Elicit: CDK4/6 Inhibition in HR+ HER2- Breast Cancer

Skip to main content

CDK4/6 Inhibition in HR+ HER2- Breast Cancer

Research reportView only

Create alertChat

May 5, 2026

What are the downstream signaling changes and biomarkers of CDK4/6 inhibition in HR+ HER2- breast cancer?

CDK4/6 inhibition induces cell cycle arrest through Rb pathway suppression and E2F downregulation, reduces immunosuppressive cells, and alters growth factor signaling, with key biomarkers including high ER and intact Rb for sensitivity versus IFN pathway activation, Cyclin D1/CDK4 overexpression, and PI3K/mTOR hyperactivation for resistance.

Abstract

CDK4/6 inhibition in HR+ HER2- breast cancer induces multiple downstream signaling changes beyond primary cell cycle arrest. Prevention of Rb phosphorylation leads to G1 arrest and downregulation of E2F-regulated genes including RRM2, TOPO2A, MKI67, MCM7, and CDK2, with treatment shifting tumors from high-risk luminal B to low-risk luminal A molecular phenotype. Growth factor signaling pathways show context-dependent alterations: baseline FGFR2 and ERBB3 expression associate with greater benefit from CDK4/6 inhibition, while acquired PI3K/mTOR hyperactivation drives resistance through Cyclin D1 and CDK4 overexpression. Immune signaling exhibits dual patterns, with beneficial reduction of immunosuppressive Tregs and MDSCs during treatment (p<0.0001) but aberrant IFN/STAT1 pathway activation associated with intrinsic and acquired resistance. Additional changes include suppression of ubiquitin-conjugating enzymes UBE2C, UBE2S, and UBE2T, downregulation of DNA repair pathways in resistant cells, and impairment of radiation-induced ERK and NF-κB/c-Myc signaling.

Predictive biomarkers of resistance include the IFN-related palbociclib-resistance signature (IRPS), overexpressed Cyclin D1 and CDK4 proteins, IL6/STAT3 pathway activation, and baseline Treg levels, while markers of sensitivity include high ER levels, Rb wild-type status, low cyclin E, and low p16. Despite comprehensive biomarker analysis in 666 patients, the PALOMA-2 trial found no single baseline marker predicting lack of benefit, suggesting broad initial efficacy. However, approximately 25-35% of patients demonstrate intrinsic resistance, and nearly all eventually acquire resistance through mechanisms including RB1 loss, cyclin E1 amplification, ERBB2/BRAF mutations, and PI3K/mTOR pathway activation. Strategies to overcome resistance include PI3K/mTOR inhibition to restore CDK4/6 inhibitor sensitivity and combined STAT3/PARP inhibition, with mTOR pathway activation correlating with clinical benefit to triplet therapy in CDK4/6 inhibitor-pretreated patients.

Methods

We analyzed 10 sources from an initial pool of 200, using 7 screening criteria. Each paper was reviewed for 7 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Population - Cancer Type and Subtype, Intervention - CDK4/6 Inhibitors, Outcomes - Mechanistic Data, Outcome Level - Molecular Focus, Population Specificity, Intervention Isolation, Publication Type

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

Press enter or space to select a node.You can then use the arrow keys to move the node around. Press delete to remove it and escape to cancel.

Press enter or space to select an edge. You can then press delete to remove it or escape to cancel.

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: “What are the downstream signaling changes and biomarkers of CDK4/6 inhibition in HR+ HER2- breast cancer?”

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:

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.

Extract study design and population characteristics for CDK4/6 inhibition research in HR+ HER2- breast cancer, including:

Extract details about CDK4/6 inhibitor treatment in HR+ HER2- breast cancer, including:

Extract all downstream signaling pathway changes and molecular alterations following CDK4/6 inhibition in HR+ HER2- breast cancer, including:

Extract all biomarker results related to CDK4/6 inhibition in HR+ HER2- breast cancer, including:

Extract specific mechanisms of resistance to CDK4/6 inhibitors in HR+ HER2- breast cancer, including:

Extract how molecular findings relate to clinical outcomes in CDK4/6 inhibitor-treated HR+ HER2- breast cancer patients, including:

Extract methodological details for molecular analyses in CDK4/6 inhibitor studies in HR+ HER2- breast cancer, including:

Results

Characteristics of Included Studies

The review included 10 studies investigating downstream signaling changes and biomarkers of CDK4/6 inhibition in HR+ HER2- breast cancer, comprising 3 clinical studies and 7 preclinical studies.

Study

Full text retrieved?

Study Type

Sample Size/Population

Disease Setting

CDK4/6 Inhibitor Used

R. Finn et al., 2019

Yes

Randomized, placebo-controlled, phase III clinical trial

666 postmenopausal women with HR+/HER2- metastatic breast cancer

Metastatic, treatment-naive

Palbociclib 125 mg daily (3 weeks on, 1 week off) plus letrozole

Carmine De Angelis et al., 2021

No

Preclinical

Cell lines (MCF7, T47D)

Not applicable

Palbociclib

Neil A. O’Brien et al., 2018

Yes

Preclinical

44 breast cancer cell lines

Not applicable

Abemaciclib (continuous daily, 50 mg/kg)

Zijie Cai et al., 2022

No

Preclinical

Palbociclib-resistant cell lines

Advanced

Palbociclib

Chih-Yi Lin et al., 2022

No

Preclinical

MCF7 and T47D cell lines

Not applicable

Palbociclib, ribociclib, abemaciclib

N. Kettner et al., 2019

No

Preclinical

MCF-7 and T47D cells

Not mentioned

Palbociclib

J. G. T. Zañudo et al., 2022

Yes

Phase I/II clinical trial

32 female patients, median age 55.5 years

Metastatic HR+ HER2-, pretreated with prior CDK4/6 inhibitors

Palbociclib 100 mg plus everolimus 5 mg plus exemestane 25 mg

E. Knudsen & A. Witkiewicz, 2016

Yes

Preclinical

MCF7 and T47D cell lines, MDA-MB-231 xenograft

Not explicitly stated

Palbociclib (in vitro: 100 nM-1 µM; in vivo: 125 mg/kg)

F. Scirocchi et al., 2022

Yes

Prospective observational study

50 consecutive women, median age 62 years

Metastatic HR+/HER2-, mostly first-line treatment

Palbociclib, ribociclib, abemaciclib with hormone therapy

Wen-Chi Yang et al., 2024

No

Not mentioned

Not mentioned

Not mentioned

Not specified

toof

Pageof

The studies investigated different aspects of CDK4/6 inhibition, including clinical efficacy, resistance mechanisms, downstream signaling pathways, and immune modulation. Three studies used palbociclib exclusively, one used abemaciclib, three tested multiple CDK4/6 inhibitors, and two did not specify the inhibitor. Among clinical studies, one was treatment-naive and one enrolled CDK4/6 inhibitor-pretreated patients.

Downstream Signaling Changes Following CDK4/6 Inhibition

Cell Cycle Pathway Changes

CDK4/6 inhibition consistently affected core cell cycle machinery across studies. The primary mechanism involved prevention of retinoblastoma (Rb) protein hyperphosphorylation, leading to G1 cell cycle arrest. This was accompanied by reduction in cell cycle progression markers including phospho-Rb, TOPOIIa, phosphohistone-H3, and FOXM1. Transcriptional analysis revealed downregulation of E2F-regulated genes including RRM2, TOPO2A, MKI67, MCM7, and CDK2, which tracked with growth inhibitory response.

CDK4/6 inhibition induced a molecular shift from high-risk luminal B to low-risk luminal A phenotype, with genes repressed by treatment strongly associated with clinical prognosis in ER+/HER2- cases. Importantly, treatment elicited induction of a comparable number of genes involved in multiple processes beyond cell cycle suppression, suggesting complex transcriptional rewiring.

In resistance settings, alterations in cell cycle machinery included loss-of-function alterations in RB1, high cyclin E1 mRNA expression, amplifications of AURKA, and CDK6 overexpression. Palbociclib-resistant cells exhibited overexpression of Cyclin D1 and CDK4 proteins due to upregulated protein synthesis, with silencing of these proteins leading to cell cycle arrest.

Growth Factor Signaling Alterations

Multiple growth factor signaling pathways showed altered activity following CDK4/6 inhibition. Tumors with increased expression of FGFR2 and ERBB3 mRNA demonstrated greater PFS gain from palbociclib addition, suggesting interplay between steroid hormone and peptide growth factor signaling drives CDK4/6 dependence.

In resistant cells, the PI3K/mTOR pathway exhibited hyper-activation, leading to increased phosphorylation of 4E-BP1 and higher levels of Cyclin D1 and CDK4 translation. Tumor samples after palbociclib progression showed significantly higher levels of Cyclin D1, CDK4, p-AKT, and p-4E-BP1. Genomic analysis revealed oncogenic pathways including PI3K/mTOR, FGFR, and ERBB were enriched in resistant samples, with consistency between oncogenic mutations and transcriptional signature activities in ER, PI3K/AKT/mTOR, and RTK/MAPK pathways.

CDK4/6 inhibition also reduced radiation-induced activation of ERK and NF-κB/c-Myc signaling pathways, suggesting broader effects on growth factor-activated cascades.

Immune Signaling Changes

Aberrant interferon (IFN) signaling emerged as a key resistance mechanism. High IFN signaling was associated with reduced CDK4/6 inhibitor sensitivity, with an “IFN-related palbociclib-resistance Signature” (IRPS) derived from transcriptomic analysis. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared to untreated counterparts. The IRPS score was significantly higher in luminal B versus luminal A subtypes and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis.

In palbociclib-resistant cells, the IL6/STAT3 pathway was induced while DNA repair and estrogen receptor pathways were downregulated. Patient samples post-palbociclib progression showed altered IL6/STAT3 signaling compared to pretreatment samples.

CDK4/6 inhibition treatment significantly downregulated circulating regulatory T cells (Tregs) and myeloid-derived suppressor cells (M-MDSCs and PMN-MDSCs) from baseline (p<0.0001 and p<0.05, respectively). The effector Treg subset (CD4+CD25+FOXP3highCD45RA-) was strongly reduced (p<0.0001). Conversely, treatment increased levels of CD4+ T cells and anti-tumor CD137+CD8+ T cells (p<0.05), suggesting relief from immunosuppression.

Lower PD-1 levels were associated with greater benefit from palbociclib plus letrozole, while the PD-1 signaling pathway was associated with reduced PFS benefit.

Ubiquitin-Proteasome Pathway Modulation

CDK4/6 inhibitors suppressed expression of three ubiquitin-conjugating enzymes: UBE2C, UBE2S, and UBE2T. Palbociclib and ribociclib decreased UBE2C at both mRNA and protein levels, though this phenomenon was not shared with abemaciclib. These E2 enzymes modulate several E3 ubiquitin ligases, including the APC/C complex which plays a role in G1/S progression.

DNA Repair and Additional Pathways

CDK4/6 inhibitors impaired the DNA-DSB repair mechanism activated by radiation. In palbociclib-resistant cells, DNA repair pathways were downregulated, and estrogen receptor pathways showed reduced activity. Patient samples post-progression demonstrated alterations in estrogen receptor, DNA repair, and IL6/STAT3 signaling compared to pretreatment.

Metabolic pathways were also affected, with increased glucose and glutamine metabolism, increased cell size, and elevated ATP levels observed following CDK4/6 inhibition. However, these metabolic changes were mitigated by concurrent endocrine therapy.

Biomarker Findings

Predictive Biomarkers of Response and Resistance

Multiple baseline markers showed associations with CDK4/6 inhibitor response. Higher CDK4 levels were associated with endocrine resistance, which was mitigated by palbociclib addition. Lower PD-1 levels at baseline associated with greater benefit from palbociclib plus letrozole.

For abemaciclib, predictive biomarkers of response included high ER levels, Rb wild-type status, high Rb total and phosphoprotein levels, lack of cyclin E amplification, low cyclin E protein, and low p16 protein levels. These markers reflected dependency on the cyclinD:CDK4/6:Rb signaling pathway.

Resistance-associated predictive biomarkers included overexpressed Cyclin D1 and CDK4 proteins, the IFN-related palbociclib-resistance Signature (IRPS), IL6/STAT3 pathway induction, and downregulation of DNA repair and estrogen receptor pathways. PI3K/AKT/mTOR mutations and high mTORC1 pathway activity correlated with clinical benefit to combined estrogen receptor, CDK4/6, and mTOR inhibition.

In the clinical trial testing triplet therapy after CDK4/6 inhibitor progression, genomic and transcriptomic features enabled identification of known or putative drivers of resistance in nearly every patient (22/23), with several patients showing transcriptomic features as sole drivers.

Prognostic Biomarkers

Higher ESR1 gene expression was associated with greater benefit from both placebo plus letrozole and palbociclib plus letrozole. Genes repressed by CDK4/6 inhibition were strongly associated with improved prognosis and reduced risk of recurrence.

The IRPS score correlated with poor prognosis in primary ER+/HER2- tumors. UBE2C/UBE2T expression levels were associated with breast cancer survival. Activating ESR1 mutations were identified in Luminal A/B subtypes while ERBB2/BRAF mutations occurred in HER2-E subtypes in mutually exclusive patterns.

Baseline Treg levels were associated with response to CDK4/6 inhibitor treatment, with Treg levels both at baseline and during treatment showing prognostic value.

Pharmacodynamic Biomarkers

CDK4 levels showed association with endocrine resistance, suggesting a role in drug activity. The pharmacodynamic biomarkers for abemaciclib included mRNA expression of RRM2, TOPO2A, MKI67, MCM7, and CDK2, which are directly regulated by E2F transcription factor downstream of Rb.

Hyper-activated PI3K/mTOR pathway markers, including p-AKT and p-4E-BP1, served as pharmacodynamic indicators. Decrease in UBE2C at mRNA and protein levels by palbociclib and ribociclib demonstrated drug activity on the ubiquitin-proteasome pathway. Activation of IFN/STAT1 signaling in resistant derivatives indicated pathway engagement.

Reduction in Tregs and MDSCs alongside increase in CD4+ and CD137+CD8+ T cells indicated immune modulation by CDK4/6 inhibitors. Impaired DNA-DSB repair mechanism and reduced ERK and NF-κB/c-Myc signaling pathways also served as pharmacodynamic markers.

Resistance Mechanisms

Intrinsic Resistance

Approximately 25-35% of patients demonstrated intrinsic resistance to CDK4/6 inhibitors. Molecular features of intrinsic resistance included high IFN signaling and the IRPS signature. High p16 protein levels and amplification of cyclin E were associated with reduced sensitivity to abemaciclib.

Baseline loss-of-function alterations in RB1, high cyclin E1 expression, low ER expression, and basal molecular subtype represented intrinsic resistance mechanisms. Higher CDK4 levels at baseline were associated with endocrine resistance, though this could be mitigated by palbociclib.

Acquired Resistance

Nearly all patients eventually acquired resistance during CDK4/6 inhibitor treatment. Acquired resistance mechanisms included activation of IFN/STAT1 signaling, dramatic upregulation compared to untreated cells. IL6/STAT3 pathway induction occurred in palbociclib-resistant cells, along with downregulation of DNA repair and estrogen receptor pathways.

Acquired genomic alterations driving resistance included activating mutations in ERBB2, BRAF, and PIK3CA, and amplifications in FGFR1 and ERBB2. Overexpression of Cyclin D1 and CDK4 proteins developed in resistant cells due to hyper-activation of the PI3K/mTOR pathway.

Dysregulation of key signaling pathways downstream of ER and/or HER2 alterations contributed to acquired resistance, particularly in metastatic settings. Aberrant mitogenic signaling pathway activation represented another acquired resistance mechanism.

Cross-Resistance Patterns

Palbociclib-resistant cells demonstrated cross-resistance to other CDK4/6 inhibitors and endocrine therapy. This included resistance to estrogen receptor downregulation. The IRPS signature correlated with increased gene expression of immune checkpoints and endocrine resistance. Patients showing resistance to CDK4/6 inhibitors also demonstrated resistance to endocrine therapy.

Strategies to Overcome Resistance

Targeting the PI3K/mTOR pathway with specific PI3Kα inhibitor (BYL719) or mTOR inhibitor (everolimus) reduced Cyclin D1 and CDK4 protein levels and restored sensitivity to palbociclib. Combined inhibition of STAT3 and PARP significantly increased cell death in palbociclib-resistant cells, suggesting this combination could effectively treat acquired resistance.

Targeting the mTOR pathway showed promise, as mTOR pathway activation correlated with clinical benefit. Selection of patients based on intact Rb pathway signaling could identify those who may benefit from abemaciclib therapy. Targeting the interplay between steroid hormone and peptide growth factor signaling was proposed to overcome resistance.

Endocrine therapy prevented compensatory growth that could contribute to resistance, and discontinuous dosing schedules may limit mitogenic signaling to prevent early resistance. Using CDK4/6 inhibitors to reduce immunosuppression and enhance checkpoint inhibitors represented another strategy.

Clinical Correlations of Molecular Findings

Response Rates and Molecular Subgroups

The PALOMA-2 trial demonstrated that palbociclib plus letrozole conferred efficacy on both luminal A and B patients, with no single biomarker or cassette of markers associated with lack of benefit from combination treatment.

In the CDK4/6 inhibitor-pretreated population, the clinical benefit rate for triplet therapy (exemestane plus everolimus plus palbociclib) was 18.8%. Among patients treated with CDK4/6 inhibitors plus aromatase inhibitors in first-line, 59% achieved complete or partial response, while in second-line with fulvestrant, 14% achieved response.

The IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6 inhibitors. Sensitivity to abemaciclib was observed predominantly in luminal ER+/HER2- and ER+/HER2+ subtypes.

Progression-Free Survival Associations

Higher CDK4 levels were associated with endocrine resistance and shorter PFS in the placebo arm, with this resistance mitigated by palbociclib. Lower PD-1 levels associated with greater PFS benefit from palbociclib plus letrozole. Active growth factor signaling, exemplified by FGFR2 and ERBB3 expression, was associated with greater PFS gain from palbociclib addition.

The IRPS score correlated with increased gene expression of immune checkpoints and poor prognosis, suggesting associations with shortened PFS. Improved PFS was associated with abemaciclib plus fulvestrant in the MONARCH-2 clinical trial.

Resistance to CDK4/6 inhibitors was associated with estrogen receptor downregulation and alterations in IL6/STAT3 and DNA damage response pathways. The hyperactivation of the PI3K/mTOR pathway in resistant cells suggested associations with shortened PFS.

CDK4/6 inhibition treatment shifted ER+/HER2- models from high-risk luminal B to low-risk luminal A molecular phenotype, associated with improved prognosis.

Immune Correlates of Response

The decrease in Treg levels was significantly greater in responder patients compared to non-responder patients. Treg levels at baseline (>T0) and Treg effector levels at T0 were significant prognostic factors, with a multivariate analysis model correctly classifying 74% of patients. The area under the ROC curve was 0.701, indicating moderate predictive accuracy for immune biomarkers.

Synthesis

The studies revealed substantial mechanistic heterogeneity in CDK4/6 inhibitor response and resistance, requiring synthesis across multiple dimensions to explain divergent findings.

Context-Dependent Mechanisms

Different resistance mechanisms dominated in distinct clinical contexts. Intrinsic resistance (25-35% of patients) was primarily characterized by baseline pathway alterations including high IFN signaling, RB1 loss, high cyclin E1, and immunosuppressive phenotypes. In contrast, acquired resistance that developed during treatment involved activation of compensatory signaling pathways not present at baseline, including IL6/STAT3 pathway induction, PI3K/mTOR hyperactivation, and ERBB2/BRAF mutations.

This temporal distinction explains why biomarker studies using baseline samples (PALOMA-2) found no single marker predicting lack of benefit, while studies examining progression samples identified multiple resistance drivers. Both observations are correct within their respective timeframes: CDK4/6 inhibitors provide broad initial benefit regardless of baseline markers, but acquired alterations subsequently drive progression.

The setting of prior CDK4/6 inhibitor exposure further modified resistance patterns. In the triplet therapy trial enrolling CDK4/6 inhibitor-pretreated patients, resistance mechanisms were identifiable in nearly all patients (22/23), with genomic drivers in RTK/MAPK, PI3K/AKT/mTOR pathways prevalent. The modest 18.8% clinical benefit rate in this pretreated population contrasted with the broad efficacy in treatment-naive patients, consistent with accumulated resistance alterations limiting subsequent treatment options.

Integration of Transcriptomic and Genomic Features

Studies employing both genomic and transcriptomic analyses revealed that genomic resistance mechanisms associated with corresponding transcriptomic signatures. ESR1 mutations linked with high estrogen receptor pathway activity in Luminal A/B subtypes, while ERBB2/BRAF mutations associated with high RTK/MAPK pathway activity in HER2-E subtypes. This concordance validated transcriptomics as complementary to genomics, with transcriptomic features identifying pathway drivers even absent genomic alterations.

The molecular shift from luminal B to luminal A phenotype following CDK4/6 inhibition provided mechanistic insight into clinical benefit. Genes repressed by CDK4/6 inhibition strongly associated with poor prognosis, explaining why their suppression improved outcomes. However, induced genes also associated with improved outcomes, suggesting dual beneficial effects beyond simple cell cycle arrest. The induction of growth-promoting pathways that could contribute to resistance was antagonized by concurrent endocrine therapy, explaining the superior efficacy of combination versus monotherapy.

Pathway Crosstalk and Therapeutic Implications

Growth factor and hormone signaling exhibited complex interdependence. Tumors with active FGFR2/ERBB3 signaling showed greater CDK4/6 inhibitor benefit, suggesting growth factor pathway activation increased CDK4/6 dependence rather than conferring resistance. Conversely, PI3K/mTOR hyperactivation in resistant cells drove Cyclin D1/CDK4 overexpression, creating CDK4/6 inhibitor insensitivity through target protein excess. This distinction explained why PI3K/mTOR pathway markers showed opposing relationships with benefit depending on context: moderate baseline activity associated with CDK4/6 inhibitor sensitivity, while acquired hyperactivation drove resistance.

The differential effects of palbociclib, ribociclib, and abemaciclib on UBE2C expression indicated mechanistic distinctions between CDK4/6 inhibitors despite shared primary targets. This heterogeneity may contribute to non-cross-resistance and sequential efficacy, though clinical validation remains limited.

Immune Modulation as Dual Mechanism

CDK4/6 inhibition’s immune effects showed apparent contradictions: treatment reduced immunosuppressive Tregs and MDSCs with benefit correlating with immune activation, yet high baseline IFN signaling associated with resistance. This paradox resolves through mechanistic separation: CDK4/6 inhibitors beneficially reduced regulatory immune populations suppressing anti-tumor immunity, while aberrant chronic IFN pathway activation represented a resistance mechanism independent of CDK4/6 inhibitor-induced immune changes. The IRPS signature’s enrichment in luminal B versus luminal A tumors and correlation with poor prognosis indicated this represented a tumor-intrinsic resistance mechanism rather than response to treatment.

Methodological Heterogeneity and Quality Weighting

Preclinical studies (7/10 sources) provided detailed mechanistic insights into signaling changes and resistance pathways but used cell lines and xenografts that may not fully recapitulate clinical heterogeneity. The phase III PALOMA-2 trial with 666 patients represented the highest-quality evidence for baseline biomarker associations, finding remarkably consistent benefit across subgroups. However, its baseline-only design limited detection of acquired resistance mechanisms identified in studies with serial sampling.

The phase I/II triplet therapy trial employed comprehensive genomic and transcriptomic profiling at progression, providing high-quality evidence for acquired resistance mechanisms despite modest sample size (32 patients). The prospective observational study with 50 patients contributed unique evidence on immune dynamics through serial blood sampling, though lacked genomic correlates. Studies with abstract-only availability provided valuable mechanistic hypotheses requiring validation.

Resistance mechanisms with convergent evidence across clinical and preclinical studies merit greatest confidence: PI3K/mTOR pathway activation was identified in both palbociclib-resistant cell lines and patient tumor samples, with clinical correlation to treatment benefit when targeted. Similarly, immune checkpoint activation was observed in both IFN-high cell lines and patient samples. Mechanisms identified solely in preclinical models (e.g., UBE2C suppression) require clinical validation.

References

R. Finn, Yuan Liu, Zhou Zhu, Miguel Martín, H. Rugo, and 8 more\ (2019).Biomarker Analyses of Response to Cyclin-Dependent Kinase 4/6 Inhibition and Endocrine Therapy in Women with Treatment-Naïve Metastatic Breast Cancer. Clinical Cancer Research

E. Knudsen, A. Witkiewicz\ (2016).Defining the transcriptional and biological response to CDK4/6 inhibition in relation to ER+/HER2- breast cancer. OncoTarget

F. Scirocchi, S. Scagnoli, A. Botticelli, A. Di Filippo, C. Napoletano, and 7 more\ (2022).Immune effects of CDK4/6 inhibitors in patients with HR+/HER2− metastatic breast cancer: Relief from immunosuppression is associated with clinical response. EBioMedicine

Wen-Chi Yang, M. Wei, Yi-Hsuan Lee, Chiun-Sheng Huang, S. Kuo\ (2024).Radiosensitizing effects of CDK4/6 inhibitors in hormone receptor-positive and HER2-negative breast cancer mediated downregulation of DNA repair mechanism and NF-κB-signaling pathway. Translational Oncology

Carmine De Angelis, Xiaoyong Fu, M. Cataldo, A. Nardone, Resel Pereira, and 25 more\ (2021).Activation of the IFN Signaling Pathway is Associated with Resistance to CDK4/6 Inhibitors and Immune Checkpoint Activation in ER-Positive Breast Cancer. Clinical Cancer Research

Neil A. O’Brien, D. Conklin, R. Beckmann, Tong Luo, Kevin Chau, and 8 more\ (2018).Preclinical Activity of Abemaciclib Alone or in Combination with Antimitotic and Targeted Therapies in Breast Cancer. Molecular Cancer Therapeutics

Zijie Cai, Jingru Wang, Yudong Li, Qianfeng Shi, Liang Jin, and 12 more\ (2022).Overexpressed Cyclin D1 and CDK4 proteins are responsible for the resistance to CDK4/6 inhibitor in breast cancer that can be reversed by PI3K/mTOR inhibitors. Science China Life Sciences

Chih-Yi Lin, Chung-Jen Yu, Chun-Yu Liu, T. Chao, Chi-Cheng Huang, and 2 more\ (2022).CDK4/6 inhibitors downregulate the ubiquitin-conjugating enzymes UBE2C/S/T involved in the ubiquitin–proteasome pathway in ER + breast cancer. Clinical and Translational Oncology

N. Kettner, S. Vijayaraghavan, M. Durak, Tuyen N Bui, Mehrnoosh Kohansal, and 17 more\ (2019).Combined Inhibition of STAT3 and DNA Repair in Palbociclib-Resistant ER-Positive Breast Cancer. Clinical Cancer Research

J. G. T. Zañudo, R. Barroso-Sousa, Esha Jain, Qing Jin, Tianyu Li, and 14 more\ (2022).Genomic and Transcriptomic Determinants of Resistance to CDK4/6 Inhibitors and Response to Combined Exemestane plus Everolimus and Palbociclib in Patients with Metastatic Hormone Receptor Positive Breast Cancer. medRxiv

Download BIBDownload RISDownload TXT

Report

Status

Gather sources

200 sources found

Details

Screen sources

10 sources included

Details

Extract data

70 data points extracted

Details

Generate report

Save PDF

BIBLaTeX, ZoteroRISZotero, MendeleyTXTAPA BibliographyPDFPDFDOCXMicrosoft Word

Chat

Got some follow-up questions?

Sign up or sign in to chat with this report.

Back

Preclinical Activity of Abemaciclib Alone or in Combination with Antimitotic and Targeted Therapies in Breast Cancer

Neil A. O’Brien, D. Conklin, R. Beckmann, Tong Luo, Kevin Chau, Jophil Thomas, Ann M Mc Nulty, C. Marchal, O. Kalous, Erika von Euw, S. Hurvitz, C. Mockbee, D. Slamon

Molecular Cancer Therapeutics·

2018·

83 citations

SourceDOI

Plain textPDF

Searching for PDF

Unable to find PDF from source

Back

Study Design

- Study type: Preclinical - Sample size and patient characteristics: Not applicable (uses 44 breast cancer cell lines) - Disease setting: Not applicable (preclinical models) - Treatment history: Not applicable (preclinical models) - Follow-up duration: Not applicable (preclinical models)

CDK4/6 Inhibitor

- Specific CDK4/6 inhibitor used: Abemaciclib - Dosing and schedule: Continuous daily treatment at 50 mg/kg - Combination therapy: Abemaciclib with tamoxifen or fulvestrant - Treatment duration: Not explicitly stated, but sustained inhibition for over 6 weeks after treatment cessation - Comparator arm: Combination therapy vs. single-agent treatments with abemaciclib or endocrine agents alone

Signaling Changes

- Cell cycle pathway changes: Loss of Rb phosphorylation leading to G1 cell-cycle arrest; reduction in cell-cycle progression markers TOPOIIa, phosphohistone-H3, and FOXM1. - Transcriptional changes: Downregulation of E2F-regulated genes RRM2, TOPO2A, MKI67, MCM7, and CDK2. - Protein expression changes: Reduction in pRb and FOXM1. - Time course of changes: Early reduction in pRb and cell-cycle markers; sustained or reversed depending on treatment regimen.

Biomarker Findings

- Predictive biomarkers: High ER levels, Rb wild-type status, high Rb total and phosphoprotein levels, lack of cyclin E amplification, low cyclin E protein, low p16 protein levels - Pharmacodynamic biomarkers: RRM2, TOPO2A, MKI67, MCM7, CDK2 (mRNA expression) - Biomarker type: Protein (Rb, ER, cyclin E, p16), gene expression (RRM2, TOPO2A, MKI67, MCM7, CDK2) - Sample source: Tumor tissue (xenograft models) - Quantitative results: Not mentioned - Clinical correlation: Not mentioned

Resistance Mechanisms

- Intrinsic resistance mechanisms: High p16 protein levels, amplification of cyclin E. - Acquired resistance mechanisms: Dysregulation of key signaling pathways downstream of ER and/or HER2 alterations. - Molecular alterations associated with resistance: Activation of Rb signaling independently of CDK4/6, high p16 protein levels, amplification of cyclin E. - Cross-resistance patterns: Not explicitly mentioned. - Proposed strategies to overcome resistance: Selection of patients based on intact Rb pathway signaling. - Temporal aspects: Resistance may occur particularly in the metastatic setting, suggesting late resistance.

Clinical Correlations

- Response rates by molecular subgroup: Sensitivity to abemaciclib is observed predominantly in luminal ER+/HER2− and ER+/HER2+ subtypes. - Progression-free survival associations with biomarkers/pathway changes: Improved PFS is associated with abemaciclib plus fulvestrant in the MONARCH-2 clinical trial. - Overall survival correlations: Not mentioned. - Toxicity correlations with molecular alterations: Not mentioned. - Predictive accuracy of molecular markers: Not explicitly detailed. - Clinical utility assessments: Not explicitly detailed.

Analysis Methods

- Sample collection timing: Not explicitly mentioned - Sample types analyzed: Snap-frozen tumor tissue - Analytical techniques used: Whole exome sequencing, comparative genomic hybridization microarray assays, reverse phase protein array (RPPA), MODAplex RNA Array multiplex qPCR - Validation methods: Not mentioned - Statistical approaches for biomarker analysis: Two-tailed paired Student t test - Quality control measures: Not mentioned

The cyclinD:CDK4/6:Rb axis is dysregulated in a variety of human cancers. Targeting this pathway has proven to be a successful therapeutic approach in ER+ breast cancer. In this study, in vitro and in vivo preclinical breast cancer models were used to investigate the expanded use of the CDK4/6 inhibitor, abemaciclib. Using a panel of 44 breast cancer cell lines, differential sensitivity to abemaciclib was observed and was seen predominately in the luminal ER+/HER2− and ER+/HER2+ subtypes. However, a subset of triple-negative breast cancer (TNBC) cell lines with intact Rb signaling were also found to be responsive. Equivalent levels of tumor growth inhibition were observed in ER+/HER2−, ER+/HER2+ as well as biomarker selected TNBC xenografts in response to abemaciclib. In addition, abemaciclib combined with hormonal blockade and/or HER2-targeted therapy induced significantly improved antitumor activity. CDK4/6 inhibition with abemaciclib combined with antimitotic agents, both in vitro and in vivo, did not antagonize the effect of either agent. Finally, we identified a set of Rb/E2F-regulated genes that consistently track with growth inhibitory response and constitute potential pharmacodynamic biomarkers of response to abemaciclib. Taken together, these data represent a comprehensive analysis of the preclinical activity of abemaciclib, used alone or in combination, in human breast cancer models. The subtypes most likely to respond to abemaciclib-based therapies can be identified by measurement of a specific set of biomarkers associated with increased dependency on cyclinD:CDK4/6:Rb signaling. These data support the clinical development of abemaciclib as monotherapy or as a combination partner in selected ER+/HER2−, HER2+/ER+, and TNBCs. Mol Cancer Ther; 17(5); 897–907. ©2018 AACR.

Introduction

Advances in our understanding of the molecular diversity of human breast cancer have led to the development of specific, molecularly targeted therapies with subsequent improvement in clinical outcomes for patients with hormone receptor positive (ER þ ) and human epidermal growth factor receptor-2 positive (HER2 þ ) disease (1)(2)(3)(4)(5). Despite these advances, a number of patients with these subtypes who receive appropriate therapies demonstrate either de novo or acquired resistance, particularly in the metastatic setting (6,7). Investigations into the mechanisms underlying therapeutic resistance have focused on dysregulation of key signaling pathways downstream of ER and/or HER2 alterations, including alterations in the PI3K/AKT/mTOR pathway and more recently the cyclin D:CDK4/6:Rb:p16 axis (8)(9)(10)(11)(12).

The cyclin-dependent kinases 4 and 6 (CDK4/6) are key regulators of the G 1 -S-phase restriction checkpoint and pharmacologically targeting these proteins in combination with hormonal blockade has been shown to provide significant therapeutic benefit to patients with advanced ER þ /HER2 À breast cancer (13)(14)(15). In ER-dependent tumors, activated ER signaling leads to increased transcription and synthesis of cyclin D1, promoting formation of activating complexes with CDK4/6 (16). Phosphorylation of the retinoblastoma protein (Rb) by the activated CDK4/6:cyclin D1 complex removes the sequestration of E2F by Rb, allowing transcription of factors that promote progression of the cell cycle through the G 1 -S-phase restriction point (17).

Mitogen-activated CDK4/6:Rb signaling can also occur independent of estradiol-mediated ER signaling. It is known that activated HER2 signaling leads to increased transcription of cyclin D1 via activation of the PI3K and MAPK signaling pathways (18). Mitogenic control of the CDK4/6:Rb axis is frequently lost through a series of molecular alterations known to occur to a greater or lesser degree, in all subtypes of breast cancer (19,20). These alterations are often complex and multifactorial, making it difficult to predict which disease subtypes will respond to CDK4/6 inhibition in the absence of biologic data.

Prior preclinical data from our laboratory demonstrated that a majority of luminal ER þ breast cancer cell lines are sensitive both in vitro and in vivo to the CDK4/6 inhibitor, palbociclib, and that a combination of this drug with hormonal blockade is therapeutically synergistic in this subtype. These data led to the clinical development and recent approval of palbociclib for use in ER þ /HER2 À metastatic breast cancer (13,(21)(22)(23) and subsequently, a second CDK4/6 inhibitor, ribociclib in the same breast cancer subtype (15). Abemaciclib (LY2835219; Eli Lilly) is also an orally available ATP-competitive inhibitor of CDK4 and CDK6, with single-digit nanomolar potency against both kinases. Based on early monotherapy clinical data (24) abemaciclib has been given "Breakthrough Therapy" status from the FDA. This drug has now completed phase II and III clinical development in advanced ER þ /HER2 À breast cancer resulting in its recent regulatory approval for ER þ /HER2 À breast cancer either alone or in combination with hormonal blockade (25,26).

In the current study, we wanted to further evaluate the preclinical activity of abemaciclib in cell lines representing each of the major therapeutic molecular subtypes of breast cancer and compare it with other approved drugs in this class. Abemaciclib is known to have increased selectivity for CDK4 over CDK6 as well as some inhibitory activity at other kinases such as CDK9, PIM1, HIPK2, and DYRK2 (27). In addition, we performed genomic and proteomic biomarker analyses in order to identify biomarkers and/or molecular profiles that correlate with sensitivity or resistance to this molecule. Finally, we interrogated the potential for therapeutically beneficial dual treatment regimens combining abemaciclib with cytotoxic therapies that are currently approved and/or commonly used in the management of breast cancer. These data provide insight into biomarkers of response, other molecular subtypes of breast cancer that may respond to CDK4/6 therapy and additional potential combination strategies that may be appropriate for clinical development.

Cell lines, cell culture, and reagents

The growth inhibitory activity of abemaciclib (LY2835219mesylate salt, provided by Richard Beckmann at Eli Lilly), palbociclib (PD-0332991-HCL; Selleck Chemicals) and ribociclib (NVP-LEE011-succinate; MedChem Express) were assessed using a panel of 44 molecularly characterized human breast cancer cell lines representing the known therapeutic subtypes of the disease (22). Cells were cultured in appropriate culture media (e.g., RPMI 1640, DMEM, L-15) supplemented with 10% to 15% heat-inactivated fetal bovine serum (FBS), 2 mmol/L glutamine, and 1% penicillin G-streptomycin-fungizone solution (PSF, Irvine Scientific) as previously described (28). Cells were routinely assessed for mycoplasma contamination using a multiplex PCR method and STR profiling by the GenePrint 10 System (Promega) was used for cell-line authentication. Trastuzumab-resistant (BT-474-TR) cells were established as previously described (12,29).

In vitro proliferation assays

Cells were seeded into 24-well plates in the relevant culture media in duplicate at 5,000 to 50,000 cells per well, as described previously (22) and the following day 10 mmol/L of abemaciclib, palbociclib, or ribociclib with 2-fold serial dilutions over 6 to 12 concentrations was added to generate dose-response curves for IC 50 determination (Supplementary Procedures). Drug combination studies using docetaxel (Taxotere, Hospira) and Carboplatin (Hospira) were performed as outlined in the Supplementary Procedures.

Molecular characterization of the breast cancer cell panel

To identify potential predictive mutational biomarkers, whole exome sequencing was performed using Agilent SureSelect hybrid capture technology paired with Illumina HiSeq sequencing according with manufacturer's protocols (Supplementary Table S1 ). The deviations from consensus "normal" sequences in these data were further filtered to enrich for mutations that are most likely to be somatic alterations with functional consequence in cancer using multiple published resources, most notably the COSMIC catalog of somatic mutations in cancer (http://www. sanger.ac.uk/science/tools/cosmic). DNA copy-number alterations were determined using comparative genomic hybridization microarray assays (a-CGH) with the Agilent 105K oligonucleotide CGH chip according with manufacturer's protocols. Known oncogenes with log 2 ratios >1 (2-fold) were considered amplified and known tumor suppressor genes log 2 ratios <0.8 were considered homozygous deletions. Baseline total and phosphoprotein levels of a list of >280 protein analytes enriched for proteins currently known to be involved in cancer biology, were determined using reverse phase protein array (RPPA) from the core service at the MD Anderson Cancer Center. Cell preparation and analysis were performed in accordance with MD Anderson published protocols. Data are presented here as log 2 (intensity) values. Molecular markers commonly tested in breast cancer, such as ER and HER2, and several biomarkers that have previously been hypothesized to play a role in response to CDK4/6 inhibition were examined for association with response to abemaciclib (listed in Supplementary Table S2 ).

In vivo efficacy studies

Xenograft models of ER þ /HER2 À , HER2 þ /ER þ , and TNBC breast cancer cell lines were established in 6-week-old CD-1 athymic nude mice (Charles River Laboratories) as described in Supplementary Materials. For ER þ /HER2 À and HER2 þ /ER À studies, 17-ß-estradiol 60-day release pellets (Innovative Research of America) were implanted subcutaneously into the left flank 7 days before tumor inoculation. For in vivo studies, Fulvestrant (Faslodex, AstraZenica), trastuzumab (Herceptin, Genentech), and docetaxel (Taxotere, Hospira) were purchased from the UCLA pharmacy and 4-hydroxytamoxifen was purchased from Sigma-Aldrich. Statistical differences between treatment arms at specific time points were performed using a two-tailed paired Student t test. Differences between groups were considered statistically significant at P < 0.05. All statistics were calculated using Microsoft Excel. All animal work was carried out under a protocol approved by IACUC and the UCLA Animal Research Committee.

siRNA knockdown of RB1

MDA-231 cells were seeded in 24-well plates at 10,000 cells per well, followed by transfection with 1.25 pmol siRNA-targeting exon 19 of RB1 gene (Thermo Fisher Scientific, cat#4390824, s522), and 1.5 mL of lipofectamine (Thermo Fisher Scientific) in Opti-MEM I Reduced Serum Medium. Silencer Select Negative Control No.1 siRNA (Thermo Fisher Scientific, cat#4390843) was used as a control in all assays. siRNA knockdown was confirmed by Western blot.

Modaplex RT-PCR assay

Total RNA was isolated from homogenized snap-frozen tumor tissue using the RNeasy kit from Qiagen. Specific cDNA was produced from the RNA using SuperScript VILO MasterMix as described by the User's Guide. mRNA expression of a set of cellcycle targets was quantified using the MODAplex RNA Array multiplex qPCR platform (Qiagen). Data were exported from the MODAplex software and analyzed in Microsoft Excel. Mean expression values for each transcript were generated from at least three replicate tumor samples. Expression values for each target transcript are represented as a ratio of the mean expression in the experimental arm relative to the vehicle control arm of the study. A "dose-dependent" response was defined as a >10% inhibition at low dose (50 mg/kg) abemaciclib accompanied by a further increase in inhibition of >10% at higher dose (75 mg/kg) of abemaciclib.

Activity of abemaciclib in the breast cancer cell line panel

The antiproliferative activity of abemaciclib was assessed in a panel of 44 breast cancer cell lines, representing the known histological subtypes of the disease (see Supplementary Table S2 for full details). The cell lines were differentially responsive to abemaciclib over a wide concentration range, with IC 50 values varying between 0.012 and 3.73 mmol/L (Fig. 1A ). On-mechanism activity of abemaciclib was confirmed by a loss of Rb phosphorylation in response to treatment (Fig. 1B ) followed by induction of G 1 cell-cycle arrest (Supplementary Fig. S1 ). No loss of pRb or induction of G 1 cell-cycle arrest was observed in the Rbdeficient MDA-468 cell line (Fig. 1B ; Supplementary Fig. S1 ).

ER þ /HER2 À and HER2-amplified breast cancer cell lines that express high levels (>median) of ER protein (i.e., HER2 þ /ER þ ; Fig. 1A ; Supplementary Table S2 ) were among the most sensitive to abemaciclib. In addition, despite relatively low levels of ER and HER2, a subset of TNBC cell lines also responded to abemaciclib at IC 50 values below 500 nmol/L (Fig. 1A ; Table 1 ). In general, sensitivity to abemaciclib was associated with high ER levels, Rb wild-type status, high Rb total, and phosphoprotein levels, lack of cyclin E amplification (normal copy number), low cyclin E protein, p16 deletion, and/or low p16 protein levels (Supplementary Table S1 ).

In the same panel of cell lines, strong correlations were identified between response to abemaciclib and two other CDK4/6 inhibitors, palbociclib and ribociclib (Fig. 1C-E ). Hormone receptor-positive cell lines (either HER2 normal or amplified) were commonly sensitive to all three molecules. In comparative analyses, abemaciclib was the most potent of the three molecules tested, followed by palbociclib and then ribociclib. In biomarkerpositive breast cancer cell lines, the average (geometric mean) IC 50 of abemaciclib was 168 nmol/L, compared with 306 nmol/L for palbociclib and 913 nmol/L for ribociclib.

Abemaciclib in combination with hormone blockade in ER þ /HER2 À breast cancer cell xenografts Combined activity of abemaciclib and hormone blockade was confirmed in two ER þ breast cancer cell lines xenografts (Fig. 2 and Supplementary Figure S2 ). Significant tumor regressions were observed with single-agent abemaciclib in MCF-7 (ER þ /HER2 À ) xenografts, and combination with either tamoxifen or fulvestrant induced a marginal increased benefit in antitumor response (Fig. 2A and B ; Supplementary Table S3 ). However, within the 1st week of drug withdrawal, tumors began to progress in the mice treated with 50 mg/kg of abemaciclib alone as well in the mice treated with either single-agent tamoxifen or fulvestrant. Conversely, sustained inhibition of tumor growth was observed for over 6 weeks after drug withdrawal in the majority of animals treated with either drug combination or with high-dose abemaciclib monotherapy at 75 mg/kg (Fig. 2C ). Molecular analysis of xenograft tissue at day 4 of dosing revealed a greater loss of pRb in response to the combination versus single-agent treatments (Fig. 2D ). This was accompanied by a reduction in mitosis as indicated by a decrease in FOXM1 protein levels (Fig. 2D ).

Western blot analysis of residual xenograft tissue collected at the end of a recovery phase of 6 to 7 weeks revealed that active cell division and Rb signaling were restored in the 50 mg/kg abemaciclib and single-agent endocrine treatments, as indicated by the recovery of FOXM1 and pRb protein levels (Fig. 2E ). In contrast, FOXM1/pRb signal remained low in xenograft tissues collected from combination arms at the same time point (Fig. 2E ). To investigate if the sustained reduction in Rb/FOXM1 signal in the combination arms was due to induction of permanent growth arrest/senescence or induction of cell death, we measured levels of the human epithelial marker protein keratin-19 (CK-19), a structural protein not directly regulated by abemaciclib treatment (Supplementary Fig. S3 ). In the MCF-7 xenograft tissues, CK-19 protein levels were significantly reduced or lost in the combination treatment arms, particularly in abemaciclib plus tamoxifen treated mice, indicating loss of human epithelial tumor cells (Fig. 2E ).

Using a ß-galactosidase-staining assay, in vitro assays of the MCF-7 cells showed increased induction of senescence in response to the combination as opposed to the single agent (Fig. 2F ).

Activity of abemaciclib in HER2 þ /ER þ breast cancer cell lines and xenografts Activity of abemaciclib single agent or combination with HER2 and ER-targeted therapy was assessed in two xenograft models of HER2 þ /ER þ breast cancer, parental BT474 cells and BT474 cells conditioned through long-term drug exposure to progress on trastuzumab therapy (BT-474-TR ( 12)). Single-agent abemaciclib induced significant tumor growth inhibition (TGI) in both models (BT-474; P ¼ 0.028, BT-474-TR; P < 0.001; Fig. 3A-C ; Supplementary Table S4 ). The combination of abemaciclib with trastuzumab induced significantly improved (P ¼ 0.0012) TGIs and tumor regressions in xenografts progressing on trastuzumab alone (Fig. 3B and C ; Supplementary Table S4 ). The triple combination of abemaciclib, trastuzumab, and tamoxifen further improved tumor regressions in both models (Fig. 3A-C ; Supplementary Table S4 ). In vitro studies confirmed that triple blockade of CDK4/6, HER2, and ER signaling leads to a greater induction of cell death in both trastuzumab-sensitive and -resistant HER2 þ /ER þ breast cancer cell lines (Fig. 3D ). Abemaciclib was also shown to combine effectively with docetaxel in these HER2amplified breast cancer cell line xenografts. The addition of docetaxel to abemaciclib did not inhibit the TGI induced by abemaciclib or docetaxel alone. Moreover, the combination of abemaciclib, trastuzumab, tamoxifen, and docetaxel was the most efficacious arm in both studies (Fig. 3A-C ; Supplementary Table S4 ). Marginal and reversible body weight loss was observed in the mice treated with docetaxel containing arms over the 4 weeks treatment period (Supplementary Table S4 ).

Western blot analysis of xenograft tissues collected after 4 days of treatment confirmed a dose-dependent loss of pRb, TOPOIIa, and pHH3 in mice treated with abemaciclib (Fig. 3E ). Further reduction in Rb signaling was observed in xenografts treated with abemaciclib plus trastuzumab or trastuzumab plus tamoxifen. The addition of docetaxel did not block the pRb knockdown induced by abemaciclib or the combination of abemaciclib with trastuzumab plus tamoxifen (Fig. 3E ).

Identification of a subset of TNBC cells sensitive to abemaciclib

In TNBC cell lines, where cell growth is independent of ER status, response to abemaciclib appears to remain dependent on intact Rb signaling. Those TNBC cell lines that have high baseline levels of total and phosphorylated pRb, accompanied by low levels of p16 protein, are among the most sensitive to abemaciclib (Table 1 ). Cell lines with copy-number gains or amplification of the CCNE1 (cyclin E1) gene are clearly less responsive (Table 1 ). Xenografts of TNBC cell lines expressing sensitive and resistant biomarker profiles were measured for response to abemaciclib single agent and combination with docetaxel, a standardof-care agent used for TNBC. Consistent with all the preceding data, induction of tumor regressions or stable disease was observed only in the MDA-231 (Fig. 4A ; Supplementary Table S5 ) and BT-20 xenografts (Supplementary Fig. S4 ) that have high levels of pRb and low levels of p16 at baseline (Fig. 4C . left). Conversely, HCC70 xenografts with low levels of pRb and high p16, continued to progress through abemaciclib monotherapy (Fig. 4B ). On-target activity of abemaciclib was confirmed in the sensitive MDA-231 xenografts by reduction in total and phosphorylated Rb and the cell-cycle progression marker, FOXM1, in response to treatment (Fig. 4C, right ). Consistent with our observations in HER2 þ /ER þ breast cancer xenografts, combination of abemaciclib with docetaxel did not antagonize the activity of either single agent (Fig. 4A and B ). Finally, the role of pRb in predicting response to abemaciclib in TNBC was confirmed by siRNA knockdown of RB1 gene expression in the MDA-231 cells. Subsequent reduction of baseline pRb levels significantly reduced the sensitivity of the MDA-231 cells to abemaciclib (Fig. 4D ).

Combined activity of abemaciclib and cytotoxic chemotherapy

The potential of abemaciclib to be used in combination with cytotoxic chemotherapy was further investigated in a cell line model of TNBC in vitro. In order to determine if the lack of antagonism observed in our in vivo models could be attributed to an effect of the timing of drug administration, we investigated dosing strategies comparing coadministration versus sequencing of these treatments.

Simultaneous treatment of MDA-231 cells with abemaciclib plus docetaxel or carboplatin resulted in increased inhibition of cell proliferation compared with single-agent treatments (Fig. 5A ). Concentrations of abemaciclib below 10 nmol/L induced profound (>60%) inhibition of cell proliferation when combined with either antimitotic agent. Pretreatment of cells with abemaciclib for 2 days reduced cell proliferation rate via induction of G 1 cell-cycle arrest (Supplementary Fig. S5A ) without impacting the activity of docetaxel or carboplatin relative to cells pretreated with vehicle control (Fig. 5B and C ). Pretreatment with abemaciclib for 24 hours also induced G 1 cell-cycle arrest (Supplement Fig. S5B ) without blocking apoptosis induced by high-dose docetaxel treatment. In contrast, a dose-dependent decrease in apoptosis induction by docetaxel was observed in response to pretreatment with palbociclib or ribociclib (Fig. 5D ). Coadministration of docetaxel with either palbociclib or ribociclib after CDK4/6-inhibitor pretreatment blocked apoptosis induction, whereas cotreatment with abemaciclib did not (Fig. 5E ).

Identification of a pharmacodynamic signature of response to abemaciclib treatment

To identify potential pharmacodynamic (PD) markers of response to abemaciclib, we used a Modaplex PCR-based platform to measure changes in the expression of a set of 22 cell-cycleassociated genes in pre-and posttreatment (day 4) samples from tumor tissues responding to abemaciclib-based therapy. In MCF-7 ER þ /HER2 À xenografts, a range of mRNA expression changes were induced by treatment with abemaciclib, hormonal blockade or a combination of the two (Fig. 6A ). A subset of 10 transcripts showed a dose-dependent reduction in expression in response to low-and high-dose abemaciclib. Single-agent treatment with either fulvestrant or tamoxifen had little effect on the expression of these 10 candidate PD biomarkers. However, combination of low-dose abemaciclib (50 mg/kg) with either fulvestrant or tamoxifen increased the inhibition of the candidate gene set compared with inhibition induced by high-dose abemaciclib (75 mg/kg; Fig. 6B ). Using the same criteria, an abemaciclib dose-dependent gene signature was identified from ER þ /HER2 þ xenografts. Similar to the ER þ /HER2 À models, expression of these transcripts was similarly regulated by CDK4/6-inhibition in combination with either trastuzumab or tamoxifen or both (Fig. 6C ). Transcripts for RRM2, TOPO2A, MKI67, MCM7, CDK2, and CDK4 were all found to be regulated by abemaciclib in a dose-dependent manner in sensitive tumors from MCF-7 (ER þ /HER2 À ), ZR-75-1 (ER þ /HER2 À ), and BT-474-TR (HER2 þ /ER þ ) xenografts (Fig. 6D ). Expression of five of these transcripts, RRM2, TOPO2A, MKI67, MCM7, and CDK2 are directly regulated by the E2Ftranscription factor immediately downstream of Rb, indicating a direct on-target regulation of these genes by CDK4/6 inhibition using abemaciclib.

Discussion

The cyclinD:CDK4/6:Rb:p16 signaling axis is frequently dysregulated in cancer. Efforts to pharmacologically target this pathway have been validated by the approval of the specific CDK4/6 inhibitor, palbociclib (PD-0332991, Pfizer), ribociclib (LEE-011, Novartis), and most recently abemaciclib (Eli Lilly), in combination with endocrine-based therapies in advanced ER þ /HER2 À breast cancer (13, 15, 21-23, 25, 26) . Abemaciclib is structurally and biologically distinct from palbociclib and ribociclib with greater selectivity for CDK4 over CDK6 (27) and clinically, abemaciclib appears to have superior single-agent activity when compared with the other approved CDK4/6 inhibitors (24,25,30,31). It is possible that the CDK4 selectivity of abemaciclib contributes to the improvement in activity by reducing the degree of neutropenia usually associated with inhibition of CDK6, which in turn avoids the need for an interruption in dosing associated with both palbociclib and ribociclib. This difference may improve the chances of forcing tumor cells into permanent growth arrest and ultimately senescence (24). In this study, we present a comprehensive analysis of the preclinical activity of abemaciclib using both in vitro and in vivo preclinical models spanning the known molecular subtypes of breast cancer. Using pre-and posttreatment data, we have identified a set of consistent biomarkers of sensitivity to abemaciclib in ER þ , HER2 þ , and TNBC subtypes. Measurement of the growth inhibitory activity of abemaciclib across a panel of 44 human breast cancer cell lines identified the ER þ /HER2 À subtype as most sensitive to CDK4/6 inhibition. Despite the wide therapeutic range of this molecule, each of the 9 cell lines classified as ER þ /HER2 À had IC 50 values below 200 nmol/L, making further stratification of response biomarkers within this subtype unnecessary. This activity was confirmed in two xenograft models of ER þ /HER2 À breast cancer by the induction of complete arrest of tumor growth using abemaciclib monotherapy. Inhibition of tumor proliferation was accompanied by dose-dependent decreases of phosphorylated Rb and induction of cell-cycle arrest as measured by loss of the cell-cycle progression markers, TOPOIIa (S-phase), phosphohistone-H3 (G 2 -M), and FOXM1 (cellular senescence). At the tested doses, abemaciclib did not inhibit CDK-9 signaling, consistent with previous data reported for this molecule (27,32). Comparison of the activity of abemaciclib, palbociclib, and ribociclib across the panel of 44 breast cancer cell lines identified a strong correlation within and between the major histologic subtypes of breast cancer for this class of molecule. However, of the three, abemaciclib appeared to be the most potent using our assays.

Our studies also confirm that abemaciclib has additive and/or synergistic activity in combination with endocrine-based therapies in cell line xenograft models of ER þ /HER2 À breast cancer. Continuous daily treatment using clinically achievable doses of abemaciclib (50 mg/kg) in combination with tamoxifen or fulvestrant was well tolerated and led to downregulation of Rb signaling and significantly improved antitumor responses compared with results with either agent alone. While tumors from single-agent-treated animals began to proliferate within days of treatment cessation, the majority of xenografts from mice treated with combination therapy showed no signs of tumor progression, despite withdrawal of treatment for more than 6 weeks. Xenografts collected during the dosing phase of the combination studies indicate an initial induction of cellular senescence as measured by loss of FOXM1 and in vitro assays showing a greater induction of senescence with the combination treatment. Xenograft materials collected at the end of the study indicate a loss of human epithelial tumor cell population. The marked tumor regressions observed in the combination arms provide further evidence of induction of tumor cell death induced by this regimen. The data reported here are consistent with those recently reported from the MONARCH-2 clinical trial (NCT02107703), which showed significant improvements in progression-free survival (PFS) and objective response rates (ORR) in patients with advanced breast cancer treated with abemaciclib plus fulvestrant versus fulvestrant alone (26).

Abemaciclib-sensitive cell lines were also identified within the HER2-amplified subgroup. There is considerable preclinical evidence to support targeting CDK4/6 in HER2-amplified breast cancers given that HER2 signaling can drive cell-cycle progression through activation of cyclinD1:CDK4/6 signaling (18,33). Cyclin D1 is required for the formation of HER2-initiated tumors in mouse models and pharmacologically targeting CDK4/6 blocks tumor formation in mice (34,35). In addition, preclinical studies show that targeting CDK4/6 overcomes resistance to HER2-directed therapy both in vitro and in vivo (9,36). The data presented here provide further insight into the specific subtypes of HER2-amplified breast cancers most likely to benefit from CDK4/6-targeted therapy. Analysis of abemaciclib response within the panel of 17 HER2-amplified breast cancer cell lines indicates that HER2amplified cell lines with higher levels of ER protein accompanied by intact downstream Rb signaling (high total Rb and pRb and no cyclin E amplification) are most sensitive to abemaciclib treatment. These data indicate that HER2-amplified breast cancer with higher levels of ER (i.e., HER2 þ /ER þ ) may be more susceptible to CDK4/6 intervention than those that are HER2 þ /ER À . The current data demonstrate that single-agent abemaciclib induces significant tumor growth inhibition as well as dose-dependent inhibition of pRb signaling and cell-cycle arrest in HER2 þ /ER þ xenografts. In addition, abemaciclib combined effectively with trastuzumab and in triple combination with tamoxifen, induced significant regressions in both trastuzumab-sensitive and -resistant HER2 þ /ER þ xenografts. In vitro studies confirmed that the triple combination of targeting CDK4/6, HER2, and ER leads to a greater induction of cell death in cells when compared with single-agent treatment. The specific mechanism by which this triple combination is effective remains to be determined. Finally, abemaciclib activity in HER2-amplified tumors progressing on trastuzumab provides encouragement that targeting CDK4/6 may be efficacious in treatment refractory HER2-amplified disease. These data also support the hypothesis that ER and downstream Rb signaling are, at least partially, driving the progression of HER2 þ /ER þ tumors. Clinical data suggest that HER2 þ /ER þ and HER2 þ /ER À breast cancers are clinically distinct subgroups based on their prognosis and response to therapy (37,38). Furthermore, preclinical studies show that crosstalk exists between ER and HER2 signaling and activation of either pathway is associated with resistance to targeting the other pathway (39)(40)(41). These data support the design of the MonarcHER trial (NCT02675231), which will evaluate the combination of abemaciclib plus trastuzumab with or without hormonal blockade (fulvestrant) in HER2 þ /ER þ breast cancer patients. The use of CDK4/6 inhibitors beyond ER þ /HER2 À breast cancer also requires investigation of combinations with standard-of-care cytotoxic chemotherapies. There are preclinical data to suggest that the mechanism of action of molecules that arrest cell cycle may be antagonistic when used with antimitotic agents (35,42). To address this question, we first investigated the in vivo activity of abemaciclib in combination with chemotherapy (docetaxel) in HER2-amplfied and TNBC xenografts. In TNBC, cotreatment with abemaciclib and docetaxel did not antagonize antitumor activity of either single agent. Moreover, the addition of docetaxel to a triple (anti-HER2, anti-ER, and anti-CDK4/6) combination in HER2 þ /ER þ xenografts increased the degree of tumor regressions observed. Analysis of tumor tissues confirmed that docetaxel did not block the induction of the cell-cycle arrest by abemaciclib. In vitro drug-sequencing experiments confirmed that induction of growth arrest by abemaciclib does not block the inhibition of cell proliferation or the apoptosis induced by docetaxel or carboplatin. In fact, either simultaneous or sequential combination of abemaciclib plus either cytotoxic leads to greater inhibition of tumor cell growth compared with singleagent treatment. The combined activity observed between these two classes of molecules could possibly be explained by fact that not all tumor cells will be uniformly arrested by abemaciclib leaving subpopulations of cells sensitive to the mechanism of action of antimitotics. It has previously been shown that abemaciclib can be combined with gemcitabine without antagonism in lung cancer xenografts with either sequential or simultaneous administration (27). Our in vitro experiments also show that abemaciclib appears to be unique among the CDK4/6 inhibitors tested. In contrast to abemaciclib, both palbociclib and ribociclib pretreatment significantly reduced the induction of apoptosis by docetaxel in a dose-dependent manner. The specific mechanisms by which abemaciclib differs from palbociclib and ribociclib in this setting requires further investigation; however, these data are encouraging for the development of abemaciclib in indications where combination with cytotoxic chemotherapies might be required.

Sensitivity to abemaciclib was also detected in a specific subgroup of TNBC cell lines matching the biomarker profile of intact Rb signaling, despite expressing low levels of ER and HER2. Of the 17 TNBC cell lines tested, the top 5 most sensitive cell lines had high levels of total and phosphorylated levels of Rb accompanied by low baseline levels of p16. Abemaciclib IC 50 values for this subset of TNBC cell lines are similar to those measured for the ER þ /HER2 À and HER2 þ /ER þ cell lines. Molecular alterations associated with resistance to CDK4/6-targeted therapy (43,44) through activation of Rb signaling independently of CDK4/6, such as high p16 protein levels or amplification of cyclin E, were found in those TNBC cell lines least sensitive to abemaciclib. Xenograft studies confirmed that TNBC cell lines selected for high baseline levels of pRb and low p16 were sensitive to abemaciclib whereas xenografts with low pRb and high p16 were unresponsive to this therapy. Reduction in pRb levels or RB1 via siRNA knockdown significantly reduced the sensitivity of TNBC cells to abemaciclib. These data support the hypothesis that Rb dependence goes beyond ER-driven breast cancers and that CDK4/6 inhibition may be effective in other subtypes with the appropriate cyclinD: CDK4/6:Rb activation profile. It is likely that the strong association between ER status and abemaciclib response in breast cancer models and patients (24) is due to the fact that ER is a useful surrogate marker of downstream dependence on cyclinD:CDK4/ 6:Rb:p16 signaling. Selection of patients based on measurement of intact Rb pathway signaling as characterized by high tumor levels of pRb, low p16, and an absence of cyclin E1 amplification, may identify additional populations of patients that could benefit from abemaciclib therapy and could provide therapeutic benefit in a subset of TNBCs where there are no current, approved targeted therapies.

Of interest, additional factors that associate with response to abemaciclib in this study include the presence of high levels of androgen receptor (AR) and the presence of PIK3CA-activating mutations. It should be noted, however, that these factors track with the luminal breast cancer subtype and as such may not be independent biomarkers of response (19,45). High levels of PTEN protein associated with abemaciclib response indicate that intact PI3K pathway signaling may increase the likelihood of response to CDK4/6-targeted therapy.

PD markers, for the early detection of response to abemaciclib therapy, were also identified in this study using a set of cell-cycleregulated genes. Expression of a core set of five E2F-regulated genes (RRM2, TOPO2A, MKI67, MCM7, and CDK2), consistently tracked with response to abemaciclib in a dose-dependent manner across three independent xenograft studies. Furthermore, the dynamic changes in expression of these transcripts tracked with an increase in antitumor response induced by abemaciclib when combined with fulvestrant, tamoxifen, or trastuzumab. The ultimate utility of this marker set should now be evaluated in tissues from abemaciclib clinical trials.

In this study, we present a comprehensive preclinical profile of abemaciclib used as a single agent or in combination with standard-of-care therapy in each of the known therapeutic molecular subtypes of breast cancer. These data further support the clinical use of abemaciclib as monotherapy as well as a possible combination partner in ER þ /HER2 À , HER2 þ /ER þ , and some TNBCs. It is likely that beyond ER þ /HER2 À breast cancer, measurement of an on-mechanism, validated set of biomarkers to confirm dependence on Rb -signaling will likely help better select patients that will benefit from abemaciclib-based therapies.

Acknowledgements

AcknowledgmentsThis study was supported by UCLA funding and a DOD Innovator Award W81XWH-11-1-0104 to Dennis J. Slamon.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.Received March 29, 2017; revised November 16, 2017; accepted February 16, 2018; published first February 26, 2018.

Funding

Veh/Veh Veh/Carb Veh/Abe Veh/Abe+Carb Abe/Veh Abe/Carb Abe/Abe Abe/Abe+Carb nmol/L Palbo/Dtx 100 nmol/L Ribo/Dtx 100 nmol/L

Failed to load PDF:

Paper sources

Abstract screening pilot

Abstract screening results

Extraction pilot

Extraction results

Research report

Modify setup