combined cdk4/6 and pi3ka inhibition is synergistic and … · checkpoint blockade resulted in...

Therapeutics, Targets, and Chemical Biology

Combined CDK4/6 and PI3Ka Inhibition IsSynergistic and Immunogenic in Triple-NegativeBreast CancerZhi Ling Teo1,2, Stephanie Versaci1, Sathana Dushyanthen1, Franco Caramia1,Peter Savas1, Chris P. Mintoff1, Magnus Zethoven1, Balaji Virassamy1, Stephen J. Luen1,Grant A. McArthur1,2,Wayne A. Phillips1,2,3,4, Phillip K. Darcy1,2,5, and Sherene Loi1,2

Abstract

New treatments for triple-negative breast cancer (TNBC) areurgently needed. Despite there being little evidence of clinicalactivity as single-agent therapies, we show that dual blockade ofPI3Ka and CDK4/6 is synergistically effective against multipleRB1-wild-type TNBC models. Combined PI3Ka and CDK4/6inhibition significantly increased apoptosis, cell-cycle arrest,and tumor immunogenicity and generated immunogenic celldeath in human TNBC cell lines. Combination treatment alsosignificantly improved disease control in human xenograftmodels compared with either monotherapy. Combined PI3Kaand CDK4/6 inhibition significantly increased tumor-infiltrat-

ing T-cell activation and cytotoxicity and decreased the fre-quency of immunosuppressive myeloid-derived suppressorcells in a syngeneic TNBC mouse model. Notably, combinedPI3Ka and CDK4/6 inhibition, along with inhibition ofimmune checkpoints PD-1 and CTLA-4, induced complete anddurable regressions (>1 year) of established TNBC tumorsin vivo. Overall, our results illustrate convergent mechanismsof PI3Ka and CDK4/6 blockade on cell-cycle progression,DNA damage response, and immune-modulation and mayprovide a novel therapeutic approach for TNBC. Cancer Res;77(22); 6340–52. �2017 AACR.

IntroductionTriple-negative breast cancer (TNBC) is an aggressive subtype of

breast cancer that is clinically characterized by its lack of expres-sion of estrogen- and progesterone receptors (ER/PR) and HER2(1). TNBChas theworst outcomeof all breast cancer subtypes (1).Cytotoxic chemotherapy is the mainstay of current treatment forTNBC patients. However, although these regimens can proveeffective for a subgroup of TNBC patients, in general, oncerelapsed, remissions are brief and are frequently followed byrapid disease progression and death. In contrast, the existence ofa tumor immune infiltrate has been reported to be a robustprognostic factor in TNBC (2). However, the causal mechanismsunderlying this immune response are unclear. It has been pro-posed that DNA damagewith resultant activation of stimulator of

interferon genes (STING) and/ormutational load and neoantigenproduction may be responsible (3, 4). Regardless, this immuneresponse seems not to be sufficient to induce primary tumorclearance in humans.

Cell-cycle control is frequently dysregulated in breast cancer(5). The transition from G1 to S phase of the cell cycle is con-trolled by interactions between cyclin-dependent kinases 4 and6 (CDK4/6), cyclinD1, and retinoblastomaprotein (RB).CDK4/6inhibition has been shown to be an effective therapeutic strategyfor ER-positive breast cancers in clinical trials (6–10). However,TNBC is a molecularly heterogeneous disease characterized bygenomic instability along with high expression of cell-cyclegenes including cyclin E1 (11) and has shown resistance tosingle-agent CDK4/6 inhibition (6, 12).

The complex biology of TNBC suggests that combinationtreatments will be required to achieve effective and durabledisease control. Combined treatment with PI3K and CDK4/6inhibitors has been shown to be effective in mitigating earlyadaption responses to single-agent PI3K and CDK4/6 inhibitorsfor overcoming single-agent inhibitor resistance in ER-positivebreast cancers (13, 14). We sought to determine whether com-bined PI3K and CDK4/6 inhibition would be a similarly usefulstrategy for TNBC. We also hypothesized that induction of celldeath could promote an immune response, given that TNBCmaybe amendable to immune approaches (2, 15–17).

Here, we show that synergistic interactions of PI3Ka andCDK4/6 inhibitors resulted in more effective disease control ofTNBC both in vitro and in vivo. Combination treatment resulted inincreased cell-cycle arrest, apoptosis, calreticulin cell-surfaceexpression, and tumor immunogenicity. Finally, we show thatcombination of PI3Ka and CDK4/6 inhibitors with immune-

1Division of Research, Peter MacCallum Cancer Centre, Melbourne, Victoria,Australia. 2Sir Peter MacCallum Department of Oncology, University of Mel-bourne, Parkville, Australia. 3Division of Cancer Surgery, Peter MacCallumCancer Centre, Melbourne, Victoria, Australia. 4Department of Surgery, St.Vincent's Hospital, University of Melbourne, Melbourne, Victoria, Australia.5Cancer Immunology Research, Peter MacCallum Cancer Centre, Melbourne,Victoria, Australia.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Sherene Loi, Sir Peter MacCallum Department ofOncology, Locked Bag 1 A'Beckett St, Melbourne, Victoria 8006, Australia.Phone: 613-8559-5935; Fax: 613-8559-5039; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-2210

�2017 American Association for Cancer Research.

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checkpoint blockade resulted in long-lasting tumor regressionin a syngeneic immunocompetent mouse model of TNBC.

Materials and MethodsCell lines and culture

All culture media were supplemented with 10% heat-inac-tivated FBS. HCC70, HCC1806, and MDA-MB-468 were kind-ly provided by Professor Roger Daly from Monash University,Australia. All other human cell lines were obtained from theATCC. The human cell lines have been authenticated by shorttandem repeat analysis and were maintained in DMEM orRPMI1640 (Gibco). AT3OVA (18) mouse TNBC cell line wasmaintained in SAFC DMEM. We have previously characterizedAT3OVA and found that it was wild-type for Rb1, Pik3ca, andTrp53 (19). All human and mouse cell lines have been verifiedto be negative for mycoplasma contamination and weremaintained at 37�C in a 5% CO2 incubator. Characteristicsof the human cell lines are further detailed in SupplementaryTable S1.

Drug compoundsBYL719 (Alpelisib) and LEE011 (Ribociclib) were kindly sup-

plied by Novartis. PD991 (Palbociclib; PD-0332991) was pur-chased from SelleckChem. All drugs for in vitro use were recon-stituted in DMSO at 10 mmol/L. BYL719, LEE011, and PD991were reconstituted in 0.5%methylcellulose (Sigma Aldrich) for invivo experiments. Immune-checkpoint antibodies and the isotypecontrol used in in vivo experiments were obtained from BioXCell:anti-mouse PD-1 mAb (RMP1-14), anti-mouse CTLA-4 mAb(9H10), and Rat IgG2a isotype control mAb (2A3). Doses usedare detailed in figure legends.

Combination treatment synergy quantitationDrug combination studies were performed according to the

Chou–Talalay method of synergy quantitation (20). MDA-MB-468, HCC1143, HCC70, HCC1806, MDA-MB-231, HS578T, andMDA-MB-453 cells were treated in vitro for 72 hours with thecombination of BYL719 and LEE011 over a range of concentra-tions held at a fixed ratio based on the GI50 (drug concentrationrequired for 50% cell growth inhibition) of each drug specific foreach cell line. CalcuSyn 2.0 (Biosoft) performs multiple drugdose-effect calculations using the Median Effects methodsdescribed by Chou and Talalay (21) and was used to determinethe combination index (CI), which offers quantitative definitionfor additive effect (CI ¼ 1), synergism (CI < 1), and antagonism(CI > 1) of drug combinations.

Flow cytometry analysisCells were plated in 24-well plates and treated the following

day with the indicated agents. Cells and tissues were lysedand processed as described in Supplementary Methods. FACSanalysis was performed on an LSR II flow cytometer (BDBiosciences).

Western blot analysisCell lines were plated in 6-well plates and treated the following

day as indicated. Cells and tissues were lysed and processed asdescribed in Supplementary Methods. Signal intensities for eachprotein of interest were quantitated using Image J (https://imagej.net) and presented in Supplementary Fig. S1.

30RNA sequencingMDA-MB-231 cells were plated in 6-well plates and treated the

following day as indicated. Two replicates were included. After 24hours, total cell RNAwas extracted using a PureLink RNAMini kit(ThermoFisher Scientific) following the manufacturer's instruc-tions. The quantity and integrity of the total RNAwere determinedusing TapeStation 2200 system (Agilent Technologies) andQubitRNA High Sensitivity assay kit (ThermoFisher Scientific). Notethat 500 ng total RNA was used for library preparation accordingto themanufacturer's instructions (QuantSeq 30 mRNA-Seq FWD;Lexogen). Indexed libraries were pooled and sequenced on aNextSeq500 (Illumina). Briefly, the library was generated withan oligo-dT containing the Illumina Read2 linker and a randomforward primer containing the Illumina Read1 linker. The librarywas then amplified with PCR primers containing sample indicesand the Illumina clustering sequences. Five to 15 million single-end 75 bp reads were generated per sample.

Gene set enrichment analysis analysisGene set enrichment analysis (GSEA) was performed using the

GSEA tool (22) on the entire normalized RNA expression countmatrix without limiting the input to only differentially expressedgenes. The Hallmark collection of 50 predefined gene sets and C5Gene Ontology (GO) collection of 5,917 predefined gene setsfrom the Molecular Signatures Database (MSigDB, Broad Insti-tute) were used for analysis. The gene sets (3,576 C5 and 49 Hall-mark gene sets) included in the analysis were limited to those thatcontained between 15 and 500 genes. Permutation (by gene sets)was conducted 1,000 times according to default weighted enrich-ment statistics and using difference of class metrics to calculateand rank genes according to their differential expression levelsbetween two treatment groups. Significant gene sets were thosedefinedwith a nominal P<0.05. Calculation of the false discoveryrate (FDR) was used to correct formultiple comparisons and geneset sizes. Significantly enriched gene sets with FDR < 0.25 wereselected for hypothesis generation.

In vivo studiesSix- to 8-week-old female Nod SCID g (NSG) mice were used

forMDA-MB-231,HCC1806, andpatient-derived xenograftmod-els. For MDA-MB-231 and HCC1806 xenografts, 2 � 106 cells(suspended in PBS:Matrigel, 1:1) were injected into the fourthmammary fat pad of the NSG mice. For experiments with thepatient-derived xenograft (PDX) model 12006, a single tumorfrom apreviously establishedNSGmouse (passage 5)was excisedand small fragments were implanted directly into the fourthmammary fat pads of 6- to 8-week-old femaleNSGmice. AT3OVA(5� 105) cells were suspended in PBS and injected into the fourthmammary fat pads of 6- to 8-week-old female immune-compe-tent C57BL/6 mice.

For all models, tumor volume (length � width2 � 0.5) wasassessed by caliper measurements every 3 to 4 days. Once tumorsreached an average of 100 to 150mm3 (human xenograftmodels)or 50 to 100mm3 (mouse syngeneic model), mice were random-ized to commence drug treatment (day 1). BYL719, LEE011, andPD991were administered via oral gavage. BYL719was given dailythroughout the duration of the experiment. LEE011 or PD991were given daily on days 1 to 21 of each 28-day cycle (3 weeks on,1 week off). Anti–PD-1 and/or anti–CTLA-4 were administeredvia intraperitoneal injections on days 1, 5, 9, and 13 only. For 7-day immune response analysis, BYL719 and LEE011 were

Combined CDK4/6 and PI3Ka Inhibition in TNBC

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administered daily with no breaks with dosing. For these experi-ments, anti–PD-1 and/or anti–CTLA-4were only administered ondays 1 and 5.

Mice were euthanized if the tumors reached ethical limit of1,400 mm3 or if the animals displayed health indicators thatmet the institutional criteria for sacrifice. All animal experi-ments were approved by the Peter MacCallum Cancer CentreAnimal Experimentation Ethics Committee (E539 and E556)and conducted in accordance with the National Health andMedical Research Council Australian Code of Practice for theCare and Use of Animals for Scientific Purposes.

Statistical analysisOne-way ANOVA with Tukey multiple comparison test was

used to compare between treatment groups. Tumor growth curveswere analyzed using two-way repeated measures ANOVA withTukey multiple comparison test. Survival differences betweentreatment groups were determined using the Kaplan–Meier logrank analysis. Statistical analyses (not including differential geneexpression or GSEA analyses) were performed using Prism 7(GraphPad). A two-tailed P < 0.05 was considered statisticallysignificant. All data are expressed as mean � SEM.

ResultsCombined PI3Ka and CDK4/6 inhibition is synergistic againstRB1-wild-type TNBC cell lines in vitro

We quantitated the level of synergy between BYL719 (PI3Kainhibitor) and LEE011 (CDK4/6 inhibitor) in sevenhumanTNBCcell lines including six RB1-wild-type and one RB1-mutant cellline. Of the six RB1-wild-type cell lines, three were classified asbasal-like, two mesenchymal-like, and one was of the luminalandrogen receptor (LAR) subtype (23). We demonstrated that theinteraction between BYL719 and LEE011 was synergistic in all sixRB1-wild-type TNBC cell lines but was antagonistic in the RB1-mutant cell line, MDA-MB-468 (Fig. 1A).

As expected from the above results, combination treatmentwith BYL719 and LEE011 significantly inhibited cell growthcompared with single-agent and vehicle treatments in the syner-gistic lines (Fig. 1B). Consistent with this, we show in HCC1806andMDA-MB-231 cell lines that BYL719 and LEE011 single-agenttreatments resulted in reduction of AKT and RB phosphorylation,respectively, compared with vehicle treatment, and bothremained suppressed with combination treatment (Fig. 1C).

Combination treatment is efficacious inTNBCxenografts in vivoThe efficacy of combined PI3Ka and CDK4/6 inhibition was

next examined in vivo. HCC1806 and MDA-MB-231 xenografttumor models were treated with BYL719, CDK4/6 inhibitor(LEE011 or PD991), the combination, or vehicle control. In bothmodels, strong synergismwas observedwith significant reductionof tumor growth of mice treated with combination therapycompared with single-agent and vehicle treatments (Fig. 1D).

We next treated a patient-derived TNBC xenograft (PDX12006)with the combination of BYL719 and LEE011. This tumor samplehad oncogenic TP53 p.S241C (COSM10709) and ARID1Ap.R2232W (COSM3724474) mutations and was classified asmesenchymal subtype (P < 0.001). Similar to what we observedin HCC1806 and MDA-MB-231 xenograft models, combinationtreatment in the PDX model exhibited significant tumor growthinhibition (Fig. 1D).

BYL719 and LEE011 treatment significantly increases apoptosisWe next examined combined BYL719 and LEE011 treatment

on cell cycle and apoptosis in vitro. Combined treatment signif-icantly increased the effect on G1 cell-cycle arrest comparedwith single-agent BYL719 and vehicle treatments in both basal-like and mesenchymal-like TNBC cell lines (Fig. 2A). In HCC70and MDA-MB-231 lines, G1 cell-cycle arrest was significantlygreater following combination treatment compared with BYL719and LEE011 single-agent treatments as well as vehicle treatment.

Notably, combined treatment with BYL719 and LEE011 sig-nificantly increased apoptosis as indicated by an increase inpercentage of Annexin Vþ/propidium iodide� cells comparedwith single-agent and vehicle treatments (Fig. 2B).

We next analyzed protein components of the PI3K andCDK4/6pathways by Western blot analysis in HCC1806 and MDA-MB-231 cells treated with vehicle, BYL719, LEE011, or the combina-tion for 72 hours in vitro (Fig. 2C). Treatment with LEE011 wasassociated with increased expression of cyclin D1, consistent withprevious reports (13, 14). Phosphorylation levels of eukaryotictranslation initiation factor 4E-binding protein-1 (4EBP1 Thr37/40), known to be a direct substrate of mTOR and one of theregulators of cyclin D1 expression (24, 25), were maintainedacross treatment groups in MDA-MB-231 and only slightlydecreased with BYL719 and LEE011 combined treatment inHCC1806 compared with vehicle treatment, suggesting incom-plete inhibition of mTOR activity. Nonetheless, we observed thatcombination treatment removed AKT inhibition on GSK3b andp21Cip1/WAF1 (p21) tumor suppressors. p-GSK3b Ser9 and p-p21Thr145 expression levels were suppressedwith combined BYL719and LEE011 treatment compared with single-agent and vehicletreatments in MDA-MB-231 cells, whereas in HCC1806 cells,suppression was observed with single-agent BYL719 and combi-nation therapy compared with vehicle treatment.

Next, we assessed the CDK2/cyclin E complex, which associateswithCDK2 and initiates CDK2 activation shortly before entry intoS phase (26). We demonstrate that p-CDK2 Thr160 levels werereduced with LEE011 treatment and further decreased with com-bination treatment in both cell lines. Furthermore, cyclin E2 levelswere markedly decreased with LEE011 and combination treat-ments compared with vehicle control; however, this was alsoaccompanied by an increase in cyclin E1 expression with LEE011and combination treatments.

These results suggest that decreased cell survival and pro-liferation due to combined PI3Ka and CDK4/6 blockade islikely mediated through multiple cell-cycle–related pathwaysin TNBC.

Synergistic effects of combination treatment on cell divisionprocesses and immune responses

To further understand the mechanism by which BYL719 andLEE011were effective in combination in an unbiasedmanner, weperformed next-generation RNA sequencing and GSEA usingMDA-MB-231 cells treated with vehicle BYL719, LEE011, or thecombination. BYL719 and LEE011 single-agent and the combi-nation treatment groups were each compared with vehicletreatment.

Not surprisingly, we observed that the "E2F targets" hallmarkgene set was the top enriched (P < 0.05) downregulated gene setacross all comparisons (Fig. 3A). Comparedwith single-agent andvehicle treatment groups, the combined therapy was associatedwith significant downregulation of E2F target genes involved in

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Figure 1.

Combined PI3Ka and CDK4/6 inhibitor treatment is synergistic in TNBC cell lines. A, Synergy quantification as indicated by CI values of BYL719 and LEE011treatment in TNBC cell lines. Gray bar, CI values indicating additive effect. � , MDA-MB-468 carries RB1 mutation; BL, basal-like subtype; MSL, mesenchymal-likesubtype; LAR, luminal androgen receptor subtype. Data depicted are the mean � SEM. B, Percent cell growth of TNBC cells relative to vehicle treatmenttreated with media containing vehicle, BYL719, LEE011, or the combination at their GI50 doses for 72 hours in vitro. Data depicted are the mean � SEM. C,Representative Western blots depicting changes in protein expression in HCC1806 and MDA-MB-231 cells following 72-hour treatment with vehicle, 1 mmol/L ofBYL719, 1 mmol/L of LEE011, or the combination. Lysates were made and probed with the indicated antibodies. D, Tumor growth of HCC1806, MDA-MB-231,and PDX12006 xenografts in vivo. HCC1806 and PDX12006 xenografts were treated with vehicle, BYL719 (15 mg/kg), LEE011 (60 mg/kg), or the combination.MDA-MB-231 xenografts were treated with vehicle, BYL719 (25 mg/kg), PD991 (120 mg/kg), or the combination. N¼ 8 mice per treatment group. Data depicted arethe mean fold change in tumor volume � SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001 by one-way ANOVA (A and B), or two-way ANOVA (D).






cell-cycle progression, consistent with our results on cell-cycleinhibition above.

The top enriched (P < 0.05) downregulated GO processes foreach comparison were all related to cell division processes, withparticular enrichment for downregulation of genes in chromo-some/chromatid segregation processes as shown in Fig. 3B.

A heatmap of the "core-enriched" genes in the top enriched (P <0.05) hallmark and GO process gene sets shows striking down-regulation of these genes (log fold change, –0.39 to –3.74;medianlog fold change ¼ –1.66) with the combination treatment com-pared with vehicle treatment (Fig. 3C). Interestingly, we notedthat genes involved in DNA damage and repair processes(highlighted in yellow in Fig. 3C) made up a sizeable proportionamong those downregulated.

Given these findings, we next investigated if there wasobjective evidence of DNA damage. Validating our observa-tions above, we found that both LEE011 single-agent andcombination treatments were associated with increased levelsof gH2AX phosphorylation on Ser139 (gH2AX, a marker forDNA damage; Fig. 3D).

Combination BYL719 and LEE011 treatment increases tumorimmunogenicity in vitro

In our GSEA analysis, we observed that immune-related path-ways were also among the top enriched (P < 0.05) upregulatedhallmarks. These included upregulation of IFNa, IFNg , TNFa, andinnate immune (complement) responses (Fig. 4A and B).We alsoobserved significant upregulation of genes (log fold change, 0.24to 1.51; median log fold change ¼ 0.55) involved in antigenpresentation that includedHLAsHLA-A (humanmajor histocom-patibility complex class I; MHCI), HLA-DMA (human MHCII),CTSD, ICAM, RELB, PSME1, and TAPBP, in response to combi-nation therapy compared with vehicle treatment (highlighted inyellow in Fig. 4B).

We next investigated whether combined BYL719 and LEE011treatment could modulate tumor immunogenicity given thetranscriptional findings above. We examined tumor cell-surfaceexpression of HLA antigens as well as the immune-checkpointligands, programmed death ligand 1 (PD-L1), and CD86 inHCC1806 and MDA-MB-231 cell lines (Fig. 4C). We found thatLEE011 single-agent as well as the combination treatments

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Combined PI3Ka and CDK4/6 inhibitor treatment induces cell-cycle arrest and pronounced apoptosis in TNBC cell lines. A, Cell-cycle analysis in TNBClines treated with vehicle, 1 mmol/L of BYL719, 1 mmol/L of LEE011, or the combination for 72 hours. Data depicted are the mean � SEM. � , P < 0.05; �� , P < 0.01;�� , P < 0.001; and �� , P < 0.0001 by one-way ANOVA comparing percentage of cells in G0–G1 between treatment groups. B, TNBC cell lines weretreated with vehicle, 1 mmol/L of BYL719, 1 mmol/L of LEE011, or the combination for 72 hours and analyzed using flow cytometry for Annexin V–positiveand propidium iodide (PI)–negative cells. Data depicted are the mean � SEM. � , P < 0.05; ��, P < 0.01; �� , P < 0.001; and �� , P < 0.0001 by one-wayANOVA. C, Representative Western blots depicting changes in protein expression in HCC1806 and MDA-MB-231 cells following 72-hour treatment withvehicle, 1 mmol/L of BYL719, 1 mmol/L of LEE011, or the combination. Lysates were made and probed with the indicated antibodies. Quantitation of relativesignal intensities of protein expression presented in Supplementary Fig. S1.

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significantly increased expression of HLA antigens (expressionwas further increased in thepresence of IFNg), aswell asCD86, theligand for immune-checkpoint cytotoxic T-lymphocyte–associat-ed protein 4 (CTLA-4), in both cell lines. BYL719 seemed to havean inhibitory effect on the expression of PD-L1, consistent withthe literature that this ligand is regulated through the PI3K/AKTsignaling pathway (27–29).

Given the above results, along with observations of increasedDNA damage and tumor immunogenicity, we next assessedwhether combined therapy could induce an increase in "immu-nogenic" cell death. We examined levels of tumor cell-surfacecalreticulin expression, given that this protein, usually presenton the endoplasmic reticulum, is translocated to the plasmamembrane surface of cells undergoing immunogenic cell deathin the early, preapoptotic phase, facilitating engulfment ofdying cells by dendritic cells (30). Consistent with our hypoth-esis, we observed pronounced increased expression of cell-surface calreticulin following combination therapy on bothHCC1806 and MDA-MB-231 cells compared with single-agentand vehicle treatments (Fig. 4D).

Collectively, these findings suggest that the combined BYL719and LEE011 treatment enhanced immunogenic cell death andcould promote tumor immunogenicity. These findings providedthe rationale to evaluate whether combination therapy couldenhance immune responses in vivo.

Combined BYL719 and LEE011 inhibition increasesT-cell activation and reduces immunosuppressive cellpopulations in vivo

AT3OVA is a well-characterized syngeneic mouse model ofTNBC and was used to examine whether immune responses wereimportant for the therapeutic effect following combined PI3KaandCDK4/6 inhibition in vivo.AT3OVA tumor–bearing immune-competent mice were treated with BYL719, LEE011, the combi-nation, or vehicle control. We observed that combined treatmentexerted greater control of AT3OVA tumor growth compared withsingle-agent or vehicle treatments (Fig. 5A).

We next characterized the effect of treatment on immune cellpopulations in the tumor microenvironment by flow cytometry.Compared with single-agent and vehicle treatment cohorts, thecombined treatment markedly enhanced infiltrating CD8þ andCD4þ T-cell activity as indicated by an increase in CD69, gran-zyme B, and IFNg expression (Fig. 5B and C). Further evidence ofenhanced T-cell activation following combined therapy was indi-cated by a significant increase in coexpression of programmed celldeath protein 1 (PD-1) and CTLA-4 immune-checkpoint proteinsinCD8þ andCD4þT cells comparedwith single-agent and vehicletreatment groups (Fig. 5B and C). Single-agent or combinationtreatments were not observed to affect proliferation or frequencyof tumor-infiltrating CD8þ and CD4þ T cells (Supplementary Fig.S2A and S2B).

In addition to activating adaptive immune cells, combinedBYL719 and LEE011 treatment significantly increased the fre-quency of tumor-infiltrating natural killer T (NKT) cells, andthere was a trend for increased mature NK cells (NK1.1þ/CD11bhigh/CD27low) compared with single-agent LEE011 andvehicle treatments (Supplementary Fig. S2C). Granzyme B expres-sion by mature NK cells was also observed to be significantlygreater with combination treatment compared with BYL719 andLEE011 single-agent and vehicle treatment groups, indicating anincrease in their cytotoxic potential (Supplementary Fig. S2C).

Interestingly, combined BYL719 and LEE011 treatmentwas effective in significantly reducing the frequency of immu-nosuppressive monocytic myeloid-derived suppressor cells(mMDSC; Fig. 5D). We also observed a significant decrease inproliferation of CD4þ FOXP3þ regulatory T cells (Treg) fol-lowing combination therapy as indicated by the significantdecrease in expression of the Ki67 proliferative marker (Sup-plementary Fig. S2B).

In summary, combined PI3Ka and CDK4/6 inhibition resultedin enhanced innate and adaptive antitumor immune responses.The increase in expression of both PD-1 and CTLA-4 on T cellssuggested that the addition of immune-checkpoint blockadecould further augment the antitumor therapeutic effect of PI3Kaand CDK4/6 inhibition.

Immune-checkpoint blockade in combination with PI3Kaand CDK4/6 inhibition results in long-lasting tumorregression in vivo

We next examined the efficacy of combining BYL719 andLEE011 with immune-checkpoint blockade in the AT3OVAmod-el. We tested anti–PD-1 and anti–CTLA-4 mAbs given that theseimmune checkpoints were found to be upregulated on T cellsfollowing PI3Ka and CDK4/6 inhibition and that they are cur-rently being evaluated in clinical trials. Anti–PD-1 and anti–CTLA-4 treatments were administered on day 1 along withBYL719 and LEE011 treatments in tumor-bearing mice. All treat-ments ceased by day 50.

We observed that tumors treated with anti–PD-1 and anti–CTLA-4 as single agents performed worse in terms of tumorgrowth control and survival compared with BYL719 and LEE011combination treatment (Fig. 6A). However, we found that thequadruple combination treatment group (comprising BYL719,LEE011, anti–PD-1, and anti–CTLA-4) performed the best amongall treatment groups (Fig. 6A). All tumors (10/10) treated withthe quadruple combination remained regressed during the50 days of treatment; whereas tumor growth continued in allother treatment groups despite being under constant drug pres-sure (Supplementary Fig. S3). Of note, 50% (5/10) of tumorstreated with the quadruple combination remained regressed formore than a year, even after treatment cessation, with resultantsignificant improvement in survival compared with BYL719 andLEE011 as well as anti–PD-1 and anti–CTLA-4 treatment groups(Fig. 6A). Notably, there was no toxicity observed in mice fol-lowing the full treatment schedule.

In terms of mechanism, we observed a significant increase inMHCI (H2KB) expression on AT3OVA tumor cells followingcombined BYL719 and LEE011 treatment in vivo (Fig. 6B),validating our results that BYL719 and LEE011 treatmentincreases tumor immunogenicity in vitro (Fig. 4C). Althoughchanges in MHCII expression were less impressive with com-bined BYL719 and LEE011 treatment, similar to resultsobtained in vitro, we show that addition of immune-checkpointblockade in the quadruple combination treatment groupresulted in a pronounced and significant increase in MHCIIas well as PDL-1 expression on AT3OVA tumor cells comparedwith other treatment groups (Fig. 6B). Moreover, we found thatgranzyme B expression in CD8þ and CD4þ T cells was signif-icantly increased following quadruple combination treatmentcompared with all other treatment groups (Fig. 6C). Theseresults support that the addition of immune-checkpoint block-ade further augments tumor immunogenicity and subsequent






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BYL719 vs. Vehicle GO Processes (Downregulated genes) ES NES NP FDRGO_CHROMOSOME_CENTROMERIC_REGION -0.506 -2.154 0.000 0.006GO_CONDENSED_CHROMOSOME -0.501 -2.112 0.000 0.007GO_PEPTIDYL_LYSINE_TRIMETHYLATION -0.718 -2.068 0.000 0.007GO_DNA_PACKAGING_COMPLEX -0.641 -2.093 0.000 0.007GO_CONDENSED_CHROMOSOME_CENTROMERIC_REGION -0.531 -2.070 0.000 0.008

LEE011 vs. VehicleGO Processes (Downregulated genes) ES NES NP FDRGO_SISTER_CHROMATID_SEGREGATION -0.816 -2.830 0.000 0.000GO_CHROMOSOME_SEGREGATION -0.789 -2.807 0.000 0.000GO_NUCLEAR_CHROMOSOME_SEGREGATION -0.802 -2.799 0.000 0.000GO_CONDENSED_CHROMOSOME -0.798 -2.746 0.000 0.000GO_DNA_REPLICATION -0.790 -2.734 0.000 0.000

BYL719+LEE011 vs. VehicleGO Processes (Downregulated genes) ES NES NP FDRGO_NUCLEAR_CHROMOSOME_SEGREGATION -0.818 -2.892 0.000 0.000GO_SISTER_CHROMATID_SEGREGATION -0.824 -2.888 0.000 0.000GO_CHROMOSOME_SEGREGATION -0.803 -2.887 0.000 0.000GO_CONDENSED_CHROMOSOME -0.818 -2.848 0.000 0.000GO_MITOTIC_NUCLEAR_DIVISION -0.762 -2.839 0.000 0.000

XPO1MTHFD2

CENPEDUT

BRCA1BRCA1CENPMBUB1BSMC1ASMC1AHELLS

PCNAPCNAMAD2L1NCAPD2SNRPBSPAG5

POLE4

CHEK2CHEK2

BRCA2BRCA2DLGAP5NUP205

POLEPOLEDDX39ACDKN1AKIF22KIF22WEE1ESPL1RB1CDKN3CDKN2CEZH2

MRE11AMRE11AUBE2S

AURKASPC25MYBL2RBBP7CCNB2DNMT1NUDT21PLK1

BARD1BARD1KIF2CPLK4POLD3POLD3CDCA8HMGB2HMGB2CDK1CDK1BUB3

RAD51AP1RAD51AP1POLA2POLD1POLD1H2AFZAURKB

NEK2

TOP2A

ZW10

CHEK1CHEK1

NUP107

SMC3SMC3

MSH2MSH2

RAD1RAD1

SMC6SMC6

RFC2RFC2

SSRP1SSRP1

UBE2TUBE2T

RPA3RPA3SKA1

HMGB3

E2F8

CDC20

MLH1MLH1

DSCC1

BIRC5

RAD51CRAD51CPDS5B

LIG1LIG1

KIF4A

SMC4

BUB1

CDK4

NUP153

RACGAP1

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RPA2RPA2RAD51RAD51PTTG1PTTG1H2AFXH2AFXMKI67

RAD21RAD21RFC3RFC3TRIP13TRIP13SKA2

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T-cell cytotoxicity responses following PI3Ka and CDK4/6inhibition in TNBC.

DiscussionTNBC remains the most challenging breast cancer subtype to

treat with the poorest prognosis andmedian survival after relapse.There are currently no targeted therapies approved for the treat-ment of TNBC, although Olaparib in BRCA1 germline-deficientbreast cancers may soon see this change (31). The majority ofTNBC, however, are BRCA1/2 wild-type, and their prognosisremains poor particularly after relapse. New therapies or drugcombinations that can achieve durable disease control are need-ed. Immunotherapy, in particular immune-checkpoint blockade,has shown remarkable success in other solid cancer types; how-ever, response rates in metastatic TNBC patients remain lowcompared with those observed for immunogenic diseases suchas melanoma (32, 33).

In this study, we have shown synergistic antitumor effects forthe combination of PI3Ka (BYL719) with CDK4/6 (LEE011,PD991) inhibitors in a variety of TNBC preclinical modelsincluding basal-like, mesenchymal, mesenchymal-like, andLAR TNBC subtypes. Cooperative increase in cell-cycle arrest,DNA damage, replicative stress, and apoptosis all contributedto the antitumor effects of combined PI3Ka and CDK4/6inhibition in TNBC.

A recent report suggested synergism for the CDK4/6 inhibitor(PD991) with two PI3K inhibitors (Pictilisib, GDC-0941 andTaselisib, GDC-0032) for only TNBC cell lines that are of theluminal-like subtype or those that harbor activating mutations inPIK3CA (encoding PI3 kinase catalytic a subunit, PI3Ka; ref. 34).However, our data suggest wide-ranging synergism in a variety ofTNBC models in vivo and in vitro, regardless of PIK3CA mutationstatus, with PI3Ka inhibitor (BYL719) togetherwith either CDK4/6 inhibitors (LEE011 or PD991). The PI3K inhibitors varymarkedly in their potency across the PI3K catalytic isoforms(PI3Ka/b/g/d), and we hypothesize that this may explain thedifferences in findings. GDC-0941 is a pan-isoform PI3K inhib-itor, whereas GDC-0032 is a b isoform–sparing PI3K inhibitor(IC50 against PI3Kb ¼ 9.1 nmol/L) and has potent inhibitoryeffects on PI3Ka (IC50¼ 0.29 nmol/L), PI3Kg (IC50¼ 0.97 nmol/L), and PI3Kd (IC50 ¼ 0.12 nmol/L) isoforms (35, 36). WhereasBYL719 is a selective PI3Ka inhibitor, exhibiting the highestpotency against PI3Ka activation (IC50 against PI3Ka ¼ 19nmol/L) compared with other isoforms such as PI3Kb (IC50 ¼1,156 nmol/L), PI3Kg (IC50 ¼ 320 nmol/L), and PI3Kd (IC50 ¼290 nmol/L; ref. 37). Furthermore, we differed in our approachesin compound synergy assessment and have additionally evaluat-ed synergistic effects on apoptosis and cell-cycle arrest.

The LAR subtype of TNBC has been previously shown to besusceptible to combined PI3K and CDK4/6 pathway inhibitiondue to the presence of PIK3CA mutations (14, 34). Moreover,tumors of the LAR subtype have been shown to be heavilyenriched in hormonally regulated pathways that include andro-gen/estrogen metabolism as well as display luminal gene expres-sion patterns, similar to ER-positive luminal breast cancers (23).RB has been shown to have a highly specific and indispensablerole in mediating antitumor responses to CDK4/6 inhibition(6, 38, 39). Consistent with other reports (34), the antagonisticinteraction of the two compounds in the RB1-mutant cell lineMDA-MB-468 further suggests that RB remains essential forsynergistic interactions following PI3K and CDK4/6 pathwayinhibitions in TNBC. In The Cancer Genome Atlas study, thepercentage of TNBC tumors where RB1 was mutated or lost wasreported to be only 20% (11), indicating that combined PI3Kaand CDK4/6 inhibitor treatment could be beneficial to themajor-ity of TNBC patients.

Gene expression analyses revealed that combined PI3Ka andCDK4/6 inhibition in TNBC significantly downregulates genesinvolved in multiple key prosurvival cellular processes includingDNA damage and repair responses. We have shown that thiscorresponds with an increase in DNA damage. The observedincreased cyclin E1 expression in addition to reduced expressionof DNA repair genes with combined PI3Ka andCDK4/6 inhibitortreatment could have contributed to further catastrophic genomicinstability in our models (40, 41).

The clinical importance of tumor-infiltrating lymphocytes(TIL) has been an emerging area of research in breast cancer,particularly in TNBC where higher levels of TILs are associatedwith both increase in response to chemotherapy and improvedsurvival (2, 15). However, many patients with advanced TNBChave few or no TILs present at diagnosis. Therefore, our novelobservation that combined PI3Ka and CDK4/6 inhibition couldeffectively promote antitumor immunity in TNBC was of highinterest. To our knowledge, we have shown for the first time thatcombined PI3Ka andCDK4/6 inhibition increases tumor antigenpresentation as well as immunogenic cell death, which wouldpotentially facilitate the eradication of tumor cells by the immunesystem (30).

Further evaluation of immune responses to PI3Ka andCDK4/6 inhibition in mouse immunocompetent environmentrevealed that combination treatment in vivo results in increasedactivation and cytotoxicity of both adaptive and innateimmune cell populations as well as decreased frequency ofimmune-suppressive MDSCs within the tumor environment.The observation of increased antitumor immunity as a result ofsuppressing PI3K and CDK4/6 pathways was intriguing as boththese pathways have been long thought to be important for

Figure 3.Combined BYL719 and LEE011 treatment downregulates cell-cycle processes and induces DNA damage in TNBC. A–C, MDA-MB-231 cells were treated in vitrofor 24 hours with vehicle, 1 mmol/L BYL719, 1 mmol/L LEE011, or the combination, after which, total RNA was extracted and analyzed via 3'RNAseq. A, GSEAenrichment plot and heatmap of differentially expressed genes in the Hallmark E2F targets gene set. V, Vehicle; B, BYL719; L, LEE011; BL, BYL719 and LEE011. B, Topfive most enriched GSEA GO processes with the largest absolute normalized enrichment score (NES) with smallest FDR P value among significantly enriched genesets with FDR < 0.25. ES, enrichment score; NP, nominal P value. C, Heatmap of core-enriched genes in top enriched downregulated Hallmark and GO process cell-cycle–related gene sets. Genes thatwere significantly differentially expressed (P<0.05) in combination treatment comparedwith vehicle treatment are shown. Genenames that are highlighted in yellow encode proteins involved with DNA damage and repair processes. D, Combined BYL719 and LEE011 treatment inducesDNA damage. HCC1806 and MDA-MB-231 cell lines were treated with 1 mmol/L of BYL719, 1 mmol/L LEE011, or the combination for 72 hours and analyzed using flowcytometry. Phosphorylated gH2AXSer139-positive cellswere quantitated as a percentage of live cells. Data depicted are themean� SEM. � ,P <0.05; �� ,P <0.01; and�� , P < 0.0001 by one-way ANOVA.






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MDA-MB-231 24 h HallmarksBYL719 vs. VehicleHallmarks (Upregulated genes) ES NES NP FDRHALLMARK_MYOGENESIS 0.439 1.843 0.000 0.013HALLMARK_HYPOXIA 0.371 1.692 0.000 0.036HALLMARK_P53_PATHWAY 0.333 1.519 0.000 0.062HALLMARK_COAGULATION 0.401 1.567 0.000 0.065HALLMARK_KRAS_SIGNALING_DN 0.414 1.530 0.013 0.069

LEE011 vs. VehicleHallmarks (Upregulated genes) ES NES NP FDRHALLMARK_COAGULATION 0.591 2.489 0.000 0.000HALLMARK_INTERFERON_ALPHA_RESPONSE 0.509 2.164 0.000 0.000HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION 0.453 2.089 0.000 0.000HALLMARK_COMPLEMENT 0.378 1.688 0.000 0.025HALLMARK_ANGIOGENESIS 0.596 1.701 0.011 0.029

BYL719+LEE011 vs. VehicleHallmarks (Upregulated genes) ES NES NP FDRHALLMARK_COAGULATION 0.586 2.343 0.000 0.000HALLMARK_INTERFERON_ALPHA_RESPONSE 0.490 1.998 0.000 0.002HALLMARK_KRAS_SIGNALING_UP 0.433 1.895 0.000 0.005HALLMARK_COMPLEMENT 0.408 1.788 0.000 0.014HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION 0.366 1.657 0.000 0.027

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CTSDCTSDTIMP1APOBEC3FIFITM2IFITM3C1SAPOBEC3GPIP4K2CUBE2L6RNASELDOCK4EPSTI1ME1MAP1LC3BTP53INP1LRP1PDP1LGALS3BPBST2BCL6PARP12FAM125ADUSP6PLATSTX4STAT2KYNULTA4HTXNIPNFKBIZIRF1TNFSF10MEF2CTGFB2IFI27

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immune cell function and proliferation (42, 43). Nonetheless,recent studies have shown that adaptive and innate effector cellfunction and survival were not affected with PI3Ka inhibition(44, 45). Moreover, our work, along with other reports, hashighlighted that various PI3K isoforms as well as some CDKscould be essential for modulation of the immune-suppressive

tumor microenvironment (46–49). These studies have demon-strated that blockade of PI3Kd or g isoforms reduces thefunction of Tregs and MDSCs to a greater extent comparedwith effector T cells, thereby maintaining better control oftumor growth (48, 49). Interestingly, in the clinical setting,neutropenia is reported as an on-target side effect of CDK4/6

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Combined PI3Ka and CDK4/6 inhibition increases tumor-infiltrating CD8þ and CD4þ T-cell activation and cytotoxic potential as well as decreases frequency ofMDSCs in AT3OVA tumors in mice. A, Tumor growth of AT3OVA. Tumor-bearing mice were treated with vehicle, BYL719 (10 mg/kg), LEE011 (40 mg/kg),or the combination. N ¼ 8 mice per treatment group. Data depicted are the mean tumor volume � SEM. B–D, AT3OVA tumor–bearing mice were treatedwith vehicle, BYL719 (10mg/kg), LEE011 (40mg/kg), or the combination for 7 days. Tumorswere then harvested, and the tumor infiltrating CD8þ T cells (B), CD4þ Tcells (C), andmMDSCs (D) were analyzed using flow cytometry.N¼ 5 to 11 mice per treatment group. Data depicted aremean� SEM. � , P < 0.05; �� , P < 0.01; �� , P <0.001; and �� , P < 0.0001 by two-way ANOVA (A) or one-way ANOVA (B–D).

Figure 4.Combined BYL719 and LEE011 treatment upregulates immune response gene expression and increases tumor immunogenicity in TNBC. A and B,MDA-MB-231 cellswere treated in vitro for 24 hourswith vehicle, 1mmol/L BYL719, 1mmol/L LEE011, or the combination, afterwhich, total RNAwas extracted and analyzed via 3'RNAseq.A, Top five upregulated enriched GSEA Hallmarks with highest positive normalized enrichment score (NES) with smallest FDR P value among significantly enrichedgene sets with FDR < 0.25. ES, enrichment score; NP, nominal P value. B,Heatmap of core-enriched genes in the top enriched upregulated Hallmark immune-relatedgene sets. Genes that were significantly differentially expressed (P < 0.05) in combination compared with vehicle treatments are shown. Gene names that arehighlighted in yellow encode proteins involved with antigen presentation. C, HCC1806 and MDA-MB-231 cell lines were treated with vehicle, 1 mmol/L of BYL719, 1mmol/L LEE011, or the combination for 72 hours and analyzed using flow cytometry for tumor cell-surface expression of human leukocyte antigens (HLA-ABC andHLA-DR), CD86, and PD-L1. Data depicted are themean� SEM. � , P <0.05; �� ,P <0.01; �� , P <0.001; and �� ,P <0.0001 by one-wayANOVA.D,HCC1806 andMDA-MB-231 cell lines were treated with vehicle, 1 mmol/L of BYL719, 1 mmol/L LEE011, or the combination for 72 hours and analyzed using flow cytometry for cell-surfacecalreticulin (CRT) expression. Data depicted are the mean � SEM. �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001 by one-way ANOVA.






inhibition (50, 51). This effect could conceivably contribute toour novel observation of decreased MDSC frequency withPI3Ka and CDK4/6 inhibition. The functional effect of com-bined therapy on regulatory cell types such MDSCs and Tregswarrants further investigation in future studies.

Taken together, our work suggests that dual inhibition ofPI3Ka and CDK4/6 acts in concert to promote synergisticdisease control, apoptosis, and antitumor immunity. Impor-tantly, further combination with immune-checkpoint blockadesignificantly increased tumor immunogenicity and T-cell cyto-toxicity, resulting in effective and durable (>1 year) tumorregression with significantly better overall survival comparedwith all other treatment groups.

In summary, our findings suggest that combined PI3Ka andCDK4/6 inhibition is synergistic and immunogenic against adiverse range of PIK3CA- and RB1-wild-type preclinical TNBCmodels. We also conclude that antitumor efficacy can be furtherenhanced with the addition of immune-checkpoint blockade.Our results have identified a novel approach that warrants eval-

uation in clinical trials for patients in critical need of new effectivetherapies.

Disclosure of Potential Conflicts of InterestS. Loi has received funding to her institution from Novartis and Pfizer.

No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: Z.L. Teo, W.A. Phillips, P.K. Darcy, S. LoiDevelopment of methodology: Z.L. Teo, B. Virassamy, S. LoiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z.L. Teo, S. Versaci, S. Dushyanthen, C.P. Mintoff,B. Virassamy, S. LoiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z.L. Teo, S. Dushyanthen, F. Caramia, P. Savas,M. Zethoven, B. Virassamy, G.A. McArthur, P.K. Darcy, S. LoiWriting, review, and/or revision of the manuscript: Z.L. Teo, P. Savas,M. Zethoven, S.J. Luen, G.A. McArthur, W.A. Phillips, P.K. Darcy, S. LoiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z.L. Teo, F. Caramia, B. Virassamy, S. LoiStudy supervision: P.K. Darcy, S. Loi

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Figure 6.

Immune-checkpoint blockade in combination with BYL719 and LEE011 treatment induces effective, long-lasting tumor regression in vivo. A, Tumor growth andsurvival of AT3OVA. Tumor-bearing mice were treated with indicated drug combinations with the following doses: BYL719 (10 mg/kg), LEE011 (40 mg/kg),anti–PD-1 (200 mg), and anti–CTLA-4 (150 mg). There were 6 mice in BYL719þ LEE011þ anti–PD-1 and BYL719þ LEE011þ anti–CTLA-4 groups. There were 10 to 14mice in all other groups. All treatments ceased by day 50, and tumor growth data are depicted in Fig. 5A. Data depicted are the mean tumor volume � SEM.B and C, AT3OVA tumor–bearing mice were treated with the indicated drug combinations, with doses as in A. Treatment was for 7 days in vivo. Tumors were thenharvested.B, Expression of tumor immunogenicitymarkers, major histocompatibility complex class I (MHCI; H2KB), MHCII and PDL-1 (C), and granzymeBexpressionby tumor-infiltrating CD8þ T and CD4þ T cells was analyzed using flow cytometry. Combination: treatment with BYL719, LEE011, anti–PD-1, and anti–CTLA-4.N ¼ 5 to 11 mice per treatment group. Data depicted are mean � SEM. � , P < 0.05; ��, P < 0.01; �� , P < 0.001; and �� , P < 0.0001 by two-way ANOVA (A) orKaplan–Meier log rank test, or one-way ANOVA (B and C).

Teo et al.





AcknowledgmentsThe authors wish to acknowledge staff from the Peter MacCallum Cancer

Centre Animal Facility, FACS Facility, andMolecular Genomics Core Facility fortheir assistance.

Grant SupportThis work was supported by a National Health and Medical Research

Council (NHMRC) of Australia Project Grant (1123208; S. Loi, P.K. Darcy,W.A. Phillips). Z.L. Teo was supported by a NHMRC Early Career Fellowship(1106967). P.K. Darcy was supported by a NHMRC Senior Research Fel-

lowship (1041828). S. Loi was supported by the Cancer Council VictoriaAustralia John Colebatch Fellowship as well as the Breast Cancer ResearchFoundation NY. S. Loi has received funding to her institution from Novartisand Pfizer.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 1, 2017; revised August 21, 2017; accepted September 12,2017; published OnlineFirst September 25, 2017.

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Teo et al.




2017;77:6340-6352. Published OnlineFirst September 25, 2017.Cancer Res Zhi Ling Teo, Stephanie Versaci, Sathana Dushyanthen, et al. Immunogenic in Triple-Negative Breast Cancer

Inhibition Is Synergistic andαCombined CDK4/6 and PI3K

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