Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor–Positive Breast Cancer James R

Home , Abemaciclib, Palbociclib, Ribociclib

Published OnlineFirst April 3, 2020; DOI: 10.1158/1078-0432.CCR-19-1872

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor–Positive Breast Cancer James R. Whittle1,2,3, Francois¸ Vaillant1,3, Elliot Surgenor1, Antonia N. Policheni3,4,Goknur€ Giner3,5, Bianca D. Capaldo1,3, Huei-Rong Chen1,HeK.Liu1, Johanna F. Dekkers1,6,7, Norman Sachs6, Hans Clevers6,7, Andrew Fellowes8, Thomas Green8, Huiling Xu8, Stephen B. Fox8,9, Marco J. Herold3,10, Gordon K. Smyth5,11, Daniel H.D. Gray3,4, Jane E. Visvader1,3, and Geoffrey J. Lindeman1,2,12

ABSTRACT ◥ Purpose: Although cyclin-dependent kinase 4 and 6 (CDK4/6) Results: Triple therapy was well tolerated and produced a super- inhibitors signiﬁcantly extend tumor response in patients with ior and more durable tumor response compared with single or þ metastatic estrogen receptor–positive (ER ) breast cancer, relapse doublet therapy. This was associated with marked apoptosis, includ- is almost inevitable. This may, in part, reﬂect the failure of CDK4/6 ing of senescent cells, indicative of senolysis. Unexpectedly, ABT- – inhibitors to induce apoptotic cell death. We therefore evaluated 199 resulted in Rb dephosphorylation and reduced G1 S cyclins, combination therapy with ABT-199 (venetoclax), a potent and most notably at high doses, thereby intensifying the fulvestrant/ selective BCL2 inhibitor. palbociclib–induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 Experimental Design: BCL2 family member expression was screen suggested that ABT-199 could mitigate loss of Rb (and assessed following treatment with endocrine therapy and the potentially other mechanisms of acquired resistance) to palbociclib. CDK4/6 inhibitor palbociclib. Functional assays were used to ABT-199 did not abrogate the favorable immunomodulatory effects þ determine the impact of adding ABT-199 to fulvestrant and palbo- of palbociclib in a syngeneic ER mammary tumor model and þ ciclib in ER breast cancer cell lines, patient-derived organoid extended tumor response when combined with anti-PD1 therapy. (PDO), and patient-derived xenograft (PDX) models. A syngeneic Conclusions: This study illustrates the potential for targeting þ ER mouse mammary tumor model was used to study the effect of BCL2 in combination with CDK4/6 inhibitors and supports inves- þ combination therapy on the immune system. tigation of combination therapy in ER breast cancer.

Introduction þ Estrogen receptor–positive (ER ) breast cancers frequently exhibit deregulation of the cyclin-dependent kinase 4 and 6 (CDK4/6)/cyclin 1Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of D1 (CCND1)/retinoblastoma (Rb) signaling pathway, resulting in Medical Research, Parkville, Victoria, Australia. 2Department of Medical Oncol- uncontrolled cellular proliferation (1, 2). CDK4/6 inhibitors are active ogy, The Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. in breast cancer where they have demonstrated synergistic activity with 3Department of Medical Biology, The University of Melbourne, Parkville, Victoria, 4 endocrine therapy, leading to improvements in progression-free sur- Australia. Immunology Division, The Walter and Eliza Hall Institute of Medical – Research, Parkville, Victoria, Australia. 5Bioinformatics Division, The Walter and vival and overall survival (3 5). As a result, combination treatment Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. 6Oncode with endocrine therapy and a CDK4/6 inhibitor is now considered þ Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences standard of care for patients with early-line metastatic ER breast (KNAW) and University Medical Centre (UMC), Utrecht, the Netherlands. 7Prin- cancer. Nevertheless, de novo or acquired resistance to therapy is 8 cess Maxima Center for Pediatric Oncology, Utrecht, The Netherlands. Depart- almost inevitable, underscoring the need for novel targeted therapies ment of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Aus- for this group of patients. tralia. 9Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia. 10Blood Cells and Blood Cancer Division, The Walter While the key mechanism of action of CDK4/6 inhibitors is to and Eliza Institute of Medical Research, Parkville, Victoria, Australia. 11School of provoke a cell-cycle arrest, they likely contribute clinically relevant Mathematics and Statistics, The University of Melbourne, Parkville, Victoria, antitumoral effects through modulation of the immune response (6, 7), Australia. 12Department of Medicine, The University of Melbourne, Parkville, induction of senescence (7, 8), or through other noncanonical func- Victoria, Australia. tions (9). Immune effects include immunogenic activation of tumor Note: Supplementary data for this article are available at Clinical Cancer cells and promotion of a switch to a less immunosuppressive state Research Online (http://clincancerres.aacrjournals.org/). through reduced regulatory T cell (Treg) numbers (7). In mammary J.E. Visvader and G.J. Lindeman contributed equally to this article tumor models, the profound cell-cycle arrest has been linked to Current address for N. Sachs: Vertex Pharmaceuticals Inc., San Diego, California. induction of a senescence program (10). Despite their potent anti- proliferative effects and demonstrated efficacy in the clinic, CDK4/6 Corresponding Author: Geoffrey J. Lindeman, The Walter and Eliza Hall Institute inhibitors do not induce apoptotic cell death in breast cancer of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia. fi Phone: 61-3-9345-2611; Fax: 61-3-9347-0852; E-mail: [email protected] cells (7, 10, 11). Indeed, recent ndings suggest that combination therapy may actually reduce apoptosis in treatment-na€ve tumors (12). Clin Cancer Res 2020;XX:XX–XX These findings are consistent with the senescent state, characterized doi: 10.1158/1078-0432.CCR-19-1872 by relatively irreversible replicative arrest and resistance to 2020 American Association for Cancer Research. apoptosis (13, 14).

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In vivo experiments using PDX models Translational Relevance þ ER PDX 50 and 315 models have been reported previously (22). Combining the BCL2 inhibitor ABT-199 (venetoclax) with PDX 50 originated from a patient following neoadjuvant chemother- endocrine therapy and a CDK4/6 inhibitor augments tumor apy (FEC-D) for her primary tumor; PDX 315 was derived from a response by eliciting a deeper cell-cycle arrest, triggering apoptosis primary tumor arising in a patient with a past history of a contralateral and enhancing the immunomodulatory response. Our findings breast tumor. She had previously received adjuvant chemotherapy suggest that venetoclax can be senolytic, supporting further inves- (FEC x 6) and endocrine therapy (tamoxifen followed by letrozole) and tigation of dual CDK4/6 and BCL2 blockade in ER-positive breast was taking letrozole at the time of her second primary tumor diagnosis. þ cancer. The resulting PDX 315 is strongly ER as reported previously (22). PDX 1105 and 1232 were derived from primary tumors in treatment- na€ve patients. NOD-SCID-IL2Rg / (NSG) mice were bred and maintained Evasion of cell death, a hallmark of cancer, can result from the according to institutional guidelines. All animal experiments were overexpression of antiapoptotic BCL2 family members. BCL2 is over- approved by the WEHI Animal Ethics Committee (2017.002). Cohorts þ expressed in the majority of primary and metastatic ER breast of 50–60 female mice were seeded with thawed single-cell suspensions cancer (15, 16). The targeting of BCL2 and other antiapoptotic proteins of early-passage human breast tumors (passage 2 or 3). Briefly, has emerged as a viable therapeutic option due to the recent devel- 150,000–250,000 cells were resuspended in 10 mL of transplantation opment of BH3-mimetic drugs that mimic endogenous antagonists of buffer (50% FCS, 10% of 0.04% Trypan blue solution and 40% PBS) BCL2 and its related family members (17–19). Venetoclax (ABT-199/ and growth factor–reduced Matrigel (BD Pharmingen) at a ratio of 3:1 GDC-0199), a potent and highly selective inhibitor of BCL2 (20), has and injected into the cleared mammary fat pads of 3- to 4-week-old demonstrated single-agent activity in chronic lymphocytic leuke- NSG female mice. Mice were monitored for tumor development three mia (21) and in combination therapy for a number of hematologic times weekly and tumor size measured using electronic Vernier þ þ malignancies (18). In preclinical models of ER BCL2 breast cancer, calipers. Tumor volume was estimated by measuring the minimum ABT-199 improved tumor response to endocrine therapy with tamox- and maximum tumor diameters using the formula: (minimum dia- ifen by enhancing apoptosis (22). These findings appear to be clinically meter)2(maximum diameter)/2. Once tumors arose, mice were ran- relevant as the addition of venetoclax to tamoxifen elicited promising domized into treatment arms. Treatment was initiated when the tumor þ clinical activity in a phase I study in women with metastatic ER volume reached 80–120 mm3. Randomization and tumor measure- þ BCL2 breast cancer (16). ments were managed using the Study Director software (v 3.0, study- On the basis of these observations, we have explored a role for dual log). Mice were sacrificed at the first measurement where tumor targeting of CDK4/6 and BCL2 signaling pathways in preclinical volume exceeded 600 mm3, or if their health deteriorated for reasons þ þ models of ER BCL2 breast cancer and found that the addition of other than disease progression or drug toxicity (censored event). ABT-199 to endocrine therapy with fulvestrant and the CDK4/6 inhibitor palbociclib substantially augmented tumor responsiveness In vivo drug treatments in vivo. Combination therapy was accompanied by a deeper cell-cycle Mice were treated for 21 days in a 28-day cycle. Fulvestrant (Clifford arrest and induction of apoptosis, including cells exhibiting a senes- Hallam) 5 mg or its vehicle was injected intraperitoneally weekly for 3 cence-associated b-galactosidase phenotype. Moreover, the favorable of 4 weeks. ABT-199 (Active Biochem) 100 mg/kg or its vehicle was palbociclib-mediated immunogenic activation of tumor cells together prepared as described previously (22) and given via oral gavage on days with reduction in Treg numbers and proliferation was not abrogated 1–5 for three out of four weeks. Palbociclib 100 mg/kg or its vehicle was by the addition of ABT-199. 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 Materials and Methods between the drugs and vehicles. Anti-PD1 (BioXcell) 200 mg/mouse or Study design isotype control was given intraperitoneally on days 1, 3, and 5. Mice This study was designed to evaluate the response of breast cancer were monitored as per institutional guidelines. cells to the combination of endocrine therapy with ABT-199 and palbociclib. We evaluated the response to single-agent or combination Preparation of 67NR cells and syngeneic transplantation therapy in breast cancer cell lines, patient-derived breast tumor experiments organoids and xenograft models, as well as a syngeneic mouse mam- Early-passage 67NR cells were maintained as described in Supple- mary tumor model. Experiments were designed to investigate the mentary Methods and cells (5 103) transplanted into the cleared mechanisms of tumor response. As outlined below, all mouse studies mammary fat pads of 3- to 4-week-old BALB/c mice. Mice bearing included randomization and blinding. The numbers of replicates 67NR tumors were euthanized at the experimental endpoint and performed for each experiment are included in the figure legends. tumors excised. To obtain a single-cell suspension for detection of cell surface and intracellular proteins, tumors and spleens were Human tissue processed as described previously (23). BALB/c mice were bred and The studies were conducted in accordance with recognized ethical maintained according to institutional guidelines. Experiments were guidelines (Declaration of Helsinki). Human breast cancer tissues were approved by the WEHI Animal Ethics Committee (2017.002). obtained from consenting patients through the Royal Melbourne Hospital Tissue Bank and the Victorian Cancer Biobank with relevant Flow cytometry institutional review board approval. Human Ethics approval was Analysis of immune cells was performed as described previous- obtained from the Walter and Eliza Hall Institute (WEHI, 05/06) ly (23). Antibodies to cell surface proteins were purchased from Human Research Ethics Committee. BioLegend unless otherwise stated: CD4 (clone GK1.5), CD8 (clone

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53-6.7), TCRb (clone H57-597), CD45 (clone 30-FII), Ki67 (clone lower BCL2 levels (Supplementary Fig. S1C). Together, these findings B56), PD1 (clone 29F.1A12), and FoxP3 (eBiosciences, clone FJK-16). suggest that BCL2 could represent a potential target in palbociclib- For intracellular detection of TNFa and IFNg, cells were isolated and treated breast cancer cells. – stimulated as described previously (23). Sample data were acquired on The link between estrogen receptor (ER) signaling and G1 S phase an LSRFortessa X-20 flow cytometer (BD Biosciences) and analyzed cell-cycle progression has informed the combined use of CDK4/6 using FlowJo software (TreeStar). inhibitors with endocrine therapy (11). To address whether combination therapy was synergistic or additive, we compared the impact of Breast cancer organoids adding either palbociclib or ABT-199 to the selective ER degrader Breast cancer organoids were generated from patient samples as fulvestrant in MCF7 cells (Fig. 1B). Doses were selected on the basis of described previously (24, 25). Briefly, breast cancer cell suspensions the induction of on-target effects, including reduced ER (for fulves- from patients undergoing surgery were prepared through mechanical trant), Rb phosphorylation (for palbociclib and fulvestrant) and disruption and enzymatic digestion. Isolated cells were then plated in generation of cleaved PARP (for ABT-199) at 24 hours and/or 7 days adherent basement membrane extract (BME; Cultrex) drops in (Supplementary Fig. S2A and S2B). As reported previously (11), uncoated 24-well plates and cultured in optimized human organoid palbociclib showed synergy when combined with endocrine therapy, medium (HOM). For PDX-derived breast cancer organoids, we as determined by cell viability using CellTiter-Glo and BLISS score obtained single-cell suspensions by digestion of primary tumors and analysis (Fig 1B, top). Synergy was also observed between fulvestrant then sorted these by flow cytometry using a FACSARIA III (BD and ABT-199 at higher doses, consistent with prior findings for þ Biosciences), as described previously (22, 26). Sorted human EpCAM tamoxifen (22). Adding ABT-199 to fulvestrant and palbociclib cells were then plated in BME drops and cultured in HOM. was neither synergistic nor antagonistic (Fig 1B, bottom), suggesting For 3D colony assays, organoids were dissociated and then seeded in that cotargeting of the BCL2 pathway might enhance response to BME in 24-well plates (four drops of 8 mL containing 1,000 cells). After fulvestrant/palbociclib. 24 hours, organoids were treated with fulvestrant (Sigma-Aldrich), To investigate the effect of drug treatment on key target proteins, ABT-199 (Active Biochemicals), palbociclib (Active Biochemicals), MCF7 cells were treated for 72 hours with fulvestrant, palbociclib, vehicle, or the indicated treatments. Treatments were replaced every ABT-199 or various combinations. As expected, combination therapy 3–4 days. After 14 days in culture, cell viability was assessed using with fulvestrant and palbociclib reduced ER and phospho-Rb (pRb) CellTiter Glo Luminescent Assay (Promega). levels, but did not induce apoptosis, as determined by cleaved PARP and cleaved caspase-3 expression (Fig. 1C). ABT-199 alone (or in Statistical analysis combination) increased expression of pro-apoptotic BIM, a measure of Statistical analyses were performed in the GraphPad Prism software primed cells (28) and induced cleaved PARP and cleaved caspase-3 version 8.0a. Kaplan–Meier (log rank test) was used to test for significant (CC3; Fig. 1C). This was unexpectedly accompanied by reduced levels – differences in the survival of mice (using the ethical end point for tumor of pRb, the G1 S cyclins D1 and E, and ER (Fig. 1C). Triple therapy size as a surrogate for death). Unpaired t tests were used to test the with fulvestrant and palbociclib induced similar changes. Interestingly, significance of differences in column means between treatments. the adaptive increase in cyclin D1 and E seen with palbociclib alone For details, see Supplementary Methods. 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 under- Results going a palbociclib-mediated cell-cycle arrest. The unexpected down- Augmented response to CDK4/6 and BCL2 inhibition in breast regulation of pRb and cyclins D1 and E could reflect selective induction cancer of apoptosis in cycling cells. Decreased ER expression is also consistent, To gain insight into the short-term effects of combined endocrine as lower ERa levels are observed in G1-arrested cells (29). and CDK4/6 inhibitor therapy on BCL2 prosurvival family members in treatment-na€ve breast cancer, we evaluated gene expression data Dephosphorylation of Rb and cell-cycle arrest in BH3 mimetic— from the NeoPalAna window study (27), in which patients with newly treated breast cancer cells þ fi – diagnosed ER breast cancer were treated with the aromatase inhibitor The unanticipated nding that ABT-199 leads to reduced G1 S anastrozole followed by the CDK4/6 inhibitor palbociclib prior to phase cyclins and Rb dephosphorylation was next investigated by cell- surgery (Fig. 1A; Supplementary Fig. S1A). As reported previously, cycle analysis using flow cytometry (Fig. 2A). MCF7 cells were treated expression of MKI67 (encodes the proliferation marker Ki67), was for 72 hours with fulvestrant (100 nmol/L), ABT-199 (5 mmol/L), and reduced by therapy but recovered when palbociclib was ceased. BCL2 palbociclib (250 nmol/L) alone or in combination. Fulvestrant and and MCL1 levels similarly declined, albeit modestly, in response to palbociclib both efficiently reduced the percentage of S-phase cells, combination therapy and rebounded once palbociclib was withdrawn. consistent with their known mechanism of action. Notably, ABT-199 Reciprocal changes in BCL2L1 (encoding BCLXL) were observed, induced similar changes, albeit to a lesser extent than observed with consistent with adaptive changes due to altered BCL2 and MCL1 palbociclib. This effect was more pronounced when ABT-199 was expression. The effect of longer-term, continuous palbociclib treat- combined with palbociclib or fulvestrant (Fig. 2A). ment on BCL2 family members was next evaluated in MCF7 and T47D We next investigated whether other BH3 mimetics could impact the breast cancer cells, which were treated for 3 months at different cell cycle. ABT-737 (which targets BCLXL, BCL2, and BCLW) and the concentrations (Supplementary Fig. S1B). A modest decrease in BCL2 MCL1 inhibitor S63845 (30) exerted similar dose-dependent effects on and MCL1 proteins was observed in MCF7 cells, while expression was pRb, cyclin D1 and ER, with all three BH3 mimetics inducing cPARP largely unchanged in T47D cells. BCLXL levels rose in both cell lines. (Fig. 2B). As expected, MCL1 levels increased in response to ABT-199, Despite this, palbociclib-treated cells (continuous treatment at high ABT-737, and S63845, the latter attributable to stabilization of the concentration) exhibited greater sensitivity to BCL2 inhibition with protein by the inhibitor (30). Minimal changes were observed in BCL2 ABT-199, consistent with either heightened cellular stress and/or or BCL-XL levels. To address whether the changes in pRb, cyclin D1, or

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Anastrozole Palbociclib A MKI67 BCL2 MCL1 BCL2L1 0.4 0.6 0.2 Log2 FC Log2 FC Log2 FC Log2 FC −0.1 0.0 0.1 0.2 −0.8 −0.6 −0.4 −0.2 0.0 −0.2 0.0 −0.15 −0.05 0.05 BL C1D1 C1D15 Surg BL C1D1 C1D15 Surg BL C1D1 C1D15 Surg BL C1D1 C1D15 Surg

BC60 50−60 50 40−50 40 30−40 VehicleFulvestrantABT-199PalbociclibFulv-199Fulv-Palbo199-PalboFulv-199-Palbo 30 20−30 20 Rb − 10−20 110 kDa 10 BLISS score 0−10 0 −10−0 pRb −110 − 0 10 1 10 Fulvestrant 250.0 0 62.5 100 31.3 125.0 7.8 (nmol/L) 15.6 3.9 ER −61

Palbociclib (nmol/L) 0 ctrl Cyclin D1 −34 − 60 50 60 50 40−50 −50 40 30−40 erocs Cyclin E 30 20−30 20 − − SSILB 10 20 26 10 0−10 BIM 0 −10−0 −10 − −20 to −10 15 −20 −30 to −20 −30 0 1 − 0.01 37

5 MCL1

52 .2 10 Fulvestrant

6.0

3.0

lrtc lrtc

0 100 1.0 (nmol/L) −

0 26 ABT-199 (µmol/L) BCL2

− 60 40 60 BCLXL −26 40 20−40

20 0−20 cPARP 0 −20−0 −89 − 20 −40 to −20 − BLISS score 40 −60 to −40 −60 CC3 −19 −80 to −60 −80 0 50 100 Fulvestrant (1 nmol/L) 5.0 10.0 2.5 + palbociclib (nmol/L) 0.6 250 1.25 0.3 Tubulin −50 0.0 0.15

ABT-199 (µmol/L) 0 ctrl

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 mmol/L), palbociclib (500 nmol/L), or the indicated combinations. Tubulin was used as a loading control.

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A Vehicle Fulvestrant ABT-199 Palbociclib 100 − G0 G1 90 − G2 M 80 S − − − − − − − − G0 G1 G2 M G0 G1 G2 M G0 G1 G2 M G0 G1 G2 M 61.5 7.33 82.2 5.92 75.6 8.20 87.5 4.95 70 60 % Live cells Fulv-199 Fulv-Palbo 199-Palbo Fulv-199-Palbo 50

-199 ehicle V ABT Fulv-199 199-Palbo − − − − − − − − Fulvestrant Palbociclib Fulv-Palbo G0 G1 G2 M G0 G1 G2 M G0 G1 G2 M G0 G1 G2 M 86.5 5.67 95.7 3.70 93.8 5.63 94.9 4.51

APC-EdU Fulv-199-Palbo

DAPI B C Vehicle ABT-199 A1331852ABT-737 S63845

VehiclePalbociclibABT-199 ABT-737S63845 BH3 mimetic - ++++++++ Lo Hi Lo Hi Lo Hi Lo Hi Q-VD-OPH -----+ +++ Rb −110 kDa Rb −110 kDa pRb −110 pRb −110 ER −61 ER −61 Cyclin D1 −34 Cyclin D1 −34 − Cyclin E −50 Cyclin E 50 MCL1 −37 MCL1 −37 BCL2 −26 BCL2 −26 BCLXL −26 BCLXL −26

cPARP −89 cPARP −89 Tubulin −50 Tubulin −50

D ABT-199 A-1331852 ABT-737 S63845

100 − G0 G1 90 − G2 M − − − − − − − − G0 G1 G2 M G0 G1 G2 M G0 G1 G2 M G0 G1 G2 M 91.0 6.67 80.5 17.6 83.1 14.6 78.0 19.7 80 S 70 ABT-199 A-1331852 ABT-737 S63845 + QVD + QVD + QVD + QVD 60 % Live cells 50

− − − − − − − − -199 G0 G1 G2 M G0 G1 G2 M G0 G1 G2 M G0 G1 G2 M T 94.7 4.35 88.5 9.84 87.4 9.51 79.2 20.0 S63845 AB ABT-737 APC-EdU -199 + QVDA-1331852 ABT ABT-737 + QVDS63845 + QVD DAPI A-1331852 + QVD

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 mmol/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 mmol/L) or “Hi” (10 mmol/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 mmol/L) with or without pan-caspase inhibitor Q-VD-OPh (20 mmol/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 mmol/L) with or without Q-VD-OPh (20 mmol/L). Quantiﬁcation shown in right panel is from three independent experiments. Data are shown as mean SEM.

ER reﬂected an “on-target” effect of BH3 mimetics or a consequence of pan-caspase inhibitor Q-VD-Oph (Fig. 2C). While apoptosis was apoptosis, MCF7 cells were treated with BH3 mimetics (including the efﬁciently blocked by Q-VD-Oph, as demonstrated by lack of PARP BCLXL inhibitor A1331852; ref. 31) in the presence or absence of the cleavage, the effects on pRb, cyclin D1, and ER were maintained,

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implying they are caspase-independent. Furthermore, cell-cycle anal- targeting 19,050 genes, and 1,864 miRNAs (6 sgRNAs per gene ysis revealed that each BH3 mimetic could inhibit proliferation and 4 sgRNAs per miRNA) at low multiplicity of infection in five independent of cell death (Fig. 2D). Together, these results suggest independent infections. Transduced cells were continuously treated that ABT-199 (and other BH3 mimetics) may have unexpected effects with either fulvestrant, fulvestrant and palbociclib, fulvestrant as inhibitors of cell-cycle progression in breast cancer cells, beyond and ABT-199, or the combination of fulvestrant, palbociclib, and their canonical role as proapoptotic agents. ABT-199 for 2 months to identify sgRNA guides that helped to promote the growth of resistant cells. Doses chosen (fulvestrant CDK4/6 inhibition primes breast cancer cells for apoptosis and 1 mmol/L, ABT-199 10 mmol/L, and palbociclib 1 mmol/L) were senolysis based on single-agent activity that exerted high selective pressure Given the potent effect of ABT-199 on apoptosis and the cell cycle, (Supplementary Fig. S3). we next evaluated the effect of single or combination therapy in When the frequency of reads for guides was compared between clonogenic cellular assays. For this, lower doses of fulvestrant and different treatments, sgRNAs for RB1 were found to be enriched in palbociclib were used to address synergy, because clonogenic activity fulvestrant–palbociclib–treated cells, with the greatest differential was readily inhibited by single-agent treatment (Supplementary observed between fulvestrant-only and fulvestrant–palbociclib Fig. S3A and S3B). T47D and MCF7 cells were treated for two weeks treated cells (Supplementary Tables S1 and S2). In contrast, RB1 with single, doublet, or triplet therapy comprising fulvestrant, palbo- sgRNAs were not over-represented in cells treated with fulvestrant– ciclib, and/or ABT-199. The addition of ABT-199 to fulvestrant and ABT-199 (Supplementary Tables S1 and S2). For fulvestrant and palbociclib significantly reduced the number and size of colonies, fulvestrant/palbociclib treatment–resistant cells, differentially repre- compared with doublet therapy (Fig. 3A and B; Supplementary sented guides (with an FDR of < 0.05) against genes and molecular Fig. S3C). Following removal of inhibitors at day 14, regrowth was pathways previously associated with CDK4/6 inhibitor resistance were apparent in cells that had been treated with doublet therapy. In identified such as PIK3C isoforms, CDK4 and FGFR members (32–34), contrast, minimal growth was observed in cells that had received mir-432-5p (35), members of the IL6–JAK–Stat pathway (IL6ST/ triplet therapy (Fig. 3A and B; Supplementary Fig. S3C). At the lower GP130, JAK1, JAK3, STAT2, and STAT4; ref. 36), E2F family members doses used for clonogenic assays, the previously observed effects on key (E2F3, E2F4, E2F6, E2F7, and E2F8; ref. 37), and Hippo pathway target proteins were maintained (Supplementary Fig. S4A and S4B). family members TEAD3 and TEAD4 (38). Interestingly, the histone These findings, together with the adaptive changes in BCL2 family H3 acetyltransferase epigenetic modifier genes from the MOZ/MORF expression in breast tumor cells treated with palbociclib (Fig. 1A; complex (KAT6B, BPRF1, and ING5) were also implicated as genes Supplementary Fig. S1B), suggest that palbociclib might prime cells for involved in resistance. Interrogation of BCL2 family members revealed apoptotic cell death with ABT-199. sgRNAs targeting MCL1 were enriched in fulvestrant-treated cells To further investigate the combinatorial effect of ABT-199 and compared with those treated with fulvestrant–palbociclib or fulves- palbociclib, MCF7 cells were treated with either vehicle or palbociclib trant–ABT-199 (Supplementary Tables S1 and S2), suggesting that this (500 nmol/L) for four days prior to switching drug treatment to could be a target for use in combination or upon progression. vehicle, ABT-199, ABT-737, or both ABT-199 and palbociclib To determine whether the addition of ABT-199 to fulvestrant and (Fig. 3C and D). Palbociclib pretreated cells were more responsive palbociclib modified mechanisms of resistance, we examined gene to either ABT-199 or ABT-737, with increased CC3 (Fig. 3C) and knockouts that were enriched or depleted between fulvestrant– reduced viability as measured by propidium iodide exclusion palbociclib and fulvestrant–palbociclib–ABT-199 triplet therapy. Nota- (Fig. 3D). As expected, palbociclib-treated cells exhibited less PCNA bly, RB1 was identified as the top candidate for cells treated with triple expression and thus proliferation. Consistent with cell-cycle analysis therapy versus fulvestrant–palbociclib while the genes identified above (Fig. 2A and D), ABT-199–treated cells also exhibited lower PCNA did not feature (Fig. 3G; Supplementary Table S2). These observations levels compared with vehicle-treated controls (Fig. 3C). suggest that treatment with ABT-199 could mitigate loss of Rb and CDK4/6 inhibitors elicit a senescence phenotype (7, 8) that can potentially other mechanisms of acquired resistance to palbociclib (34). result in cells becoming more resistant to apoptosis (13, 14). In keeping with this finding, palbociclib induced a dose-dependent senescence- Triple therapy substantially inhibits the growth of breast tumor like phenotype characterized by increased cell size, cell flattening, and organoids b-galactosidase expression (Supplementary Fig. S5A), while no dis- Given their distinct molecular effects and augmented response cernible change in morphology or b-galactosidase expression was observed with CDK4/6 and BCL2 inhibition, we next studied observed with ABT-199 alone. Cells treated with palbociclib for 4 days patient-derived breast tumor organoid models, which more accurately maintained this senescent-like phenotype following drug withdrawal reflect tumor heterogeneity and are emerging as promising preclinical þ (Fig. 3E and F). This phenotype, accompanied by the increased models to predict drug response (24, 25). ER tumor organoids were þ vulnerability to apoptosis observed following treatment with a BH3 generated from primary ER breast tumors or patient-derived xeno- mimetic (Fig. 3C), suggests that ABT-199 (and ABT-737) can aug- graft (PDX) models (Fig. 4A; Supplementary Table S3). Primary breast ment CDK4/6 inhibition through the induction of senolysis of cells tumor organoids were generated as recently described (24, 25) and that have undergone growth arrest. were found to retain ER and BCL2 expression (Supplementary Fig. S7). They exhibited mutational profiles expected in luminal breast tumors ABT-199 may modulate mechanisms of palbociclib resistance (Supplementary Table S4). For PDX organoids, single-cell suspensions To address potential mechanisms of response or resistance to were prepared from PDX tumors, then sorted by flow cytometry and various combinations, we performed a genome-wide CRISPR/Cas9 plated in organoid medium optimized for breast tumors (24, 25). These screen in T47D cells (Fig. 3G; Supplementary Fig. S6A and S6B; retained the histologic features of the parental tumor, including ER and Supplementary Tables S1 and S2). Cas9-expressing T47D cells were BCL2 expression (Supplementary Fig. S8A and S8B). The four PDX transduced with a pooled human genome-wide small guide RNA tumors exhibited variable levels of BCL2 and cell-cycle proteins, with (sgRNA) lentiviral library containing 123,411 unique sgRNAs notable absence of pRb expression in PDX 50 (Fig. 4B). PDX 50 was

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A T47D B Vehicle Fulvestrant ABT-199 Palbociclib 2 wk 1 wk off **** * ** 60 *** * Fulv-199 Fulv-Palbo 199-Palbo Fulv-199-Palbo **** * 2 wk ** 40 * ** ns 20 * Fulv-199 Fulv-Palbo 199-Palbo Fulv-199-Palbo % Surface area stained 0 1 wk off

ehicle T-199 V AB Fulv-199 Fulv-199 Fulvestrant Palbociclib Fulv-Palbo199-Palbo Fulv-Palbo199-Palbo Fulv-199-Palbo Fulv-199-Palbo C MCF7 D MCF7 ns d1-4 Vehicle Palbociclib ns 100 * *** d5-8 75 **** Vehicle 199-Palbo Vehicle ABT-199 Palbociclib ABT-199 ABT-737 50 Rb −110 kDa 25

pRb Viability (% PI negative) 0 − Vehicle 110 d1-4 +++++− − − − −−−++− + − +Palbociclib PCNA −30 d5-8 -737 -737 -199 T T T Vehicle Vehicle ABT-199 AB AB AB 199-Palbo 199-Palbo Palbociclib Cyclin D1 −34 E − Cyclin E 50 (d1-4) cPARP −89 Vehicle

Vehicle Palbociclib ABT-199 ABT-737 199-Palbo − CC3 19

− (d1-4) Tubulin 50 Palbociclib

Palbociclib Vehicle ABT-199 ABT-737 199-Palbo

F ** **** G ns *** Genome-wide CRISPR screen ns Non−DE

**** 15 Up ns Down 100 **** ns 10 * ns Positive 50 L 50 -GA %

0 −5 Vehicle Palbociclib Log fold change d1-4 RB1 RB1 RB1 d5-8 −15 −10 RB1 Vehicle Vehicle ABT-199 ABT-737 ABT-199 ABT-737 Palbo-199 Palbo-199 Palbociclib Palbociclib 0 5 10 15 Average log CPM

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 mmol/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 mmol/L), ABT-737 (10 mmol/L), palbociclib (500 nmol/L), or the indicated combinations for 4 days. (Continued on the following page.)

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found to harbor a heterozygous RB1 mutation (c.1215þ1G>A) known with the same regimen used for the PDX models, comprising 3 weeks to be pathogenic (ClinVar). of therapy followed by a 1-week “rest” period. Triple therapy was well PDX-derived breast tumor organoids were treated with fulvestrant, tolerated, with mice maintaining normal body weight (Supplementary ABT-199, palbociclib, or combination therapy to evaluate tumor Fig. S11A, left), similar to the findings in NSG tumor-bearing mice response (Fig. 4; Supplementary Fig. S9) using a parallel approach (Supplementary Fig. S11A, right). As anticipated, a reduction in to that described for MCF7 and T47D cells. Doses were selected on the neutrophils and lymphocytes was observed at three weeks (cycles 1 basis of the dose–response observed for single-agent treatment (Sup- and 2, day 21), but these recovered after a 1-week rest period (cycles 1 plementary Fig. S9A and S9B). As illustrated for PDX 1105 (Fig. 4C–F), and 2, day 28; Supplementary Fig. S11B). No perturbation in urea, treatment for 14 days reduced tumor organoid formation (Fig. 4C) creatinine, or liver enzymes was observed at day 28 of cycles 1 and 2 and cell viability (Fig. 4D), with the greatest reduction observed upon (Supplementary Fig. S11C). These findings suggest that combination triple therapy. Recovery in growth and viability was observed after a therapy might be safely administered in humans. 1 week wash-out period for organoids treated with doublet combinations, whereas minimal tumor organoids were observed after triple Combination therapy induces an immune response in mice þ therapy (Fig. 4C and D). Similar findings were observed in the other bearing ER tumors PDX organoid models (Supplementary Fig. S9C). As expected, fulves- CDK4/6 inhibitors have been recently shown to promote antitumor trant and palbociclib reduced cellular proliferation, as measured by immunity (6, 7), while venetoclax has been associated with lympho- Ki67 (Fig. 4E; Supplementary Fig. S9D), while ABT-199 (as a single penia in patients with breast cancer (16), raising the possibility that agent or in combination) augmented cell death, as determined by CC3 combination therapy could adversely impact the immune response. expression by Western blot analysis (Fig. 4F) or immunostaining We therefore evaluated immune modulation in a syngeneic mouse (Supplementary Fig. S9E). Similar effects of fulvestrant, ABT-199, and mammary tumor model using the 67NR cell line, which is a non- palbociclib on cell viability were observed in three tumor organoid metastasizing subclone derived from a spontaneous BALB/cfC3H models that were directly derived from primary breast tumors mammary tumor (39). This cell line expresses nuclear ER (40) and (Fig. 4G). is BCL2 positive (Supplementary Fig. S12A). We first confirmed that the growth of 67NR cells was endocrine dependent through siRNA- Dual CDK4/6 and BCL2 inhibition enhances tumor response mediated knockdown of ESR1, which significantly reduced cell via- in vivo bility (Fig. 6A; Supplementary Fig. S12B). These cells also displayed a The in vivo response to combination therapy was next evaluated in dose-dependent response to fulvestrant (Supplementary Fig. S12C). þ þ the four ER BCL2 PDX models, whereby NSG mice bearing PDX Thus, 67NR cells represent a potentially useful syngeneic model to tumors were treated with up to 3 cycles of combination therapy. A investigate the in vivo immune effects of palbociclib and ABT-199. treatment cycle was defined as 28 days, with fulvestrant administered To determine whether ABT-199 modulated immunogenic activa- weekly for 3 weeks (on day 1, 8, 15). Palbociclib and ABT-199 were tion of tumor cells by a CDK4/6 inhibitor, 67NR cells were treated with administered for 3 weeks (on days 1–5, 8–12, 15–19) followed by a fulvestrant or combinations containing palbociclib and/or ABT-199. 1-week rest. Fulvestrant alone marginally delayed tumor growth As previously described in human cells for MHCI, and in a mouse compared with vehicle in all models. Tumor response was improved mammary tumor model (7), palbociclib in combination with fulves- with either fulvestrant–palbociclib or fulvestrant–ABT-199 combina- trant increased expression of MHCI molecules (H-2Kd). Intriguingly, tions. Triple therapy that included ABT-199 further attenuated tumor H-2Kd levels were also increased by ABT-199 and were further growth during the treatment period (3 cycles of therapy for PDX 315 elevated when both drugs were combined (Fig. 6B), with similar and 1232) and improved survival to the experimental ethical endpoint, findings for b2-microglobulin (Supplementary Fig. S12D and S12E). including the 315 model, which was derived from a patient with In keeping with enhanced IFN signaling, we also observed increased endocrine refractory breast cancer (Fig. 5A; Supplementary expression of phospho-STAT1 in 67NR and MCF7 cells in response to Fig. S10A). Mechanistically, this response was accompanied by ABT-199 treatment (Supplementary Fig. S12F and S12G). These data reduced cellular proliferation and increased apoptosis, as determined suggest that ABT-199 further enhances the immunogenicity of 67NR by Ki67 and CC3 levels, respectively (Fig. 5B–D). As expected in the cells when combined with palbociclib. RB1-deficient PDX 50 model, palbociclib did not improve tumor We next transplanted 67NR cells into the cleared mammary fat pads response to fulvestrant, although the fulvestrant–ABT-199 combina- of BALB/c recipient mice. Mice bearing small tumors 10–14 days after tion was efficacious in vivo (Supplementary Fig. S10B). engraftment were randomized into one of five treatment arms: (i) Because palbociclib is associated with neutropenia (3–5) and vene- vehicle, (ii) fulvestrant, (iii) fulvestrant plus ABT-199, (iv) fulvestrant toclax produces “on-target” lymphopenia and low-grade neutrope- plus palbociclib, or (v) fulvestrant plus ABT-199 and palbociclib. nia (16), we next determined whether myelotoxicity was problematic While single-agent treatment with fulvestrant produced modest atten- in vivo. As NSG mice are leukopenic, safety studies were conducted on uation in growth, there was no benefit to the addition of palbociclib or healthy BALB/c mice (Supplementary Fig. S11). Mice were treated ABT-199. However, similar to PDX models (Fig. 5), triple therapy

(Continued.) 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 b-galactosidase staining for cellular senescence. F, Quantiﬁcation of b-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 mmol/L), fulvestrant and ABT-199 (10 mmol/L), fulvestrant and palbociclib (1 mmol/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.

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Figure 4. A B 315 1232 1105 50 ERþ breast cancer organoids. A, Schematic overview of the genera- Rb −110 kDa tion of breast cancer organoids from pRb −110 primary breast cancer samples or −61 patient-derived xenograft (PDX) ER models. B, Western blot analysis of − Tissue digest 24 well Cyclin D1 34

Rb, pRb (S780), ER, Cyclin D1, CDK4, Patient-derived − CDK6, and BH3 family member CDK4 30 (MCL1, BCL2, and BCLXL) expression CDK6 −36 þ in ER PDX models (two independent EpCAM − Media BME MCL1 37 tumors per PDXs are shown). Tubulin was used as a loading control. C, BCL2 −26 PDX-derived CD49f Clonogenic assays of PDX 1105 cancer BCLXL −26 organoids treated continuously for 14 days with vehicle, fulvestrant Tubulin −50 (1 mmol/L), ABT-199 (2.5 mmol/L), palbociclib (50 nmol/L), and the indi- CDPDX 1105 organoids cated combinations. Bottom, cells Vehicle Fulvestrant ABT-199 Palbociclib 2 wk 1 wk off treated for 14 days before drugs ) 125 300 were washed out and organoid ted 100 **** colonies allowed to recover in media 200 ** 75 for seven days. Scale bar, 5 mm. D, *** Fulv-199 Fulv-Palbo 199-Palbo Fulv-199-Palbo 50 * Quantiﬁcation of C using CellTiter- 100 2 wk ty (% untrea ***

Glo. Bars represent mean SEM 25 *** Proliferation (%) iabili for at least three independent experi- V 0 0 ments. Unpaired two-tailed t tests Vehicle Fulv-199 ( , P < 0.05; , P < 0.01; , Fulv-199 Fulv-Palbo 199-Palbo Fulv-199-Palbo 199-Palbo Fulv-199 Fulv-Palbo Fulv-Palbo199-Palbo P < 0.001; , P < 0.0001). E, Rep- Fulv-199-Palbo Fulv-199-Palbo ABT-199 Palbociclib resentative sections of PDX 1105 Fulvestrant organoids from immunostained for 1 wk off Ki67 after 24 hours with the indicated treatments (C). Scale bar, 50 mm. F, Western blot showing expression of E Vehicle Fulvestrant ABT-199 Palbociclib F Rb, pRb, and cleaved caspase 3 (CC3) T-199 ulv-199-Palbo in PDX 1105 cancer organoids treated VehicleFulvestrantAB PalbociclibFulv-199Fulv-Palbo199-PalboF with the indicated drugs for 24 hours. Rb −110 kDa Tubulin was used as a loading control. −110 G, Primary breast tumor organoids Fulv-199 Fulv-Palbo 199-Palbo Fulv-199-Palbo pRb were cultured for 14 days in organoid Ki67 CC3 medium with the indicated drugs −19 before assessment of cell viability using CellTiter-Glo. Results are pre- − Tubulin 50 sented as percentages of untreated cells. Data represent mean SEM for n ¼ 3 independent experiments. G Unpaired two-tailed t tests is shown Patient tumor organoids for doublet combination compared 100 Hu # 4 with single agent, or triple therapy Hu # 6 compared to doublet combination Hu # 10 ( , P < 0.05; , P < 0.01; , 50 P < 0.0001). * ** **** * **** **** **

Viability (% untreated) 0

Vehicle ABT-199 Fulv-199 Fulvestrant Palbociclib Fulv-Palbo 199-Palbo Fulv-199-Palbo

þ elicited an improved response and survival benefit compared with are associated with poor survival in ER breast cancer (41). Con- doublet therapy with either fulvestrant–palbociclib or fulvestrant– sistent with this observation, Tregs were also less proliferative ABT-199 (Fig. 6C; Supplementary Fig. S12H). (Fig. 6F), suggesting the impact of pRb and BCL2 inhibition in this To explore the intratumoral immune response to combination setting. The reduction in Tregs resulted in a modest but significant therapy, analysis was performed after a short treatment of 9 days, increase in the CD8/Treg ratio (Supplementary Fig. S13D). Reduced when most vehicle-treated tumors reached ethical endpoint expression of the immune checkpoint receptor PD1 in Tregs was also (Fig. 6D). Flow cytometric analysis of the tumor-infiltrating cells observed (Fig. 6G), consistent with previous findings in a breast showed that combinations of fulvestrant–palbociclib resulted in a cancer mouse model using the CDK4/6 inhibitor abemaciclib (7). þ reduction in the proportion and absolute number of FOXP3 Tregs ThesystemicimpactofBCL2andCDK4/6inhibitionwasmost (Fig. 6E; Supplementary Fig. S13A–S13C), elevated levels of which pronounced in the tumor microenvironment, but modest effects of

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Figure 5. BCL2 and CDK 4/6 inhibition with endocrine therapy attenuates growth of ERþ 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 Sup- plementary Fig. S10A. B, Representa- tive 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 mm. C, Quantiﬁcation of Ki67 and CC3 immunostaining from B. Values represent mean SEM (n ¼ 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.

combination therapy on Treg cells were observed in the spleen To further investigate the immunomodulatory effects of CDK4/6 (Supplementary Fig. S14A–S14D). Furthermore, increased produc- and BCL2 inhibition, we measured PDL1 expression in 67NR cells þ þ tion of tumor necrosis factor (TNFa)byCD4 and CD8 T cells in vitro and noted that ABT-199 increased PDL1 levels in 67NR cells (Fig. 6H) and a trend toward increased IFNg were evident (Supple- (Fig. 6I). A similar effect was observed in MCF7 cells (Supplementary mentary Fig. S14E and S14F), indicative of enhanced effector func- Fig S14G). The favorable intratumoral immune microenvironment tion. Together, these findings confirm that increased tumor immu- and upregulation of PDL1 expression in 67NR cells suggested that nogenicity and intratumoral immune cell activation are provoked by immune checkpoint blockade might further improve the efficacy of palbociclib and indicate that ABT-199 does not antagonize these triple therapy. We therefore treated 67NR tumor–bearing mice with effects, but may further enhance them. either (i) vehicle (which included an isotype control antibody);

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A 67NR BC67NR 67NR **** 600 * 100 * P = 0.0025 l

Mock NontargetingESR1 FI) 400 (M ER −61 kDa 50 200 H-2Kd Actin −42 Percent surviva 0 0 0102030

Vehicle Time (days) Fulv-199 Fulv-Palbo Fulvestrant Vehicle Fulv-199-Palbo D Fulvestrant Day 0 1 2−8 9 Fulvestrant-199 Seed 67NR Baseline Fulvestrant 199/Palbo Endpoint Fulvestrant-Palbociclib Fulvestrabt-199-Palbociclib 14 days

Cleared fat pad Harvest tumors and spleen E Tregs (tumor) FGTregs Ki67 (tumor) Tregs PD1 (tumor) * 80 20 *** 100 **

) *** * + * ) ) + * + ** 80

60 15

* + + 60 (of CD4 FOXP3 + 40 FOXP3

10 + + 40 % PD-1 % Ki67 20 5 20 (of CD4 (of CD4 % FOXP3 0 0 0

ehicle Vehicle Vehicle V Fulv-199 Fulv-199 Fulv-199 Fulv-Palbo Fulv-Palbo Fulvestrant Fulvestrant Fulv-Palbo Fulvestrant Fulv-199-Palbo Fulv-199-Palbo Fulv-199-Palbo H CD4+ TNFα+ CD8+ TNFα+ I 67NR **** 0.5 3 ** **** 600

) ** ) + + **** * * 4

0.4 ** * * *** 2 400 0.3 (of CD8 (of CD + + 0.2

F 1 200 PDL1 (MFI) 0.1 % TN % TNF 0.0 0 0

ehicle Vehicle V Fulv-199 Fulv-199 Vehicle Fulv-Palbo Fulv-Palbo Fulv-199 Fulvestrant Fulvestrant Fulvestrant Fulv-Palbo Fulv-199-Palbo Fulv-199-Palbo Fulv-199-Palbo J 1,000 67NR 67NR 100 800 ) 3 m 600 Vehicle Fulvestrant-199 400 50 Fulvestrant-199-anti-PD1

olume (m Fulvestrant-199-Palbociclib V 200 Percent survival Fulvestrant-199-Palbociclib-anti-PD1 P = 0.01 0 0 01020300102030 Time (days) Time (days)

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(ii) fulvestrant and ABT-199; (iii) fulvestrant, ABT-199, and anti-PD1; imposed by BH3 mimetics and preferential killing of cycling cells. (iv) fulvestrant plus ABT-199 and palbociclib; or (v) fulvestrant, ABT- Further studies will be required to clarify this, including on BAX/BAK- 199, palbociclib, and anti-PD1. Consistent with the data shown deficient cells. The downregulation of ER following ABT-199 treat- in Fig. 6C, triple therapy improved tumor response and survival, ment would be consistent with the resultant G1 arrest. The precise compared with fulvestrant plus ABT-199. The addition of anti-PD1 mechanism underpinning the reduction in G1 cyclins (cyclin D and E) therapy to this triple combination further attenuated tumor growth is unclear but this could alleviate resistance to CDK4/6 inhibitors given and improved survival (Fig. 6J; Supplementary Fig. S14H). 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 Discussion resistance. þ Despite the majority of patients with ER breast cancer responding As reported previously (7, 8), palbociclib induced a senescence-like to endocrine therapy, the duration of response is highly variable due to phenotype, characterized by distinctive morphologic changes that multiple mechanisms that result in innate or acquired resistance (42). included cell flattening and enlargement of growth-arrested cells that The recent emergence of CDK4/6 inhibitors has changed the landscape were ß-gal–positive and more resistant to apoptosis. Notably, short- þ for treatment of ER breast cancer, with remarkable improvements in and long-term palbociclib-treated cells appeared to be more vulnerable survival (3–5) for a subset of patients. Resistance to therapy, however, to ABT-199–mediated apoptotic cell death. Previous reports have is almost inevitable. In patients with breast cancer, the combination of shown that BCL-XL is a senolytic target (14). Our findings indicate endocrine therapy with CDK4/6 inhibition is predominantly cyto- that BCL2 is also a target and suggest that venetoclax could be a static, likely accounting for reduced apoptosis (12). Combination senolytic agent when combined with CDK4/6 inhibitor therapy in therapy that overcomes resistance through the induction of tumor breast tumors. cell death could therefore be beneficial. Using breast cancer cell lines, Data from clinical trial samples are helping to elucidate the organoids, and PDX models, we show that inhibition of BCL2 and pleiotropic mechanisms of acquired resistance to CDK4/6 inhibi- CDK4/6 in combination with endocrine therapy can inhibit prolifer- tors. Due to the requirement for intact Rb tumor suppressor protein ation and induce apoptosis of cancer cells, including of phenotypically function, RB1 loss or mutation has been identified as a robust senescent cells, leading to enhanced responsiveness in vivo. Notably, marker of resistance to CDK4/6 inhibition. Notably, patients who we also show that ABT-199 augments the CDK4/6 inhibitor response progressed on fulvestrant and palbociclib in the PALOMA-2 clinical – RB1 through dephosphorylation of Rb and reduced G1 S cyclins. In trial developed mutations, although at relatively low preva- addition, pharmacologic inhibition of both CDK4/6 and BCL2 in a lence (34). ABT-199 may provide an alternate means of targeting syngeneic model reduced the proportion and proliferation of Tregs in these tumors, as demonstrated in vitro and in vivo in PDX model 50, the tumor microenvironment. This was accompanied by T-cell acti- which harbored a RB1 mutation. Other resistance mechanisms that vation, with improved antitumor activity observed in vivo upon PD1 have been identified in patient tumors include perturbations in the blockade. PI3K/AKT and mTOR, FGFR, IL6–JAK–Stat,E2F,andHippo The unexpected observation that ABT-199 and other BH3 mimetics signaling pathways (32–38). Our CRISPR/Cas9 screen of fulves- led to Rb dephosphorylation (or possibly degradation of pRb), and trant–palbociclib versus fulvestrant-treated cells produced differ- – reduced G1 S cyclins potentially accounts for ABT-199 enhancing the ential enrichment of sgRNA guides in these pathways, supporting effect of fulvestrant and palbociclib. As this was more pronounced at those findings. Interestingly, we observed differentially enriched higher doses, the clinical significance of this finding is unclear. sgRNA guides for epigenetic modifier genes in the MOZ/MORF Although this was a caspase-independent effect (indicating that the complex, such as KAT6B, suggesting that these may represent novel phenotype is independent of the final “demolition” stage of apoptosis), targets. When we evaluated sgRNA guides that were enriched in it remains possible that the effect is due to mitochondrial stress fulvestrant–palbociclib–venetoclax versus fulvestrant–palbociclib–

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 TNFa in splenic CD4þ and CD8þ 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 mg 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 ¼ 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).

OF12 Clin Cancer Res; 2020 CLINICAL CANCER RESEARCH

Targeting BCL2 and CDK4/6 in ER-Positive Breast Cancer

þ treated cells, RB1 mutations were under-represented with triple ity of relapsing ER tumors are BCL2 positive (16), underscoring its therapy, while targets identified above did not feature. This, togeth- potential utility as a therapeutic target. er with the reduction in cyclin E levels noted above, suggests that The combination of ABT-199 with tamoxifen has shown prom- þ cotreatment with venetoclax could mitigate some of the mechan- ising clinical activity in patients with metastatic ER breast can- isms of acquired resistance to CDK4/6 inhibitors. cer (16). These findings are currently being further investigated in a While immune checkpoint inhibitors are showing promise for randomized phase II study of fulvestrant with or without venetoclax þ þ triple-negative breast cancer, ER breast cancers contain fewer TILs in patients with ER breast cancer who progress on a CDK4/6 and are considered to be far less immunogenic (44). It is notewor- inhibitor (NCT03584009). Here we show that combining BCL2 and thy, however, that anti-PDL1 therapy led to responses in a subset of CDK4/6 inhibition with endocrine therapy elicits potent activity in þ þ þ patients with advanced ER PDL1 breast cancer (45). Intriguingly, endocrine-sensitive and refractory models of ER breast cancer. CDK4/6 inhibitors have been shown to modulate the tumor This combination overcomes the cytostatic effects of endocrine and immune microenvironment by increasing antigen presentation, CDK4/6 inhibitor therapy to trigger cell death in vivo, and induces a reducing inhibitory Treg cells and stimulating increased PDL1 favorable tumor immune microenvironment, together supporting expression (6, 7). These findings imply that the therapeutic effect further investigation of this combination in the clinic, currently of these agents may be mediated, in part, through overcoming underway (NCT03900884). immune evasion, although the magnitude of this effect remains þ uncertain. Utilizing a syngeneic model of ER breast cancer, we Disclosure of Potential Conflicts of Interest fi con rmed these observations and showed that ABT-199 augments The Walter and Eliza Hall Institute receives milestone and royalty payments CD4 and CD8 T-cell activation and does not abrogate palbociclib- related to venetoclax, and employees (J.R. Whittle, F. Vaillant, E. Surgenor, mediated effects in reducing Tregs. Although ABT-199 induces a A.N. Policheni, G. Giner, B.D. Capaldo, H. Chen, H.K. Liu, M.J. Herold, G.K. Smyth, peripheral lymphopenia, this is largely due to a reduction in B D.H.D. Gray, J.E. Visvader, and G.J. Lindeman) may be eligible for benefits related to cells (16). Our data suggest that ABT-199 may favorably contribute 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 to the intratumoral immune microenvironment rather than H. Clevers are listed as co-inventors on two patents that relate to improved culture adversely impact immune signaling systemically. The additional methods for expanding epithelial stem cells and obtaining organoids, which is owned response we observed with PD1 checkpoint inhibition supports this by the Royal Netherlands Academy of Arts and Sciences (KNAW, Koninklijke observation. These data are also consistent with recent findings Nederlandse Akademie van Wetenschappen) and licensed to the HUB Foundation using a MYC-driven model of breast cancer where adjuvant ABT- for Organoid Technology. H. Clevers is a paid advisory board member for Roche 199 plus anti-PD1 therapy produced a survival benefit(46). 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 Despite the promising responses observed in preclinical models fi þ of Medical Research and reports receiving bene ts related to venetoclax. of ER breast cancer, it remains to be determined if the triple G.J. Lindeman is an employee of The Walter and Eliza Hall Institute of Medical combination will be effective and can be safely delivered in the research and reports receiving benefits related to venetoclax; is a paid advisory board clinic. Both CDK4/6 inhibitors and venetoclax are metabolized by member of AbbVie; reports receiving commercial research grants from AbbVie, CYP3A4 and can induce neutropenia and lymphopenia, although Roche/Genentech, Pfizer, and Servier; reports receiving speakers bureau honoraria this has not proven to be problematic when each drug is admin- 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. istered 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 Authors Contributions appeared to facilitate leukocyte recovery. This regimen could also 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, potentially prime tumor cells for cell death, given that BCL2 levels G.J. Lindeman rose following palbociclib withdrawal. Acquisition of data (provided animals, acquired and managed patients, provided The specificity of ABT-199 for BCL2 is advantageous for reducing facilities, etc.): J.R. Whittle, F. Vaillant, E. Surgenor, A.N. Policheni, B.D. Capaldo, possible toxicity, given the limitation of current BCLXL inhibitors due H.-R. Chen, H.K. Liu, J.F. Dekkers, A. Fellowes, T. Green, H. Xu, S.B. Fox, to their induction of on-target thrombocytopenia (50). However, this J.E. Visvader, G.J. Lindeman could increase the risk of resistance due to upregulation of prosurvival Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.R. Whittle, F. Vaillant, A.N. Policheni, G. Giner, proteins, including BCLXL and MCL1. We observed that prolonged A. Fellowes, T. Green, H. Xu, M.J. Herold, G.K. Smyth, D.H.D. Gray, exposure to high concentrations of palbociclib increased BCLXL J.E. Visvader, G.J. Lindeman expression in both MCF7 and T47D cells. However, they remained Writing, review, and/or revision of the manuscript: J.R. Whittle, F. Vaillant, sensitive to ABT-199, presumably due to increased cellular stress. The G. Giner, H.K. Liu, H. Clevers, A. Fellowes, S.B. Fox, G.K. Smyth, D.H.D. Gray, downregulation of MCL1 following palbociclib treatment, together J.E. Visvader, G.J. Lindeman with its potential role in inducing resistance to standard therapy 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 observed in our CRISPR/Cas9 screen, indicates that MCL1 is also a Study supervision: J.E. Visvader, G.J. Lindeman possible target. This is consistent with findings by others that MCL1 is þ a potential target in ER breast cancer (51, 52). Further investigation of Acknowledgments combinatorial (if clinically tolerated) or sequential BH3 mimetic We are grateful to G.B. Mann, L. Graham, and staff at the Royal Melbourne therapy may therefore be warranted. Hospital Tissue Bank for support and thank the Animal, Histology and FACS services It will be important to determine whether our preclinical findings at WEHI. We thank A. Strasser for helpful discussions. Coded breast tumor samples þ translate in the clinic for patients with ER breast cancer. BCL2 is often were provided by the Victorian Cancer Biobank (which is supported by the Victorian þ BCL2 Government). This work was supported by the National Health and Medical Research expressed at high levels in ER tumors, presumably because is an Council, Australia (NHMRC 1054618, 1113133, 1153049), NHMRC IRIISS, Victo- estrogen-responsive gene. This could account for the apparent para- rian State Government Operational Infrastructure Support, Australian Cancer dox of BCL2 being considered a favorable prognostic marker, while Research Foundation, National Breast Cancer Foundation (NT-13-06, IIRS-19- also contributing to treatment resistance (19). Importantly, the major- 004), Breast Cancer Research Foundation (BCRF-18-182), Cancer Australia

AACRJournals.org Clin Cancer Res; 2020 OF13

Whittle et al.

(1165878), Qualtrough Cancer Research Fund, Joan Marshall Breast Cancer Research The costs of publication of this article were defrayed in part by the payment of page Fund, and Collie Foundation (2017F001). J.R. Whittle was supported by an NHMRC/ charges. This article must therefore be hereby marked advertisement in accordance NBCF Research Fellowship and the Royal Australasian College of Physicians, with 18 U.S.C. Section 1734 solely to indicate this fact. 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. Received June 7, 2019; revised February 20, 2020; accepted March 31, 2020; ﬁ Visvader (1037230), and G.J. Lindeman (1078730 and 1175960). published rst April 3, 2020.

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Targeting BCL2 and CDK4/6 in ER-Positive Breast Cancer

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AACRJournals.org Clin Cancer Res; 2020 OF15

Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor−Positive Breast Cancer

James R. Whittle, François Vaillant, Elliot Surgenor, et al.

Clin Cancer Res Published OnlineFirst April 3, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-1872

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