Xue Yang

1Department of Medical Oncology,Tianjin Medical University General Hospital,Tianjin 300052,China.

Abstract Endocrine therapy (ET) is the therapy backbone of hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer.However,there are about 20% HR positive patients with no response to ET due to primary or acquired ET resistance.In this background,many agents have been studied to overcome ET resistance and of which the important agents are cyclin-dependent kinase 4/6 (CDK4/6) inhibitors.The prognosis of advanced breast cancer has been improved by combing ET with CDK4/6 inhibitors.In this review,we mainly focused on the CDK4/6 inhibitors in the treatment of HR-positive,HER2-negative advanced breast cancer and discussed the action mechanisms of CDK4/6 inhibitors alone or combined with ET.We also summarized several molecular features that would predict response or resistance to CDK4/6 inhibitors.In addition,we put forward possible strategies to overcome CDK4/6 inhibitor resistance according to the latest research.

Keywords:Breast cancer,CDK4/6 inhibitors,Endocrine resistance,Palbociclib,Ribociclib,Abemaciclib

Background

Breast cancer (BC) is the most common cancer in women globally with an estimated 2.4 million new cases being reported yearly [1].It is also the leading cause of cancer-related death in females worldwide with over 500,000 deaths annually [2].BC is categorized into three subtypes according to estrogen receptor (ER),progesterone receptor (PR) and HER2 status:HR-positive,HER2-positive and triple negative subtypes [3].Of these,HR-positive breast cancers constitute approximately 60%-70% [4].

Endocrine therapy (ET),including selective estrogen receptor modulators (SERMs),aromatase inhibitors(AIs),and a selective estrogen receptor degrader(SERD),is considered to be the mainstay therapy for HR-positive breast cancer [5].A major obstacle in the treatment of HR-positive advanced BC is intrinsic or acquired resistance to ET.The resistance mechanisms are complicated and include but not limited to increased expression of Cyclin-dependent kinase 4 and 6 (CDK4/6),activation of the PI3K/AKT/mTOR signaling pathway andESR1mutations [6].Here,we pay more attention to the effects of the CDK4/6-Cyclin D1-Rb pathway in ET resistance and discussed the action mechanisms of CDK4/6 inhibitors alone or combined with ET.We also summarized several molecular features that would predict response or resistance to CDK4/6 inhibitors.In addition,we put forward possible strategies to overcome CDK4/6 inhibitor resistance according to the latest research.Well understanding of resistant molecule mechanisms and predictive biomarkers are helpful to judge which patients will be more likely benefit from CDK4/6 inhibitors.

The underlying combination therapy mechanisms of ET and CDK4/6 inhibitors

The progression of cell cycle from the G1 phase to the S phase is a key checkpoint in protecting the cell from abnormal replication.Studies show that estrogen facilitates the G1 to S phase transition by means of the activation and binding of cyclin D1 to dimerized CDK4/6 [7].Cyclin D1 associates with CDK4/6 kinases to phosphorylate retinoblastoma proteins (Rb)and dissociates them from the E2F transcription factor,which activates transcription of genes required for DNA replication [8,9].In addition,E2F transcription factor induces the transcription of S phase specific proteins,such as CDK2.CDK2 forms a complex with cyclin E and triggers greater Rb phosphorylation and inactivation,therefore driving the cell towards an irreversible and mitogenic signals independent cell division [10].

Cyclin D1 is a direct target gene of the ER [11].CCND1(encoding cyclin D1) mRNA expression is 1.4 fold increase (P=1.76E-2) in ductal carcinoma in situ compared with benign breast hyperplasia (Figure 1).Moreover,CCND1mRNA expression is approximately 2.79 fold increase (P=4.38E-5) in luminal-like subtype of invasive breast carcinomavsbasal-like subtype(Figure 2).Coincidentally,The Cancer Genome Atlas(TCGA) reported that amplification of cyclin-D1 occurred in 29% of luminal A and 58% of luminal B breast cancer [12].Studies have implicated cyclin D1 amplification was associated with ET resistance.In vitro studies demonstrated that HR-positive BC cells continued to grow in the presence of tamoxifen after inducible overexpression of cyclin D1 [13]and cyclin D1 is necessary for the proliferation of tamoxifen resistant BC cells [14].Furthermore,Cyclin D1 expression is also an independent poor prognostic factor in women with early-stage,HR-positive BC who received adjuvant tamoxifen-based therapy [15].

Preclinically,cell lines representing luminal ER+subtype (including those that are HER2 amplified)were most sensitive to growth inhibition by palbociclib,a CDK4/6 inhibitor,while nonluminal/basal subtypes were most resistant.The underlying mechanism analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines.Moreover,palbociclib enhanced sensitivity to tamoxifen in cell lines with ET resistance [16].Furthermore,the combination of palbociclib with fulvestrant or bazedoxifene proved to effectively inhibit the growth of MCF-7 cells (tamoxifen resistant cell line) and tumour xenografts [17].

Thus,the CDK4/6-Cyclin D1-Rb pathway plays crucial roles in HR-positive BC and the CDK4/6 inhibitor is of great attractive for ET therapy resistance(Figure 3).

The CDK4/6 inhibitors

Three CDK4/6 inhibitors have now been approved by the FDA for the treatment of HR-positive,HER2-negative advanced BC in combination with ET:palbociclib (PD0332991),ribociclib (LEE011) and abemaciclib (LY835219).The addition of these agents to ET has resulted in improved progression-free survival) in luminal A breast cancer [18-20].In addition,abemaciclib also obtained an approval as single-agent second line therapy in patients with HR-positive,HER2-negative metastatic BC who experienced disease progression following ET and prior chemotherapy [21,22].The clinical trials data,including trial design,primary outcome and adverse effects,of these three CDK4/6 inhibitors have been well summarized in previous reviews [6,23].In this review,we mainly focused on the unique action mechanisms,efficacy predictive factors,and the resistance mechanisms of palbociclib,ribociclib and abemaciclib (Table1).

Studies on the structure-activity relationship suggest that the presence of the 2-amino-pyridine group and the piperazine ring are important molecular determinants for ATP-competitive inhibition and CDK4/6 selectivity [39,40].Inhibition of CDK4/6 will block its binding site to cyclin D1 [41].Palbociclib and ribociclib potently inhibit both CDK4 and CDK6 activities,with the IC50 for CDK4/cyclin D1 complex is 11 and 10nmol/L,respectively and CDK6/cyclin D1 complex is 16 and 39nmol/L,respectively [42].While abemaciclib shows a greater selectivity for the complexes CDK4/cyclin D1 (IC50=2nmol/L) than CDK6/cyclin D1 (IC50=10nmol/L).

At the same time,the chemical structures of the three CDK4/6 inhibitors all contain N-ethyl piperazine ring,which is expected to undergo metabolism generating reactive iminium intermediates that can initiate several toxic side effects through previously reported mechanisms as in the case of brigatinib and ponatinib that bind covalently to a DNA base [43,44].A study suggests that blocking or isosteric replacement of α-carbon to the tertiary nitrogen atoms of piperazine ring can aid in reducing toxic side effects of abemaciclib [45].

Palbociclib

Palbociclib is the first approved CDK4/6 inhibitor by the US Food and Drug Administration (FDA) based on the results of three pivotal randomized clinical trials,the PALOMA-1,PALOMA-2 and PALOMA-3[46-48].Inhibition of Rb phosphorylation and subsequent G1 cell cycle arrest is the primary action mechanisim of CDK4/6 inhibitors including palbociclib [24,25].However,the effect of Rb status on palbociclib activity is controversial.On the one hand,palbociclib has no activity against Rb-negative cells for the IC 50 is 1 to 2 orders of magnitude higher than the IC 50 in Rb-positive tumor cells [49].In addition,Rb downregulation is common at the time of acquired resistance to palbociclib in virious BC cell lines.On the other hand,a clinical study shows that no significant interaction is found between palbociclib treatment and the expression levels ofRB1mRNA [29].Meanwhile,study shows that the inhibition of Rb phosphorylation by palbociclib is completely reversible after removal of the drug [49],and this phenomenon could specify that the adverse effects such as neutropenia,leukopenia caused by palbociclib is also reversible with the schedule of 3-weeks-on/1week-off.

Table 1 The unique action mechanisms,efficacy predictive factors,and the resistance mechanisms of palbociclib,ribociclib and abemaciclib

The expression level of Cyclin E1 is of importance for palbociclib resistance.Overexpression of cyclin E1 is associate with resistance to palbociclib [26].Recently,Guarducci C et al.found that the combination ofCCNE1andRB1might serve as a better maker to predict the resistance to palbociclib compared to the single biomarkersCCNE1andRB1used separately [27].Eric Raspé et al.found the presence of T172-phosphorylated CDK4 was proved to predict the sensitivity to palbociclib in breast cancer,which could help to select a subset of drug sensitive patients in case of inefficacy [28].

Ribociclib

Ribociclib was the second specific CDK4/6 inhibitor approved by FDA in combination with ET for the firstline treatment of postmenopausal women with metastatic HR+/HER2-BC [50].The approval was based on the results of the phase III MONALEESA-2 trial showing a significantly longer progression-free survival in the ribociclib group than in the placebo group [19].In a chemoproteomics study of CDK4/6 inhibitor activity in lung cancer cell lines and primary tumor samples,ribociclib showed significantly more selective towards CDK4/6 than palbociclib [30].

Updated results from MONALEESA-2 showed that Ribociclib treatment benefit was maintained irrespective of PIK3CA or TP53 mutation status,total Rb,Ki67,or p16 protein expression,and CDKN2A,CCND1,or ESR1 mRNA levels.Ribociclib benefit was more pronounced in patients with wild-type versus altered receptor tyrosine kinase genes [31].Jansen VM et al.highlighted a role for the PI3K-PDK1(3-phosphoinositide-dependent protein kinase 1)signaling pathway in mediating acquired resistance to ribociclib [32].Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines [32].Cornell et al.demonstrated a acquired mechanism that palbociclib resistance was mediated by increased exosomal miR-432-5p causing a downregulation of TGF-β pathway signaling,which in turn results in increased expression of CDK6,allowing cells to overcome G1 arrest and promote cell survival[33].In addition,C Lenihan et al.found ribociclib resistant clones of BC cell lines presented with unchanged or even decreased CDK2 expression,but demonstrated notable increase in E2F1,which indicated the requirement of a non-canonical CDK-cyclin pairing independent of CDK2 [34].

Abemaciclib

Abemaciclib has the highest CDK4/CDK6 selectivity ratio within the class and its selectivity for CDK4 is higher compared with CDK6 [35].Furthermore,the unique ability of abemaciclib is targeting on CDK9 at clinically relevant concentrations [37].These properties may account,at least partially,for single-agent activity of abemaciclib in breast cancer patients [51],whereas the other CDK4/6 inhibitors showed comparable efficacy in combination with hormonal therapy [52].

In addition,compared to other CDK4/6 inhibitors,the effect of abemaciclib on brain metastases seems unique.Abemaciclib was shown to cross the blood-brain barrier,with drug concentrations in the cerebrospinal fluid comparable to those found in plasma [36,53,54].

The resistance mechanisms of CDK4/6 inhibitors and coping strategies

CDK4/6 inhibitors are cytostatic rather than cytotoxic drugs.In this regards,primary and acquired resistance to these drugs would occur sooner or later.As mentioned above,activation of the PI3K signaling is another prominent mechanism for resistant to ET in addition to the Cyclin D1-CDK4/6-Rb pathway.In this case,sequential therapy as single-agent or in combination with other targeted drugs would still be worthy of deep reflection.Herrera-Abreu MT et al.explored the synergistic therapies effects by targeting of both CDK4/6 and PI3K.They showed that combined inhibition of both CDK4/6 and PI3K triggered BC cells apoptosis,resulting in tumor regression and improved disease control.Furthermore,a triple combination of ET,CDK4/6,and PI3K inhibition was more effective than paired combinations.Besides,this study also found that cells acquiring resistance to CDK4/6 inhibitors due toCCNE1amplification could be resensitized by targeting CDK2[26].Thus,multiple targeted drugs combination with ET would be the therapy tendency for BC in the future.

Aberrant FGFR signaling is a potential mechanism of escape from ET plus CDK4/6 inhibitors.One study showed that FGFR1 overexpression or amplification reduced the sensitivity to ET ± palbociclib or ribociclib.Addition of FGFR TKIs or CCND1 siRNA to the combination reversed drug resistance.Thus,triple pharmacological inhibition of FGFR1,ERα and CDK4/6 maybe a choice for HR+/HER2-BC patients with FGFR1 overexpression or amplification[55].

Malorni L et al.established a gene expression signature of Rb loss-of-function (RBsig) by identifying genes correlated with E2F1 and E2F2 expression in BCs within TCGA.The RBsig was associated with RB1 genetic status.And they found that the RBsig was able to identify palbociclib resistant and sensitive breast cancer cells.Therefore,RBsig might be a helpful maker in personalizing treatment of patients with HR+/HER2-BC [56].

Another study uncovered a tumor suppressor function of Hippo signaling in ER+breast cancer and establishedFAT1loss as a mechanism of resistance to CDK4/6 inhibitor.This study showedFAT1loss led to marked elevations in CDK6,the suppression of which restored sensitivity to CDK4/6 inhibitor.The induction of CDK6 was mediated by the Hippo pathway with accumulation of YAP and TAZ transcription factors on the CDK6 promoter.Genomic alterations in other Hippo pathway components were also found to promote CDK4/6 inhibitor resistance [57].

Lysosomal trapping has been considered as a mechanism of resistance for some drugs [58].These drugs accumulate into lysosomes and other cellular acidic compartments,a process known as lysosomal trapping [59].Indeed,palbociclib also undergoes lysosomal trapping because it is shown that palbociclib concentrates in intracellular acidic vesicles in human melanoma cell line SK-Mel-103.Ribociclib and abemaciclib also present a similar behavior as palbociclib [59].As a result of lysosomal trapping,the cytosolic and nuclear concentration of palbociclib is reduced,which may be responsible for the increased cellular and therapeutic IC50 with regard to the biochemical value.It has been shown that weakly basic drugs can alter the capacity of lysosomes to accumulate co-administered lysosomotropic drugs [22,44].Llanos S et al.also found that the combination of palbociclib with lysosomotropic drugs (chloroquine and NH4Cl) could improve its therapeutic efficacy[59].

Conclusions

In conclusion,CDK4/6 inhibitors are effective for the treatment of ER-positive HER2-negative breast cancer alone or in combination with ET.However,drug resistance is a frustrating problem needing to solve as soon as possible.The resistance mechanisms of CDK4/6 inhibitors have been explored and combination therapy strategies maybe one optional solution to solve this problem.Regarding biomarkers for the prediction of treatment efficiency of CDK4/6 inhibitors,we cannot draw a conclusion although many studies have engaged in this problem.But the results of the current studies would help us to give personalized treatment of patients with HR+/HER2-BC.