Targeting the cyclin D–cyclin-dependent kinase (CDK)4/6–retinoblastoma pathway with selective CDK 4/6 inhibitors in hormone receptor-positive breast cancer: rationale, current status, and future directions

Abstract

Dysregulation of the cyclin D–cyclin-dependent kinase (CDK)4/6–INK4–retinoblastoma (Rb) pathway is an important contributor to endocrine therapy resistance. Recent clinical development of selective inhibitors of CDK4 and CDK6 kinases has led to renewed interest in cell cycle regulators, following experience with relatively nonselective pan-CDK inhibitors that often resulted in limited activity and poor safety profiles in the clinic. The highly selective oral CDK 4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219) are able to inhibit the proliferation of Rb-positive tumor cells and have demonstrated dose-dependent growth inhibition in ER+ breast cancer models. In metastatic breast cancer, all three agents are being explored in combination with endocrine therapy in Phase III studies. Results so far indicate promising efficacy and manageable safety profiles, and led to the FDA approval of palbociclib. Phase II–III studies of these agents, in combination with endocrine therapy, are also underway in early breast cancer in the neoadjuvant and adjuvant settings. Selective CDK 4/6 inhibitors are also being investigated with other targeted agents or chemotherapy in the advanced setting. This article reviews the rationale for targeting cyclin D–CDK 4/6 in hormone receptor-positive (HR+) breast cancer, provides an overview of the available preclinical and clinical data with CDK 4/6 inhibitors in breast cancer to date, and summarizes the main features of ongoing clinical trials of these new agents in breast cancer. Future trials evaluating further combinations strategies with CDK 4/6 backbone and translational studies refining predictive biomarkers are needed to help personalize the optimal treatment regimen for individual patients with ER+ breast cancer.

Introduction

Breast cancer is a diverse disease, comprising several molecular subgroups with distinct tumor biology. Approximately 75% of breast cancers express the estrogen receptor (ER) and rely upon ER signaling for growth and survival (Nadji et al., 2005). Endocrine therapy, which antagonizes the growth-promoting activity of estrogen, remains the predominant front-line treatment for these patients (Cardoso et al., 2014). However, not all patients respond (de novo resistance), and a proportion of patients who do respond eventually progress (acquired resistance) (Osborne et al., 2011). Multiple mechanisms for endocrine resistance have been proposed, including the disruption of various components of the ER pathway itself and alterations in the cell cycle and cell survival signaling molecules (Osborne et al., 2011). Among these, dysregulation of the cyclin D–cyclin-dependent kinase (CDK)4/6–INK4–retinoblastoma (Rb) pathway has been associated with a poor response to endocrine therapy (Thangavel et al., 2011) and alterations in this pathway are frequently observed in hormone receptor-positive (HR+) breast cancer (Cancer Genome Atlas Network, 2012). Thus, CDK4 and CDK6 are considered valid targets for therapeutic intervention. This review will focus on the rationale for targeting cyclin D–CDK 4/6 in HR+ breast cancer, and will provide an overview of the preclinical and clinical data with CDK 4/6 inhibitors to date.

Overview of the cyclin D–CDK 4/6–INK4–Rb pathway

The cell cycle constitutes a series of tightly controlled events that drive DNA replication and cell division (Caldon et al., 2006). The cell cycle is divided into phases: G0 (quiescence) followed by G1 (pre-DNA synthesis), S (DNA synthesis), G2 (pre-division), and M (cell division) (Caldon et al., 2006). The progression from G1 to S is a critical checkpoint in protecting the cell from abnormal replication, and a key regulator of this process is the cyclin D–CDK 4/6–INK4–Rb pathway (Fig. 1) (Lange et al., 2011). A variety of mitogenic signaling pathways, including steroid hormones (such as the ER pathway), PI3K/AKT/mTOR, MAPKs, wnt/β-catenin, STATs, and NF-κB/IKK, upregulate the expression of cyclin D, which associates with CDK 4/6 (Lange et al., 2011). Activation of the cyclin D-CDK 4/6 complex contributes to the hyperphosphorylation of the Rb protein, which causes inactivation of its growth-inhibitory function by decoupling it from E2F transcription factors. The newly released E2F transcription factors allow the transcription of genes promoting entry into the S phase and thus cell-cycle progression (Lange et al., 2011). Association of cyclin D with CDK 4/6 is tightly regulated by inhibitors, such as INK4 (p16^INK4A, p15^INK4B, p18^INK4C, p19^INK4D), Cip (21^Cip1), and Kip (p27^kip1, p57^kip2) proteins (Fig. 1) (Ortega et al., 2002).

Fig. 1. Targeting the Cyclin D–CDK 4/6–INK4–Rb pathway.

CDK cyclin-dependent kinase, E2F E2 transcription factor, ER estrogen receptor, GRB2 growth factor receptor-bound protein 2, HR+ hormone receptor-positive, mTOR mammalian target of rapamycin, PI3K phosphatidylinositol 3-kinase, Rb retinoblastoma, RTK receptor tyrosine kinase, S6K S6 kinase

Dysregulation of the cyclin D–CDK 4/6–INK4–Rb pathway in breast cancer

The cyclin D–CDK 4/6–INK4–Rb pathway is critical to cell cycle entry and the majority of cancers disrupt this pathway to promote cell proliferation through mechanisms including overexpression, amplification, and chromosomal translocations of D cyclins; mutations and amplification of CDK 4/6; and loss of inhibitors such as INK4 proteins (Ortega et al., 2002; Asghar et al., 2015; Shapiro, 2006). Loss of the tumor suppressor p16 is one of the most common abnormalities in breast cancer and has been observed in 49% of archival breast tumor specimens (Geradts and Wilson, 1996). Cyclin D1 amplification and overexpression also play important roles (Lundgren et al., 2012), and amplification of the CCND1 gene, which encodes cyclin D1, has been identified in 29–58% of breast cancers (Cancer Genome Atlas Network, 2012). In addition, cyclin D1 protein overexpression, whether due to gene amplification or transcriptional or post-transcriptional dysregulation, is found in up to 50% of breast cancers, where it is believed to drive aberrant phosphorylation and inactivation of Rb protein (Lundgren et al., 2012; Abraham et al., 2014). Notably, a deficiency in Rb is associated with the evolution of a CDK 4/6-independent state, poor prognosis in response to hormonal therapy, and resistance to CDK 4/6 inhibition (Ertel et al., 2009; Dean et al., 2010). However, the majority (>90%) of ER+ breast cancers express functional Rb (Abraham et al., 2014).

Inhibiting CDK 4/6 in HR+ breast cancer

In the past, therapeutic targeting of CDK activity has been limited to pan-CDK inhibitors, which generally resulted in limited activity and challenging safety profiles (Dickson, 2014). However, the development of selective ATP inhibitors of CDK4 and CDK6 kinase activity has the potential to cause dose-dependent G1 arrest in human breast cancers with an improved safety profile. Three CDK 4/6 inhibitors are currently in clinical development: palbociclib (PD0332991; Pfizer Inc., FDA-approved), ribociclib (LEE011; Novartis), and abemaciclib (LY2835219; Lilly, FDA Breakthrough Therapy status).

Preclinical data for single-agent CDK 4/6 inhibitors

Determining the selectivity of CDK 4/6 inhibitors has been an important step in establishing the effectiveness of these compounds versus pan-CDK inhibitors (Fry et al., 2004). Palbociclib, ribociclib, and abemaciclib are highly selective CDK 4/6 inhibitors with IC₅₀ values against CDK4 and CDK6 of <40 nM (Table 1). All three agents inhibit cell proliferation in Rb-positive cells and have demonstrated dose-dependent growth inhibition in tumor xenograft models (Fry et al., 2004; Gelbert et al., 2014; Kim et al., 2013).

Table 1.

IC₅₀ values of CDK 4/6 inhibitors

	IC50 (nM)
	Palbociclib (Fry et al., 2004)	Ribociclib (Kim et al., 2013)	Abemaciclib (Gelbert et al., 2014)
CDK4-cyclin D1	11	10	2
CDK6-cyclin D1/2/3	16	39	10
CDK1-cyclin B	>10,000	113,000	1627
CDK2-cyclin A/E	>10,000	76,000	504
CDK9-cyclin T	NR	NR	57

NR not reported

Preclinical studies have provided rationale for the clinical development of CDK 4/6 inhibitors in specific molecular subgroups of breast cancer. Among a panel of 47 and 50 breast cancer cell lines exposed to palbociclib and ribociclib, respectively, those that were ER+ were the most sensitive to growth inhibition, while basal subtypes were shown to be the most resistant to palbociclib (Finn et al., 2009; O’Brien et al., 2014). Notably, responses were also seen in HER2-amplified cell lines, though primarily those with luminal features (O’Brien et al., 2014). Palbociclib has been shown to slow tumor progression, not only by exerting cell-cycle control, but also via the suppression of epithelial–mesenchymal transition and stem-like properties of cancer cells (Lamb et al., 2013; Arima et al., 2013). In addition, elevated expression of cyclin-D1, Rb and reduced p16 expression have been associated with sensitivity towards palbociclib (Finn et al., 2009).

Clinical data for single-agent CDK 4/6 inhibitors

Safety and preliminary clinical activity

The safety and preliminary antitumor activity of CDK 4/6 inhibitors as single agents have been investigated in several clinical trials. These are summarized in Table 2 and select trials discussed below.

Table 2.

Clinical trials of single-agent CDK 4/6 inhibitors

Study drug	NCT number/reference	Phase	Patient population	Safety	Clinical activity
Palbociclib	NCT00141297/ (Flaherty et al., 2012)	I	Advanced solid tumors n = 41	DLTs observed in 5 (12%) patients Hematologic AEs after Cycle 1: G3 neutropenia (12%), anemia (7%), and leukopenia (2%) Most common (>10%) non-hematologic AEs after Cycle 1: fatigue (24%), diarrhea (15%) and nausea, dyspnea, arthralgia (12% each)	SD >2 cycles: 35% of patients SD ≥4 cycles: 27% of patients SD ≥10 cycles: 16.2% of patients, including 1 patient with breast cancer
	NCT01037790/ (DeMichele et al., 2015)	II	Refractory solid tumors n = 37 (MBC cohort)	G3/4 toxicities: transient neutropenia (51%), thrombocytopenia (22%) and anemia (5%) One episode of neutropenic sepsis occurred in Cycle 1 in patient with 6 prior chemotherapy regimens Cytopenias managed by dose reduction All non-hematological toxicities were G1/2	Overall (n = 37) PR: 5% (2/37) SD <6 months: 38% (14/37) SD ≥6 months: 14% (5/37) PD: 43% (16/37) PFS: 3.7 months HR+ patients (n = 33) PR: 6% (2/33) SD <6 months: 39% (13/33) SD ≥6 months: 16% (5/33) PD: 39% (13/33) PFS: 3.8 months
Ribociclib	NCT01237236/ (Infante et al., 2014)	I	Advanced solid tumors or lymphomas n = 128	Most common (>10%) treatment-related G3/4 AEs: neutropenia (29%), leukopenia (21%), and lymphopenia (18%)	Three PRs: One patient with a head and neck acinar carcinoma and CDKN2A loss One patient with PIK3CA-mutant, CCND1-amplified estrogen receptor-positive breast cancer One patient with BRAF/NRAS wild-type, CCND1-amplified melanoma
Abemaciclib	NCT01394016/ (Shapiro et al., 2013; Patnaik et al., 2014)	I	Advanced cancer (NSCLC, glioblastoma, breast cancer, melanoma, and colorectal cancer) n = 55	The most common (>10%) study drug-related AEs were diarrhea (52%, including 5% G3), nausea (30%, 4% G3), fatigue (21%, 7% G3), vomiting (18%, 2% G3), and neutropenia (16%, 7% G3)	One patient with ovarian cancer had a durable CA-125 response with >50% decrease for 16 cycles One patient with KRAS mutant NSCLC had a 27% decrease by RECIST One patient with CDKN2A−/− NRAS mutant melanoma had a confirmed PR MBC (n = 47) Confirmed PR: 9 SD: 24 PD: 11 DCR: 70% PFS: 5.8 months HR+ MBC (n = 36) Confirmed PR: 9 ORR: 25% SD: 20 DCR: 81% PFS: 9.1 months

AE adverse event, DCR disease control rate, DLT dose-limiting toxicity, G Grade, HR+ hormone receptor-positive, MBC metastatic breast cancer, NSCLC non-small cell lung cancer, ORR overall response rate, PD progressive disease, PFS progression-free survival, PR partial response, RECIST Response Evaluation Criteria In Solid Tumors, SD stable disease

Palbociclib

Early clinical trials have investigated the safety and clinical activity of palbociclib as a single agent (Table 2). The most common adverse event associated with palbociclib is neutropenia, however, it is distinct from that observed with cytotoxic agents in that it is rapidly reversible, reflecting a cytostatic effect on neutrophil precursors in the bone marrow (Asghar et al., 2015). CDK6 appears to be important in promoting the proliferation of hematologic precursors, and CDK 4/6 inhibition in mice has been associated with a transient growth arrest in hematopoietic precursor cells (Asghar et al., 2015; Johnson et al., 2010). Non-hematologic adverse events related to treatment with palbociclib include nausea, fatigue, diarrhea, stomatitis, and asthenia (Flaherty et al., 2012; Pfizer Inc., 2015; DeMichele et al., 2015).

In the Phase I first-in-human dose-escalation study, patients with advanced solid tumors received oral palbociclib once daily for 21 of 28 days (NCT00141297). Neutropenia was the major toxicity observed and was also dose-limiting. The maximum-tolerated dose (MTD) and recommended Phase II dose (RP2D) were declared as 125 mg given once daily on a 3-weeks-on/1-week-off schedule every 28 days. Ten (27%) of 37 evaluable patients achieved stable disease (SD) for ≥4 cycles, six of whom derived prolonged benefit (>10 cycles) (Flaherty et al., 2012).

Ribociclib

As with palbociclib, hematologic adverse events, including neutropenia, are also common with ribociclib and therefore a 1-week resting period is incorporated into dosing regimens in most trials (Asghar et al., 2015). In the Phase I, first-in-human study, patients with advanced solid tumors or lymphomas received escalating doses of single-agent ribociclib either as part of a 3-weeks-on/1-week-off schedule, or as part of a continuous 28-day schedule (NCT01237236). The MTD and recommended dose for expansion were declared as 900 mg/day and 600 mg/day on a 3-weeks-on/1-week-off dosing schedule, respectively. Out of 110 evaluable patients, three partial responses (PRs) were observed and stable disease (SD) for ≥4 and ≥6 cycles was observed in 24% and 15% of patients, respectively (Table 2) (Infante et al., 2014).

Abemaciclib

Hematologic adverse events, including neutropenia, are somewhat less common in patients receiving abemaciclib. Gastrointestinal-related toxicity appears to be more predominant with abemaciclib, with typical adverse events being nausea, fatigue, diarrhea, and vomiting (Asghar et al., 2015; Shapiro et al., 2013; Patnaik et al., 2014). Notably, abemaciclib is administered on a continuous dosing schedule.

A Phase I first-in-human study is investigating abemaciclib, taken orally every 12 or 24 hours, in patients with advanced cancer in five tumor types, including breast cancer (NCT01394016) (Shapiro et al., 2013). In the expansion cohort, patients with HR+ breast cancer were administered abemaciclib continuously at 150–200 mg orally every 12 hours. Nine of 36 patients with HR+ disease had confirmed PRs with an overall response rate of 25%, and 20 (56%) patients had SD (Patnaik et al., 2014).

Combination approaches with CDK 4/6 inhibitors

Combination with endocrine therapy

Despite the clinical efficacy observed with endocrine therapy, resistance remains a major obstacle in ER+ breast cancer. Treatment of endocrine therapy-resistant cells with palbociclib has been shown to suppress proliferation effectively (Thangavel et al., 2011). Results of key and ongoing combination studies are described in Table 3 with select studies discussed below.

Table 3.

HR+ breast cancer clinical trials of CDK 4/6 inhibitors in combination with other agents

Combination therapy/setting	Study drug	Trial reference/ NCT number	Phase	Patient population	Combination agent(s)	Primary endpoint	Results (primary endpoint)
Combination with endocrine therapy
Metastatic BC setting
Letrozole	Palbociclib	PALOMA-1/TRIO-18; NCT00721409	II	ER+, HER2− advanced BC	Letrozole	PFS	Median PFS was 20.2 mos with palbociclib + letrozole vs 10.2 mos with letrozole (p = 0.0004)
		PALOMA-2; NCT01740427	III	Postmenopausal women with ER+, HER2− advanced BC	Letrozole	PFS	N/A
		PALOMA-4; NCT02297438	III	Postmenopausal women with ER+/HER2− advanced BC who have not received prior systemic anticancer therapies for advanced/metastatic disease	Letrozole	PFS	N/A
	Ribociclib	MONALEESA-2; NCT01958021	III	Postmenopausal women with HR+, HER2− advanced BC	Letrozole	PFS	N/A
	Abemaciclib	MONARCH-3; NCT02246621	III	Postmenopausal women with BC	Anastrozole or letrozole	PFS	N/A
Fulvestrant	Palbociclib	PALOMA-3; NCT01942135	III	HR+, HER2− metastatic BC whose disease has progressed after prior endocrine therapy	Fulvestrant	PFS	Median PFS was 9.2 mos with palbociclib + fulvestrant vs 3.8 mos with fulvestrant (p < 0.001)
	Ribociclib	MONALEESA-3; NCT02422615	III	Postmenopausal women with HR+, HER2− advanced BC, who have had no or only one line of prior endocrine treatment	Fulvestrant	PFS	N/A
	Abemaciclib	MONARCH-2; NCT02107703	III	HR+, HER2− locally advanced or metastatic breast cancer, who have had no or only one line of prior endocrine treatment	Fulvestrant	PFS	N/A
		NCT01394016	I	HR+ metastatic BC (among 5 different tumor types)	Fulvestrant	Safety	Grade 3 AEs (no Grade 4 AE) Diarrhea: 8% Fatigue: 8% Neutropenia: 31% Leukopenia: 23%
Tamoxifen	Ribociclib	MONALEESA-7; NCT02278120	III	Pre- or peri-menopausal women with HR+, HER2− advanced BC	Tamoxifen or letrozole or anastrozole Goserelin	PFS	N/A
Other/various therapies	Palbociclib	PEARL; NCT02028507	III	HR+, HER2− metastatic BC with resistance to NSAIs	Capecitabine Exemestane	PFS	N/A
	Abemaciclib	NCT02057133	Ib	HR+, HER2−/HER2+ metastatic BC	Letrozole Anastrozole Tamoxifen Everolimus + exemestane Trastuzumab	Safety	N/A
Early BC setting
Neoadjuvant	Palbociclib	NCT01723774	II	Women with Stage II/III ER+, HER2− BC	Anastrozole Goserelin	pCR Ki67 levels	N/A
	Palbociclib	PALLET; NCT02296801	II	Postmenopausal women with ER+, primary BC	Letrozole	Change in Ki67 from baseline to 14 weeks Clinical complete response	N/A
	Abemaciclib	neoMONARCH; NCT02441946	II	Postmenopausal women with HR+, HER2−, early-stage BC	Anastrozole	Ki67 levels	N/A
Adjuvant	Palbociclib	PENELOPE-B; NCT01864746	III	HR+, HER2− negative BC with residual disease after neoadjuvant chemotherapy	Standard anti-hormonal therapy	iDFS	N/A
	Palbociclib	NCT02040857	II	HR+/HER2− invasive BC	Letrozole, anastrozole, or exemestane	Treatment discontinuation rate	N/A
	Palbociclib	ABCSG-42; BIG 03/14	III	HR+/HER2− Stage II/III BC	Endocrine therapy	N/A	N/A
Combination with targeted signaling pathway inhibitors
mTOR inhibitors	Ribociclib	NCT01857193	Ib	Advanced/metastatic ER+ BC	Exemestane Everolimus	Incidence of DLTs	DLTs with triplet therapy: Grade 3 febrile neutropenia (n = 1) Grade 3 ALT elevation (n = 2)
			II	Advanced/metastatic ER+ BC	Exemestane Everolimus	PFS	N/A
PI3K inhibitors	Ribociclib	NCT01872260	Ib (dose escalation/expansion)	Advanced ER+ BC (dose expansion phase only includes patients in the first-line setting)	Letrozole Alpelisib	Incidence of DLTs	1 DLT with ribociclib + letrozole (Grade 4 neutropenia)
			II	Advanced ER+ BC (first-line only)	Letrozole Alpelisib	PFS	N/A
	Palbociclib	NCT02389842	Ib	PIK3CA-mutant BC	Taselisib (GDC-0032) or Pictilisib (GDC-0941) Fulvestrant	RP2D Safety Preliminary antitumor assessments	N/A
Combination with chemotherapy
	Palbociclib	NCT01320592	I	Rb-positive metastatic BC	Paclitaxel	Safety	DLT was Grade 3 AST and ALT elevations Grade 3/4 neutropenia: 10/15 patients

ALT alanine transaminase, AST aspartate transaminase, BC breast cancer, DLT dose-limiting toxicity, ER+ estrogen receptor-positive, HER2− human epidermal growth factor receptor 2-negative, HR+ hormone receptor-positive, iDFS invasive disease-free survival, PFS progression-free survival, NSAI non-steroidal aromatase inhibitors, Rb retinoblastoma, RP2D recommended Phase II dose

In the metastatic setting

Combination with letrozole

Letrozole is an orally administered non-steroidal aromatase inhibitor (NSAI) (Novartis, 2015). Preclinical studies of CDK 4/6 inhibitors in combination with letrozole have shown enhanced clinical activity (O’Brien et al., 2014). The combination of ribociclib and letrozole has also demonstrated sustained tumor control in a PIK3CA-wildtype, ER+ breast cancer model (Parasuraman et al., 2014).

Clinical results

Palbociclib

Perhaps the biggest clinical validation of the CDK 4/6 pathway as an important therapeutic target was provided by the results of the PALOMA-1 trial, a Phase II open-label, randomized trial in advanced ER+, HER2− breast cancer. Patients treated with palbociclib plus letrozole in the first-line setting had a median PFS of 20.2 months (versus 10.2 months with letrozole only; one-sided p = 0.0004; NCT00721409; Table 3) (Finn et al., 2015). Based on these results, palbociclib (Ibrance^®; Pfizer), in combination with letrozole, was approved by the United States Food and Drug Administration in February 2015 as a front-line endocrine-based therapy for the treatment of postmenopausal women with ER+, HER2− advanced breast cancer (Pfizer Inc., 2015). Additional subanalyses from PALOMA-1 indicated that the PFS benefit for palbociclib plus letrozole also occurred in patients ≥65 years of age and in those who had not received systemic therapy (Crown et al., 2015; Finn et al., 2015). Clinically meaningful delays in progression in the bone were observed and long-term safety analyses (≥24 months) suggest that the palbociclib–letrozole combination is not associated with cumulative or late-onset toxicities (Slamon et al., 2015). A Phase III confirmatory study is ongoing (NCT01740427; PALOMA-2; Table 3).

Ribociclib

In a Phase Ib/II study of ribociclib in combination with letrozole, an acceptable safety profile and preliminary clinical activity was observed in postmenopausal women with ER+, HER2− advanced breast cancer (NCT01872260; Table 3). The RP2D of ribociclib was declared as 600 mg/day (3-weeks-on/1-week-off) in combination with continuous letrozole 2.5 mg/day (Munster et al., 2014). A Phase III study is currently ongoing (MONALEESA-2; NCT01958021; Table 3).

Abemaciclib

The combination of abemaciclib and either letrozole or anastrozole is being evaluated in the ongoing Phase III, randomized, double-blind, placebo-controlled MONARCH-3 study in postmenopausal women with recurrent or metastatic HR+ breast cancer, who have had no prior systemic therapy for this setting (NCT02246621; Table 3).

Combination with fulvestrant

Fulvestrant is a selective estrogen receptor degrader approved for the treatment of HR+ metastatic breast cancer in postmenopausal women whose disease has progressed following antiestrogen therapy (Howell, 2006; AstraZeneca, 2015).

Clinical results

Palbociclib

The combination of palbociclib and fulvestrant in patients whose disease has progressed after prior endocrine therapy was explored in a Phase III trial (PALOMA-3; NCT01942135; Table 3). After a pre-planned interim analysis, PALOMA-3 was stopped based on an efficacy assessment by an independent data monitoring committee. Addition of palbociclib to fulvestrant significantly prolonged median investigator-assessed PFS compared with fulvestrant alone (9.2 vs 3.8 months; p < 0.001). Interestingly, the palbociclib and fulvestrant combination demonstrated a PFS benefit across all pre-specified patient subgroups, including menopausal status, site of metastatic disease (visceral or non-visceral) and sensitivity to prior hormonal therapy (Turner et al., 2015).

Ribociclib

The combination of ribociclib and fulvestrant is being evaluated in the ongoing Phase III, randomized, double-blind, placebo-controlled MONALEESA-3 study in postmenopausal women with HR+, HER2− advanced breast cancer, who have had one or less prior lines of endocrine treatment (NCT02422615; Table 3).

Abemaciclib

Abemaciclib in combination with fulvestrant was evaluated in a Phase I study, demonstrating an acceptable safety profile and evidence of efficacy (NCT01394016; Table 3) (Patnaik et al., 2014). This combination is now being explored in a Phase III trial (MONARCH-2; NCT02107703; Table 3).

Combination with tamoxifen

Tamoxifen is an estrogen receptor antagonist that has been used for the treatment of HR+ breast cancer for over 30 years (Howell, 2006).

Clinical results

Ribociclib

The combination of ribociclib and tamoxifen is being investigated in peri- or premenopausal women with HR+, HER2− advanced breast cancer. This population typically receives tamoxifen or NSAIs with ovarian function suppression as standard first-line therapy; however, resistance to endocrine therapy and disease progression can occur. The Phase III MONALEESA-7 study is a randomized, double-blind, placebo-controlled study of this population investigating ribociclib combined with either tamoxifen and goserelin or a NSAI and goserelin (NCT02278120) (Tripathy et al., 2015).

Combination with other/various therapies in metastatic breast cancer

In addition to the above studies, a number of trials are investigating CDK 4/6 inhibitors in combination with a variety of other agents (Table 3). One Phase Ib study is evaluating abemaciclib in six patient cohorts where it is combined with letrozole, anastrozole, tamoxifen, exemestane, exemestane plus everolimus, or trastuzumab (NCT02057133; Table 3). Combinations of abemaciclib and endocrine therapy demonstrated manageable safety and early clinical evidence of antitumor activity (Tolaney et al., 2015). A separate Phase III study is underway to compare the efficacy and safety of palbociclib in combination with exemestane with that of capecitabine in postmenopausal women with HR+ metastatic breast cancer whose disease was refractory to prior NSAI (PEARL; NCT02028507; Table 3).

In early breast cancer

In addition to studies in advanced HR+ breast cancer, trials are exploring combinations of CDK 4/6 inhibitors with hormonal agents in early HR+ breast cancer (Table 3).

Neoadjuvant therapy

The potential utility of palbociclib plus anastrozole in the neoadjuvant setting is being investigated in a Phase II trial of women with clinical Stage II/III ER+, HER2− breast cancer (NCT01723774). The ongoing PALLET trial is investigating the combination of palbociclib and letrozole as neoadjuvant therapy in postmenopausal women with ER+ breast cancer (NCT02296801). Another neoadjuvant trial on the horizon is the neoMONARCH Phase II study of neoadjuvant abemaciclib in combination with anastrozole (NCT02441946).

Adjuvant therapy

The ongoing PENELOPE-B trial (NCT01864746) is a Phase III study of palbociclib and standard anti-hormonal therapy in women with HR+, HER2− early breast cancer who did not obtain a pathological complete response after taxane-containing neoadjuvant chemotherapy, and who are at high risk of relapse. A Phase II single-arm trial evaluating the feasibility of 2 years of treatment with palbociclib in combination with adjuvant endocrine therapy (letrozole, anastrozole, or exemestane) in patients with HR+ breast cancer is also ongoing (NCT02040857). The Phase III, double-blind, randomized PALLAS study is evaluating adjuvant palbociclib for 2 years plus endocrine treatment for 5 years versus 5 years of endocrine therapy alone in 4,600 patients with HR+, HER2− Stage II/III breast cancer.

Combination with targeted signaling pathway inhibitors

PI3K-targeted agents are currently being investigated in several Phase III trials in patients with HR+ breast cancer. The rationale for combining these agents with CDK 4/6 inhibitors in patients with HR+ breast cancer is attributed to the increase in cyclin D expression as a result of PI3K/AKT/mTOR pathway activation (Takuwa et al., 1999). In addition, preclinical and clinical data suggest that inhibiting CDK 4/6 activity or the PI3K/AKT/mTOR signaling axis may delay the development of endocrine resistance (Shapiro, 2006). Addition of a pan-PI3K inhibitor (buparlisib [BKM120] or a PI3K-alpha inhibitor (alpelisib [BYL719]) in a triplet combination with ribociclib and an antiestrogen resulted in enhanced, robust tumor regressions, without inducing significant toxicity in preclinical model (O’Brien et al., 2014). In another study, when PIK3CA-mutant breast cancer mouse models, both sensitive and resistant to alpelisib, were exposed to ribociclib, or alpelisib, or a combination of both, the combination regimen led to more enhanced regression, relative to single-agent therapy. The pan-PI3K inhibitor, pictilisib (GDC-0941), in combination with ribociclib, elicited tumor regression in MCF7 and CAL51 xenografts, whereas single-agent treatment did not (Vora et al., 2014). Due to the non-overlapping side effects of PI3K and CDK 4/6 inhibition, combination of these agents was predicted to be well tolerated in patients. Ongoing clinical trials of such combination therapies are summarized in Table 3 and select studies are discussed below.

A Phase Ib/II study of the triplet combination of ribociclib, alpelisib, and letrozole in postmenopausal women with ER+, HER2− advanced breast cancer is currently ongoing (NCT01872260; Table 3). Preliminary pharmacodynamic analyses suggest that, in some patients, alpelisib may help prevent compensatory PI3K/AKT/mTOR pathway activation following treatment with ribociclib and letrozole (Juric et al., 2014). A Phase Ib study is evaluating palbociclib in combination with the PI3K pathway inhibitors taselisib (GDC-0032) or pictilisib (GDC-0941), with the subsequent addition of fulvestrant in patients with PIK3CA-mutant breast cancers (NCT02389842; Table 3). An ongoing Phase Ib/II study is evaluating ribociclib, exemestane, and the mTOR inhibitor everolimus in patients with advanced/metastatic ER+ breast cancer refractory to the NSAIs letrozole or anastrozole (NCT01857193; Table 3). Preliminary results have demonstrated that the triplet combination is feasible, with encouraging signs of clinical activity (Bardia et al., 2014).

Combination with chemotherapy

Co-administration of CDK 4/6 inhibitors with chemotherapy has yielded contrasting results. In Rb-positive triple-negative breast cancer cell lines and xenografts, palbociclib and doxorubicin had an additive cytostatic effect by eliciting a G1 arrest or G2/M arrest, respectively. However, doxorubicin-mediated cell death signaling was also inhibited. Therefore, combination regimens of CDK 4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects may need to be approached with caution (McClendon et al., 2012). A Phase I trial investigating the combination of paclitaxel and palbociclib in patients with Rb-positive metastatic breast cancer demonstrated that the combination was well tolerated, and elicited prolonged tumor responses, though dose reductions and interruptions were common due to neutropenia (Clark et al., 2014); NCT01320592; Table 3).

Future challenges

Preclinical and clinical data suggest that CDK 4/6 inhibitors have significant potential in the treatment of breast cancer. In addition, combining CDK 4/6 inhibitors with other well-established therapies has demonstrated enhanced efficacy. Therefore, determining the ideal combinations of CDK 4/6 inhibitors with other targeted agents is an important challenge.

The use of biomarkers may have a role in helping to identify those patients who are most likely to respond to treatment with CDK 4/6 inhibitors. Various clinical trials are investigating the use of biomarkers, such as changes in copy number or expression/activation levels of genes including CCND1, CDKN2A, RB, and changes in the proliferation marker Ki67; however, as yet no potential biomarkers predictive of response have been identified. Results from the PALOMA-1 study demonstrated that both biomarker-positive (CCND1/CEP11 ratio ≥1.5 and/or CDKN2A/CEP9 ratio <0.8) and biomarker-negative patients benefited similarly from treatment with palbociclib and letrozole. In addition, Ki67 staining did not identify a subgroup that benefited more from this treatment combination (Jiang et al., 2014).

Conclusions

Although endocrine therapy is currently the mainstay of treatment for ER+ breast cancer, there is a requirement for novel treatment approaches due to the de novo and acquired resistance that occurs in many patients with advanced disease. CDK 4/6 inhibitors have demonstrated promising antitumor activity, and ongoing studies are exploring the combination of these agents with existing endocrine treatments and with inhibitors of upstream and downstream signaling molecules. Identifying optimal treatment combinations, and refining biomarkers that will predict patients’ responses to treatment, remains a key challenge.

List of abbreviations

ATP

Adenosine triphosphate

CDK

Cyclin-dependent kinase

DLT

Dose-limiting toxicity

ER

Estrogen receptor

ER+

Estrogen receptor-positive

ER−

Estrogen receptor-negative

HER2−

Human epidermal growth factor receptor 2-negative

HR+

Hormone receptor-positive

MTD

Maximum tolerated dose

mTOR

Mammalian target of rapamycin

NSAI

Non-steroidal aromatase inhibitor

NSCLC

Non-small cell lung carcinoma

PFS

Progression-free survival

PI3K

Phosphatidylinositol 3-kinase

Rb

RP2D, RetinoblastomaRecommended Phase II dose