ARTICLE Received 1 Mar 2017 | Accepted 12 May 2017 | Published 27 Jun 2017 DOI: 10.1038/ncomms15916 OPEN CDK4/6 and autophagy inhibitors synergistically induce senescence in Rb positive cytoplasmic cyclin E negative cancers Smruthi Vijayaraghavan1,2, Cansu Karakas1, Iman Doostan1,2, Xian Chen1, Tuyen Bui1, Min Yi3, Akshara S. Raghavendra4, Yang Zhao5, Sami I. Bashour4, Nuhad K. Ibrahim4, Meghan Karuturi4, Jing Wang5, Jeffrey D. Winkler6, Ravi K. Amaravadi7, Kelly K. Hunt3, Debu Tripathy4 & Khandan Keyomarsi1,2 Deregulation of the cell cycle machinery is a hallmark of cancer. While CDK4/6 inhibitors are FDA approved (palbociclib) for treating advanced estrogen receptor-positive breast cancer, two major clinical challenges remain: (i) adverse events leading to therapy discontinuation and (ii) lack of reliable biomarkers. Here we report that breast cancer cells activate autophagy in response to palbociclib, and that the combination of autophagy and CDK4/6 inhibitors induces irreversible growth inhibition and senescence in vitro, and diminishes growth of cell line and patient-derived xenograft tumours in vivo. Furthermore, intact G1/S transition (Rb-positive and low-molecular-weight isoform of cyclin E (cytoplasmic)-negative) is a reli- able prognostic biomarker in ER positive breast cancer patients, and predictive of preclinical sensitivity to this drug combination. Inhibition of CDK4/6 and autophagy is also synergistic in other solid cancers with an intact G1/S checkpoint, providing a novel and promising bio- marker-driven combination therapeutic strategy to treat breast and other solid tumours. 1 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, 6565 MD Anderson Boulevard, Houston, Texas 77030, USA. 2 Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, 6767 Bertner Avenue, Houston, Texas 77030, USA. 3 Department of Breast Surgical Oncology, The University of Texas MD Anderson Cancer Center, 1400 Pressler Street, Houston, Texas 77030, USA. 4 Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, 1155 Pressler Street, Houston, Texas 77030, USA. 5 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. 6 Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 7 Division of Hematology Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. Correspondence and requests for materials shouldbe addressed to K.K. (email: [email protected]). NATURE COMMUNICATIONS | 8:15916 | DOI: 10.1038/ncomms15916 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15916 eregulation of the cell cycle checkpoint proteins, such as inhibited growth of ER þ cells in a time- and dose-dependent cyclin-dependent kinases CDK4 and CDK6, is a key manner, MCF10A was more resistant to this drug (Fig. 1a,b and Dhallmark of cancer, resulting in uncontrolled cellular Supplementary Fig. 2a–c). To examine their ability to recover proliferation and tumorigenesis. Selective CDK4/6 inhibitors, from palbociclib-mediated growth inhibition, cells were treated palbociclib, ribociclib and abemaciclib, have shown promising for 6 days and cultured in the absence of drug for 4 days preclinical and clinical activities in numerous solid tumours1. (Supplementary Fig. 2d). Treatment with palbociclib at doses In particular, palbociclib (PD-0332991 or Ibrance) has shown of 1 mM or less resulted in reversible G1 arrest and growth clear benefits in Phase II (PALOMA-1) and III (PALOMA-2 and -3) inhibition, while only higher doses (42.5 mM) resulted in clinical trials in advanced estrogen receptor-positive (ER þ ) irreversible inhibition of growth and cell cycle progression in the breast cancers, doubling the progression-free survival (PFS) ER þ cells but not in MCF10A (Fig. 1c and Supplementary compared to letrozole or fulvestrant alone2–4. Palbociclib was Fig. 2e–h). Palbociclib did not trigger apoptosis (Supplementary recently approved by the U.S. Food and Drug Administration for Fig. 3a–d), but significantly increased senescence-associated both these indications and is currently being evaluated clinically -galactosidase (SA- gal) activity and cellular complexity in a in other solid tumours5. Moreover, numerous Phase III clinical dose-dependent manner, both characteristics of cells undergoing trials are currently underway with ribociclib (LEE011) and senescence (Fig. 1d and Supplementary Fig. 3e,f). Further, abemaciclib (LY2835219) in breast and other solid tumours5. western blot analysis showed a dose-dependent decrease in Despite these promising clinical advances with CDK4/6 known palbociclib effectors, pRb, FOXM1 (ref. 8) and total Rb inhibitors, there are three major limitations for this treatment: levels, a consequence of G1 arrest, as previously reported in breast (i) adverse events (Grade 3&4 neutropenia (56%) and leukopenia cancer15,16 (Supplementary Fig. 3g). To confirm the specificity of (25.2%)), which lead to interruption and/or discontinuation of palbociclib-mediated growth inhibition and examine potential treatment, possibly attenuating therapeutic benefit2, (ii) B16% off-target effects, MCF7 and T47D cells with shRNA- or (ref. 3) of the ER þ cancer patients do not respond to palbociclib siRNA-mediated CDK4 and/or CDK6 knockdown were treated or exhibit progression within 24 weeks, and half the patients with palbociclib. Combined knockdown of CDK4 and CDK6 was develop clinical resistance with progression within 25 months, able to recapitulate the drug effect at 1 mM but not 5 mM, resulting in no overall survival benefit and (iii) lack of reliable indicating that the irreversible grown inhibition and high levels of biomarkers that can be used as prognostic indicators for senescence observed at 5 mM could be due to off-target effects at advanced ER þ breast cancer. Clinical investigation of higher palbociclib concentrations (Fig. 1e and Supplementary prognostic biomarkers to-date, have failed to show a correlation Fig. 3h). to PFS in patients treated with palbociclib2,6. A stress response signal mediated by cell cycle arrest is Mechanistically, palbociclib induces G1 arrest and cellular autophagy, a catabolic process that can degrade ROS and mediate senescence in vitro and inhibits growth of tumour xenografts reversal of G1 arrest and senescence12,14,17. Hence, it was in vivo7,8. Palbociclib also induces autophagy in fibroblasts and hypothesized that CDK4/6 inhibition-mediated cell cycle arrest leukemic cells9,10. Autophagy is a stress tolerance mechanism in (on-target effect at 1 mM palbociclib) triggers autophagy as a cancer that recycles cellular constituents by engulfing them into a stress response, which prevents the induction of senescence at double-membrane vesicle called autophagosome, which these doses. To test this hypothesis, we assessed autophagy by: eventually fuses with lysosomes to facilitate degradation of the (i) monodansylcadavarine (MDC) staining18, (ii) transmission cellular constituents and generation of energy for survival11. electron microscopy (TEM)19, (iii) GFP-LC3 puncta, (iv) western Autophagy mediates resistance to numerous cancer-targeting blot analysis of LC3B-II (autophagosomal surface protein20) and agents12,13, making it a promising co-target and leading to the p62 (SQSTM1, an autophagic substrate21) and (v) reverse-phase recent development of numerous clinical trials with autophagy protein array (RPPA) analysis of key autophagy proteins. siRNA inhibitors, chloroquine (CQ) and hydroxychloroquine (HCQ)14. against CDK4/6 or palbociclib treatment (low doses) of MCF7 In this study we address the aforementioned limitations of and T47D cells significantly increased levels of MDC, LC3B-II palbociclib therapy by interrogating the mechanisms by which the and other key autophagy proteins (Atg-7, Beclin-1, BNIP3), while drug’s action can be enhanced in breast cancer. Our studies decreasing BCl2 (a known inhibitor of autophagy22) and p62 at suggest that the selectivity and efficacy of palbociclib is improved low doses (Fig. 1f,g and Supplementary Fig. 4a–d). Further, through a biomarker-driven combination treatment approach TEM and GFP-LC3 assays showed significant accumulation of that targets CDK4/6 and autophagy in breast and other solid double-membrane electron-dense autophagosomes and GFP-LC3 tumours. puncta, respectively, in Palbociclib (1 mM)-treated cells (Fig. 1h,j and Supplementary Figs 4e, 5d). We next examined the presence of an intact autophagic flux following palbociclib treatment by: (i) Results flux ratio (LC3B-II to LC3B-I and LC3B-II to p62), (ii) treatment CDK4/6 inhibition induces ROS-mediated senescence and with lysosomal block CQ23, (iii) GFP-LC3 puncta with CQ autophagy. Analysis of The Cancer Genome Atlas’s (TCGA) treatment and (iv) RFP-GFP-LC3 dual-reporter assay24. ER þ breast tumour cohort revealed alterations in the Treatment with low-dose (1 mM) palbociclib, exhibited higher CDK4/CDK6/cyclin D pathway in about 35% of the patients, flux ratios (Supplementary Fig. 5a), elevated MDC and LC3B-II making them an ideal population for targeting CDK4 and CDK6 levels with CQ treatment (Fig. 1i and Supplementary Fig. 5b,c), (Supplementary Fig. 1a). To interrogate the biological effect of significantly higher GFP-LC3 puncta with CQ treatment (Fig. 1j downregulating these proteins, we knocked down