The evolution of cyclin dependent kinase inhibitors in the treatment of cancer

ABSTRACT

Introduction: The cell cycle cyclin-dependent kinases (CDKs) play a critical role in controlling the transition between cell cycle phases, as well as cellular transcription. Aberrant CDK activation is common in cancer, and deregulation of the cell cycle a key hallmark of cancer. Although CDK4/6 inhibitors are now a standard-of-care option for first- and second-line HR+/HER2- metastatic breast cancer, resistance inevitably limits their clinical benefit.

Areas covered: Early pan-CDK inhibitors targeted the cell cycle and RNA polymerase II phosphorylation, but were complicated by toxicity, providing a rationale and need for the development of selective CDK inhibitors. In this review, we highlight selected recent literature to provide a narrative review summarizing the current CDK inhibitor therapeutic landscape. We detail the challenges associated with targeting CDKs for the treatment of breast and other cancers and review emerging biomarkers that may aid response prediction. We also discuss the risk-benefit ratio for CDK therapy and explore promising combination approaches.

Expert opinion: Although CDK inhibitors may stem the proliferation of cancer cells, resistance remains an issue, and currently there are limited biomarkers to predict response to therapy. Ongoing research investigating CDK inhibitors in cancer is of paramount importance to define appropriate and effective treatment regimens.

KEYWORDS:

• Breast
• biomarkers
• cancer
• cell cycle
• cyclin dependent kinase inhibitor
• oncology
• resistance

1. Introduction

Following the discovery of the crucial role of cyclin-dependent kinases (CDKs) in controlling the cell cycle, many agents targeting CDK activity have emerged. Translating scientific knowledge of CDKs into the clinical development of successful CDK inhibitors was initially challenging, with a number demonstrating disappointing results. However, over the 5 years after the US Food and Drug Administration (FDA) approved the first CDK4/6 inhibitor, palbociclib, for the treatment of hormone receptor-positive (HR+) human epidermal growth factor receptor 2-negative (HER2–) breast cancer, two additional CDK4/6 inhibitors, abemaciclib and ribociclib, entered the market. Although widely utilized in the treatment of breast cancer, their use is associated with intrinsic and acquired drug resistance. Additionally, for patients who derive benefit, it is unclear what the optimal therapeutic options are after development of resistance.

This review focuses on cell cycle CDK biology and the safety and clinical activity of selective CDK inhibitors, and the current understanding and challenges of these inhibitors in clinical oncology, including biomarkers for predicting response and resistance to treatment. We also discuss early-stage disease, neoadjuvant therapy, and future directions.

2. Biology of cell cycle CDKs

2.1. Cell cycle progression

CDKs and cyclins are crucial for driving cell cycle transitions and cell division [1,2]. The cell cycle is tightly choreographed, involving sequential activation of several cyclin–CDK complexes. To proliferate, cells must activate CDKs via binding to a cyclin subunit, whose expression oscillates throughout different stages of the cell cycle, as well as via phosphorylation by CDK-activated kinase (CAK). CDK4/6 is typically bound to a D-type cyclin; cyclin D-CDK4/6 complexes also require a CIP/KIP protein in the proper stoichiometry for assembly, such as p21^Waf1/Cip1 or p27^Kip1, and are inhibited by INK4 proteins [3].

Active cyclin D-CDK4/6 holoenzymes control G1 progression during the cell cycle. Cyclin D-CDK4/6 and cyclin E-CDK2 complexes sequentially phosphorylate the retinoblastoma (RB) protein, a key tumor suppressor of the G1 to S phase transition [4], leading to its inactivation and release of sequestered E2F family members, which govern gene transcription necessary for S phase progression [5]. In normal cell signaling of breast cells expressing the estrogen receptor (ER) and progesterone receptor, upstream mitogenic signaling pathways, including ER, MEK/ERK, and PI3K/AKT/mTOR [mammalian target of rapamycin] activate transcriptional and translational regulation of CCND1 messenger RNA (mRNA) and cyclin D1 protein, forming cyclin D1-CDK4/6 complexes (Figure 1). Concomitant targeting of ER signaling and CDK4/6 affords vertical pathway inhibition to suppress proliferation.

Figure 1. CDK4/6 inhibition in the cell cycle

While cyclin D-CDK4/6 and cyclin E-CDK2 promote G1 progression and the G1/S transition, cyclin A-CDK2 plays a key role in progression through S phase [6] and cyclin B-CDK1 is required for entry into and progression through mitosis [7]. Cell cycle checkpoints, including those at the G1/S boundary, those active during S phase and those operating at the G2/M boundary, limit CDK activity to prevent premature progression.

2.2. CDKs and cancer

Dysregulation of cell-cycle machinery is a hallmark of cancer leading to overactivation of CDKs and uncontrolled proliferation. Although some cancer cells lose RB, the majority retain expression. Cancer cells may overcome RB-dependent growth suppression through overactivation of cyclin D-CDK4/6 complexes, resulting in constitutive RB phosphorylation and inactivation. Amplification of the genes encoding D-cyclins or CDK4/6, and deletion, mutation, or methylation of INK4 genes may all contribute to oncogenesis [8]. Additionally, cyclin E-CDK2 activity may also be dysregulated, particularly by CCNE1 amplification or CIP/KIP protein loss, also leading to RB hyperphosphorylation [9,10].

Analyses of the Cancer Genome Atlas Pan-Cancer Atlas using cBioPortal (https://www.cbioportal.org/) identifies genes that reach a specified cutoff for alterations in given indications. When evaluating the cancer genome landscape for alterations in cell cycle-related genes in an indication agnostic sense, unsurprisingly seven genes in particular are commonly perturbed: CDKN2A and CDKN2B (encoding p16^INK4A and p15^INK4B, respectively), RB1, CCND1, CCNE1, CDK4, and FBXW7 (inactivation of which can lead to cyclin E and MYC stabilization) (Figure 2(a)) [11–13]. Each of these genes reach an arbitrary threshold of >5% altered across all tumor lineages, with the exception of CDK4, where there is predominant amplification in glioblastoma (12.0%) and in liposarcoma (90%) [14,15]. The remaining genes are modified in a predictable fashion given its known function (i.e. CDKN2A, CDKN2B, RB1, and FBXW7 are primarily deleted or mutated, whereas CCND1 and CCNE1 are commonly amplified). Genetic insults for these cell cycle genes may favor a given tumor lineage, similar to the case for CDK4, for example, CDKN2A/2B deletion in glioblastoma [16], non–small-cell lung (NSCLC) [17], and breast cancers [18], RB1 deletion or mutation primarily in small-cell lung cancer (SCLC) [19], CCND1 amplification in HR+ breast cancer [20], and CCNE1 amplification in ovarian [21] and triple-negative breast cancers (TNBC) [22]. Even with the observed lineage enrichment, cell cycle dysregulation due to genetic alteration can account for ≥50% of patients across a wide variety of carcinomas, including pancreatic, melanoma, squamous cell lung cancer, head and neck/esophageal squamous cell carcinoma, glioblastoma and bladder cancers (Figure 2(b)). Considering the overall survival (OS) of cancer patients with and without genomic alterations in these cell cycle genes, it is apparent that direct modification of the cell cycle machinery can result in a more aggressive phenotype that manifests in poorer overall survival (OS) (Figure 2(c)).

Figure 2. Genes commonly perturbed in cancer (a); Cancer types and alteration frequency (b); Poorer overall survival associated with gene alterations (c)

Selective CDK4/6 inhibitors have marked efficacy in HR+ breast cancer combined with hormonal therapy, raising the possibility that other lineage-specific combinations could yield similar impact, including those with KRAS G12C inhibitors in NSCLC or with anti-androgen therapies in prostate cancer. Furthermore, the discovery and development of next-generation CDK inhibitors with novel selectivity profiles might be effective in tumors unresponsive to CDK4/6 inhibition, including CCNE1 amplification or RB loss.

3. CDK inhibitors – early clinical development and HR+ breast cancer

3.1. Early generation of CDK inhibitors

Several pan-CDK inhibitors have been clinically tested in various tumor types. Alvocidib (Flavopiridol®, Tolero Pharmaceuticals, Inc., Lehi, UT) was one of the first-generation pan-CDK inhibitors and the most extensively studied. Alvocidib targets CDK1, CDK2, CDK4, CDK6, CDK7, and CDK9 [23], with particular potency against CDK9. Although encouraging results were observed in phase I trials in chronic lymphocytic leukemia [24], likely related to transcriptional CDK inhibition, tumor lysis syndrome was a life-threatening dose-limiting toxicity. Additionally, phase II studies showed little evidence of single-agent activity in solid tumors, although it does have orphan drug status in acute myeloid leukemia, with ongoing trials in acute myeloid leukemia (NCT03969420) and myelodysplastic syndromes (NCT03593915). Second-generation inhibitors of multiple CDKs were subsequently explored clinically, such as dinaciclib [25–27], which inhibits CDK1, CDK2, CDK5, CDK9, CDK12, with less activity against CDK4, CDK6, and CDK7 [23,28]. Dinaciclib-mediated inhibition of CDK1 and CDK9 may result in cytotoxicity in MYC-driven cells [29,30].

Dinaciclib induces immunogenic cell death with modulation of the immune microenvironment [31], prompting combination with pembrolizumab in hematological malignancies and TNBC (NCT02684617; NCT01676753). In TNBC, toxicities were generally manageable and in 29/32 patients evaluable for response, one patient had complete response, four had partial response, and six stable disease, with MYC expression correlating with response [32]. Nonetheless, dinaciclib has produced disappointing results in randomized phase II trials [33,34] and substantial toxicity at doses associated with pharmacodynamic effects. Use of non-CDK4/6 selective drugs, typically inhibiting CDK9, can result in neutropenia based on MCL1 depletion [35], and therefore can be complicated by toxicity.

3.2. CDK4/6 inhibitors

More recently, a newer generation of oral, potent, and highly selective reversible inhibitors of CDK4 and CDK6 have emerged, including palbociclib, ribociclib, and abemaciclib, established as the standard of care for advanced HR+/HER2– breast cancer combined with hormonal therapy. Pharmacodynamically, CDK4/6 inhibition is demonstrated by reduced RB phosphorylation at Ser⁷⁸⁰ or Ser^807/811 in HR+ breast cancer and mantle cell lymphoma (MCL) [36,37], correlating with reduced proliferation assessed by Ki67 staining. In breast cancer, the serum activity of thymidine kinase 1, the product of an E2F-1-responsive gene secreted into serum by cancer cells, was also shown to be decreased in response to CDK4/6 inhibition [38]. During early treatment, a lack of reduction of these markers can be indicative of intrinsic resistance.

3.2.1. Approved CDK4/6 inhibitors in HR+ breast cancer

In HR+/HER2- breast cancer, combining CDK4/6 inhibition and ER-targeted therapy likely augments a senescence response. Use of CDK4/6 inhibitors with aromatase inhibitors (AIs) or fulvestrant shows substantial increases in progression-free survival (PFS) and overall response rate (ORR), similar across agents and clinical trials. A summary of the key trials [39–47] leading to FDA approval for palbociclib, ribociclib, and abemaciclib is provided in Table 1. A pooled analysis of seven phase III randomized breast cancer trials of CDK inhibitors, including endocrine therapy (ET), demonstrated the median PFS difference was 8.8 months, favoring CDK inhibition plus ET versus placebo plus ET [48]. Consequently, palbociclib and abemaciclib are approved in combination with an AI for initial endocrine-based therapy or with fulvestrant after disease progression on ET. Ribociclib is approved combined with an AI or fulvestrant as initial endocrine-based therapy or with fulvestrant following disease progression on ET [47]. Abemaciclib is also approved as monotherapy in disease progression following ET and chemotherapy in the metastatic setting [47]. In such patients with heavily pretreated HR+/HER2- breast cancer, the addition of tamoxifen to abemaciclib improved OS compared with abemaciclib monotherapy [49], although this combination treatment is not on the label.

Table 1. Summary of key clinical trials leading to FDA approval

Trial	Reference	FDA approval date	Study arms	N	Menopausal status	Median PFS (months)	ORR (%)
1st line
PALOMA-1 TRIO-18 NCT00721409	[39]	Feb 2015	Palbociclib + letrozole vs. letrozole	165	Post	20.2 vs. 10.2	55.0 vs. 39.0
PALOMA-2 NCT01740427	[40]	Feb 2016	Palbociclib + letrozole vs. letrozole	666	Post	24.8 vs. 14.5	55.3 vs. 44.4
MONALEESA-2 NCT01958021	[41]	Mar 2017	Ribociclib + letrozole vs. letrozole	668	Post	25.3 vs. 16.0	52.7 vs. 37.1
MONALEESA-7 NCT02278120	[42]		Ribociclib + OFS + tamoxifen/AI vs. OFS + tamoxifen/AI	660	Pre	23.8 vs. 13.0	51.0 vs. 44.0
MONARCH-3 NCT02246621	[43]	Feb 2018	Abemaciclib + AI vs. AI	493	Post	NR vs. 14.7	59.0 vs. 44.0
1st & 2nd line
MONARCH-2 NCT02107703	[44]	Sep 2017	Abemaciclib + fulvestrant vs. fulvestrant	669	Post	16.4 vs. 9.3	48.1 vs. 21.3
MONALEESA-3 NCT02422615	[45]	Jul 2018	Ribociclib + fulvestrant vs. fulvestrant	726	Post	20.5 vs. 12.8	32.4 vs. 21.5
n^th line
PALOMA-3 NCT01942135	[46]	Feb 2016	Palbociclib + fulvestrant ± OFS vs. fulvestrant ± OFS	521	Post and pre	9.5 vs. 4.6	24.6 vs. 15.0
MONARCH-1 NCT02102490	[47]	Sep 2017	Abemaciclib single agent	132	Post	6.0	19.7

AI, aromatase inhibitor; FDA, US food and drug administration; OFS, ovarian function suppression; ORR, overall response rate; PFS, progression-free survival; NR, not reached

Recent data indicate improvements in OS following addition of CDK4/6 inhibition to hormonal therapy. In MONALEESA-3, utilizing ribociclib plus fulvestrant versus placebo plus fulvestrant in the postmenopausal setting, median OS was not reached with ribociclib plus fulvestrant but was 40.0 months for placebo plus fulvestrant, representing a 28% risk reduction [50]. In the early relapse, second-line setting, ribociclib plus fulvestrant led to a survival benefit of 7.7 months and a 27% reduction in risk [50]. These results mirrored those of MONALEESA-7 in the premenopausal setting [42]. In MONARCH 2, abemaciclib plus fulvestrant led to a median 9.4-month OS benefit and a 24% reduction in risk in patients with disease progression with prior ET [51]. Hazard ratios were consistent between ribociclib and abemaciclib, and survival benefits observed across subgroups, including patients with poor prognostic factors, including visceral metastases and primary endocrine resistance [51]. A statistically significant OS benefit was not reported in the phase 3 PALOMA-3 trial evaluating palbociclib plus fulvestrant compared with fulvestrant plus placebo [52]. However, the patient population was more heavily pretreated with 34% having received ≥1 prior systemic chemotherapy and 35% ≥2 prior systemic therapies for metastatic disease compared to 0% in MONARCH 2 and MONALEESA-3. Regardless, the HRs in these trials ranged between 0.7 and 0.8, highlighting a class effect.

Studies have also focused on patients with breast cancer with poor prognostic factors, including high-grade, progesterone receptor-negative status or hepatic metastases (proMONARCH, NCT03988114 [abemaciclib], study withdrawn); and comparing with chemotherapy in patients with visceral metastases (NCT04031885 [abemaciclib], study terminated; NCT03905343 [ribociclib]), older age (NCT03956654, NCT03477396 [ribociclib]), and in specific ethnicities (PALINA, NCT02692755 [palbociclib]) [53]. Abemaciclib is of interest in patients with brain metastases because it penetrates the blood–brain barrier, resulting in comparable concentrations in cerebrospinal fluid and plasma [54] with intracranial responses demonstrated (NCT02308020), warranting further evaluation with novel abemaciclib-based combinations.

3.3. CDK4/6 inhibition in the adjuvant or neoadjuvant setting

Prevention of metastatic disease in HR+/HER2– breast cancer remains an unmet need, warranting further research into novel approaches and identification of high-risk subgroups who may benefit from adjuvant treatment with CDK inhibitors. In the phase III monarchE trial (NCT03155997), 5,637 patients with high-risk, node-positive, early stage, HR+/HER2– breast cancer were randomized to receive abemaciclib (2 years) plus standard adjuvant ET or standard adjuvant ET alone. At a preplanned interim efficacy analysis, after a median follow-up of 15.5 months, a statistically significant improvement in invasive disease-free survival (IDFS) was shown with abemaciclib plus ET versus ET alone (p = .0096, HR: 0.747, 95% CI: 0.598, 0.932), corresponding to a 25.3% reduction in the risk of an IDFS event [55]. In contrast, the PALbociclib CoLlaborative Adjuvant Study (PALLAS; NCT02513394) is a phase III trial investigating the addition of 2 years of palbociclib to standard adjuvant ET versus standard adjuvant ET alone in HR+/HER2– early (stage 2 and 3) breast cancer. After a median follow-up of 23.7 months, IDFS was similar for both arms [56]. The lower risk population, early discontinuation rate, and intermittent dosing are hypothesized as possible contributing factors. In the phase III PENELOPE-B trial (NCT01864746), addition of 1 year of palbociclib to standard adjuvant ET was investigated versus 1 year of placebo plus standard adjuvant ET in pre- and postmenopausal women with HR+/HER2– early breast cancer who did not achieve pathological complete response to neoadjuvant taxane-containing chemotherapy. After a median follow-up of 42.7 months, the trial failed to meet the primary endpoint of improved IDFS [57]. Analyses are ongoing to evaluate factors contributing to this outcome. The ongoing phase III NATALEE trial (NCT03701334) is evaluating the addition of 3 years ribociclib to adjuvant ET versus adjuvant ET alone in HR+/HER2– early breast cancer.

Although a number of smaller neoadjuvant trials, such as neoMONARCH (NCT02441946) [58], NeoPalAna (NCT01723774) [59], and NeoPAL (NCT02400567) [60], reported more patients with tumors achieving complete cell cycle arrest with the addition of CDK4/6 inhibitors to ET, compared with ET alone, substantial improvements in pathological complete response rates were not observed by adding a CDK4/6 inhibitor. A summary of recently completed or active adjuvant/neoadjuvant trials are listed in Table 2.

Table 2. Selected ongoing or completed (neo) adjuvant clinical trials in breast cancer for approved CDK4/6 inhibitors

Molecule	Description of trial	Clinical phase	ClinicalTrials.gov identifier
Palbociclib	PALLAS: Palbociclib + standard adjuvant ET vs. standard adjuvant ET alone in HR+/HER2– early (stage 2 and 3) BC. Recent press release reported that the combination therapy is unlikely to show a statistically significant improvement in the primary endpoint of invasive disease-free survival	III	NCT02513394
Palbociclib	POLAR: Palbociclib with standard ET vs. standard ET in patients with HR+/HER2– resected isolated locoregional recurrence of BC.	III	NCT03820830
Palbociclib	Feasibility study of palbociclib with adjuvant ET in HR+/HER2– early BC; 63% of patients completed 2 years of therapy. The safety profile was congruent with that observed in metastatic disease	II	NCT02040857
Palbociclib	Appalaches study: Efficacy of the combination of ≥5-year ET and 2-year palbociclib as adjuvant systemic treatment instead of adjuvant chemotherapy followed by ET in older patients with stage II–III ER+/HER2– early BC.	II	NCT03609047
Palbociclib	Palbociclib combined with letrozole vs. epirubicin combined with cyclophosphamide and sequential docetaxel as neoadjuvant chemotherapy in postmenopausal patients with ER+ locally advanced BC with low Ki67 expression.	IV	NCT04137640
Palbociclib	PENELOPE-B: Palbociclib in pre- and postmenopausal women with HR+/HER2– early BC with a high-risk of relapse. Aim was to show that, in the background of standard antihormonal therapy, palbociclib improves invasive disease-free survival in patients not achieving pathological complete response to neoadjuvant taxane-containing chemotherapy. The trial did not meet the primary endpoint of improved IDFS	III	NCT01864746
Palbociclib	NeoPalAna: Palbociclib and anastrozole in HR+/HER2– BC. Aim was to assess if the pathologic complete response rate is improved compared with historical control data of single-agent AIs.	II	NCT01723774
Palbociclib	NeoPAL: Letrozole with palbociclib as neoadjuvant treatment of luminal BC, in postmenopausal women. The combination was associated with poor pathologic response	II	NCT02400567
Palbociclib	NA-PHER2: Women with invasive unilateral non-metastatic ER+/HER2+ BC received trastuzumab + pertuzumab + palbociclib ± fulvestrant. ORR was high in two cohorts with or without fulvestrant (ORR ~90%), and pathological complete response was seen in about one fourth of patients in these 2 cohorts. A third cohort of patients with tumors with HER2 low (1+/2+, no amplification) also received fulvestrant in the combination and had ORR of 78.3% with an absence of pathological complete response	II	NCT02530424
Palbociclib	PALTAN: Palbociclib with letrozole and trastuzumab in ER+/HER2+ early stage BC.	II	NCT02907918
Ribociclib	LEADER: Ribociclib with adjuvant ET in ER+ BC. Patients had completed surgery and were on adjuvant ET with ≥1 year of treatment remaining. Serious AEs with 1 year of adjuvant ribociclib were low but 24/81 patients discontinued adjuvant ribociclib.	II	NCT03285412
Ribociclib	NATALEE: Ribociclib with ET as adjuvant treatment in HR+/HER2– early BC. Expected to complete in Dec 2025.	III	NCT03701334
Ribociclib	EarLEE-1: Evaluates preliminary safety and tolerability of ribociclib with standard adjuvant ET in patients with HR+/HER2– high-risk early BC.	II	NCT03078751
Ribociclib	FELINE: Ribociclib in combination with letrozole vs. placebo with letrozole for 24 weeks as neoadjuvant ET to assess any increase in the proportion of women with pre-operative Endocrine Prognostic Index score 0 at surgery vs. patients treated with placebo/letrozole.	II	NCT02712723
Ribociclib	NEOLBC: To test if chemotherapy can be replaced by ribociclib + letrozole as neo-adjuvant therapy in patients with HR+/HER2– luminal BC.	II	NCT03283384
Abemaciclib	monarchE: Abemaciclib (with or without standard adjuvant ET) in patients with high-risk, node-positive, early stage, HR+/HER2– BC.	III	NCT03155997
SHR6390	MULAN: Umbrella-shaped clinical study in patients with HR+/HER2– endocrine-resistant advanced BC. In this multiple arms and multiple treatments study, patients receiving SHR6390 are gBRCA-mutated and will receive SHR6390 with the PARP inhibitor SHR3162.	II	NCT04355858
Abemaciclib neo	neoMONARCH: Abemaciclib + anastrozole vs. abemaciclib alone and anastrozole alone in postmenopausal women with HR+/HER2– BC. Abemaciclib + anastrozole was significantly more effective than anastrozole alone. Greater reductions in Ki67 by abemaciclib was not associated with disease stage, baseline lymph node involvement, tumor grade, tumor size, or PIK3CA mutation status.	II	NCT02441946
SHR6390 neo	Open-label, single-center, Bayesian adaptive design, umbrella study evaluates the efficacy and safety of neo-adjuvant therapy in patients with BC. SHR6390 will be given in combination with various combination partners in patients with HR+/HER2– BC with or without PI3K-AKT pathway mutation.	I/II	NCT04215003

AI, aromatase inhibitor; BC, breast cancer; CDK, cyclin-dependent kinase; ER, estrogen receptor; ET, endocrine therapy; HER2, human epidermal growth factor receptor 2; HR, hormone receptor; IDFS, invasive disease-free survival; ORR, overall response rate; PARP, poly (ADP-ribose) polymerase

3.4. Optimal endocrine backbone in metastatic disease

The phase III FALCON trial (NCT01602380) reported that fulvestrant monotherapy prolonged PFS compared with anastrozole monotherapy in postmenopausal women with HR+/HER2– locally advanced or metastatic breast cancer without prior hormonal therapy [61]. In the phase II FLIPPER trial, adding palbociclib to fulvestrant as first-line therapy in postmenopausal women with endocrine-sensitive HR+/HER2– metastatic breast cancer significantly prolonged median PFS (31.8 versus 22.0 months) [62]. Following the FALCON trial, the PARSIFAL trial (NCT02491983) compared fulvestrant with letrozole, each combined with palbociclib in patients with endocrine-sensitive HR+/HER2– metastatic breast cancer, to determine which hormonal treatment is most effective combined with CDK4/6 inhibition [63]. Participants had no prior treatment for metastatic breast cancer or hormonal therapy for early-stage disease for at least 12 months prior to enrollment. No significant difference in PFS was observed between treatment arms, with generally similar side effects [63]. These results suggest that, in combination with CDK4/6 inhibitors, the endocrine backbone may not impact efficacy.

Additionally, the role of ESR1 mutation in the response to combined ET and CDK4/6 inhibition is under study. In the first-line PADA-1 trial (NCT03079011) patients with ER+/HER2– metastatic breast cancer considered AI-sensitive (no previous adjuvant AI or a disease-free interval of >12 months after adjuvant AI) were treated with palbociclib + letrozole. ESR1 mutations were rare at baseline, but more common among those with prior AI in the adjuvant setting [64,65]. The presence of baseline ESR1 mutation was associated with a worse prognosis: median PFS was 11.0 months versus 26.7 months in wild-type disease (hazard ratio = 2.3). Clearance of the mutation after 1 month of treatment improved the prognosis of patients initially ESR1-positive, whose median PFS was 24.1 months, compared with 7.4 months for patients not clearing the mutation. With serial monitoring for ESR1 mutations in ctDNA, the second part compares continued palbociclib + AI versus a switch to palbociclib + fulvestrant [65]. This study will help guide decisions on choosing the ET-combination partner with palbociclib in patients who may have received prior AI and/or carry ESR1 mutations.

4. Risk–benefit ratio for CDK inhibitors

4.1. Neutropenia

Efficacy data indicate a favorable risk–benefit ratio for CDK4/6 inhibitors in HR+/HER2– breast cancer. Neutropenia has emerged as the dose-limiting toxicity observed with palbociclib and ribociclib [66,67], and in an analysis of clinical trials, including 4557 patients with breast cancer, CDK 4/6 inhibitors significantly contributed to hematologic toxicities and febrile neutropenia [68]. Significant neutropenia may be managed by dose modification

The PALOMA-2 trial of palbociclib plus letrozole [40], and the PALOMA-3 trial of palbociclib plus fulvestrant reported a manageable safety profile [69]. Over half of palbociclib-treated patients experienced neutropenia lasting a median 7 days, with 65% experiencing grade 3–4 neutropenia [69]. However, febrile neutropenia was infrequent, (0.9% palbociclib versus 0.6% placebo) [69]. Fewer than 2% of patients given palbociclib had grade 3‒4 neutropenia and grade 3‒4 infections concurrently [69]. Dose modifications (dose reduction, interruption, or cycle delay) did not impact PFS [69].

This finding was explained in a Translational Breast Cancer Research Consortium study comparing palbociclib 100 mg versus the standard 125-mg dose combined with hormonal therapy in patients with metastatic breast cancer [70]. Paired pre- and on-treatment tumor and skin biopsies among 70 patients demonstrated similar reductions in phospho-RB and Ki67 [70], irrespective of the starting dose, suggesting they are pharmacodynamically equivalent; PFS and clinical benefit rate were also similar. Hematologic toxicity was ameliorated at the 100-mg dose, with fewer instances of grade 3/4 neutropenia, fewer dose reductions, and dosing delays [70].

Neutropenia following palbociclib treatment was characterized with a pharmacokinetic/pharmacodynamic model assessing the time course of absolute neutrophil count and the exposure–response relationship for neutropenia across three clinical trials in advanced cancer [71]. Neutropenia was rapidly reversible, cytostatic, noncumulative, and had a weaker antiproliferative effect on hematologic precursor cells when compared to cytotoxic chemotherapies [71]. The reversibility and noncumulative characteristics of neutropenia may explain the low incidence of fever or infection observed in clinical studies of palbociclib, despite transient grade 3 or 4 neutropenia [71].

Another analysis (N = 1827) included three randomized studies of CDK4/6 inhibitors with an AI as initial treatment in postmenopausal women with HR+/HER2– metastatic breast cancer [72]. Women aged ≥75 years showed similar PFS as younger women, but older women experienced higher rates of toxicity (grade 3–4 adverse events: 88.8% versus 73.4%) and decreased quality of life [72]. Reduced doses that achieve similar biological antiproliferative effects may be important in this population.

A meta-analysis of 3743 patients reported similar grade 3–4 toxicity profiles for palbociclib, ribociclib, and abemaciclib [73]. CDK6 is especially involved in the differentiation of hematologic precursor cells. Inhibition of this kinase leads to these cells becoming incapable of further propagation, resulting in neutropenia. Abemaciclib, however, is known to have greater selectivity for CDK4 than CDK6, which may explain lower rates of neutropenia reported in clinical trials and the ability to treat on a continuous schedule [54].

4.2. Other adverse events

Palbociclib and ribociclib are reported to have a lower risk for diarrhea compared with abemaciclib, potentially owing to off-target activity, including inhibition of GSK3β [74]. Adverse events often associated with CDK4/6 inhibition are summarized in Table 3 [43,44,46,50,75,76].

Table 3. Common adverse events observed with CDK4/6 inhibitors

	Palbociclib+ letrozole (PALOMA-2)		Palbociclib+ fulvestrant (PALOMA-3)		Ribociclib+ letrozole (MONALEESA-2)		Ribociclib+ fulvestrant (MONALEESA-3)		Abemaciclib+ letrozole/anastrozole (MONARCH-3)		Abemaciclib+ fulvestrant (MONARCH-2)
Common adverse events, %	All grades	Grades 3 + 4	All grades	Grades 3 + 4	All grades	Grades 3 + 4	All grades	Grades 3 + 4	All grades	Grades 3 + 4	All grades	Grades 3 + 4
Hematologic toxicities
Neutropenia	79.5	66.4	80.9	64.6	74.3	59.3	69.6^a	53.4^a	41.3	21.1	46	26.5
Febrile neutropenia	1.8	0.2	0.9	0.9	1.5	NR			0.3	NR	0.9	0
Thrombocytopenia	15.5	1.6	21.2	2.3	NR	NR	NR	NR	36.2	1.8	15.6	3.4
GI toxicities
Fatigue	37.4	1.8	39.1	2.3	36.5	2.4	31.5	1.7	40.1	1.8	39.9	2.7
Diarrhea	26.1	1.4	21.4	0	35	1.2	29	0.6	81.3	9.5	86.4	13.4
Nausea	35.1	0.2	32.5	0	51.5	2.4	45.3	1.4	38.5	0.9	45.1	2.7
Vomiting	15.5	0.5	16.8	0.3	29.3	3.6	26.7	1.4	28.4	1.2	25.9	0.9
Hepatobiliary
ALT increase	NR	0.2	5.5	1.7	15.6	9.3	NR	8.5	47.6	6.7	13.4	4.1
AST increase	NR	NR	7.0	2.6	15	5.7	NR	6.0	36.7	3.8	12.2	2.3
Creatinine increase	NR	0.2	NR	NR	NR	NR	NR	NR	19.0	2.1	11.8	0.9

^aNeutropenia includes neutropenia, decreased neutrophil count, febrile neutropenia, and neutropenic sepsis.

ALT, alanine aminotransferase; AST, aspartate aminotransferase; CDK, cyclin-dependent kinase; GI, gastrointestinal; NR, not reported.

Abemaciclib has been associated with venous thromboembolism [77], increased creatinine [78] and elevated liver transaminases [43]. Ribociclib has been associated with corrected QT (QT_c) interval prolongation [41] and hepatobiliary toxicity [79], which requires additional monitoring. The risk of QT_c prolongation is reported to be significantly lower for palbiciclib than ribociclib [73].

In September 2019, following a review of cases of interstitial lung disease (ILD) and pneumonitis with CDK 4/6 inhibitors identified in the manufacturers’ clinical trials and post-market safety databases, the FDA issued a warning for the risk of ILD and/or pneumonitis [80]. Across clinical trials of the three CDK 4/6 inhibitors, 1% – 3% of patients had ILD/pneumonitis of any grade and <1% had fatal outcomes. The FDA maintained that ‘the overall benefit of CDK4/6 inhibitors is still greater than the risks when used as prescribed’ [80]. With proper supportive care and close monitoring, improved efficacy outcomes and manageable toxicities with CDK 4/6 inhibitors are observed [81]. Safety signals also appear to be similar in the adjuvant setting.

5. Biomarkers of response and resistance

5.1. Predictive biomarkers in breast cancer

No predictive biomarkers reliably identify patients with HR+/HER2– breast cancer who may benefit from the addition of a CDK4/6 inhibitor to ET. At present, the only biomarker predicting antitumor response is the expression of ERs.

Preclinical data have suggested p16^INK4A (CDKN2A) loss or overexpression of cyclin D1 (CCND1) and the presence of RB may define cancers more reliant on the CDK4/6 pathway and more susceptible to its suppression. However, in PALOMA-1, improvements with combined palbociclib plus letrozole vs. letrozole alone were similar in unselected patients and in those whose tumors harbored CCND1 amplification or CDKN2A loss [39]. Similar results were found in an analysis of 568 tumor tissues from 666 patients (PALOMA-2), where no single biomarker or cassette of biomarkers was associated with lack of benefit from combination treatment [82,83]. Although expression of RB, was not predictive of palbociclib benefit in combination with letrozole, ER-positivity itself classifies breast cancers into a higher RB expression group.

Endocrine resistance associated with higher CDK4 levels was allayed by palbociclib, and its addition associated with longer PFS in tumors with increased growth factor signaling and higher expression of ERBB3 and fibroblast growth factor receptor 2 (FGFR2) mRNA [83]. In an update to the MONALEESA-2 study, baseline ctDNA samples analyzed and sequenced in 494/668 patients with HR+/HER2– advanced breast cancer indicated ribociclib prolonged PFS irrespective of PIK3CA or TP53 mutation status [75]. Also, a biomarker analysis from the phase III randomized MONALEESA-7 trial reported generally consistent PFS benefits for ribociclib combined with goserelin (ovarian function suppression), tamoxifen, and an AI, irrespective of baseline expression of Ki67, total RB, p16 protein expression, CCND1, CDKN2A (p16), and ESR1 mRNA levels [84]. An analysis of the MONARCH-1 trial of single-agent abemaciclib in HR+/HER2– metastatic breast cancer found (by single-marker analysis of tumors) only higher mRNA levels of RB correlated with efficacy, including disease control rate, PFS, and treatment duration [85]. However, as a single-agent study, it is difficult to draw conclusions.

5.2. Intrinsic and acquired resistance

Substantial efforts are underway to understand mechanisms of intrinsic and acquired resistance to inform treatment strategies after CDK4/6 inhibitor failure.

5.2.1. RB loss

Although RB loss of function may predict intrinsic resistance to CDK4/6 inhibition, few HR+/HER2– breast tumors have RB1 gene alteration. Nonetheless, RB loss likely accounts for acquired resistance in a subset of patients, and is reported in a number of studies [86–88].

Although RB loss is infrequent, strategies to address this alteration as cells acquire CDK4/6 inhibitor resistance are in development. For example, RB loss has recently been demonstrated to be synthetic lethal with either aurora kinase A (AURKA) or B deficiency [89], and HR+ breast cancer cells treated to CDK4/6 inhibitor resistance that demonstrated RB loss was found to be exquisitely sensitive to LY3295668, a novel, selective AURKA inhibitor [88].

5.2.2. CDK6 overexpression

Mining of tumor genomic data (MSK-IMPACT™, https://www.mskcc.org/msk-impact#) of 348 patients with breast cancer was obtained prior to treatment with CDK4/6 inhibitors. Loss of the tumor suppressor FAT1 was strongly associated with shorter PFS (2.4 versus 10.1 months), consistent with intrinsic resistance and poor clinical outcome while on CDK4/6 inhibitors [90]. Preclinical studies demonstrated that FAT1 loss in ER+/HER2– breast cancer cells led to resistance to CDK4/6 inhibition, mediated by increased CDK6 [90]. Consistent with this finding, significantly higher levels of CDK6 transcripts were found in FAT1-deleted ER+ breast tumors in The Cancer Genome Atlas, compared with tumors with wild-type FAT1 [90]. Additionally, FAT1-negative tumors showed strong CDK6 expression, whereas tumors with FAT1 wild-type had high FAT1 and low CDK6 expression [90].

CKD6 overexpression has emerged in preclinical models of acquired resistance via gene amplification [91] or via microRNA (miRNA)-mediated suppression of the transforming growth factor beta pathway. Increased expression of the implicated miRNA (mIR-432-5p), targeting Smad4, correlated with intrinsic or acquired resistance in primary breast cancer samples from patients receiving CDK4/6 inhibitors [92]. miRNA expression mediating increased CDK6 expression was reversible after removal of CDK4/6 inhibition, suggesting that, in some cases, a drug holiday may result in re-sensitization to CDK4/6 inhibition[92]. Additionally, strategies to reduce CDK6 levels in overexpressing cells are under investigation, including the development of selective degraders and use of bromodomain inhibitors [93,94].

5.2.3. Cyclin E1 and E2 amplification or overexpression

Potential biomarkers (10 genes relevant to the G1 checkpoint) predicted to confer intrinsic resistance to the combination of palbociclib/fulvestrant were examined in the PALOMA-3 trial [95]. Archival tumor tissue samples were analyzed vs. a panel of 2500 genes. The median PFS for patients with low CCNE1 levels was 14.1 months (palbociclib) vs. 4.8 months (placebo). For patients with high CCNE1 levels, median PFS was 7.6 months versus 4.0 months. The role of high cyclin E1 in resistance to CDK4/6 inhibition is supported by preclinical data where palbociclib-resistant MCF-7 breast cancer cells are re-sensitized to CDK4/6 inhibition by genetic knockdown of CCNE1 [96].

Cyclin E activates CDK2, which has been shown to drive the cell cycle independently of CDKs 4 and 6 [97]. Upon CDK4/6 inhibition in breast cancer cells with depleted cyclin E1, adaptation occurs as early as 72 hours, resulting from CDK2 activation via non-canonical activation of cyclin D1/CDK2 complexes, and increased cyclin E2 expression, leading to S-phase entry [96]. These data affirm the strong predilection of CDK4/6 inhibited cells to activate CDK2. Additionally, whole-exome sequencing data from patients with progressive disease after exposure to CDK4/6 inhibition demonstrated amplification of CCNE2 in 6 of 41 cases [88].

The association of amplification and/or overexpression of cyclin E1 and cyclin E2 with CDK4/6 inhibitor resistance has renewed interest developing CDK2 inhibitors [98], which could overcome CDK4/6 inhibitor resistance, whether intrinsic or acquired. This has prompted the development of PF-06873600 (Pfizer; New York, NY), a CDK2/4/6 inhibitor under study (NCT03519178) as monotherapy and combined with ET. The activity of CDK2 inhibitor agents in CDK4/6 inhibitor-resistant breast cancer and to determine whether cyclin E levels dictate sensitivity will be critical to assess. However, CDK2 inhibition may have limitations, because cyclin E can promote proliferation via CDK2-dependent and -independent mechanisms [99], and CDK2 activity can be compensated by CDK1 [100,101].

CDK2 activity, and other cell cycle CDKs, may also be downregulated by CDK7 inhibition, which serves as a transcriptional CDK and a component of CAK [102]. The utility of CDK2 or CAK (CDK7) inhibition may not be restricted to CDK4/6-inhibitor–resistant cells with high levels of cyclin E1 or cyclin E2. Following RB phosphorylation and the release and activation of E2F-1, E2F-1 activity must be downregulated for proper S-G2 progression, mediated by cyclin A-CDK2. CDK2 inhibition therefore maintains the inappropriately high levels of E2F-1 activity found in transformed cells, which reaches a threshold capable of inducing apoptosis [103]. CDK2 inhibition may produce selective cytotoxicity in other CDK4/6-inhibitor–resistant states expected to increase E2F-1 activity, including those governed by RB loss [104] and possibly by CDK6 overexpression.

CCNE1 amplification and cyclin E overexpression, related to CDK2-dependent and -independent activities, have been associated with a state of replicative stress and susceptibility to inhibitors of the ATR-CHK1 pathway [105]. An MCF-7 palbociclib-resistant derivative was highly sensitive to the CHK1 inhibitor prexasertib [88], which has demonstrated preliminary activity in high-grade serous ovarian cancers with CCNE1 amplification and/or high expression of cyclin E at the mRNA or protein level [106,107].

5.2.4. FGFR, ERBB2, and RAS-mediated signaling

Intrinsic resistance to CDK4/6 inhibition in HR+/HER2– breast cancer can be mediated by aberrant FGFR signaling. A study involving the expression of a library of 559 sequence-validated kinase open-reading frame clones in ER+ MCF-7 cells reported FGFR1 overexpression reduced cell sensitivity to ribociclib/fulvestrant [108]. Resistance was reversed by addition of the FGFR inhibitor lucitanib. In vivo, addition of the pan-FGFR inhibitor erdafitinib to fulvestrant and ribociclib induced complete responses in inhibiting growth of FGFR-amplified HR+/HER2– patient-derived xenografts [108]. Importantly, patients from the MONALEESA-2 study with baseline FGFR1 amplification detected in ctDNA or high FGFR1 mRNA expression in archival tumor tissue had shorter PFS compared with patients without FGFR amplification or overexpression [108]. Also, ctDNA analysis in 34 patients after progression on a CDK4/6 inhibitor identified FGFR1/2 amplification of activating mutation in 14 of 34 (41%) post-progression specimens [108]. Among 14 patients who demonstrated a CDK4/6-inhibitor–resistant phenotype, there were three instances of FGFR2 abnormalities with intrinsic resistance [108].

An open-label, multicenter, phase Ib trial (NCT03238196) evaluating the safety and tolerability and antitumor activity of fulvestrant, palbociclib, and erdafitinib in ER+/HER2–/FGFR-amplified metastatic breast cancer demonstrated disease stabilization with prolonged benefit noted in those with high FGFR1 amplification or those with FGFR3 amplification [109]. Another phase II trial (NCT04024436) is evaluating the safety and efficacy of the FGFR inhibitor TAS-120 ± fulvestrant in metastatic breast cancer.

The acquisition of RAS pathway-activating mutations or amplifications in four of 41 samples (9.8%), including KRAS G12D, KRAS Q61L, HRAS K117N, and focal amplification of NRAS, and activating mutations and amplifications of ERBB2 (5/41 cases; 12.2%), have been demonstrated in patients with HR+ breast cancer experiencing acquired resistance to CDK4/6 inhibitors [88]. The activation of FGFR, RAS, and ERBB2 in suggests inhibition of MAP kinase signaling may address a large proportion of CDK4/6 inhibitor-resistant breast cancers.

Notably, FGFR and mitogen-activated protein kinase (MAPK) activation may be a generalized mechanism of CDK4/6 inhibition extending beyond breast cancer [110]. KRAS-mutant NSCLC cell lines are initially sensitive to CDK4/6 inhibition, but acquire resistance associated with increased expression of CDK6, D-type cyclins, and cyclin E [110]. Resistant cells demonstrated increased ERK1/2 activity and sensitivity to MEK and ERK inhibitors, suggesting CDK4/6 inhibitor resistance results in MAP kinase dependence [110]. Similar results are shown in models of prostate cancer [111]. Moreover, MEK inhibition reduced the expression and activity of cell cycle proteins mediating CDK4/6 inhibitor resistance [110]. In resistant cells, ERK activated mTOR, driven in part by upstream FGFR1 signaling resulting from the extracellular secretion of FGF ligands. A mouse model of KRAS-mutant NSCLC initially sensitive to palbociclib similarly developed acquired resistance with increased expression of cell cycle mediators, ERK1/2, and FGFR1. In this model, resistance was delayed with combined palbociclib and MEK inhibitor treatment. These findings implicate an FGFR1-MAP kinase-mTOR pathway resulting in increased expression of D-cyclins and CDK6 conferring CDK4/6 inhibitor resistance [110]. These findings have translated clinically, with prolonged PFS with combination of palbociclib and the MEK inhibitor mirdametinib (PD-0325901) in KRAS-mutant NSCLC (NCT02022982) [112].

5.2.5. PTEN and AKT

The loss of PTEN can lead to resistance to CDK4/6 inhibition. In one report, PTEN loss was found in two of five progression tumor biopsies in patients treated with ribociclib/letrozole [113]. Mechanistically, PTEN loss impairs p27 nuclear localization; as PTEN regulates AKT, PTEN loss leads to AKT-mediated phosphorylation of p27 on Thr157, impairing p27 nuclear import [114], leading to increased CDK4 and CDK2 activation [113]. Preclinically, knockdown of PTEN led to loss of tumor growth inhibition by ribociclib, with sensitivity restored by AKT inhibition [113]. AKT1 activating mutation and amplification have also been observed in 5 of 41 cases (12.2%) of patients with HR+ breast cancer that progressed on CDK4/6 inhibitor therapy [88]. The AKT inhibitors capivasertib and ipatasertib are being studied in combination with ET and/or CDK4/6 inhibition in HR+ breast cancer (NCT04305496, NCT04060862, and NCT03959891).

5.2.6. 3-phosphoinositide dependent protein kinase 1 (PDK-1)

A kinome-wide small-interfering RNA (siRNA) screen identified kinases that, when downregulated, afford sensitivity to CDK4/6 inhibitors [115]. PDK1 is an important modifier of sensitivity to ribociclib in ER+ MCF-7 breast cancer cells, and its inhibition combined with ribociclib or palbociclib increased apoptosis and synergistic inhibition of proliferation in ER+ breast cancer cell lines [115]. Additionally, cells rendered resistant by chronic exposure to ribociclib had increased levels of PDK1 and AKT pathway activation, and CDK4/6 inhibitors did not induce senescence or cell-cycle arrest [115]. Resistant cells had upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT; PDK-1 inhibition or exposure to dinacicilb reversed these changes and reestablished the sensitivity of resistant cells to CDK4/6 inhibition [115].

5.2.7. Combined CDK4/6 and PI3K inhibition

Deregulation of cyclin E/CDK2 or PI3K/AKT/mTOR signaling may allow a therapeutic bypass to CDK4/6 inhibitors, and PI3K inhibition reduced cyclin D1 expression and prevent early adaptation [96]. In a study to identify how to improve the effectiveness of PI3K inhibitor (PI3Ki)-based therapy by reducing adaptive resistance and improving the initial response, two models of resistance to PI3Ki were discovered [116]. In one model, residual AKT signaling permits the continued activity of mTOR complex (mTORC) and its effectors. Suppressing AKT signaling blocked phosphorylation of downstream nodes such as mTORC and synergized with the inhibition of PI3K. In the other model, PI3K remains strongly inhibited whereas mTORC activity continues, likely by input from other pathways. These models demonstrated there is no extra benefit of AKT inhibition in combination with a PI3Ki [116]. Targeting downstream at CDK4/6 sensitized both models to PI3Ki, suggesting clinical utility of the dual vertical inhibition combination in cancers that may not be responsive to PI3Ki owing to varied mechanisms [116].

The interaction of the CDK4/6 and PI3K–mTOR pathways provides a hypothesis for investigating the inhibition of both pathways [116–118]. Hyperactivation of the PI3K pathway stabilized the cyclin D protein and the cyclin D-CDK4/6 complex [119], and, preclinically, CDK4/6 inhibition sensitized PIK3CA-mutant breast cancer to PI3K inhibition [116]. In ER+ breast cancer cells, two in vitro analyses reported that combined PI3K and CDK4/6 inhibition overcame resistance to CDK4/6 inhibition, owing to cyclin D1 downregulation [96,116]. The combination resulted in cancer cell apoptosis in vitro and in patient-derived xenograft (PDX) mice [96]. Furthermore, a triplet regime involving CDK4/6 and PI3Ki with endocrine treatment was effective in vitro and in PDX models, with tumor regression and stabilization of disease [96]. Emerging data suggest that triplet therapy, combining ET plus CDK4/6 inhibitors with inhibitors of the PI3K/AKT/mTOR pathway, may better prevent acquired CDK4/6 resistance than doublet regimens [96]; clinical studies investigating this are ongoing (NCT03006172, NCT01872260, NCT02389842, NCT02732119, NCT02154776, NCT02088684, NCT02753686, NCT03959891, and NCT01857193).

TRINITI-1 (NCT02732119), a clinical trial involving triplet therapy of everolimus (mTOR inhibitor) with ribociclib and exemestane (AI) in patients with HR+/HER2– and locally advanced/metastatic breast cancer following progression on CDK 4/6 inhibitors, is recruiting [120]. Early data demonstrated clinical benefit at Week 24 in 39 (41.1%) patients, exceeding the predefined primary endpoint threshold of ≥10%. The ORR was 8.4% by investigator assessment, median PFS 5.7 months, and 1-year PFS was 33% [120,121].

5.2.8. MYC addiction

Tumor metabolic reprogramming appears to be an important factor in adaptive responses to drug‐induced stress and may reveal weaknesses of cancer cells [122,123]. A study combining metabolic analysis with transcriptomic data found metabolic changes associated with CDK4/6 depletion, reporting that MYC upregulation and its downstream network, which includes mTOR signaling and glutaminolysis, is an adaptation to CDK4/6 inhibition [124]. MYC overexpression is associated with drug resistance, and reduction or inhibition of CDK4/6 in cancer cells can result in de novo addiction to MYC, and to glutaminase and mTOR signaling, and a compromised adaptation to hypoxia [124]. Of particular note, Hydbring et al. described direct phosphorylation of MYC by CDK2/Cyclin E as a mechanism for overcoming Ras driven oncogene induced senescence [125,126]. In addition, CDK2 knockout mice displayed prolonged survival in the Emu-MYC background, correlating with increased cellular senescence [127]. Therapeutic combinations of CDK4/6 inhibitors with agents depleting MYC mRNA or protein may exploit these dependencies. MYC stabilization can be mediated by AURKA activity where it drives G2/M cell cycle progression and inhibition of AURKA results in degradation of MYC protein [128]. In 11/41 HR+ breast cancer patients who were refractory to CDK4/6 inhibitor therapy, AURKA amplification was observed and likely contributed to CDK4/6 inhibitor resistance [88]. Amplification and overexpression of MYC may also be targeted by CDK1 inhibition, CDK9 inhibition, or BET bromodomain inhibition [29,30,129,130].

5.2.9. Summary of intrinsic and acquired resistance mechanisms

Figure 3 shows the main drivers of resistance to CDK inhibition and strategies to circumvent resistance. Mechanisms of resistance are heterogeneous and sometimes overlapping. Current data suggest that a resistance mechanism can be assigned to approximately two-thirds of metastatic HR+ breast cancer cases [88]. Biopsies at the time of acquired resistance and serial ctDNA analyses will be critical to guide prospective treatment options.

Figure 3. Possible mechanisms of resistance to CDK4/6 inhibitors

6. New CDK4/6 inhibitors in development for HR+ breast cancer

Continued research aims to improve efficacy, safety, added differentiation through new modes of action, and addressing resistance with expansion into other tumor types. A summary of clinical trials of various CDK inhibitors that are in development and not yet on the market across a range of CDK targets and indications are listed in Table 4.

Table 4. Ongoing or planned clinical trials of investigational CDK inhibitors

Molecule	Selectivity	Cancer indications in clinical studies	Clinical phase	ClinicalTrials.gov identifier
CS3002	CDK4/6	Advanced solid tumors	I	NCT04162301
SHR6390	CDK4/6	Advanced BC, BC, GC, solid tumors, advanced melanoma, MBC, NSCLC, CRC, HCC	I, II or III	NCT03480256^a, NCT02684266, NCT03993964^a, NCT03601598^b, NCT03966898^c, NCT03927456^d, NCT03481998^e, NCT03772353^f, NCT04236310^g, NCT04095390^a
Lerociclib (G1T38)	CDK4/6	Locally advanced or MBC, eGFR-mutant NSCLC	I/II	NCT02983071^d, NCT03455829^h
Trilaciclib	CDK4/6	SCLC, MTNBC	II	NCT03041311^i, NCT02514447^j, NCT02978716^k
FCN-437	CDK4/6	Advanced solid tumors	I	NCT03951116
PF-06873600	CDK2/4/6	HR+/HER2– MBC, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, TNBC, male BC	II	NCT03519178
AT7519M	CDK1/2/4–6/9	Solid tumors	I	NCT02503709^l
PF-07104091	CDK2	SCLC, ovarian cancer, TNBC, NSCLC, HR+/HER2 – breast cancer	II	NCT04553133
Dinaciclib	CDK1/2/5/9	rrAML, advanced solid tumors, Advanced BC, melanoma	I/II	NCT03484520^m, NCT01434316^n, NCT01676753°, NCT00937937
PF-07220060	CDK4	Advanced solid tumors	I	NCT04557449
SEL120	CDK8/19	AML, high-risk MDS	I	NCT04021368
CT7001	CDK7	Advanced malignancies	I/II	NCT03363893^d
LY3405105	CDK7	Advanced cancer	I	NCT03770494
SY-5609	CDK7	Advanced solid tumors	I	NCT04247126
Alvocidib	CDK9	r/r AML, AML	I/II	NCT03441555^m, NCT03969420, NCT03298984^p, NCT03593915^q, NCT02520011^r
TP-1287	CDK9	Advanced solid tumors	I	NCT03604783
AZD4573	CDK9	r/r hematologic malignancies	I	NCT03263637
CYC065	CDK2/5/9	r/rAML, rrMDS, r/rCLL, advanced cancers	I	NCT04017546^m, NCT03739554^m, NCT02552953

^ain combination with pyrotinib, ^bin combination with SHR-1210 (anti-PD1), ^cin combination with letrozole or anastrozole, ^din combination with fulvestrant, ^ein combination with letrozole, anastrozole or fulvestrant, ^fin combination with letrozole and pyrotinib, ^gin combination with anastrozole, pyrotinib, and trastuzumab, ^hin combination with osimertinib, ⁱin combination with carboplatin, etoposide, and atezolizumab, ^jin combination with topotecan, ^kin combination with gemcitabine and carboplatin, ^lin combination with onalespib, ^min combination with venetoclax, ⁿin combination with veliparib, ^oin combination with pembrolizumab, ^pin combination with cytarabine/daunorubicin, ^qin combination with decitabine or azacitidine, ^rincombination with cytarabine/mitoxantrone,

AML, acute myeloid leukemia; BC, breast cancer; CLL, chronic lymphocytic leukemia; CRC, colorectal cancer; eGFR, estimated glomerular filtration rate; GC, gastric cancer; HCC, hepatocellular carcinoma; HER, human growth factor receptor; HR, hormone receptor; MBC, metastatic breast cancer; MDS, myelodysplastic syndrome; NSCLC, non small-cell lung cancer; MTNBC, metastatic triple-negative breast cancer, r/r, relapsing/remitting; SCLC, small cell lung cancer; TNBC, triple negative breast cancer

Lerociclib (G1T38) (G1 Therapeutics, Durham, NC) is a potent and selective oral CDK4/6 inhibitor with efficacy in several preclinical models of HR+ breast cancer [131]. An ongoing-phase Ib/II study (NCT02983071) of continuous lerociclib dosing with fulvestrant was well tolerated, with a low rate of grade 4 neutropenia and efficacy consistent with other studies of CDK4/6 inhibition in combination with fulvestrant [132]. Another CDK4/6 inhibitor, CS3002 (CStone Pharmaceuticals, Suzhou, China), when combined with programmed cell death 1 (PD-1) monoclonal antibody (mAb) therapy or ET improved tumor suppressing activities versus monotherapies [133]. The CDK4/6 inhibitor SHR6390 (Jiangsu HengRui Medicine, Lianyungang, Jiangsu, China) is being investigated in a number of clinical trials.

7. CDK4/6 inhibitors beyond HR+ breast cancer: HER2+ and TNBC and other cancers

7.1. HER2+ disease

Substantial preclinical data support the use of CDK4/6 inhibition in HER2+ breast cancer. In mouse models of HER2/neu-driven mammary carcinoma, disease can be prevented by ablation of CCND1 or CDK4 [134]. The continued presence of CDK4-associated kinase activity is also required to maintain tumorigenesis [135,136]. Cyclin D1-CDK4 activity also mediates resistance to HER2-directed therapies and the targeting of resistant tumor cells with a CDK4/6 inhibitor re-sensitizes them to anti-HER2 therapy [117]. Inhibition of CDK4/6 reduces RB phosphorylation and TSC2 phosphorylation, partially attenuating TORC1 and S6K activities [117]. This relieves feedback suppression of epidermal growth factor receptor (EGFR) family kinases and re-sensitizes tumors to EGFR/HER2 blockade. In transgenic mouse models and in vitro, combined HER2 and CDK4/6 inhibition synergistically inhibits cell proliferation, promotes senescence, controls tumor growth in vivo, and delays tumor recurrence [76,137].

To translate these findings, a phase Ib/II trial (NCT02657343) investigated ribociclib and trastuzumab in patients with advanced HER2+ breast cancer previously treated with trastuzumab, pertuzumab, or trastuzumab emtansine [138]. However, limited efficacy was observed suggesting further study of CDK4/6 inhibitor/anti-HER2 combinations should focus on patients who are not heavily pretreated or capitalize on the combined effects of CDK4/6 inhibition and hormonal therapy in the HR+ population. This was addressed in the monarcHER trial (NCT02675231) where 237 patients with HR+/HER2+ breast cancer with unresectable, locally advanced, recurrent, or metastatic disease received abemaciclib/trastuzumab/fulvestrant versus abemaciclib/trastuzumab versus standard-of care-chemotherapy/trastuzumab [139]. With a median follow-up of 19 months, the study met its primary endpoint, with a statistically significant difference in PFS between the triplet and the standard-of-care arms (8.3 versus 5.7 months; hazard ratio 0.67) [139]. However, no differences in PFS between abemaciclib/trastuzumab and the standard-of-care arms were observed, and absence of a fulvestrant/trastuzumab arm allowed no firm conclusions to be drawn. Nonetheless, these results suggest a chemotherapy-free regimen may be a treatment option for recurrent HR+/HER2+ disease.

The PATINA trial (NCT02947685) is investigating whether patients with HR+/HER2+ metastatic breast cancer gain benefit from addition of palbociclib to first-line treatment (trastuzumab/pertuzumab plus ET). Following 6–8 cycles of induction chemotherapy (taxane or vinorelbine) with anti-HER2 therapy, patients are randomized to anti-HER2 therapy (trastuzumab/pertuzumab plus ET) ± palbociclib until disease progression. The study is still recruiting, and results not yet reported. The PATRICIA 2 trial (NCT02448420) in patients with previously treated HR+/HER2+ advanced or metastatic breast cancer with PAM50 luminal A or B intrinsic subtype tumors is investigating palbociclib + trastuzumab and ET (Cohort C1), or treatment of physician’s choice: T-DM1 or chemotherapy (gemcitabine, vinorelbine, capecitabine, eribulin, paclitaxel, or docetaxel) plus trastuzumab (cohort C2). The ET options are either an AI, fulvestrant, or tamoxifen ± ovarian suppression [140]. This trial follows the PATRICIA phase II trial where PAM50 luminal disease was associated with a larger and clinically meaningful PFS following treatment with palbociclib, trastuzumab, and ET compared with PAM50 non-luminal disease [141].

The FDA-approved HER2 small-molecule inhibitor tucatinib is being studied in a Phase 1b trial (NCT03054363) in combination with palbociclb and letrozole in HR+/HER2+ metastatic breast cancer. The combination has shown an acceptable safety profile with encouraging antitumor activity [142] and a Phase II trial is ongoing.

7.2. TNBC

TNBC has been largely resistant to CDK4/6 inhibition. This subset is most likely to be RB- and TP53-deficient. Among HR+ breast cancers, TP53 mutations were enriched among those with a resistance phenotype (n = 24/41 [58.5%]) [88], although engineered TP53 inactivation was insufficient to drive CDK4/6 inhibitor resistance, so that its role in resistance is not clear.

The CDK4/6 inhibitor trilaciclib (G1T28) is in development for administration prior to myelotoxic chemotherapy, with the goal of transiently inducing G1 cell cycle arrest in hematopoietic stem and progenitor cells, preserving them from chemotherapy-induced damage. Proof-of-principle for myelopreservation using this approach was successful in a randomized trial where trilaciclib was administered alongside etoposide/carboplatin for first-line treatment of metastatic SCLC, a uniformly RB-negative tumor, where CDK4/6 inhibition is not expected to antagonize chemotherapy [143]. The use of trilaciclib resulted in myelopreservation across multiple hematopoietic lineages, fewer supportive care interventions and dose reductions, improved safety profile, and no detriment to antitumor efficacy [143].

A similar randomized study (NCT02978716) was conducted in TNBC in which gemcitabine/carboplatin was administered ±trilaciclib, utilized on two different schedules surrounding the chemotherapy. There were no significant differences in myelosuppression between groups [144]. However, those administered trilaciclib demonstrated higher responses rates, improved PFS and OS (12.6 months for control and 20.1 and 17.8 months for trilaciclib versus gemcitabine/carboplatin) [144]. Pulsatile administration of a CDK4/6 inhibitor did not compromise the efficacy of chemotherapy. Trilaciclib may have limited lysosomal sequestration, contributing to direct antitumor activity, or has favorable effects on the tumor microenvironment, enhancing a long-term favorable outcome.

Synergistic effects have also been observed with PI3K and CDK4/6 inhibitors in TNBC. Preclincally, palbociclib/taselisib or ribociclib/alpelisib have greater activity in comparison with each drug alone [145,146]. The inhibition of the PI3K signaling pathway also increased the sensitivity of cancer cells to palbociclib, thought to be mediated in part by the suppression of post-mitotic CDK2 activity [145].

8. Effects of CDK4/6 inhibition on the immune microenvironment

Data suggest biological effects for CDK 4/6 inhibitors on T cells and other immune cells. Mechanisms include increased tumor cell antigen presentation [147], effector T-cell activation [148,149], and reduced propagation of regulatory T-cells [147].

These findings have stimulated multiple studies in which CDK4/6 inhibition is combined with immune checkpoint blockade. For example, the MORPHEUS HR+BC trial (NCT03280563) is recruiting patients with HR+/HER2– breast cancer who have progressed on or after treatment with CDK 4/6 inhibitors in first- or second-line therapy. The phase II palbociclib after CDK and Endocrine Therapy (PACE) trial (NCT03147287) investigates palbociclib, fulvestrant, and avelumab in patients with HR+/HER2– metastatic breast cancer who stopped responding to prior palbociclib and ET. Another trial (NCT02778685) of pembrolizumab, letrozole, and palbociclib is recruiting postmenopausal women with newly diagnosed metastatic breast cancer. These latter trials will help determine whether combined CDK4/6 inhibition and PD-L1 blockade are better tolerated than PD-1 blockade. A trial (NCT03294694) of ribociclib with the anti-PD1 mAb spartalizumab with or without fulvestrant (PDR001; Novartis, Basel, Switzerland) reported toxicity issues with grade 3/4 neutropenia (50%), increased aspartate aminotransferase (25%), and increased alanine aminotransferase (25%) among 16 patients enrolled (n = 15 with metastatic breast cancer, 1 with ovarian cancer). Two patients with ER+ disease who had previously received CDK4/6 inhibitor-based treatment + ET achieved PR and SD > 24 weeks, respectively [150].

A summary of phase II/III clinical trials in breast cancer for approved CDK4/6 inhibitors with immunotherapy are shown in Table 5.

Table 5. Phase II/III clinical trials in breast cancer for approved CDK4/6 inhibitors with immunotherapy

Molecule	Description of trial	Clinical phase	ClinicalTrials.gov identifier
Palbociclib	Palbociclib, fulvestrant and avelumab in patients with HR+/HER2– metastatic BC who stopped responding to prior palbociclib and ET.	II	NCT03147287
Palbociclib	Combination of TDM1^a with palbociclib vs. single-agent TDM1 in patients with metastatic HER2+ BC.	II	NCT03530696
Palbociclib	Pembrolizumab + letrozole and palbociclib in postmenopausal women with newly diagnosed metastatic stage IV ER+ BC.	II	NCT02778685
Palbociclib	Palbociclib + trastuzumab, with or without letrozole, in post-menopausal women with HER2+ locally advanced or metastatic BC.	II	NCT02448420
Palbociclib	Palbociclib with anti-HER2 therapy + ET vs. anti-HER2-based therapy + ET alone in HR+/HER2+ metastatic BC.	II	NCT02947685
Ribociclib	Ribociclib, letrozole and trastuzumab in postmenopausal women with HR+/HER2+ advanced BC.	II	NCT03913234
Ribociclib	Ribociclib with either one of TDM1, trastuzumab or tratuzumab + fulvestrant in HR+/HER2– BC.	II	NCT02657343
Ribociclib	Chemotherapy vs. endocrine therapy in combination with dual therapy (trastuzumab, pertuzumab) + ribociclib in HR+/HER2+ metastatic BC.	III	NCT02344472
Abemaciclib	Abemaciclib with or without TDM1 in HER2+ metastatic BC.	II	NCT04351230
Abemaciclib	Abemaciclib + trastuzumab with or without fulvestrant vs. trastuzumab + physician’s choice standard of care chemotherapy in HR+/HER2+ locally advanced or metastatic BC after prior exposure to ≥ HER2-directed therapies.	II	NCT02675231
Abemaciclib	Abemaciclib, atezolizumab and fulvestrant in participants with inoperable locally advanced or metastatic HR+/HER2– BC who have progressed during or following treatment with a CDK4/6 inhibitor in the first- or second-line setting.	II	NCT03280563

^aTDM1 is an antibody-drug conjugate consisting of the humanized monoclonal antibody trastuzumab covalently linked to the cytotoxic agent DM1

AI, aromatase inhibitor; BC, breast cancer; CDK, cyclin-dependent kinase; DM1, mertansine; ER, estrogen receptor; ET, endocrine therapy; HER2, human epidermal growth factor receptor 2; HR, hormone receptor; TDM1, trastuzumab emtansine

9. CDK7 and CDK9 inhibition

Transcriptional CDKs (tCDKs) are central to transcription and co-ordination of processes including RNA capping, splicing, 3ʹ end formation, export and regulation of the chromatin landscape [151]. Research has shown there is significant crosstalk amongst tCDKs, and each kinase impacts multiple points of the transcription cycle [151]. CDK7 is an important regulator of transcription and cell cycle progression, and is elevated in breast cancer, associated with a poor prognosis and response to endocrine treatment [152]. It is an attractive target, as inhibition of CDK7 decreases transcript levels of oncogenic transcription factors [153] and provides the means to influence cell cycle progression and transcription at the same time.

Increased expression of CDK7 is reported to confer resistance to CDK4/6 inhibitors [154], and CDK7 inhibition may reverse resistance either alone or in combination with fulvestrant in models resistant to CDK4/6 inhibition, fulvestrant, or both. CDK7 inhibition overcame resistance to palbociclib in a cell model of palbociclib resistance with loss of RB [155]. Another study reporte potent synergy of CDK7 inhibition with lapatinib (an HER2 inhibitor) in HER2-inhibitor–resistant breast cancer cells in vitro, and the induction of tumor regression in HER2-inhibitor–resistant breast cancer xenograft models in vivo [156].

Several CDK7 inhibitors are in development, including ATP competitive inhibitors, and covalent inhibitors that leverage cysteine binding remote from the ATP pocket to confer substantial selectivity. For example, LY3405105 (Eli Lilly and Company, Indianapolis, IN) is currently in a phase I trial (NCT03770494) and a phase I clinical trial (NCT04247126) of the CDK7 inhibitor SY-5609 (Syros Pharmaceuticals, Cambridge, MA) in patients with select advanced solid tumors began recruiting patients in January 2020.

Similar to dinaciclib, other CDK2 inhibitors also inhibit the transcriptional CDK9. Fadraciclib (CYC-065) (Cyclacel Pharmaceuticals, Berkeley Heights, NJ) is a highly selective second-generation inhibitor of CDK2/5/9, currently investigated as monotherapy in advanced cancers in a phase 1 study (NCT02552953) [157,158]. Inhibition of CDK9 induces apoptotic tumor cell death via transcriptional downregulation of cancer cell survival pathways, including MCL1, which is overexpressed in many cancers [159]. CYC-065 is being combined with venetoclax in hematologic malignancies (NCT04017546, NCT03739554) to circumvent MCL1-mediated resistance to BCL2 inhibition; this combination may also have relevance in solid tumors, including breast cancer.

In contrast to the role of various CDKs in cell cycle progression, an increased understanding of the role of transcriptional CDKs has allowed a refocus on the development of drugs, such as alvocidib (Sumitomo Dainippon Pharma Oncology, Osaka, Japan), a potent inhibitor of CDK9; clinical activity for this agent is shown in various solid tumor types and in hematologic malignancies [160].

10. Conclusions

The use of CDK4/6 inhibitors in patients with breast cancer has provided dramatic improvements in PFS and these effects are relatively consistent for palbociclib, ribociclib, and abemaciclib. Drug resistance is complex, the known resistance mechanisms are quite varied, and it is unclear which biomarkers may predict a lack of response. Continued profiling of post-CDK4/6 tumors will hopefully provide insights to aid treatment decisions. As monotherapy rarely works, novel agents and/or rational combinations are required to improve efficacy, reduce toxicities, and overcome resistance to CDK4/6 inhibition. More research into response and resistance mechanisms is needed, with several promising biomarkers such as Cyclin E on the horizon.

11. Expert opinion

CDK4/6 inhibitors have transformed the treatment of advanced HR+ breast cancer and are poised to impact the treatment of patients with early-stage disease with a high risk of early relapse. As clinical trials and basic research in this area progresses, new drugs and research into novel drug combinations involving CDK inhibition will pave the way for improved treatment options. The adoption of learnings from clinical trials will help develop treatment guidelines, and guide a better understanding of the mechanisms involved in the response to therapy. Additionally, we may see a similar benefit of CDK4/6 inhibitor therapy beyond progression in HER2+ disease, and it is likely that specific CDK4/6 inhibitors will become a part of standard therapy for high-risk early-stage disease.

Resistance is common and there are limited options for patients who develop resistance to treatment. A better understanding of the mechanisms of resistance is a key area for improvement, and ongoing and future research must remain focused on overcoming or circumventing resistance. Clinical studies and basic research are yielding some results, but drug resistance remains complex and currently not well understood. A better understanding of the resistance mechanisms underlying CDK inhibitors will lead to the development of novel therapies focused against such putative resistance biomarkers, for example, CDK2, as well as the combination of CDK4/6 inhibitors with relevant antitumor agents that may overcome monotherapy drug resistance. Regular re-testing of genomic alterations from the tumor and/or plasma after progression on CDK4/6 inhibitor therapy will afford us the ability to offer more individualized therapy with rational combinations based on resistance mechanisms.

Ongoing and future research may lead to the development of a collection of highly selective agents against specific CDKs, and a better understanding of relevant biomarkers with improved diagnostic approaches to enable the selection of patient populations most sensitive to a particular agent. Such specific CDKs may also allow improvements in drug safety and tolerability, enabling a wider therapeutic index. This will in turn enable the development of rational combinations with other antitumor agents. Although many studies are in progress, further research is required to understand optimal treatment sequencing and to develop novel combinations involving CDK inhibitors. The generation of the data to reach this point will take time and will involve an improved understanding of the interplay between pharmacology and biology to provide a basis for rational drug combinations. Together, these advances would be anticipated to have a positive impact on the quality of life for patients in clinical practice, with the hope of improved efficacy and safety over current marketed approved CDK4/6 inhibitors. The approach of targeting CDKs in cancer is an attractive one, and this area will continue to be important in the treatment of breast and other cancers.

Article highlights

• Aberrant CDK activation is common in cancer, and deregulation of the cell cycle is one of the key hallmarks of cancer

• Early pan-CDK inhibitors targeted both the cell cycle and RNA polymerase II phosphorylation, but such approaches were complicated by toxicity, providing a rationale for exploring the utility of selective CDK inhibitors

• Although small-molecule inhibitors of CDK4/6 are now established as a standard-of-care therapeutic option for first- and second-line HR+/HER2- metastatic breast cancer, resistance remains an issue

• Selective CDK inhibitors are in development for a range of cancer indications, including further options for breast cancer with molecules that target other distinct CDKs in addition to or instead of CDK4/6

• Drug resistance is complex, the known resistance mechanisms to the approved CDK4/6 inhibitors are varied, and it is unclear which biomarkers may robustly predict a lack of response

• The identification of biomarkers measured at baseline or on-treatment that are predictive of response is of key importance for choosing the most appropriate therapy, and further research in this area is warranted

• Identifying novel agents and/or appropriate combination approaches involving CDK inhibitors are required to improve efficacy, reduce toxicities, and overcome resistance

Declaration of interest

K Jhaveri has served as a consultant and on the advisory boards for Novartis, Spectrum Pharmaceuticals, ADC Therapeutics, Pfizer, BMS, Jounce Therapeutics, Taiho Oncology, Genentech, Synthon, AbbVie, AstraZeneca, Lilly Pharmaceuticals, Intellisphere, Seattle Genetics, Blueprint Medicines; research funding from Novartis, Clovis Oncology, Genentech, AstraZeneca, ADC Therapeutics, Novita Pharmaceuticals, Debio Pharmaceuticals, Pfizer, Lilly Pharmaceuticals, Zymeworks, Immunomedics, Puma Biotechnology.

HA Burris 3^rd has received consulting fees paid to institution from AstraZeneca, FORMA Therapeutics, Celgene, Incyte, research funding paid to institution: Roche/Genentech, Bristol-Myers Squibb, Incyte, AstraZeneca, MedImmune, Macrogenics, Novartis, Boehringer Ingelheim, Lilly, Seattle Genetics, Merck, Agios, Jounce Therapeutics, Moderna Therapeutics, CytomX Therapeutics, GlaxoSmithKline, Verastem, Tesaro, BioMed Valley Discoveries, TG Therapeutics, Vertex, eFFECTOR Therapeutics, Janssen, Gilead Sciences, BioAtla, CicloMed, Harpoon Therapeutics, Arch, Arvinas, Revolution Medicines, Array BioPharma, Bayer, BIND Therapeutics, Kyocera, miRNA Therapeutics, Pfizer, Takeda/Millenium, Foundation Medicine, Expert Testimony for Novartis.

TA Yap has received research support from AstraZeneca, Bayer, Pfizer, Tesaro, Jounce, Lilly, Seattle Genetics, Kyowa, Constellation, and Vertex Pharmaceuticals; consultancies from Aduro, Almac, AstraZeneca, Atrin, Bayer, Bristol-Myers Squibb, Calithera, Clovis, Cybrexa, EMD Serono, Ignyta, Jansen, Merck, Pfizer, Roche, Seattle Genetics, and Vertex Pharmaceuticals.

E Hamilton has received consulting fees paid to institution only (no personal fees) from Pfizer, Genentech/Roche, Eli Lilly and Company (Lilly), Puma Biotechnology, Daiichi Sankyo, Mersana Therapeutics, Boehringer Ingelheim, AstraZeneca, Novartis, Silverback Therapeutics, and Black Diamond; and research/clinical trial support paid to institution only (no personal fees) from AstraZeneca, Hutchinson MediPharma, OncoMed, MedImmune, StemCentrx, Genentech/Roche, Curis, Verastem, Zymeworks, Syndax, Lycera, Rgenix, Novartis, Mersana, Millenium, TapImmune, Cascadian, Lilly, BerGenBio, Medivation, Pfizer, Tesaro, Boehringer Ingelheim, Eisai, H3 Biomedicine, Radius Health, Acerta, Takeda, Macrogenics, AbbVie, Immunomedics, FujiFilm, Effector, Merus, Nucana, Regeneron, Leap Therapeutics, Taiho Pharmaceutical, EMD Serono, Daiichi Sankyo, ArQule, Syros, Clovis, Cytomx, InventisBio, Deciphera, Unum Therapeutics, Sermonix Pharmaceuticals, Sutro, Aravive, Zenith Epigenetics, Arvinas, Torque, Harpoon, Fochon, Black Diamond, Orinove, Molecular Templates, and Silverback Therapeutics.

HS Rugo has received research support to the University of California San Francisco from Eisai, Genentech, Lilly, MacroGenics, Merck, Novartis, OBI Pharma, Odonate Therapeutics, Immunomedics, Daiichi-Sankyo, Daichi, Sermonix, and Pfizer; has served in a consulting role with Samsung and Puma; and has received travel support from Pfizer, Novartis, AstraZeneca, and Roche.

JW Goldman has received research grants from Pfizer and Lilly, and consultant fees from Pfizer.

S Dann is an employee of Pfizer and holds stock in Pfizer.

F Liu is an employee of Pfizer and holds stock in Pfizer.

GY Wong is a former employee of Pfizer and holds stock in Pfizer; is employee of BeiGene and holds stock in BeiGene.

H Krupka is a former employee of Pfizer and holds stock in Pfizer.

GI Shapiro has received research funding from Lilly, Merck KGaA/EMD-Serono, Merck, and Sierra Oncology; served on advisory boards for Pfizer, Lilly, G1 Therapeutics, Roche, Merck KGaA/EMD-Serono, Sierra Oncology, Bicycle Therapeutics, Fusion Pharmaceuticals, Cybrexa Therapeutics, Astex, Almac, Ipsen, Bayer, Angiex, Daiichi Sankyo, Seattle Genetics, Boehringer Ingelheim, ImmunoMet, Asana, Artios, Atrin, and Concarlo Holdings; and holds a patent for ‘Dosage Regimen for Sapacitabine and Seliciclib,’ also issued to Cyclacel Pharmaceuticals, and a pending patent, ‘Compositions and Methods for Predicting Response and Resistance to CDK4/6 Inhibition,’ together with Liam Cornell.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Acknowledgments

We thank Janeen Azare PhD, Pfizer, for reviewing manuscript content.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by Pfizer. Medical writing support was provided by David Sunter PhD, CMPP of Engage Medical Affairs and was funded by Pfizer. This work was supported by National Institutes of Health awards P30 CA008748