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 Ó2020 American Association for Cancer Research. apoptosis (13, 14). AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst April 3, 2020; DOI: 10.1158/1078-0432.CCR-19-1872 Whittle et al. 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. À À 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