Elicit: Mechanism of Abemaciclib in CDK4/6 Inhibition
Mechanism of Abemaciclib in CDK4/6 Inhibition
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May 5, 2026
How does abemaciclib inhibit CDK4/6 to block G1-to-S cell-cycle progression?
Abemaciclib competitively binds the ATP pocket of CDK4/6 to catalytically inhibit Rb phosphorylation while simultaneously displacing p21 from CDK4 complexes to indirectly inhibit CDK2, collectively blocking G1-to-S cell cycle progression.
Abstract
Abemaciclib blocks G1-to-S cell cycle progression through multiple complementary mechanisms operating at different timescales. The drug functions as an ATP-competitive, reversible inhibitor with 13-15 fold selectivity for CDK4 over CDK6. Crystal structure analysis revealed direct binding to the ATP pocket through hydrogen bonds with the hinge region and key residues. Mechanistically, abemaciclib both catalytically inhibits CDK4/6 kinase activity and non-catalytically displaces p21 from CDK4 complexes, with the displaced p21 redistributing to CDK2 complexes to provide immediate indirect CDK2 inhibition. This dual mechanism prevents Rb phosphorylation, blocking release of E2F transcription factors necessary for S phase entry. The inhibition of Rb phosphorylation occurs within 16 hours and manifests as G1 arrest with increased 2n cell populations by flow cytometry.
Beyond transient cell cycle arrest, prolonged continuous treatment induces irreversible cellular outcomes including senescence (marked by β-galactosidase accumulation and heterochromatin foci) and apoptosis, with effects becoming significant by day 9 of treatment. Abemaciclib also triggers a unique form of cell death characterized by lysosomal dysfunction and cytoplasmic vacuole formation. Sustained drug binding is critical, as rapid rebound of Rb phosphorylation occurs upon withdrawal, necessitating continuous dosing to maintain pathway inhibition and achieve terminal cellular outcomes. Abemaciclib demonstrates broader polypharmacology than other CDK4/6 inhibitors, including inhibition of CDK2, CDK1, and GSK3β, which may contribute to its ability to restore sensitivity in palbociclib-resistant models and its distinct clinical profile permitting continuous rather than intermittent dosing.
Methods
We analyzed 10 sources from an initial pool of 200, using 8 screening criteria. Each paper was reviewed for 6 key aspects that mattered most to the research question. More on methods
Records from Elicit search
n = 200
Papers screened using: Abemaciclib Focus, CDK4/6 Pathway Investigation, Mechanistic Outcomes, Experimental Study Design, Appropriate Controls, Study Type, Mechanistic Data Inclusion, Publication Type
n = 200
Papers screened out
n = 190
Papers included for extraction
n = 10
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Paper search
We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.
We ran this query: “How does abemaciclib inhibit CDK4/6 to block G1-to-S cell-cycle progression?”
The search returned 200 total results from Elicit.
We retrieved 200 papers most relevant to the query for screening.
Screening
We screened in sources based on their abstracts that met these criteria:
- Abemaciclib Focus: Does this study investigate abemaciclib as the primary intervention (either alone or in comparison with other CDK4/6 inhibitors)?
- CDK4/6 Pathway Investigation: Does this study examine CDK4/6 pathway inhibition or cell cycle effects related to G1-to-S progression?
- Mechanistic Outcomes: Does this study report molecular, biochemical, or cellular outcomes related to cell cycle progression mechanisms?
- Experimental Study Design: Is this an in vitro, in vivo, or ex vivo experimental study with appropriate mechanistic design?
- Appropriate Controls: Does this study include appropriate controls (untreated, vehicle-treated, or comparator groups)?
- Study Type: Is this an original research article, systematic review, or meta-analysis with primary mechanistic data?
- Mechanistic Data Inclusion: Does this study include mechanistic data beyond solely clinical efficacy or safety outcomes?
- Publication Type: Is this study a full publication (not a conference abstract, case report, case series, editorial, or commentary without original mechanistic data)?
We considered all screening questions together and made a holistic judgement about whether to screen in each paper.
Data extraction
We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.
- CDK4/6 Binding Mechanism:
Extract detailed information about how abemaciclib interacts with CDK4 and/or CDK6 kinases, including:
Binding mode or mechanism (competitive, non-competitive, allosteric)
CDK4 vs CDK6 selectivity with specific data (IC50 values, fold differences)
Binding affinity or potency measurements
Structural insights (crystal structures, binding sites, key residues)
ATP competition data
Any comparison of binding characteristics vs other CDK4/6 inhibitors
G1-S Transition Blockade:
Extract specific details about how abemaciclib blocks G1-to-S cell cycle progression, including:
Mechanism of G1 arrest (cell cycle checkpoint activation, duration)
Effects on CDK4/6-cyclin D complex formation or activity
Impact on Rb phosphorylation and E2F transcription factors
Cell cycle phase distribution changes (flow cytometry data)
Timeline of cell cycle arrest onset
Reversibility or persistence of G1 block
Downstream Signaling Effects:
Extract information about cellular signaling pathways affected by abemaciclib’s CDK4/6 inhibition, including:
Changes in downstream effector proteins (p21, p27, Rb protein family)
mTOR pathway modulation
Transcriptional changes related to cell cycle control
Effects on DNA synthesis and replication machinery
Any pathway reactivation or compensatory mechanisms observed
Biomarkers of CDK4/6 inhibition (pT172 CDK4, etc.)
Cellular Consequences:
Extract details about the ultimate cellular outcomes of abemaciclib treatment beyond cell cycle arrest, including:
Induction of senescence (markers, morphology, timing)
Apoptosis or cell death mechanisms
Autophagy effects or lysosomal dysfunction
Formation of cytoplasmic vacuoles or other morphological changes
Duration of treatment needed for different outcomes
Cell viability and proliferation effects over time
Mechanistic Distinctions:
Extract information comparing abemaciclib’s mechanism to other CDK4/6 inhibitors (palbociclib, ribociclib), including:
Differences in CDK4 vs CDK6 selectivity
Unique binding characteristics or structural interactions
Differences in cellular outcomes or potency
Continuous vs intermittent dosing effects on mechanism
Distinct effects on normal vs cancer cells
Any mechanistic advantages or unique properties of abemaciclib
Experimental Methods:
Extract details about experimental approaches used to study abemaciclib’s mechanism of CDK4/6 inhibition, including:
- Cell-free kinase assays and biochemical methods
- Cell lines or models used (cancer types, normal cells)
- Structural biology techniques (X-ray crystallography, NMR)
- Cell cycle analysis methods (flow cytometry, microscopy)
- Concentrations or doses tested
- Duration of treatment in mechanistic studies
Results
Characteristics of Included Studies
Study
Full text retrieved?
Study focus
Cell lines/models
Methods
Torres-Guzmán et al., 2017
Yes
Preclinical characterization of abemaciclib mechanisms
MCF7, T-47D, ZR-75-1, EFM-19, MDA-MB-361 (ER+ breast cancer)
Biochemical assays, flow cytometry, xenograft models
Schaer et al., 2018
Yes
Immune microenvironment effects and anti-PD-L1 combination
CT26, MC38, EMT6 (murine); MCF-7, MDA-MB-468 (human)
Gene expression profiling, syngeneic tumor models
Gharbi et al., 2022
Yes
Crystal structure and mechanism of active CDK4-cyclin D complex
T47D, KPL1 breast cancer cells
X-ray crystallography, cell-free kinase assays, high-content imaging
Knudsen et al., 2017
Yes
Biological specificity and dose-response relationship
MCF7, T47D, MB231, MB468, BT549, AW23 (breast cancer)
Gene expression analysis, BrdU ELISA, RB gene editing
Cousins et al., 2017
No
Competitive kinase enrichment proteomics
Not specified
Multiplexed inhibitor bead mass spectrometry (MIB/MS)
Torres-Guzmán et al., 2021
No
Pharmacological properties and CDK4 selectivity
Breast cancer cell lines, palbociclib-resistant models, bone marrow progenitor cells
Cell-free assays, high-content imaging, flow cytometry
Torres-Guzmán et al., 2022
Yes
Continuous treatment effects on proliferation inhibition
Panel of 37 breast cancer cell lines
TR-FRET, FB assays, flow cytometry, high-content imaging
Hafner et al., 2019
Yes
Multiomics profiling and polypharmacology
Seven breast cancer cell lines, MCF7 cells
mRNA sequencing, phosphoproteomics, KINOMEscan, immunofluorescence
Pack et al., 2021
Yes
Non-catalytic p21 displacement from CDK4 complexes
MCF-10A, T-47D cells
FRAP, live-cell imaging, IP-kinase assays
Hino et al., 2020
No
Atypical cell death with lysosomal dysfunction
A549 non-small cell lung cancer cells
Live-cell imaging, transmission electron microscopy, cycloheximide chase assay
toof
Pageof
The included studies employed diverse experimental approaches to characterize abemaciclib’s mechanism of action. Seven of ten studies had full-text availability. The studies predominantly used breast cancer cell lines, reflecting abemaciclib’s primary clinical indication in hormone receptor-positive breast cancer. Experimental methods ranged from biochemical kinase assays and structural biology to advanced proteomics and live-cell imaging approaches. Three studies were conference abstracts or had limited availability, potentially constraining the depth of mechanistic information extractable from those sources.
CDK4/6 Binding Mechanism and Selectivity
Abemaciclib functions as an ATP-competitive, reversible inhibitor of CDK4 and CDK6. The first crystal structure of active CDK4-cyclin D3 bound to abemaciclib revealed direct hydrogen bonds between the pyridyl group and H96 in the hinge region, as well as between the benzimidazole ring’s free amine and K35. This structural characterization confirmed that abemaciclib binds to the ATP pocket regardless of CDK4 T172 phosphorylation status.
A defining feature of abemaciclib is its selectivity for CDK4 over CDK6. Biochemical studies reported Ki ATP values of 0.6 nmol/L for CDK4/cyclin D1 compared to 8.2 nmol/L for CDK6/cyclin D3, representing 14-fold higher potency for CDK4. Additional studies confirmed this preferential CDK4 selectivity, with reported selectivity ratios of 13-fold to 15-fold. Cell-free assays demonstrated IC50 values of 0.94 nM for CDK4/Cyclin D1 versus 14 nM for CDK6/CyclinD3. This selectivity manifested functionally, as abemaciclib preferentially inhibited proliferation of cells dependent on CDK4 rather than CDK6.
Interestingly, one study using multiomics profiling reported that the IC50 for CDK4 was approximately 2.5-fold greater than for CDK6, suggesting slightly greater potency toward CDK6. This discrepancy may reflect differences in assay conditions or complex composition, highlighting the importance of experimental context in measuring kinase selectivity.
Abemaciclib exhibited broader kinase inhibition beyond CDK4/6. Competitive proteomics revealed 83 target kinases, with GSK3α/β and CAMKIIδ/γ among the most potently inhibited at low nanomolar concentrations. This polypharmacology distinguishes abemaciclib from other CDK4/6 inhibitors and may contribute to its unique clinical profile.
G1-S Transition Blockade
Abemaciclib blocks G1-to-S cell cycle progression primarily through inhibition of CDK4/6-mediated Rb phosphorylation. Treatment of ER+ breast cancer cells led to decreased phosphorylation of Rb at Ser780 within 16 hours. This inhibition of Rb phosphorylation prevents release of E2F transcription factors, which are necessary for S phase entry.
The mechanism proved RB-dependent at clinically achievable concentrations, with potent suppression of RB/E2F-regulated genes associated with cell cycle progression. Gene expression analysis revealed downregulation of genes facilitating G1 to S phase progression, including Mcm7, Rrm2, Topo2a, Cdk2, Cdk4, Ccna2, Ccnb1, Aura, and Mki67. Flow cytometry demonstrated increased accumulation of cells in the 2n (G1) subpopulation and decreased 4n (G2/M) subpopulation, confirming G1 arrest.
A mechanistically important finding was that abemaciclib stabilizes the primed (pT172) CDK4-cyclin D complex while selectively displacing p21 from CDK4 but not CDK6 complexes. This p21 displacement occurred immediately upon treatment with all three clinical CDK4/6 inhibitors (abemaciclib, palbociclib, ribociclib). The displaced p21 redistributed to CDK2 complexes, providing a non-catalytic mechanism for indirect CDK2 inhibition. This dual mechanism—direct catalytic inhibition of CDK4/6 plus non-catalytic inhibition of CDK2 via p21 redistribution—enabled rapid blockade of cell cycle entry.
The persistence of G1 arrest following abemaciclib withdrawal varied. Some studies reported durable inhibition of pRb and TopoIIα expression maintained after drug removal, while others demonstrated rapid rebound of Rb phosphorylation and CDK4 signaling upon abemaciclib withdrawal. This discrepancy suggests that sustained treatment is necessary for prolonged cell cycle inhibition.
Downstream Signaling Effects
Beyond direct CDK4/6 inhibition, abemaciclib triggered multiple downstream signaling alterations. Transcriptional changes included suppression of E2F-dependent genes and increased expression of D cyclins (Ccnd2, Ccnd3). The inhibition of Rb phosphorylation served as a key biomarker of CDK4/6 inhibition, though pT172 CDK4 phosphorylation was maintained despite pathway inhibition.
The displacement of p21 from CDK4-cyclin D complexes represented a critical non-catalytic mechanism affecting downstream CDK2 activity. However, this displacement was selective for CDK4 over CDK6 complexes, and involved p21 but not p27 redistribution.
One study reported mTORC1 inhibition following abemaciclib treatment, though this appeared linked to lysosomal dysfunction rather than direct pathway modulation. Effects on DNA synthesis and replication machinery included decreased DNA synthesis and inhibition of genes involved in S phase and mitosis through effects on CDK1 and CDK2.
Pathway reactivation emerged as a concern. Rapid rebound of Rb phosphorylation occurred upon abemaciclib withdrawal, with stabilization of active CDK4-cyclin D1 complex potentially leading to pathway reactivation following alternate dosing regimens. This finding emphasized the importance of sustained drug exposure for maintaining pathway inhibition.
Cellular Consequences Beyond Cell Cycle Arrest
Prolonged abemaciclib treatment induced cellular senescence, evidenced by β-galactosidase accumulation, senescence-associated heterochromatin foci formation, and decreased FOXM1-positive cells. Morphological changes included cells becoming larger and flatter. The timing of senescence induction varied with concentration and treatment duration, with β-gal staining increasing over 4, 7, and 10 days. Sustained inhibition of cell proliferation and maintained Rb phosphorylation inhibition after compound removal supported senescence induction.
Apoptosis occurred following continuous abemaciclib exposure, with dose-dependent increases in annexin V and TUNEL staining. Time course experiments showed early apoptotic effects at Days 2 and 6, becoming significant by Day 9. Continuous treatment promoted greater apoptosis compared to intermittent dosing. At higher concentrations (0.3 mM and above), abemaciclib induced cell death indicated by negative growth rate values.
A unique finding was abemaciclib’s induction of atypical cell death characterized by formation of multiple cytoplasmic vacuoles derived from swollen, dysfunctional lysosomes. This phenotype differed from classical apoptosis or necroptosis. Live-cell imaging and transmission electron microscopy revealed vacuoles containing undigested debris and organellar remnants. Abemaciclib blocked autophagic flux, leading to autophagosome accumulation, and caused partial lysosomal membrane permeabilization and mTORC1 inhibition. Inhibitors of vacuolar-type ATPase (V-ATPase) such as bafilomycin A1 prevented both vacuole formation and cell death.
Notably, at doses of 250 nM to 1 µM, abemaciclib induced cell death in RB-deficient cell lines accompanied by a multi-vacuolar phenotype indicative of lysosomal membrane permeabilization. This effect occurred within 6 hours and could be ameliorated with chloroquine. This RB-independent cytotoxicity was not observed with palbociclib and likely reflected off-target inhibition of casein, DYRK, and HIPK kinases.
Mechanistic Distinctions from Other CDK4/6 Inhibitors
Abemaciclib demonstrated several mechanistic differences from palbociclib and ribociclib. While all three inhibitors are ATP-competitive and reversible, abemaciclib showed greater CDK4 selectivity with 13-15 fold preference for CDK4 over CDK6, compared to more balanced inhibition by palbociclib and ribociclib.
Cellular potency differed substantially. In a panel of 37 breast cancer cell lines, abemaciclib demonstrated higher average potency than palbociclib or ribociclib. Multiomics profiling revealed that abemaciclib was the most potent inhibitor of CDK4 and CDK6, with broader inhibitory activities against additional kinases including CDK2/cyclin A/E and CDK1/cyclin B—kinases implicated in resistance to CDK4/6 inhibition. This polypharmacology enabled abemaciclib to cause cell cycle arrest in both G1 and G2 phases at higher concentrations.
In cellular outcomes, abemaciclib induced senescence and apoptosis earlier and at lower concentrations compared to palbociclib and ribociclib. It demonstrated the most potent cytotoxic effect among the three inhibitors and proved more efficacious in inhibiting and killing pRb-proficient cells. The unique vacuole-forming cell death phenotype was specific to abemaciclib.
Effects on normal cells diverged notably. Abemaciclib showed lesser impact on myeloid maturation in human bone marrow progenitor cells compared to palbociclib and ribociclib when tested at clinically relevant unbound concentrations. This differential toxicity profile enabled continuous dosing of abemaciclib (versus the intermittent dosing required for palbociclib and ribociclib), with the continuous dosing schedule potentially contributing to its unique mechanism and sustained pathway inhibition.
Abemaciclib possessed unique off-target activities. Unlike palbociclib or ribociclib, it directly inhibited GSK3α/β and CAMKIIγ/δ at low nanomolar concentrations, and potently activated β-catenin-dependent WNT signaling. In palbociclib-resistant cell lines, abemaciclib combined with fulvestrant restored CDK4/6 sensitivity, leading to senescence and cell death, suggesting potential mechanistic advantages in overcoming resistance.
All three inhibitors shared the ability to displace p21 from CDK4 complexes, though differences in their broader mechanistic profiles suggested they may not be entirely interchangeable clinically.
Synthesis
The mechanistic characterization of abemaciclib reveals a complex inhibitor with both on-target CDK4/6 effects and polypharmacological properties. Several apparent contradictions in the literature require reconciliation.
CDK4 vs CDK6 Selectivity
Most studies consistently reported 13-15 fold selectivity for CDK4 over CDK6 in cell-free assays, while one multiomics study found 2.5-fold greater potency toward CDK6. This discrepancy likely reflects differences in assay methodology. The cell-free biochemical assays measured inhibition of isolated CDK4-cyclin D1 versus CDK6-cyclin D3 complexes, whereas the multiomics study may have assessed inhibition in more complex cellular contexts. Both findings may be correct within their respective experimental systems. The functional consequence—preferential inhibition of CDK4-dependent versus CDK6-dependent cell proliferation—supports meaningful CDK4 selectivity in relevant biological contexts.
RB-Dependent vs RB-Independent Effects
At clinically relevant concentrations (low nanomolar to ~250 nM), abemaciclib’s antiproliferative activity was strictly RB-dependent. However, at higher concentrations (250 nM - 1 µM), RB-independent cell death occurred through lysosomal membrane permeabilization. This concentration-dependent mechanism explains why abemaciclib can induce cell death in RB-deficient lines at suprapharmacological doses while functioning as a cytostatic agent at therapeutic concentrations. The RB-independent effects likely reflect off-target inhibition of casein, DYRK, and HIPK kinases, representing a distinct mechanism from canonical CDK4/6 inhibition.
Persistence of Cell Cycle Arrest
Studies diverged on whether G1 arrest persisted after drug withdrawal. Some reported durable inhibition maintained after compound removal, while others showed rapid rebound of Rb phosphorylation upon withdrawal. This apparent contradiction resolves through consideration of treatment duration and dosing schedules. Prolonged continuous treatment (the clinically approved regimen for abemaciclib) triggered senescence and apoptosis—irreversible cellular states that would persist after drug removal. In contrast, brief treatment followed by immediate washout allowed rapid pathway reactivation. The divergent findings thus reflect different experimental paradigms: durable effects required sustained treatment to induce terminal outcomes (senescence/apoptosis), while transient cell cycle arrest reversed quickly without such prolonged exposure.
Continuous vs Intermittent Dosing
The importance of continuous dosing emerged as a consistent theme. Sustained abemaciclib binding prevented rebound activation of downstream signaling, and continuous treatment promoted greater apoptosis and senescence compared to intermittent exposure. The lower impact on neutrophil maturation enabled this continuous dosing schedule, creating a therapeutic advantage. This contrasts with palbociclib and ribociclib, which require intermittent dosing due to myelotoxicity. The mechanistic implication is that abemaciclib’s full therapeutic effect—including induction of senescence and apoptosis beyond simple cytostasis—requires sustained CDK4/6 inhibition achievable only through continuous dosing.
Polypharmacology and Clinical Differentiation
Abemaciclib’s broader kinase inhibition profile—including CDK2, CDK1, GSK3β, and others—represents both a mechanistic distinction and potential clinical advantage. Inhibition of CDK2/cyclin E may overcome resistance mechanisms related to cyclin E amplification, while effects on CDK1 contribute to cell killing and potentially enhance tumor immunogenicity. The activation of WNT signaling through GSK3β inhibition adds another layer of complexity whose clinical significance remains unclear. However, the ability to restore sensitivity in palbociclib-resistant models suggests this polypharmacology may be therapeutically beneficial rather than merely representing off-target toxicity.
In summary, abemaciclib functions through multiple mechanisms operating at different concentration ranges and time scales: (1) immediate p21 displacement from CDK4 complexes enabling rapid CDK2 inhibition; (2) sustained catalytic inhibition of CDK4 (and to lesser extent CDK6) blocking Rb phosphorylation; (3) prolonged treatment inducing irreversible senescence and apoptosis; and (4) at higher concentrations, polypharmacological effects including lysosomal dysfunction. The continuous dosing schedule enabled by its myeloid-sparing profile allows sustained pathway inhibition necessary for optimal efficacy.
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Preclinical characterization of abemaciclib in hormone receptor positive breast cancer
Raquel Torres-Guzmán, Bruna Calsina, Ana Hermoso, Carmen Baquero, Beatriz Álvarez, Joaquín Amat, A. Mcnulty, Xueqian Gong, K. Boehnke, Jian Du, A. de Dios, R. Beckmann, S. Buchanan, M. Lallena
OncoTarget·
2017·
168 citations
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CDK4/6 Binding Mechanism
- Binding mode or mechanism: Competitive inhibition implied by Ki ATP values. - CDK4 vs CDK6 selectivity: 14-fold higher potency for CDK4/cyclin D1 over CDK6/cyclin D3. - Binding affinity or potency measurements: Ki ATP = 0.6 nmol/L for CDK4/cyclin D1, Ki ATP = 8.2 nmol/L for CDK6/cyclin D3. - Structural insights: Not mentioned. - ATP competition data: Not explicitly mentioned. - Comparison with other CDK4/6 inhibitors: Not mentioned.
G1-S Transition Blockade
- Mechanism of G1 arrest: Inhibition of CDK4 and CDK6 activity, leading to decreased Rb phosphorylation and cell cycle checkpoint activation. - Effects on CDK4/6-cyclin D complex formation or activity: Abemaciclib inhibits CDK4/ cyclin D1 complexes with a Ki ATP = 0.6 nmol/L ± 0.3 nmol/L and CDK6/cyclin D3 complexes with a Ki ATP = 8.2 ± 1.1 nmol/L. - Impact on Rb phosphorylation and E2F transcription factors: Decrease in Rb phosphorylation prevents E2F release, blocking cell cycle progression. - Cell cycle phase distribution changes: Increase in 2n subpopulation and decrease in 4n subpopulation, indicating G1 arrest. - Timeline of cell cycle arrest onset: Within 16 hours of treatment. - Reversibility or persistence of G1 block: Persistent even after compound removal.
Downstream Signaling Effects
- Changes in downstream effector proteins: Decrease in phosphorylation of Rb, leading to G1 arrest and decreased cell proliferation. - mTOR pathway modulation: Implicated through Rb's role in modulating nutrient availability and energy production. - Transcriptional changes related to cell cycle control: Inhibition of E2F-dependent genes. - Effects on DNA synthesis and replication machinery: Decrease in DNA synthesis. - Pathway reactivation or compensatory mechanisms: Induction of senescence and apoptosis. - Biomarkers of CDK4/6 inhibition: Decrease in pRb and TopoIIα expression.
Cellular Consequences
- Induction of senescence: Markers include β-galactosidase accumulation and senescence-associated heterochromatin foci. Morphological changes include cells becoming larger and flatter. Timing varies with concentration and duration. - Apoptosis or cell death mechanisms: Indicated by dose-dependent increases in annexin V and TUNEL staining. - Autophagy effects or lysosomal dysfunction: Not mentioned. - Formation of cytoplasmic vacuoles or other morphological changes: Cells become larger and flatter. - Duration of treatment needed for different outcomes: Senescence and apoptosis observed after continuous exposure. - Cell viability and proliferation effects over time: Decrease in cell proliferation and increase in cell death with longer treatment duration.
Mechanistic Distinctions
- Differences in CDK4 vs CDK6 selectivity: Abemaciclib shows a 14-fold specificity for CDK4/cyclin D1 over CDK6/cyclin D3 complexes. - Unique binding characteristics or structural interactions: Not explicitly mentioned in the paper. - Differences in cellular outcomes or potency: Abemaciclib induces senescence and apoptosis earlier and at lower concentrations compared to palbociclib and ribociclib. - Continuous vs intermittent dosing effects on mechanism: Continuous treatment with abemaciclib is essential for sustained inhibition of CDK4/6 and optimal efficacy. - Distinct effects on normal vs cancer cells: Not explicitly mentioned in the paper. - Mechanistic advantages or unique properties: Abemaciclib's sustained effects on cell cycle arrest and metabolism, as well as its ability to induce senescence and apoptosis at lower concentrations, are unique properties.
Experimental Methods
- Cell-free kinase assays and biochemical methods: Abemaciclib inhibits CDK4 and CDK6 activity with specificity over other CDKs in vitro. Ki ATP values for CDK4/cyclin D1 and CDK6/cyclin D3 complexes were determined. - Cell lines or models used: MCF7, T-47D, ZR-75-1, EFM-19, MDA-MB-361 (ER+ breast cancer cell lines). - Structural biology techniques: Not mentioned. - Cell cycle analysis methods: Flow cytometry to assess cell cycle progression and senescence markers. - Concentrations or doses tested: 0.027 µM to 20 µM in cell-based assays; 50 to 75 mg/kg in vivo. - Duration of treatment: Varied from 24 hours to several days (e.g., 2 DT, 3 DT).
Abemaciclib is an ATP-competitive, reversible kinase inhibitor selective for CDK4 and CDK6 that has shown antitumor activity as a single agent in hormone receptor positive (HR+) metastatic breast cancer in clinical trials. Here, we examined the mechanistic effects of abemaciclib treatment using in vitro and in vivo breast cancer models. Treatment of estrogen receptor positive (ER+) breast cancer cells with abemaciclib alone led to a decrease in phosphorylation of Rb, arrest at G1, and a decrease in cell proliferation. Moreover, abemaciclib exposure led to durable inhibition of pRb, TopoIIα expression and DNA synthesis, which were maintained after drug removal. Treatment of ER+ breast cancer cells also led to a senescence response as indicated by accumulation of β-galactosidase, formation of senescence-associated heterochromatin foci, and a decrease in FOXM1 positive cells. Continuous exposure to abemaciclib altered breast cancer cell metabolism and induced apoptosis. In a xenograft model of ER+ breast cancer, abemaciclib monotherapy caused regression of tumor growth. Overall these data indicate that abemaciclib is a CDK4 and CDK6 inhibitor that, as a single agent, blocks breast cancer cell progression, and upon longer treatment can lead to sustained antitumor effects through the induction of senescence, apoptosis, and alteration of cellular metabolism.
INTRODUCTION
Components of the cyclin-dependent kinase 4 (CDK4)/CDK6/Cyclin D/retinoblastoma (Rb)/p16INK4a pathway function in multiple processes implicated in cancer, including cell proliferation, senescence, apoptosis, and cellular metabolism [1][2][3][4]. The catalytic proteins CDK4 and CDK6, work with their activating partners, D type cyclins, to phosphorylate the Rb tumor suppressor protein. This phosphorylation of Rb by CDK4/CDK6/ Cyclin D, and subsequently by CDK2/Cyclin E, leads to the dissociation of the transcription factor E2F, and release of transcriptional repression of genes important for DNA synthesis and S phase progression [3,5].
Evidence from breast cancer mouse models suggests that Cyclin D1, and CDK4 and CDK6 activation contribute to suppressing senescence, as Cyclin D1 ablation in these models after tumor formation led to an onset of senescence in the isolated tumor cells, as did treatment with a CDK4 and CDK6 inhibitor [6]. In KRAS G12V NSCLC mouse models, conditional knockout of CDK4, but not of CDK2 or CDK6, led to senescence in lung cells, suggesting that certain cell types with particular genetic alterations may be reliant on a specific CDK family member to carry out selected cellular functions [7]. At least one of the downstream mechanisms by which CDK4 and CDK6 suppress senescence is by activating and stabilizing the forkhead transcription factor, FOXM1, through multi-site phosphorylation [8].
Research Paper
Rb also has both pro-apoptotic and anti-apoptotic roles, depending on the cellular context and the balance between Rb activity and the relative activity of other apoptotic or proliferation-related proteins in the cell [1]. The Rb family has also been implicated in glutamine synthesis and mitochondrial function; thus, one additional avenue through which the CDK4/CDK6/Cyclin D/Rb/ p16INK4a pathway can control proliferation may be by modulating nutrient availability and the production of cellular energy [2].
The direct contributions of mutations or alterations in this pathway to tumor establishment and/or maintenance are supported by several studies [6,[9][10][11]. Moreover, evidence suggests that certain tumor types may depend on specific CDK family members for establishment and/ or maintenance of tumorigenesis. For example, in mouse models of breast cancers driven by the HER2/neu oncogene, loss of CDK4 activity is sufficient to inhibit the formation and proliferation of tumors [10,12]. Together the available preclinical and emerging clinical data suggest that CDK4 and CDK6 are promising targets for the development of anticancer drugs [13][14][15].
Abemaciclib (LY2835219) is a small molecule that inhibits CDK4 and CDK6 activity with marked specificity over other CDKs in vitro and induces G1 arrest in Rb-proficient cells [16]. In a Phase 1 clinical study (NCT01394016), abemaciclib as single agent had a safety profile which enabled dosing on a continuous schedule, and responses were observed in previously treated patients with HR + metastatic breast cancer (MBC), NSCLC, and melanoma; in addition, confirmation of antitumor activity of abemaciclib as a single agent in HR+ HER2-MBC has been demonstrated in a phase 2 trial [17,18].
Here we report a mechanistic exploration of the effects of abemaciclib on breast cancer cells. We demonstrate that abemaciclib inhibits Rb phosphorylation and arrests cells in G1 both in vitro and in murine models bearing human ER+ breast cancer xenografts. We further show that prolonged and continuous exposure to abemaciclib is accompanied by altered cell metabolism and a substantial increase in markers of senescence and apoptosis in ER+ breast cancer.
Abemaciclib is a potent inhibitor of CDK4 and CDK6 that inhibits proliferation of ER+ breast cancer cells
Abemaciclib (LY2835219) is an orally available small molecule pyrimidine-benzimidazole inhibitor discovered at Eli Lilly and Company [16]. In biochemical assays, abemaciclib inhibits the kinase activity of CDK4/ cyclin D1 complexes with a Ki ATP = 0.6 nmol/L ± 0.3 nmol/L [16] and of CDK6/cyclin D3 complexes with a Ki ATP = 8.2 ± 1.1 nmol/L, indicating that in in vitro cell-free assays, abemaciclib shows specificity of approximately 14fold for CDK4/cyclin D1 over CDK6/cyclin D3 complexes (Figure 1A ). Results of enzymatic profiling experiments comparing the inhibitory activity of abemaciclib on several other kinases confirmed its selectivity for CDK4 and CDK6 complexes over other kinases, including CDK9/ cyclin T1 complexes (Figure 1A ) [16].
To test if abemaciclib also specifically inhibited the CDK4 and CDK6 pathways in cells, we treated MCF7, T-47D, and ZR-75-1 cells for 24 hours with concentrations of abemaciclib ranging from 0.027 µM to 20 µM. Consistent with the mechanism of action of abemaciclib as a CDK4 and CDK6 inhibitor [16], we observed a decrease in levels of phosphorylated Rb (pRb) at S807/811 (Figure 1B ). However, even at the highest concentration of abemaciclib, no changes were observed in levels of pCTD_ s2 or MCL1, indicating that CDK9-dependent processes were not affected (Figure 1B ), consistent with previous observations in U2OS cells [16]. Thus, biochemical and cellular data demonstrate that abemaciclib is a potent and selective inhibitor of CDK4 and CDK6.
Since levels of pRb decreased in ER+ breast cancer lines treated with abemaciclib, and early phase clinical data suggested preferential antitumor activity in patients with HR+ disease [17], we further examined the effects of abemaciclib in ER+ cell lines. A concentrationdependent decrease in viable cell number was observed in ER+ breast cancer cell lines treated with abemaciclib (Supplementary Table 2 ). Treatment with abemaciclib for 3 DT also led to a concentration-dependent decrease in BrdU incorporation (Figure 1C ), with an EC 50 of 0.178 µM ± 0.0655 in MCF7 cells (Figure 1D ). Ki-67 levels decreased in a concentration-dependent manner, in both EFM-19 and MCF7 cells after treatment with abemaciclib for 2 DT, with EC 50 s of 0.0167 µM ± 0.002 and 0.0421 µM ± 0.002, respectively (Figure 1E ). These results show that abemaciclib inhibits cellular proliferation in multiple cell lines.
Abemaciclib inhibited Rb phosphorylation and led to G1/S arrest in ER+ breast cancer cell lines
The effects of abemaciclib on Rb phosphorylation and the cell cycle were assessed in vitro in the luminal ER+ breast cancer cell lines EFM-19 and MDA-MB-361. Treatment of these cell lines with abemaciclib for 16 hours, led to a decrease in Rb phosphorylation at Ser 780, in a concentration-dependent manner (Figure 2A , 2B; Supplementary Table 2 ). Moreover, treatment of EFM-19 and MDA-MB-361 cells with abemaciclib for 2 DT led to an increase in the percentage of cells in the 2n subpopulation and a corresponding decrease in the percentage of cells in the 4n subpopulation, which correlated with the concentration-dependent inhibition of pRb (Figure 2A , 2B; Supplementary Table 2 ). Together, these results demonstrate that abemaciclib is a potent (A) Kinetic parameters (K i ATP ) and selectivity ratio of abemaciclib for cyclinD1/CDK4 and cyclinD3/CDK6 complexes in vitro. Data reported as average of two independent determinations (n = 2) ± standard deviation (SD) (B) ZR-75-1, T-47D and MCF7 cells were treated for 24 hours with DMSO vehicle or with 3-fold dilutions of either abemaciclib or flavopiridol starting at 20 µM and ending at 27 nM. Protein lysates were prepared from the cells and analyzed for the expression of various biomarkers by immunoblotting. The biomarkers included phosphor-Rb at serines 807/811,phosphorylation of serine 2 on the C-terminal repeat domain on RNA polymerase II (pCTD) and MCL1. Images of Western blots were cropped to denote the relevant band(s) for clarity. (C, D) MCF7 cells were incubated for 3DT with abemaciclib at the concentrations indicated in (C). % BrdU signal was normalized using a PI3K/mTOR inhibitor (BEZ235) as a positive control for inhibiton of cell proliferation and 0.2% DMSO as a negative control (C) and the corresponding IC 50 curve is shown in (D). (E) Ki-67 expression level in MCF7 and EFM-19 cells upon abemaciclib treatment at the indicated concentrations for 2DT. Ki-67 positive events were obtained by gating versus background signal. The percentage of Ki-67 positive cells was calculated among the total number of cells and is expressed as mean (n = 2 for samples treated with abemaciclib, n = 4 for untreated samples).
inhibitor of CDK4 and CDK6 that effectively inhibits the phosphorylation of Rb, leading to cell cycle arrest in G1 with a corresponding decrease in cell proliferation.
Moreover, the observed decreases in pRb and cell cycle progression, as monitored by levels of TopoIIα, were sustainable with continuous exposure of breast cancer cells (T-47D) to abemaciclib, with increased effects observed with longer initial treatment times of abemaciclib (Figure 2C ). DNA synthesis, as measured by EdU incorporation, was preferentially reduced in HR+ compared to HR-cell lines, and this inhibition was sustained even after short-term treatment with abemaciclib (Supplementary Figure 1B-1D ). HR-cell lines with RB1 mutation or loss including MDA-MB-468, BT549, BT20, HCC1937, HCC70 [21] [22][23] were insensitive to these effects of abemaciclib (Supplementary Figure 1B-1D ). Palbociclib treatment also led to a decrease in proliferation preferentially for HR+ cell lines; however, this effect was only observed after longer treatment times (Supplementary Figure 1B-1D ). Those data suggest that long term treatment with abemaciclib could lead to sustained response measured by pathway engagement (pRB, Topo IIα) and cell proliferation inhibition. Further experiments should be done to investigate the failure of cells to entry in cell cycle and re-gain proliferative ability. Abemaciclib induced senescence in ER+ breast cancer cell lines Since the CDK4/CDK6/CyclinD/Rb/p16INK4a pathway plays a role in preventing senescence, we explored whether treatment with abemaciclib could induce senescence. We observed that MCF7, MDA-MB-361 and EFM-19 cells treated for 3 DT with 0.5 µM abemaciclib were larger and flatter compared to vehicletreated cells in all three cell lines (Figure 3A , 3B and Supplementary Figure 2A-2D ). Moreover, morphological characteristics, as assessed by side scatter measurements using flow cytometry, indicated an increase in overall cellular complexity, consistent with a senescent phenotype (Figure 3C and Supplementary Figure 2E, 2F ).
Increased β-gal staining was also detected in MCF7 cells treated with 0.5 µM or 2 µM abemaciclib for 4,7 and 10 days, compared to cells treated with DMSO (Figure 3D -3F, Supplementary Figure 2G, 2H ). Overall, the percentage of cells with detectable β-gal increased with higher concentrations of abemaciclib such that at 2 µM, 55.4 +/-18.8% cells were β-gal positive (Supplementary Table 3 ). Notably, the increase in β-gal staining was apparent even after treatment duration as short as 4 hours with concentrations similar to those observed in plasma in humans (data not shown). Longer incubation times generally led to an increase in the percentage of β-gal positive cells observed, although at the highest concentration (2 µM) and longest incubation time (10 days), a decrease was observed (Figure 3D -3F, Supplementary Table 3 ), along with a concomitant reduction in viable cell number (data not shown).
Consistent with cells having entered a senescent state, a concentration-dependent reduction in FOXM1 positive EFM-19 cells (IC 50 =0.008 µM ± 0.005) was observed after 1 DT (Figure 3G ). These data are consistent with observations that a decrease in FOXM1 levels due to CDK4 and CDK6 inhibition is involved in promoting senescence [8]. We further assessed senescence-related chromatin changes in cells treated with abemaciclib, and observed higher levels of trimethylation of histone H3 at lysine 9 (H3K9me3) in MCF7 and EFM-19 cells after 2 DT of treatment with 500 nM abemaciclib compared to cells treated with 0.2% DMSO (vehicle), consistent with formation of senescence-associated heterochromatin foci (SAHF) (Figures 3H and Supplementary Figure 2I , Supplementary Table 4 ). This staining showed that vacuolization was increased during treatment with abemaciclib, demonstrating an additional feature of senescence. PI staining also indicated fewer viable cells in the samples treated with abemaciclib, again indicating an impact on cell viability (Figures 3H and Supplementary Figure 2I ). The increase in H3K9me3 in cells treated with abemaciclib was concentration-dependent up to 2 µM, achieving maximum levels in MCF7 cells at clinicallyachievable concentrations (Figure 3I , Supplementary Figure 2J ). In EFM-19 cells, abemaciclib led to increased expression of H3K9me3 up to 20 µM for this marker with a potency of 0.023 µM (Supplementary Figure 2J and Supplementary Table 4 ). These observations indicate that abemaciclib induces senescence, as signified by characteristic morphological changes, expression of β-gal, and increases in H3K9me3.
Programmed cell death in breast cancer cell lines treated with abemaciclib
The observations described above suggest that as the concentration and duration of abemaciclib treatment increased, an increase in cell death was occurring. Consistent with this observation, dose-dependent increases in annexin V staining were observed in MCF7 and MDA-MB-361 cells treated with abemaciclib for 3 DT (Figure 4 ). In untreated MCF7 cells, 11.1% of cells were positive for annexin V, whereas 41.4% and 66.7% of cells were positive for annexin V when treated with 0.5 µM and 2 µM of abemaciclib, respectively (Figure 4A, 4C ). Likewise, in untreated MDA-MB-361 cells, 45.7% were annexin V-positive, compared with 84.6% and 88.8% upon 3 DT of treatment with 0.5 µM and 2 µM abemaciclib, respectively (Figure 4B, 4D ).
Next, the fragmentation of DNA ends was assessed using TUNEL staining, concurrently with senescence and total viable cell number A decrease in cell proliferation concurrent with an increase in percentages of β-gal positive and TUNEL positive cells was observed in MCF7 cells treated with abemaciclib in a time and concentrationdependent manner, with the exception that β-gal levels decreased at the highest concentration of abemaciclib at 5 DT in MCF7 cells (Figure 5A ). Under the conditions tested, a similar decrease in viable cell number was observed with treatment of palbociclib compared with abemaciclib; however, the percentage of cells with β-gal staining was lower for palbociclib (as well as ribociclib) than for abemaciclib (Figure 5 ). An increase in TUNEL staining was observed by 2DT for cells treated with abemaciclib, while only minor increases in TUNEL staining were observed and only after longer treatment times in cells treated with palbociclib and ribociclib (Figure 5 ). Taken together, the data suggest that abemaciclib leads to the cell cycle arrest of ER+ breast cancer cells, and which becomes irreversible to the induction of senescence and apoptosis.
Abemaciclib promoted metabolic alterations in metastatic HR+ breast cancer cell lines
The growth of MCF7 cells was decreased under glutamine starvation, an effect that was enhanced upon concurrent treatment with abemaciclib (Figure 6A -6D, Supplementary Figure 4 ). Metabolic profiling was carried out in MCF7 cells treated with abemaciclib at 0.5 and 2 µM, over a time course, to profile glutamate and several additional metabolites including aspartate, citrate, isocitrate, α-ketoglutarate, succinate, fumarate, malate, lactate and pyruvate (Figure 6E and Supplementary Tables 1, 5 ). The level of metabolites began decreasing at 1 DT and 2 DT (data not shown), and became more pronounced at 3 DT. A drop in the concentration of the metabolites α-ketoglutarate, fumarate and malate was observed after treatment with 0.5 µM abemaciclib in relation to vehicle-treated cells, and a minor decrease in succinate concentration was also observed, which may be related to alterations in mitochondrial function (Figure 6E and Supplementary Table 5 ). Α concentration-dependent decrease in α-ketoglutarate was observed at abemaciclib concentrations ranging from 20 µM to 32 nM (Supplementary Table 5 ).
Antitumor activity of abemaciclib in vivo
The in vivo anti-tumor activity of abemaciclib was assessed in mice implanted with ZR-75-1 xenograft, a model for human luminal ER+ breast cancer. Dosedependent anti-tumor activity was observed following treatment with abemaciclib as single agent, with a measured reduction in tumor volume (regression) at the highest dose (75 mg/kg) (Figure 7A-7C ). Notably, the analysis of the tumors isolated from mice treated for 5 days showed that the inhibition of tumor growth correlated with inhibition of phospho-(Ser780/807/811)-Rb as well as the inhibition of cell cycle progression as indicated by the inhibition of the expression of Topo IIα and phospho-(Ser10)-H3 observed on Western blots (Figure 7D ). The lack of inhibition of either phosphorylation of serine 2 on the C-terminal domain (pCTD) repeat on RNA polymerase II or MCL1 expression, indicated that CDK9 was not inhibited at any dose of abemaciclib (Figure 7D ), thereby providing additional evidence for antitumor activity through inhibition of CDK4 and CDK6.
This specificity of abemaciclib for CDK4 and CDK6 is further supported by mRNA expression studies which showed that treatment with abemaciclib resulted in a significant inhibition of expression of various genes involved in cell cycle regulation (Supplementary Figure 3 ). In particular, treatment with either 50 or 75 mg/kg abemaciclib resulted in the suppression of the expression of several genes encoding for proteins required for progression through the S or G2/M phases of the cell cycle including Cyclin E, MCM7, CDKN2C (p18), PTEN, Aurora A (AURA), cyclin B1 (CCNB1), FOXM1, ribonucleotide reductase subunit M2 (RRM2), Ki67 (MKI67) and topoisomerase II-alpha (Topo IIα). Notably the transcription of these genes, with the exception of PTEN and AURA, are dependent upon E2F1, a transcription factor that is negatively regulated by Rb. Since Rb is the principal kinase target for CDK4 and CDK6, these results provide further evidence that cell cycle inhibition and subsequent inhibition of growth by abemaciclib resulted directly from the inhibition of these two kinases. Together, the data show that abemaciclib is a CDK4 and CDK6 inhibitor that demonstrates both in vitro and in vivo antitumor activity in breast cancer models.
DISCUSSION
Several CDK4 and CDK6 inhibitors are currently being studied in clinical trials, including abemaciclib, and FDA-approved palbociclib and ribociclib [13][14][15]24]. Here, we explored the detailed mechanisms of action of abemaciclib as a single agent in ER+ breast cancer cell lines and in vivo. In cell-free biochemical assays, abemaciclib potently inhibited the kinase activity of both CDK4 and CDK6, with a 14-fold higher potency for CDK4/cyclinD1 complexes than CDK6/cyclinD3. The difference in potencies between these CDK4 and CDK6 complexes with specific D-type cyclins may contribute, to the differential anti-proliferative activity of abemaciclib observed in cell lines derived from various tissue types where distinct CDKs or D-type cyclins may play a distinct role [25]. CDK4 and Cyclin D1 may be especially relevant for breast cancer, since evidence suggests that both are important oncogenic drivers for this disease [4,9,10,12,26]. As previously described, abemaciclib is specific for CDK4 and CDK6 [16], a result which is confirmed in this current study in breast cancer cell lines.
Treatment with abemaciclib inhibited cell proliferation and led to G1 cell cycle arrest in a concentration-dependent manner, concurrent with inhibition of Rb phosphorylation and of expression of the biomarkers Topo IIα and pHH3; after longer term treatment (more than 2 DT) These changes were maintained after compound removal, in cells treated with abemaciclib for at least 2DT. Consistent with these observations, longer treatment of breast cancer cells with abemaciclib was associated with a loss of replicative capacity, particularly for HR+ compared with HR-breast cancer cells. The higher anti-proliferative activity of abemaciclib in HR+ cells is consistent with known alterations in many of these cell lines, wherein CCND1 amplification indicates that HR+ breast cancers may be especially reliant on this pathway for tumorigenesis [27].
Treatment with abemaciclib induced a concentration-and time-dependent senescence response, as shown by multiple markers, including the formation of heterochromatin foci and β-gal expression. Additionally, apoptosis was observed in Rb-proficient breast cancer cell lines following prolonged treatment with abemaciclib, as indicated by annexin V and TUNEL. Promotion of apoptosis after treatment with CDK4 and CDK6 inhibitor has been described in leukemic cells [6]. Continuous, longterm treatment with abemaciclib also altered primary cell metabolism as indicated by the observed TCA metabolite profiles as well as increased cell death upon glutamine deprivation. Thus, abemaciclib treatment induced potent, stable inhibition of breast cancer proliferation, most likely through the induction of long-term sustained changes in cells including senescence and apoptosis.
Abemaciclib induced tumor regression in vivo in xenografts of HR+ breast cancer, with corresponding changes of molecular markers of cell cycle inhibition, including phosphorylation of Rb and TopoIIα expression in tumors from mice administered abemaciclib. Transcriptional profiling studies from tumors isolated from mouse xenografts of breast cancer after abemaciclib treatment, show that growth inhibition by abemaciclib correlates with inhibition of expression of genes which are dependent on the E2F family of transcription factors for expression. The expression levels of FOXM1, RRM2, TOPO2A, MKI67, MCM7, CDKN2C, CCNE1 (Cyclin E1), and CCNB1 (cyclin B1) mRNA were all significantly reduced following treatment with abemaciclib (Supplementary Figure 3 ). These results are again consistent with the well-defined roles of CDK4 and CDK6 as kinases which reverse the Rb-mediated suppression of E2F-dependent transcription. Known targets of CDK9, including the CTD of RNA Polymerase II and expression of MCL1, were not affected by abemaciclib either in cell lines or in xenograft tumors, further indicating that CDK4 and CDK6 are the principal targets of abemaciclib.
The current studies indicate that abemaciclib induces concentration-dependent changes in cellular metabolism, senescence and apoptosis which are timedependent indicating that the ability to maintain sustained inhibition of CDK4 or CDK6 in addition to the potent inhibition of these targets may be essential for achieving optimal efficacy. Interestingly, the loss of replicative capacity in HR+ breast cancer cells occurred earlier upon treatment with abemaciclib when compared to treatment with the same concentration of palbociclib (Supplementary Figure 1 ). Moreover, both senescence and apoptosis occurred earlier and at lower concentrations of abemaciclib, compared to treatment with either palbociclib or ribociclib (Figure 5 ). The results of the studies presented here suggest a relationship between the potencies of CDK4 and CDK6 inhibitors and the onset of senescence. Promotion of senescence after treatment with CDK4 and (study day 55). The text at the top of each panel of bars provides a description of each of the treatment groups. Growth inhibition is observed in those instances where the calculated changes in %DT/C are less than 100% whereby greater inhibition results in smaller %DT/C values. Change in %DT/C values that are less than zero indicate regressions whereby the tumor volumes on day 55 are smaller than the baseline tumor volume measured on the day prior to the initiation of dosing (baseline day = day 27). The bars for the individual animals are colored on the basis of overall response such that the blue bars indicate progressive disease (PD), the red bars indicate stable disease (SD), and the green bars indicate a partial response (PR). The tumor volumes measured on day 27 served as the baseline volumes for the calculation of %DT/C. Note that one animal in the vehicle control group was sacrificed prior to day 55 due to tumor growth that exceeded the ethical size limits. (D) Mice (n = 5/group) implanted with ZR-75-1 xenograft tumors were treated once-daily for 5 days (QD x 5) with vehicle or 50 or 75 mg/kg of abemaciclib beginning on Day 27 after tumor implantation. The xenograft tumors were collected 24 hours later and analyzed by immunoblotting for inhibition of CDK4/CDK6 by assessing the inhibition of phosphorylated Rb (pRb) at serine sites 780 (ser780) or 807/811 (ser807/811), Topo IIα, and phosphorylated histone H3 (pHH3) at serine 10 (ser10). Potential CDK9 inhibition was assessed by measuring the expression of MCL1 or phosphorylation of serine 2 on the C-terminal domain repeat on RNA polymerase II (pCTD). Images of Western blots were cropped to denote the relevant band(s) for clarity; β-actin was used as a loading control.
CDK6 inhibitors or CDK4 ablation has been described in other cancer cell types [6,7,8] Altogether, the data demonstrate that abemaciclib was effective at blocking breast cancer cell progression and viability through multiple mechanisms which include cell cycle arrest, the induction of senescence as well as upon prolonged exposure, apoptosis and altered energetic metabolism. Moreover, these studies also provide some preliminary mechanistic insights regarding the singleagent activity that has been observed in clinical trials with abemaciclib [17,18], and may distinguish this particular CDK4 and CDK6 inhibitor from other CDK4 and CDK6 inhibitors currently in clinical use or undergoing clinical evaluation.
Ethics statement
All animal studies were performed in accordance with American Association for Laboratory Animal Care institutional guidelines, and all protocols were approved by the Eli Lilly and Company Animal Care and Use Committee.
Synthesis and naming of compounds
All compounds were synthesized at Lilly Research Laboratories (LRL). Unless otherwise indicated, all preclinical data described herein for abemaciclib and palbociclib were obtained using the methanesulfonate salt of each compound; ribociclib was synthesized as a succinate salt. Full chemical names have been previously published [16]
Antibodies
Mouse anti-Rb antibodies from BD Pharmigen (cat# 558385), or Cell Signaling Technology (cat# 8516s, or 9308s) were used for detection of Rb phosphorylated at S780, or S807/811, respectively. Other antibodies used were: topoisomerase (topo) II α (Cell Signaling Technology, cat# 12286s), histone H3 (tri methyl K9) (Abcam cat# ab8898), phospho-Histone H3 (Ser10) (Millipore cat# 06-570), phospho-CTD (Abcam cat# 5095), Mcl-1 (Cell Signaling Technology cat# 5453), FOXM1 (Abcam cat# ab175798), Ki67 (Abcam cat# ab16667), β-galactosidase (Abcam cat# ab9361), goat anti-mouse IgG-Alexa Fluor ® 488 conjugated antibody (Life Technologies cat# A11017), goat anti-rabbit IgG-Alexa Fluor ® 488 conjugated antibody (Life Technologies cat# A11008), and goat anti-chicken IgY H&L-Alexa Fluor ® 647 conjugated antibody (Abcam cat# ab150171). For TUNEL assays, Roche cat#11767291910, 11767305001 and 11966006001 were used, and an annexin V-FITC kit was used for flow cytometry (Miltenyi Biotec cat# 130-092-052).
Cell lines and culture conditions
EFM-19 cells were purchased from DSMZ (#ACC 231) and grown in RPMI-1640 medium (Gibco #52400), supplemented with 10% FBS (Gibco #16000) and 1% Penicillin/Streptomycin (Gibco #15140-122). MCF7 (#HTB-22), T-47D (#HTB-133) and MDA-MB-361 (#HTB-27) cells were purchased and authenticated by ATCC. MCF7 were grown in DMEM (Sigma #D5796), supplemented with 10% FBS, 1% Penicillin/Streptomycin and 0.01 mg/mL human insulin (Sigma #I9278). MDA-MB-361 were grown in RPMI-1640, supplemented with 20% FBS and 1% Penicillin/Streptomycin; additional information can be found in the Supplemental Methods. Except indicated otherwise, all cell lines were grown as indicated by the ATCC. For most experiments, cells were plated (prior to becoming 70% confluent) in 96well flat-bottom plates at a density of 1000 cells per well in 100 µL (exceptions are indicated below). Cells were incubated overnight in a cell culture incubator (5% CO 2 , 95% Relative Humidity (RH) and 37°C) and allowed to attach to the plate (approximately 14-24 hours after seeding) before treatment. The doubling times (DT) of cell lines were determined by growing cells in 6-well plates and counting cells every 24h after seeding using Vi-cell. Cell number was obtained and fitted to the following exponential growth equation regression: DT=T ln2/ln(Xe/ Xb); where T is the incubation time, Xb is the cell number at the beginning of the incubation time and Xe is the cell number at the end of the incubation time [19]. Doubling times were calculated to be approximately 48 hours (2 days) for MCF7 cells, 72 hours (3 days) for EFM-19 cells and 120 hours (~5 days) for MDA-MB-361 cells.
Determination of IC 50 /EC 50 values
Relative IC 50 /EC 50 values were determined by curve fitting to a four parameter logistic equation: Y = bottom + ((top-bottom)/ (1+exp(HillSlope*(X -LnIC 50 )), where X is the natural log of dose/concentration, Y is response, decreasing as X increases, top and bottom are plateaus in the same units as Y, lnIC 50 is the natural log of potency, in the same log units as X, and HillSlope measures the steepness of the curve [20]. Data were plotted using GraphPad Prism.
Enzyme kinetics
Kinetic parameters for CDK4/cyclinD1 and CDK6/ cyclinD1 complexes as well as IC 50 values for CDK9/ cyclinT1 were obtained as previously described [16].
SA-β-gal immunofluorescence
MCF7 cells seeded in 24 well plates were treated with 0.5 µM, or 2 µM of abemaciclib or vehicle (0.2% v/v DMSO) and incubated for 4, 7 or 10 days for single detection of β-gal. For multiplexing experiments, cells seeded in 96 wells plates treated with 0.5 µM or 0.125 µM of abemaciclib or vehicle and treated for 1, 2, 3.5, or 5 DT. After treatment, cells were fixed, washed with PBS, permeabilized and β-gal was detected using anti-βgalactosidase antibody; for multiplexed assays, TUNEL staining was carried out using Roche TUNEL reagents as indicated above. Nuclei were also stained with PI for total cell counting. Plates were read in an Acumen Explorer Ex3 (TTP Labtech) and raw data were analyzed with Cellista software. Outputs were expressed as percentages of each identified subpopulation 1) positive β-gal, and 2) total cell number. The percentages of β-gal positive cells, TUNEL positive cells, and cell numbers were plotted in Microsoft Excel.
TCA metabolite profiling by LC/MS
Cells were incubated with a 5-fold dilution series of abemaciclib ranging from 20 µM to 0.032 µM final concentration in 0.4% v/v DMSO; vehicle-treated cells (0.4% v/v DMSO in growth media) were used as a control. MCF7 cells were plated at 20,000 cells/well in 24-well plate; approximately 14-24 hours after seeding, cells were treated with DMSO (vehicle) or the indicated compounds diluted in DMSO. Cells were incubated for three different incubation times: 48 hours (1 DT), 96 hours (2 DT), and 6 days (3 DT). After treatment, cells were harvested and the collected pellet was stored at -80ºC. For additional details see Supplemental Methods and Supplementary Table 1 .
Mouse xenograft tumor implantation and treatment
Female NOD SCID mice (Harlan) 18-20 grams in size were used for these studies. For subcutaneous tumors, growth was initiated by subcutaneous injection of 5 × 10 6 human ZR-75-1 cells in a 1:1 mixture of HBSS and Matrigel TM (BD Bioscience, Franklin Lakes, NJ) in the rear flank of each subject animal. Since growth of this model is dependent upon a source of estradiol, the mice were implanted with 17-B estradiol 90-day release pellets (0.72 mg, Innovation Research of America) 1 day prior to the implantation of the tumor cells. When mean tumor volumes reached approximately 200 mm 3 in size, the animals were randomized by tumor size and body weight and placed into their respective treatment groups. Abemaciclib (lot# E29-H71350-002) was formulated in 1% hydroxyethyl cellulose (HEC) in 25 mM phosphate buffer (PB) pH = 2. For the efficacy studies, abemaciclib was administered by oral gavage once-daily for 28 days (PO, QD × 28) at doses of 50 or 75 mg/kg using 0.2 mL/dose. The control group was given 1% HEC vehicle according to the same schedule for abemaciclib. A parallel set of tumor-bearing mice used for the in vivo target inhibition (IVTI) studies were treated daily with the same doses of abemaciclib for 5 days. Additional details are provided in Supplemental Methods.
annex
www.impactjournals.com/oncotarget Additional details for CDK6/cyclinD3 activity are provided in Supplemental Methods.
High content imaging cell based assays for monitoring phosphorylation of Rb (Ser780), G1 arrest and cell proliferation
MDA-MB-361 or EFM-19 cells were incubated for 16 hours with 10 different concentrations of abemaciclib (starting with 20 µM and using a 3-fold dilution series down to 0.001 µM). As controls, cells were treated with 10 µM palbociclib or vehicle ( 0.2 % v/v DMSO). Additional details for cell-based assays are provided in the Supplemental Methods.
Cell immunolabeling with 5-bromodeoxyuridine (BrdU)
MCF7 cells were plated in a 96-well format and treated for 3 DT with 2 µM (or 3-fold dilutions) of abemaciclib. After treatment, an ELISA for BrdU detection was performed (Roche, Cat. No. 11 669 915 001) following the vendor's manual. Envision ® (PerKinElmer) was used to measure the light emission of the samples and raw data were processed with Microsoft Excel and relative EC 50 values were determined as described above. For normalization, the mean value obtained from cells treated with 2 µM µM doxorubicin (Sigma #D1515) was set as the minimum, and that obtained from treatment with the vehicle (0.2% DMSO) was set as the maximum.
FOXM1 and Ki-67 protein expression
EFM-19 cells were treated with 2 µM (or a series of 5 additional 3-fold dilutions) of abemaciclib. After 72 hours, cells were stained with an anti-FOXM1 antibody and a secondary goat anti-rabbit IgG, Alexa Fluor ® 488 conjugate; nuclei were stained with propidium iodide (PI). Alternatively, for assessment of Ki-67 levels, cells were treated for 2 DT and then stained with Ki-67 and a secondary goat anti-rabbit IgG, Alexa Fluor ® 488 conjugate. Plates were read in an Acumen Explorer Ex3 (TTP Labtech) using Cellista software and the percentages of FOXM1 positive and Ki-67 positive cells were calculated over the background signal. Data were normalized in Excel using the mean value obtained from treatment with 2 µM BEZ235 as the minimum and the mean value obtained from treatment with 0.2% DMSO as the maximum.
Side scatter and detection of annexin V by flow cytometry
Changes in complexity and morphology of cells treated with 0.2% DMSO or 0.5 µM of abemaciclib 3 DT for MCF7, EFM-19, or MDA-MB-361 were monitored by side scatter (SSC-A) measurements using a MACSQUANT ® Analyzer 10 (Miltenyi Biotec). Annexin V/PI staining was performed following the vendor's specifications in MCF7 and MDA-MB-361 cells. Cells were treated with 0.2% DMSO or 0.5 µM and 2 µM of abemaciclib overnight for 3 DT, and assessed by flow cytometry (MACSQuant ® Analyzer 10, Miltenyi Biotec). Raw data were analyzed with FlowJo X software. Events were gated for debris exclusion and singlets selection. A minimum of 10,000 cells were analyzed per sample.
Acknowledgements
ACKNOLWEDGMENTSKaren T. Smith, Nathalie Godinot and Anastasia Perkowski, employees of Eli Lilly and Company, provided medical writing and editing support.
Funding
FINANCIAL SUPPORTThis work was supported by Eli Lilly and Company.
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