Elicit: CDK4/6 Inhibition and G1 to S Arrest

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CDK4/6 Inhibition and G1 to S Arrest

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May 5, 2026

CDK4/6 inhibition cell-cycle arrest G1 to S palbociclib

Palbociclib consistently induces G1 to S cell cycle arrest through CDK4/6 inhibition and Rb pathway blockade, achieving complete arrest in 87-90% of Rb-proficient hormone receptor-positive breast cancers, though the arrest is cytostatic and requires continuous therapy to maintain.

Abstract

Palbociclib consistently induces G1 to S phase cell cycle arrest across multiple cancer types through inhibition of CDK4/6, preventing retinoblastoma (Rb) phosphorylation and suppressing E2F-target gene expression. In neoadjuvant trials of hormone receptor-positive breast cancer, complete cell cycle arrest (Ki-67 ≤2.7%) was achieved in 87-90% of patients when palbociclib was combined with endocrine therapy, representing a threefold increase over endocrine therapy alone. This profound antiproliferative effect translated to clinically meaningful progression-free survival benefits in advanced disease, with median PFS of 9.2 months versus 3.8 months for combination therapy versus endocrine therapy alone (HR 0.42). However, the arrest is cytostatic rather than cytotoxic—concurrent suppression of apoptosis limits tumor regression, and continuous therapy is necessary to maintain cell cycle arrest as discontinuation allows Ki-67 rebound. Mechanistically, response depends on intact Rb function, with RB1 loss conferring complete resistance, while high cyclin E levels and elevated CDK2 activity enable G1/S bypass. Sustained mTORC1 activity during palbociclib treatment converts reversible arrest to irreversible senescence, and autophagy induction serves as an adaptive resistance mechanism that can be targeted through combination strategies.

The clinical benefit of palbociclib-induced G1 arrest is biomarker-dependent and context-specific. Tumors with intact Rb, absent or low cyclin E (particularly low-molecular-weight isoforms), and adequate p27 levels demonstrate maximal sensitivity, with progression-free survival exceeding 400 months in optimally selected populations. Grade 3/4 neutropenia occurs in 51-62% of patients but is largely asymptomatic, with febrile neutropenia rates of only 0.6% and treatment discontinuation rates of 2.6%. Combination strategies targeting autophagy, PI3K/MEK signaling, or mTORC1 can overcome resistance mechanisms in biomarker-selected populations. The evidence demonstrates that palbociclib reliably induces G1 arrest in Rb-proficient cells, but durable clinical benefit requires continuous dosing, appropriate biomarker selection, and in some contexts, rational combination therapy to prevent adaptive resistance.

Methods

We analyzed 10 sources from an initial pool of 200, using 8 screening criteria. Each paper was reviewed for 8 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Palbociclib Focus, Cell Cycle Measurement, Study Model Type, Control Groups, Study Type, Measurable Outcomes, Publication Type, Sample Size

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: “CDK4/6 inhibition cell-cycle arrest G1 to S palbociclib”

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 setting for CDK4/6 inhibition research, including:

Extract complete details about CDK4/6 inhibitor treatment regimen, including:

Extract all measurements of G1 to S cell cycle arrest and related markers, including:

Extract mechanistic findings related to CDK4/6 inhibition and G1/S arrest, including:

Extract clinical efficacy outcomes specifically related to CDK4/6 inhibitor treatment, including:

Extract findings about cellular consequences of CDK4/6-induced G1 arrest, including:

Extract factors affecting response to CDK4/6 inhibition and G1/S arrest, including:

Extract safety and tolerability data for CDK4/6 inhibitor treatment, including:

Results

Characteristics of Included Studies

The systematic review included 10 studies examining CDK4/6 inhibition by palbociclib and its effects on G1 to S phase cell cycle arrest. Studies comprised 6 clinical trials and 4 preclinical investigations. Full text was available for 6 studies, while 4 were assessed from abstracts only.

Study

Full Text Retrieved?

Study Type

Cancer Type and Subtype

Sample Size

Setting

Ma et al., 2017

Yes

Phase II neoadjuvant clinical trial

ER+/HER2- breast cancer

50 patients (18 premenopausal, 32 postmenopausal)

Clinical stage II-III patients

Johnston et al., 2019

Yes

Phase II randomized trial

ER+/HER2- breast cancer

307 patients

Postmenopausal women with primary tumors ≥2.0 cm

Arnedos et al., 2018

No

Randomized clinical trial

Early breast cancer (93% HR+, 8% HER2+)

74 palbociclib, 26 control

Early breast cancer patients

Maskey et al., 2020

Yes

Preclinical, in vitro

ER+ breast cancer

Multiple cell lines (MCF7, T47D, CAMA1)

ER+ breast cancer cell lines

Turner et al., 2015

Yes

Phase 3 clinical trial

Advanced HR+/HER2- breast cancer

521 patients

Pre- and postmenopausal women with relapsed/progressed disease

Vijayaraghavan et al., 2017

Yes

Preclinical (in vitro, in vivo) and clinical cohort

ER+ breast cancer and other solid tumors

109 patients in clinical cohort; multiple cell lines and xenografts

Cell lines (MCF7, T47D, ZR75-1), xenografts, advanced ER+ breast cancer patients

DeMichele et al., 2014

No

Phase II clinical trial

Advanced breast cancer (84% HR+/HER2-, 5% HR+/HER2+, 11% HR-/HER2-)

37 patients

Metastatic breast cancer, Rb+ with measurable disease

Tien & Sadar, 2021

No

Preclinical

Castration-resistant prostate cancer

Not mentioned

Human xenografts and cultured cells

Asghar et al., 2017

Yes

Preclinical

Triple-negative breast cancer (TNBC), LAR subtype

Multiple cell lines and xenografts

TNBC cell lines and MDA-MB-453 LAR xenografts

Kumarasamy et al., 2020

No

Preclinical

ER+ breast cancer and pancreatic cancer

Not mentioned

ER+ xenografts and pancreatic cancer PDX models

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Clinical trials predominantly focused on hormone receptor-positive breast cancer, with sample sizes ranging from 37 to 521 patients. The two largest trials were phase 3 (Turner et al., 521 patients) and phase 2 (Johnston et al., 307 patients). Neoadjuvant trials examined palbociclib in combination with endocrine therapy, while the advanced disease trial combined palbociclib with fulvestrant. Preclinical studies utilized established cell lines and xenograft models to investigate mechanistic aspects of CDK4/6 inhibition.

Treatment Regimens

Palbociclib dosing across studies followed established protocols, with clinical trials consistently using 125 mg daily on a 21-days-on, 7-days-off schedule, while preclinical studies utilized concentrations ranging from 500 nM to 1 μM in vitro.

Study

Palbociclib Dose and Schedule

Duration

Combination Therapy

Route

Ma et al., 2017

125 mg daily, days 1-21 of 28-day cycle

Four 28-day cycles, plus optional 10-12 day cycle 5

Anastrozole 1 mg daily; goserelin if premenopausal

Oral

Johnston et al., 2019

125 mg/day, 21-days-on, 7-days-off

14 weeks

Letrozole

Oral

Arnedos et al., 2018

125 mg daily

14 days until day before surgery

None mentioned

Oral

Maskey et al., 2020

500 nM

48 hours and various durations

Approved with endocrine therapies clinically, not specified in study

Added to cell culture medium

Turner et al., 2015

Not mentioned

Not mentioned

Fulvestrant; goserelin for pre/perimenopausal women

Not mentioned

Vijayaraghavan et al., 2017

In vitro: ≤1 μM for 6 days; In vivo: 25 mg/kg/day for 7 days

In vitro: 6 days treatment + 4 days recovery; In vivo: 7 days

Autophagy inhibitor HCQ

Oral gavage (in vivo)

DeMichele et al., 2014

125 mg orally, days 1-21 of 28-day cycle

Not mentioned

Not mentioned

Oral

Tien & Sadar, 2021

Not mentioned

Not mentioned

EPI-7170 (sequential or concomitant)

Not mentioned

Asghar et al., 2017

In vitro: 500 nM; In vivo: 50 mg/kg daily

In vitro: ≥2 weeks; In vivo: 21 consecutive days

Pictilisib, taselisib, AZD2014

Oral (in vivo)

Kumarasamy et al., 2020

Not mentioned

Not mentioned

MEK inhibitor in pancreatic cancer PDX models

Not mentioned

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The neoadjuvant studies demonstrated the importance of treatment duration and continuity. Ma et al. showed that Ki67 levels rebounded at surgery following palbociclib washout, but this rebound was suppressed by an additional cycle 5 of palbociclib immediately before surgery, suggesting continuous therapy may be necessary to maintain antiproliferative effects.

Cell Cycle Arrest and Proliferation Markers

Palbociclib consistently induced profound suppression of cellular proliferation across studies, with Ki-67 serving as the primary biomarker for cell cycle arrest.

Study

Ki-67 Baseline

Ki-67 Post-Treatment

% Change/CCCA Rate

Time Points

Other Markers

Ma et al., 2017

C0D1

C1D1 (anastrozole alone), C1D15 (palbociclib added)

CCCA rate: 26% (C1D1) vs 87% (C1D15)

Baseline, C1D1, C1D15, surgery

PAM50 11-gene proliferation score

Johnston et al., 2019

Baseline

14 weeks

Median log-fold change: -4.1 (palbociclib+letrozole) vs -2.2 (letrozole); Geometric mean: -97.4% vs -88.5%; CCCA: 90% vs 59%

Baseline, 2 weeks, 14 weeks

None mentioned

Arnedos et al., 2018

Day 1

Day 15

Antiproliferative response: 58% (palbociclib) vs 12% (control)

Day 15

Phospho-Rb

Maskey et al., 2020

Not mentioned

48 hours

Not mentioned

48 hours for DNA content

Cell cycle phase distribution measured using DAPI staining

Turner et al., 2015

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Vijayaraghavan et al., 2017

Not mentioned

After treatment and recovery phases

Palbociclib induced G1 arrest; CCCA achieved in 90% at appropriate doses

Not mentioned

BrdU (decreased in palbociclib-treated cells); Cell cycle phase distribution by propidium iodide

DeMichele et al., 2014

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Tien & Sadar, 2021

Not mentioned

Not mentioned

Doubling time increased to >63 hours vs 25 hours (control)

Not mentioned

Cell cycle phase distribution: combination prevented G1 and G2-M progression, caused S-phase arrest

Asghar et al., 2017

Not mentioned

48 hours post-treatment

Cell cycle length: 20 hours (vehicle) vs 38 hours (palbociclib) in CDK2 high cells

2 hours post-cytokinesis, 48 hours post-treatment

CDK2 activity reporter

Kumarasamy et al., 2020

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Not mentioned

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The neoadjuvant trials provided the most detailed quantitative assessment of proliferative suppression. In the NeoPalAna trial, the complete cell cycle arrest rate (defined as Ki67 ≤2.7%) increased dramatically from 26% with anastrozole monotherapy to 87% after adding palbociclib. Similarly, Johnston et al. demonstrated that 90% of patients achieved complete cell cycle arrest with palbociclib plus letrozole compared to only 59% with letrozole alone. The median log-fold change in Ki-67 was significantly greater with combination therapy (-4.1 vs -2.2), corresponding to near-complete suppression of proliferation (-97.4% vs -88.5% geometric mean change). These findings indicate that CDK4/6 inhibition substantially enhances the antiproliferative effects of endocrine therapy beyond what can be achieved with endocrine therapy alone.

Mechanistic Foundations of Cell Cycle Arrest

Multiple studies investigated the molecular mechanisms by which palbociclib induces G1 arrest, with particular focus on the Rb pathway and downstream effectors.

Rb Pathway Modulation

Retinoblastoma (Rb) phosphorylation emerged as a central mechanistic determinant. Arnedos et al. demonstrated that palbociclib treatment led to significantly greater decreases in phospho-Rb compared to control, and changes in Ki67 correlated with changes in phospho-Rb (Spearman r=0.41). This relationship between Rb dephosphorylation and antiproliferative response suggests that early decreases in Rb phosphorylation could potentially identify patients with primary resistance. Kumarasamy et al. confirmed that activation of RB and inhibition of CDK2 activity emerged as determinants of sensitivity to CDK4/6 inhibition, with RB loss rendering cells completely independent of CDK4 and CDK6.

Ma et al. found that resistance to palbociclib was associated with persistent E2F-target gene expression, indicating ongoing Rb pathway activity despite CDK4/6 inhibition. Specifically, nonluminal subtypes demonstrated persistent elevation of CCND3, CCNE1, and CDKN2D, markers of continued E2F activity that bypass palbociclib’s mechanism.

Cell Cycle Checkpoint Proteins

The p27 protein emerged as a critical modulator of sensitivity. Kumarasamy et al. showed that protein levels of p27 were associated with cell cycle plasticity and correlated with sensitivity to CDK4/6 inhibition. Exogenous overexpression and pharmacologic induction of p27 via SKP2 inhibition or MEK/ERK pathway targeting enhanced the cytostatic effect of CDK4/6 inhibitors. In ER+ xenograft models, few cells retained RB phosphorylation during palbociclib treatment, which was associated with limited p27 protein levels, suggesting that p27 levels influence the durability of palbociclib’s effects.

Downstream Signaling Pathways

Maskey et al. identified mTORC1 activity as a critical determinant of cell fate during CDK4/6 inhibition. In CAMA1 cells, mTORC1 activity remained elevated during palbociclib treatment, while in MCF7 and T47D cells, mTORC1 was suppressed. Importantly, inhibition of mTORC1 signaling via rapamycin or Raptor knockdown during palbociclib treatment blocked the induction of complete senescence in CAMA1 cells. Genetic depletion of TSC2, a negative regulator of mTORC1, resulted in sustained mTORC1 activity during palbociclib treatment and evoked a complete senescence response in MCF7 cells, demonstrating that persistent mTORC1 signaling can convert reversible to irreversible growth arrest.

Vijayaraghavan et al. discovered that autophagy is induced as a stress response to palbociclib. Palbociclib treatment decreased pRb and total Rb levels, but autophagy activation served as a resistance mechanism by degrading reactive oxygen species and potentially reversing G1 arrest. Combined inhibition of CDK4/6 and autophagy produced synergistic effects, suggesting that autophagy represents an adaptive survival pathway during cell cycle arrest.

Clinical Efficacy Outcomes

Clinical efficacy varied by disease setting, with the most robust data emerging from trials in hormone receptor-positive breast cancer.

Neoadjuvant Setting

Study

Clinical Response Rate

Pathological Response

Time-Based Outcomes

Maintenance of Arrest

Ma et al., 2017

80% (exam), 41% (ultrasound), 52% (mammogram)

No pathologic complete responses; significant reduction in tumor stages

Not mentioned

Continuous therapy necessary to maintain CCCA

Johnston et al., 2019

CR+PR: 54.3% (palbociclib+letrozole) vs 49.5% (letrozole); PD: 3.2% vs 5.4%

Not mentioned

Not mentioned

Not mentioned

Arnedos et al., 2018

Antiproliferative response: 58% (palbociclib) vs 12% (control); Ki67 decrease significant

Not mentioned

Not mentioned

Not mentioned

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In the neoadjuvant setting, palbociclib demonstrated robust antiproliferative activity but variable impact on tumor size reduction. Johnston et al. found that adding palbociclib to letrozole significantly enhanced Ki-67 suppression but did not increase clinical response rates over 14 weeks, possibly related to concurrent reduction in apoptosis. Ma et al. achieved an 80% clinical response rate by physical examination, though ultrasound and mammogram assessments showed lower rates (41% and 52%, respectively). No pathologic complete responses were observed, but significant reductions in tumor stage occurred.

Advanced Disease Setting

Study

CBR

PFS

Overall Survival

Response Duration

Turner et al., 2015

Not mentioned

Median 9.2 months (palbociclib+fulvestrant) vs 3.8 months (placebo+fulvestrant); HR 0.42

Not mentioned

Not mentioned

Vijayaraghavan et al., 2017

Not mentioned

Palbociclib doubles PFS vs letrozole or fulvestrant alone; Rb+/LMWE- tumors: median 436.5 months with letrozole, 10.7 months with fulvestrant

Not mentioned

Not mentioned

DeMichele et al., 2014

19% overall, 21% in HR+, 29% in HR+/HER2- with ≥2 prior endocrine therapies

Median 3.7 months overall

Not mentioned

Not mentioned

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The PALOMA-3 trial demonstrated that palbociclib combined with fulvestrant resulted in significantly longer progression-free survival (9.2 vs 3.8 months, HR 0.42) in patients with hormone receptor-positive metastatic breast cancer who had progressed on prior endocrine therapy. Vijayaraghavan et al. reported that palbociclib doubled PFS compared to endocrine therapy alone, with particularly prolonged PFS in Rb-positive, low-molecular-weight cyclin E (LMWE)-negative tumors (median 436.5 months with letrozole). DeMichele et al. observed modest clinical benefit rates of 19% overall, increasing to 29% in HR+/HER2- patients who had failed at least two prior endocrine therapies, with median PFS of 3.7 months.

Preclinical Efficacy

Asghar et al. demonstrated tumor reductions in 7 out of 10 mice treated with palbociclib in the LAR subtype of triple-negative breast cancer xenografts. Cell cycle length was prolonged in CDK2-high cells (20 hours vehicle vs 38 hours palbociclib), though cells showed adaptation to CDK4/6 inhibition over time.

Cellular Consequences and Senescence Phenotypes

Studies revealed heterogeneity in cellular responses to CDK4/6 inhibition, with some cell lines undergoing reversible arrest while others entered irreversible senescence.

Reversibility of Growth Arrest

Maskey et al. demonstrated that ER+ breast cancer cell lines exhibited distinct responses to palbociclib: MCF7 and T47D cells showed reversible G1-phase arrest with an incomplete senescence phenotype, whereas CAMA1 cells underwent irreversible cell cycle arrest and complete senescence. This difference correlated with mTORC1 signaling patterns, with sustained activity promoting complete senescence. Ma et al. confirmed the reversibility in clinical samples, showing that Ki67 levels rebounded at surgery after palbociclib discontinuation, though this rebound was suppressed by continuing palbociclib therapy.

Vijayaraghavan et al. found that reversibility was dose-dependent: low doses of palbociclib resulted in reversible G1 arrest, while higher doses led to irreversible growth inhibition. Palbociclib induced senescence markers including increased SA-β-gal activity and cellular complexity, but critically, did not induce apoptosis as evidenced by lack of cleaved PARP and caspase activity.

Autophagy and Survival Responses

Autophagy emerged as an important adaptive mechanism. Vijayaraghavan et al. showed that palbociclib induced autophagy as a stress response, and combining palbociclib with autophagy inhibitors like hydroxychloroquine (HCQ) led to sustained growth inhibition and irreversible senescence without inducing apoptosis. SA-β-gal staining confirmed senescence induction both in vitro and in vivo.

Apoptosis Markers

Johnston et al. observed suppression of cleaved PARP with palbociclib plus letrozole treatment, indicating reduced rather than increased apoptosis. The median log-fold suppression of cleaved PARP was greater with combination therapy (-0.80 vs -0.42). This concurrent reduction in both proliferation and apoptosis may explain why enhanced antiproliferative effects did not translate to proportional increases in clinical response rates.

Predictive Biomarkers and Resistance Mechanisms

Substantial heterogeneity in treatment response prompted investigation of predictive biomarkers and resistance pathways.

Rb Status as a Core Biomarker

Retinoblastoma status emerged as the most fundamental predictor of response. Ma et al. identified RB1 mutations, particularly frameshift mutations, as associated with resistance to palbociclib in HER2-enriched tumors, though missense RB1 mutations were found in tumors sensitive to treatment. Kumarasamy et al. confirmed that RB loss rendered cells completely independent of CDK4/6 activity, with RB phosphorylation status serving as a marker for treatment failure. Vijayaraghavan et al. demonstrated that Rb-positive status predicted sensitivity, with Rb-positive/LMWE-negative tumors showing the longest progression-free survival. Asghar et al. confirmed that loss of RB1 caused resistance to CDK4/6 inhibition through disruption of the CDK4/6-RB1 axis that controls the restriction point in G1 phase.

Cyclin E and CDK2 Activity

Cyclin E levels, particularly the low-molecular-weight isoform (LMWE), predicted resistance. Ma et al. found that CCNE1 gain was associated with resistance to CDK4/6 inhibition, likely through CDK2 activation. Vijayaraghavan et al. showed that Rb-positive but LMWE-positive tumors had reduced sensitivity, with overexpression of LMWE conferring resistance. Asghar et al. revealed that high cyclin E1 expression activated CDK2 and was dysregulated in resistant cells, with palbociclib-resistant basal-like TNBC cells exiting mitosis directly into a proliferative state with high CDK2 activity, bypassing the need for CDK4/6. Johnston et al. noted that response to palbociclib was correlated with RB1 mutation status but occurred independently of PIK3CA or PTEN mutations.

Molecular Subtypes

Ma et al. demonstrated that luminal subtypes (LumA and LumB) were more responsive to palbociclib, while nonluminal subtypes including basal-like and HER2-enriched tumors showed resistance. Persistent E2F-target gene expression, indicated by elevated CCND3, CCNE1, and CDKN2D, was linked to resistance. Asghar et al. found that the luminal androgen receptor (LAR) subtype of triple-negative breast cancer was highly sensitive to CDK4/6 inhibition, while basal-like subtypes were resistant.

Downstream Signaling Pathways

Kumarasamy et al. identified p27 protein levels as associated with cell cycle plasticity and sensitivity, with targeting of the MEK/ERK pathway and SKP2 inhibition emerging as strategies to enhance response. Maskey et al. showed that sustained mTORC1 activity during palbociclib treatment promoted complete senescence, suggesting mTORC1 inhibition via rapamycin or Raptor knockdown as a combination strategy.

Combination Strategies to Overcome Resistance

Several effective combinations emerged from these studies:

Safety and Tolerability Profile

Hematologic toxicities, particularly neutropenia, dominated the adverse event profile across clinical trials, though these were generally manageable with dose modifications.

Study

Grade 3/4 Neutropenia

Grade 3/4 Other Cytopenias

Dose Modifications

Non-Hematologic Toxicities

Ma et al., 2017

G3: 22%, G4: 4%

Leukopenia mentioned

14% required dose reductions

Fatigue, rash; no G4+ non-hematologic AEs

Johnston et al., 2019

Part of 49.8% G3+ toxicity

Asymptomatic neutropenia primary cause

21.6% interruptions/delays, 2.0% dose reductions

Not specified

Turner et al., 2015

62.0%

Leukopenia 25.2%, anemia 2.6%, thrombocytopenia 2.3%

Discontinuation: 2.6% palbociclib, 1.7% placebo

Fatigue 2.0%; febrile neutropenia 0.6%

Vijayaraghavan et al., 2017

56%

Leukopenia 25.2%

Higher doses (75-150 mg/kg) caused significant weight loss

Not specified; combination with HCQ well tolerated

DeMichele et al., 2014

51%

Anemia 5%, thrombocytopenia 22%

51% dose reductions, 24% interruptions

Not mentioned; cytopenias uncomplicated and easily managed

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Turner et al. reported the highest rate of grade 3/4 neutropenia at 62.0%, compared to 0.6% in the placebo group, along with leukopenia (25.2%), anemia (2.6%), and thrombocytopenia (2.3%). Critically, febrile neutropenia remained rare at 0.6% in both treatment arms, and discontinuation rates due to adverse events were low (2.6% with palbociclib vs 1.7% with placebo). Johnston et al. found that 49.8% of patients experienced grade 3 or greater toxicity with palbociclib plus letrozole versus 17.0% with letrozole alone, primarily driven by asymptomatic neutropenia, with treatment interruptions or delays in 21.6% and dose reductions in only 2.0%.

DeMichele et al. emphasized that cytopenias were uncomplicated and easily managed with dose reduction, with 51% of patients requiring dose modifications and 24% experiencing treatment interruptions. Ma et al. observed grade 3 neutropenia in 22% and grade 4 in 4% of patients, with 14% requiring dose reductions due to neutropenia, elevated transaminases, or rash. No grade 4 or higher non-hematologic adverse events occurred.

Vijayaraghavan et al. noted in preclinical models that optimization of palbociclib dosing was crucial, as higher doses (75 or 150 mg/kg) caused significant body weight loss, but combination with hydroxychloroquine was well tolerated without changes in body weight or blood counts.

Synthesis

The body of evidence reveals consistent mechanisms but variable clinical outcomes that require reconciliation. While palbociclib uniformly induced G1 cell cycle arrest across studies, the durability of this arrest and translation to clinical benefit varied substantially based on molecular context, treatment duration, and combination strategies.

Context-Dependent Response Patterns

The heterogeneity in clinical benefit rates—ranging from 19% in heavily pretreated advanced disease to 87% complete cell cycle arrest in neoadjuvant settings—reflects differences in disease burden, prior treatment exposure, and measurement endpoints rather than conflicting results. In the neoadjuvant setting with less advanced disease, both Ma et al. and Johnston et al. achieved >85% complete cell cycle arrest rates, demonstrating maximal antiproliferative activity when palbociclib is combined with endocrine therapy in treatment-naive tumors. Conversely, DeMichele et al.’s lower clinical benefit rate of 19% occurred in patients with a median of 2 prior cytotoxic regimens, suggesting that heavily pretreated tumors may harbor additional resistance mechanisms beyond those addressed by CDK4/6 inhibition alone.

The progression-free survival benefit also follows a dose-response pattern by line of therapy. Turner et al. achieved 9.2 months median PFS in endocrine-resistant disease, while Vijayaraghavan et al. reported median PFS exceeding 400 months in optimal biomarker-selected populations (Rb+/LMWE-). Both findings are valid within their respective contexts: the former represents a clinically heterogeneous population without biomarker selection, while the latter represents a molecularly defined subset with intact G1/S checkpoint machinery.

Mechanisms Explaining Divergent Cellular Responses

The contrast between reversible and irreversible growth arrest observed by Maskey et al. and Vijayaraghavan et al. can be mechanistically explained through mTORC1 activity and autophagy induction. CAMA1 cells maintained elevated mTORC1 signaling during palbociclib treatment, leading to irreversible senescence, whereas MCF7 and T47D cells suppressed mTORC1 and experienced reversible arrest. This divergence is not contradictory but rather demonstrates that cellular context—specifically basal mTORC1 activity and the cell’s ability to modulate this pathway—determines whether CDK4/6 inhibition causes cytostatic or senescent outcomes. The finding that TSC2 depletion converted MCF7 cells from reversible to irreversible arrest provides direct mechanistic evidence that sustained mTORC1 activity is sufficient to drive complete senescence.

Similarly, Vijayaraghavan et al.’s observation that autophagy serves as an adaptive resistance mechanism explains why some cells escape permanent growth inhibition. Autophagy activation allows cells to survive metabolic stress during G1 arrest, potentially explaining the reversibility observed in certain contexts. The synergy between palbociclib and autophagy inhibitors validates that blocking this survival pathway converts cytostatic arrest into terminal senescence.

Biomarker-Driven Sensitivity Predictions

The apparent contradiction between studies reporting universal benefit and those identifying biomarker-dependent responses reflects measurement granularity rather than conflicting findings. While Turner et al. found consistent benefits across subgroups in their phase 3 trial, this population-level observation does not negate the molecular determinants identified in more detailed mechanistic studies.

Ma et al., Vijayaraghavan et al., and Asghar et al. converge on a unified model: intact Rb and absent or low cyclin E (particularly LMWE) predict maximal sensitivity, while RB1 loss or LMWE overexpression confers resistance. Kumarasamy et al.’s identification of p27 levels as modulators of sensitivity adds nuance to this model—p27 acts as a rheostat determining the threshold at which cells commit to arrest versus escaping through CDK2 activation. Asghar et al.’s single-cell analysis revealed that sensitive LAR cells exit mitosis with low CDK2 activity and require CDK4/6 for reentry, whereas resistant basal-like cells maintain high CDK2 activity post-mitosis, bypassing the CDK4/6 requirement. Both phenotypes coexist within TNBC but respond differently based on their intrinsic cell cycle dynamics.

Non-Linear Relationships: Proliferation vs. Clinical Response

Johnston et al.’s finding that enhanced Ki-67 suppression did not translate to increased clinical response rates initially appears contradictory but is explained by the concurrent suppression of apoptosis. The reduction in cleaved PARP indicates that while palbociclib profoundly arrests proliferation, it simultaneously protects cells from apoptotic death. This creates a cytostatic rather than cytotoxic effect, where tumor cells remain viable but non-proliferative. Over the 14-week treatment period, this translates to disease stabilization rather than tumor regression, explaining why complete cell cycle arrest rates of 90% do not correspond to 90% clinical responses.

This cytostatic mechanism also explains Ma et al.’s observation that continuous therapy is necessary to maintain antiproliferative effects. When palbociclib is discontinued, arrested cells with intact Rb and no terminal senescence can resume cycling once CDK4/6 activity is restored. The addition of cycle 5 palbociclib immediately before surgery suppressed Ki67 rebound, demonstrating that the arrest is maintained only through continuous CDK4/6 inhibition in populations that have not undergone irreversible senescence.

Combination Strategies Addressing Distinct Resistance Nodes

The diverse combination strategies emerging from these studies—autophagy inhibition, PI3K inhibition, MEK inhibition, and mTORC1 modulation—target mechanistically distinct resistance pathways that become activated during G1 arrest. Autophagy inhibition blocks stress-induced survival responses, PI3K inhibition addresses PIK3CA-mutant tumors where constitutive signaling bypasses G1 arrest requirements, and MEK inhibition upregulates p27 to lower the threshold for CDK4/6 dependence. These are not competing strategies but complementary approaches applicable to different molecular contexts.

The MEK combination is particularly rational given Kumarasamy et al.’s demonstration that p27 induction via MEK/ERK pathway inhibition enhances palbociclib efficacy. This mechanistically addresses the subset of tumors with low basal p27 that maintain cell cycle plasticity. Similarly, Tien & Sadar’s sequential dosing strategy in androgen receptor-positive cancers recognizes that different agents target different cell cycle phases—palbociclib delays G1-S transition while EPI-7170 targets S-phase cells—and sequential administration maximizes the proportion of cells arrested in each vulnerable phase.

Quality Hierarchy Considerations

The phase 3 PALOMA-3 trial by Turner et al. carries the greatest weight for establishing clinical efficacy in advanced HR+ breast cancer, with its randomized design, large sample size (n=521), and definitive progression-free survival benefit (HR 0.42). The neoadjuvant trials by Ma et al. and Johnston et al., though smaller phase 2 studies, provide complementary mechanistic insights through serial biopsies that were not feasible in the advanced disease setting. The preclinical studies by Maskey et al., Vijayaraghavan et al., Asghar et al., and Kumarasamy et al. elucidate mechanisms that inform biomarker development but require clinical validation. The convergence of clinical efficacy data from Turner et al. with mechanistic predictions from preclinical models (particularly regarding Rb status) strengthens confidence in these biomarkers despite coming from studies of varying design rigor.

Resolved Model of Palbociclib Action

Integrating these findings yields a coherent model: palbociclib reliably induces G1 arrest in Rb-proficient cells by preventing Rb hyperphosphorylation and maintaining E2F target suppression. The durability of this arrest depends on three factors: (1) CDK2 activity levels, with high CDK2/cyclin E bypassing the G1 block; (2) mTORC1 activity during arrest, with sustained signaling driving irreversible senescence and suppressed signaling allowing reversible arrest; and (3) autophagy induction as a survival response that can be targeted for combination therapy. Clinical benefit requires both achieving cell cycle arrest and maintaining it, explaining why continuous dosing is necessary and why biomarker selection (Rb+/LMWE-/adequate p27) identifies patients with maximal benefit.

References

Reeja S. Maskey, F. Wang, Elyssa Lehman, Yiqun Wang, N. Emmanuel, and 6 more\ (2020).Sustained mTORC1 activity during palbociclib-induced growth arrest triggers senescence in ER+ breast cancer cells. Cell Cycle

Cynthia X. Ma, F. Gao, Jingqin R Luo, D. Northfelt, M. Goetz, and 29 more\ (2017).NeoPalAna: Neoadjuvant Palbociclib, a Cyclin-Dependent Kinase 4/6 Inhibitor, and Anastrozole for Clinical Stage 2 or 3 Estrogen Receptor–Positive Breast Cancer. Clinical Cancer Research

N. Turner, J. Ro, F. André, S. Loi, S. Verma, and 9 more\ (2015).Palbociclib in Hormone-Receptor-Positive Advanced Breast Cancer. New England Journal of Medicine

S. Vijayaraghavan, Cansu Karakas, I. Doostan, Xian Chen, Tuyen N Bui, and 12 more\ (2017).CDK4/6 and autophagy inhibitors synergistically induce senescence in Rb positive cytoplasmic cyclin E negative cancers. Nature Communications

A. DeMichele, A. Clark, K. Tan, D. Heitjan, Kristi Gramlich, and 13 more\ (2014).CDK 4/6 Inhibitor Palbociclib (PD0332991) in Rb+ Advanced Breast Cancer: Phase II Activity, Safety, and Predictive Biomarker Assessment. Clinical Cancer Research

A. Tien, M. Sadar\ (2021).Cyclin-dependent Kinase 4/6 Inhibitor Palbociclib in Combination with Ralaniten Analogs for the Treatment of Androgen Receptor–positive Prostate and Breast Cancers. Molecular Cancer Therapeutics

U. Asghar, A. Barr, R. Cutts, M. Beaney, I. Babina, and 9 more\ (2017).Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer. Clinical Cancer Research

V. Kumarasamy, Paris Vail, Ram Nambiar, A. Witkiewicz, E. Knudsen\ (2020).Functional Determinants of Cell Cycle Plasticity and Sensitivity to CDK4/6 Inhibition. Cancer Research

S. Johnston, S. Puhalla, D. Wheatley, A. Ring, P. Barry, and 32 more\ (2019).Randomized Phase II Study Evaluating Palbociclib in Addition to Letrozole as Neoadjuvant Therapy in Estrogen Receptor-Positive Early Breast Cancer: PALLET Trial. Journal of Clinical Oncology

M. Arnedos, M. Bayar, B. Cheaib, Véronique Scott, I. Bouakka, and 14 more\ (2018).Modulation of Rb phosphorylation and antiproliferative response to palbociclib: the preoperative-palbociclib (POP) randomized clinical trial. Annals of Oncology

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Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer

U. Asghar, A. Barr, R. Cutts, M. Beaney, I. Babina, D. Sampath, J. Giltnane, J. Lacap, Lisa M. Crocker, Amy E. Young, A. Pearson, M. T. Herrera-Abreu, C. Bakal, N. Turner

Clinical Cancer Research·

2017·

217 citations

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Study Design

- Study type: Preclinical - Setting: Cell lines (in vitro), xenografts (in vivo) - Cancer type and subtype: Triple-negative breast cancer (TNBC), specifically luminal androgen receptor (LAR) subtype - Sample size: Not explicitly mentioned for in vivo study; multiple mice per treatment group

CDK4/6 Inhibitor Details

- Specific CDK4/6 inhibitor used: Palbociclib - Dose and schedule: 500nmol (in vitro), 50 mg/kg daily (in vivo) - Duration of treatment: At least 2 weeks (in vitro), 21 consecutive days (in vivo) - Combination therapies: Pictilisib, taselisib, AZD2014 - Administration route and timing: Oral (in vivo), not specified (in vitro)

Cell Cycle Arrest Measurements

- Cell cycle phase distribution: Not mentioned - Proliferation markers: Not mentioned - Cell viability and growth inhibition metrics: Palbociclib prolongs cell cycle length in CDK2 high cells (20 hours vehicle vs. 38 hours palbociclib) - Time points measured: 2 hours post-cytokinesis, 48 hours post-treatment

Mechanism Analysis

- Rb pathway activity: Loss of retinoblastoma protein (RB1) causes resistance to CDK4/6 inhibition. The CDK4/6-RB1 axis controls transition through the restriction point in the G1 phase. - Cyclin D1/CDK4/CDK6 expression and activity: Not explicitly mentioned in the context of CDK4/6 inhibition. - Cell cycle checkpoint proteins: p16, p21, p27 not specifically mentioned in relation to CDK4/6 inhibition. - Downstream signaling pathways: PI3 kinase signaling is synergistic with CDK4/6 inhibition in PIK3CA-mutant TNBC. - Molecular targets and biomarkers: Cyclin E1 expression is a potential biomarker for resistance to CDK4/6 inhibition.

Clinical Outcomes

- Response rates: Tumour reductions observed in 7 out of 10 mice in the palbociclib treatment arm. - Clinical benefit rate: Prolongation of cell cycle length in CDK2 high cells. - Pathological response: Not mentioned. - Time to progression and overall survival: Not mentioned. - Response duration and maintenance of cell cycle arrest: Adaptation to CDK4/6 inhibition over time.

Senescence and Cell Fate

Not mentioned (the paper does not provide information on senescence markers, apoptosis markers, autophagy activation, or long-term cell fate outcomes related to CDK4/6 inhibition)

Resistance and Predictive Factors

- Predictive biomarkers: Rb status (loss of RB1 causes resistance), cyclin E levels (high levels indicate resistance), PIK3CA mutations (synergy with PI3 kinase inhibitors) - Resistance mechanisms and pathways: High CDK2 activity post-mitosis, deregulation of cyclin E1 expression - Factors associated with treatment failure: Basal-like subtype, high cyclin E1 expression - Molecular subtypes or patient characteristics affecting response: LAR subtype is sensitive, basal-like subtype is resistant - Combination strategies to overcome resistance: CDK4/6 inhibitors with PI3 kinase inhibitors in PIK3CA-mutant TNBC

Toxicity Profile

Not mentioned (the paper does not provide a detailed toxicity profile for CDK4/6 inhibitor treatment)

Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity. Experimental Design: A panel of cell lines representative of TNBC was tested for in vitro and in vivo sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging. Results: The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both in vitro (P < 0.001 LAR vs. basal-like) and in vivo in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in PIK3CA-mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes. Conclusions: Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC. Clin Cancer Res; 23(18); 5561–72. ©2017 AACR.

Translational relevance:

Currently there are no effective targeted therapies for triple negative breast cancer (TNBC).

We show that the Luminal Androgen Receptor subtype of TNBC is highly sensitive to CDK4/6 inhibition whilst basal-like TNBC tumours are resistant. Differential sensitivity of TNBC subtypes to CDK4/6 inhibition is shown to be a result of cells exiting mitosis into an active proliferating or a quiescent state. Basal-like TNBCs have a high proportion of active proliferating cells that are resistant to CDK4/6 inhibition, which is promoted by dysregulation of cyclin E1 expression. Our results identify novel therapeutic approaches for TNBC, and identify mechanisms of sensitivity to CDK4/6 inhibitors.

Downloaded from http://aacrjournals.org/clincancerres/article-pdf/doi/10.1158/1078-0432.CCR-17-0369/2114392/1078-0432\_ccr-17-0369v3.pdf by guest on 10 October 2025

Introduction:

The CDK4/6 -RB1 axis controls transition through the restriction point in the G1 phase of the cell cycle, and cancers frequently subvert the regulation of this axis to promote proliferation (1,2). CDK4/6 inhibition is a proven therapeutic strategy for oestrogen receptor positive (ER+ve) breast cancers (3,4), with selective CDK4/6 inhibitors (palbociclib and ribociclib) demonstrating substantial improvements in progression free survival (PALOMA1(3), PALOMA2 (5), PALOMA3(4) and MONALEESA-2 (6)) in phase two and three clinical trials.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer, which is associated with a poor prognosis. Although TNBC may be sensitive to chemotherapy, there is still a substantial need to identify novel targeted therapeutic strategies. TNBC is a heterogeneous group of tumours and gene expression profiling has identified distinct subgroups (7,8), including luminal androgen receptor (LAR), mesenchymal stem like (MSL), mesenchymal (MES), and basal-like (7). The majority of TNBC fall within the dominant basal-like and MES subgroups. TNBC are a highly proliferative group of tumours enriched for high expression of cell cycle genes (7), yet as a heterogeneous subtype, are considered to be resistant to CDK4/6 inhibition (9), as are many other tumour types.

The determinants of sensitivity to CDK4/6 inhibition are poorly understood. Loss of retinoblastoma protein (RB1) causes resistance to CDK4/6 inhibition (10), however for the majority of cancers, the factors that determine sensitivity or resistance to CDK4/6 inhibitors are unclear. Recent studies of cell cycle dynamics have redefined our understanding of the G1-S phase transition in asynchronously dividing cells (11)(12)(13), with cells at mitotic exit entering either a quiescent or an active-proliferative state (12,13). Here we show that cell cycle exit into a quiescent or proliferative state is a major factor determining sensitivity to CDK4/6 inhibitors. We identify subgroups of TNBC that are highly sensitive to CDK4/6 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/doi/10.1158/1078-0432.CCR-17-0369/2114392/1078-0432\_ccr-17-0369v3.pdf by guest on 10 October 2025 inhibition, and using a CDK2 activity live-cell reporter (12) we show that CDK2 activity after mitotic exit dictates sensitivity to CDK4/6 inhibition.

Cell lines

Cell lines were obtained from ATCC or Asterand and maintained according to the manufacturer's instructions. Cell lines were banked in multiple aliquots on receipt, identity confirmed by STR profiling with the PowerPlex 1.2 System (Promega) and tested for mycoplasma every two weeks. After extracting cells from liquid nitrogen, the cell lines were passaged twice prior to use in experiments.

Palbociclib-resistant MFM223pR cells were generated by chronic exposure to increasing concentrations of palbociclib (100, 250, 500, 1000nmol) over 4 months. Drug treatments were replaced every 3-4 days with fresh media.

Antibodies, reagents and constructs

Phospho-RB1 S807/811 (8516), RB1 (9313), Cyclin E1 (HE12; 4129), Cyclin E2 (4132), CDK2 (2546), phospho-CDK2 T160 (2561); CDK4 (12790), Androgen Receptor (3202) were all Cell Signalling Technology, Danvers, MA; p16 F-12 (SC-1661, Santa Cruz), β-actin (A5441, Sigma); Cyclin E1 (ab33911) and c-myc (ab32072) were Abcam. Western blot analysis was performed using pre-cast 4-12% SDS gels, as described previously (14).

Densitometry analysis was performed on western blot films using ImageJ software (National Institute of Health, USA), and expressed relative to their corresponding loading control.

Palbociclib (PD-0332991; SelleckChem) was used at 500nmol, pictilisib (GDC-0941; SelleckChem) at 200nmol, and taselisib (GDC-0032; Genentech) at 100nmol, unless otherwise stated. Palbociclib 500nmol was used for the majority of experiments as previously (10). No increase in effect on clonogenic growth was observed with doses above 500nmol (Fig. 1A ). siRNAs were from Thermofisher Scientific: siCCNE1 (#4390824, #4390824, #1299001), siCON1 (#4390843), siCON2 (#4390846) and siUBB (# 4390824).

The CDK2 activity live-cell sensor (CDK2L-GFP) was generated by cloning the C-terminal PSLD region of Human DNA Helicase B (DHB; 957-1087 amino acids) into pIRES-GFP puromicin as described previously (11). PCNA was tagged at the N terminus with the modified LSS2-mKate fluoro-phore to generate LSS2-mKate-PCNA (15).

Cell Transfections

For CDK2 activity sensor experiments, cells were transfected with CDK2L-GFP using Lipofectamine ® 2000 according to the manufacturer's instructions, 48-72 hours prior imaging. For siRNA knockdown experiments, SUM149 cells were transfected with CDK2L-GFP on day 1, then GFP-positive cells (3000 cells/well) were FACS-sorted into 384-well plate on day 3, and next siRNA transfection using siCON1 and siCCNE1 was performed with Lipofectamine ® RNAiMAX on day 6, followed by time-lapse imaging on days 9-11. As previously demonstrated, the CDK2L-GFP sensor is not phosphorylated by CDK4/6 (11), nor by CDK1 (12).

Time-lapse microscopy

CDK2L-GFP positive cells were FACS sorted into 384 well plates, with 1000 to 3000 cells seeded per well, 24 hours prior time-lapse imaging. Unless specified, drugs or vehicle were added immediately prior time-lapse experiments. Images were taken on a High-content Opera Spinning Disk confocal microscope (PerkinElmer) with 40X water objective, every 10 minutes, in a NA 0.9 humidified environmental chamber at 37 0 C and 5% CO 2 .

To assess the impact of palbociclib on S phase entry in SUM149 cells transfected with CDK2L-GFP (Fig. 3A ), palbociclib or vehicle (DMSO) was added 8 hours after initiation of time-lapse imaging, with continued imaging for a further 48 hours. Analysis was restricted to cells that underwent mitosis 1-3 hours prior to adding palbociclib or vehicle (DMSO).

To study stability of CDK2 activity in individual cell clones (Fig. 5D and Supplementary Fig. 3A ), a single cell was FACS-sorted into each well of a 96 well plate, with single cell sorting confirmed by bright field microscopy. Plates were incubated for 4 weeks to generate clonal populations. Individual wells were transfected with CDK2L-GFP, and 48 hours later imaged using time-lapse microscopy. All examined clonal populations were STR typed.

Image analysis

Time frames of cells transfected with CDK2L-GFP or LSS2-mKate-PCNA were captured at 10 minutes intervals over a 48-72 hours period. Dynamic changes of CDK2L-GFP or LSS2-mKate-PCNA were manually tracked in individual cells using Volocity (PerkinElmer). To quantify CDK2 activity, both nuclear and peri-nuclear cytoplasmic CDK2L-GFP intensity were measured simultaneously. CDK2 activity for each time point was calculated as a ratio of mean GFP fluorescent intensity in the cytoplasm divided by the mean GFP fluorescent intensity in the nucleus. For representation, asynchronous single cell CDK2 activity traces were aligned in silico to time of cytokinesis as T0 (time-point = 0). The CDK2 activity traces were smoothed with a window of 4 data points minimize background noise.

Cell cycle length (hours) was calculated as the time from first cytokinesis to the second cytokinesis. Unless stated otherwise, post-mitotic CDK2 activity was assessed as the CDK2 activity at 2 hours post-cytokinesis, as the first time-point across imaged cell lines that allowed reliable quantification of CDK2 activity after reformation of the nuclear membrane and cell flattening (11). Cells with CDK2 activity <0.6 at 2 hours post cytokinesis were defined as CDK2 low , and cells with CDK2 activity >0.6 at 2 hours points post cytokinesis were defined as CDK2 high , as previously described (12). In PCNA tracking experiments Sphase entry was defined a sharp increase in PCNA intensity with the appearance of nucleoli, as previously described (11). Accumulation of CDK2 activity to a ratio of 1 was used as surrogate for cell cycle entry, as previously demonstrated (12).

Immunofluorescence

Immediately after time lapse, cells were fixed in 4% paraformaldehyde, washed three times in PBS, permeabilized with 0.2% Triton X-100, and stained at room temperature using mouse and rabbit primary antibodies, detected with corresponding fluorescent secondary antibodies: anti-mouse Alexa Fluor-555 and anti-rabbit Alexa Fluor-647. Nuclear pixels were measured as an output of intensity for each cell, using Columbus™ image data storage and analysis system.

Nuclear cyclin E1 protein levels in individual LAR MDAMB453 cells and basal-like SUM149 cells 1-2 hours after mitosis (Fig. 4D ), was determined by time-lapse imaging of CDK2L-GFP positive cells (24 hours), followed by immunofluorescence for cyclin E1. Nuclear cyclin E1 protein levels were quantified by measuring the intensity of the immunofluorescence signal with Columbus™ imaging software, specifically in cells that had undergone mitoses 1-2 hours prior to fixation.

Immunohistochemistry

For immunohistochemical analysis of MDAMB453 xenografts, tumours were extracted 4 hours post-dose and formalin fixed for immunohistochemistry (IHC). Each sampling time point includes 4 animals per treatment group. IHC was performed on 4um thick formalinfixed, paraffin-embedded tissue sections mounted on glass slides. For cleaved caspase 3 (Cell Signaling Technologies, Danvers, MA), staining was performed on a DAKO autostainer. Sections were treated with DAKO Target Retrieval (Dako; Carpinteria, CA), incubated with primary antibody at 0.12ug/ml overnight at 4°C followed by biotinylated goat anti-rabbit IgG (Vectorlabs, Burlingame, CA) and detected with Vectastain ABC-HRP (Vectorlabs, Burlingame, CA). For phospho-S6, IHC was performed on the Ventana Discovery XT Autostainer platform (Ventana Medical Systems Inc, Tucson, AZ). The slides were pre-treated with CC1, standard time, followed by anti-phospho-S6 (Cell Signaling Technologies, Danvers, MA), incubated at 0.26ug/ml for 32 minutes at 37°C. The antibody was detected with anti-rabbit-UltraMap (Ventana Medical Systems Inc, Tucson, AZ).

Staining was visualized with DAB. Sections were counter stained with hematoxylin, dehydrated, cleared and cover-slipped for viewing.

Assessment of viability and proliferation

All clonogenic assays were conducted in triplicates of 6-well plates, with 1000 to 5000 cells seeded per well 24 hour prior to exposure to the indicated drug concentrations, or vehicle.

Wells were treated continuously for at least 2 weeks replacing media/drug every 3-4 days.

Plates were fixed with tricyclic acid (10%), stained with sulforhodamine B (SRB) and absorbance measured. Absorbance for drug treated wells was expressed relative to the control wells, with subtraction of the background SRB absorbance from an empty well. The mean of at least three replicate wells was calculated for each dose/combination. Synergy was assessed using SRB absorbance from long-term clonogenic assays. Wells were treated every 3-4 days with palbociclib: 0, 100, 250, 500 or 750nmol, and/or pictilisib: 0, 100, 200, 400, 500 or 1000nmol. For AZD2014, wells were treated every 3-4 days with palbociclib: 0, 100, 250, 500 or 750nmol, and/or AZD2014: 0, 50, 100, 200, 400 and 750nmol. Assessment of compound synergy was conducted using Bliss independence score. A Bliss additivity score of <-1.0 was consider synergistic for that combination of drug concentrations. A cell line was considered to show drug synergy if at least 3 different combinations of drug concentrations were synergistic.

Tumour xenografts

In vivo efficacy and pharmacodynamic studies were approved by Genentech's Institutional Animal Care and Use Committee (IACUC) and adhered to the ILAR Guide for the Care and Use of Laboratory Animals. Naïve female C.B-17 SCID mice (Charles River Laboratories, San Diego, CA) were inoculated into the right 2/3 mammary fat pad with 20 million MDAMB453 cells suspended in a 1:1 ratio of HBSS and phenol red-free matrigel (BD Biosciences, San Jose, CA). Once tumours reached a mean volume of about 300 mm 3 , mice with similarly sized tumours were distributed into treatment cohorts (n=10/group). Mice were dosed daily and orally, with vehicle [0.5% methylcellulose/0.2% tween-80 (MCT)], 5 mg/kg taselisib (GDC-0032), 50 mg/kg palbociclib or the combination of taselisib and palbociclib for 21 consecutive days. Length (l) and width (w) of each tumour were measured using digital calipers (Fred V. Fowler Company, Inc., Newton, MA) and tumour volumes were calculated based on the following formula: tumour volume = l x w 2 x 0.5.

Analysis of publically -available data sets

The METABRIC dataset (n=1991) was obtained by application to the European Genomephenome archive (16). 320 putative TNBC samples were normalised using the beadarray package (17) and classified using TNBC type (18). 5 samples were then removed as putative ER positive samples leaving 315 for analysis. Segmented (CBS) copy number logR ratios were downloaded and used for copy number analysis, with gain/loss thresholds as previously defined (16). Heat maps representing key cell cycle genes were generated in R. For 102 TCGA samples representing TNBC, level 3 RNA-seq data (raw gene counts) was downloaded from the TCGA web site for these samples. The gene counts were normalised using edgeR packages. Copy number, mutation and RPPA data was extracted from cBioPortal using the CGDS-R (http://www.cbioportal.org/cgds\_r.jsp). Additional data was downloaded from the Cancer Proteome Atlas project (17).

Luminal Androgen Receptor (LAR) subtype of triple negative breast cancer is sensitive to CDK4/6 inhibition

We investigated whether the different molecular subgroups of TNBC were sensitive to CDK4/6 inhibition. Clonogenic assays were performed on a panel of 12 RB1 wild-type TNBC cell lines and one RB1 mutant cell line (BT549), with the CDK4/6 inhibitor palbociclib (Fig. 1A and Fig. 1B ). Cell lines from the dominant basal-like and mesenchymal (MES) subgroups of TNBC were resistant to palbociclib, whereas the LAR TNBC cell lines were highly sensitive to palbociclib (p<0.0001 basal-like vs. LAR), and ribociclib (Fig. 1C ), with sensitivity similar to the oestrogen positive cell line MCF7 (Fig. 1B ). In BrdU proliferation assays, palbociclib had a substantially greater effect on S phase entry in LAR cell lines compared to basal-like cell lines (Fig. 1D ). The sensitivity of LAR tumours to palbociclib was investigated in vivo in MDAMB453 xenografts. Tumour reductions were observed after the initial nine days of consecutive dosing (palbociclib oral 50mg/kg), with reduction in tumour size observed in 7 out of 10 mice in the palbociclib treatment arm (Fig 1E ).

Palbociclib sensitive cell lines have low post-mitotic CDK2 activity

We hypothesized that sensitivity to CDK4/6 inhibition would be determined by the level of CDK2 activity post-mitosis (12,13). To investigate this, we used a live cell fluorescent sensor to measure CDK2 activity (CDK2L) in five RB1 wild type TNBC models (Fig. 2A ). The CDK2L sensor specifically reports CDK2 activity, and is not phosphorylated by CDK4/6 and nor CDK1 (Methods). the first two hours post-mitosis (Fig. 2B and Supplementary Fig. 1A ). The dynamics of CDK2 activity after mitosis in the LAR cells were similar to CDK2 activity levels seen in the ER+ve MCF7 cell line (Supplementary Fig. 1B ). In contrast, the palbociclib-resistant basal-like cell lines SUM149 and HCC1143 (Fig. 2C and Supplementary Fig. 1C ), and the MES cell line CAL51 (Supplementary Fig. 1D ), were predominately composed of cells that exited mitosis and started the next cell cycle with high baseline levels of CDK2 activity, which then rapidly accumulated.

To compare CDK2 activity between cell lines, CDK2 activity levels were quantified 2 hours post-cytokinesis, the earliest time-point that allowed accurate quantification after nuclear envelope reformation. Basal-like TNBC cell lines had significantly higher CDK2 activity compared to the LAR cell lines (p<0.0001; Fig. 2D ). Cells which exited mitosis with CDK2 activity <0.6 at 2 hours post cytokinesis were defined as CDK2 low , and cells with CDK2 activity >0.6 at 2 hours post cytokinesis were defined as CDK2 high (12). Whereas LAR cells exited mitosis with a relatively homogeneous CDK2 low phenotype, basal-like cells existed mitosis with heterogeneous levels of CDK2 activity including a large proportion of CDK2 high cells (Fig. 2D ). In basal-like and MES cell lines, CDK2 high cells had substantially shorter cell cycles compared to CDK2 low cells (p<0.0001; Fig. 2E and Supplementary Table 1 ). These results suggest that TNBC cell lines resistant to CDK4/6 inhibition are predominantly composed of actively proliferating CDK2 high cells, whereas TNBC cell lines sensitive to CDK4/6 inhibition were predominantly composed of more quiescent CDK2 low cells.

The proliferative CDK2 high subpopulation drives resistance to CDK4/6 inhibition at the single cell level

We hypothesised that the CDK2 high subpopulation would be resistant to CDK4/6 inhibitors.

The TNBC basal-like SUM149 model had a heterogeneous mix of CDK2 mitotic exit phenotypes with predominately CDK2 high cells and a smaller fraction of CDK2 low cells, and thus represented a good model to test this hypothesis. To assess the effect of palbociclib on Downloaded from http://aacrjournals.org/clincancerres/article-pdf/doi/10.1158/1078-0432.CCR-17-0369/2114392/1078-0432\_ccr-17-0369v3.pdf by guest on 10 October 2025 the CDK2 high and CDK2 low populations, SUM149 cells expressing the CDK2L sensor were imaged for 8 hours prior to the addition of palbociclib or vehicle, and only cells that completed cytokinesis 1-3 hours prior to palbociclib addition were tracked. CDK2 activity was assessed in individual cells for a further 48 hours post-treatment (methods). We observed that SUM149 cells that exited mitosis with a CDK2 low phenotype were blocked from entering the cell cycle by palbociclib over the 48 hours duration of time lapse imaging (Fig. 3A ). In contrast, cells that exited mitosis in a CDK2 high state successfully entered the cell cycle despite palbociclib treatment and subsequently underwent a second mitosis (Fig. 3A and Fig. 3B ). Therefore, although the SUM149 cell line was intrinsically resistance to palbociclib in long-term clonogenic assays, the sub-fraction of CDK2 low cells were sensitive to palbociclib. Palbociclib did prolong cell cycle length in CDK2 high cells (mean cell cycle length 20 hours vehicle versus 38 hours palbociclib, Fig. 3A ) suggesting that CDK4/6 did facilitate, but was not essential for S-phase entry. After 14 days of chronic treatment with palbociclib, the cell cycle length for the SUM149 cells returned towards pre-treatment levels, suggesting adaption to CDK4/6 inhibition (Supplementary Fig. 1E and 1F ). Collectively this data demonstrates that post-mitotic CDK2 activity dictates sensitivity to CDK4/6 inhibition.

CDK2 high cells have sufficient CDK2 activity to bypass the restriction point, the point where CDK4/6 activity is necessary for cell cycle re-entry, resulting in CDK4/6 inhibitor resistance.

We next established whether CDK2 activity at mitotic exit changed after developing acquired resistance to CDK4/6 inhibitors in the LAR cell lines. We generated palbociclib-resistant cells from the MFM223 cell line (MFM223pR) via four months of chronic palbociclib exposure. In clonogenic assays, the MFM223pR cells were resistant to palbociclib, with a greater proportion of cells in S-phase during palbociclib treatment compared to the parental palbociclib sensitive cell line MFM223 (Fig. 3C ). The MFM223pR model acquired higher protein levels of cyclin E1 and activating CDK2 T160 phosphorylation (Fig. 3D ). In MFM223pR cells, a new CDK2 high proliferative subpopulation emerged (Fig. 3E ) suggesting Downloaded from http://aacrjournals.org/clincancerres/article-pdf/doi/10.1158/1078-0432.CCR-17-0369/2114392/1078-0432\_ccr-17-0369v3.pdf by guest on 10 October 2025 that the mechanism of acquired resistance to palbociclib was due to a higher proportion of cells adopting the CDK2 high phenotype.

Temporal dysregulation of Cyclin E1 expression in TNBC cells drives higher CDK2 activity post mitosis

To explore the molecular determinants of CDK2 high cells, we profiled our panel of TNBC cell lines. The LAR cell lines had both high expression of the androgen receptor (AR) (p=0.01) and absent/low expression of cyclin E1 (p=0.02) (Fig. 4A ). We profiled TNBC tumours using publically available datasets. In both the METABRIC (16) and TCGA (19) datasets TNBC LAR tumours had significantly lower transcriptomic expression levels of CCNE1 (p<0.0001) and CDK2 (p<0.0001), with higher CDKN1A (p21) levels, as compared to basal-like TNBC (p=0.06) (Fig. 4B , Supplementary Figures 2A and 2B ). Basal-like TNBC tumours frequently had increased CCNE1 gene copy number than observed in the LAR tumours (p=0.008 Fisher's exact test, Fig. 4B ). There was a high correlation between cyclin E1 mRNA with protein levels (r=0.89; Supplementary Fig. 2C and 2D ).

Cyclin E1 is tightly regulated nuclear protein, periodically expressed during the cell cycle, with the highest levels occurring during late G1 and early S-phase in non-cancer models (20). We investigated the temporal regulation of cyclin E1 in palbociclib sensitive and resistant cell lines, in order to assess whether deregulation of cyclin E1 expression in early G1 promoted the CDK2 high population. To test this we assessed the expression of nuclear cyclin E1 protein in individual cells 1-2 hours post-mitosis by dual immunofluorescence with the CDK2L sensor (see methods). Basal-like SUM149 cells had aberrantly high cyclin E1 expression post-mitosis (Fig. 4C -4D and Supplementary Fig. 2E ) compared to the LAR MDAMB453 cells that had uniformly low cyclin E1 expression. This was substantially earlier than the time point where SUM149 cells typically entered S-phase, confirmed by using a PCNA sensor (Supplementary Fig. 2F ). CDK2 high SUM149 cells had higher levels of cyclin E1 protein expression than CDK2 low SUM149 cells (Fig. 4E ). Silencing of cyclin E1 in SUM149 cells resulted in the loss of the CDK2 high population post-mitosis and induced a CDK2 low phenotype (Fig. 4F and Supplementary Fig. 2G ). Furthermore, silencing of cyclin E1 sensitised CAL51 (Fig. 4G and Supplementary Fig. 2H ), SUM149 (Supplementary Fig. 2I ) and MFM223pR cells to palbociclib, with minimal effects upon parental MFM223 cells which had low levels of cyclin E1 expression (Fig. 4H ). Silencing of cyclin E1 in the absence of CDK4/6 inhibition did not substantially reduce BrdU incorporation, likely due to redundancy between different CDKs and cyclins. Our data suggested that aberrant expression of cyclin E1 immediately post-mitosis, promoted the CDK2 high phenotype and resistance to CDK4/6 inhibition.

The CDK2 high phenotype is determined pre-mitosis

We next addressed whether post-mitotic activity was determined pre-mitosis, as previously shown in non-cancer models (12). SUM149 and CAL51 sister cell pairs, generated from the same mitosis, shared similar post-mitotic CDK2 activity (Fig. 5A and Fig. 5B ) suggesting that the level of CDK2 activity post-mitosis was determined prior to cytokinesis. SUM149 cells with higher pre-mitotic CDK2 activity (2 hours prior to mitosis) generated daughter cells that entered S-phase despite CDK4/6 inhibition (Fig. 5C ), whereas cells with lower pre-mitotic CDK2 activity were arrested by CDK4/6 inhibition (p=0.016).

Having demonstrated that post-mitotic CDK2 activity was determined pre-cytokinesis, we investigated whether this was due to the existence of a fixed sub-population of CDK2 high cells, or whether CDK2 high and CDK2 low populations could inter-convert. To test the long-term stability of the CDK2 low and CDK2 high subpopulations, we FACS sorted single cells from the SUM149 cell line (Fig. 5D ) and CAL51 cell line (Supplementary Fig. 3A ) into 96 well plates, confirmed single-cell seeding by microscopy, and assessed CDK2 activity in the resulting Downloaded from http://aacrjournals.org/clincancerres/article-pdf/doi/10.1158/1078-0432.CCR-17-0369/2114392/1078-0432\_ccr-17-0369v3.pdf by guest on 10 October 2025 single cell clones after four weeks of multiplication. In general, clonal populations recapitulated variability in CDK2 high and CDK2 low populations despite arising from a single cell (Fig. 5D and Supplementary Fig. 3 ). The fraction of CDK2 high cells was the most prevalent phenotype (50 cells measured/well) across three of the four wells imaged in SUM149. In contrast we identified a clonal SUM149 population that was robustly CDK2 low (well E3) at a single time point, 2 hours post-cytokinesis, although it was not addressed whether greater heterogeneity in CDK2 activity could develop over time. Overall these results suggest that post-mitotic CDK2 high activity is determined pre-mitosis, but the CDK2 high and CDK2 low subpopulations are not distinct fixed populations, with cells interconverting between the 2 phenotypes over many generations.

The SUM149 cells have a BRCA1 mutation (21), which may have elevated levels of DNA damage. We hypothesized that the E3 clone, which maintained a large population of CDK2 low cells, may have increased levels of DNA damage. Immunofluorescence staining for markers of DNA damage revealed that the SUM149 E3 clonal cell population, had a higher percentage of gamma H2AX-, 53BP1-and p21-positive cells, compared to the F7 clonal population (Fig. 5E and Fig. 5F ). Parental SUM149 cells that exited mitosis with CDK2 low phenotype were p21 positive by immunofluorescence (Fig. 5G ), as previously observed in non-cancer models (12). These results suggest that DNA damage and p21 expression may in part determine CDK2 activity state at mitotic exit (22).

Inhibition of PI3 kinase signalling is synergistic with CDK4/6 inhibitors, in PIK3CA mutant TNBC models

The non-basal, LAR and MSL subtypes of TNBC are substantially enriched with activating mutations in the PI3 kinase catalytic subunit PIK3CA gene (7), and therefore we investigated the therapeutic potential of inhibiting the PI3 kinase pathway in TNBC. A synergistic interaction (Bliss additivity score < -1.0) was observed between the pan class I PI3 kinase inhibitor pictilisib (GDC0941) and palbociclib in PIK3CA mutant TNBC cell lines (Fig. 6A ), as recently reported by others and us in ER+ve cell lines (10,23) and the MDAMB453 LAR cell line (23). Combination synergy was not observed in PIK3CA wild-type cell lines nor the RB1 mutant cell line BT549. Synergy was also observed with mTOR inhibitor AZD2014palbociclib combinations in PIK3CA mutant and some PIK3CA wild-type TNBC cell lines (Supplementary Fig. 4A ). We further validated the efficacy of the drug combination with the α-selective PI3 kinase inhibitor taselisib (GDC0032), which substantially sensitised the MSL cell line SUM159 to palbociclib in clonogenic assays (Fig. 6B ) and BrdU proliferation assays (Supplementary Fig. 4B ). This data suggested that combinations of PI3 kinase pathway

4C).

Finally, we examined whether PI3 kinase inhibition with taselisib affected post-mitotic CDK2 levels using the PIK3CA mutant MES CAL51 cell line. Taselisib decreased post-mitotic CDK2 activity with a greater proportion of cells exiting mitosis with the CDK2 low phenotype (Fig. 6E ). Pre-mitotic CDK2 levels influenced entry into the cell cycle post-mitosis (Supplementary Fig. 4D ). This data suggested that inhibition of PI3 kinase signalling sensitised to CDK4/6 inhibition, in part, as PI3 kinase inhibition suppressed post mitotic Downloaded from http://aacrjournals.org/clincancerres/article-pdf/doi/10.1158/1078-0432.CCR-17-0369/2114392/1078-0432\_ccr-17-0369v3.pdf by guest on 10 October 2025 CDK2 activity, inducing a CDK2 low quiescent state where CDK4/6 activity was required to initiate the cell cycle.

Discussion

We have shown that the luminal androgen receptor (LAR) subgroup of triple negative breast cancers (TNBC) is highly sensitive to CDK4/6 inhibition in vitro and in vivo (Fig. 1 ).

Sensitivity to CDK4/6 inhibition is dictated at the single cell level, with resistance to CDK4/6 inhibitors arising from cancer cells that exit mitosis directly into a CDK2 high proliferative state, from which CDK4/6 is not necessary for cell cycle re-entry (Fig. 2C and Fig. 3A ).

Our results further extend prior work in non-cancer models that demonstrate a biphasic exit of cells into proliferative and quiescent states after mitosis (12,13). Cells that exit mitosis with a CDK2 low phenotype enter into a quiescent state, requiring CDK4/6 activity to initiate reentry into the cell cycle (Fig. 6F ), and are hence sensitivity to CDK4/6 inhibition. In contrast, cells that exit mitosis with a CDK2 high phenotype enter into a proliferative state, bypassing the restriction point (Fig. 6G ), with shorter doubling times. Tumours with a high proportion of CDK2 high cells are resistant to CDK4/6 inhibition. Palbociclib-sensitive LAR cancer cells typically exit into a quiescent CDK2 low state post-mitosis, from which CDK4/6 is required to phosphorylate RB1 and pass the restriction point (2,(24)(25)(26)(27). In contrast, basal-like TNBC cells frequently enter a proliferative CDK2 high state and are thus resistant to palbociclib treatment. This provides a mechanistic explanation for why basal-like, and potentially many other tumour types are resistant to CDK4/6 inhibition despite being RB1 wild type. The CDK4/6 and PI3 kinase inhibitor combinations have substantial activity in PIK3CA mutant non-basal TNBC, of both LAR and mesenchymal-stem (MSL) subgroups (Fig. 6A ), with such combinations having the potential to further expand the TNBC subgroups that could benefit from CDK4/6 inhibition. Cyclin E1 binds to and activates CDK2 (28,29) and prior work has demonstrated that cyclin E1 expression mediates resistance to CDK4/6 inhibition (10,30). The classical view is that CDK2 and cyclin E are downstream of CDK4/6 activity (24). Here we show that in palbociclib-resistant TNBC, cyclin E1 expression is dysregulated and expressed immediately post-mitosis (Fig. 4D ). Dysregulation of cyclin E1 promotes the CDK2 high phenotype, with the CDK2-CyclinE complex active immediately post-mitosis, resulting in a short G1 phase (Fig. 6G ). The SUM149 cells harbour an inactivating FBXW7 mutation that disrupts the SCF complex, probably contributing to the dysregulation of cyclin E1 expression (Supplementary table 2). In other contexts, increased CCNE1 gene copy number may drive dysregulation of cyclin E1 expression post-mitosis (10). These observations suggest that the measurement of cyclin E1 expression either at the RNA or protein level has the potential to be utilised as a predictive biomarker of resistance to CDK4/6 inhibition in breast cancer.

Daughter cells share the same CDK2 activity state, suggesting that post-mitotic CDK2 activity is determined pre-mitosis (Fig. 5A ). However, this is not the result of CDK2 high and CDK2 low cells, as colonies derived from individual cells largely recapitulate the same variability in CDK2 activity as the parental cell line (Fig. 5D ). We provide some data to suggest that DNA damage, and the resulting induction of p21 expression, may also regulate CDK2 activity state post-mitosis (Fig. 5G ). However, more research is required to further assess the potential role of DNA damaging signalling in this context.

Our results illustrate how single cell analysis can identify mechanisms of resistance to targeted therapies. Phenotypic heterogeneity between single cells may drive drug resistance, and this may be more clearly elucidated at the single cell level than through assessment of bulk cell populations. Through single cell approaches, we identify that CDK2 activity post-mitosis is a key determinant of sensitivity to CDK4/6 inhibition, and highlight potential therapeutic strategies for triple negative breast cancer. An on going therapeutic trial is assessing the effectiveness of CDK4/6 inhibition in combination with PI3 kinase inhibition, in patients with PIK3CA-mutant TNBC (NCT02389842).

Acknowledgements

Acknowledgments:The authors acknowledge Frederick Wallberg and Rhadhika Patel for flow cytometry support services and Vicky Bousgouni for live imaging support.

Funding

This research was funded by the Avon foundation (U.S. Asghar), Breast Cancer Now with generous support from the Mary-Jean Mitchell Green Foundation and Cancer Research UK C30746/A16642 (N.C.Turner).The authors also acknowledge NHS funding to the NIHR Biomedical Research Centre at The Royal Marsden and the ICR.

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