The Ongoing Search for Biomarkers of CDK4/6 Inhibitor Responsiveness in Breast Cancer Scott F

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 2020 American Association for Cancer Research. DNA in S phase (ref. 14; Fig. 2). DK4/6 is actually a ternary complex

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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

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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. Cyclin D is a direct transcriptional target of oncogenic signaling pathways (Fig. 1). It has a very short half-life, and upon assembly with CDK4, the complex rapidly dissociates unless it is stabilized by p27 or p21. p27 binds in two alternative conformations, a closed, inactive conformation or an open, active conformation, and this transition is mediated by Y phosphorylation of p27 itself by a nonreceptor bound Y kinase, such as Brk (Breast tumor Related Kinase; ref. 69). p27 causes the translocation of the complex to the nucleus via its NLS, where the open p27 Y phosphorylated-cyclin D–CDK4 complex is a substrate for additional phosphorylation by CAK. CAK phosphorylates CDK4 on reside T172, causing another conformational change and fully activating the complex. Only this modified complex can phosphorylate RB. all approved for the same indications and all target CDK4/6, tran- cancers, so named because their gene-expression profiles resemble scriptional and proteomic data now suggest that they have different those of luminal breast epithelial cells, are characterized by expression target specificities. Ribociclib has a similar efficacy and toxicity profile of ER-related genes. Within this group, differences in expression of to palbociclib, inhibiting both CDK4 and CDK6 (16). Abemaciclib on ER-related and proliferation-related genes define the primary sub- the other hand may have a greater affinity for CDK4 than CDK6, but types, Luminal A and Luminal B. The nonluminal subtypes are also inhibits CDK9, DYRK/HIPK kinases, and GSK3 a/b, resulting in a grouped into the HER2-enriched tumors and basal-like tumors. different toxicity profile, with fatigue as the dose-limiting toxicity and Molecular subtyping is used clinically to guide treatment in certain diarrhea as a common AE (17, 18). Abemaciclib also has inhibitory scenarios; however, IHC remains the standard for typing breast cancer activity against CDK2 and CDK1, suggesting that it may more closely into the major clinically relevant categories: HRþ, HER2þ, triple- resemble the previous pan-cdk inhibitors (18). In addition, abemaci- positive, and triple-negative (TN). There is a general but imprecise clib is approved as monotherapy, as well as in combination with correlation between molecular subtypes and receptor phenotype: letrozole or aromatase inhibitors, as opposed to palbociclib and Luminal A and B subtypes are typically HRþ/HER2-, whereas the ribociclib, which are only approved in combination (19). It is also nonluminal HER2-enriched tumors are typically HR-/HER2þ, and dosed continuously, which may offer kinetic advantages in some nonluminal basal-like tumors are frequently TN. HRþ/HER2þ patients. At the current time, all three agents are under clinical tumors remain the subject of some debate (22–24). Receptor status investigation for a variety of other tumor types, including in combi- is important for guiding both initial treatment and metastatic disease. nation with other agents. In the metastatic setting, HRþ tumors are treated with hormonal therapy, often now with the addition of anti-CDK4/6 therapy. HER2þ Molecular subtypes of breast cancer tumors are treated with an anti-HER2 monoclonal antibody, frequent- Molecular analysis has identified two major groups of breast cancer, ly with the addition of chemotherapy, whereas TN tumors are treated luminal subtypes, and nonluminal subtypes (20, 21). Luminal subtype with chemotherapy alone and now in some settings with atezolizumab

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(anti–PD-LI; ref. 25). Furthermore, receptor status helps guide treat- derived benefit. In another study using abemaciclib, 29 of 36 HRþ ment options in the neoadjuvant and adjuvant settings. patients (81%) derived benefit (response or stable disease by RECIST v1.1 criteria), compared with 3 of 9 HR- patients (33%; ER expression ref. 17). It is worth noting, however, that the benefit experienced þ The initial rationale for using palbociclib in ER/PR patients was by HR- patients was only stable disease, not regression. The based on the preclinical work of Finn and colleagues, who discovered PALOMA-2 and PALOMA-3 trials, which included only HRþ, that luminal ERþ cell lines responded better to palbociclib treatment, HER2- patients, stratified patients by the level of ER positivity on in vitro, than nonluminal subtypes (26). This observation is consistent IHC, but no statistically significant correlation between ER staining with the mechanism of action of palbociclib, as CCND1, the gene intensity and response to palbociclib was found (7, 30). Further- coding for Cyclin D1, is a transcriptional target of ER (27). Thus, more, despite only enrolling HRþ patients, 20% of patients exhib- overactivation of ER could lead to increased expression of Cyclin D1, ited primary resistance (<6 months PFS) in the PALOMA-3 study, causing DK4/6-mediated cellular proliferation. and an additional 50% developed resistance within 2 years of Subsequent completed trials of palbociclib in breast cancer have starting therapy (7). Thus, hormone-receptor status by IHC may only included patients with ER/PRþ, HER2- tumors; thus, there is a be an imprecise predictor of palbociclib responsiveness. Many HRþ sparsity of clinical data to directly support the hypothesis that patients do not benefit, whereas other non-HRþ patients, who palbociclib is only active in HRþ tumors without HER2 (6, 7, 28). could potentially derive benefit, do not receive treatment. Investi- The limited data come from a phase II study by DeMichele and gation into CDK4/6i biomarkers focuses on two interrelated issues: colleagues, which evaluated palbociclib monotherapy in patients (1) the inability to identify the actionable, active CDK4 target and with advanced breast cancer, most of whom had been heavily (2) preexisting or acquired resistance mechanisms that render the pretreated (29). Thirty-three of 37 patients in that study were drugs ineffective, despite having the CDK4 target. The remainder of HRþ/HER2-, and 4 were TN. Although 7 of the HRþ patients this review will summarize the status of the most promising experienced a clinical benefit (21%), none of the 4 TN patients potential biomarkers in both classes (Fig. 3).

Biomarkers to identify Biomarkers to identify CDK4/6 target secondary resistance

p27 CDK4 ON P Cyclin D CDK2

Cyclin E DCAF P RB

p27 CDK6 ON P Cyclin D p27

SequestrationSequestrat on

FAT

Figure 3. Potential biomarkers for CDK4/6i response. Left: Biomarkers to identify the CDK4 target would need to identify the fully active complex. CDK4T172 phosphorylation (orange P) or p27 pY phosphorylation (red P) delineates the active complex. Increased cyclin D levels should increase the cyclin D–CDK4 complex and additionally increase sequestration of p27 away from CDK2, activating that complex (14, 61). Alternatively, detection of DCAF could identify cells with genetic aberrations leading to increased cyclin D-CDK4 activity. Right: Biomarkers that identify resistance mechanisms would include loss of RB, which would render the presence of CDK4 unnecessary, or increased cyclin E-CDK2 activity due to increased cyclin E or reduced p27 inhibition. FAT, which increases cyclin D levels, might increase the CDK4/6 target directly or alternatively sequester p27 away from CDK2 complexes, increasing p27-free CDK2, so it might fall into both classes of biomarkers.

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Biomarkers to Identify the CDK4/6 suggest that this result was not surprising as CDKN2A levels may be too Target low in HRþ cells to be predictive (38). They note that all of the high CDKN2A-expressing cell lines from Finn's 2009 panel were HR-, CCND1 fi Cyclin D1, ampli cation, and D-Cyclin activating features suggesting that selecting for HRþ tumors may eliminate the predictive CCND1 The gene encodes Cyclin D1, one of the three D-type value of CDKN2A. However, this biomarker may still have value for cyclins. Cyclin D1 appears to be the most important of the three in the patient exclusion in other tumor types. normal and malignant proliferation of breast tissue, though this may fi only be due to tissue-speci c expression patterns, not a functional Phosphorylation status of CDK4 difference (31, 32). In addition to being a transcriptional target of ER, CDK4 activity is primarily regulated posttranslationally, by phos- Cyclin D1 overexpression can also be driven by several upstream phorylation and association with other proteins, such as Cyclin D and mitogenic signals, which have been proposed as potential mechanisms p27. It has been shown that phosphorylation of the threonine 172 CCND1 of endocrine therapy resistance (33). In Finn's preclinical data, residue (pThr172) is the rate-limiting step in CDK4 activation (39). It transcriptome expression was also found to correlate with different cell is thus reasonable to hypothesize that tumor cells lacking Thr172 ’ in vitro lines sensitivity to palbociclib, , suggesting it may serve as a phosphorylation would progress through the cell cycle in a CDK4- biomarker for response (26). independent manner, and thus be resistant to palbociclib. This was CCND1 fi The PALOMA-1 trial evaluated gene ampli cation by demonstrated by Raspe and colleagues, who found that pThr172 status FISH, as well as Cyclin D1 protein expression by IHC (28, 30). Using correlated with sensitivity to palbociclib in 20 breast cancer cell either parameter, gene copy number or protein expression, there was lines (40). However, use of pThr172 as a clinical biomarker is limited no correlation with sensitivity to palbociclib; it was similar in all by the difficulty in detecting it in formalin-fixed, paraffin-embedded CCND1 fi groups. The lack of predictive value of gene ampli cation by (FFPE) tissue, so Raspe and colleagues identified a gene expression FISH was also seen in the DeMichele study (29). The PALOMA-2 profile, which correlated with pThr172, to predict response to palbo- study, the largest clinical trial of palbociclib to date, also included an ciclib. Using the profile, they were able to predict sensitivity to array of possible biomarkers as one of its secondary endpoints. Cyclin palbociclib in 49 of 52 breast cancer cell lines tested. Again, clinical CCND1 D1 IHC was included in this panel, as well as mRNA studies will be needed to see if the profile is able to predict response to fi expression, and neither showed any prognostic signi cance for pal- palbociclib in vivo. bociclib treatment (28, 34). Thus, although CCND1 levels were predictive biomarkers in cell lines, they were not in patients. Phosphorylation status of p27 at tyrosine-88 Further insight into the genetic aberrations responsible for Cyclin p27 is an important regulator of DK4/6, serving as either an – ’ D mediated proliferation is provided by Gong and colleagues analysis inhibitor or activator of the complex depending on its phosphorylation “ ” of D-Cyclin Activating Features (DCAF). Gong and colleagues status at the tyrosine-88 residue (pY88; ref. 41). Patel and colleagues looked at 560 cell lines from a variety of cancer types to identify other hypothesized that p27 pY88 status could thus be indicative of DK4/6 tumor types that might be sensitive to CDK4/6 inhibition (35, 36). axis activity, indicating potential sensitivity to CDK4/6 inhibitors. This CCND1 They found that cells with DCAF, including translocation, hypothesis was supported in cell culture experiments, as the level of CCND1 -3 30 UTR loss (resulting in mRNAs lacking destabilizing pY88 directly correlated with CDK4 activity and Palbociclib respon- CCND2 CCND3 fi CCNK elements), or ampli cation, (encoding siveness: highly responsive MCF7 cells had lower levels of CDK4 FBX031 Kaposi's sarcoma virus D-type cyclin), or loss (encoding a activity and pY88, whereas Palbociclib-insensitive HCC1954 cells had ubiquitin ligase controlling cyclin D1 stability), tended to be sensitive much higher levels of pY88 and CDK4 activity (42). To further test this fi to abemaciclib. Given the disparity between the prognostic signi cance hypothesis, Gottesman and colleagues developed a dual IHC assay, CCND1 of mRNA levels in cell lines and in patients, it is unclear if staining for both total p27 and pY88 specifically. Using FFPE biopsy looking at the DCAF themselves in patients will provide useful clinical tissue, they demonstrated that pY88, which is negative in benign breast fi information. If it does, it may still be a dif cult biomarker to use, as epithelium, was differentially expressed in otherwise pathologically multiple analysis tools would need to be involved to identify responsive identical HRþ/Her2- tumors. This difference in pY88 stratified patients. tumors in the Her2þ and TN subgroups as well (43). Lack of pY88 suggested that DK4 was inactive, and that these samples would not p16 and CDKN2A amplification have the active CDK4 target. CDKN2A, the gene encoding p16INK4A, was also assessed for To demonstrate the predictive value of pY88 staining for palbociclib amplification by FISH in the PALOMA-1 trial. p16INK4A is a critical response, Gottesman and colleagues obtained fresh tumor tissue tumor-suppressor protein which inhibits the activity of CDK4/6, and samples from patients undergoing surgery and grew them in explant its expression has been shown to correlate with an improved prognosis culture. The explants were stained for Ki-67 as a marker of prolifer- in breast cancer (37). Low expression of CDKN2A, and thus p16INK4A, ation at the onset of culture and following 60 hours of treatment with was hypothesized to correlate with increased CDK4/6 activity, and palbociclib or control. In parallel, they stained archival biopsy material increased sensitivity to palbociclib. Finn's preclinical data supported from these patients with p27 and pY88 to stratify them into pY88þ or this hypothesis; however, other preclinical data from Gong and pY88- groups. Explants from the pY88þ group demonstrated a colleagues suggested that CDK4/6 inhibition in CDKN2A-deficient decrease in the percentage of Ki-67þ cells, indicating palbociclib- cells only resulted in transient arrest, with resistance likely mediated by mediated growth arrest, whereas explants from the pY88- group had CDK2 (26, 35). Ultimately, the PALOMA-1 trial showed that unchanged Ki-67 levels in the presence of palbociclib, indicating that CDKN2A copy number was not predictive of response to treat- they were resistant to Palbociclib-mediated arrest. Interestingly, pY88- ment (28, 30). p16INK4A expression was assessed by IHC in the positive tumors were seen in Her2þ and TN subtypes, supporting the PALOMA-2 and DeMichele trials, and did not predict response to idea that there may be subgroups of patients in those populations who therapy either (6, 29). CDKN2A mRNA was also looked at in the could benefit from CDK4/6 inhibitors. Although this study demon- PALOMA-2 study, and was nonpredictive (34). Green and colleagues strated that the pY88 marker might be used for target identification

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and might predict primary resistance, additional work using patients proliferation despite continually suppressed CDK4 activity. Using an treated clinically with CDK4/6i therapy will be required to assay the in vitro kinase assay, Patel and colleagues showed that this was due to strength of this marker. Recently, this group reported stratification increased CDK2-mediated phosphorylation of Rb. Interestingly, of patients who had previously been treated with palbociclib and CDK2 and Cyclin E protein levels were unchanged, suggesting a letrozole into responsive and nonresponsive subgroups based on this posttranslational mechanism responsible for increased CDK2 activity. biomarker (44). Patel and colleagues found that p27 levels were also decreased in these samples and hypothesized that this may be responsible for increased Biomarkers to Identify Resistance CDK2 activity. In addition to forming the ternary complex with Cyclin D and CDK4/6, p27 can also serve as an inhibitor of the Cyclin Mechanisms E–CDK2 complex. Thus, ubiquitin-mediated degradation of p27 could Loss of Rb allow for compensatory CDK2 activity in the setting of CDK4/6 Although the above-described biomarkers might help to identify inhibition. patients with the active CDK4 target, resistance to CDK4/6i may exist a Guarducci and colleagues generated palbociclib-resistant cell lines priori and be independent of the presence of the CDK4 target. For from 7 parental breast cancer cell lines sensitive to the drug, and example, our knowledge about cell-cycle progression suggests that loss performed gene expression profiling and Western blot to characterize of Rb should confer resistance to Palbociclib. Without Rb, E2F changes associated with resistance (53). Although they observed a high transcription factors are free and active, regardless of CDK4/6 sta- degree of molecular heterogeneity in resistant cells, CCNE1 amplifi- tus (14). Preclinical data support the hypothesis that cell lines with cation and loss of RB1 were common events at time of resistance. lower baseline Rb expression are less sensitive to palbociclib, and that Furthermore, these molecular changes were consistent with increased downregulation of Rb is a mechanism of secondary palbociclib resis- Cyclin E and decreased Rb protein expression by Western blot. tance, in vitro (26, 45). Loss of Rb at the DNA level is rare in HRþ Guarducci and colleagues then considered the CCNE1/RB1 ratio as breast cancer, though it does occur in about one third of TN breast a marker of palbociclib resistance, in order to take both into account as cancers, conferring a worse prognosis overall but increased sensitivity one biomarker. They found the ratio to be a better predictor of to chemotherapy (46). Consistent with this, less than 10% of patients in palbociclib resistance than either marker alone. In addition, they the PALOMA-1 and 2 trials, which only enrolled HRþ patients, did found that a high CCNE1/RB1 ratio predicted resistance to palbociclib not express Rb by IHC (30, 47). Li and colleagues’ study, which will be in the publicly available NeoPalAna clinical trial dataset (described discussed later, found RB1 loss in 9 of 338 patients subsequently treated later), as well as poor overall survival in the METABRIC dataset, with CDK4/6i (48). Despite this small number of RB1 loss patients, independent of molecular subtype. they found a statistically significant difference in PFS on CDK4/6i Turner and colleagues recently reported retrospective gene expres- therapy, of 3.6 months compared with 10.1 months, for patients with sion profiling on 302 archival FFPE samples from the PALOMA-3 intact RB1. trial (54). Fifty-three percent of the samples were collected from Given that Rb expression was found to correlate with palbociclib primary tumors at the time of initial presentation, whereas 47% were response in preclinical models, Malorni and colleagues identified a collected from metastatic sites immediately prior to study entry, where panel of 87 genes whose expression corresponds to an Rb loss of the patients had already been exposed to at least one line of endocrine function phenotype, despite an intact RB1 gene (49). They found that therapy. mRNA expressions of 2,534 cancer-related genes were ana- this signature (RBsig) was able to reliably discriminate between cell lyzed. Looking specifically at the metastatic samples, patients with low lines that are sensitive versus resistant to palbociclib treatment, with CCNE1 expression had a median PFS of 14.1 months when treated resistant cells exhibiting higher levels of RBsig (indicating decreased with palbociclib plus fulvestrant, compared with 4.8 months for Rb function), a composite score of genetic changes in Rb-related genes. placebo plus fulvestrant, a statistically significant change. In the high Similar approaches using different panels have previously identified CCNE1-expressing patients, the median PFS was 7.6 months for cells with genetic alterations in the Rb pathway, and it is not clear if any palbociclib plus fulvestrant-treated patients, compared with of these offers benefits over each other (50, 51). The panel will need to 4.0 months for placebo plus fulvestrant-treated patients, and did not be applied to tumor samples from palbociclib-treated patients to see if reach statistical significance. Thus, the addition of Palbociclib provided it can predict response clinically. a median PFS benefit, but the degree of benefit was decreased in the high CCNE1-expressing patients. A more modest benefit was seen in Cyclin E–CDK2 low CCNE1-expressing patients for the primary tumor samples, which Given that the Cyclin E–CDK2 complex (EK2) is able to phos- was not statistically significant, consistent with this being a marker of phorylate Rb and release E2F, some have speculated that upregulation evolved resistance. In the NeoPalAna trial, a study of combined of Cyclin E or CDK2 may compensate for CDK4/6 inhibition, allowing hormonal therapy with palbociclib in the neoadjuvant setting for – progression through the G1 S restriction point (42, 45, 52). Herrera- HRþ patients, increased expression of CCNE1, as well as CCND2 Abreu and colleagues generated palbociclib-resistant MCF-7 breast and CCND3 (encoding Cyclin D2 and D3, respectively) also correlated cancer cells via chronic exposure to the drug and showed that these with treatment response (55). Interestingly, a recent in vitro study of cells harbored amplifications of the CCNE1 gene, which codes for 10 gastric cancer cell lines treated with palbociclib found Cyclin E Cyclin E1 (52). Although inhibition of CDK2 or Cyclin E1 expression protein expression by Western blot, but not CCNE1 amplification, to using siRNA was ineffective at inhibiting proliferation on its own, the be predictive of resistance to palbociclib (56). Thus, there may be combination of CDK2 or CCNE1 siRNA knockdown with palbociclib multiple mechanisms responsible for Cyclin E upregulation, and caused cell-cycle arrest in palbociclib-resistant MCF-7 cells, suggesting multiple assay methods may have to be employed for this to develop that EK2 activation was the mechanism responsible for DK4/6 bypass. into a viable biomarker. Patel and colleagues also treated MCF-7 cells with palbociclib to Vijayaraghavan and colleagues also found that CCNE1 gene explore induced resistance mechanisms (42). With ongoing palboci- expression in breast cancer cell lines correlated with palbociclib clib treatment, MCF-7 cells became resistant by day 6, with continued resistance (57). When they induced full-length Cyclin E, or

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low-molecular-weight Cyclin E (LMWE) overexpression, in previous- expression, and when they examined the MCF-7–resistant cell lines ly sensitive cell lines, however, they found that only LMWE caused the generated by Yang and colleagues, they found that the increased CDK6 cells to become resistant to palbociclib. LMWE is an oncogenic isoform in these lines correlated with reduced FAT1 levels. whose expression leads to RB hyperphosphorylation, bypassing FAT1 is a member of the cadherin superfamily, which has been CDK4-dependent phosphorylation, and correlates with a worse prog- found to interact with the Beta-catenin and Hippo signaling pathways. nosis (57, 58). It is created by posttranslational modification of intact By assessing transcriptional targets of these pathways in parental and Cyclin E; thus, there would not be differences in the CCNE1 mRNA. FAT1 knockout cell lines, Li and colleagues determined that although When they looked at protein expression in a set of TN cell lines, using the Beta-catenin axis was unchanged, FAT1 knockout cells exhibited Western blot, they found that palbociclib-resistant cell lines generally downregulation of Hippo-pathway signaling, which subsequently lead expressed LMWE, whereas sensitive cell lines expressed intact Cyclin to increased CDK6 expression. Induced expression of the intracellular E. They performed IHC staining on archival tumor tissue from 109 domain of FAT1 was sufficient to restore Hippo signaling and reduce patients with advanced breast cancer who were subsequently treated CDK6 expression. Although this seems to be a viable mechanism of with palbociclib, in combination with either letrozole or fulvestrant. resistance, FAT1 mutations are only observed in approximately 6% of Presumably because there are no antibodies specific for LMWE, they metastatic breast cancers prior to CDK4/6i treatment, so they are likely used cytoplasmic Cyclin E staining as a surrogate marker, because it to represent just a small portion of patients with primary resistance, so had previously been shown to correlate with LMWE expression (59). its usefulness as a predictive biomarker will have to be determined. Fifty-four of the 109 patients (49.5%) were found to have cytoplasmic Cyclin E staining, which was associated with a worse prognosis (P ¼ 0.01). Excluding the 7 Rb- patients, patients with cytoplasmic Cyclin E Posttreatment Analysis staining had a median PFS of 13.4 months, compared with 36.5 months Given the difficulty in developing a predictive biomarker for for patients with negative cytoplasmic Cyclin E staining. Given that CDK4/6 inhibitors, alternative approaches to guide therapy have been cytoplasmic Cyclin E staining is a negative prognostic factor regardless explored. One such approach is to dose patients with a short course of of treatment, prospective randomized studies will be needed to deter- CDK4/6i and to assess response. mine whether it is indeed a useful prospective marker of palbociclib resistance. Ki-67 expression Ki-67 is a proliferative marker that has been shown to predict CDK6 amplification and loss of the FAT1 tumor suppressor sensitivity to chemotherapy in HRþ breast cancer. Preclinical models Similar to the studies described above using palbociclib, Yang and have shown that palbociclib has activity against breast cancer cell lines colleagues generated abemaciclib-resistant MCF-7 breast cancer cell with a wide range of proliferation rates, which was also seen in Dean lines, and assayed for induced genetic changes associated with treat- and colleagues’ ex vivo analysis of breast tumors treated with palbo- ment. After 21 weeks of exposure, they obtained resistant clones with ciclib in culture (62). Thus, it was not surprising that pretreatment an IC50 nearly 10-fold higher than the parental clone (60). FISH Ki-67 expression did not correlate with response in the PALOMA-1 or demonstrated that this was due to gene amplification of the CDK6 PALOMA-2 trials (30, 47). However, changes in Ki-67 expression in locus, which resulted in a 7-fold increase in the levels of CDK6 mRNA response to palbociclib treatment have been used as a surrogate marker and an associated increase in CDK6 protein levels. Knockdown of of drug sensitivity, and thus can be used to assess the prognostic value CDK6 using siRNA restored sensitivity to abemaciclib, and induced of other biomarkers without needing to wait for follow-up data to overexpression of CDK6 in abemaciclib-sensitive cells was sufficient to mature (55, 63). The POP trial found significant decreases in Ki-67 cause resistance. Surprisingly, this effect was not seen with induced staining after just 14 days of palbociclib treatment before surgery, overexpression of CDK4, suggesting that the increased CDK6 may not suggesting that it may be possible to determine primary resistance after merely be increasing CDK4/6 kinase activity. Increased sequestration just a short course of therapy, provided pre- and posttreatment tumor of p27 or p21 away from CDK2 may also account for this resistance, samples are available (63). and thus results of this class of biomarkers may correlate with the increased Cyclin E-CDK2 category described above (14, 61). Yang and Early ctDNA changes with PI3KCA and ESR1 mutations colleagues also generated abemaciclib-resistant T47D and CAMA-1 Examination of relative changes in the level of circulating cell-free cell lines: The T47D cells exhibited a similar increase in CDK6 mRNA, tumor DNA in the plasma (ctDNA) has also shown promise in but also had a prominent decrease in RB1 mRNA. The CAMA-1 cells predicting CDK4/6i response as well. O’Leary and colleagues com- had minimal changes in CDK6, with more significant changes appear- pared ctDNA levels from patients in the PALOMA-3 trial at baseline ing in RB1 and CCNE1, akin to Guarducci and colleagues and Dean and on cycle 1 day 15 of treatment. They chose to look at two loci, and colleagues’ data. Thus, different mechanisms of resistance may PI3KCA and ESR1, which are frequently mutated in breast cancer (64). develop in different cell lines, under the same experimental conditions, For patients with identifiable PI3KCA mutations at baseline, there was but the same classes appear to persist. a significant reduction in PI3KCA ctDNA levels at C1D15 compared Li and colleagues performed genetic sequencing, using the MSK- with baseline, for both palbociclib þ fulvestrant and placebo þ IMPACT platform, on pretreatment ERþ/HER2- biopsy specimens fulvestrant arms; however, the decrease was significantly greater in from 348 patients subsequently treated with palbociclib, ribociclib, or the palbociclib arm. Furthermore, the level of decrease predicted abemaciclib, and correlated the results with PFS. Patients with a response to therapy (P ¼ 0.0013). Though ESR1 ctDNA levels also deleterious FAT1 mutation had a significantly decreased median PFS decreased significantly at C1D15, they did not predict response to of 2.4 months, compared with 10.1 months for patients without FAT1 therapy. The authors found that this difference was because PI3KCA is mutations (48). Loss of FAT1 in FAT1 knockout MCF7 and CAMA-1 usually a truncal, or originating mutation, present throughout the cell lines did not promote accelerated growth in the absence of tumor, whereas ESR1 mutations are usually subclonal, arising in treatment; however, it did render cell lines resistant to all three response to endocrine therapy (of note, patients in PALOMA-3 had CDK4/6i. In this setting, loss of FAT1 significantly increased CDK6 already received prior endocrine therapy). The limitation of this

AACRJournals.org Mol Cancer Ther; 19(1) January 2020 9

approach is the need to identify a truncal mutation for each patient, for patients with the IHC HRþ/HER2þ phenotype. We eagerly await and in the PALOMA-3 samples, only 22% of the patients had a the publication of their results, but it is likely that responses will be PI3KCA mutation. highly variable given the variety of molecular subtypes present in the trial cohorts. mRNA and proteomic signatures Hafner and colleagues and others have reported that the different CDK4/6i drugs induce specific mRNA and proteomic signatures in Conclusion treated cells, which in theory could be used to monitor drug response to The introduction of CDK4/6 inhibitors into the clinic has been a assess treatment at the molecular level (18). These techniques would significant step forward in the treatment of HRþ metastatic breast have to be adapted to work on circulating tumor cells in order to cancer. The two main issues surrounding their use are (1) acquired practically monitor initial treatment response. However, it is unclear drug resistance which develops in the majority of patients and (2) the how secondary resistance, which may overlay new signatures on top of lack of predictive biomarkers to determine who will benefit from old signatures, will be assessed. Green and colleagues have used an treatment. These two issues are interrelated, as solving both requires an ADP acyl phosphate probe to measure CDK4 target engagement and understanding of resistance mechanisms that develop before and demonstrate that target engagement correlates to palbociclib sensi- during treatment. We have summarized the data from a number of tivity (38). Again, how this would be clinically used and how secondary biomarkers, which have been assessed clinically, as well as others which resistance, which may occur on top of original CDK4/6i target show promise in the laboratory. Unfortunately, to date there does not engagement, will be determined, is still unclear. appear to be any one specific genetic aberration indicative of CDK4/6i sensitivity, as exists for other upstream, targetable enzyme mutations. Biomarkers in Other Breast Cancer Unlike many other kinases, which are therapeutic targets, the activation of DK4/6 is too complex for the levels of either of these proteins to Subgroups be predictive. We have classified biomarker research into two cate- Will a biomarker that predicts response in HRþ breast cancer gories: those that identify the actionable CDK4/6 target, such as translate into the other breast cancer subgroups, as we hope use of the changes in cyclin D or the phosphorylation status of CDK4 or p27, CDK4/6i therapies does? Foidart and colleagues showed that TN cell the lack of which may indicate primary resistance, and those that lines which expressed EGFR could be sensitized to palbociclib treat- delineate resistance mechanisms, such as loss of RB or increased cyclin ment after transfection with membrane-type-4 matrix metalloprotei- E-CDK2 activity, which can be defined as secondary resistance, even nase (MT4-MMP) DNA (65). MTR-MMP is a cell surface protein that when they appear in the pretreatment condition. Most biomarkers for has been shown to partner with EGFR and enhance its signaling upon targeted therapies in the oncology field fall into the first category, but binding ligand. They hypothesized that TN tumors which express identification of biomarkers of resistance remains a problem univer- EGFR and MT4-MMP might be more sensitive to CDK4/6 inhibition, sally. For example, although Her2 staining identifies tumors that may as long as RB remained intact. They subsequently analyzed a series of respond to trastuzumab, de novo or acquired resistance occurs clin- 72 TN tissue samples, and 37 patient-derived xenografts (PDX), with ically in 66% to 88% of Her2þ metastatic breast cancer (68). Thus, a IHC staining for MT4-MMP, EGFR, and RB. They found that focus on both classes of biomarkers should continue as it is likely that approximately 50% of the TN samples and xenografts stained positive some combined use of a marker against the CDK4 target and against for all three biomarkers. They then treated select PDX-mice, repre- resistance mechanisms will produce the most accurate response data. sentative of each biomarker combination (EGFR , MTR-MMP ,Rb Future clinical trials should include rational biomarker analyses, and ) with vehicle, palbociclib, erlotinib (EGFR inhibitor), or the com- additional experimental tissue collection, where possible, to facilitate bination, and assessed tumor growth. PDX positive for MT4-MMP, application of these experimental biomarkers to clinical samples. EGFR, and RB demonstrated significantly decreased proliferation Another factor will be ease of usage, and the confidence ratio, as a when exposed to palbociclib treatment as compared with the other companion diagnostic must be highly predictive or we continue to run groups. Cotreatment with palbociclib and erlotinib yielded an additive the risk of including and excluding patients, similar to the current effect. The data indicate that a subset of TN patients, potentially as high scenario seen with the lack of any biomarker at all. Finally, many of the as 50%, may derive benefit from treatment with CDK4/6 inhibitors, current trials for the expanded use of CDK4/6i therapy combines and use of these markers might aid in identifying them. them with additional cytotoxic chemotherapies or radiotherapy, and Likewise, the PATRICIA trial is an ongoing study of palbociclib in this may necessitate a different set of biomarkers to predict response. combination with trastuzumab for patients with previously treated However, identification of the actionable CDK4/6i target or the HRþ, HER2þ, advanced breast cancer (66). Previous work had shown markers responsible for secondary resistance described in this review that patients with the HRþ, HER2þ phenotype by IHC can fall into will be a good first start to increase our usage of these therapies. one of several molecular subtypes, including Luminal A or B, or HER2- enriched (67). Given palbociclib's effectiveness in luminal subtypes, Disclosure of Potential Conflicts of Interest the authors hypothesized that patients with the Luminal A or B subtype S.W. Blain has ownership interest (including patents) in Concarlo Holdings, LLC, might be more sensitive to this combination strategy than patients with and has consultant/advisory board relationship with Princeton University. No fl other subtypes, such as HER2-enriched. This hypothesis was con- potential con icts of interest were disclosed by the other authors. fi fi rmed in the rst stage of the trial, which showed a PFS of 10.37 months Acknowledgments for patients with luminal disease, compared with 3.53 months for The authors thank Susan Gottesman, Vladislav Tsiperson, and Christopher P patients with other subtypes, with a value of 0.023. Thus, molecular Roman for critical reading of this article. This work was supported by NIH subtyping might be prudent to select which Her2þ patients are most R01CA201536 (to S.W. Blain). likely to benefit from this combination strategy. This finding is especially relevant given the multiple clinical trials which are currently Received May 29, 2019; revised August 2, 2019; accepted September 5, 2019; ongoing utilizing anti-CDK4/6 and anti-HER2 combination strategies published first January 3, 2020.

10 Mol Cancer Ther; 19(1) January 2020 MOLECULAR CANCER THERAPEUTICS

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12 Mol Cancer Ther; 19(1) January 2020 MOLECULAR CANCER THERAPEUTICS

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