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Targeting the Cyclin-Dependent Kinases (CDK) 4/6 in Estrogen Receptor-Positive Breast Cancers Richard S

Targeting the Cyclin-Dependent Kinases (CDK) 4/6 in Estrogen Receptor-Positive Breast Cancers Richard S

Finn et al. Breast Research (2016) 18:17 DOI 10.1186/s13058-015-0661-5

REVIEW Open Access Targeting the -dependent (CDK) 4/6 in -positive breast Richard S. Finn1*, Alexey Aleshin1 and Dennis J. Slamon2

Abstract Despite significant advances in early detection and treatment, still remains a major cause of morbidity and mortality for women. Our understanding of the molecular heterogeneity of the disease has significantly expanded over the past decade and the role of cycle signaling in both breast cancer oncogenesis and anti-estrogen resistance has gained increasing attention. The mammalian is driven by a complex interplay between and their associated cyclin-dependent (CDK) partners, and dysregulation of this process is one of the hallmarks of cancer. Despite this, initial results with broadly acting CDK inhibitors were largely disappointing. However, recent preclinical and phase I/II clinical studies using a novel, oral, reversible CDK4/6 inhibitor, (PD-0332991), have validated the role of CDK4/6 as a potential target in estrogen receptor-positive (ER+) breast cancers. This review highlights our current understanding of CDK signaling in both normal and malignant breast tissues, with special attention placed on recent clinical advances in inhibition of CDK4/6 in ER+ disease.

Background regulators of some 40 years ago [2]. Breast cancer is a global disease, with a yearly incidence of subsequently found the human homologues to these over 1.3 million, accounting for over 23 % of all malignan- and named the family cyclin-dependent kinases (CDKs) [3]. cies [1]. Our knowledge of the molecular diversity and In the early 1980s Tim Hunt discovered cyclin molecules in drivers of specific subtypes of breast cancer has paved the his studies of sea urchin egg division [4]. These molecules way for the rational design and clinical development of were named on the basis of their cyclical appearance and targeted agents. These are designed to increase efficacy were found to play an important role in binding and while sparing many of the traditional toxicities associated activating CDK . This critical array of activators with and the success of this approach has and kinases is now known to be central in regulating cell been clearly demonstrated by the development of anti- division and these important accomplishments were recog- estrogens and HER2-targeted agents for receptor- nized by the 2001 Noble Prize in Physiology and Medicine. positive and HER2-amplified breast cancers, respectively. Today the cell cycle is viewed as an orderly progres- Despite these advances in our treatment armamentarium, sion of distinct phases (G1, S, G2, M), with various many patients still develop resistance to both targeted and cyclin/CDK combinations being essential in regulating non-targeted therapeutics, ultimately developing fatal this process. Pursuant to these pivotal observations, disease and underscoring the need for new therapeutic multiple studies have linked alterations in cell cycle approaches. biology to cancer. In breast cancer, alterations in sev- Using temperature-sensitive mutants, Lee Hartwell eral cell cycle regulatory proteins have been described, first identified cell division cycle (CDC) genes as key including various cyclins, CDKs, and the RB product (pRb) [5–7]. Evidence indicates that dysregu- * Correspondence: [email protected] lation of the :CDK4/6 axis has a role in 1 Geffen School of Medicine at UCLA, Department of Medicine, Division of breast cancer, with some tumors overexpressing cyclin Hematology Oncology, 2825 Santa Monica Blvd, Santa Monica, CA 90404, USA D1 [5]. Additionally, while not necessary for normal Full list of author information is available at the end of the article

© 2016 Finn et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Finn et al. Breast Cancer Research (2016) 18:17 Page 2 of 11

mammary gland development, CDK4 and cyclin D1 component of the CDK-activating complex, and tran- are required for induction of breast malignancies in scriptional CDKs (CDK8, CDK9) [11–13]. Unlike mouse models, suggesting that CDK4 inhibition may CDKs, cyclins are an extremely diverse family of pro- inhibit breast cancer cells while sparing healthy tis- teins, subdivided into four classes (A-, B-, D-, E-type sues [6, 7]. The above data seemed to suggest that cyclins) that act as regulatory subunits of the CDK- pharmacological inhibition of the cyclin D1:CDK4/6 cyclin holoenzyme [11]. Despite the large number of axis in cancers may be both efficacious and relatively CDKs and cyclins, only a few have been strongly non-toxic. However, the initial clinical experience with implicated in breast cancer pathogenesis. This review broad specificity, first-generation CDK inhibitors focuses primarily on CDK4 and CDK6, which have proved to be disappointing, yielding poor efficacy and largely overlapping though not entirely identical speci- significant toxicity and raising the question of ficity, as well as cyclin D1, the most characterized whether these agents failed due to poor phamacologic member of the D-type (D1, D2, D3) cyclin family [14]. characteristics and/or specificities of the compounds Typically, repression of cell cycle progression is or a less essential role of CDK signaling in cancer. maintained via sequestration of the family of Additionally, lack of appropriate patient selection factors by the retinoblastoma gene and/or lack of predictive markers of response may product (pRb), and other so-called pocket proteins, have also contributed to these initial clinical failures. including p107 and p130 [15]. Upon entering the cell Recently, the development of more specific CDK in- cycle, however, quiescent cells synthesize cyclin D1 hibitors has renewed interest in targeting the cell in response to specific mitogenic and adhesion sig- cycle as a novel therapeutic approach in cancer. In a nals. Newly synthesized cyclin D1 goes on to form series of preclinical studies using cell line models of activating complexes with CDK4/CDK6, which then human breast cancers, we demonstrated significant initiate phosphorylation of pRb. The process of growth inhibitory activity of palbociclib (PD-0332991), phosphorylation mediated by the cyclin D1:CDK4/6 which is a highly selective inhibitor of CDK4/6 [8]. complex lifts pRb’s transcriptional repression of E2F, These observations were followed by a logical transla- resulting in transcription of S-phase-specific target tion of the laboratory findings into a phase I/II clin- genes. One of these genes encodes , which ical study that has now demonstrated significant associates with CDK2 and further phosphorylates clinical activity in patients with advanced estrogen pRb as well as other key mediators of the G1/S receptor-positive (ER+) breast cancer [9]. checkpoint. This process sets up a In this review, we further describe the role of loop committing cells to irreversibly undergo the cyclin:CDK activity in regulating the cell cycle and G1–S transition (the so called '') and focus on the central role of :CDK4/6 activity to continue the cell cycle in a -independent in both normal and malignant tissues. Finally, we manner [16, 17]. In addition to pRb phosphorylation, discuss the preclinical and clinical experience with recent studies have implicated cyclin D:CDK4 as CDK inhibitors with particular emphasis on selective directly acting on pathways involved in proliferation, CDK4/6 inhibitors. migration, and response to DNA damage [18–21] by phosphorylating targets such as SMAD2, Cdt1, Role of CDK4/6 in cell cycle control MARCKS,FOXM1,andPRMT5–MEP50 complex The basic regulatory framework of the cell cycle has [18, 21–24]. been extensively investigated and reported in the In late CDK2 is further activated by cyclin literature. It is more extensively reviewed elsewhere A2, enabling transition from S phase to . [10, 11] but a brief summary of these important prior Lastly, CDK1 is activated by A-type and B-type cyclins findings follows. to facilitate the onset and progression of the actual The mammalian cell cycle is classically partitioned process of [11]. into four distinct phases, termed G1, S, G2, and M. An orderly progression between these phases is tightly Negative regulators of CDK4/6 signaling controlled at 'checkpoints' by the interplay of various CDK4/6 activity is negatively regulated by two families cyclins and their associated CDKs [12] (Fig. 1). At of cyclin kinase inhibitors (CKIs), the INK4 (, p15, least 12 separate genetic loci are known to code for p18, p19) and CIP/KIP (, p27, p57) families the CDKs and belong to a well conserved family of [11, 25]. These inhibitors, while largely undetectable serine/ protein kinases. This family includes in cycling cells, are rapidly upregulated in response to three CDKs (CDK2, CDK4, CDK6), one inhibitory signals, including transforming growth mitotic CDK (CDK1, previously known as CDC2), and factor-β, contact inhibition, or senescence [26, 27]. a number of regulatory CDKs, such as CDK7, a The effect of the CIP/KIP family on the cell cycle Finn et al. Breast Cancer Research (2016) 18:17 Page 3 of 11

Fig. 1 The cyclin D/cyclin-dependent kinase (CDK)4/6/retinoblastoma (Rb) Pathway and the cell cycle. The mammalian cell cycle is tightly regulated. In the context of breast cancer, both steroid and peptide growth factors drive proliferation through cyclin D/CDK4/6 activation. This results in the hyper-phosporylation of pRb as G1 progresses. When (pRb) is hyper-phosphorylated, the E2F is released and the cell cycle progresses through S phase. Small molecule kinase inhibitors of CDK4/6 aim to block the hyper-phosphorylation of pRb inducing a G1 arrest and preventing proliferation. ER estrogen receptor

machinery is complex and can be both activating and Non-catalytic functions of the cyclin D:CDK4/6 inhibitory under different circumstances [10]. Interest- pathway ingly, tamoxifen is known to upregulate p21 as well as Not all effects of the cyclin D:CDK4/6 pathway are p27 and the loss of these cell cycle inhibitors has been driven by phosphorylation, and a non-catalytic role of implicated in anti-estrogen resistance [28]. Within the cyclin D1 is being increasingly recognized. Cyclin D1 is INK4 (inhibitors of CDK4) family of proteins, p16 now also implicated in transcriptional regulation of seems to be most directly implicated in the pathogen- many genes by acting directly at regions and esis of many malignancies and when bound to CDK4/ regulating histone and methylation [31–33]. 6 abrogates the ability of cyclin D1 to bind effectively, Cyclin D1 has been shown to interact with ER-alpha, thereby triggering a G1 cell cycle arrest [29]. enhancing its activity, while inhibiting the activity of an- Additionally, p16 has been implicated in activation of drogen receptor (AR), thyroid hormone receptor-β and cellular senescence defined as a stable and long-term peroxisome proliferator activated receptor-γ (PPARγ) loss of proliferative capacity and is another process [34]. Another well described non-catalytic function of that is frequently dysregulated in cancer [30]. cyclin D1 is the sequestration of p21 and p27, leading to Finn et al. Breast Cancer Research (2016) 18:17 Page 4 of 11

CDK4/6-independent effects on migration and the DNA pathogenesis derives from studies demonstrating that damage response [35, 36]. The relative degrees to which the cyclin D1:CDK4/6 axis is critical for breast cancer these non-catalytic functions of cyclin D1 are physiolo- maintenance and progression. This is based on data gically relevant in the cell cycle specifically and regula- showing ErbB2-driven tumor arrest and senescence tion of cell division and motility are still being in vivo in response to acute cyclin D1 ablation or determined but they should be considered when evaluat- targeted inhibition of CDK4/6 [47]. ing the effects of inhibition of CDK4/6-mediated While cyclin D1:CDK4/6 complexes have a central signaling. role in regulating the initiation of the cell cycle, activat- ing in CDK4/6 are exceedingly rare in cancer. Role of CDK4/6 in normal development Nevertheless, amplification of CDK4 and cyclin D1 have Given the inherent linearity of CDK–cyclin activation been reported in upwards of 15–25 % of breast cancers, during the cell cycle, it was long believed that loss of an while overexpression of cyclin D1 has been reported to individual CDK would have deleterious effects on cellu- occur in over half of all breast cancers in some published lar proliferation and embryonic development. This no- studies [44, 45, 50, 51]. The recent Cancer Genome tion has been called into question by separate mouse Atlas publication presented data from 510 tumor speci- knockouts of CDK2, CDK3, CDK4, and CDK6, all of mens from 507 patients for which a comprehensive gen- which are viable [37–40]. However, double knockouts of omic analysis was performed [52]. When analyzed by CDK4 and CDK6 and triple knockouts of cyclin D1, D2, intrinsic subtype of breast cancer, alterations in cell cycle and D3 develop largely normally but die in mid/late genes varied, with cyclin D1 amplification being found gestation from severe anemia and heart abnormalities, most frequently in the luminal A, B and HER2 enriched respectively. Embryonic fibroblasts from these mice subtypes at frequencies of 29 %, 58 %, and 38 %, respect- essentially proliferate normally, despite an increased ively. Conversely, amplification of was more mitogen requirement and slower S phase entry, but they common in the basal subtype. Similar to cyclin D1, gains also display less propensity for oncogenic transformation in CDK4 were more common in the luminal A, B and [41, 42]. These data indicate that CDK4 and CDK6 activ- HER2 enriched subgroups: 14 %, 25 %, and 24 %, re- ity may be dispensable in some developmental and spectively. Additional alterations that would be hypothe- normal cellular functions, suggesting that targeted inhib- sized to antagonize CDK4/6 dependence, such as lower ition could be relatively well tolerated by normal tissues. pRb expression or RB loss/, were common in the basal type as well (20 % for mutation/loss). Role of CDK4/6 in breast cancer pathogenesis Amplification of both cyclin D1 and CDK4 is espe- Alterations in the mechanisms governing the cell cycle cially high in luminal B (58 % and 25 %, respectively) are considered a 'hallmark of cancer' and result in un- and HER2-expressing subtypes (38 % and 24 %, respect- controlled cellular proliferation [43]. Numerous lines of ively), intermediate in luminal A (29 % and 14 %, re- evidence point to an important role of a dysregulated spectively), and lower in basal-like tumors that tend to cyclin D1:CDK4/6 complex in both the initiation and also have frequent loss of pRb [52]. In retrospect, other progression of many cancers, including breast cancer. alterations that would antagonize CDK4/6 dependence, Dysregulation of the cyclin D1:CDK4/6 axis appears to such as lower RB expression or RB loss/mutation, are be an early step in breast cancer pathogenesis given that more common in the basal subtype as well. 'overexpression' of cyclin D1 is frequently found as early as ductal carcinoma in situ and maintained in metastatic Interplay of CDK4/6 and endocrine signaling in lesions but is absent in the earliest lesions such as atyp- breast cancer ical ductal hyperplasias [44, 45]. The D-type cyclins are The cross-talk between peptide growth factor and ster- known to be dispensable during mammary gland devel- oid hormone signaling has been an area of active re- opment, but are required for efficient tumor initiation as search in breast cancer and a focus of clinical research evidenced by the fact that mice lacking functional cyclin studies. ER and HER2 signaling appear to be putative D1 are resistant to cancers initiated by ErbB-2/HER2/ 'drivers' in the biology of about 60 % and 20–25 % of neu and ras , while null animals are breast cancers, respectively [53]. While the therapeutic refractory to Notch1-driven T cell acute lymphoblastic approaches to these subtypes focus on these respective [7, 46, 47]. Additionally, it appears that cyclin receptors, the two pathways potentially converge, ultim- D1 and D3 can compensate for one another in driving ately exerting their downstream effects on the cyclin tumor initiation and progression [48]. Similarly, CDK4 D:CDK4/6 pathway. expression appears to be required for ErbB-2 tumorigen- ER+ breast cancers are largely dependent on estrogen esis, but is dispensable for wnt-induced oncogenesis signaling for proliferation and survival [54], with ER inhib- [49]. Further evidence for their role in malignant ition leading to reduced tumor cell viability and cell cycle Finn et al. Breast Cancer Research (2016) 18:17 Page 5 of 11

arrest in the [55, 56]. ER signaling is known to [62]. At least a portion of this toxicity is attributable to upregulate cyclin D1 levels and potentiate multiple signal- the inhibition of transcription by the compounds effects ing pathways largely culminating in upregulation of CDK4/ on CDK9 and possibly CDK7 that lead to depletion of 6 activity [57, 58]. Not surprisingly, hormone-based thera- short-lived cell cycle and anti-apoptotic mRNA transcripts peutic strategies form the backbone of treatment of ER+ [63]. Though this likely contributes to the in vitro efficacy breast cancers. However, not all ER+ cancers respond to of flavopiridol on tumors dependent on the expression of this approach and, among those that do, acquired resistance such transcripts, off-target effects in healthy tissues would is not uncommon. Data indicate that this may be mediated, contribute to the severe anti-proliferative toxicity ob- at least in some of these cancers, by deregulation of mul- served in multiple clinical trials of this compound [64]. tiple alternative mitogenic pathways (for example, HER2, Other examples of pan-CDK inhibitors include UCN- PI3K/AKT, and so on) that can potentiate cyclin D1:CDK4/ 01 and R-Rescovitine (seliciclib; Cyclacel). UCN-01 is a 6 signaling in an ER-independent fashion. Also, as men- staurosporine analog with broad activity against CDKs, tioned above, cyclin D1 can independently activate ER and AKT, Chk1, and . This drug showed a majority of cyclin D1 overexpressing breast cancers are good G1/S phase cell cycle arrest, induction of p21 and ER+ [51, 59]. These findings suggest a potential role for hypophosphorylation of pRb in preclinical models but cyclin D:CDK4/6-mediated signaling in the estrogen inde- phase I studies showed several dose-limiting toxicities, pendence of ER+ breast cancers [60]. including hyperglycemia, arrhythmia, and pulmonary dysfunction [65, 66]. Results of phase II studies in breast Therapeutic targeting of the cyclin D:CDK4/6 cancer were unimpressive [67]. pathway Cell cycle regulation has been identified as an attractive Second-generation CDK inhibitors target for targeted drug . Given their kinase ac- As mentioned, until recently, CDK inhibitors have shown tivity, the CDKs were pursued as drug targets. A large largely disappointing results in terms of clinical efficacy, number of drug discovery programs have yielded potent safety, and tolerability. One of the main issues associated small molecule CDK inhibitors, with several compounds with first-generation inhibitors is the low specificity toward successfully entering preclinical and early clinical trials. the target kinases, which can explain their unpredictable Until relatively recently, however, many CDK inhibitors and serious side effect profiles. Additionally, some of these have shown poor clinical activity accompanied by an agents suffered from suboptimal dosing schedules, typically undesirable adverse event profile. In general, CDK inhib- focusing on intravenous bolus administration that may be itors can be broken down into two classes: first- insufficient for many solid tumor types that have doubling generation inhibitors such as flavopiridol, R-roscovitine, times in the order of days. Based on these observations, so- and UCN-01, which tended to be less specific and broad called second-generation CDK inhibitors were developed in in their ability to block a number of CDKs (pan-CDK the late 1990s and early 2000s that showed preferential in- inhibitors); and second-generation agents that are more hibition of specific CDK subtypes. Initial efforts focused specific to certain CDKs. The latter group of compounds mainly on CDK2 inhibition, given the availability of X-ray has now shown more potent activity against their targets crystallographic structures of CDK2 (CDK4 has subse- and a more favorable safety profile. quently been crystalized) [68].

The first-generation CDK inhibitors Specific CDK4/6 inhibitors As mentioned, most of the first-generation compounds Recently, a number of inhibitors specific for CDK4 and are not specific for any single CDK and act pri- CDK6 have entered clinical testing (Table 1). Palbociclib marily as pan-CDK inhibitors. Despite initial enthusiasm generated by preclinical studies, however, many of these compounds suffered from low activity and/or toxicity in Table 1 Current CDK4/6 inhibitors in clinical development clinical studies. Compound Manufacturer Phase CDK4/6 in vitro inhibition profile (IC50, nM) Flavopiridol (National Cancer Institute) is the most Palbociclib Pfizer, Inc. Approved CDK4 (cyclinD1): 11 studied of all first-generation CDK inhibitors, and is a (PD-0332991) classic pan-CDK inhibitor. In phase I and II studies, CDK4 (cyclinD3): 9 flavopiridol showed minimal single agent efficacy and CDK6 (cyclinD2): 15 was associated with several toxicities more typical of III CDK4 (cyclinD1): 10 (LEE011) traditional cytotoxic agents, including infusion site irrita- CDK6 (cyclinD2): 40 tion, gastrointestinal toxicity, and severe neutropenia Eli Lilly III CDK4 (cyclinD1): 2 [61]. In metastatic breast cancers in particular, flavopiri- (LY2835219) CDK6 (cyclinD1): 9.9 dol generated unacceptably high rates of neutropenia Finn et al. Breast Cancer Research (2016) 18:17 Page 6 of 11

(PD 0332991; Pfizer) is furthest along in clinical develop- compare progression-free survival (PFS) as its primary ment, having received US Food and Drug Administration endpoint with safety and overall survival as secondary (FDA) approval on 3 February 2015 for the first-line treat- endpoints and randomized patients to receive either ment of advanced post-menopausal ER+, HER2-negative alone or the combination of letrozole and breast cancer in combination with letrozole. It is an orally palbociclib. The study consisted of two parts that en- bioavailable, potent CDK4/6 inhibitor with an in vitro rolled sequentially: part 1 required that patient tu- kinase IC50 of 0.01 μM and high selectivity when evaluat- mors be ER+, the sole biomarker for study entry; part ing 36 other kinases including CDK2 (IC50 > 5 μM) [69]. 2 enrolled the same population but patient tumors Preclinical studies have shown that palbociclib behaves were also required to have either CCND1 (cyclin D1) very much like an agent specifically targeting CDK4/6. It amplification by fluorescence in situ hybridization exhibits potent inhibition of tumor cell proliferation ac- (FISH) or CDKN2A (p16) loss by FISH as selection companied by a pure G1 arrest, and dephosphorylation of biomarkers in addition to the ER+ biomarker. While pRb as well as a decrease in E2F-dependent gene expres- the preclinical data did not suggest that these gen- sion [70]. Further evidence of palbociclib's targeted design omic markers were required for augmented response, is the fact that it is completely inactive in pRb-negative part 2 of the study was designed to determine tumor cell lines and xenografts [9, 60, 70]. In phase I clin- whether the presence of these biomarkers might fur- ical studies palbociclib showed excellent bioavailability ther enrich the responsive patient population. with a generally mild to moderate adverse events profile Results from part 1 were presented at the IMPAKT with the major dose limiting toxicities being related meeting in 2012 [73]. About half the women in each mainly to myelosuppression [71]. arm had not received any prior neoadjuvant or adju- Using an unbiased screening approach we performed vant systemic treatment for their diagnosis but about preclinical work aimed at identifying breast cancers that a third had received prior anti-estrogen therapy in might be growth inhibited by palbociclib and predictive early breast cancer settings. There was a significant markers of drug response. This was done by evaluating improvement in PFS in part 1 with the median PFS palbociclib’s growth inhibition effects in a large panel of increasing from 5.7 months with letrozole alone to molecularly characterized human breast cancer cell lines. over 18 months with the combination, resulting in a This study identified that cell lines representing either hazard ratio (HR) of 0.35 (95 % confidence interval the luminal, ER+ or HER2-amplified subtypes were most (CI) 0.17–0.72, P = 0.06). In addition, in patients with sensitive to palbociclib inhibition while those represent- measurable disease the response rate increased from ing the non-luminal subtypes were most resistant [9]. 32 to 52 % and the clinical benefit rate increased This work also demonstrated consistent synergistic from 47 to 76 %. Dose reductions and delays were growth inhibitory activity between palbociclib and tam- common in the palbociclib arm, but again, the most oxifen or in ER+ and HER2-amplified cell common treatment-related adverse events were models, respectively. Lastly, the drug showed activity in leukopenia, neutropenia, and fatigue, although no in- a model of acquired tamoxifen resistance leading to the stances of neutropenic fevers were reported. Retro- concept that it may be clinically active in hormone- spective biomarker analysis for CCND1 amplification resistant, ER+ breast cancers. and p16 loss was performed in the 66 patients from These data were used to support the clinical develop- part1.Thoughthegroupsweresmall,theHRsfor ment of palbociclib in a phase I/II study of frontline each group demonstrated a consistent benefit regard- treatment of advanced ER+ post-menopausal breast can- less of the presence or absence of these biomarkers; cer with a combination of palbociclib and letrozole. The biomarkers present (n = 21) HR = 0.37 (95 % CI 0.10–1.40, phase I portion enrolled 12 patients and was designed to P = 0.13), biomarkers absent (n = 25) HR = 0.19 (95 % CI evaluate the safety of a dosing regimen consisting of 0.05–0.67, P < 0.01), biomarker unknown (n = 20) HR = 125 mg palbociclib orally given daily on a 3-week on/1- 0.59 (95 % CI 0.11–3.08, P = 0.53). These data support the week off regimen in combination with daily letrozole preclinical observation that ER positivity may be the best [72]. There were no treatment-related serious adverse selection biomarker for patients likely to benefit from events and the most common treatment emergent ad- CDK4/6 inhibition. verse events were leukopenia, neutropenia, and fatigue. An interim analysis combining parts 1 and 2, based However, there were no instances of neutropenic fever on 50 % of events of the 114 needed for the final and there were no dose–dose interactions between pal- PFS analysis, was presented at the 2012 San Antonio bociclib and letrozole. Breast Cancer Symposium and the final results have The phase II study was developed as an open label been published [73, 74]. These analyses included 165 trial in post-menopausal women with advanced ER+, patients and confirmed the benefit and safety profile frontline metastatic breast cancer. It was designed to observed initially in part 1. Specifically, the final Finn et al. Breast Cancer Research (2016) 18:17 Page 7 of 11

results demonstrated that median PFS increased from have progressed on or within 1 month of prior aroma- 10.2 months with letrozole alone to 20.2 months with tase inhibitor for advanced disease, or within 12 months the combination (HR = 0.488 (95 % CI 0.319–0.748, P of completion or discontinuation of therapy for adju- < 0.001)). In cohort 1, median PFS was 5.7 months in vant therapy. This study also allowed pre-menopausal the letrozole alone arm and was 26.1 months in the women that received goserelin as well. The safety combinationarm;incohort2thesenumberswere profile looked very similar to what was seen in the 11.1 months and 18.1 months, respectively. HRs for PALOMA-1/TRIO18 study. both cohorts, 0.299 for cohort 1 and 0.508 for cohort Single-agent activity of palbociclib has also been 2, confirmed a benefit regardless of the presence of evaluated in a single arm phase II trial of palbociclib cyclin D1 amplification or p16, suggesting the most in advanced, heavily pre-treated breast cancer [79]. important determinant for benefit in this study is be- Despite being tested in a heavily pre-treated cohort of ing ER+. The objective response rate for patients with patients (median lines of therapy = 3), single-agent measurable disease was increased from 39 to 54 % activity was noted (clinical benefit 21 %, stable disease with the addition of palbociclib and the clinical bene- >6 months 14 %). Importantly, as the preclinical data fit rate (complete response, partial response, and suggested, this activity was seen in women with ER+ stable disease >6 months) for the intent-to-treat or HER2-amplified breast cancers. Myelosuppression population improved from 58 to 81 %. The adverse again was the most frequently observed adverse event, event profile remained essentially the same. While the with 46 % of patients requiring dose reductions and incidence of grade 3 and 4 neutropenia was 48 % and 25 % requiring dose interruptions. 6 %, respectively, there were no cases of neutropenic In addition to palbociclib, two other small molecule complications (that is, febrile neutropenia or serious CDK4/6 inhibitors are currently in early clinical devel- infections). The lack of serious complications from opment. Both have had their development programs the neutropenia may be explained by the cytostatic expedited, going from phase I to phase III based on the effect of CDK4/6 inhibition on the bone marrow palbociclib experience. The molecules and ongoing which, compared with cytotoxic chemotherapy, results trials in breast cancer are highlighted in Tables 1 and 2, in a relatively short of neutropenia. In respectively. Phase I data with LY2835219 (abemaciclib; addition, no mucositis or skin toxicity was associated Eli Lilly) in patients with advanced malignancies was with palbociclib, which are often considered sources presented at the ASCO 2013 meeting [80]. In this dose of infection with chemotherapy-associated neutro- escalation study it was determined that the doses in the penia. Preclinical studies suggest that CDK4/6 inhib- expansion phase were to be 150 mg and 200 mg twice a ition induces a reversible pharmacologic quiescence in day continuously, without a dosing break like with pal- hematopoietic stem/progenitor cells that differs sig- bociclib. They concluded that it had an acceptable nificantly from cytotoxic effects and may explain the safety profile and early signals of clinical efficacy were clinical observation [75]. seen. Data on an expansion cohort of advanced breast Together, the safety and efficacy data from this study cancer patients have been presented as well [81, 82]. resulted in palbociclib receiving a 'Breakthrough Ther- Two cohorts were examined, one with single-agent abe- apy' designation from the US FDA and more recently maciclib and one with abemaciclib and for accelerated approval for advanced ER+ breast cancer ER+ disease. In the single-agent cohort, 47 patients [76, 77]. A phase III, double-blind, placebo-controlled with all subtypes of breast cancer were enrolled, but study designed to confirm the phase II observations has significant single-agent activity was seen only in women completed accrual and results are awaited (PALOMA- with ER+ breast cancer. The median lines of prior 2/TRIO-22, NCT01740427). Results of the PALOMA-3 therapy in this group were 7 (2–16). The overall re- study have recently been published and again demon- sponse rate in the 36 patients with ER+ disease was strate a significant improvement in PFS when palboci- 33 % and the disease control rate was 80.6 %. Median clib is used in combination with endocrine therapy PFS was 8.8 months for the ER+ cohort compared with [78]. In this large phase III, placebo-controlled, double- 1.1 months in the ER-negative group. In the combin- blind study, palbociclib and fulvestrant was compared ation cohort, patients with ER+ metastatic breast to fulvestrant and placebo. The study demonstrated a cancer (n = 18) were treated with the combination abe- doubling of PFS. The PFS in the treatment arm was maciclib plus fulvestrant. Patients received abemaciclib 9.2 months (95 % CI 7.5–not estimable) compared to at 200 mg orally every 12 hours on a continuous sched- 3.8 months (95 % CI 3.5–5.5) in the control arm. Un- ule. Patients also received 500 mg fulvestrant intramus- like the PALOMA-1/TRIO18 and PALOMA-2/TRIO22 cularly every month. Patients in this cohort had a studies, this population of patients had a more median of four lines of prior therapy. The disease endocrine-resistant disease, with the requirement to control rate in the latter cohort was 72.2 %. Like Finn et al. Breast Cancer Research (2016) 18:17 Page 8 of 11

Table 2 Currently registered clinical studies with CDK4/6 inhibitors in breast cancer Compound Setting Trial primary endpoint Combination N Phase ClinicalTrials.gov identifier Palbociclib First-line PFS Letrozole 450 III NCT01740427 (PALOMA-2) (PD-0332991) metastatic Metastatic PFS Fulvestrant 417 III NCT01942135 (PALOMA-3) High-risk adjuvant iDFI Anti-hormonal 800 III NCT01864746 (PENELOPE-B) Neo-adjuvant pCR Anastrozole 29 II NCT01723774 Pre-operative ORR Letrozole 45 II NCT01709370 Metastatic MTD 20 I NCT01320592 Neo-adjuvant Biomarker cCR Letrozole 306 II NCT02296801 (PALLET) Metastatic PFS Exemestane versus 348 III NCT02028507 (PEARL) Adjuvant Treatment Letrozole 160 II NCT02028507 discontinuation Metastatic Dose/toxicity TDM-1 17 Ib NCT01976169 Neoadjuvant RCB Letrozole versus FEC-3 132 II NCT02400569 (NeoPAL) Adjuvant iDFS SAT 4600 III NCT02513394 (PALLAS) Ribociclib (LEE011) First-line PFS Letrozole 450 III NCT01958021 metastatic Pre-surgical PD Letrozole 120 II NCT01919229 (MONALEESA-1) Metastatic DLT, PFS BYL719, letrozole 300 I/II NCT01872260 Metastatic DLT, PFS Exemestane, 185 Ib/II NCT01857193 Metastatic PFS Letrozole 650 III NCT01958021 (MONALEESA-2) Metastatic DLT Letrozole, buparlisib 13 I NCT02154776 Metastatic PFS Tamoxifen, NSAI 660 III NCT2278120 (pre-menopausal) (MONALEESA-7) Metastatic DLT/PFS BYL719a or BKM120 216 I/II NCT01872260 Metastatic PFS Fulvestrant 660 III NCT02422615 (MONALEESA-3) Abemaciclib Neoadjuvant Biomarker Anastrozole 220 II NCT02441946 (LY2835219) (NeoMONARCH) Brain metastasis Response Single agent 120 II NCT02308020 Metastatic PFS NSAI 450 III NCT02246621 (MONARCH-3) Metastatic Response Single agent 128 II NCT021024490 (MONARCH-1) Metastatic PFS Fulvestrant 630 III NCT02107703 (MONARCH-2) Metastatic PFS NSAI, tamoxifen, exemestane, 102 I NCT02057133 everolimus, trastuzumab aBYL719 (Novartis) is a phosphoinositide 3-kinase α-specific inhibitor. cCR clinical complete response, DLT dose limiting toxicity, FEC 5-, , ; iDFS invasive disease-free interval, MTD maximum tolerated dose, NSAI non-steroidal , ORR objective response rate, pCR pathologic complete response rate, PD pharmacodynamic, PFS progression-free survival, RCB residual cancer burden, SAT standard adjuvant therapy, TDM-1 ado- palbociclib, neutropenia was seen in 40 % of all-grade off. Limited data in breast cancer have been pre- cases, and 21 % of grade 3/4 cases. There was 66 % all- sented. In a large phase I study of advanced pRb + grades diarrhea reported, of which there were only 6 % solid tumors, single-agent activity was seen in patients grade 3 cases and no grade 4 cases. This side effect with breast cancer [83]. The most common grade 3/4 seems to indicate some differences between palbociclib toxicities at the recommended dose for expansion and abemaciclib. The dose in phase III breast cancer were neutropenia (26 %), leukopenia (16 %), and lym- studies is 150 mg daily every 12 hours, continuously. phonepnia (16 %). LEE011 is now moving ahead into Like palbociclib, LEE011 (ribociclib; Novartis) is be- more advanced studies in breast and other cancers. In ing dosed at 600 mg daily, 3 weeks on and 1 week addition, it is being evaluated in combination with Finn et al. Breast Cancer Research (2016) 18:17 Page 9 of 11

hypothesize that, given appropriate selection bio- Note markers, subgroups of patients with other tumor types This article is part of a series on ‘Recent advances in breast cancer may benefit from CDK4/6 inhibition. treatment’ , edited by Jenny Chang. Other articles in this series Abbreviations can be found at http://breast-cancer-research.com/series/ CDK: Cyclin-dependent kinase; CI: Confidence interval; ER: Estrogen receptor; FDA: Food and Drug Administration; FISH: Fluorescence in situ hybridization; treatment. HR: Hazard ratio; PFS: Progression-free survival; pRb: Retinoblastoma protein; RB: Retinoblastoma. the p110α-specific phosphoinositide 3-kinase inhibitor Competing interests (BYL719) and letrozole and in combination RSF and DJS received research support from Pfizer and have served as consultants to the same company. DJS has common stock in Pfizer. AA with everolimus plus exemestane. More mature data declares no competing interests. with both these compounds are eagerly awaited. Authors’ contributions All authors read and approved the final manuscript. Conclusion The translational road to effectively targeting the cell Acknowledgments cycle has been a long journey from basic science stud- Supported in part by the Revlon/UCLA Women’s Cancer Research Program, the Wittich Family Project for Emerging in Breast Cancer at UCLAs ies to eventual clinical testing. The challenges to this Jonsson Comprehensive Cancer Center, and the Department of Defense process have been highlighted, and include the identi- Innovator Award (DJS) W81XWH-05-1-0395. fication of the most relevant biologic targets, the de- Author details velopment of effective, clinical grade inhibitors of 1Geffen School of Medicine at UCLA, Department of Medicine, Division of those targets, and ultimately the identification of the Hematology Oncology, 2825 Santa Monica Blvd, Santa Monica, CA 90404, 2 appropriate target population to pursue for clinical de- USA. Stanford School of Medicine, 291 Campus Drive, Stanford, CA 94305, USA. velopment. The preclinical observation that palboci- clib (PD-0332991) had preferential activity in cell line Received: 31 October 2013 Accepted: 10 December 2015 models that represented the ER+ as well as HER2- amplified subgroups has led to very promising phase References II efficacy/safety data in ER+ breast cancers. While 1. 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