Cyclin-dependent kinase 4 and 6 inhibitors for hormone receptor-positive breast cancer: past, present, and future

Laura M Spring, Seth A Wander, Fabrice Andre, Beverly Moy, Nicholas C Turner, Aditya Bardia

The development and approval of cyclin-dependent kinase (CDK) 4 and 6 inhibitors for hormone receptor-positive and human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer represents a major milestone in cancer therapeutics. Three different oral CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, have significantly improved progression-free survival by a number of months when combined with endocrine therapy. More recently, improvement in overall survival has been reported with ribociclib and abemaciclib. The toxicity profile of all three drugs is well described and generally easily manageable with dose reductions when indicated. More myelotoxicity is observed with palbociclib and ribociclib, but more gastrointestinal toxicity is observed with abemaciclib. Emerging data is shedding light on the resistance mechanisms associated with CDK4/6 inhibitors, including cell cycle alterations and activation of upstream tyrosine kinase receptors. A number of clinical trials are exploring several important questions regarding treatment sequencing, combinatorial strategies, and the use of CDK4/6 inhibitors in the adjuvant and neoadjuvant settings, thereby further expanding and refining the clinical application of CDK4/6 inhibitors for patients with breast cancer.

Lancet 2020; 395: 817–27 Department of Medical Oncology, Massachusetts General Hospital Cancer Center, Harvard Medical School,
Boston, MA, USA
(L M Spring MD, S A Wander MD, B Moy MD, A Bardia MD); Department of Medical Oncology, Institut Gustave Roussy, Villejuif, France
(Prof F Andre MD); and Department of Medical Oncology, Royal Marsden Hospital, Institute of Cancer

Introduction
The approval of cyclin-dependent kinase (CDK) 4 and 6 inhibitors for hormone receptor-positive and human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer has transformed the treatments landscape of breast oncology, arguably representing one of the most important new treatment in breast oncology
1–11 of the three approved CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, established the combinatorial strategy of endocrine therapy plus targeted therapy as the preferred upfront treatment approach for most patients with hormone receptor-positive and HER2-negative metastatic breast cancer. Given the success of CDK4/6 inhibitors for hormone receptor-positive, HER2-negative metastatic breast cancer, these drugs are being explored in other settings, including for early stage hormone receptor- positive and HER2-negative breast cancer, and for other clinical breast cancer subtypes. Substantial progress is also being made in understanding which patients derive the most benefit, and the mechanisms of resistance to therapy. In this review we detail the clinical development of CDK4/6 inhibitors for breast cancer, summarise the key developments, and discuss future directions.

Biological rationale for CDK4/6 inhibitors CDK4/6–retinoblastoma protein axis and the cell cycle Transition through the G1 phase of the cell cycle, and the commitment to enter the S phase and divide, is modulated by a complex network of regulatory proteins including the
12 RB1 is a crucial negative regulator of the cell cycle acting through the assembly of multiprotein complexes that prevent premature cell division by binding the E2F transcription
12 RB1 is regulated by phosphorylation: the protein is fully active when not phosphorylated early in the G1 phase of the cell cycle and when cells are quiescent in G0. During these quiescent
phases of the cell cycle, growth signals promote expres- sion of D type cyclins, that complex with and activate with either CDK4 or CDK6, which facilitates the addition
12,13 Therapeutically, CDK4 and CDK6 are considered functionally equivalent in their ability to phosphorylate RB1 and, although kinase dominance depends on tissue type, inhibition by both kinases is essential because one might compensate for
14 The phosphorylation of RB1 by CDK4/6 initiates an intricate process of phosphorylation-mediated deactivation of RB1 function that releases E2F to drive expression of genes required for S phase entry and
12

Dysregulation of the CDK4/6–RB1 pathway in breast cancer
Given the central role of the CDK4/6–RB1 pathway in mediating cellular proliferation, this pathway is frequently dysregulated in cancer cells. Dysregulation of the CDK4/6–RB1 pathway is one of the central features of breast cancer biology, and the mechanisms through which this dysregulation occurs are fundamentally different between breast cancer subtypes. Although multiple molecularly distinct subtypes of breast cancer

Search strategy and selection criteria
We searched PubMed from inception until Dec 31, 2019, for articles using the search terms “breast cancer”, “cyclin- dependent kinases 4 and 6”, “CDK 4/6”, and “palbociclib;
ribociclib; abemaciclib”. 490 results were found, the abstracts of which were reviewed to identify pivotal clinical and preclinical studies based on author consensus. The reference lists of relevant articles were also searched for additional articles and abstracts of interest. Abstracts of selected major oncology annual meetings were also searched from
Jan 1, 2016, to Dec 31, 2019.
Research, London, UK (Prof N C Turner MD) Correspondence to:
Dr Aditya Bardia, Department of Medical Oncology,
Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA [email protected]. edu

breast cancers, and cyclin D1 protein overexpression

A

1.Upstream oncogenic signalling leads to activation of cyclin D–CDK4/6 complex
Growth factor receptors

MAPK signalling PI3K/AKT/mTOR signalling
15,16
Although hormone receptor-positive breast cancers usually retain a functional RB1, loss of RB1 expression is commonly seen in triple negative breast cancer and is generally associated with poorer differentiation and
17–19

FAT1–hippo complex
P
YAP

CDK4/6 inhibitors

Cyclin D–CDK4/6
complex

Oestrogen mediated signalling

Biologic rationale for targeting CDK4/6 in hormone receptor-positive breast cancer
The cyclin D1–CDK4/6–RB1 complex is the major

2.Activation of cyclin D–CDK4/6 complex leads to phosphorylation of RB1 which contributes to E2F release and transition of cell cycle from G1 to S phase
P
RB1

RB1

E2F
Cyclin E–CDK2
complex
mediator of cellular proliferation mediated by oestrogen
20–22 Oestrogen induces expression of cyclin 23–25 and thereby promotes CDK4/6 activity in hormone
receptor-positive breast cancers, leading to hyperphos-
26

M G1 E2F Decreasing oestrogen signalling has been shown to

Cyclin B–CDK1
complex
Cell cycle
reduce CDK–cyclin complexing, followed by a subse-
23 Therefore, hormone

B
Aurora kinase A
G2 S

Growth factor receptors
receptor-positive breast cancer is characterised by fre- quent dysregulation of CDK4/6 activity because of the
15,16
but usually retains a functional RB1 that governs cellular
16

1.Upstream oncogenic alterations
•FGFR amplification
•ERBB2 mutation
•AKT hyperactivation
•FAT1 loss

MAPK signalling PI3K/AKT/mTOR signalling
Key preclinical studies of CDK4/6 inhibitors for hormone receptor-positive breast cancer
The pathogenesis of hormone receptor-positive breast cancer is dependent on promoting cyclin D1 expression

FAT1–hippo complex
P
YAP

CDK4/6 inhibitors

Cyclin D–CDK4/6
complex

Oestrogen mediated signalling
and CDK4/6 activity. Although it is difficult to attribute one key experiment that led to the success of CDK4/6 inhibitors, some of the most important obser-
27,28
showed that ablation of cyclin D1 and CDK4 in mouse

2.Cell cycle alterations
•RB1 loss
•Cyclin E overexpression
P
RB1

RB1

E2F
models prevented breast tumours, highlighting the essential role of this axis in oncogenesis. A few years

•Aurora kinase A alteration
Cyclin E–CDK2
complex
29,30 investigated in-vitro sensitivity to palbociclib—the highly selective reversible inhibitor of

M
G1
E2F
CDK4/6—in multiple molecularly characterised human breast cancer cell lines and reported that sensitivity to

Aurora kinase A
Cyclin B–CDK1
complex
Cell cycle G2

S
palbociclib varied on the basis of molecular pheno-
30 Hormone receptor-positive cell lines with luminal features, which usually retain a functional RB1, were found to be most sensitive and basal cell triple negative lines, which usually have functional RB1 loss, were

Figure 1: The CDK4/6 pathway and mechanisms of resistance to CDK4/6 inhibitors
(A) Central role of the cyclin D–CDK4/6 complex in mediating cellular proliferation. (B) Proposed mechanisms driving resistance to CDK4/6 inhibitors. Factors that promote cell cycle are denoted in red, and factors that inhibit cell cycle are denoted in green. AKT=protein kinase B. CDK=cyclin-dependent kinase. FGFR=fibroblast growth factor receptor. mTOR=mammalian target of rapamycin. P=phosphate. PI3K=phosphoinositide 3-kinase. RB1=retinoblastoma tumour suppressor protein.

exists, there are broadly three clinically important sub- groups based on the presence or absence of molecular markers: hormone receptor-positive and HER2-negative breast cancer, HER2-positive (also known as ERBB2) breast cancer, and triple negative breast cancer. Amplifi- cation of the CCND1 gene is estimated to occur in 15% of
30 Of note, growth inhibition was also seen in HER2-amplified cell lines,
30 Overall,
30The combination of palbociclib with tamoxifen was tested in vitro, in oestrogen receptor-positive human breast
30
This work led to an investigator-initiated clinical trial evaluating the combination of palbociclib with antioestrogen therapy in hormone receptor-positive breast cancer, which was later expanded to the pivotal
31The results of the

trial led to the US Food and Drug Administration (FDA) approval of the first CDK4/6 inhibitor, palbociclib,
31

Pivotal trials of CDK4/6 inhibitors in metastatic breast cancer
Approved CDK4/6 inhibitors for breast cancer
There are currently three approved, orally bioavailable, highly selective, small molecule inhibitors of CDK4/6, palbociclib , abemaciclib and ribociclib, for the treatment of advanced hormone receptor-positive and HER2-
32–36 The selectivity of all three compounds is theorised to reflect structural preference for the specialised ATP-binding pocket of CDK4/6 because of specific interactions with residues in
37The specificity of these drugs in targeting CDK4/6 is essential for establishing a therapeutic window through preferential inhibition of oncogenic events without significant toxicity in normal tissue, unlike pan CDK inhibitors, which were found to be overtly toxic
38For reasons that are incompletely understood, CDK4/6 is not essential for proliferation in many cell types, probably because CDK2 can compensate for inhibition of CDK4/6

39Although the three drugs have similar mechanisms of action, key differences exist between them with regard to pharmacokinetics and toxicity, most notably with abemaciclib (table 1). All three drugs are approved in combination with aromatase inhibitors and with the selective oestrogen receptor degrader, fulvestrant for patients with hormone receptor-positive and HER2-negative metastatic breast cancer. Abemaciclib is also approved as monotherapy in all patients with hormone receptor-positive and HER2-negative metastatic breast cancer. Pivotal trials are summarised in table 1.

CDK4/6 inhibitors plus non-steroidal aromatase inhibitors
Palbociclib, ribociclib, and abemaciclib have all been approved for use in combination with a non-steroidal aromatase inhibitor in the first-line setting, for women with metastatic hormone receptor-positive and HER2- negative breast cancer. Pivotal trials investigating these drugs all showed significant improvement in progression- free survival with the addition of CDK4/6 inhibitor to non-steroidal aromatase inhibitor, with almost identical consistent hazard ratios (HRs) across the three trials
1 0·55,2 and 0·58;3 table 1).

CDK4/6 inhibitor

Setting and study population

Sample size

Median progression- free survival vs placebo (months)

Median overall survival vs placebo (months)

Trials in combination with non-steroidal aromatase inhibitors
PALOMA-21 Palbociclib Postmenopausal women with hormone receptor-positive and HER2-negative advanced breast cancer; no previous systemic treatment for advanced breast cancer; and neoadjuvant endocrine therapy permitted if disease-free interval >12 months from therapy completion 666 24·8 vs 14·5 (HR 0·58; p<0·001) Not reported
MONALEESA-22 Ribociclib Postmenopausal women with hormone receptor-positive and HER2-negative advanced breast cancer; no previous systemic treatment for advanced breast cancer; and (neo)adjuvant endocrine therapy permitted if disease-free interval >12 months from therapy completion 668 25·3 vs 16·0 (HR 0·57; p<0·001) Not reported
MONARCH 33 Abemaciclib Postmenopausal women with hormone receptor-positive and HER2-negative advanced breast cancer; no previous systemic treatment for advanced breast cancer; (neo)adjuvant endocrine therapy permitted if disease-free interval >12 months from therapy completion 493 Not reached vs 14·7 (HR 0·54; p<0·001) Not reported
Trials in combination with fulvestrant
PALOMA-34,40 Palbociclib Women with hormone receptor-positive and HER2-negative advanced breast cancer that relapsed or progressed during endocrine therapy; any menopausal status; ≤1 line of chemotherapy for advanced disease* 521 9·5 vs 4·6 (HR 0·46; p<0·001) 34·9 vs 28·0 (HR 0·81; p=0·09)
MONALEESA-38 Ribociclib Postmenopausal women and men with hormone receptor-positive and HER2-negative advanced breast cancer; 0–1 line of endocrine therapy for advanced breast cancer† 726 20·5 vs 12·8 (HR 0·60; p<0·001) Not reached vs 40·0 (HR 0·72; p=0·005)
MONARCH 26 Abemaciclib Women with hormone receptor-positive and HER2-negative advanced breast cancer that had progressed during previous endocrine therapy; any menopausal status, ≤1 endocrine therapy; no previous chemotherapy for advanced disease‡ 669 16·4 vs 9·3 (HR 0·55; p<0·001) 46·7 vs 37·3 (HR 0·76; p=0·014)
Trials in combination with tamoxifen or non-steroidal aromatase inhibitor plus goserelin
MONALEESA-710 Ribociclib Premenopausal and perimenopausal women with hormone receptor-positive and
HER2-negative advanced breast cancer; no previous endocrine therapy for advanced disease; ≤1 line of chemotherapy for advanced disease 672 23·8 vs 13·0 (HR 0·55; p<0·001) Not reached vs 40·9 (HR 0·71; p=0·01)
Palbociclib dose was 125 mg daily orally and ribociclib dose was 600 mg daily orally on a 3 week on, 1 week off schedule in all studies. The abemaciclib final dose was 150 mg orally twice a day continuously. CDK=cyclin-dependent kinase. HER2=human epidermal growth factor receptor 2. HR=hazard ratio. *Patients with postmenopausal advanced breast cancer must have had progression during a previous aromatase inhibitor treatment. Premenopausal and perimenopausal patients must have had progression during a previous endocrine therapy. Premenopausal and perimenopausal participants received goserelin for the duration of study treatment starting ≥4 weeks before randomisation and continuing every 28 days. †First-line (ie, treatment-naive for patients with metastatic breast cancer): relapse >12 months after neoadjuvant endocrine therapy for localised breast cancer or de-novo metastatic breast cancer with no previous endocrine therapy. Second-line setting and patients who relapsed early: early relapse when receiving or ≤12 months after (neo)adjuvant endocrine therapy or relapse >12 months after (neo)adjuvant endocrine therapy with progressive disease after one line endocrine therapy for metastatic breast cancer or metastatic breast cancer with progression after one line endocrine therapy for metastatic breast cancer. ‡Premenopausal and perimenopausal participants received a gonadotropin-releasing hormone agonist.
Table 1: Summary of randomised phase 3 clinical trials evaluating CDK4/6 inhibitors in hormone receptor-positive and HER2-negative metastatic breast cancer

CDK4/6 inhibitors plus fulvestrant
Palbociclib, ribociclib, and abemaciclib have also been approved in combination with fulvestrant for patients whose tumours progressed while receiving previous endocrine therapy. Some key differences exist between the phase 3 trials in this setting, particularly regarding eligibility criteria; however, all the trials showed significant improvements in progression-free survival.
4 compared fulvestrant plus palbociclib with placebo in both premenopausal (goserelin added to the regimen for premenopausal women) and postmenopausal women with hormone receptor-positive and HER2-negative metastatic breast cancer who had relapsed or progressed during previous endocrine treatment, with no limit on the number of
4 PALOMA-3 reported a significant improvement in progression-free survival with addition of palbociclib to fulvestrant (4·6 months to 9·5 months; p<0·01), and a numerical increase in overall survival that did not reach statistical significance
5
Of note, a more substantial overall survival effect was reported in patients that had been sensitive to previous lines of endocrine therapy (overall survival 29·7 months for patients in the placebo group vs 39·7 months in those who were in the palbociclib group), but because this was a retrospective subset analysis the findings should be considered hypothesis-generating and needs validation in additional studies. The MONARCH 2
6 (fulvestrant plus abemaciclib or placebo) included women of any menopausal status (with a gonadotropin- releasing hormone agonist added to the regimen for premenopausal women) with hormone receptor-posi- tive and HER2-negative metastatic breast cancer who had progressed during a previous endocrine therapy regimen, but no more than one line of previous therapy was permissible. The addition of abemaciclib resulted in an improvement in progression-free survival from
6 with similar HRs to those reported in PALOMA-3 (HR 0·46; p<0·001), and a higher absolute progression-free sur- vival in the control group in the study (9·3 months) compared with PALOMA-3 (4·6 months), reflecting the much less pretreated patient population. The study also reported positive overall survival results, favouring the abemaciclib group (46·7 months in the abemaciclib
7
8 (fulvestrant with ribociclib or placebo) included postmenopausal women and men with hormone receptor-positive and HER2-negative metastatic breast cancer who had received no or one previous first-line endocrine therapy for advanced disease, and therefore included both first-line and
8 The addition of ribociclib resulted in an improvement in progression-free survival from 12·8 months to 20·5 months (HR 0·60; p<0·001),
8 The study also reported positive

overall survival results favouring the ribociclib group (HR 0·724; p<0·01) in both the first-line and second-line
9

CDK4/6 inhibitors in premenopausal women Premenopausal women with hormone receptor-positive breast cancer commonly have an aggressive tumour biology with higher predilection for visceral metastasis and shorter survival. Although some of the pivotal CDK4/6 inhibitor trials included both premenopausal
10,11 was a phase 3 trial that focused on premenopausal women with hormone receptor-positive and HER2-negative metastatic breast cancer, with patients randomly assigned to receive either tamoxifen and goserelin or a non-steroidal aroma- tase inhibitor and goserelin plus ribociclib or placebo, as a first-line endocrine-based therapy for metastatic breast cancer. The addition of ribociclib significantly improved progression-free survival (HR 0·55; p<0·001) and overall
10,11 An exploratory analysis of all subgroups suggested a greater overall survival benefit was seen in people of Asian origin, in whom the HR was 0·40 (95% CI 0·22–0·72) compared with an HR of 0·91 (0·64–1·30) in people not of Asian origin, but this is a preliminary finding that requires
11

CDK4/6 inhibitors versus chemotherapy
Although endocrine-based therapy is the preferred first-line therapy option for hormone receptor-positive metastatic breast cancer, chemotherapy is sometimes used as a first-line therapy for patients with aggressive hormone receptor-positive disease in whom a quick response is needed—such as patients with visceral disease, particularly the ones in visceral crisis. However, the advent of CDK4/6 inhibitors has challenged this paradigm. In a small randomised phase 2 study (n=189) median progression-free survival after endocrine therapy with palbociclib (19·0 months) was superior to capecitabine (11·3 months; p=0·0493) in premenopausal women with hormone receptor-positive metastatic
41 Several studies are exploring regimens using a CDK4/6 inhi- bitor versus chemotherapy in various settings, including how CDK4/6 inhibitors compare with chemotherapy for those with impending or actual visceral crisis (RIGHT Choice study, NCT03839823).

CDK4/6 inhibitors as monotherapy
Unlike other CDK4/6 inhibitors, abemaciclib has also shown single-agent activity in endocrine-resistant
42) that recruited women with heavily pretreated hormone receptor-positive and HER2-negative metastatic breast cancer (disease progression following both endocrine therapy and at least one but no more than two lines of chemotherapy) confirmed objective response rate

of 19·7% and a median progression-free survival of 6·0 months was noted with single-agent abemaciclib leading to approval of abemaciclib monotherapy at a dose
42 Although the study included heavily pretreated patients, the participants were all CDK4/6 naive, and the activity of abemaciclib mono- therapy in patients who have received a previous CDK4/6 inhibitor is unclear. However, preliminary data from retrospective multi-institutional studies suggests that abemaciclib retains activity in a subset of patients who have previously received CDK4/6 inhibitors possibly because of an absence of cross-resistance with other
43,44

Safety and tolerability
In general, the CDK4/6 inhibitors are well tolerated and adverse events are typically easily managed with dose modification and supportive care measures. Haemato- logical toxicities, primarily neutropenia (although febrile neutropenia is rare), are commonly seen with all three inhibitors, but they are more frequent with palbociclib and ribociclib than abemaciclib. The cytopenias seen with CDK4/6 inhibitors are considered an on-target effect because CDK6 plays a key role in the proliferation
37 Unlike cytotoxic chemo- therapy, which causes apoptosis, the CDK4/6 inhibitors impede neutrophil precursors through a cytostatic action resulting in cellular quiescence that is often rapidly
39 Palbociclib and ribociclib are dosed intermittently (ie, 21 days on followed by 7 days off) to allow for the recovery of haematological precursors. Abemaciclib, which is more selective for CDK4 than CDK6, can be dosed continuously because it is associated with a lower prevalence of haematological
32For all three agents, the FDA has issued a 45
A detailed list of toxicities reported in the phase 3 clinical trials of CDK4/6 inhibitors and aromatase inhibitors is displayed in table 2.
Alongside the haematological toxicities, there are some distinct side-effects associated with the different CDK4/6 inhibitors. For example, ribociclib might cause hepatotoxicity more frequently than the others, and has also been associated with reversible, concentration-
2Of note, abemaciclib results in higher prevalence of diarrhoea and fatigue in comparison with palbociclib and ribociclib, and
3
Venous thromboembolic events were reported in more patients who received abemaciclib compared with pla-
3(5% vs 0·9%) and MONARCH 36 (5% vs 0·6%). An increased concentration of serum creatinine can be seen with abemaciclib as a result of inhibition of several tubular secretion transporters. Of note, this does not affect glomerular function and is not
42 All three CDK4/6 inhibitors are subject to interactions with other drugs primarily

mediated by modification of the cytochrome P450 (CYP) pathway because the CDK4/6 inhibitors are all major sub-
46 Consequently, moderate or strong inhibitors or inducers of CYP3A4 must be avoided for patients receiving these inhibitors.

Unanswered questions
When should CDK4/6 inhibitors be used (first line or later)?
A number of important questions exist surrounding CDK4/6 inhibitors (appendix p 1). For patients who relapse early during or shortly after completion of the adjuvant endocrine therapy, the general consensus is that CDK4/6 inhibitors should be used as the first-line regimen because progression-free survival to endocrine monotherapy might only range from 4 to 6 months. However, considerable debate exists over whether all patients should receive a CDK4/6 inhibitor in the first- line setting when patients have de-novo metastatic disease or have relapsed years after completion of adjuvant endocrine therapy. Although such patients could do well with endocrine monotherapy, particularly
48 these patients could do even better with the addition of CDK4/6 inhibitors.
With the absolute magnitude of improvement in progression-free survival greater in the first-line setting than in the second-line setting in studies of the first-line setting and the emerging data related to improvements in overall survival from CDK4/6 inhibitors, including
9,11 we believe the pendulum is swinging towards CDK4/6 inhibitors as a first-line therapy for hormone receptor-positive and HER2-negative metastatic breast cancer. However, the effect of CDK4/6 sequence is unclear and at least one study is randomising drug sequence to directly address the issue in a randomised clinical trial (SONIA, NCT03425838; appendix p 1).

Do all patients with hormone receptor-positive and HER2-negative metastatic breast cancer benefit?
There is also an absence of predictive biomarkers to guide who might benefit from CDK4/6 inhibitors. The only validated biomarker predictive of response is expression of the oestrogen receptor. Because the pivotal phase 3 trials of CDK4/6 inhibitors in the first-line setting all show a similar near-doubling of progression-
1–3 and the benefit was seen across subgroups (including patients with bone-only disease), we recommend upfront combination therapy with endocrine therapy plus a CDK4/6 inhibitor, if consistent with the patient’s goals of care. Older patients also appear to benefit equally from CDK4/6
49

What is the best endocrine therapy backbone?
For patients who do not relapse on adjuvant aromatase inhibitor, generally a combination with an aromatase inhibitor is used in the first-line setting and fulvestrant is

See Online for appendix

Palbociclib Ribociclib Abemaciclib Palbociclib Ribociclib Abemaciclib

50 (nM)
Cyclin D1–CDK4 11 10 2
Cyclin 16 39 10 D1/2/3–CDK6
Cyclin B–CDK1 >10 000 113 000 1627
Cyclin A/E–CDK2 >10 000 76 000 504
Cyclin T–CDK9 NR NR 57
Mean terminal 26 36 17–38 half-life (h)
Standard dosing 125 mg 600 mg 150 mg twice daily
daily (3 weeks daily 3 weeks in combination with
on, 1 week on, 1 week antioestrogen;
off) off) 200 mg twice daily as monotherapy
Adverse event frequency in combination with non-steroidal aromatase inhibitor in phase 3 trials (%)
Neutropenia
Any grade 80 75 41
Grade 3–4 66 60 22 Anaemia
Any grade 24 18 28
Grade 3–4 5 1 6 Thrombocytopenia
Any grade 16 29* 10
Grade 3–4 1 1* 2 Nausea

Table 2: CDK4/6 inhibitors: key pharmacologic features and adverse
1–3,14,32,46,47

Any grade 35 52 39
Grade 3–4 <1 2 <1

Vomiting Any grade Grade 3–4
Abdominal pain
Any grade NR
Grade 3–4 NR Diarrhoea
Any grade Grade 3–4
Decreased appetite
Any grade Grade 3–4
Cough
Any grade Grade 3–4
Stomatitis Any grade Grade 3–4

16
1

26
1

15
1

25
0

30
1

29
4

11
1

35
1

19
2

23
0

12
<1

28
1

35
2

81
9

27
1

13
0

15
<1
typically chosen in the second-line setting. Although the pivotal phase 3 trials of CDK4/6 inhibitors in combination with non-steroidal aromatase inhibitors were first-line
8 trial—which investigated the use of fulvestrant with or without ribociclib—did include first-line patients with hormone receptor-positive and HER2-negative metastatic breast cancer. Both patients with de-novo metastatic breast cancer and patients who relapsed more than a year after (neo)adjuvant endocrine therapy for localised breast cancer, showed significant improvement in both progression-free survival and overall
8 Comparing the various first-line CDK4/6 trials, the absolute progression-free survival with fulvestrant and CDK 4/6 inhibitor appears to be higher than that with non-steroidal aromatase inhibitors; however, cross- trial comparisons should be interpreted with caution and further validation is required, ideally in a randomised controlled trial.

Peripheral oedema

Any grade NR 12 12
Grade 3–4 NR 0 0 Rash
Any grade 18 17 11
Grade 3–4 1 1 1
(Table 2 continues in next column)
What is the best treatment after progression on a CDK4/6 inhibitor?
Another major question is choice of second-line therapy and optimal sequencing strategy following disease progression on first-line CDK4/6 inhibitor therapy. At present, there are no prospective data to support the use of continued CDK4/6 inhibitor therapy after disease progression if CDK4/6 therapy was used in the first-line setting, including abemaciclib monotherapy in patients

who have previously received a CDK4/6 inhibitor regi- men. Similarly, it is unclear if CDK4/6 inhibitors change tumour biology and alter how other targeted therapies, such as everolimus, which were FDA approved in the
50will perform post-CDK4/6 inhibitor, although existing post-progression trial evidence from
5 suggests that, in general, therapies have similar efficacy after progression. However, we believe this is probably context-dependent and the response might vary depending on whether clinical progression is due to resistance to the endocrine therapy partner, the CDK4/6 inhibitor, or both. Furthermore, resistance to one targeted therapy could confer cross-resistance to another targeted therapy. For example, based on the
51the α-specific phosphoinositide 3-kinase inhibitor (PI3K), alpelisib, was approved in combination with endocrine therapy for phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit α (PIK3CA) mutated hormone receptor-positive and HER2-negative metastatic breast cancer, but relatively few patients in
51
Preliminary translational work suggests that tumours exposed to CDK4/6 inhibitors could acquire phos- phatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN) loss, which can then result in cross-resistance to PI3K inhibitors, showing how previous exposure to CDK4/6 inhibitors could affect the patient’s response to subsequent ther-
52Similarly, fibroblast growth factor receptor 1 (FGFR1) amplification can mediate broad cross-resistance to multiple targeted inhibitors, including CDK4/6 and PI3K inhibitors, but retains sensitivity to mTOR inhi-
53Several biomarker-driven clinical trials are trying to answer these important questions and will shed light on optimal sequencing in the post CDK4/6 second-line setting and beyond.

Understanding response and resistance
Understanding patients’ response to and resistance to CDK4/6 inhibitors is a major unmet need. At present, the only established predictive biomarker is oestrogen
30 A number of studies have explored resistance to CDK4/6 inhibitors and can broadly be divided into two major categories: cell cycle alterations or activation of upstream tyrosine kinase receptors and PI3K/mitogen-activated protein kinase 1 signalling (figure 1B).
Because RB1 is the primary phosphorylation target of the cyclin D–CDK4/6 complex, it is not surprising that loss of RB1 is one of the potential mechanisms of resistance to CDK4/6 inhibitors. In vitro, loss of RB1 is associated with de-novo CDK4/6 inhibitor resistance, and, similarly, loss of RB1 expression over time in the presence of palbociclib in hormone receptor-positive breast cancer cells and in a patient-derived xenograft model with prolonged exposure to ribociclib has been
54Furthermore, emergence of acquired RB1

mutations have been identified through serial analysis of circulating tumour DNA as a potential mechanism of clinical resistance to CDK4/6 inhibitors in patients with metastatic breast cancer, with an estimated frequency of
55,56 Inactivating mutations in the RB1 gene before treatment might also be predictive of little benefit
57Besides RB1 loss, ectopic overexpression of cyclin E1 and cyclin E2 can result in a bypass track and promote resistance to antioestrogen therapies and palbociclib monotherapy in vitro, through
58A large-panel tissue-based gene
59cohort showed that palbociclib efficacy was lower in patients with high median cyclin E1 (CCNE1) mRNA expression (median progression-free survival in the palbociclib group was 7·6 months [high cyclin E1] vs 14·1 months [low cyclin E1]; placebo group 4·0 months [high cyclin E1]
vs 4·8 months [low cyclin E1]; false discovery rate-adjusted p=0.0238). Of note, early evidence suggests overex- pression of CDK6 might cause resistance to CDK4/6
60,61 CDK6 overexpression also led to reduced expression of the oestrogen receptor and con- comitant resistance to antioestrogen agents, suggesting a mechanism by which resistance occurs to both the
60
Similarly, molecular alterations in aurora kinase A (AURKA), that can lead to enhanced G2 to M phase cell cycle transition, have been observed in tumour specimens
62 Overall, there is mounting evidence that bypass cell cycle alterations probably have a key role in resistance to CDK4/6 inhibitors.
In addition to cell cycle alterations, additional evidence suggests upstream oncogenic signal transduction altera- tions can also promote resistance. Whole exome analysis of tumour specimens resistant to CDK4/6 inhibitors showed a broad array of potential resistance mechanisms, including upstream alterations in AKT1, KRAS, HRAS,
62 These alterations provoke resistance to CDK4/6 inhibitors in vitro and, although response to antioestrogens in vitro was variable, these findings suggest that, in some instances, a single driver might provoke resistance to both drugs in the combination whereas in others a cooperating event might be required. Acquired point mutations in ERBB2 have been observed to cause resistance to antioestrogens and palbociclib in vitro that could be reversed with the addition of the HER2
63,64 Overexpression of FGFR1 or FGFR2 is another potential mechanism of resistance to CDK4/6 inhibitors, and FGFR inhibition has been
53,65,66 Finally, rare inactivating mutations in the FAT atypical cadherin 1 (FAT1) gene have also been observed as a potential resistance mechanism, with its loss leading to activation of YAP1 via the Hippo pathway and subsequent elevation
67
Given these findings, there is significant interest in exploring combinatorial therapy to overcome resistance

important set of trials are those assessing whether

Metastatic hormone receptor-positive and HER2-negative breast cancer

Disease recurrence during or within 12 months of adjuvant endocrine therapy
De-novo metastatic disease, or disease recurrence >12 months after adjuvant endocrine therapy

Premenopausal Postmenopausal Premenopausal Postmenopausal

metastases only
Visceral Bone metastases

NSAI plus CDK4/6 inhibitor* with LHRH agonist NSAI plus CDK4/6 inhibitor† NSAI with or without CDK4/6 inhibitor‡ Fulvestrant plus CDK4/6 inhibitor with LHRH agonist Fulvestrant plus CDK4/6 inhibitor
CDK4/6 inhibitors can improve survival in women treated for primary breast cancer, and CDK4/6 inhibitors have been, and continue to be, explored in the neoadjuvant and adjuvant setting for hormone receptor-positive and HER2-negative localised breast cancer. There are also studies of CDK4/6 inhibitors in HER2-positive breast cancer and triple negative breast cancer. Combination studies with immunotherapy are also ongoing, with a preclinical rationale suggesting that CDK4/6 exposure
69

Neoadjuvant trials
Because the majority of hormone receptor-positive and HER2-negative metastatic breast cancer presents as recurrent disease and late recurrences are common, the exploration of new strategies to reduce recurrence risk in
70Multiple neoadjuvant studies have shown that CDK4/6 inhibitors substantially increase cell-cycle arrest compared with that achieved

with hormone therapy alone. The phase 2 neoadjuvant

Figure 2: Initial treatment approach for hormone receptor-positive and HER2-negative metastatic breast cancer
HER2=human epidermal growth factor receptor 2. NSAI=non-steroidal aromatase inhibitor. LHRH=luteinising hormone-releasing receptor agonist. *Level 1 evidence exists in the premenopausal setting with ribociclib only; however, CDK4/6 inhibitors are often used interchangeably. †Improvement in progression-free survival and overall survival also noted with fulvestrant and ribociclib in the first-line setting, which could be used as the therapeutic regimen. ‡Although the addition of CDK4/6 inhibitors is associated with improvement in progression-free survival, even in patients who have bone metastases only, patients might also do well for a long time with endocrine monotherapy with less frequent monitoring, and detailed discussion with the patient about pros and cons needs to be considered.

to CDK4/6 inhibitors. On the basis of heterogeneus genomic landscape of resistance, it is clear that a more personalised approach will be needed and many of the implicated resistance mediators can be targeted with emerging therapeutic agents in the clinic. The TAKTIC trial (NCT03959891) began accrual and is exploring the use of the AKT1 inhibitor, ipatasertib, with an antioestrogen with or without continued CDK4/6 block- ade in hormone receptor-positive and HER2-negative metastatic breast cancer patients with previous progres- sion on CDK4/6-directed therapy. Additional strategies might include the use of CDK2 inhibitors in patients with CCNE1/2 amplified or overexpressing tumours, aurora kinase inhibitors in AURKA amplified tumours, neratinib and other HER2 kinase inhibitors in ERBB2 mutant patients, FGFR-pathway inhibitors in FGFR1/2 mutant or amplified tumours, and ERK inhibitors
53,62,63 There is also growing interest in selective CDK7 inhibitors because CDK7 is
68

Future directions
Given the success of CDK4/6 inhibitors for hormone receptor-positive and HER2-negative metastatic breast cancer, there is great interest in exploring these drugs in other settings—selected ongoing and completed trials are summarised in the appendix (p 2). The most
71showed that the addition of CDK4/6 inhibitors caused enhanced antiproliferative activity in hormone receptor-positive and HER2-negative early breast cancer, with 87% complete cell-cycle arrest with palbociclib plus anastrozole compared with a 26% com- plete arrest with anastrozole alone (p<0·001). In the
72the addition of abemaciclib to anastrozole was also shown to significantly reduce Ki67 compared with anastrozole alone, with similar results
73Neoadju- vant trials comparing endocrine therapy and CDK 4/6 inhibitor versus chemotherapy have shown equivalent
74,75 In addition, the neoadjuvant approach, including window of opportunity studies, might also shed light on predictive biomarkers. For example, the
76 a window of opportunity clinical trial comparing 14 days of palbociclib with placebo before surgery for early breast cancer, found that early decrease of RB1 phosphorylation correlates with the drug’s effect on cell proliferation, suggesting a potential approach to
76Results from another neoadjuvant trial (NCT02712723) are anticipated and will help clarify the potential role of CDK4/6 inhi- bitors in this setting.

Adjuvant trials
All three CDK4/6 inhibitors are being studied in the adjuvant setting in phase 3 trials. Two of the trials have completed enrolment: the PALLAS trial (NCT02513394)— studying the addition of palbociclib to standard of care endocrine therapy in stage 2 and 3 hormone receptor- positive breast cancer—and the monarchE trial (NCT03155997)—which is exploring the adjuvant use of abemaciclib. The NATALEE trial (NCT03701334), assessing the adjuvant use of ribociclib, is currently enrolling participants. The phase 3 PENELOPE-B trial

(NCT01864746) is studying the addition of palbociclib to standard endocrine therapy for patients with hormone receptor-positive residual disease after neoadjuvant che- motherapy. If these studies are positive, a very important question will be the optimal duration of CDK4/6 inhibitor therapy because these drugs have been given for variable durations in the different studies outlined in this review. Timing might also be critical because many of the trials are assessing progression-free survival and events that might occur late in disease course.

Continued CDK4/6 blockade
The question of continued CDK4/6 blockade (ie, using CDK4/6 inhibitors after disease progression and the utility of combinatorial strategies) is also being explored in a number of trials of metastatic breast cancer. For example, the TRINITI study (NCT02732119) was a phase 1/2 study that explored triplet therapy with ribociclib plus the mTOR inhibitor everolimus and the steroidal aromatase inhibitor exemestane following disease progression after a previous CDK4/6 inhibitor-based therapy. In the study, a median progression-free survival of 5·7 months was observed, but progression-free survival varied substan- tially with the baseline biomarker profile ranging from 3·2 months in people with ESR1 and PIK3CA mutations to 9·9 months in people with wild-type ESR1 and PIK3CA, highlighting the importance of biomarker strategies
77Similarly, the phase 2 PACE study (NCT03147287) is evaluating fulvestrant monother- apy against fulvestrant plus palbociclib with or without avelumab in the post CDK4/6 regimen setting. The trial aims to shed more light on biomarkers to guide optimal sequencing.
As the amount of data from CDK4/6 inhibitor studies increases, academic collaboration will be important to better understand which subgroups of patients benefit most from continued CDK4/6 blockade, and to establish the optimal sequencing strategy for patients with breast cancer.

CDK4/6 inhibitors in other breast cancer subtypes
A growing number of trials are exploring CDK4/6 inhibitors in HER2-positive breast cancer, particularly hormone receptor-positive and HER2-positive breast
78exploring abemaciclib with other therapies in hormone receptor- positive and HER2-positive metastatic breast cancer reported improved progression-free survival with abemaciclib plus fulvestrant and trastuzumab compared with trastuzumab and investigator’s choice of chemo-
78 Use of CDK4/6 inhibitors is more challenging in triple negative breast cancer because of frequent RB1 loss; however, subsets of patients might be sensitive, particularly patients with androgen receptor (AR)-positive triple negative breast cancer because there
79
and ongoing studies (NCT02605486; NCT03090165) are

exploring CDK4/6 inhibitors with AR antagonists in patients with triple negative breast cancer. Furthermore, there is emerging interest in the use of CDK4/6 inhibitors to induce cell-cycle arrest in bone marrow cells before starting chemotherapy, consequently protecting the bone
80

Conclusions
The integration of CDK4/6 inhibitors in the treatment of hormone receptor-positive and HER2-negative metastatic breast cancer represents a major advancement in breast cancer therapy. The addition of CDK4/6 inhibitors to endocrine therapy appears to benefit all clinicopatho- logical subgroups with hormone receptor-positive and HER2-negative metastatic breast cancer, as suggested by
81 Not surprisingly, these drugs have entered widespread clinical practice and have emerged as the standard of care for the initial treatment of hormone receptor-positive and HER2- negative metastatic breast cancer (figure 2). Preclinical and translational work suggests that the landscape of resistance to these agents is heterogeneous, implicating a variety of cell cycle regulators and oncogenic drivers at the time of progression. Much remains to be learned about the optimal use of CDK4/6 inhibitors for patients with breast cancer. A number of ongoing clinical and translational studies will help to answer important questions about response and resistance to CDK4/6 inhibition, treatment sequencing and the effect of drug sequence on early breast cancer, combinatorial strategies, and the role of CDK4/6 in other subtypes, which could establish CDK4/6 inhibitors as the cornerstone of breast cancer therapy in various settings.
Contributors
LMS searched the literature and wrote the first draft of the manuscript. All authors edited the manuscript and approved the final version.
Declaration of interests
LMS reports consultant and advisory board fees from Novartis, Lumicell, and Puma Biotechnology; travel funds from Merck and Tesaro; and research grants to their institution from Merck and Tesaro. SAW reports
consultant and advisory board fees from Foundation Medicine, InfiniteMD, Eli Lilly, and PumaBiotechnology; equity from InfiniteMD; and a research grant to their institution from Genentech. BM reports consultant and advisory board fees paid to their spouse from MOTUS GI; and a research grant paid to their institution from Puma Biotechnology. FA reports research grants paid to their institution from AstraZeneca, Lilly, Novartis, Pfizer, Daiichi, and Roche. NCT reports consultant and advisory board fees from AstraZeneca, Bristol-Myers Squibb, Lilly, Merck Sharpe and Dohme, Novartis, Pfizer, Roche/Genentech, Tesaro, Bicycle Therapeutics, and Taiho; and research grants paid to their institution from AstraZeneca, BioRad, Pfizer, Roche/Genentech, Clovis, Merck Sharpe and Dohme, and Guardant Health. AB reports consultant and advisory board fees from Genentech/
Roche, Immunomedics, Novartis, Pfizer, Merck, Radius Health, Spectrum Pharma, Taiho Pharm, Diiachi, Sanofi, Puma Biotechnology, Phillips, and Eli Lilly; a research grant from Biothernostics; and research grants paid to their institution from Genentech/Roche, Immunomedics, Novartis, Pfizer, Merck, Radius Health, Sanofi, and Mersana.
Acknowledgments
LMS is supported by National Cancer Institute grant KL2 TR002542,
a Terri Brodeur Breast Cancer Foundation grant, and a Massachusetts General Hospital Electronic Space Systems Corporation Breast Cancer Research Fund grant.

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