Purpose:

Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor–positive (ER+) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor.

Experimental Design:

BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER+ breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER+ mouse mammary tumor model was used to study the effect of combination therapy on the immune system.

Results:

Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G1–S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib–induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER+ mammary tumor model and extended tumor response when combined with anti-PD1 therapy.

Conclusions:

This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER+ breast cancer.

Translational Relevance

Combining the BCL2 inhibitor ABT-199 (venetoclax) with endocrine therapy and a CDK4/6 inhibitor augments tumor response by eliciting a deeper cell-cycle arrest, triggering apoptosis and enhancing the immunomodulatory response. Our findings suggest that venetoclax can be senolytic, supporting further investigation of dual CDK4/6 and BCL2 blockade in ER-positive breast cancer.

Estrogen receptor–positive (ER+) breast cancers frequently exhibit deregulation of the cyclin-dependent kinase 4 and 6 (CDK4/6)/cyclin D1 (CCND1)/retinoblastoma (Rb) signaling pathway, resulting in uncontrolled cellular proliferation (1, 2). CDK4/6 inhibitors are active in breast cancer where they have demonstrated synergistic activity with endocrine therapy, leading to improvements in progression-free survival and overall survival (3–5). As a result, combination treatment with endocrine therapy and a CDK4/6 inhibitor is now considered standard of care for patients with early-line metastatic ER+ breast cancer. Nevertheless, de novo or acquired resistance to therapy is almost inevitable, underscoring the need for novel targeted therapies for this group of patients.

While the key mechanism of action of CDK4/6 inhibitors is to provoke a cell-cycle arrest, they likely contribute clinically relevant antitumoral effects through modulation of the immune response (6, 7), induction of senescence (7, 8), or through other noncanonical functions (9). Immune effects include immunogenic activation of tumor cells and promotion of a switch to a less immunosuppressive state through reduced regulatory T cell (Treg) numbers (7). In mammary tumor models, the profound cell-cycle arrest has been linked to induction of a senescence program (10). Despite their potent antiproliferative effects and demonstrated efficacy in the clinic, CDK4/6 inhibitors do not induce apoptotic cell death in breast cancer cells (7, 10, 11). Indeed, recent findings suggest that combination therapy may actually reduce apoptosis in treatment-naïve tumors (12). These findings are consistent with the senescent state, characterized by relatively irreversible replicative arrest and resistance to apoptosis (13, 14).

Evasion of cell death, a hallmark of cancer, can result from the overexpression of antiapoptotic BCL2 family members. BCL2 is overexpressed in the majority of primary and metastatic ER+ breast cancer (15, 16). The targeting of BCL2 and other antiapoptotic proteins has emerged as a viable therapeutic option due to the recent development of BH3-mimetic drugs that mimic endogenous antagonists of BCL2 and its related family members (17–19). Venetoclax (ABT-199/GDC-0199), a potent and highly selective inhibitor of BCL2 (20), has demonstrated single-agent activity in chronic lymphocytic leukemia (21) and in combination therapy for a number of hematologic malignancies (18). In preclinical models of ER+ BCL2+ breast cancer, ABT-199 improved tumor response to endocrine therapy with tamoxifen by enhancing apoptosis (22). These findings appear to be clinically relevant as the addition of venetoclax to tamoxifen elicited promising clinical activity in a phase I study in women with metastatic ER+ BCL2+ breast cancer (16).

On the basis of these observations, we have explored a role for dual targeting of CDK4/6 and BCL2 signaling pathways in preclinical models of ER+ BCL2+ breast cancer and found that the addition of ABT-199 to endocrine therapy with fulvestrant and the CDK4/6 inhibitor palbociclib substantially augmented tumor responsiveness in vivo. Combination therapy was accompanied by a deeper cell-cycle arrest and induction of apoptosis, including cells exhibiting a senescence-associated β-galactosidase phenotype. Moreover, the favorable palbociclib-mediated immunogenic activation of tumor cells together with reduction in Treg numbers and proliferation was not abrogated by the addition of ABT-199.

Study design

This study was designed to evaluate the response of breast cancer cells to the combination of endocrine therapy with ABT-199 and palbociclib. We evaluated the response to single-agent or combination therapy in breast cancer cell lines, patient-derived breast tumor organoids and xenograft models, as well as a syngeneic mouse mammary tumor model. Experiments were designed to investigate the mechanisms of tumor response. As outlined below, all mouse studies included randomization and blinding. The numbers of replicates performed for each experiment are included in the figure legends.

Human tissue

The studies were conducted in accordance with recognized ethical guidelines (Declaration of Helsinki). Human breast cancer tissues were obtained from consenting patients through the Royal Melbourne Hospital Tissue Bank and the Victorian Cancer Biobank with relevant institutional review board approval. Human Ethics approval was obtained from the Walter and Eliza Hall Institute (WEHI, 05/06) Human Research Ethics Committee.

In vivo experiments using PDX models

ER+ PDX 50 and 315 models have been reported previously (22). PDX 50 originated from a patient following neoadjuvant chemotherapy (FEC-D) for her primary tumor; PDX 315 was derived from a primary tumor arising in a patient with a past history of a contralateral breast tumor. She had previously received adjuvant chemotherapy (FEC x 6) and endocrine therapy (tamoxifen followed by letrozole) and was taking letrozole at the time of her second primary tumor diagnosis. The resulting PDX 315 is strongly ER+ as reported previously (22). PDX 1105 and 1232 were derived from primary tumors in treatment-naïve patients.

NOD-SCID-IL2Rγ−/− (NSG) mice were bred and maintained according to institutional guidelines. All animal experiments were approved by the WEHI Animal Ethics Committee (2017.002). Cohorts of 50–60 female mice were seeded with thawed single-cell suspensions of early-passage human breast tumors (passage 2 or 3). Briefly, 150,000–250,000 cells were resuspended in 10 μL of transplantation buffer (50% FCS, 10% of 0.04% Trypan blue solution and 40% PBS) and growth factor–reduced Matrigel (BD Pharmingen) at a ratio of 3:1 and injected into the cleared mammary fat pads of 3- to 4-week-old NSG female mice. Mice were monitored for tumor development three times weekly and tumor size measured using electronic Vernier calipers. Tumor volume was estimated by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter)2(maximum diameter)/2. Once tumors arose, mice were randomized into treatment arms. Treatment was initiated when the tumor volume reached 80–120 mm3. Randomization and tumor measurements were managed using the Study Director software (v 3.0, studylog). Mice were sacrificed at the first measurement where tumor volume exceeded 600 mm3, or if their health deteriorated for reasons other than disease progression or drug toxicity (censored event).

In vivo drug treatments

Mice were treated for 21 days in a 28-day cycle. Fulvestrant (Clifford Hallam) 5 mg or its vehicle was injected intraperitoneally weekly for 3 of 4 weeks. ABT-199 (Active Biochem) 100 mg/kg or its vehicle was prepared as described previously (22) and given via oral gavage on days 1–5 for three out of four weeks. Palbociclib 100 mg/kg or its vehicle was given via oral gavage on days 1–5 for 3 out of 4 weeks. Palbociclib was dissolved in 50 mmol/L sodium lactate, pH 4. Palbociclib was administered at least 4 hours after ABT-199 to avoid negative interaction between the drugs and vehicles. Anti-PD1 (BioXcell) 200 μg/mouse or isotype control was given intraperitoneally on days 1, 3, and 5. Mice were monitored as per institutional guidelines.

Preparation of 67NR cells and syngeneic transplantation experiments

Early-passage 67NR cells were maintained as described in Supplementary Methods and cells (5 × 103) transplanted into the cleared mammary fat pads of 3- to 4-week-old BALB/c mice. Mice bearing 67NR tumors were euthanized at the experimental endpoint and tumors excised. To obtain a single-cell suspension for detection of cell surface and intracellular proteins, tumors and spleens were processed as described previously (23). BALB/c mice were bred and maintained according to institutional guidelines. Experiments were approved by the WEHI Animal Ethics Committee (2017.002).

Flow cytometry

Analysis of immune cells was performed as described previously (23). Antibodies to cell surface proteins were purchased from BioLegend unless otherwise stated: CD4 (clone GK1.5), CD8 (clone 53-6.7), TCRβ (clone H57-597), CD45 (clone 30-FII), Ki67 (clone B56), PD1 (clone 29F.1A12), and FoxP3 (eBiosciences, clone FJK-16). For intracellular detection of TNFα and IFNγ, cells were isolated and stimulated as described previously (23). Sample data were acquired on an LSRFortessa X-20 flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).

Breast cancer organoids

Breast cancer organoids were generated from patient samples as described previously (24, 25). Briefly, breast cancer cell suspensions from patients undergoing surgery were prepared through mechanical disruption and enzymatic digestion. Isolated cells were then plated in adherent basement membrane extract (BME; Cultrex) drops in uncoated 24-well plates and cultured in optimized human organoid medium (HOM). For PDX-derived breast cancer organoids, we obtained single-cell suspensions by digestion of primary tumors and then sorted these by flow cytometry using a FACSARIA III (BD Biosciences), as described previously (22, 26). Sorted human EpCAM+ cells were then plated in BME drops and cultured in HOM.

For 3D colony assays, organoids were dissociated and then seeded in BME in 24-well plates (four drops of 8 μL containing 1,000 cells). After 24 hours, organoids were treated with fulvestrant (Sigma-Aldrich), ABT-199 (Active Biochemicals), palbociclib (Active Biochemicals), vehicle, or the indicated treatments. Treatments were replaced every 3–4 days. After 14 days in culture, cell viability was assessed using CellTiter Glo Luminescent Assay (Promega).

Statistical analysis

Statistical analyses were performed in the GraphPad Prism software version 8.0a. Kaplan–Meier (log rank test) was used to test for significant differences in the survival of mice (using the ethical end point for tumor size as a surrogate for death). Unpaired t tests were used to test the significance of differences in column means between treatments.

For details, see Supplementary Methods.

Augmented response to CDK4/6 and BCL2 inhibition in breast cancer

To gain insight into the short-term effects of combined endocrine and CDK4/6 inhibitor therapy on BCL2 prosurvival family members in treatment-naïve breast cancer, we evaluated gene expression data from the NeoPalAna window study (27), in which patients with newly diagnosed ER+ breast cancer were treated with the aromatase inhibitor anastrozole followed by the CDK4/6 inhibitor palbociclib prior to surgery (Fig. 1A; Supplementary Fig. S1A). As reported previously, expression of MKI67 (encodes the proliferation marker Ki67), was reduced by therapy but recovered when palbociclib was ceased. BCL2 and MCL1 levels similarly declined, albeit modestly, in response to combination therapy and rebounded once palbociclib was withdrawn. Reciprocal changes in BCL2L1 (encoding BCLXL) were observed, consistent with adaptive changes due to altered BCL2 and MCL1 expression. The effect of longer-term, continuous palbociclib treatment on BCL2 family members was next evaluated in MCF7 and T47D breast cancer cells, which were treated for 3 months at different concentrations (Supplementary Fig. S1B). A modest decrease in BCL2 and MCL1 proteins was observed in MCF7 cells, while expression was largely unchanged in T47D cells. BCLXL levels rose in both cell lines. Despite this, palbociclib-treated cells (continuous treatment at high concentration) exhibited greater sensitivity to BCL2 inhibition with ABT-199, consistent with either heightened cellular stress and/or lower BCL2 levels (Supplementary Fig. S1C). Together, these findings suggest that BCL2 could represent a potential target in palbociclib-treated breast cancer cells.

Figure 1.

BCL2 family expression in response to endocrine therapy, CDK4/6 inhibition, and ABT-199. A, Plots showing relative expression (log2 fold change) of MKI67, BCL2, MCL1, and BCL2L1 in response to sequential treatment with anastrozole (red line) and palbociclib (blue line) from the NeoPalAna study conducted by Ellis and colleagues (27). Time points are baseline (BL), anastrozole alone (C1D1), anastrozole and palbociclib in combination (C1D15), and surgery (Surg). Multiple microarray probes for the same gene are shown as separate lines. Mean and variance of representative probes are shown in Supplementary Fig. S1A. B, MCF7 cells were treated with increasing concentrations of fulvestrant and palbociclib (top), fulvestrant and ABT-199 (middle), and the combination of fulvestrant, ABT-199, and palbociclib (bottom) for 72 hours and then subjected to viability assessment using CellTiter-Glo followed by BLISS score analysis. BLISS synergy values are > 0.0 on the vertical axis. C, Western blot analysis showing expression of Rb, pRb (S780), estrogen receptor (ER), Cyclin D1, Cyclin E, BIM, MCL1, BCL2, BCLXL, cleaved PARP (cPARP), and cleaved caspase 3 (CC3) in MCF7 cells treated for 72 hours with fulvestrant (100 nmol/L), ABT-199 (10 μmol/L), palbociclib (500 nmol/L), or the indicated combinations. Tubulin was used as a loading control.

Figure 1.

BCL2 family expression in response to endocrine therapy, CDK4/6 inhibition, and ABT-199. A, Plots showing relative expression (log2 fold change) of MKI67, BCL2, MCL1, and BCL2L1 in response to sequential treatment with anastrozole (red line) and palbociclib (blue line) from the NeoPalAna study conducted by Ellis and colleagues (27). Time points are baseline (BL), anastrozole alone (C1D1), anastrozole and palbociclib in combination (C1D15), and surgery (Surg). Multiple microarray probes for the same gene are shown as separate lines. Mean and variance of representative probes are shown in Supplementary Fig. S1A. B, MCF7 cells were treated with increasing concentrations of fulvestrant and palbociclib (top), fulvestrant and ABT-199 (middle), and the combination of fulvestrant, ABT-199, and palbociclib (bottom) for 72 hours and then subjected to viability assessment using CellTiter-Glo followed by BLISS score analysis. BLISS synergy values are > 0.0 on the vertical axis. C, Western blot analysis showing expression of Rb, pRb (S780), estrogen receptor (ER), Cyclin D1, Cyclin E, BIM, MCL1, BCL2, BCLXL, cleaved PARP (cPARP), and cleaved caspase 3 (CC3) in MCF7 cells treated for 72 hours with fulvestrant (100 nmol/L), ABT-199 (10 μmol/L), palbociclib (500 nmol/L), or the indicated combinations. Tubulin was used as a loading control.

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The link between estrogen receptor (ER) signaling and G1–S phase cell-cycle progression has informed the combined use of CDK4/6 inhibitors with endocrine therapy (11). To address whether combination therapy was synergistic or additive, we compared the impact of adding either palbociclib or ABT-199 to the selective ER degrader fulvestrant in MCF7 cells (Fig. 1B). Doses were selected on the basis of the induction of on-target effects, including reduced ER (for fulvestrant), Rb phosphorylation (for palbociclib and fulvestrant) and generation of cleaved PARP (for ABT-199) at 24 hours and/or 7 days (Supplementary Fig. S2A and S2B). As reported previously (11), palbociclib showed synergy when combined with endocrine therapy, as determined by cell viability using CellTiter-Glo and BLISS score analysis (Fig 1B, top). Synergy was also observed between fulvestrant and ABT-199 at higher doses, consistent with prior findings for tamoxifen (22). Adding ABT-199 to fulvestrant and palbociclib was neither synergistic nor antagonistic (Fig 1B, bottom), suggesting that cotargeting of the BCL2 pathway might enhance response to fulvestrant/palbociclib.

To investigate the effect of drug treatment on key target proteins, MCF7 cells were treated for 72 hours with fulvestrant, palbociclib, ABT-199 or various combinations. As expected, combination therapy with fulvestrant and palbociclib reduced ER and phospho-Rb (pRb) levels, but did not induce apoptosis, as determined by cleaved PARP and cleaved caspase-3 expression (Fig. 1C). ABT-199 alone (or in combination) increased expression of pro-apoptotic BIM, a measure of primed cells (28) and induced cleaved PARP and cleaved caspase-3 (CC3; Fig. 1C). This was unexpectedly accompanied by reduced levels of pRb, the G1–S cyclins D1 and E, and ER (Fig. 1C). Triple therapy with fulvestrant and palbociclib induced similar changes. Interestingly, the adaptive increase in cyclin D1 and E seen with palbociclib alone was abrogated by ABT-199 cotreatment (Fig. 1C). Together, these findings indicate that combining ABT-199 with endocrine therapy and palbociclib can trigger apoptosis in breast cancer cells that are undergoing a palbociclib-mediated cell-cycle arrest. The unexpected downregulation of pRb and cyclins D1 and E could reflect selective induction of apoptosis in cycling cells. Decreased ER expression is also consistent, as lower ERα levels are observed in G1-arrested cells (29).

Dephosphorylation of Rb and cell-cycle arrest in BH3 mimetic—treated breast cancer cells

The unanticipated finding that ABT-199 leads to reduced G1–S phase cyclins and Rb dephosphorylation was next investigated by cell-cycle analysis using flow cytometry (Fig. 2A). MCF7 cells were treated for 72 hours with fulvestrant (100 nmol/L), ABT-199 (5 μmol/L), and palbociclib (250 nmol/L) alone or in combination. Fulvestrant and palbociclib both efficiently reduced the percentage of S-phase cells, consistent with their known mechanism of action. Notably, ABT-199 induced similar changes, albeit to a lesser extent than observed with palbociclib. This effect was more pronounced when ABT-199 was combined with palbociclib or fulvestrant (Fig. 2A).

Figure 2.

BH3 mimetics induce caspase-independent cell-cycle arrest and dephosphorylation of Rb. A, Cell-cycle analysis of MCF7 cells treated with fulvestrant (100 nmol/L), ABT-199 (5 μmol/L), palbociclib (200 nmol/L), or the indicated combinations for 72 hours. Quantification shown in right panel is from three independent experiments. Data are shown as mean ± SEM. B, MCF7 cells were treated for 72 hours with palbociclib “Lo” (50 nmol/L) or “Hi” (500 nmol/L) doses or the indicated BH3 mimetics at “Lo” (1 μmol/L) or “Hi” (10 μmol/L) doses. Cells were then subjected to Western blot analysis for Rb, pRb (S780), estrogen receptor (ER), Cyclin D1, Cyclin E, MCL1, BCL2, BCLXL, and cleaved PARP (cPARP). Tubulin was used as a loading control. C, MCF7 cells were treated for 72 hours with the indicated BH3 mimetics (10 μmol/L) with or without pan-caspase inhibitor Q-VD-OPh (20 μmol/L). Cells were then subjected to Western blot analysis for Rb, pRb (S780), estrogen receptor (ER), Cyclin D1, Cyclin E, MCL1, BCL2, BCLXL, and cleaved PARP (cPARP). Tubulin was used as a loading control. D, Cell-cycle analysis of MCF7 cells treated for 72 hours with the indicated BH3 mimetics (10 μmol/L) with or without Q-VD-OPh (20 μmol/L). Quantification shown in right panel is from three independent experiments. Data are shown as mean ± SEM.

Figure 2.

BH3 mimetics induce caspase-independent cell-cycle arrest and dephosphorylation of Rb. A, Cell-cycle analysis of MCF7 cells treated with fulvestrant (100 nmol/L), ABT-199 (5 μmol/L), palbociclib (200 nmol/L), or the indicated combinations for 72 hours. Quantification shown in right panel is from three independent experiments. Data are shown as mean ± SEM. B, MCF7 cells were treated for 72 hours with palbociclib “Lo” (50 nmol/L) or “Hi” (500 nmol/L) doses or the indicated BH3 mimetics at “Lo” (1 μmol/L) or “Hi” (10 μmol/L) doses. Cells were then subjected to Western blot analysis for Rb, pRb (S780), estrogen receptor (ER), Cyclin D1, Cyclin E, MCL1, BCL2, BCLXL, and cleaved PARP (cPARP). Tubulin was used as a loading control. C, MCF7 cells were treated for 72 hours with the indicated BH3 mimetics (10 μmol/L) with or without pan-caspase inhibitor Q-VD-OPh (20 μmol/L). Cells were then subjected to Western blot analysis for Rb, pRb (S780), estrogen receptor (ER), Cyclin D1, Cyclin E, MCL1, BCL2, BCLXL, and cleaved PARP (cPARP). Tubulin was used as a loading control. D, Cell-cycle analysis of MCF7 cells treated for 72 hours with the indicated BH3 mimetics (10 μmol/L) with or without Q-VD-OPh (20 μmol/L). Quantification shown in right panel is from three independent experiments. Data are shown as mean ± SEM.

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We next investigated whether other BH3 mimetics could impact the cell cycle. ABT-737 (which targets BCLXL, BCL2, and BCLW) and the MCL1 inhibitor S63845 (30) exerted similar dose-dependent effects on pRb, cyclin D1 and ER, with all three BH3 mimetics inducing cPARP (Fig. 2B). As expected, MCL1 levels increased in response to ABT-199, ABT-737, and S63845, the latter attributable to stabilization of the protein by the inhibitor (30). Minimal changes were observed in BCL2 or BCL-XL levels. To address whether the changes in pRb, cyclin D1, or ER reflected an “on-target” effect of BH3 mimetics or a consequence of apoptosis, MCF7 cells were treated with BH3 mimetics (including the BCLXL inhibitor A1331852; ref. 31) in the presence or absence of the pan-caspase inhibitor Q-VD-Oph (Fig. 2C). While apoptosis was efficiently blocked by Q-VD-Oph, as demonstrated by lack of PARP cleavage, the effects on pRb, cyclin D1, and ER were maintained, implying they are caspase-independent. Furthermore, cell-cycle analysis revealed that each BH3 mimetic could inhibit proliferation independent of cell death (Fig. 2D). Together, these results suggest that ABT-199 (and other BH3 mimetics) may have unexpected effects as inhibitors of cell-cycle progression in breast cancer cells, beyond their canonical role as proapoptotic agents.

CDK4/6 inhibition primes breast cancer cells for apoptosis and senolysis

Given the potent effect of ABT-199 on apoptosis and the cell cycle, we next evaluated the effect of single or combination therapy in clonogenic cellular assays. For this, lower doses of fulvestrant and palbociclib were used to address synergy, because clonogenic activity was readily inhibited by single-agent treatment (Supplementary Fig. S3A and S3B). T47D and MCF7 cells were treated for two weeks with single, doublet, or triplet therapy comprising fulvestrant, palbociclib, and/or ABT-199. The addition of ABT-199 to fulvestrant and palbociclib significantly reduced the number and size of colonies, compared with doublet therapy (Fig. 3A and B; Supplementary Fig. S3C). Following removal of inhibitors at day 14, regrowth was apparent in cells that had been treated with doublet therapy. In contrast, minimal growth was observed in cells that had received triplet therapy (Fig. 3A and B; Supplementary Fig. S3C). At the lower doses used for clonogenic assays, the previously observed effects on key target proteins were maintained (Supplementary Fig. S4A and S4B). These findings, together with the adaptive changes in BCL2 family expression in breast tumor cells treated with palbociclib (Fig. 1A; Supplementary Fig. S1B), suggest that palbociclib might prime cells for apoptotic cell death with ABT-199.

Figure 3.

ABT-199 augments response to palbociclib. A, Clonogenic survival assays of T47D cells treated continuously for 14 days with vehicle, fulvestrant (100 pmol/L), ABT-199 (5 μmol/L), palbociclib (50 nmol/L), and the indicated combinations. Bottom row shows cells treated for 14 days before drugs were washed out and colonies allowed to recover in media for seven days. Scale bar, 5 mm. MCF7 cells are shown in Supplementary Fig. S3C. B, Quantification of A for three independent experiments (mean ± SEM). Unpaired two-tailed t tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). C, MCF7 cells were treated for 4 days with vehicle or palbociclib (500 nmol/L) prior to washing and then treatment switch to vehicle, ABT-199 (10 μmol/L), ABT-737 (10 μmol/L), palbociclib (500 nmol/L), or the indicated combinations for 4 days. Cells were then subjected to Western blot analysis for Rb, pRb (S780), PCNA, Cyclin D1, Cyclin E, cleaved PARP (cPARP), and cleaved caspase 3 (CC3). Tubulin was used as a loading control. D, MCF7 cells were treated as described in C before viability analysis with propidium iodide staining. Results are presented as a percentage of untreated cells and represent two technical replicates for two independent experiments. Unpaired two-tailed t tests (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). E, MCF7 cells were treated as described in C before β-galactosidase staining for cellular senescence. F, Quantification of β-galactosidase staining from E. Shown is mean ± SEM for four independent experiments. Unpaired two-tailed t tests (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). G, Genome-wide CRISPR/Cas9 screen in T47D cells. T47D cells were infected with a genome-wide lentiviral sgRNA library and then treated with fulvestrant (1 μmol/L), fulvestrant and ABT-199 (10 μmol/L), fulvestrant and palbociclib (1 μmol/L), or the combination of fulvestrant, ABT-199, and palbociclib. The pooled analysis from five independent infections, displaying normalized values for fulvestrant–palbociclib or fulvestrant–ABT-199–palbociclib combinations is shown. The sgRNA enriched or depleted in the fulvestrant–ABT-199–palbociclib treatments are shown as red and blue dots, respectively. RB1 is the top depleted gene. See Supplementary Table S1 for differentially enriched sgRNAs in the treated pools and Supplementary Table S2 for differentially enriched genes.

Figure 3.

ABT-199 augments response to palbociclib. A, Clonogenic survival assays of T47D cells treated continuously for 14 days with vehicle, fulvestrant (100 pmol/L), ABT-199 (5 μmol/L), palbociclib (50 nmol/L), and the indicated combinations. Bottom row shows cells treated for 14 days before drugs were washed out and colonies allowed to recover in media for seven days. Scale bar, 5 mm. MCF7 cells are shown in Supplementary Fig. S3C. B, Quantification of A for three independent experiments (mean ± SEM). Unpaired two-tailed t tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). C, MCF7 cells were treated for 4 days with vehicle or palbociclib (500 nmol/L) prior to washing and then treatment switch to vehicle, ABT-199 (10 μmol/L), ABT-737 (10 μmol/L), palbociclib (500 nmol/L), or the indicated combinations for 4 days. Cells were then subjected to Western blot analysis for Rb, pRb (S780), PCNA, Cyclin D1, Cyclin E, cleaved PARP (cPARP), and cleaved caspase 3 (CC3). Tubulin was used as a loading control. D, MCF7 cells were treated as described in C before viability analysis with propidium iodide staining. Results are presented as a percentage of untreated cells and represent two technical replicates for two independent experiments. Unpaired two-tailed t tests (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). E, MCF7 cells were treated as described in C before β-galactosidase staining for cellular senescence. F, Quantification of β-galactosidase staining from E. Shown is mean ± SEM for four independent experiments. Unpaired two-tailed t tests (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). G, Genome-wide CRISPR/Cas9 screen in T47D cells. T47D cells were infected with a genome-wide lentiviral sgRNA library and then treated with fulvestrant (1 μmol/L), fulvestrant and ABT-199 (10 μmol/L), fulvestrant and palbociclib (1 μmol/L), or the combination of fulvestrant, ABT-199, and palbociclib. The pooled analysis from five independent infections, displaying normalized values for fulvestrant–palbociclib or fulvestrant–ABT-199–palbociclib combinations is shown. The sgRNA enriched or depleted in the fulvestrant–ABT-199–palbociclib treatments are shown as red and blue dots, respectively. RB1 is the top depleted gene. See Supplementary Table S1 for differentially enriched sgRNAs in the treated pools and Supplementary Table S2 for differentially enriched genes.

Close modal

To further investigate the combinatorial effect of ABT-199 and palbociclib, MCF7 cells were treated with either vehicle or palbociclib (500 nmol/L) for four days prior to switching drug treatment to vehicle, ABT-199, ABT-737, or both ABT-199 and palbociclib (Fig. 3C and D). Palbociclib pretreated cells were more responsive to either ABT-199 or ABT-737, with increased CC3 (Fig. 3C) and reduced viability as measured by propidium iodide exclusion (Fig. 3D). As expected, palbociclib-treated cells exhibited less PCNA expression and thus proliferation. Consistent with cell-cycle analysis (Fig. 2A and D), ABT-199–treated cells also exhibited lower PCNA levels compared with vehicle-treated controls (Fig. 3C).

CDK4/6 inhibitors elicit a senescence phenotype (7, 8) that can result in cells becoming more resistant to apoptosis (13, 14). In keeping with this finding, palbociclib induced a dose-dependent senescence-like phenotype characterized by increased cell size, cell flattening, and β-galactosidase expression (Supplementary Fig. S5A), while no discernible change in morphology or β-galactosidase expression was observed with ABT-199 alone. Cells treated with palbociclib for 4 days maintained this senescent-like phenotype following drug withdrawal (Fig. 3E and F). This phenotype, accompanied by the increased vulnerability to apoptosis observed following treatment with a BH3 mimetic (Fig. 3C), suggests that ABT-199 (and ABT-737) can augment CDK4/6 inhibition through the induction of senolysis of cells that have undergone growth arrest.

ABT-199 may modulate mechanisms of palbociclib resistance

To address potential mechanisms of response or resistance to various combinations, we performed a genome-wide CRISPR/Cas9 screen in T47D cells (Fig. 3G; Supplementary Fig. S6A and S6B; Supplementary Tables S1 and S2). Cas9-expressing T47D cells were transduced with a pooled human genome-wide small guide RNA (sgRNA) lentiviral library containing 123,411 unique sgRNAs targeting 19,050 genes, and 1,864 miRNAs (6 sgRNAs per gene and 4 sgRNAs per miRNA) at low multiplicity of infection in five independent infections. Transduced cells were continuously treated with either fulvestrant, fulvestrant and palbociclib, fulvestrant and ABT-199, or the combination of fulvestrant, palbociclib, and ABT-199 for 2 months to identify sgRNA guides that helped to promote the growth of resistant cells. Doses chosen (fulvestrant 1 μmol/L, ABT-199 10 μmol/L, and palbociclib 1 μmol/L) were based on single-agent activity that exerted high selective pressure (Supplementary Fig. S3).

When the frequency of reads for guides was compared between different treatments, sgRNAs for RB1 were found to be enriched in fulvestrant–palbociclib–treated cells, with the greatest differential observed between fulvestrant-only and fulvestrant–palbociclib treated cells (Supplementary Tables S1 and S2). In contrast, RB1 sgRNAs were not over-represented in cells treated with fulvestrant–ABT-199 (Supplementary Tables S1 and S2). For fulvestrant and fulvestrant/palbociclib treatment–resistant cells, differentially represented guides (with an FDR of < 0.05) against genes and molecular pathways previously associated with CDK4/6 inhibitor resistance were identified such as PIK3C isoforms, CDK4 and FGFR members (32–34), mir-432-5p (35), members of the IL6–JAK–Stat pathway (IL6ST/GP130, JAK1, JAK3, STAT2, and STAT4; ref. 36), E2F family members (E2F3, E2F4, E2F6, E2F7, and E2F8; ref. 37), and Hippo pathway family members TEAD3 and TEAD4 (38). Interestingly, the histone H3 acetyltransferase epigenetic modifier genes from the MOZ/MORF complex (KAT6B, BPRF1, and ING5) were also implicated as genes involved in resistance. Interrogation of BCL2 family members revealed sgRNAs targeting MCL1 were enriched in fulvestrant-treated cells compared with those treated with fulvestrant–palbociclib or fulvestrant–ABT-199 (Supplementary Tables S1 and S2), suggesting that this could be a target for use in combination or upon progression.

To determine whether the addition of ABT-199 to fulvestrant and palbociclib modified mechanisms of resistance, we examined gene knockouts that were enriched or depleted between fulvestrant–palbociclib and fulvestrant–palbociclib–ABT-199 triplet therapy. Notably, RB1 was identified as the top candidate for cells treated with triple therapy versus fulvestrant–palbociclib while the genes identified above did not feature (Fig. 3G; Supplementary Table S2). These observations suggest that treatment with ABT-199 could mitigate loss of Rb and potentially other mechanisms of acquired resistance to palbociclib (34).

Triple therapy substantially inhibits the growth of breast tumor organoids

Given their distinct molecular effects and augmented response observed with CDK4/6 and BCL2 inhibition, we next studied patient-derived breast tumor organoid models, which more accurately reflect tumor heterogeneity and are emerging as promising preclinical models to predict drug response (24, 25). ER+ tumor organoids were generated from primary ER+ breast tumors or patient-derived xenograft (PDX) models (Fig. 4A; Supplementary Table S3). Primary breast tumor organoids were generated as recently described (24, 25) and were found to retain ER and BCL2 expression (Supplementary Fig. S7). They exhibited mutational profiles expected in luminal breast tumors (Supplementary Table S4). For PDX organoids, single-cell suspensions were prepared from PDX tumors, then sorted by flow cytometry and plated in organoid medium optimized for breast tumors (24, 25). These retained the histologic features of the parental tumor, including ER and BCL2 expression (Supplementary Fig. S8A and S8B). The four PDX tumors exhibited variable levels of BCL2 and cell-cycle proteins, with notable absence of pRb expression in PDX 50 (Fig. 4B). PDX 50 was found to harbor a heterozygous RB1 mutation (c.1215+1G>A) known to be pathogenic (ClinVar).

Figure 4.

ER+ breast cancer organoids. A, Schematic overview of the generation of breast cancer organoids from primary breast cancer samples or patient-derived xenograft (PDX) models. B, Western blot analysis of Rb, pRb (S780), ER, Cyclin D1, CDK4, CDK6, and BH3 family member (MCL1, BCL2, and BCLXL) expression in ER+ PDX models (two independent tumors per PDXs are shown). Tubulin was used as a loading control. C, Clonogenic assays of PDX 1105 cancer organoids treated continuously for 14 days with vehicle, fulvestrant (1 μmol/L), ABT-199 (2.5 μmol/L), palbociclib (50 nmol/L), and the indicated combinations. Bottom, cells treated for 14 days before drugs were washed out and organoid colonies allowed to recover in media for seven days. Scale bar, 5 mm. D, Quantification of C using CellTiter-Glo. Bars represent mean ± SEM for at least three independent experiments. Unpaired two-tailed t tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). E, Representative sections of PDX 1105 organoids from immunostained for Ki67 after 24 hours with the indicated treatments (C). Scale bar, 50 μm. F, Western blot showing expression of Rb, pRb, and cleaved caspase 3 (CC3) in PDX 1105 cancer organoids treated with the indicated drugs for 24 hours. Tubulin was used as a loading control. G, Primary breast tumor organoids were cultured for 14 days in organoid medium with the indicated drugs before assessment of cell viability using CellTiter-Glo. Results are presented as percentages of untreated cells. Data represent mean ± SEM for n = 3 independent experiments. Unpaired two-tailed t tests is shown for doublet combination compared with single agent, or triple therapy compared to doublet combination (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

Figure 4.

ER+ breast cancer organoids. A, Schematic overview of the generation of breast cancer organoids from primary breast cancer samples or patient-derived xenograft (PDX) models. B, Western blot analysis of Rb, pRb (S780), ER, Cyclin D1, CDK4, CDK6, and BH3 family member (MCL1, BCL2, and BCLXL) expression in ER+ PDX models (two independent tumors per PDXs are shown). Tubulin was used as a loading control. C, Clonogenic assays of PDX 1105 cancer organoids treated continuously for 14 days with vehicle, fulvestrant (1 μmol/L), ABT-199 (2.5 μmol/L), palbociclib (50 nmol/L), and the indicated combinations. Bottom, cells treated for 14 days before drugs were washed out and organoid colonies allowed to recover in media for seven days. Scale bar, 5 mm. D, Quantification of C using CellTiter-Glo. Bars represent mean ± SEM for at least three independent experiments. Unpaired two-tailed t tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). E, Representative sections of PDX 1105 organoids from immunostained for Ki67 after 24 hours with the indicated treatments (C). Scale bar, 50 μm. F, Western blot showing expression of Rb, pRb, and cleaved caspase 3 (CC3) in PDX 1105 cancer organoids treated with the indicated drugs for 24 hours. Tubulin was used as a loading control. G, Primary breast tumor organoids were cultured for 14 days in organoid medium with the indicated drugs before assessment of cell viability using CellTiter-Glo. Results are presented as percentages of untreated cells. Data represent mean ± SEM for n = 3 independent experiments. Unpaired two-tailed t tests is shown for doublet combination compared with single agent, or triple therapy compared to doublet combination (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

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PDX-derived breast tumor organoids were treated with fulvestrant, ABT-199, palbociclib, or combination therapy to evaluate tumor response (Fig. 4; Supplementary Fig. S9) using a parallel approach to that described for MCF7 and T47D cells. Doses were selected on the basis of the dose–response observed for single-agent treatment (Supplementary Fig. S9A and S9B). As illustrated for PDX 1105 (Fig. 4CF), treatment for 14 days reduced tumor organoid formation (Fig. 4C) and cell viability (Fig. 4D), with the greatest reduction observed upon triple therapy. Recovery in growth and viability was observed after a 1 week wash-out period for organoids treated with doublet combinations, whereas minimal tumor organoids were observed after triple therapy (Fig. 4C and D). Similar findings were observed in the other PDX organoid models (Supplementary Fig. S9C). As expected, fulvestrant and palbociclib reduced cellular proliferation, as measured by Ki67 (Fig. 4E; Supplementary Fig. S9D), while ABT-199 (as a single agent or in combination) augmented cell death, as determined by CC3 expression by Western blot analysis (Fig. 4F) or immunostaining (Supplementary Fig. S9E). Similar effects of fulvestrant, ABT-199, and palbociclib on cell viability were observed in three tumor organoid models that were directly derived from primary breast tumors (Fig. 4G).

Dual CDK4/6 and BCL2 inhibition enhances tumor response in vivo

The in vivo response to combination therapy was next evaluated in the four ER+ BCL2+ PDX models, whereby NSG mice bearing PDX tumors were treated with up to 3 cycles of combination therapy. A treatment cycle was defined as 28 days, with fulvestrant administered weekly for 3 weeks (on day 1, 8, 15). Palbociclib and ABT-199 were administered for 3 weeks (on days 1–5, 8–12, 15–19) followed by a 1-week rest. Fulvestrant alone marginally delayed tumor growth compared with vehicle in all models. Tumor response was improved with either fulvestrant–palbociclib or fulvestrant–ABT-199 combinations. Triple therapy that included ABT-199 further attenuated tumor growth during the treatment period (3 cycles of therapy for PDX 315 and 1232) and improved survival to the experimental ethical endpoint, including the 315 model, which was derived from a patient with endocrine refractory breast cancer (Fig. 5A; Supplementary Fig. S10A). Mechanistically, this response was accompanied by reduced cellular proliferation and increased apoptosis, as determined by Ki67 and CC3 levels, respectively (Fig. 5BD). As expected in the RB1-deficient PDX 50 model, palbociclib did not improve tumor response to fulvestrant, although the fulvestrant–ABT-199 combination was efficacious in vivo (Supplementary Fig. S10B).

Figure 5.

BCL2 and CDK 4/6 inhibition with endocrine therapy attenuates growth of ER+ tumors. A, Tumor growth volume curves (left) and Kaplan–Meier survival curves (right) for PDX models 315, 1232, and 1105. Mice were treated for 21 days in a 28-day cycle, with vehicle alone (black line), fulvestrant (5 mg intraperitoneally weekly on days 1, 8, and 15) plus vehicle for ABT-199 and vehicle for palbociclib (orange line), fulvestrant plus ABT-199 (100 mg/kg oral gavage days 1–5, 8–12, 15–19) plus vehicle for palbociclib (green line), fulvestrant plus palbociclib (100 mg/kg oral gavage days 1–5, 8–12, 15–19) plus vehicle for ABT-199 (blue line), or combination fulvestrant plus ABT-199 and palbociclib (red line). Horizontal black bars at top of plots indicate treatment cycles. Data for mean ± SEM are shown. Log-rank (Mantel–Cox) P value is shown for triple combination therapy versus doublet combination of fulvestrant and palbociclib. Tumor growth curves for individual mice from PDX models 315, 1232, and 1105 are shown in Supplementary Fig. S10A. B, Representative tumor sections from PDX 1232 tumors immunostained for Ki67 and cleaved caspase 3 (CC3) after treatment with the indicated treatments as described in A. Tumors were collected after 48 hours of ABT-199 and/or palbociclib treatment. Scale bar, 50 μm. C, Quantification of Ki67 and CC3 immunostaining from B. Values represent mean ± SEM (n = 3 tumors per condition; 3 fields per tumor). D, Western blot of protein lysates from B for pRb, cleaved caspase 3 (CC3), and cleaved PARP (cPARP). Tubulin was used as a loading control.

Figure 5.

BCL2 and CDK 4/6 inhibition with endocrine therapy attenuates growth of ER+ tumors. A, Tumor growth volume curves (left) and Kaplan–Meier survival curves (right) for PDX models 315, 1232, and 1105. Mice were treated for 21 days in a 28-day cycle, with vehicle alone (black line), fulvestrant (5 mg intraperitoneally weekly on days 1, 8, and 15) plus vehicle for ABT-199 and vehicle for palbociclib (orange line), fulvestrant plus ABT-199 (100 mg/kg oral gavage days 1–5, 8–12, 15–19) plus vehicle for palbociclib (green line), fulvestrant plus palbociclib (100 mg/kg oral gavage days 1–5, 8–12, 15–19) plus vehicle for ABT-199 (blue line), or combination fulvestrant plus ABT-199 and palbociclib (red line). Horizontal black bars at top of plots indicate treatment cycles. Data for mean ± SEM are shown. Log-rank (Mantel–Cox) P value is shown for triple combination therapy versus doublet combination of fulvestrant and palbociclib. Tumor growth curves for individual mice from PDX models 315, 1232, and 1105 are shown in Supplementary Fig. S10A. B, Representative tumor sections from PDX 1232 tumors immunostained for Ki67 and cleaved caspase 3 (CC3) after treatment with the indicated treatments as described in A. Tumors were collected after 48 hours of ABT-199 and/or palbociclib treatment. Scale bar, 50 μm. C, Quantification of Ki67 and CC3 immunostaining from B. Values represent mean ± SEM (n = 3 tumors per condition; 3 fields per tumor). D, Western blot of protein lysates from B for pRb, cleaved caspase 3 (CC3), and cleaved PARP (cPARP). Tubulin was used as a loading control.

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Because palbociclib is associated with neutropenia (3–5) and venetoclax produces “on-target” lymphopenia and low-grade neutropenia (16), we next determined whether myelotoxicity was problematic in vivo. As NSG mice are leukopenic, safety studies were conducted on healthy BALB/c mice (Supplementary Fig. S11). Mice were treated with the same regimen used for the PDX models, comprising 3 weeks of therapy followed by a 1-week “rest” period. Triple therapy was well tolerated, with mice maintaining normal body weight (Supplementary Fig. S11A, left), similar to the findings in NSG tumor-bearing mice (Supplementary Fig. S11A, right). As anticipated, a reduction in neutrophils and lymphocytes was observed at three weeks (cycles 1 and 2, day 21), but these recovered after a 1-week rest period (cycles 1 and 2, day 28; Supplementary Fig. S11B). No perturbation in urea, creatinine, or liver enzymes was observed at day 28 of cycles 1 and 2 (Supplementary Fig. S11C). These findings suggest that combination therapy might be safely administered in humans.

Combination therapy induces an immune response in mice bearing ER+ tumors

CDK4/6 inhibitors have been recently shown to promote antitumor immunity (6, 7), while venetoclax has been associated with lymphopenia in patients with breast cancer (16), raising the possibility that combination therapy could adversely impact the immune response. We therefore evaluated immune modulation in a syngeneic mouse mammary tumor model using the 67NR cell line, which is a nonmetastasizing subclone derived from a spontaneous BALB/cfC3H mammary tumor (39). This cell line expresses nuclear ER (40) and is BCL2 positive (Supplementary Fig. S12A). We first confirmed that the growth of 67NR cells was endocrine dependent through siRNA-mediated knockdown of ESR1, which significantly reduced cell viability (Fig. 6A; Supplementary Fig. S12B). These cells also displayed a dose-dependent response to fulvestrant (Supplementary Fig. S12C). Thus, 67NR cells represent a potentially useful syngeneic model to investigate the in vivo immune effects of palbociclib and ABT-199.

Figure 6.

Combination therapy induces an immune response, augmented by PD1 inhibition. A, Western blot analysis of ER expression in 67NR cells following siRNA-mediated knockdown of ESR1. Expression for mock, a nontargeted (NT) control guide and ESR1 are shown. Actin was used as a loading control. B, FACS analysis of H-2KD on the 67NR cell line after 72 hours treatment with vehicle, fulvestrant, fulvestrant plus ABT-199, fulvestrant plus palbociclib or the combination. Data represent median fluorescence intensity (MFI) relative to vehicle for 8 independent experiments. Error bars, mean ± SEM. P values are from paired t tests on the log(MFI) scale. C, Kaplan–Meier survival curves for 67NR tumors. Mice were treated for 21 days in a 28-day cycle, with vehicles alone (black line), fulvestrant (5 mg s.c. once weekly) plus vehicle for ABT-199 plus vehicle for palbociclib (orange line), fulvestrant plus ABT-199 (100 mg/kg oral gavage continuously) plus vehicle for palbociclib (green line), fulvestrant plus palbociclib (100 mg/kg oral gavage continuously) plus vehicle for ABT-199 (blue line), or combination fulvestrant, ABT-199, and palbociclib (red line). Tumor growth curves are shown in Supplementary Fig. S12H. Log-rank (Mantel–Cox) is shown for triple combination therapy versus fulvestrant and palbociclib. D, Schematic diagram of the experimental protocol for short-term studies. 67NR cells were transplanted into the cleared fat pads of syngeneic (BALB/c) mice and allowed to form tumors. Tumors and spleens were then collected 9 days after treatment. Quantification of Tregs in tumors (E), their proliferative activity (Ki67; F), and their PD1 expression (G). H, Quantification of TNFα in splenic CD4+ and CD8+ T cells following in vitro stimulation with ionomycin, phorbol 12-myristate 13-acetate, and Golgistop. I, FACS analysis of PDL1 expression in 67NR cells after 72-hour treatment with vehicle, fulvestrant, fulvestrant plus ABT-199, fulvestrant plus palbociclib, or the combination. Data represent median fluorescence intensity (MFI) relative to vehicle for six independent experiments. Error bars, mean ± SEM. P values are from paired t tests on the log (MFI) scale. J, Tumor growth volume curves (left) and Kaplan–Meier survival curves (right) for 67NR tumor model. Mice were treated for 21 days in a 28-day cycle, with vehicle (including an isotype control antibody for anti-PD1) alone (black line), fulvestrant plus ABT-199 (100 mg/kg oral gavage continuously) plus vehicle for palbociclib plus vehicle including an isotype control antibody for anti-PD1 (orange line), fulvestrant plus ABT-199 plus anti-PD1 (200 μg i.p. days 1, 3, 5) plus vehicle for palbociclib (green line), fulvestrant plus ABT-199 plus palbociclib (100 mg/kg oral gavage continuously) plus vehicle including an isotype control antibody for anti-PD1 (blue line), or combination fulvestrant, ABT-199, palbociclib, and anti-PD1 (red line). Log-rank (Mantel–Cox) P value is shown for quadruple combination therapy with anti-PD1 versus the triple combination comprising fulvestrant, ABT-199, isotype control, and palbociclib. Tumor growth curves are shown in Supplementary Fig. S14H. For E–H, flow cytometric analysis is representative of two independent experiments with n = 5–6 mice per group. Mean ± SEM are shown with analysis by unpaired two-tailed t tests (E–I; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 6.

Combination therapy induces an immune response, augmented by PD1 inhibition. A, Western blot analysis of ER expression in 67NR cells following siRNA-mediated knockdown of ESR1. Expression for mock, a nontargeted (NT) control guide and ESR1 are shown. Actin was used as a loading control. B, FACS analysis of H-2KD on the 67NR cell line after 72 hours treatment with vehicle, fulvestrant, fulvestrant plus ABT-199, fulvestrant plus palbociclib or the combination. Data represent median fluorescence intensity (MFI) relative to vehicle for 8 independent experiments. Error bars, mean ± SEM. P values are from paired t tests on the log(MFI) scale. C, Kaplan–Meier survival curves for 67NR tumors. Mice were treated for 21 days in a 28-day cycle, with vehicles alone (black line), fulvestrant (5 mg s.c. once weekly) plus vehicle for ABT-199 plus vehicle for palbociclib (orange line), fulvestrant plus ABT-199 (100 mg/kg oral gavage continuously) plus vehicle for palbociclib (green line), fulvestrant plus palbociclib (100 mg/kg oral gavage continuously) plus vehicle for ABT-199 (blue line), or combination fulvestrant, ABT-199, and palbociclib (red line). Tumor growth curves are shown in Supplementary Fig. S12H. Log-rank (Mantel–Cox) is shown for triple combination therapy versus fulvestrant and palbociclib. D, Schematic diagram of the experimental protocol for short-term studies. 67NR cells were transplanted into the cleared fat pads of syngeneic (BALB/c) mice and allowed to form tumors. Tumors and spleens were then collected 9 days after treatment. Quantification of Tregs in tumors (E), their proliferative activity (Ki67; F), and their PD1 expression (G). H, Quantification of TNFα in splenic CD4+ and CD8+ T cells following in vitro stimulation with ionomycin, phorbol 12-myristate 13-acetate, and Golgistop. I, FACS analysis of PDL1 expression in 67NR cells after 72-hour treatment with vehicle, fulvestrant, fulvestrant plus ABT-199, fulvestrant plus palbociclib, or the combination. Data represent median fluorescence intensity (MFI) relative to vehicle for six independent experiments. Error bars, mean ± SEM. P values are from paired t tests on the log (MFI) scale. J, Tumor growth volume curves (left) and Kaplan–Meier survival curves (right) for 67NR tumor model. Mice were treated for 21 days in a 28-day cycle, with vehicle (including an isotype control antibody for anti-PD1) alone (black line), fulvestrant plus ABT-199 (100 mg/kg oral gavage continuously) plus vehicle for palbociclib plus vehicle including an isotype control antibody for anti-PD1 (orange line), fulvestrant plus ABT-199 plus anti-PD1 (200 μg i.p. days 1, 3, 5) plus vehicle for palbociclib (green line), fulvestrant plus ABT-199 plus palbociclib (100 mg/kg oral gavage continuously) plus vehicle including an isotype control antibody for anti-PD1 (blue line), or combination fulvestrant, ABT-199, palbociclib, and anti-PD1 (red line). Log-rank (Mantel–Cox) P value is shown for quadruple combination therapy with anti-PD1 versus the triple combination comprising fulvestrant, ABT-199, isotype control, and palbociclib. Tumor growth curves are shown in Supplementary Fig. S14H. For E–H, flow cytometric analysis is representative of two independent experiments with n = 5–6 mice per group. Mean ± SEM are shown with analysis by unpaired two-tailed t tests (E–I; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

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To determine whether ABT-199 modulated immunogenic activation of tumor cells by a CDK4/6 inhibitor, 67NR cells were treated with fulvestrant or combinations containing palbociclib and/or ABT-199. As previously described in human cells for MHCI, and in a mouse mammary tumor model (7), palbociclib in combination with fulvestrant increased expression of MHCI molecules (H-2Kd). Intriguingly, H-2Kd levels were also increased by ABT-199 and were further elevated when both drugs were combined (Fig. 6B), with similar findings for β2-microglobulin (Supplementary Fig. S12D and S12E). In keeping with enhanced IFN signaling, we also observed increased expression of phospho-STAT1 in 67NR and MCF7 cells in response to ABT-199 treatment (Supplementary Fig. S12F and S12G). These data suggest that ABT-199 further enhances the immunogenicity of 67NR cells when combined with palbociclib.

We next transplanted 67NR cells into the cleared mammary fat pads of BALB/c recipient mice. Mice bearing small tumors 10–14 days after engraftment were randomized into one of five treatment arms: (i) vehicle, (ii) fulvestrant, (iii) fulvestrant plus ABT-199, (iv) fulvestrant plus palbociclib, or (v) fulvestrant plus ABT-199 and palbociclib. While single-agent treatment with fulvestrant produced modest attenuation in growth, there was no benefit to the addition of palbociclib or ABT-199. However, similar to PDX models (Fig. 5), triple therapy elicited an improved response and survival benefit compared with doublet therapy with either fulvestrant–palbociclib or fulvestrant–ABT-199 (Fig. 6C; Supplementary Fig. S12H).

To explore the intratumoral immune response to combination therapy, analysis was performed after a short treatment of 9 days, when most vehicle-treated tumors reached ethical endpoint (Fig. 6D). Flow cytometric analysis of the tumor-infiltrating cells showed that combinations of fulvestrant–palbociclib resulted in a reduction in the proportion and absolute number of FOXP3+ Tregs (Fig. 6E; Supplementary Fig. S13A–S13C), elevated levels of which are associated with poor survival in ER+ breast cancer (41). Consistent with this observation, Tregs were also less proliferative (Fig. 6F), suggesting the impact of pRb and BCL2 inhibition in this setting. The reduction in Tregs resulted in a modest but significant increase in the CD8/Treg ratio (Supplementary Fig. S13D). Reduced expression of the immune checkpoint receptor PD1 in Tregs was also observed (Fig. 6G), consistent with previous findings in a breast cancer mouse model using the CDK4/6 inhibitor abemaciclib (7). The systemic impact of BCL2 and CDK4/6 inhibition was most pronounced in the tumor microenvironment, but modest effects of combination therapy on Treg cells were observed in the spleen (Supplementary Fig. S14A–S14D). Furthermore, increased production of tumor necrosis factor (TNFα) by CD4+ and CD8+ T cells (Fig. 6H) and a trend toward increased IFNγ were evident (Supplementary Fig. S14E and S14F), indicative of enhanced effector function. Together, these findings confirm that increased tumor immunogenicity and intratumoral immune cell activation are provoked by palbociclib and indicate that ABT-199 does not antagonize these effects, but may further enhance them.

To further investigate the immunomodulatory effects of CDK4/6 and BCL2 inhibition, we measured PDL1 expression in 67NR cells in vitro and noted that ABT-199 increased PDL1 levels in 67NR cells (Fig. 6I). A similar effect was observed in MCF7 cells (Supplementary Fig S14G). The favorable intratumoral immune microenvironment and upregulation of PDL1 expression in 67NR cells suggested that immune checkpoint blockade might further improve the efficacy of triple therapy. We therefore treated 67NR tumor–bearing mice with either (i) vehicle (which included an isotype control antibody); (ii) fulvestrant and ABT-199; (iii) fulvestrant, ABT-199, and anti-PD1; (iv) fulvestrant plus ABT-199 and palbociclib; or (v) fulvestrant, ABT-199, palbociclib, and anti-PD1. Consistent with the data shown in Fig. 6C, triple therapy improved tumor response and survival, compared with fulvestrant plus ABT-199. The addition of anti-PD1 therapy to this triple combination further attenuated tumor growth and improved survival (Fig. 6J; Supplementary Fig. S14H).

Despite the majority of patients with ER+ breast cancer responding to endocrine therapy, the duration of response is highly variable due to multiple mechanisms that result in innate or acquired resistance (42). The recent emergence of CDK4/6 inhibitors has changed the landscape for treatment of ER+ breast cancer, with remarkable improvements in survival (3–5) for a subset of patients. Resistance to therapy, however, is almost inevitable. In patients with breast cancer, the combination of endocrine therapy with CDK4/6 inhibition is predominantly cytostatic, likely accounting for reduced apoptosis (12). Combination therapy that overcomes resistance through the induction of tumor cell death could therefore be beneficial. Using breast cancer cell lines, organoids, and PDX models, we show that inhibition of BCL2 and CDK4/6 in combination with endocrine therapy can inhibit proliferation and induce apoptosis of cancer cells, including of phenotypically senescent cells, leading to enhanced responsiveness in vivo. Notably, we also show that ABT-199 augments the CDK4/6 inhibitor response through dephosphorylation of Rb and reduced G1–S cyclins. In addition, pharmacologic inhibition of both CDK4/6 and BCL2 in a syngeneic model reduced the proportion and proliferation of Tregs in the tumor microenvironment. This was accompanied by T-cell activation, with improved antitumor activity observed in vivo upon PD1 blockade.

The unexpected observation that ABT-199 and other BH3 mimetics led to Rb dephosphorylation (or possibly degradation of pRb), and reduced G1–S cyclins potentially accounts for ABT-199 enhancing the effect of fulvestrant and palbociclib. As this was more pronounced at higher doses, the clinical significance of this finding is unclear. Although this was a caspase-independent effect (indicating that the phenotype is independent of the final “demolition” stage of apoptosis), it remains possible that the effect is due to mitochondrial stress imposed by BH3 mimetics and preferential killing of cycling cells. Further studies will be required to clarify this, including on BAX/BAK-deficient cells. The downregulation of ER following ABT-199 treatment would be consistent with the resultant G1 arrest. The precise mechanism underpinning the reduction in G1 cyclins (cyclin D and E) is unclear but this could alleviate resistance to CDK4/6 inhibitors given that elevated cyclin E levels have been clinically reported (43). These findings indicate the potential of triple therapy as early line therapy for patients with endocrine sensitive tumors, aimed at delaying adaptive resistance.

As reported previously (7, 8), palbociclib induced a senescence-like phenotype, characterized by distinctive morphologic changes that included cell flattening and enlargement of growth-arrested cells that were ß-gal–positive and more resistant to apoptosis. Notably, short- and long-term palbociclib-treated cells appeared to be more vulnerable to ABT-199–mediated apoptotic cell death. Previous reports have shown that BCL-XL is a senolytic target (14). Our findings indicate that BCL2 is also a target and suggest that venetoclax could be a senolytic agent when combined with CDK4/6 inhibitor therapy in breast tumors.

Data from clinical trial samples are helping to elucidate the pleiotropic mechanisms of acquired resistance to CDK4/6 inhibitors. Due to the requirement for intact Rb tumor suppressor protein function, RB1 loss or mutation has been identified as a robust marker of resistance to CDK4/6 inhibition. Notably, patients who progressed on fulvestrant and palbociclib in the PALOMA-2 clinical trial developed RB1 mutations, although at relatively low prevalence (34). ABT-199 may provide an alternate means of targeting these tumors, as demonstrated in vitro and in vivo in PDX model 50, which harbored a RB1 mutation. Other resistance mechanisms that have been identified in patient tumors include perturbations in the PI3K/AKT and mTOR, FGFR, IL6–JAK–Stat, E2F, and Hippo signaling pathways (32–38). Our CRISPR/Cas9 screen of fulvestrant–palbociclib versus fulvestrant-treated cells produced differential enrichment of sgRNA guides in these pathways, supporting those findings. Interestingly, we observed differentially enriched sgRNA guides for epigenetic modifier genes in the MOZ/MORF complex, such as KAT6B, suggesting that these may represent novel targets. When we evaluated sgRNA guides that were enriched in fulvestrant–palbociclib–venetoclax versus fulvestrant–palbociclib–treated cells, RB1 mutations were under-represented with triple therapy, while targets identified above did not feature. This, together with the reduction in cyclin E levels noted above, suggests that cotreatment with venetoclax could mitigate some of the mechanisms of acquired resistance to CDK4/6 inhibitors.

While immune checkpoint inhibitors are showing promise for triple-negative breast cancer, ER+ breast cancers contain fewer TILs and are considered to be far less immunogenic (44). It is noteworthy, however, that anti-PDL1 therapy led to responses in a subset of patients with advanced ER+ PDL1+ breast cancer (45). Intriguingly, CDK4/6 inhibitors have been shown to modulate the tumor immune microenvironment by increasing antigen presentation, reducing inhibitory Treg cells and stimulating increased PDL1 expression (6, 7). These findings imply that the therapeutic effect of these agents may be mediated, in part, through overcoming immune evasion, although the magnitude of this effect remains uncertain. Utilizing a syngeneic model of ER+ breast cancer, we confirmed these observations and showed that ABT-199 augments CD4 and CD8 T-cell activation and does not abrogate palbociclib-mediated effects in reducing Tregs. Although ABT-199 induces a peripheral lymphopenia, this is largely due to a reduction in B cells (16). Our data suggest that ABT-199 may favorably contribute to the intratumoral immune microenvironment rather than adversely impact immune signaling systemically. The additional response we observed with PD1 checkpoint inhibition supports this observation. These data are also consistent with recent findings using a MYC-driven model of breast cancer where adjuvant ABT-199 plus anti-PD1 therapy produced a survival benefit (46).

Despite the promising responses observed in preclinical models of ER+ breast cancer, it remains to be determined if the triple combination will be effective and can be safely delivered in the clinic. Both CDK4/6 inhibitors and venetoclax are metabolized by CYP3A4 and can induce neutropenia and lymphopenia, although this has not proven to be problematic when each drug is administered alone with endocrine therapy (16, 47–49). The 28-day regimen used in our studies included a 1-week break from both drugs (currently in clinical use for palbociclib and ribociclib) and appeared to facilitate leukocyte recovery. This regimen could also potentially prime tumor cells for cell death, given that BCL2 levels rose following palbociclib withdrawal.

The specificity of ABT-199 for BCL2 is advantageous for reducing possible toxicity, given the limitation of current BCLXL inhibitors due to their induction of on-target thrombocytopenia (50). However, this could increase the risk of resistance due to upregulation of prosurvival proteins, including BCLXL and MCL1. We observed that prolonged exposure to high concentrations of palbociclib increased BCLXL expression in both MCF7 and T47D cells. However, they remained sensitive to ABT-199, presumably due to increased cellular stress. The downregulation of MCL1 following palbociclib treatment, together with its potential role in inducing resistance to standard therapy observed in our CRISPR/Cas9 screen, indicates that MCL1 is also a possible target. This is consistent with findings by others that MCL1 is a potential target in ER+ breast cancer (51, 52). Further investigation of combinatorial (if clinically tolerated) or sequential BH3 mimetic therapy may therefore be warranted.

It will be important to determine whether our preclinical findings translate in the clinic for patients with ER+ breast cancer. BCL2 is often expressed at high levels in ER+ tumors, presumably because BCL2 is an estrogen-responsive gene. This could account for the apparent paradox of BCL2 being considered a favorable prognostic marker, while also contributing to treatment resistance (19). Importantly, the majority of relapsing ER+ tumors are BCL2 positive (16), underscoring its potential utility as a therapeutic target.

The combination of ABT-199 with tamoxifen has shown promising clinical activity in patients with metastatic ER+ breast cancer (16). These findings are currently being further investigated in a randomized phase II study of fulvestrant with or without venetoclax in patients with ER+ breast cancer who progress on a CDK4/6 inhibitor (NCT03584009). Here we show that combining BCL2 and CDK4/6 inhibition with endocrine therapy elicits potent activity in endocrine-sensitive and refractory models of ER+ breast cancer. This combination overcomes the cytostatic effects of endocrine and CDK4/6 inhibitor therapy to trigger cell death in vivo, and induces a favorable tumor immune microenvironment, together supporting further investigation of this combination in the clinic, currently underway (NCT03900884).

The Walter and Eliza Hall Institute receives milestone and royalty payments related to venetoclax, and employees (J.R. Whittle, F. Vaillant, E. Surgenor, A.N. Policheni, G. Giner, B.D. Capaldo, H. Chen, H.K. Liu, M.J. Herold, G.K. Smyth, D.H.D. Gray, J.E. Visvader, and G.J. Lindeman) may be eligible for benefits related to these payments. J.R. Whittle is an employee of The Walter and Eliza Hall Institute of Medical Research and reports receiving benefits related to venetoclax. N. Sachs and H. Clevers are listed as co-inventors on two patents that relate to improved culture methods for expanding epithelial stem cells and obtaining organoids, which is owned by the Royal Netherlands Academy of Arts and Sciences (KNAW, Koninklijke Nederlandse Akademie van Wetenschappen) and licensed to the HUB Foundation for Organoid Technology. H. Clevers is a paid advisory board member for Roche Holding. D.H.D. Gray reports receiving commercial research grants from Servier Pharmaceuticals. J.E. Visvader is an employee of The Walter and Eliza Hall Institute of Medical Research and reports receiving benefits related to venetoclax. G.J. Lindeman is an employee of The Walter and Eliza Hall Institute of Medical research and reports receiving benefits related to venetoclax; is a paid advisory board member of AbbVie; reports receiving commercial research grants from AbbVie, Roche/Genentech, Pfizer, and Servier; reports receiving speakers bureau honoraria from Amgen and Genentech; and is an unpaid consultant/advisory board member for Roche. No potential conflicts of interest were disclosed by the other authors.

Conception and design: J.R. Whittle, F. Vaillant, J.E. Visvader, G.J. Lindeman

Development of methodology: J.R. Whittle, N. Sachs, D.H.D. Gray, J.E. Visvader, G.J. Lindeman

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.R. Whittle, F. Vaillant, E. Surgenor, A.N. Policheni, B.D. Capaldo, H.-R. Chen, H.K. Liu, J.F. Dekkers, A. Fellowes, T. Green, H. Xu, S.B. Fox, J.E. Visvader, G.J. Lindeman

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.R. Whittle, F. Vaillant, A.N. Policheni, G. Giner, A. Fellowes, T. Green, H. Xu, M.J. Herold, G.K. Smyth, D.H.D. Gray, J.E. Visvader, G.J. Lindeman

Writing, review, and/or revision of the manuscript: J.R. Whittle, F. Vaillant, G. Giner, H.K. Liu, H. Clevers, A. Fellowes, S.B. Fox, G.K. Smyth, D.H.D. Gray, J.E. Visvader, G.J. Lindeman

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.R. Whittle, S.B. Fox, J.E. Visvader, G.J. Lindeman

Study supervision: J.E. Visvader, G.J. Lindeman

We are grateful to G.B. Mann, L. Graham, and staff at the Royal Melbourne Hospital Tissue Bank for support and thank the Animal, Histology and FACS services at WEHI. We thank A. Strasser for helpful discussions. Coded breast tumor samples were provided by the Victorian Cancer Biobank (which is supported by the Victorian Government). This work was supported by the National Health and Medical Research Council, Australia (NHMRC 1054618, 1113133, 1153049), NHMRC IRIISS, Victorian State Government Operational Infrastructure Support, Australian Cancer Research Foundation, National Breast Cancer Foundation (NT-13-06, IIRS-19-004), Breast Cancer Research Foundation (BCRF-18-182), Cancer Australia (1165878), Qualtrough Cancer Research Fund, Joan Marshall Breast Cancer Research Fund, and Collie Foundation (2017F001). J.R. Whittle was supported by an NHMRC/NBCF Research Fellowship and the Royal Australasian College of Physicians, A.N. Policheni by a Cancer Council Victoria Postdoctoral Research Fellowship, J.F. Dekkers by a Marie-Curie Postdoctoral Fellowship, and NHMRC Research Fellowships to D.H.D. Gray (1090236 and 1158024), G.K. Smyth (1058892), J.E. Visvader (1037230), and G.J. Lindeman (1078730 and 1175960).

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.

1.
Musgrove
EA
,
Caldon
CE
,
Barraclough
J
,
Stone
A
,
Sutherland
RL
. 
Cyclin D as a therapeutic target in cancer
.
Nat Rev Cancer
2011
;
11
:
558
72
.
2.
O'Leary
B
,
Finn
RS
,
Turner
NC
. 
Treating cancer with selective CDK4/6 inhibitors
.
Nat Rev Clin Oncol
2016
;
13
:
417
30
.
3.
Im
SA
,
Lu
YS
,
Bardia
A
,
Harbeck
N
,
Colleoni
M
,
Franke
F
, et al
Overall survival with ribociclib plus endocrine therapy in breast cancer
.
N Engl J Med
2019
;
381
:
307
16
.
4.
Sledge
GW
 Jr.
,
Toi
M
,
Neven
P
,
Sohn
J
,
Inoue
K
,
Pivot
X
, et al
The effect of abemaciclib plus fulvestrant on overall survival in hormone receptor-positive, ERBB2-negative breast cancer that progressed on endocrine therapy-MONARCH 2: a randomized clinical trial
.
JAMA Oncol
2020
;
6
:
116
24
.
5.
Turner
NC
,
Slamon
DJ
,
Ro
J
,
Bondarenko
I
,
Im
SA
,
Masuda
N
, et al
Overall survival with palbociclib and fulvestrant in advanced breast cancer
.
N Engl J Med
2018
;
379
:
1926
36
.
6.
Deng
J
,
Wang
ES
,
Jenkins
RW
,
Li
S
,
Dries
R
,
Yates
K
, et al
CDK4/6 inhibition augments antitumor immunity by enhancing T-cell activation
.
Cancer Discov
2018
;
8
:
216
33
.
7.
Goel
S
,
DeCristo
MJ
,
Watt
AC
,
BrinJones
H
,
Sceneay
J
,
Li
BB
, et al
CDK4/6 inhibition triggers anti-tumour immunity
.
Nature
2017
;
548
:
471
5
.
8.
Herrera-Abreu
MT
,
Palafox
M
,
Asghar
U
,
Rivas
MA
,
Cutts
RJ
,
Garcia-Murillas
I
, et al
Early adaptation and acquired resistance to CDK4/6 inhibition in estrogen receptor-positive breast cancer
.
Cancer Res
2016
;
76
:
2301
13
.
9.
Hydbring
P
,
Malumbres
M
,
Sicinski
P
. 
Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases
.
Nat Rev Mol Cell Biol
2016
;
17
:
280
92
.
10.
Choi
YJ
,
Li
X
,
Hydbring
P
,
Sanda
T
,
Stefano
J
,
Christie
AL
, et al
The requirement for cyclin D function in tumor maintenance
.
Cancer Cell
2012
;
22
:
438
51
.
11.
Finn
RS
,
Dering
J
,
Conklin
D
,
Kalous
O
,
Cohen
DJ
,
Desai
AJ
, et al
PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro
.
Breast Cancer Res
2009
;
11
:
R77
.
12.
Johnston
S
,
Puhalla
S
,
Wheatley
D
,
Ring
A
,
Barry
P
,
Holcombe
C
, et al
Randomized phase II study evaluating palbociclib in addition to letrozole as neoadjuvant therapy in estrogen receptor-positive early breast cancer: PALLET trial
.
J Clin Oncol
2019
;
37
:
178
89
.
13.
Wang
E
. 
Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved
.
Cancer Res
1995
;
55
:
2284
92
.
14.
Yosef
R
,
Pilpel
N
,
Tokarsky-Amiel
R
,
Biran
A
,
Ovadya
Y
,
Cohen
S
, et al
Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL
.
Nat Commun
2016
;
7
:
11190
.
15.
Dawson
SJ
,
Makretsov
N
,
Blows
FM
,
Driver
KE
,
Provenzano
E
,
Le Quesne
J
, et al
BCL2 in breast cancer: a favourable prognostic marker across molecular subtypes and independent of adjuvant therapy received
.
Br J Cancer
2010
;
103
:
668
75
.
16.
Lok
SW
,
Whittle
JR
,
Vaillant
F
,
Teh
CE
,
Lo
LL
,
Policheni
AN
, et al
A phase Ib dose-escalation and expansion study of the BCL2 inhibitor venetoclax combined with tamoxifen in ER and BCL2-positive metastatic breast cancer
.
Cancer Discov
2019
;
9
:
354
69
.
17.
Cory
S
,
Roberts
AW
,
Colman
PM
,
Adams
JM
. 
Targeting BCL-2-like proteins to kill cancer cells
.
Trends Cancer
2016
;
2
:
443
60
.
18.
Leverson
JD
,
Sampath
D
,
Souers
AJ
,
Rosenberg
SH
,
Fairbrother
WJ
,
Amiot
M
, et al
Found in translation: how preclinical research is guiding the clinical development of the BCL2-selective inhibitor venetoclax
.
Cancer Discov
2017
;
7
:
1376
93
.
19.
Merino
D
,
Lok
SW
,
Visvader
JE
,
Lindeman
GJ
. 
Targeting BCL-2 to enhance vulnerability to therapy in estrogen receptor-positive breast cancer
.
Oncogene
2016
;
35
:
1877
87
.
20.
Souers
AJ
,
Leverson
JD
,
Boghaert
ER
,
Ackler
SL
,
Catron
ND
,
Chen
J
, et al
ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets
.
Nat Med
2013
;
19
:
202
8
.
21.
Roberts
AW
,
Davids
MS
,
Pagel
JM
,
Kahl
BS
,
Puvvada
SD
,
Gerecitano
JF
, et al
Targeting BCL2 with venetoclax in relapsed chronic lymphocytic leukemia
.
N Engl J Med
2016
;
374
:
311
22
.
22.
Vaillant
F
,
Merino
D
,
Lee
L
,
Breslin
K
,
Pal
B
,
Ritchie
ME
, et al
Targeting BCL-2 with the BH3 mimetic ABT-199 in estrogen receptor-positive breast cancer
.
Cancer Cell
2013
;
24
:
120
9
.
23.
Nolan
E
,
Savas
P
,
Policheni
AN
,
Darcy
PK
,
Vaillant
F
,
Mintoff
CP
, et al
Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated breast cancer
.
Sci Transl Med
2017
;
9
:
eaal4922
.
24.
Dekkers
JF
,
Whittle
JR
,
Vaillant
F
,
Chen
HR
,
Dawson
C
,
Liu
K
, et al
Modeling breast cancer using CRISPR/Cas9-mediated engineering of human breast organoids
.
J Natl Cancer Inst
2020
;
112
:
540
4
.
25.
Sachs
N
,
de Ligt
J
,
Kopper
O
,
Gogola
E
,
Bounova
G
,
Weeber
F
, et al
A living biobank of breast cancer organoids captures disease heterogeneity
.
Cell
2018
;
172
:
373
86
.
26.
Oakes
SR
,
Vaillant
F
,
Lim
E
,
Lee
L
,
Breslin
K
,
Feleppa
F
, et al
Sensitization of BCL-2-expressing breast tumors to chemotherapy by the BH3 mimetic ABT-737
.
Proc Natl Acad Sci U S A
2012
;
109
:
2766
71
.
27.
Ma
CX
,
Gao
F
,
Luo
J
,
Northfelt
DW
,
Goetz
M
,
Forero
A
, et al
NeoPalAna: neoadjuvant palbociclib, a cyclin-dependent kinase 4/6 inhibitor, and anastrozole for clinical stage 2 or 3 estrogen receptor-positive breast cancer
.
Clin Cancer Res
2017
;
23
:
4055
65
.
28.
Letai
A
,
Bassik
MC
,
Walensky
LD
,
Sorcinelli
MD
,
Weiler
S
,
Korsmeyer
SJ
. 
Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics
.
Cancer Cell
2002
;
2
:
183
92
.
29.
JavanMoghadam
S
,
Weihua
Z
,
Hunt
KK
,
Keyomarsi
K
. 
Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion
.
Cell Cycle
2016
;
15
:
1579
90
.
30.
Kotschy
A
,
Szlavik
Z
,
Murray
J
,
Davidson
J
,
Maragno
AL
,
Le Toumelin-Braizat
G
, et al
The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models
.
Nature
2016
;
538
:
477
82
.
31.
Leverson
JD
,
Phillips
DC
,
Mitten
MJ
,
Boghaert
ER
,
Diaz
D
,
Tahir
SK
, et al
Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy
.
Sci Transl Med
2015
;
7
:
279ra40
.
32.
Finn
RS
,
Liu
Y
,
Zhu
Z
,
Martin
M
,
Rugo
HS
,
Dieras
V
, et al
Biomarker analyses of response to cyclin-dependent kinase 4/6 inhibition and endocrine therapy in women with treatment-naive metastatic breast cancer
.
Clin Cancer Res
2020
;
26
:
110
21
.
33.
Formisano
L
,
Lu
Y
,
Servetto
A
,
Hanker
AB
,
Jansen
VM
,
Bauer
JA
, et al
Aberrant FGFR signaling mediates resistance to CDK4/6 inhibitors in ER+ breast cancer
.
Nat Commun
2019
;
10
:
1373
.
34.
O'Leary
B
,
Cutts
RJ
,
Liu
Y
,
Hrebien
S
,
Huang
X
,
Fenwick
K
, et al
The genetic landscape and clonal evolution of breast cancer resistance to palbociclib plus fulvestrant in the PALOMA-3 trial
.
Cancer Discov
2018
;
8
:
1390
403
.
35.
Cornell
L
,
Wander
SA
,
Visal
T
,
Wagle
N
,
Shapiro
GI
. 
MicroRNA-mediated suppression of the TGF-beta pathway confers transmissible and reversible CDK4/6 inhibitor resistance
.
Cell Rep
2019
;
26
:
2667
80
.
36.
Kettner
NM
,
Vijayaraghavan
S
,
Durak
MG
,
Bui
T
,
Kohansal
M
,
Ha
MJ
, et al
Combined inhibition of STAT3 and DNA repair in palbociclib-resistant ER-positive breast cancer
.
Clin Cancer Res
2019
;
25
:
3996
4013
.
37.
Guarducci
C
,
Bonechi
M
,
Benelli
M
,
Biagioni
C
,
Boccalini
G
,
Romagnoli
D
, et al
Cyclin E1 and Rb modulation as common events at time of resistance to palbociclib in hormone receptor-positive breast cancer
.
NPJ Breast Cancer
2018
;
4
:
38
.
38.
Li
Z
,
Razavi
P
,
Li
Q
,
Toy
W
,
Liu
B
,
Ping
C
, et al
Loss of the FAT1 tumor suppressor promotes resistance to CDK4/6 inhibitors via the Hippo pathway
.
Cancer Cell
2018
;
34
:
893
905
.
39.
Aslakson
CJ
,
Miller
FR
. 
Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor
.
Cancer Res
1992
;
52
:
1399
405
.
40.
Johnstone
CN
,
Smith
YE
,
Cao
Y
,
Burrows
AD
,
Cross
RS
,
Ling
X
, et al
Functional and molecular characterisation of EO771.LMB tumours, a new C57BL/6-mouse-derived model of spontaneously metastatic mammary cancer
.
Dis Model Mech
2015
;
8
:
237
51
.
41.
Shang
B
,
Liu
Y
,
Jiang
SJ
,
Liu
Y
. 
Prognostic value of tumor-infiltrating FoxP3+ regulatory T cells in cancers: a systematic review and meta-analysis
.
Sci Rep
2015
;
5
:
15179
.
42.
Turner
NC
,
Neven
P
,
Loibl
S
,
Andre
F
. 
Advances in the treatment of advanced oestrogen-receptor-positive breast cancer
.
Lancet
2017
;
389
:
2403
14
.
43.
Turner
NC
,
Liu
Y
,
Zhu
Z
,
Loi
S
,
Colleoni
M
,
Loibl
S
, et al
Cyclin E1 expression and palbociclib efficacy in previously treated hormone receptor-positive metastatic breast cancer
.
J Clin Oncol
2019
;
37
:
1169
78
.
44.
Luen
SJ
,
Savas
P
,
Fox
SB
,
Salgado
R
,
Loi
S
. 
Tumour-infiltrating lymphocytes and the emerging role of immunotherapy in breast cancer
.
Pathology
2017
;
49
:
141
55
.
45.
Rugo
HS
,
Delord
JP
,
Im
SA
,
Ott
PA
,
Piha-Paul
SA
,
Bedard
PL
, et al
Safety and antitumor activity of pembrolizumab in patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer
.
Clin Cancer Res
2018
;
24
:
2804
11
.
46.
Haikala
HM
,
Anttila
JM
,
Marques
E
,
Raatikainen
T
,
Ilander
M
,
Hakanen
H
, et al
Pharmacological reactivation of MYC-dependent apoptosis induces susceptibility to anti-PD-1 immunotherapy
.
Nat Commun
2019
;
10
:
620
.
47.
Finn
RS
,
Martin
M
,
Rugo
HS
,
Jones
S
,
Im
SA
,
Gelmon
K
, et al
Palbociclib and letrozole in advanced breast cancer
.
N Engl J Med
2016
;
375
:
1925
36
.
48.
Goetz
MP
,
Toi
M
,
Campone
M
,
Sohn
J
,
Paluch-Shimon
S
,
Huober
J
, et al
MONARCH 3: abemaciclib as initial therapy for advanced breast cancer
.
J Clin Oncol
2017
;
35
:
3638
46
.
49.
Hortobagyi
GN
,
Stemmer
SM
,
Burris
HA
,
Yap
YS
,
Sonke
GS
,
Paluch-Shimon
S
, et al
Updated results from MONALEESA-2, a phase III trial of first-line ribociclib plus letrozole versus placebo plus letrozole in hormone receptor-positive, HER2-negative advanced breast cancer
.
Ann Oncol
2018
;
29
:
1541
7
.
50.
Mason
KD
,
Carpinelli
MR
,
Fletcher
JI
,
Collinge
JE
,
Hilton
AA
,
Ellis
S
, et al
Programmed anuclear cell death delimits platelet life span
.
Cell
2007
;
128
:
1173
86
.
51.
Campbell
KJ
,
Dhayade
S
,
Ferrari
N
,
Sims
AH
,
Johnson
E
,
Mason
SM
, et al
MCL-1 is a prognostic indicator and drug target in breast cancer
.
Cell Death Dis
2018
;
9
:
19
.
52.
Williams
MM
,
Lee
L
,
Hicks
DJ
,
Joly
MM
,
Elion
D
,
Rahman
B
, et al
Key survival factor, Mcl-1, correlates with sensitivity to combined Bcl-2/Bcl-xL blockade
.
Mol Cancer Res
2017
;
15
:
259
68
.