Stromal Senescence by Prolonged CDK4/6 Inhibition Potentiates Tumor Growth Xiangnan Guan1, Kyle M

Published OnlineFirst December 30, 2016; DOI: 10.1158/1541-7786.MCR-16-0319

Cell Cycle and Senescence Molecular Cancer Research Stromal Senescence By Prolonged CDK4/6 Inhibition Potentiates Tumor Growth Xiangnan Guan1, Kyle M. LaPak1,2, Rebecca C. Hennessey2, Christina Y.Yu2,3, Reena Shakya4, Jianying Zhang3, and Christin E. Burd1,2,4

Abstract

Senescent cells within the tumor microenvironment (TME) blasts, melanoma cell lines exhibited genotype-dependent pro- adopt a proinflammatory, senescence-associated secretory phe- liferative responses. However, in vivo, PD-0332991–treated notype (SASP) that promotes cancer initiation, progression, fibroblasts enhanced the growth of all melanoma lines tested and therapeutic resistance. Here, exposure to palbociclib (PD- and promoted the recruitment of Gr-1–positive immune cells. 0332991), a CDK4/6 inhibitor, induces senescence and a These data indicate that prolonged CDK4/6 inhibitor treatment robust SASP in normal fibroblasts. Senescence caused by pro- causes normal fibroblasts to enter senescence and adopt a longed CDK4/6 inhibition is DNA damage–independent and robust SASP. Such senescent cells suppress the antitumor associated with Mdm2 downregulation, whereas the SASP immune response and promote melanoma growth in immu- elicited by these cells is largely reliant upon NF-kB activation. nocompetent, in vivo models. Based upon these observations, it was hypothesized that the exposure of nontransformed stromal cells to PD-0332991 Implications: The ability of prolonged CDK4/6 inhibitor treat- would promote tumor growth. Ongoing clinical trials of ment to induce cellular senescence and a robust SASP in primary CDK4/6 inhibitors in melanoma prompted a validation of this cells may hinder therapeutic efficacy and promote long-term, hypothesis using a suite of genetically defined melanoma cells gerontogenic consequences that should be considered in clin- (i.e., Ras mutant, Braf mutant, and Ras/Braf wild-type). When ical trials aiming to treat melanoma and other cancer types. cultured in the presence of CDK4/6i-induced senescent fibro- Mol Cancer Res; 15(3); 237–49. 2016 AACR.

Introduction fore, senescent cells are not phenotypic equivalents and likely to contribute to distinct biological outcomes in vivo. Cellular senescence is a process in which cells with the potential Emerging data reveal the presence of senescent cells within the to divide permanently exit the cell cycle, but remain viable and tumor microenvironment. For example, using a genetically mod- metabolically active. Senescent cells are characterized by numer- ified mouse model wherein activation of the senescence biomark- ous cellular phenotypes, including insensitivity to mitogenic er, p16INK4a, induces firefly luciferase expression, the accumula- stimuli, flattened morphology, increased senescence-associated tion of senescent stromal cells can be visualized both in auto- ß-galactosidase activity (SA-ß-gal), shortened telomeres, elevated chthonous tumor transplants and spontaneous neoplasms (4). cyclin-dependent kinase inhibitor expression, changes in chro- In humans, p16INK4a-positive stromal cells accumulate around matin structure, pervasive DNA damage foci, and resistance to large-cell lung carcinomas and ductal carcinoma in situ lesions apoptosis and activation of the proinflammatory senescence- of the breast and pancreas (5, 6). Of note, stromal p16INK4a associated secretory phenotype (SASP; refs. 1, 2). Notably, not expression in breast cancer is more predictive of disease recurrence every senescent cell exhibits all of these characteristics. Instead, the than HER2, PR, or ER status (7), suggesting that senescent stromal triggering event (e.g., oncogene activation, telomere attrition, cells are indicative of poor prognosis. prolonged CDKi expression, or DNA damage) and originating Numerous in vitro coculture studies indicate that the SASP of cell type appear to dictate which phenotypes ensue (2, 3). There- senescent stromal cells influences cancer initiation, progression, and therapeutic response; however, few studies extend 1Department of Molecular Genetics, The Ohio State University, Columbus, Ohio. these observations to in vivo models (8–11). Of the publica- 2Department of Cancer Biology and Genetics, The Ohio State University, Columbus, tions that do address how senescent stromal cells influence Ohio. 3Department of Biomedical Informatics, The Ohio State University, Columbus, 4 tumor growth in vivo, only one has been conducted in an Ohio. The Ohio State University Comprehensive Cancer Center - Arthur G. James immunoproficient mouse model (12). Consequently, mechan- Cancer Hospital and Richard J. Solove Research Institute, Columbus, Ohio. isms by which paracrine SASP signals emanating from the Note: Supplementary data for this article are available at Molecular Cancer senescent tumor stroma might alter malignant cell clearance Research Online (http://mcr.aacrjournals.org/). by the immune system are understudied, a particularly perti- Corresponding Author: Christin E. Burd, The Ohio State University, Biomedical nent point given increasing interest in combining established Research Tower, Rm 586, 460 W. 12th Avenue, Columbus, OH 43210. Phone: 614- treatment modalities (i.e., chemotherapy, radiotherapy, and 688-7569; Fax: 614-292-6356; E-mail: [email protected] molecularly targeted therapies) with immunotherapy (13). doi: 10.1158/1541-7786.MCR-16-0319 Senescence induced by pharmaceutical inhibitors of CDK4/6 2016 American Association for Cancer Research. is of growing biological and clinical relevance. Developed to

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combat frequent inactivation of the CDK/Cyclin-RB signaling sequence inserted between the BamHI and NotI restriction sites. axis in human cancer, these compounds induce a permanent GFP from pEGFP-N3 (Clontech) was inserted into the resulting cell-cycle arrest in many tumor-derived cell lines. CDK4/6 multiple cloning sequence. Transduced tumor cells were selected inhibitors (e.g., LY2835219, LEE011, G1T28, and P1446A- with 3 mg/mL puromycin. 05) are under clinical investigation for a variety of tumor types, Cell lines were tested for mycoplasma using Mycoplasma Plus including melanoma. Given early results suggesting that CDK4/ PCR Primers (Agilent Technologies) and identity-verified at mul- 6 inhibitors enhance the efficacy of other targeted melanoma tiple time points during the study. Identity verification was con- therapies (14–16), it is likely these drugs will be soon approved ducted by PCR for all alleles unique to the study cell lines (e.g., for use in patients with metastatic melanoma. One potential alterations in Stk11, Ink4a/Arf, NRas, and Braf). In addition, concern surrounding the use of these drugs stems from recent BrafV600E and NRasQ61R mutations were sequence verified using in vitro data showing that extended exposure to PD-0332991 PCR products generated from genomic DNA. can trigger cellular senescence in normal fibroblasts (17). Given the known tumor-promoting effects of the SASP (2) as well Senescence induction as the contribution of senescent cells to biological aging (1), it To generate senescent MEFs, fibroblasts cryopreserved two days is logical to examine the effects of these drugs on normal after isolation were thawed, grown in culture for 48 hours, and tissues. However, no study to date has extensively characterized then plated at a density of 4 105 cells per 10-cm plate. Two days the phenotype of CDK4/6 inhibitor–induced senescence in later, cells were treated to induce senescence. For UV-induced normal fibroblasts or determined the effect of these stromal senescence, MEFs were irradiated with two doses of 3 mJ/cm2 UV cells on tumor growth. administered 48 hours apart using a Stratalinker 1800 (Strata- Here, we set out to determine how stromal senescence gene). MEFs were allowed to recover for 48 hours under normal induced by prolonged PD-0332991 treatment influences mel- growth conditions prior to any experimental assessments. For anoma cell proliferation both in vitro and in vivo.Tothisend,we mitomycin C–induced senescence, MEFs were exposed to 10 mg/ extensively compared the phenotypes of fibroblasts triggered to mL mitomycin C (Abcam) for 2.5 hours and then cultured in enter senescence via PD-0332991 treatment to those triggered to growth media for 4 days to establish senescence. For CDK4/6 enter senescence by other, melanoma-relevant signals (i.e., UV inhibitor–induced senescence, MEFs were treated with 4 mmol/L irradiation and DNA-damaging chemotherapy). Then, employ- PD-0332991 (Sigma, 827022-33-3) for 8 days, adding new drug ing a panel of syngeneic murine melanoma cell lines represent- and media on day 4. During in vitro assays, PD-0332991–treated ing major genetic subtypes of human melanoma [BRAF mutant cells were trypisinized, washed with PBS, and then plated in (40%–60% of melanomas), NRAS mutant (15%–30% of mel- normal growth media for at least 24 hours before the start of anomas), and NRAS/BRAF wild-type (<20% of melanomas)], any experiments. Prior to in vivo injections, PD-0332991–treated the ability of these senescent fibroblasts to influence cancer cell cells were trypsinized and thoroughly washed with PBS to remove proliferation was assessed both in vitro and in an immunocom- any residual drug. petent murine model. Our results reveal that CDK4/6 inhibitor– To test the role of NF-kB in establishing the SASP, retrovirus induced stromal senescence triggers a robust, DNA damage– encoding IkBa S32A/S36A (i.e., NF-kB super-repressor; ref. 21) or independent SASP and that these cells can foster the growth of control (empty pBabe-PURO vector) was generated in HEK293T melanoma in vivo via alterations in immune cell infiltration. cells using standard procedures. Next, passage 2 fibroblasts were These data provide insight relevant to the clinical implementa- transduced with virus and rapidly selected in 3 mg/mL puromycin tion of CDK4/6 inhibitors, suggesting that the drug efficacy for 72 hours. Following selection, cells were treated with either might be enhanced by protecting stromal cells from senescence. vehicle alone or 4 mmol/L PD-0332991 as described above. Cells Moreover, we propose that the ability of these drugs to drive were then split and seeded at subconfluence for SA-b-gal staining biological aging should be considered and monitored during or processed for real-time PCR analysis as described below (See clinical trials. Supplementary Table S1 for a list of validated primer sets).

Materials and Methods Direct and indirect coculture Cell lines and culture procedures For direct coculture assays, 3 104 passage 3 or 3.5 104 B16-F1 (CRL-6323) and B16-F10 (CRL-6475) mouse melano- senescent MEFs were seeded in 96-well plates (Thermo Scientific, ma cell lines were purchased from ATCC at the onset of this study. 165305). The next day, 1,500 (NL212, NL216, B16-F1, B16-F10 NL212 and NL216 cells were derived from TpLN61R/61R melano- and TRIA) or 2,000 (4434 and 21015) GFP-labeled, melanoma mas (18). The TRIA cell line was generated from a Tyr-HRas12V cells were seeded on top of the MEFs. Cultures were grown for 4 Ink4a/Arf / melanoma (19). Murine BrafV600E melanoma cell days, at which time the media were replaced with 50 mL PBS and lines 4434 and 21015 were kindly provided by Dr. R. Marais fluorescence determined on the Bio-Tek Synergy HT (excitation ¼ (Cancer Research UK, London, United Kingdom; ref. 20). Cells 485 nm; emission ¼ 528 nm). Fluorescent signal from empty were cultured in DMEM supplemented with 10% FBS, 1% pen- wells or wells with feeder cells alone was subtracted from exper- icillin–streptomycin, and 2 mmol/L L-glutamine. MEFs were imental values to correct for background. These values were then isolated from E13.5 mouse embryos as described in ref. 4. normalized to the average reading for wells containing only tumor To generate GFP-labeled NL212, NL216, TRIA, B16-F1, B16- cells. For statistical analyses, average values for biological repli- F10, 4434, and 21015 cultures, cells were transduced with pLenti- cates, measured in triplicate, were first log2 transformed. Linear puro-GFP lentivirus using 10 mg/mL polybrene. The pLenti-puro mixed modeling was then employed to account for the random vector is a derivative of pTRIPZ (Open Biosystems) in which block factor, experimental date, and to compare between treat- turboRFP and rtTA3 were removed and a multiple cloning ment groups.

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For indirect coculture assays, conditioned medium (CM) from Cytokine arrays either cocultures or feeder cells alone was collected 96 hours after To generate CM, MEF cultures were thoroughly washed and plating. Following centrifugation at 500 g for 5 minutes, 100 mL incubated in serum-free DMEM supplemented with 1% peni- of cleared, CM was added to tumor cells preseeded in 100 mL fresh cillin–streptomycin and 2 mmol/L L-glutamine. After 24 hours DMEM. After 4 days of culture, fluorescence was determined as of incubation, CM was collected and MEFs counted on a hemo- described above. cytometer. CM was centrifuged to remove any residual cells and then analyzed using Proteome Profiler Mouse XL Cytokine SA-b-gal staining Arrays (R&D Systems; catalog no. ARY028). Array signals were MEFs were seeded on coverslips and SA-b-gal activity measured visualized on an Odyssey CLX system (LI-COR) using IRDye using the Senescent b-galactosidase Staining Kit (Cell Signaling 800CW Streptavidin (1:2000; LI-COR, catalog no. 926-32230) Technology). NIH ImageJ was used to count total and SA-b-gal– secondary antibody. Signal intensity from each spot was deter- positive cell numbers. At least three independent views from two mined using Image Studio Software Ver5.2 (LI-COR) and cor- biological replicates were quantified for each treatment group. rected for background by subtracting the local median (intensity of grid border pixels for each spot). For each cytokine on the array, the average signal (pixel density) from duplicate spots on Cell-cycle analysis the membrane was first calculated. Next, the average signal from MEFs were seeded at subconfluence in normal growth media negative control spots on the array was subtracted. Finally, the and 10 mmol/L Edu added for 24 hours prior to analysis. EdU corrected signal intensity for each cytokine was normalized to incorporation was measured using the Click-iT Plus EdU Alexa cell number. The resulting data is expressed as fold change of Fluor 488 Flow Cytometry Assay Kit (Life Technologies). Cells CDK4/6i over presenescent (p4) cells. were analyzed on a FlowSight cytometer (Amnis) with non-EdU– labeled cells serving as the control. Immunoblotting MEF lysates were run on a 12% SDS-PAGE gel, transferred, and fl Immuno uorescence immunoblotted for p65 (1:1,000; Santa Cruz Biotechnology sc- fi Cells grown on glass coverslips were xed in 4% formaldehyde 109), p-p65 (1:1,000; Abcam, ab-86299), p38 (1:1,000; Santa for 15 minutes and then permeabilized with 0.2% Triton X-100 Cruz Biotechnology sc-535), p-p38 (1:1,000; Cell Signaling Tech- for 10 minutes. Coverslips were blocked in 5% BSA and incubated nology #4511), p21 (1:1,000; Santa Cruz Biotechnology sc-471), overnight with anti-gH2AX (1:400; Cell Signaling Technology) or Mdm2 (1:750; Santa Cruz Biotechnology sc-965), p53 (1:1,000; anti-53BP1 (1:1,000; Bethyl Laboratories) antibody. Primary Leica Biosystems CM5), or b-actin (1:10,000; Cell Signaling antibody was visualized using Alexa Fluor-555 secondary anti- Technology #3700S). Blots of biological replicates were scanned bodies (1:500; Molecular Probes). Slides were mounted in Pro- on Odyssey CLX system and quantified using Image Studio long Gold Anti-fade reagent containing DAPI (Molecular Probes). Software Ver5.2 (LI-COR). Cells with >3 gH2AX or 53BP1 foci per nucleus were considered positive and at least 100 cells were assessed per biological repli- Syngeneic tumorigenesis assays fi cate. Quanti cation was performed by two independent Animal experiments were performed in compliance with pro- reviewers, blinded to the slide information. tocols approved by the Ohio State University Institutional Animal Care and Use Committee (IACUC, protocol #2012A00000134). Real-time PCR analyses C57BL/6J mice (6–10 weeks old) were injected subcutaneously Total RNA was isolated from cells using the NucleoSpin RNA with 5 105 tumor cells plus either vehicle or 5 105 p4, UV, II kit (Macherey-Nagel Ltd.) and cDNA generated using the MMC, or CDK4/6i fibroblasts. Once established, tumors were Improm II Reverse Transcription Kit (Promega). To asses measured at least every other day by calipers until euthanasia p16INK4a levels, real-time PCR was performed in triplicate using was required. Tumor size was calculated as tumor width previously described TaqMan gene expression assays for (mm) length (mm). p16INK4a and 18S (4). p16INK4a levels were calculated using the DDCt method with 18S as the reference (4). To validate differ- Tumor IHC entially expressed genes identified in the TaqMan OpenArray Formalin-fixed, paraffin-embedded tumor sections were Mouse Inflammation Panel as well as 10 additional targets stained with anti-Gr-1 (1:200; BioLegend 108401), anti-F4/ selected from the literature (Bmp6,Bmp7,Ccl5,Ccl6,Ccl17, 80 (1:100; Thermo Scientific MF48000) or anti-FoxP3 (1:800; Ccl20, Cd40, Csf1, Csf3,andMmp3; refs. 8, 9), 100 ng of RNA Abcam ab-54501). Baked slides were dewaxed and rehydrated was reverse transcribed and SYBR-Green–based real-time PCR prior to antigen retrieval in a steamer for 40 minutes using analyses performed. Relative transcript levels for each target DAKO Antigen Retrieval Buffer (pH 6.10, catalog no. S1699). were calculated using the DDCt method with Gusb as the Primary antibody staining was detected using the DAKO Liquid reference. In the NF-kB super repressor experiments, 500 ng DAB and Substrate Chromogen System (#K3468), and all slides of RNA was reverse transcribed and SYBR-Green–based real- counterstained with hematoxylin. To remove the melanin pres- time PCR performed in triplicate to assess key targets (IL6, Ccl6, ent in B16-F10 tumors, slides were incubated in 10% H2O2 at Ccl8, Ccl11, c3, Cxcl5, Mmp3,andLbp). Relative transcript 60C for 90 minutes following DAB staining. Staining for CD3 levels for each target were calculated using the DDCt method was conducted on a Leica Bond autostainer using anti-CD3 with Gusb as the reference. All SYBR-Green–based assays used (1:100; Abcam ab-16669) antibody and the Bond Polymer SensiFast SYBR (Bioline). Primer sets were validated by gel Refine Red Detection System (Leica). For all immunohisto- electrophoresis of real-time PCR products and are listed in chemical stains, five distinct fields per tumor were acquired Supplementary Table S1. on Vectra Imaging System (Perkin Elmer) and the percent

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positivity determined using Inform2.1 software (Caliper Life platform. This approach revealed distinctions among the inflam- Sciences). matory gene expression profiles of all four fibroblast lines (p4, CDK4/6i, UV, and MMC; Supplementary Dataset S1). Of particular interest, CDK4/6i cells, with little evidence of DNA Results damage (Table 1; Fig. 1D and E), exhibited a more robust proin- PD-0332991–triggered senescence exhibits a robust SASP flammatory gene expression profile than fibroblasts triggered to without DNA damage enter senescence via UV or MMC (Supplementary Dataset S1). Prolonged PD-0332991 exposure is reported to induce cellular Compared with presenescent (p4) fibroblasts, 115 proinflam- senescence in normal fibroblasts, yet the phenotypic character- matory mRNAs were upregulated 2.5-fold in CDK4/6i MEFs, istics of this form of senescence are largely unknown (17). To whereas only 89 and 62, respectively, were induced in UV- and address this gap in knowledge, we comprehensively characterized MMC-treated cells. the phenotypes of fibroblasts triggered to enter senescence by We used real-time PCR to validate 69 hits from the Open- long-term PD-0332991 exposure and compared these results Array platform that differed by 2.5-fold in CDK4/6i cells with those of other, well-characterized forms of senescence ini- compared with UV- and MMC-treated fibroblasts. Simulta- tiated by either environmental DNA damaging or chemothera- neously, we assessed the expression of 10 additional senes- peutic agents (i.e., mitomycin C, UV light). To establish senescence-associated mRNAs reported in the literature (See Materi- cence, presenescent MEFs (passage 4/p4) were treated with either als and Methods). In three biological replicates, we confirmed PD-0332991 (4 mmol/L for 8 days), UV irradiation (3 mJ/cm2 that 51 of 69 targets identified by the OpenArray platform were twice at a 48-hour interval), or mitomycin C (10 mg/mL for distinctly altered in PD-0332991–induced senescence (33/47 2.5 hours). Herein, these treated fibroblast populations are up- and 18/22 downregulated 2-fold in comparison with referred to as CDK4/6i, UV, and MMC cells, respectively. Prior UV- and MMC-treated cells; Fig. 2A; Supplementary Dataset to any in vitro assessments, cells were removed from treatment and S2).Ofthe69mRNAsassessed,39wereexpressedathigher replated in normal growth media for at least 24 hours. levels in CDK4/6i fibroblasts than in proliferating MEFs, where- To confirm that these fibroblast cultures were no longer as 10 were lower in CDK4/6i cells (Supplementary Dataset S2). responsive to mitogenic stimuli, cells were seeded at subcon- Fewer transcripts selected from the literature validated in our fluence in normal growth medium and, one day later, incu- assays—only4of10transcriptsweregreaterinCDK4/6i bated with the thymidine analogue, EdU, for a period of 24 cells than in normal, proliferating MEFs, and 2 of these targets hours. Using flow cytometry to detect EdU incorporation, over were similarly upregulated in UV- and MMC-treated cells (Sup- 76% of p4 cells were positive (Table 1; Fig. 1A). In contrast, less plementary Dataset S2). Together, these data validate that pro- than 10% of CDK4/6i, UV, and MMC cells incorporated EdU longed PD-0332991 treatment leads to the robust induction of during the labeling period. Next, we measured SA-b-gal posi- a variety of proinflammatory genes. tivity in these cells, revealing that 11% of p4 cells, 74% CDK4/ To determine whether changes in proinflammatory gene 6i–treated cells, 58% of UV-treated cells, and 72% of MMC- transcription alter the secretome of CDK4/6i fibroblasts, we treated cells were SA-b-gal positive (Table 1; Fig. 1B). Marked employed a membrane-based antibody array. Using this accumulation of p16INK4a mRNA also occurred in the CDK4/6i-, approach, 111 cytokines were simultaneously measured in CM UV-, and MMC-treated cells, with respective increases of 4.4-, from p4 and CDK4/6i cells. On a per-cell basis, cytokine levels 5.4-, and 3.0-fold over presenescent (p4) fibroblasts (Table 1; were 1.1 to 17.8 times higher in CM from CDK4/6i MEFs than Fig. 1C). Consistent with publications suggesting that DNA from p4 MEFs (Supplementary Dataset S3). Cross-comparison damage is requisite for the formation of senescence-associated of 34 targets common to our mRNA and cytokine panels genomic lesions (22), UV- and MMC-treated cells showed revealed a strong correlation between mRNA and secreted pronounced foci containing 53BP1 (58% and 56%, respective- protein levels for most targets analyzed. However, several ly) and gH2AX (73% and 81%, respectively; Table 1; Fig. 1D targets showed transcriptional activation that was not paral- and E). CDK4/6i cells and p4 fibroblasts showed only basal leled by protein secretion (i.e., Eoxtaxin, Cxcl5, IL1a, Tnfsf13b, levels of DNA damage (i.e., 53BP1 and gH2AX foci) likely and PTX-3; Supplementary Dataset S3). Other targets were associated with replication (5.0% 53BP1 and 5.6% gH2AX not transcriptionally induced, but showed increased secretion positive; Table 1; Fig. 1D and E). in CDK4/6i fibroblasts compared with p4 controls (i.e., Vegf, To determine whether PD-0332991–induced fibroblast senes- Ccl5, Cd14; Supplementary Dataset S3). Thus, while post- cence triggers the SASP, we analyzed proinflammatory gene transcriptional mechanisms influence the production and expression in total MEF RNA using the TaqMan OpenArray export of some proinflammatory cytokines associated with

Table 1. Phenotypic distinctions amongst p4, UV, MMC, and CDK4/6i cells Culture condition Edu Labelinga %SA-b-galb p16INK4a mRNAc % 53BP1d % gH2AXd p4 76.77 5.55 11.08 1.53 1 0.09 6.59 2.08 4.59 0.67 UV 9.68 2.4 58.15 3.26 5.39 0.25 58.23 3.90 72.56 2.77 MMC 2.72 1.38 71.97 3.98 3.03 0.07 56.05 0.21 80.91 4.12 CDK4/6i 9.11 3.96 74.45 4.12 4.36 0.22 4.99 1.30 5.62 0.70 aAverage and SD from three independent assays are shown. bAverage and SD from at least three views in two biological replicates. cp16INK4a mRNA levels in UV, MMC, CDK4/6i cells were normalized to p4. Data indicate the average and SD of three biological replicates. dCells with >3 nuclear foci were scored as positive. Average and SD from three independent assays are shown. Bold values indicate CDK4/6i values that are statistically different from both UV and MMC MEFs (P < 0.001).

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Figure 1. Prolonged PD-0332991 treatment induces senescence in primary fibroblasts. A, Representative histograms of MEFs labeled with EdU for 24 hours in normal growth medium. The percentage of EdU-positive cells is indicated in each histogram, with quantification from three biological replicates shown in Table 1. B, Representative images of presenescent (p4), UV-, MMC-, and CDK4/6i-treated cells stained for SA-b-gal. Scale bar, 100 mm. Percent positivity from three biological replicates appears in Table 1. C, 18s normalized p16INK4a mRNA levels measured using a TaqMan-based strategy. Fold differences relative to p4 were calculated using the DDCt method. Data bars represent the mean of three biological replicates performed in triplicate. Error bars show the SD. D and E, Representative immunofluorescent staining for 53BP1 (C)orgH2AX (D) foci. Scale bar, 100 mm. Quantification of three biological replicates appears in Table 1.

PD-0332991–induced senescence, most proinflammatory longed PD-0332991 exposure promotes Mdm2 degradation, mRNAs induced in CDK4/6i cells are also elevated in the fibro- leading to increased p21Cip1/Waf1 stability, cell-cycle arrest, and blast secretome. Together, these data demonstrate that PD- subsequent senescence in normal fibroblast populations. 0332991–induced senescence activates a diverse and potent SASP. NF-kB activity promotes the SASP of CDK4/6i cells To determine how CDK4/6i fibroblasts elicit a SASP in the Loss of Mdm2 expression triggers CDK4/6i-induced senescence absence of DNA damage, we examined the p38 and NF-kB We searched for a DNA damage–independent mechanism pathways, each of which has been linked to SASP gene expres- to explain how CDK4/6 inhibitor treatment triggers senescence sion (24, 25). Although elevated in MMC-treated fibroblasts, in primary fibroblasts. Similar to a recent study by Kovatcheva p38 was not activated in senescent CDK4/6i cells (Supple- and colleagues in liposarcoma cells lines (23), we found that mentary Fig. S1). In contrast, activating phosphorylation of prolonged PD-0332991 treatment promoted a reduction in the NF-kB subunit, p65, was elevated in CDK4/6i, UV, Mdm2 and p53 protein levels (Fig. 2B). However, unlike what and MMC fibroblasts when compared with control (Fig. 3A). is observed in liposarcomas, p21Cip1/Waf1 protein levels were To further confirmtheroleofNF-kBindrivingtheSASPof elevated in CDK4/6i MEFs in comparison with p4 cells CDK4/6i fibroblasts, MEFs were transduced with retrovirus (Fig. 2B). Together, these data support a model wherein pro- encoding NF-kB super-repressor (SR, i.e., IkBa S32A/S36A) or

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Figure 2. Loss of Mdm2 induces senescence in CDK4/6i fibroblasts and is accompanied by a robust SASP. A, Heatmap depicting proinflammatory gene expression levels relative to actively proliferating fibroblasts (p4). Transcripts were measured by real- time PCR in three biological replicates and fold change values

log2-transformed. Blue hues indicate decreased gene expression relative to p4. Red hues represent genes with elevated expression compared with p4. B, Top, representative immunoblots of Mdm2, p53, p21Cip1/Waf1,andb-actin expression in ﬁbroblasts. Bottom, quantiﬁcation of Mdm2, p53, and p21Cip1/Waf1 signal intensity relative to b-actin. Bars represent the average fold change over p4 from three biological replicates. Error bars, SD. , P < 0.05; , P < 0.01 compared with p4.

a control (EV, empty vector). After rapid selection for stably CDK4/6i fibroblasts trigger genotype-dependent proliferative transduced clones, cells were subjected to PD-0332991 or responses in melanoma cocultures vehicle treatment as described above. As evidenced by SA-b-gal To investigate the potential effects of CDK4/6i fibroblasts on staining, CDK4/6 inhibitor–induced senescence was unaffected melanoma growth, we first utilized a 2D coculture system. Pre- by expression of the NF-kB super-repressor (Fig. 3B). In con- senescent (p4) or senescent (CDK4/6i, UV, or MMC) MEFs were trast, the induction of many SASP-associated transcripts (i.e., seeded into 96-well plates to produce confluent monolayers IL6, MMP3, Ccl6, Ccl8,andCcl11) in CDK4/6i MEFs was (Fig. 4A). One day after seeding, murine melanoma cell lines completely blocked by NF-kBinhibition(Fig.3C).Other stably expressing GFP were added to each well. Melanoma cell SASP-associated transcripts (C3, Cxcl5,andLbp) showed lines selected for this study are primarily syngeneic to C57Bl/6 reduced activation in both control and CDK4/6i fibroblasts and carry alterations in Ras (4, 18, 19), Braf (20), or neither of transduced with the NF-kB super-repressor. However, the mag- these genes (i.e., B16 cell lines). nitude by which NF-kB super-repressor decreased gene expres- While prior literature suggests that only senescent fibroblasts sion was significantly higher in fibroblasts treated with CDK4/6 stimulate the proliferation of tumor cells (10, 26, 27), we found inhibitor versus untreated controls (Fig. 3D). Notably, expres- that both senescent and presenescent fibroblasts influenced sion of our endogenous control gene, Gusb, did not change in melanoma cell growth in vitro (Fig. 4B–H). Interestingly, the cells harboring the NF-kB super-repressor (Fig. 3C). These data direction and magnitude of how fibroblast coculture changed provide strong evidence that NF-kBactivitypromotesthe melanoma cell proliferation was genotype-dependent. For induction of a wide variety of proinflammatory transcripts example, the BrafV600E cell lines 4434 and 21015 proliferated associated with the SASP of CDK4/6i fibroblasts. faster in the presence of any form of MEFs, whereas other

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Figure 3. NF-kB activity triggers the SASP in CDK4/6i fibroblasts. A, Top, representative immunoblots showing p-p65 and p65 expression in fibroblast lysates. Bottom, quantification of p-p65 a signal intensity relative to p65. Bars represent the average fold change over p4 from three biological replicates. Error bars, SD. , P < 0.05; , P < 0.01; , P < 0.001 compared with p4. B, Representative images of vehicle or PD-03329991–treated pBabe-NFkB-SR or pBabe-EV–transduced fibroblasts stained for SA-b-gal. Scale bar, 100 mm. C and D, Gusb normalized gene expression levels measured by real-time PCR. Fold differences relative to vehicle treated pBabe-EV cells were calculated using the DDCt method. Shown is one representative of two independent biological replicates. Data bars represent the mean of one biological experiment performed in triplicate. Error bars, SD. , P < 0.05; , P < 0.01; , P < 0.001. melanoma cells responded only to specific types of presenes- carrying a Ras mutation (TRIA, NL216, NL212; Fig. 5B–D; cent and/or senescent fibroblasts (Fig. 4B–H). N-Ras–mutant indicated by ''), Braf mutant (4434 and 21015) and wild-type cell lines (NL212, NL216) grew slower when plated on CDK4/ (B16-F1 and B16-F10) melanoma cell lines were nonresponsive 6i fibroblasts (Fig. 4C and D), and the proliferation of B16-F1 (Fig. 5E–H). In all cases, the in vitro effectsofCMfromCDK4/6i and B16-F10 cells only changed in response to presenescent MEFs paralleled those caused by media collected from p4, UV, and MMC-treated fibroblasts (Fig. 4G and H). Of note, in no or MMC fibroblasts (Fig. 5B–H). case did we find that CDK4/6i fibroblasts stimulated the Intercellular communication is hypothesized to augment growth of melanoma cells better than normal, proliferating paracrine SASP signals through the establishment of feed-for- MEFs (Fig. 4B–H). Together, these data indicate that the in vitro ward signaling loops (28). For this reason, we repeated our proliferative response of melanoma cells to CDK4/6i fibro- indirect coculture studies, transferring CM from our direct blasts is genotype-dependent and frequently parallels what is coculture setup instead of senescent fibroblasts alone (Supple- seen with other forms of senescent and/or presenescent feeders. mentary Fig. S2A). Paralleling our results in Fig. 5, all of the Ras- Senescent cells affect surrounding tissues and/or tumor cells mutant melanoma cell lines showed increased proliferation in via paracrine signaling. Such effects include the induction of the presence of CDK4/6i coculture CM (Supplementary neighboring cell senescence, disruption of epithelial organiza- Fig. S2B–S2D; indicated by ''); however, in contrast to CM tion, and promotion of premalignant epithelial cell growth collected from CDK4/6i fibroblasts alone, Braf-mutant cells (1, 3). To determine whether CDK4/6i CM was sufficient to proliferated in response to coculture CM (Supplementary Fig. alter melanoma cell proliferation in vitro,weperformedindirect S2E and S2F). This same media had no proliferative effect on coculture assays. Conditioned medium from p4, UV, MMC, the B16-F1 or B16-F10 cell lines (Supplementary Fig. S2G and and CDK4/6i MEFs was transferred onto melanoma cell cul- S2H). Together, these direct and indirect in vitro coculture tures and changes in proliferation assessed (Fig. 5A). While studies reveal assay-dependent genetic trends in how CDK4/ CDK4/6i CM promoted the growth of melanoma cell lines 6i fibroblasts influence melanoma cell proliferation. To discern

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Figure 4. CDK4/6i ﬁbroblasts exert genotype-dependent effects on melanoma growth in vitro. A, Procedure for direct 2D coculture experiments. B–H, GFP-labeled Ras mutant (TRIA, NL212, NL216), Braf mutant (4434 and 21015), and wild-type (B16-F1 and B16-F10) melanoma cells were cultured for 4 days on presenescent (p4) or senescent (UV, MMC, CDK4/6i) MEF monolayers. Relative cell numbers were determined by measuring GFP intensity. Individual dots represent the mean of a single biological replicate performed in triplicate. Error bars represent the SD from 4 independent experiments performed in triplicate. , Statistical comparison with no MEFs, P < 0.001; †, Statistical comparison with p4 cells, P < 0.001; D, Statistical comparison with CDK4/6i cells, P < 0.001.

which in vitro assay best represents the physiologic conse- Tumor cells and senescent fibroblasts were established in vitro quences of PD-0332991–induced stromal senescence, in vivo (See Materials and Methods), trypsinized, washed, and resus- experiments were performed. pended in PBS for subcutaneous injection. While NL212 (Nras mutant) cells efficiently formed melanomas in the absence of CKD4/6i fibroblasts promote melanoma growth in vivo coinjected fibroblasts, tumor growth was significantly augmented Prior publications rely predominantly upon xenograft mod- by the coinjection of either presenescent or CDK4/6i fibroblasts els to show that senescent fibroblasts, triggered by a variety of (Fig. 6A; tumor take rate 100% in all groups). Similarly, 4434 (Braf DNA-damaging stimuli, promote the growth of human and mutant) cells formed tumors and grew better when coinjected murine tumors in vivo (8, 10, 17, 27, 29, 30). However, as these with any type of fibroblasts, including CDK4/6i cells (Fig. 6B). In studies employ immunocompromised mice, they do not fully contrast, B16-F10 and B16-F1 (Nras and Braf wild-type) cells address the potential effects of senescent cells on immune formed tumors efficiently and grew rapidly in vivo, but were infiltration and tumor rejection. Here, we sought to determine unaffected by the coinjection of presenescent fibroblasts how CDK4/6i fibroblasts in the tumor stroma impact melano- (p4; Fig. 6C; Supplementary Fig. S3; tumor take rate 100% in all ma growth using an immunoproficient, syngeneic mouse mod- groups). B16-F10 and B16-F1 tumors did, however, show accel- el. Based upon the genotype-dependent proliferative responses erated growth when coinjected with senescent cells (including seen in our coculture assays, we selected four representative CDK4/6i fibroblasts; Fig. 6C; Supplementary Fig. S3; P < 0.01 melanoma lines for assessment (i.e., NL212, 4434, B16-F1, and comparing each MEF population to p4). Together, these data B16-F10). show that in contrast to our in vitro findings, all senescent

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Figure 5. The response of melanoma cells to ﬁbroblast CM is genotype dependent. A, Procedure for indirect coculture experiments. B–H, Melanoma cells were cultured for 4 days in fresh (FR) or CM from presenescent (p4) or senescent (UV, MMC, CDK4/6i) ﬁbroblasts, after which relative cell numbers were determined by measuring GFP intensity. Individual dots represent the mean of a single biological replicate performed in triplicate. Error bars represent the SD from 4 experiments performed in triplicate. , Statistical comparison with no MEFs, P < 0.001; †, Statistical comparison with p4 cells, P < 0.001; D, Statistical comparison with CDK4/6i cells, P < 0.001.

fibroblasts, including those established via prolonged CDK4/6i immune infiltrates that might explain the in vivo growth phe- treatment, can promote melanoma growth in vivo. However, notypes observed in each melanoma cell line. In tumors from whether CKD4/6i fibroblasts have stronger proliferative effects all three cell lines, the coinjection of CDK4/6i fibroblasts led than presenescent fibroblasts is dependent upon the melanoma to significant increases in Gr-1–positive immune cell infiltrates cell line assayed. as measured by IHC (Fig. 6D–F). In contrast, the number of Decades of studies have established that SASP components FoxP3 (T-regulatory cells) and F4/80 (macrophages) positive (i.e., IL6, IL8, CXCL-1, VEGF) promote the proliferation cells remained constant among tumors coinjected with prese- of tumor cells in vitro (8,9,11).However,astheSASPalso nescent (p4) or CDK4/6i fibroblasts (Supplementary Fig. S4). contains numerous proinflammatory factors capable of stim- Notably, total CD3-positive cells decreased in all CDK4/6i ulating immune influx into the tumor microenvironment (31, coinjected tumors, suggesting that the infiltrating Gr-1–positive 32), we sought to determine whether there were differences in cells are myeloid-derived suppressor cells (MDSC; Fig. 6G–I).

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Figure 6. CDK4/6i senescent fibroblasts recruit Gr-1–positive immune cells to promote syngeneic tumor growth. A–C, 5 105 NL212 (A), 4434 (B) or B16-F10 (C) melanoma cells were injected alone into C57BL/6 mice, or coinjected with 5 105 presenescent (p4) or senescent (UV, MMC, CDK4/6i) fibroblasts. Tumor growth was measured until exclusion criteria were met by any study group. Average tumor sizes, relative to the nadir, are plotted with error bars representing the SEM. For NL212 tumors, n ¼ 8 in the no MEFs and p4 groups and n ¼ 10 for UV, MMC and CDK4/6i groups. For 4434 and B16-F10 tumors, n ¼ 12 except for the 4434 no MEFs group where n ¼ 4 due to decreased tumor take. D–F, The percentage of tumor-associated cells that stain Gr-1 positive under each experimental condition is indicated. Five views per tumor were quantified,witheachdot representing an individual tumor. Error bars, SEM. , P < 0.05; , P < 0.01. G–I, The percentage of tumor-associated cells that stain Gr-1 positive under each experimental condition is indicated. Five views per tumor were quantified, with each dot representing an individual tumor. Error bars, SEM. , P < 0.05; , P < 0.01.

fi These ndings support a model in which stromal senescence Discussion induced by prolonged PD-0332991 exposure leads to MDSC accumulation and enhanced tumor growth. When MDSC num- It is well-established that components of the SASP (e.g., IL6, bers are already elevated, as seen in NL212 and 4434 cells IL8, CXCL-1, VEGF, MMP-3) drive tumor proliferation both coinjected with p4 fibroblasts, the additive effects of CDK4/6i in vitro and in xenograft assays (8–11). However, the recent cells on melanoma growth are negligible (Fig. 6). However, in discovery that primary cell cultures enter senescence following tumors that are less immunogenic (i.e., B16-F10 and B16-F1), sustained PD-0332991 exposure (17) prompted us to investi- increases in MDSC numbers suppress the immune system gate whether CDK4/6 inhibitor treatment can activate the sufficiently to promote an added growth advantage (Fig. 6; SASP in normal tissues. Here, we report that PD-0332991– Supplementary Fig. S3). induced fibroblast senescence is characterized by a robust, DNA

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damage-independent SASP (Table 1; Figs. 1B–Eand2A).While direct and indirect coculture techniques, which, as evidenced by in vitro assays reveal that these senescent fibroblasts stimulate our findings, often yield conflicting results (Figs. 4 and 5; Sup- the growth of melanoma cells in a genotype-dependent manner plementary Fig. S2). Nevertheless, we were intrigued by the in vitro (Figs. 4 and 5; Supplementary Fig. S2), this observation does observation that CDK4/6i fibroblasts alter melanoma cell prolif- not hold true in a more biologically relevant setting. Speci- eration in a genotype-dependent manner (Figs. 4 and 5; Supple- fically, melanoma cells lines grow better in C57Bl/6 mice when mentary Fig. S2). Upon moving these studies into a more phys- coinjected with CDK4/6i fibroblasts versus vehicle alone iologically relevant model, we found that none of our in vitro (Fig. 6A–C; Supplementary Fig. S3). CDK4/6i fibroblasts also systems could accurately model in vivo conditions. In fact, regard- promote the accumulation of Gr-1–positive MDSCs within the less of their mode of induction, senescent fibroblasts equally tumor microenvironment, thereby dampening the antitumor promoted the growth of four genetically distinct melanoma cell immune response. Together, these data suggest that the impact lines in immunocompetent, syngeneic mice (Fig. 6A–C; Supple- of CDK4/6 inhibitor therapy on normal cells should be con- mentary Fig. S3). This finding underscores the importance of sidered when treating cancer patients, as it may limit therapeu- studying the non-cell–autonomous effects of senescent cells on tic efficacy and promote senescent cell accumulation through- tumor growth in vivo rather than in vitro. out the body. Here, we report the first studies to characterize how different forms of senescent stromal cells influence melanoma growth in PD-0332991 triggers a robust SASP in the absence of DNA a syngeneic, immunocompetent murine model. Using this damage experimental approach, we were able to determine how the The SASP of senescent cells is often triggered by cell-intrinsic antitumor immune system is influenced by stromal senescence. DNA damage, an observation supported by the fact that senes- While all melanoma cell lines grew better in the presence of cent cells established by exogenous p16INK4a expression lack senescent fibroblasts than vehicle alone, we were surprised to the SASP (33). For this reason, we were surprised to find find that the growth of NL212 (NRas mutant) and 4434 (Braf that primary fibroblasts treated with the CDK4/6 inhibitor, mutant) tumors was also accelerated in the presence of pre- PD-0332991 (i.e., CDK4/6i fibroblasts), exhibit a robust SASP senescent MEFs (Fig. 6; Supplementary Fig. S3). Oswald and in the absence of DNA damage (Table 1; Figs. 1D and E and 2A; colleagues recently report a similar finding, observing that the Supplementary Dataset S1–S3). Prior studies link SASP gene coinjection of normal human fibroblasts and H1437 non– activation to transcriptional programs initiated by NF-kB, small cell lung cancer cells exacerbates tumorigenesis and CCAAT/Enhancer-Binding Protein Beta (C/EBPb), MacroH2A1, reduces the survival of experimental mice (38). While these mTOR, and GATA4 (34–36). Here, we find that CDK4/6i data might suggest that fibroblast factors independent of the fibroblasts activate NF-kB to the same extent as cells triggered SASP stimulate melanoma growth, analysis of infiltrating to enter senescence via UV or MMC treatment (Fig. 3A). immune cells suggests a different possibility. Furthermore, we show that inhibition of NF-kB signaling pre- We consistently see higher numbers of Gr-1–positive cells vents the induction of classic SASP genes (e.g., IL6, Mmp3, Ccl6) within the tumor microenvironment of melanomas coinjected without altering the ability of prolonged PD-0332991 treat- with CDK4/6i fibroblasts versus those injected with presenescent ment to establish senescence (Fig. 3B and C). These data suggest MEFs (Fig. 6D–F). These Gr-1–positive cell infiltrates are likely to that the CDK4/6 inhibitor–inducedSASPofsenescentstromal be MDSCs as evidenced by the decreased numbers of CD3- cells might be mitigated by dampening NF-kBactivity. positive T cells within these tumors. Unfortunately, fresh tissue Mechanisms by which CDK4/6 inhibitors block tumor cell was not available to confirm MDSC identity using a dual Gr-1, proliferation are well-studied, yet our understanding of how Cd11b staining protocol. In the context of an already elevated these drugs affect nontransformed cells is limited. CDK4/6i antitumor response like that observed in p4 coinjected 4434 and MEFs lack evidence of traditional senescence mechanisms (i.e., NL212 melanomas, the effect PD-0332991–induced stromal DNA damage and p38 activation; Table 1, Fig. 1D and E; senescence on melanoma growth appears negligible (Fig. 6A, B, and Supplementary Fig. S1), but show reductions in Mdm2 D, and E). Conversely, when Gr-1–positive cells are naturally low and p53, paralleling that of senescent, PD-0332991–treated within the tumor microenvironment, stromal senescence induced liposarcoma cell lines (Fig. 2B; ref. 23). Distinct from what by CDK4/6 inhibition promotes tumor growth in vivo (Fig. 6C and is seen in senescent liposarcoma cell lines, CDK4/6i MEFs F; Supplementary Fig. S3). This model is supported by multiple exhibit posttranscriptional increases in the CDK inhibitor, publications. For instance, a similar connection between stromal p21Cip1/Waf1 (Fig. 2B). As Mdm2 is known to destabilize senescence and MDSC recruitment has been shown to occur in the p21Cip1/Waf1 via an E3-ubiquitin ligase–independent mecha- setting of Pten-null prostate cancers (39). Here, the onset of nism (37), our findings suggest that PD-0332991 promotes senescence in murine prostate tumors promotes the accumulation fibroblast senescence through Mdm2 destabilization and sus- of Gr-1–positive cells, leading to reduced antitumor immunity tained p21Cip1/Waf1 expression. and enhanced cancer growth (39). In another recent publication, senescent fibroblasts established by exogenous p27Kip1 expres- CDK4/6i fibroblasts promote genotype-dependent sion are shown to enhance tumorigenesis in an immunocompe- melanoma growth tent, syngeneic mouse model (12). Mirroring our findings again, After observing that prolonged PD-0332991 treatment induces the mechanism by which these senescent fibroblasts promote senescence accompanied by a robust SASP in primary fibroblasts, tumor growth is attributed to infiltrating granulocytic MDSCs we wondered how stromal cells exposed to CDK4/6 inhibitors (12). Alongside these articles, our data strongly suggest that PD- might influence melanoma growth. Numerous publications 0332991–induced stromal senescence promotes the establish- examine the mechanisms by which senescent cells promote tumor ment of a tumor-permissive environment that could reduce the growth in vitro. Unfortunately, these studies rely largely upon overall efficacy of therapies targeting CDK4/6 activity.

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Implications for the therapeutic administration of CDK4/6 CDK4/6 inhibitors be augmented by preventing SASP activity inhibitors in melanoma and/or protecting normal cells from drug exposure?, and (ii) are After relatively few passages in culture, oxidative stress induces there long-term consequences of CDK4/6 inhibitor therapy that the senescence of primary fibroblasts (1). Thus, to ensure that the may decrease a patient's probability of responding to immune phenotype of CDK4/6i cells is PD-0332991–dependent, it was checkpoint inhibitors or promote age-related disease? It is our necessary to select a dose of CDK4/6 inhibitor that rapidly induces hope that this work will encourage ongoing and future clinical senescence. We examined a variety of PD-0332991 concentrations trials to assess the global consequences of CDK4/6 inhibition, to determine the lowest dose meeting this requirement (>90% of rather than focusing on the tumor alone. cells EdU negative; data not shown), and decided to move forward with a dose of 4 mmol/L. While this dose is approximately 10-fold Disclosure of Potential Conflicts of Interest higher than observed in the plasma of treated breast cancer No potential conflicts of interest were disclosed. patients (ClinicalTrials.gov; NCT00721409), it is unlikely that off-target effects contribute to our findings. Notably, the IC50 of PD-0332991 for other CDKs exceeds 10 mmol/L (40). In clinical Authors' Contributions trials, PD-0332991 has a long half-life (26.7 hours) and can Conception and design: X. Guan, C.E. Burd effectively depot in human peripheral tissues (41). Whether Development of methodology: X. Guan, C.E. Burd Acquisition of data (provided animals, acquired and managed patients, CDK4/6 inhibitors accumulate over multiple treatment cycles to provided facilities, etc.): X. Guan, K.M. LaPak, R.C. Hennessey, R. Shakya, promote senescence in nontumor cells has yet to be examined; C.E. Burd however, common side effects of PD-0332991 (i.e., neutropenia Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and thrombocytopenia) certainly demonstrate that nontrans- computational analysis): X. Guan, K.M. LaPak, C.Y. Yu, J. Zhang, C.E. Burd formed cell types are affected by this drug. Based upon our Writing, review, and/or revision of the manuscript: X. Guan, K.M. LaPak, R.C. work and that of others (41, 42), the therapeutic window for Hennessey, C.Y. Yu, C.E. Burd fi — Administrative, technical, or material support (i.e., reporting or organizing PD-0332991 should be suf cient to spare normal cells the IC50 data, constructing databases): X. Guan, C.E. Burd of PD-0332991 is at least 3-fold lower in drug-sensitive cancer cell Study supervision: C.E. Burd lines (0.04–0.17 mmol/L) than in primary cells (>0.5 mmol/L; ref. 43). However, emerging data suggest that senescent stromal Acknowledgments cells predict cancer recurrence (5), and together with our findings, The authors thank members of The Ohio State University Comprehensive provide a strong case for studies to assess the impact of CDK4/6 Cancer Center's Genomics and Target Validation Shared Resources as well as inhibitor therapies on normal, proliferating cells. While potential R. Kladney, D. Guttridge, M. Ostrowski, X. Liu, and N. Dhomen for technical side effects may be negligible for patients with drug-sensitive support and reagents. In addition, we acknowledge A. Holderbaum and tumors, senescent cells could accumulate in those with nonre- C. J. Burd for critical reading of the manuscript. sponding tumors, limiting future therapeutic benefit and promoting frailty. Grant Support fi In sum, our ndings imply that the consequences of CDK4/6 This work was supported by the NIH (R00AG036817; to C.E. Burd; and inhibitors on normal, proliferating cell types could hinder ther- T15LM11270-4, to C.Y. Yu), Melanoma Research Alliance (309669; to C.E. apeutic efficacy and lead to premature biological aging. As single Burd), American Federation for Aging Research (to C.E. Burd), and Pelotonia agents in melanoma, CDK4/6 inhibitors do not display dramatic (to K.M. LaPak and R.C. Hennessey). clinical activity (41, 42, 44–47). However, combination therapies The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked employing CDK4/6 inhibitors with drugs targeting MEK or BRAF advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate look promising, showing improved disease control and pro- this fact. longed progression-free survival (14, 15, 48, 49). Our data regarding the effects of CDK4/6 inhibition on normal cells raise Received September 20, 2016; revised December 6, 2016; accepted December two questions for the field: (i) could the efficacy of single-agent 8, 2016; published OnlineFirst December 30, 2016.

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www.aacrjournals.org Mol Cancer Res; 15(3) March 2017 249

Stromal Senescence By Prolonged CDK4/6 Inhibition Potentiates Tumor Growth

Xiangnan Guan, Kyle M. LaPak, Rebecca C. Hennessey, et al.

Mol Cancer Res 2017;15:237-249. Published OnlineFirst December 30, 2016.

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