patients with the HRþ, HER2- phenotype in the metastatic frequently marketed with the drug. A classic example is HER2 setting (2). In the pivotal PALOMA-2 study, palbociclib, in combi- testingbyIHCorFISH,asonlypatientswithdocumentedHER2þ nation with letrozole, was shown to increase median progression-free status are offered treatment with trastuzumab. At the current time, survival (PFS) from 14.5 to 24.8 months, compared with letrozole no clinically available biomarkers, other than ER/PR expression, are alone, in patients who had received no prior therapy for metastatic used to prescribe CDK4/6 inhibitors (9–13). Therefore, many disease (6). The PALOMA-3 trial subsequently evaluated palbociclib patients receive treatment with these agents but do not benefit, in combination with fulvestrant in patients with metastatic disease, and there are likely also many patients who could benefit, but are whom had progressed on prior endocrine therapy. The addition of never offered treatment with one of these agents. One difficulty with palbociclib to fulvestrant increased median PFS from 4.6 to 9.5 months, the identification of a CDK4/6i biomarker is the fact that CDK4/6 leading to FDA approval of palbociclib in this second-line setting as activity is due to a phosphorylated multiprotein complex, and simple assessment of the complex'scomponentshasproveninef- fective in predicting response. 1College of Medicine, SUNY Downstate Medical Center, Brooklyn, New York. 2Departments of Pediatrics and Cell Biology, SUNY Downstate Medical Center, Brooklyn, New York. Mechanism of action of CDK4/6 inhibitors CDK4 and CDK6 have been sought-after drug targets since their Corresponding Author: Stacy W. Blain, SUNY Downstate Medical Center, 450 discovery in the early 1990s. As cell-cycle regulatory proteins, they Clarkson Ave., BSB 7-80, Brooklyn, NY 11203. Phone: 718-270-4471; Fax: 718- fi 270-1985; E-mail: [email protected] exist downstream of other oncogenic pathways, as a nal common hub in cell-cycle progression (Fig. 1). Cyclin D complexes with CDK4 or Mol Cancer Ther 2020;19:3–12 – CDK6 (DK4/6) to regulate the G1 S phase transition, allowing for doi: 10.1158/1535-7163.MCT-19-0253 progression through this checkpoint and the subsequent replication of Ó2020 American Association for Cancer Research. DNA in S phase (ref. 14; Fig. 2). DK4/6 is actually a ternary complex AACRJournals.org | 3 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Schoninger and Blain EGFR CDK4/6i RAS Erb2/Her2 palbociclib ribociclib abemaciclib RAF P ERK Hormone receptors Y CDK4/6 ER ER ON AR p16 Cyclin D P P P P CDK2 RB E2F S phase genes RB Cyclin E E2F Cyclin E CDK2 Figure 1. All roads lead to cyclin D-CDK4/6, and as such has been a long sought-after therapeutic target. Cyclin D-CDK4/6 is downstream of most oncogenic signaling pathways, including tyrosine kinases, such as EGFR and Erb2/Her2, cytokine receptors, the Wnt and Hedgehog pathways, cadherin and integrin pathways, and the hormone receptors, estrogen (ER) and androgen (AR). Cyclin D is a direct transcriptional target of these pathways. Cyclin D-CDK4/6 phosphorylates RB, which permits cyclin E transcription, which then partners with CDK2 to fully phosphorylate RB, causing S phase–specific transcription. p16 is a member of the INK4A family and a specific CDK4/6 inhibitor. The active CDK4 complex is actually a trimer: cyclin D-CDK4/6-p27, as described in Fig. 2. The CDK4/6i, palbociclib, ribociclib, and abemaciclib are specific CDK4/6 inhibitors. and is only stable when associated with p27Kip1 or p21Cip1. p27 and Initial attempts at developing CDK inhibitors yielded relatively possibly p21 must be phosphorylated on residue Y88/89 (p27) or Y76 nonspecific drugs which inhibited multiple CDKs and were toxic (p21) to convert the closed ternary complex into an open, active above low doses. Palbociclib was the first highly specific CDK4/6 complex (15). The cyclin D-CDK4/6-p27 complex is translocated to inhibitor, with an affinity for CDK4 and CDK6 over 1,000 times greater the nucleus by virtue of p27's Nuclear Localization Signal (NLS). The than its affinity for other CDKs, such as CDK2 or CDK1 (14). open complex now permits ATP access to the CDK4 active site, and Significant adverse events (AE) in early clinical trials included neu- also renders CDK4 competent to be phosphorylated on residue T172 tropenia and thrombocytopenia, due to the reliance of bone marrow by the Cyclin Activating Kinase (CAK), which fully activates CDK4 progenitors on CDK6. Limited efficacy was seen in phase I studies in a activity. Active DK4/6 can phosphorylate the retinoblastoma protein variety of solid tumors and mantle cell lymphoma, a cyclin D1 over- (Rb), causing the release of E2F transcription factors, which in turn expressing tumor type. Based on preclinical data suggesting increased lead to the transcription of genes required for DNA replication and sensitivity in HRþ breast cancer cell lines, which will be discussed later, cell-cycle progression, including Cyclin E. This leads to a positive subsequent clinical trials were initiated in the HRþ, HER2- metastatic feedback loop where E2F-mediated expression of Cyclin E drives breast cancer population, eventually leading to approval of the drug in activation of CDK2, which further phosphorylates Rb and causes the 2015. Two other CDK4/6 inhibitors, ribociclib and abemaciclib, have full release of E2F. also been approved for this population. Although these three drugs are 4 Mol Cancer Ther; 19(1) January 2020 MOLECULAR CANCER THERAPEUTICS Downloaded from mct.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Biomarkers for CDK4/6 Inhibitors Assembly 2 Association Y kinase (Brk) 3 with p27 or p21 BRK NLS CDK4/6 P OFF NLS p27 Y CDK4/6 CDK4/6 CDK4/6 OFF OFF ON Cyclin D Cyclin D Cyclin D NLS Cyclin D Catalytic Substrate binding p27 Y domain domain 4 phosphorylation Synthesis 1 of cyclin D 5 Nuclear translocation P CAK phosphorylation NLS Y 6 CDK4/6 CAK ON P P Cyclin D RB CDK7 Cyclin H E2F ACTIVE Figure 2. The activation of cyclin D-CDK4/6 is complex, explaining why the levels of cyclin D or CDK4 are not predictive of CDK4/6i response.