stromal senescence by prolonged cdk4/6 inhibition ...€¦ · 30.12.2016 · 49 cellular...

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1

Stromal senescence by prolonged CDK4/6 inhibition potentiates 2

tumor growth 3

4 Xiangnan Guan1, Kyle M. LaPak1,2, Rebecca C. Hennessey2, Christina Y. Yu2,3, Reena Shakya4, Jianying 5 Zhang3 and Christin E. Burd1,2,4 6 1Departments of Molecular Genetics, 2Cancer Biology and Genetics, and 3Biomedical Informatics, The Ohio State University, Columbus, Ohio, 43210, USA. 4The Ohio State James Comprehensive Cancer Center, Columbus, Ohio, 43210, USA.

RUNNING TITLE 7 CDK4/6i–induced stromal senescence aids melanoma 8

KEY WORDS 9 Melanoma, PD-0332991, Senescence, Tumor Stroma, SASP 10 ADDITIONAL INFORMATION 11 Financial support 12 This work was supported by the NIH (R00AG036817; C.E.B. and T15LM11270-4 C.Y.Y.), Melanoma Research Alliance (309669; C.E.B.), American Federation for Aging Research (C.E.B) and Pelotonia (K.M.L., R.C.H.).

Address correspondence to: Christin E. Burd Biomedical Research Tower, Rm 586 The Ohio State University Columbus, Ohio 43210, USA Phone: (614)688-7569 Fax: (614)292-6356 Email: [email protected] 13 Conflict of interest statement: 14 The authors declare no affiliations and/or financial interests related to the contents of this manuscript. 15 16 Article metrics: 17

Word count: 6,188 18 Citation count: 50 19 6 Figures 20 1 Table 21 4 Supplementary Figures 22 1 Supplementary Table 23 3 Supplementary Datasets 24

on March 26, 2021. © 2016 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 30, 2016; DOI: 10.1158/1541-7786.MCR-16-0319

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Abstract 25

Senescent cells within the tumor microenvironment (TME) adopt a pro-inflammatory, 26

senescence-associated secretory phenotype (SASP) that promotes cancer initiation, progression and 27

therapeutic resistance. Here, exposure to Palbociclib (PD-0332991), a CDK4/6 inhibitor, induces senescence 28

and a robust SASP in normal fibroblasts. Senescence caused by prolonged CDK4/6 inhibition is DNA 29

damage-independent and associated with Mdm2 downregulation, whereas the SASP elicited by these cells is 30

largely reliant upon NF-κB activation. Based upon these observations, it was hypothesized that the exposure 31

of non-transformed stromal cells to PD-0332991 would promote tumor growth. Ongoing clinical trials of 32

CDK4/6 inhibitors in melanoma prompted a validation of this hypothesis using a suite of genetically defined 33

melanoma cells (i.e. Ras mutant, Braf mutant, and Ras/Braf wild type). When cultured in the presence of 34

CDK4/6i-induced senescent fibroblasts, melanoma cell lines exhibited genotype-dependent proliferative 35

responses. However, in vivo, PD-0332991-treated fibroblasts enhanced the growth of all melanoma lines 36

tested and promoted the recruitment of Gr-1-positive immune cells. These data indicate that prolonged 37

CDK4/6 inhibitor treatment causes normal fibroblasts to enter senescence and adopt a robust SASP. Such 38

senescent cells suppress the anti-tumor immune response and promote melanoma growth in 39

immunocompetent, in vivo models. 40

41

Implications: The ability of prolonged CDK4/6 inhibitor treatment to induce cellular senescence and a robust 42

SASP in primary cells may hinder therapeutic efficacy and promote long-term, gerontogenic consequences 43

that should be considered in clinical trials aiming to treat melanoma and other cancer types. 44

45

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47

INTRODUCTION 48

Cellular senescence is a process in which cells with the potential to divide permanently exit the cell 49

cycle, but remain viable and metabolically active. Senescent cells are characterized by numerous cellular 50

phenotypes, including insensitivity to mitogenic stimuli, flattened morphology, increased 51

senescence-associated ß-galactosidase activity (SA-ß-gal), shortened telomeres, elevated cyclin-dependent 52

kinase inhibitor expression, changes in chromatin structure, pervasive DNA damage foci, resistance to 53

apoptosis and activation of the pro-inflammatory senescence-associated secretory phenotype (SASP) (1, 2). 54

Notably, not every senescent cell exhibits all of these characteristics. Instead, the triggering event (e.g. 55

oncogene activation, telomere attrition, prolonged CDKi expression, or DNA damage) and originating cell 56

type appear to dictate which phenotypes ensue (2, 3). Therefore, senescent cells are not phenotypic 57

equivalents and likely to contribute to distinct biological outcomes in vivo. 58

Emerging data reveal the presence of senescent cells within the tumor microenvironment. For example, 59

using a genetically modified mouse model wherein activation of the senescence biomarker, p16INK4a, induces 60

firefly luciferase expression, the accumulation of senescent stromal cells can be visualized both in 61

autochthonous tumor transplants and spontaneous neoplasms (4). In humans, p16INK4a-positive stromal cells 62

accumulate around large cell lung carcinomas and ductal carcinoma in situ lesions of the breast and pancreas 63

(5, 6). Of note, stromal p16INK4a expression in breast cancer is more predictive of disease recurrence than 64

HER2, PR or ER status (7), suggesting that senescent stromal cells are indicative of poor prognosis. 65

Numerous in vitro co-culture studies indicate that the SASP of senescent stromal cells influences cancer 66

initiation, progression and therapeutic response; however, few studies extend these observations to in vivo 67

models (8-11). Of the publications that do address how senescent stromal cells influence tumor growth in 68




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vivo, only one has been conducted in an immune-proficient mouse model (12). Consequently, mechanisms 69

by which paracrine SASP signals emanating from the senescent tumor stroma might alter malignant cell 70

clearance by the immune system are understudied—a particularly pertinent point given increasing interest in 71

combining established treatment modalities (i.e. chemotherapy, radiotherapy and molecularly targeted 72

therapies) with immunotherapy (13). 73

Senescence induced by pharmaceutical inhibitors of CDK4/6 is of growing biological and clinical 74

relevance. Developed to combat frequent inactivation of the CDK/Cyclin-RB signaling axis in human cancer, 75

these compounds induce a permanent cell cycle arrest in many tumor-derived cell lines. CDK4/6 inhibitors 76

(e.g. LY2835219, LEE011, G1T28, and P1446A-05) are under clinical investigation for a variety of tumor types, 77

including melanoma. Given early results suggesting that CDK4/6 inhibitors enhance the efficacy of other 78

targeted melanoma therapies (14-16), it is likely these drugs will be soon approved for use in patients with 79

metastatic melanoma. One potential concern surrounding the use of these drugs stems from recent in vitro 80

data showing that extended exposure to PD-0332991 can trigger cellular senescence in normal fibroblasts 81

(17). Given the known tumor-promoting effects of the SASP (2) as well as the contribution of senescent cells 82

to biological aging (1), it is logical to examine the effects of these drugs on normal tissues. However, no study 83

to date has extensively characterized the phenotype of CDK4/6 inhibitor-induced senescence in normal 84

fibroblasts or determined the effect of these stromal cells on tumor growth. 85

Here, we set out to determine how stromal senescence induced by prolonged PD-0332991 treatment 86

influences melanoma cell proliferation both in vitro and in vivo. To this end, we extensively compared the 87

phenotypes of fibroblasts triggered to enter senescence via PD-0332991 treatment to those triggered to 88

enter senescence by other, melanoma-relevant signals (i.e. UV irradiation and DNA damaging 89

chemotherapy). Then, employing a panel of syngeneic murine melanoma cell lines representing major 90




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genetic subtypes of human melanoma (BRAF mutant (40-60% of melanomas), NRAS mutant (15-30% of 91

melanomas), and NRAS/BRAF wild-type (< 20% of melanomas)), the ability of these senescent fibroblasts to 92

influence cancer cell proliferation was assessed both in vitro and in an immunocompetent murine model. 93

Our results reveal that CDK4/6 inhibitor-induced stromal senescence triggers a robust, 94

DNA-damage-independent SASP and that these cells can foster the growth of melanoma in vivo via 95

alterations in immune cell infiltration. These data provide insight relevant to the clinical implementation of 96

CDK4/6 inhibitors, suggesting that drug efficacy might be enhanced by protecting stromal cells from 97

senescence. Moreover, we propose that the ability of these drugs to drive biological aging should be 98

considered and monitored during clinical trials. 99

100

101

METHODS 102

Cell lines and culture procedures 103

B16-F1 (CRL-6323) and B16-F10 (CRL-6475) mouse melanoma cell lines were purchased from ATCC at 104

the onset of this study. NL212 and NL216 cells were derived from TpLN61R/61R melanomas (18). The TRIA cell 105

line was generated from a Tyr-HRas12V Ink4a/Arf-/- melanoma (19). Murine BrafV600E melanoma cell lines 4434 106

and 21015 were kindly provided by Dr. R. Marais (Cancer Research UK) (20). Cells were cultured in DMEM 107

supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 2mM L-glutamine. MEFs were 108

isolated from E13.5 mouse embryos as described (4). 109

To generate GFP-labeled NL212, NL216, TRIA, B16-F1, B16-F10, 4434, and 21015 cultures, cells were 110

transduced with pLenti-puro-GFP lentivirus using 10μg/mL polybrene. The pLenti-puro vector is a derivative 111

of pTRIPZ (Open Biosystems) in which turboRFP and rtTA3 were removed and a multiple cloning sequence 112




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inserted between the BamHI and NotI restriction sites. GFP from pEGFP-N3 (Clontech) was inserted into the 113

resulting multiple cloning sequence. Transduced tumor cells were selected with 3µg/mL puromycin. 114

Cell lines were tested for mycoplasma using Mycoplasma Plus PCR Primers (Agilent Technologies) and 115

identity-verified at multiple time points during the study. Identity verification was conducted by PCR for all 116

alleles unique to the study cell lines (e.g. Alterations in Stk11, Ink4a/Arf, NRas, and Braf). In addition, 117

BrafV600E and NRasQ61R mutations were sequence verified using PCR products generated from genomic DNA. 118

119

Senescence induction 120

To generate senescent MEFs, fibroblasts cryopreserved two days after isolation were thawed, grown in 121

culture for 48 hours, and then plated at a density of 400,000 cells per 10cm plate. Two days later, cells were 122

treated to induce senescence. For UV-induced senescence, MEFs were irradiated with two doses of 3 mJ/cm2 123

UV administered 48 hours apart using a Stratalinker 1800 (Stratagene). MEFs were allowed to recover for 48 124

hours under normal growth conditions prior to any experimental assessments. For mitomycin C-induced 125

senescence, MEFs were exposed to 10µg/mL mitomycin C (Abcam) for 2.5 hours and then cultured in growth 126

media for 4 days to establish senescence. For CDK4/6 inhibitor-induced senescence, MEFs were treated with 127

4µM PD-0332991 (Sigma, 827022-33-3) for 8 days, adding new drug and media on day 4. During in vitro 128

assays, PD-0332991-treated cells were trypisinized, washed with PBS and then plated in normal growth 129

media for at least 24 hours before the start of any experiments. Prior to in vivo injections, 130

PD-0332991-treated cells were trypsinized and thoroughly washed with PBS to remove any residual drug. 131

To test the role of NF-κB in establishing the SASP, retrovirus encoding IκBα S32A/S36A (i.e. NF-κB 132

super-repressor; (21)) or control (empty pBabe-PURO vector) was generated in HEK293T cells using standard 133

procedures. Next, passage 2 fibroblasts were transduced with virus and rapidly selected in 3μg/mL 134




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puromycin for 72 hours. Following selection, cells were treated with either vehicle alone or 4µM PD-0332991 135

as described above. Cells were then split and seeded at sub-confluence for SA-β-gal staining or processed for 136

real-time PCR analysis as described below (See Supplementary Table 1 for a list of validated primer sets). 137

138

Direct and indirect co-culture 139

For direct co-culture assays, 30,000 passage 3 or 35,000 senescent MEFs were seeded in 96-well plates 140

(Thermo, 165305). The next day, 1,500 (NL212, NL216, B16-F1, B16-F10 and TRIA) or 2,000 (4434 and 21015) 141

GFP-labeled, melanoma cells were seeded on top of the MEFs. Cultures were grown for 4 days, at which time 142

the media was replaced with 50µl PBS and fluorescence determined on the Bio-Tek Synergy HT (excitation = 143

485 nm; emission = 528 nm). Fluorescent signal from empty wells or wells with feeder cells alone was 144

subtracted from experimental values to correct for background. These values were then normalized to the 145

average reading for wells containing only tumor cells. For statistical analyses, average values for biological 146

replicates, measured in triplicate, were first log2 transformed. Linear mixed modeling was then employed to 147

account for the random block factor, experimental date, and to compare between treatment groups. 148

For indirect co-culture assays, conditioned medium from either co-cultures or feeder cells alone was 149

collected 96 hours after plating. Following centrifugation at 500g for 5 min, 100µl of cleared, conditioned 150

media was added to tumor cells pre-seeded in 100μL fresh DMEM. After 4 days of culture, fluorescence was 151

determined as described above. 152

153

SA-β-galactosidase staining 154

MEFs were seeded on coverslips and SA-β-gal activity measured using the Senescent β-galactosidase 155

Staining Kit (Cell Signaling). NIH ImageJ was used to count total and SA-β-gal positive cell numbers. At least 156




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three independent views from 2 biological replicates were quantified for each treatment group. 157

158

Cell cycle analysis 159

MEFs were seeded at sub-confluence in normal growth media and 10μM Edu added for 24 hours prior 160

to analysis. EdU incorporation was measured using the Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry 161

Assay Kit (Life Technologies). Cells were analyzed on a FlowSight cytometer (Amnis) with non-EdU labeled 162

cells serving as the control. 163

164

Immunofluorescence 165

Cells grown on glass coverslips were fixed in 4% formaldehyde for 15 min and then permeabilized with 166

0.2% Triton X-100 for 10 min. Coverslips were blocked in 5% BSA and incubated overnight with anti-γH2AX 167

(1:400; Cell Signaling) or anti-53BP1 (1:1000; Bethyl Labs) antibody. Primary antibody was visualized using 168

Alexa Fluor-555 secondary antibodies (1:500; Molecular Probes). Slides were mounted in Prolong Gold 169

Anti-fade reagent containing DAPI (Molecular Probes). Cells with >3 γH2AX or 53BP1 foci per nucleus were 170

considered positive and at least 100 cells were assessed per biological replicate. Quantification was 171

performed by two independent reviewers, blinded to the slide information. 172

173

Real-time PCR analyses 174

Total RNA was isolated from cells using the NucleoSpin RNA II kit (Macherey-Nagel Ltd.) and cDNA 175

generated using the Improm II Reverse Transcription Kit (Promega). To asses p16INK4a levels, real-time PCR 176

was performed in triplicate using previously described TaqMan gene expression assays for p16INK4a and 18S 177

(4). p16INK4a levels were calculated using the ΔΔCt method with 18S as the reference (4). To validate 178




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differentially expressed genes identified in the TaqMan® OpenArray® Mouse Inflammation Panel as well as 179

10 additional targets selected from the literature (Bmp6, Bmp7, Ccl5, Ccl6, Ccl17, Ccl20, Cd40, Csf1, Csf3, and 180

Mmp3 (8, 9)), 100ng of RNA was reverse transcribed and SYBR-Green based real-time PCR analyses 181

performed. Relative transcript levels for each target were calculated using the ΔΔCt method with Gusb as the 182

reference. In the NF-κB super repressor experiments, 500 ng of RNA was reverse transcribed and 183

SYBR-Green-based real-time PCR performed in triplicate to assess key targets (IL6, Ccl6, Ccl8, Ccl11, c3, Cxcl5, 184

Mmp3, and Lbp). Relative transcript levels for each target were calculated using the ΔΔCt method with Gusb 185

as the reference. All SYBR-Green based assays used SensiFastTM SYBR (Bioline). Primer sets were validated by 186

gel electrophorese of real-time PCR products and are listed in Supplementary Table 1. 187

188

Cytokine arrays 189

To generate conditioned medium (CM), MEF cultures were thoroughly washed and incubated in 190

serum-free DMEM supplemented with 1% penicillin-streptomycin and 2mM L-glutamine. After 24 hours of 191

incubation, CM was collected and MEFs counted on a hemocytometer. CM was centrifuged to remove any 192

residual cells and then analyzed using Proteome Profiler Mouse XL Cytokine Arrays (R&D Systems; 193

Cat#ARY028). Array signals were visualized on an Odyssey CLX system (LiCor) using IRDye 800CW Streptavidin 194

(1:2000; LI-COR, Catalog #926-32230) secondary antibody. Signal intensity from each spot was determined 195

using Image Studio Software Ver5.2 (LiCor) and corrected for background by subtracting the local median 196

(intensity of grid border pixels for each spot). For each cytokine on the array the average signal (pixel density) 197

from duplicate spots on the membrane was first calculated. Next, the average signal from negative control 198

spots on the array was subtracted. Finally, the corrected signal intensity for each cytokine was normalized to 199

cell number. The resulting data is expressed as fold change of CDK4/6i over pre-senescent (p4) cells. 200




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201

Immunoblotting 202

MEF lysates were run on a 12% SDS-PAGE gel, transferred and immunoblotted for p65 (1:1000; Santa 203

Cruz sc-109), p-p65 (1:1000; Abcam, ab-86299), p38 (1:1000; Santa Cruz sc-535), p-p38 (1:1000; Cell 204

Signaling #4511), p21 (1:1000; Santa Cruz sc-471), Mdm2 (1:750; Santa Cruz sc-965), p53 (1:1000; Leica 205

Biosystems CM5), or β-actin (1:10000; Cell Signaling #3700S). Blots of biological replicates were scanned on 206

Odyssey CLX system and quantified using Image Studio Software Ver5.2 (LiCor). 207

208

Syngeneic tumorigenesis assays 209

Animal experiments were performed in compliance with protocols approved by the Ohio State 210

University Institutional Animal Care and Use Committee (IACUC, Protocol #2012A00000134). C57BL/6J mice 211

(6-10 weeks old) were injected subcutaneously with 5×105 tumor cells plus either vehicle or 5×105 p4, UV, 212

MMC, or CDK4/6i fibroblasts. Once established, tumors were measured at least every other day by calipers 213

until euthanasia was required. Tumor size was calculated as tumor width (mm) x length (mm). 214

215

Tumor immunohistochemistry 216

Formalin fixed, paraffin embedded tumor sections were stained with anti-Gr-1 (1:200; BioLegend 108401), 217

anti-F4/80 (1:100; Thermo Sci MF48000) or anti-FoxP3 (1:800; Abcam ab-54501). Baked slides were dewaxed 218

and rehydrated prior to antigen retrieval in a steamer for 40 min using DAKO Antigen Retrieval Buffer (pH 219

6.10, Catalog #S1699). Primary antibody staining was detected using the DAKO Liquid DAB and Substrate 220

Chromogen System (#K3468), and all slides counterstained with hematoxylin. To remove the melanin present 221

in B16-F10 tumors, slides were incubated in 10% H2O2 at 60°C for 90 min following DAB staining. Staining for 222




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CD3 was conducted on a Leica Bond autostainer using anti-CD3 (1:100; Abcam ab-16669) antibody and the 223

Bond Polymer Refine Red Detection System (Lieca). For all immunohistochemical stains, five distinct fields 224

per tumor were acquired on Vectra Imaging System (Perkin Elmer) and the percent positivity determined 225

using Inform2.1 software (Caliper Life Sciences). 226

227

RESULTS 228

PD-0332991-triggered senescence exhibits a robust SASP without DNA damage 229

Prolonged PD-0332991 exposure is reported to induce cellular senescence in normal fibroblasts, yet the 230

phenotypic characteristics of this form of senescence are largely unknown (17). To address this gap in 231

knowledge, we comprehensively characterized the phenotypes of fibroblasts triggered to enter senescence 232

by long-term PD-0332991 exposure and compared these results to those of other, well-characterized forms 233

of senescence initiated by either environmental DNA damaging or chemotherapeutic agents (i.e. mitomycin 234

C, ultraviolet light). To establish senescence, pre-senescent mouse embryonic fibroblasts (MEFs; passage 235

4/p4) were treated with either PD-0332991 (4 µM for 8 days), UV irradiation (3 mJ/cm2 twice at a 48-hour 236

interval) or mitomycin C (10 µg/mL for 2.5 hours). Herein, these treated fibroblast populations are referred to 237

as CDK4/6i, UV and MMC cells, respectively. Prior to any in vitro assessments, cells were removed from 238

treatment and re-plated in normal growth media for at least 24 hours. 239

To confirm that these fibroblast cultures were no longer responsive to mitogenic stimuli, cells were 240

seeded at sub-confluence in normal growth medium and, one day later, incubated with the thymidine 241

analog, EdU, for a period of 24 hours. Using flow cytometry to detect EdU incorporation, over 76% of p4 cells 242

were positive (Table 1, Fig. 1A). By contrast, less than 10% of CDK4/6i, UV, and MMC cells incorporated EdU 243

during the labeling period. Next, we measured SA-β-gal positivity in these cells, revealing that 11% of p4 244




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cells, 74% CDK4/6i treated cells, 58% of UV treated cells and 72% of MMC treated cells were SA-β-gal positive 245

(Table 1 and Fig. 1B). Marked accumulation of p16INK4a mRNA also occurred in the CDK4/6i, UV, and MMC 246

treated cells, with respective increases of 4.4, 5.4 and 3.0-fold over pre-senescent (p4) fibroblasts (Table 1, 247

Fig. 1C). Consistent with publications suggesting that DNA damage is requisite for the formation of 248

senescence-associated genomic lesions (22), UV and MMC treated cells showed pronounced foci containing 249

53BP1 (58% and 56%, respectively) and γH2AX (73% and 81%, respectively) (Table 1, Fig. 1D and E). CDK4/6i 250

cells and p4 fibroblasts showed only basal levels of DNA damage (i.e. 53BP1 and γH2AX foci) likely associated 251

with replication (5.0% 53BP1 and 5.6% γH2AX positive; Table 1, Fig. 1D and E). 252

To determine if PD-0332991-induced fibroblast senescence triggers the SASP we analyzed 253

pro-inflammatory gene expression in total MEF RNA using the TaqMan® OpenArray® platform. This approach 254

revealed distinctions amongst the inflammatory gene expression profiles of all four fibroblast lines (p4, 255

CDK4/6i, UV and MMC; Dataset S1). Of particular interest, CDK4/6i cells, with little evidence of DNA damage 256

(Table 1, Fig. 1D and E), exhibited a more robust pro-inflammatory gene expression profile than fibroblasts 257

triggered to enter senescence via UV or MMC (Dataset S1). Compared to pre-senescent (p4) fibroblasts, 115 258

pro-inflammatory mRNAs were upregulated ≥2.5-fold in CDK4/6i MEFs, whereas only 89 and 62, respectively, 259

were induced in UV and MMC-treated cells. 260

We used real-time PCR to validate 69 hits from the OpenArray® platform that differed by ≥2.5-fold in 261

CDK4/6i cells compared to UV and MMC-treated fibroblasts. Simultaneously, we assessed the expression of 262

10 additional senescence-associated mRNAs reported in the literature (See Materials and Methods). In three 263

biological replicates, we confirmed that 51/69 targets identified by the OpenArray® platform were distinctly 264

altered in PD-0332991-induced senescence (33/47 up- and 18/22 down-regulated ≥2-fold in comparison to 265

UV and MMC treated cells) (Fig. 2A, Dataset S2). Of the 69 mRNAs assessed, 39 were expressed at higher 266




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levels in CDK4/6i fibroblasts than in proliferating MEFs, whereas 10 were lower in CDK4/6i cells (Dataset S2). 267

Fewer transcripts selected from the literature validated in our assays—only 4 of 10 transcripts were greater 268

in CDK4/6i cells than in normal, proliferating MEFs, and 2 of these targets were similarly upregulated in UV 269

and MMC treated cells (Dataset S2). Together, these data validate that prolonged PD-0332991 treatment 270

leads to the robust induction of a variety of pro-inflammatory genes. 271

To determine if changes in pro-inflammatory gene transcription alter the secretome of CDK4/6i 272

fibroblasts, we employed a membrane-based antibody array. Using this approach, 111 cytokines were 273

simultaneously measured in conditioned medium (CM) from p4 and CDK4/6i cells. On a per-cell basis, 274

cytokine levels were 1.1 to 17.8 times higher in CM from CDK4/6i MEFs than from p4 MEFs (Dataset S3). 275

Cross-comparison of 34 targets common to our mRNA and cytokine panels revealed a strong correlation 276

between mRNA and secreted protein levels for most targets analyzed. However, several targets showed 277

transcriptional activation that was not paralleled by protein secretion (i.e. Eoxtaxin, Cxcl5, IL-1α, Tnfsf13b, 278

and PTX-3; Dataset S3). Other targets were not transcriptionally induced, but showed increased secretion in 279

CDK4/6i fibroblasts compared to p4 controls (i.e. Vegf, Ccl5, Cd14; Dataset S3). Thus, while 280

post-transcriptional mechanisms influence the production and export of some pro-inflammatory cytokines 281

associated with PD-0332991-induced senescence, most pro-inflammatory mRNAs induced in CDK4/6i cells 282

are also elevated in the fibroblast secretome. Together, these data demonstrate that PD-0332991-induced 283

senescence activates a diverse and potent SASP. 284

285

Loss of Mdm2 expression triggers CDK4/6i-induced senescence 286

We searched for a DNA-damage-independent mechanism to explain how CDK4/6 inhibitor treatment 287

triggers senescence in primary fibroblasts. Similar to a recent study by Kovatcheva et al. in liposarcoma cells 288




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lines (23), we found that prolonged PD-0332991 treatment promoted a reduction in Mdm2 and p53 protein 289

levels (Fig. 2B). However, unlike what is observed in liposarcomas, p21Cip1/Waf1 protein levels were elevated in 290

CDK4/6i MEFs in comparison to p4 cells (Fig. 2B). Together, these data support a model wherein prolonged 291

PD-0332991 exposure promotes Mdm2 degradation, leading to increased p21Cip1/Waf1 stability, cell cycle arrest 292

and subsequent senescence in normal fibroblast populations. 293

294

NF-κB activity promotes the SASP of CDK4/6i cells 295

To determine how CDK4/6i fibroblasts elicit a SASP in the absence of DNA damage, we examined the 296

p38 and NF-κB pathways, each of which has been linked to SASP gene expression (24, 25). Although elevated 297

in MMC-treated fibroblasts, p38 was not activated in senescent CDK4/6i cells (Supplementary Fig. S1). By 298

contrast, activating phosphorylation of the NF-κB subunit, p65, was elevated in CDK4/6i, UV and MMC 299

fibroblasts when compared to control (Fig. 3A). To further confirm the role of NF-κB in driving the SASP of 300

CDK4/6i fibroblasts, MEFs were transduced with retrovirus encoding NF-κB super-repressor (SR, i.e. IκBα 301

S32A/S36A) or a control (EV, empty vector). After rapid selection for stably transduced clones, cells were 302

subjected to PD-0332991 or vehicle treatment as described above. As evidenced by SA-β-gal staining, CDK4/6 303

inhibitor-induced senescence was unaffected by expression of the NF-κB super-repressor (Fig. 3B). By 304

contrast, the induction of many SASP-associated transcripts (i.e. IL6, MMP3, Ccl6, Ccl8, and Ccl11) in CDK4/6i 305

MEFs was completely blocked by NF-κB inhibition (Fig. 3C). Other SASP-associated transcripts (C3, Cxcl5, and 306

Lbp) showed reduced activation in both control and CDK4/6i fibroblasts transduced with the NF-κB 307

super-repressor. However, the magnitude by which NF-κB super-repressor decreased gene expression was 308

significantly higher in fibroblasts treated with CDK4/6 inhibitor versus untreated controls (Fig. 3D). Notably, 309

expression of our endogenous control gene, Gusb, did not change in cells harboring the NF-κB 310




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super-repressor (Fig. 3C). These data provide strong evidence that NF-κB activity promotes the induction of a 311

wide variety of pro-inflammatory transcripts associated with the SASP of CDK4/6i fibroblasts. 312

313

CDK4/6i fibroblasts trigger genotype-dependent proliferative responses in melanoma co-cultures 314

To investigate the potential effects of CDK4/6i fibroblasts on melanoma growth, we first utilized a 2D 315

co-culture system. Pre-senescent (p4) or senescent (CDK4/6i, UV or MMC) MEFs were seeded into 96-well 316

plates to produce confluent monolayers (Fig. 4A). One day after seeding, murine melanoma cell lines stably 317

expressing GFP were added to each well. Melanoma cell lines selected for this study are primarily syngeneic 318

to C57Bl/6 and carry alterations in Ras (4, 18, 19), Braf (20) or neither of these genes (i.e. B16 cell lines). 319

While prior literature suggests that only senescent fibroblasts stimulate the proliferation of tumor cells 320

(10, 26, 27), we found that both senescent and pre-senescent fibroblasts influenced melanoma cell growth in 321

vitro (Fig. 4B-H). Interestingly, the direction and magnitude of how fibroblast co-culture changed melanoma 322

cell proliferation was genotype-dependent. For example, the BrafV600E cell lines 4434 and 21015 proliferated 323

faster in the presence of any form of MEFs, whereas other melanoma cells responded only to specific types 324

of pre-senescent and/or senescent fibroblasts (Fig. 4B-H). N-Ras mutant cell lines (NL212, NL216) grew 325

slower when plated on CDK4/6i fibroblasts (Fig. 4C-D), and the proliferation of B16-F1 and B16-F10 cells only 326

changed in response to pre-senescent and MMC-treated fibroblasts (Fig. 4G and H). Of note, in no case did 327

we find that CDK4/6i fibroblasts stimulated the growth of melanoma cells better than normal, proliferating 328

MEFs (Fig. 4B-H). Together, these data indicate that the in vitro proliferative response of melanoma cells to 329

CDK4/6i fibroblasts is genotype-dependent and frequently parallels what is seen with other forms of 330

senescent and/or pre-senescent feeders. 331

Senescent cells affect surrounding tissues and/or tumor cells via paracrine signaling. Such effects 332




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include the induction of neighboring cell senescence, disruption of epithelial organization, and promotion of 333

premalignant epithelial cell growth (1, 3). To determine whether CDK4/6i conditioned media was sufficient to 334

alter melanoma cell proliferation in vitro, we performed indirect co-culture assays. Conditioned medium 335

from p4, UV, MMC and CDK4/6i MEFs was transferred onto melanoma cell cultures and changes in 336

proliferation assessed (Fig. 5A). While CDK4/6i conditioned media promoted the growth of melanoma cell 337

lines carrying a Ras mutation (TRIA, NL216, NL212; Fig. 5B-D; indicated by ‘*’), Braf mutant (4434 and 21015) 338

and wildtype (B16-F1 and B16-F10) melanoma cell lines were non-responsive (Fig. 5E-H). In all cases, the in 339

vitro effects of conditioned media from CDK4/6i MEFs paralleled those caused by media collected from p4, 340

UV, or MMC fibroblasts (Fig. 5B-H) 341

Intercellular communication is hypothesized to augment paracrine SASP signals through the 342

establishment of feed-forward signaling loops (28). For this reason, we repeated our indirect co-culture 343

studies, transferring conditioned media from our direct co-culture setup instead of senescent fibroblasts 344

alone (Supplementary Fig. S2A). Paralleling our results in Figure 5, all of the Ras mutant melanoma cell lines 345

showed increased proliferation in the presence of CDK4/6i co-culture conditioned media (Supplementary Fig. 346

S2B-D; indicated by ‘*’); however, in contrast to conditioned media collected from CDK4/6i fibroblasts alone, 347

Braf mutant cells proliferated in response to co-culture conditioned media (Supplementary Fig. S2E-F). This 348

same media had no proliferative effect on the B16-F1 or B16-F10 cell lines (Supplementary Fig. S2G-H). 349

Together, these direct and indirect in vitro co-culture studies reveal assay-dependent genetic trends in how 350

CDK4/6i fibroblasts influence melanoma cell proliferation. To discern which in vitro assay best represents the 351

physiological consequences of PD-0332991 induced stromal senescence, in vivo experiments were 352

performed. 353

354




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CKD4/6i fibroblasts promote melanoma growth in vivo. 355

Prior publications rely predominantly upon xenograft models to show that senescent fibroblasts, 356

triggered by a variety of DNA damaging stimuli, promote the growth of human and murine tumors in vivo (8, 357

10, 17, 27, 29, 30). However, since these studies employ immunocompromised mice, they do not fully 358

address the potential effects of senescent cells on immune infiltration and tumor rejection. Here, we sought 359

to determine how CDK4/6i fibroblasts in the tumor stroma impact melanoma growth using an 360

immune-proficient, syngeneic mouse model. Based upon the genotype-dependent proliferative responses 361

seen in our co-culture assays, we selected four representative melanoma lines for assessment (i.e. NL212, 362

4434, B16-F1 and B16-F10). 363

Tumor cells and senescent fibroblasts were established in vitro (See Materials and Methods), trypsinized, 364

washed and resuspended in PBS for subcutaneous injection. While NL212 (Nras mutant) cells efficiently 365

formed melanomas in the absence of co-injected fibroblasts, tumor growth was significantly augmented by 366

the co-injection of either pre-senescent or CDK4/6i fibroblasts (Fig. 6A; tumor take rate 100% in all groups). 367

Similarly, 4434 (Braf mutant) cells formed tumors and grew better when co-injected with any type of 368

fibroblasts, including CDK4/6i cells (Fig. 6B). By contrast, B16-F10 and B16-F1 (Nras and Braf wild type) cells 369

formed tumors efficiently and grew rapidly in vivo, but were unaffected by the co-injection of pre-senescent 370

fibroblasts (p4) (Fig. 6C and Supplementary Fig. S3; tumor take rate 100% in all groups). B16-F10 and B16-F1 371

tumors did, however, show accelerated growth when co-injected with senescent cells (including CDK4/6i 372

fibroblasts) (Fig. 6C and Supplementary Fig. S3; p <0.01 comparing each MEF population to p4). Together, 373

these data show that in contrast to our in vitro findings, all senescent fibroblasts, including those established 374

via prolonged CDK4/6i treatment, can promote melanoma growth in vivo. However, whether CKD4/6i 375

fibroblasts have stronger proliferative effects than pre-senescent fibroblasts is dependent upon the 376




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melanoma cell line assayed. 377

Decades of studies have established that SASP components (i.e. IL-6, IL-8, CXCL-1, VEGF) promote the 378

proliferation of tumor cells in vitro (8, 9, 11). However, since the SASP also contains numerous 379

pro-inflammatory factors capable of stimulating immune influx into the tumor microenvironment (31, 32), 380

we sought to determine if there were differences in immune infiltrates that might explain the in vivo growth 381

phenotypes observed in each melanoma cell line. In tumors from all three cell lines, the co-injection of 382

CDK4/6i fibroblasts led to significant increases in Gr-1 positive immune cell infiltrates as measured by 383

immunohistochemistry (Fig. 6D-F). By contrast, the number of FoxP3 (T-regulatory cells) and F4/80 384

(macrophages) positive cells remained constant amongst tumors co-injected with pre-senescent (p4) or 385

CDK4/6i fibroblasts (Supplementary Fig. S4). Notably, total CD3 positive cells decreased in all CDK4/6i 386

co-injected tumors, suggesting that the infiltrating Gr-1 positive cells are myeloid-derived suppressor cells 387

(MDSCs; Fig. 6G-I). These findings support a model in which stromal senescence induced by prolonged 388

PD-0332991 exposure leads to MDSC accumulation and enhanced tumor growth. When MDSC numbers are 389

already elevated, as seen in NL212 and 4434 cells co-injected with p4 fibroblasts, the additive effects of 390

CDK4/6i cells on melanoma growth are negligible (Fig. 6). However, in tumors that are less immunogenic (i.e. 391

B16-F10 and B16-F1), increases in MDSC numbers suppress the immune system sufficiently to promote an 392

added growth advantage (Fig. 6 and Supplementary Fig. S3). 393

394

395

DISCUSSION 396

It is well-established that components of the SASP (e.g. IL-6, IL-8, CXCL-1, VEGF, MMP-3) drive tumor 397

proliferation both in vitro and in xenograft assays (8-11). However, the recent discovery that primary cell 398




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cultures enter senescence following sustained PD-0332991 exposure (17) prompted us to investigate if 399

CDK4/6 inhibitor treatment can activate the SASP in normal tissues. Here, we report that 400

PD-0332991-induced fibroblast senescence is characterized by a robust, DNA damage-independent SASP 401

(Table 1, Fig. 1B-E, 2A). While in vitro assays reveal that these senescent fibroblasts stimulate the growth of 402

melanoma cells in a genotype-dependent manner (Figs. 4-5 and Supplementary Fig. S2), this observation 403

does not hold true in a more biologically relevant setting. Specifically, melanoma cells lines grow better in 404

C57Bl/6 mice when co-injected with CDK4/6i fibroblasts versus vehicle alone (Fig. 6A-C and Supplementary 405

Fig. S3). CDK4/6i fibroblasts also promote the accumulation of Gr-1 positive MDSCs within the tumor 406

microenvironment, thereby dampening the anti-tumor immune response. Together, these data suggest that 407

the impact of CDK4/6 inhibitor therapy on normal cells should be considered when treating cancer patients, 408

as it may limit therapeutic efficacy and promote senescent cell accumulation throughout the body. 409

410

PD-0332991 triggers a robust SASP in the absence of DNA damage 411

The SASP of senescent cells is often triggered by cell-intrinsic DNA damage—an observation supported 412

by the fact that senescent cells established by exogenous p16INK4a expression lack the SASP (33). For this 413

reason, we were surprised to find that primary fibroblasts treated with the CDK4/6 inhibitor, PD-0332991 414

(i.e. CDK4/6i fibroblasts), exhibit a robust SASP in the absence of DNA damage (Table 1, Fig. 1D-E, 2A, Dataset 415

S1-3). Prior studies link SASP gene activation to transcriptional programs initiated by Nuclear Factor (NF)-κB, 416

CCAAT/Enhancer-Binding Protein Beta (C/EBPβ), MacroH2A1, Mammalian Target of Rapamycin (mTOR), and 417

GATA4 (34-36). Here, we find that CDK4/6i fibroblasts activate NF-κB to the same extent as cells triggered to 418

enter senescence via UV or MMC treatment (Fig. 3A). Furthermore, we show that inhibition of NF-κB 419

signaling prevents the induction of classic SASP genes (e.g. IL-6, Mmp3, Ccl6) without altering the ability of 420




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prolonged PD-0332991 treatment to establish senescence (Fig. 3B-C). These data suggest that the CDK4/6 421

inhibitor-induced SASP of senescent stromal cells might be mitigated by dampening NF-κB activity. 422

Mechanisms by which CDK4/6 inhibitors block tumor cell proliferation are well-studied, yet our 423

understanding of how these drugs affect non-transformed cells is limited. CDK4/6i MEFs lack evidence of 424

traditional senescence mechanisms (i.e. DNA damage and p38 activation; Table 1, Fig. 1D-E, and 425

Supplementary Fig. S1), but show reductions in Mdm2 and p53, paralleling that of senescent, 426

PD-0332991-treated liposarcoma cell lines (Fig. 2B and (23)). Distinct from what is seen in senescent 427

liposarcoma cell lines, CDK4/6i MEFs exhibit post-transcriptional increases in the CDK inhibitor, p21Cip1/Waf1 428

(Fig. 2B). Since Mdm2 is known to destabilize p21Cip1/Waf1 via an E3-ubiquitin ligase independent mechanism 429

(37), our findings suggest that PD-0332991 promotes fibroblast senescence through Mdm2 destabilization 430

and sustained p21Cip1/Waf1 expression. 431

432

CDK4/6i fibroblasts promote genotype-dependent melanoma growth 433

After observing that prolonged PD-0332991 treatment induces senescence accompanied by a robust 434

SASP in primary fibroblasts, we wondered how stromal cells exposed to CDK4/6 inhibitors might influence 435

melanoma growth. Numerous publications examine the mechanisms by which senescent cells promote 436

tumor growth in vitro. Unfortunately, these studies rely largely upon direct and indirect co-culture 437

techniques, which, as evidenced by our findings, often yield conflicting results (Figs. 4, 5 and Supplementary 438

Fig. S2). Nevertheless, we were intrigued by the in vitro observation that CDK4/6i fibroblasts alter melanoma 439

cell proliferation in a genotype-dependent manner (Figs. 4, 5 and Supplementary Fig. S2). Upon moving these 440

studies into a more physiologically relevant model, we found that none of our in vitro systems could 441

accurately model in vivo conditions. In fact, regardless of their mode of induction, senescent fibroblasts 442




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equally promoted the growth of four genetically distinct melanoma cell lines in immunocompetent, 443

syngeneic mice (Fig. 6A-C and Supplementary Fig. S3). This finding underscores the importance of studying 444

the non-cell autonomous effects of senescent cells on tumor growth in vivo rather than in vitro. 445

Here, we report the first studies to characterize how different forms of senescent stromal cells influence 446

melanoma growth in a syngeneic, immunocompetent murine model. Using this experimental approach, we 447

were able to determine how the anti-tumor immune system is influenced by stromal senescence. While all 448

melanoma cell lines grew better in the presence of senescent fibroblasts than vehicle alone, we were 449

surprised to find that the growth of NL212 (NRas mutant) and 4434 (Braf mutant) tumors was also 450

accelerated in the presence of pre-senescent MEFs (Fig. 6 and Supplementary Fig. S3). Oswald et al. recently 451

report a similar finding, observing that the co-injection of normal human fibroblasts and H1437 non-small 452

cell lung cancer cells exacerbates tumorigenesis and reduces the survival of experimental mice (38). While 453

these data might suggest that fibroblast factors independent of the SASP stimulate melanoma growth, 454

analysis of infiltrating immune cells suggests a different possibility. 455

We consistently see higher numbers of Gr-1 positive cells within the tumor microenvironment of 456

melanomas co-injected with CDK4/6i fibroblasts versus those injected with pre-senescent MEFs (Fig. 6D-F). 457

These Gr-1 positive cell infiltrates are likely to be myeloid-derived suppressor cells (MDSCs) as evidenced by 458

the decreased numbers of CD3 positive T-cells within these tumors. Unfortunately, fresh tissue was not 459

available to confirm MDSC identity using a dual Gr-1, Cd11b staining protocol. In the context of an already 460

elevated anti-tumor response like that observed in p4 co-injected 4434 and NL212 melanomas, the effect 461

PD-0332991-induced stromal senescence on melanoma growth appears negligible (Fig. 6A, B, D and E). 462

Conversely, when Gr-1 positive cells are naturally low within the tumor microenvironment, stromal 463

senescence induced by CDK4/6 inhibition promotes tumor growth in vivo (Fig. 6C, F and Supplementary Fig. 464




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S3). This model is supported by multiple publications. For instance, a similar connection between stromal 465

senescence and MDSC recruitment has been shown to occur in the setting of Pten null prostate cancers (39). 466

Here, the onset of senescence in murine prostate tumors promotes the accumulation of Gr-1 positive cells, 467

leading to reduced anti-tumor immunity and enhanced cancer growth (39). In another recent publication, 468

senescent fibroblasts established by exogenous p27Kip1 expression are shown to enhance tumorigenesis in an 469

immunocompetent, syngeneic mouse model (12). Mirroring our findings again, the mechanism by which 470

these senescent fibroblasts promote tumor growth is attributed to infiltrating granulocytic MDSCs (12). 471

Alongside these articles, our data strongly suggest that PD-0332991-induced stromal senescence promotes 472

the establishment of a tumor permissive environment that could reduce the overall efficacy of therapies 473

targeting CDK4/6 activity. 474

475

Implications for the therapeutic administration of CDK4/6 inhibitors in melanoma 476

After relatively few passages in culture, oxidative stress induces the senescence of primary fibroblasts 477

(1). Thus, in order to ensure that the phenotype of CDK4/6i cells is PD-0332991-dependent, it was necessary 478

to select a dose of CDK4/6 inhibitor that rapidly induces senescence. We examined a variety of PD-0332991 479

concentrations to determine the lowest dose meeting this requirement (>90% of cells EdU negative; data not 480

shown), and decided to move forward with a dose of 4 μM. While this dose is ~10-fold higher than observed 481

in the plasma of treated breast cancer patients (ClinicalTrials.gov; NCT00721409), it is unlikely that off-target 482

effects contribute to our findings. Notably, the IC50 of PD-0332991 for other CDKs exceeds 10 μM (40). In 483

clinical trials, PD-0332991 has a long half-life (26.7 hours) and can effectively depot in human peripheral 484

tissues (41). Whether CDK4/6 inhibitors accumulate over multiple treatment cycles to promote senescence 485

in non-tumor cells has yet to be examined, however, common side effects of PD-0332991 (i.e. neutropenia 486




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and thrombocytopenia) certainly demonstrate that non-transformed cell types are affected by this drug. 487

Based upon our work and that of others (42, 43), the therapeutic window for PD-0332991 should be 488

sufficient to spare normal cells—the IC50 of PD-0332991 is at least 3-fold lower in drug-sensitive cancer cell 489

lines (0.04—0.17 μM) than in primary cells (>0.5 μM) (44). However, emerging data suggest that senescent 490

stromal cells predict cancer recurrence (5), and together with our findings, provide a strong case for studies 491

to assess the impact of CDK4/6 inhibitor therapies on normal, proliferating cells. While potential side effects 492

may be negligible for patients with drug-sensitive tumors, senescent cells could accumulate in those with 493

non-responding tumors, limiting future therapeutic benefit and promoting frailty. 494

In sum, our findings imply that the consequences of CDK4/6 inhibitors on normal, proliferating cell types 495

could hinder therapeutic efficacy and lead to premature biological aging. As single agents in melanoma, 496

CDK4/6 inhibitors do not display dramatic clinical activity (42, 43, 45-48). However, combination therapies 497

employing CDK4/6 inhibitors with drugs targeting MEK or BRAF look promising, showing improved disease 498

control and prolonged progression-free survival (14, 15, 49, 50). Our data regarding the effects of CDK4/6 499

inhibition on normal cells raise two questions for the field: 1. Could the efficacy of single agent CDK4/6 500

inhibitors be augmented by preventing SASP activity and/or protecting normal cells from drug exposure?, 501

and 2. Are there long-term consequences of CDK4/6 inhibitor therapy that may decrease a patient’s 502

probability of responding to immune checkpoint inhibitors or promote age-related disease? It is our hope 503

that this work will encourage ongoing and future clinical trials to assess the global consequences of CDK4/6 504

inhibition, rather than focusing on the tumor alone. 505

506

507

508




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509

510

ACKNOWLEDGEMENTS 511

The authors thank members of the Ohio State’s Comprehensive Cancer Center Genomics and Target 512

Validation Shared Resources as well as R. Kladney, D. Guttridge, M. Ostrowski, X. Liu and N. Dhomen for 513

technical support and reagents. In addition, we acknowledge A. Holderbaum and C. J. Burd for critical 514

reading of the manuscript. This work was supported by grants from the National Institute of Health 515

(R00AG036817 to C.E.B.), Melanoma Research Alliance (309669 to C.E.B.), American Federation for Aging 516

Research (C.E.B.), and Pelotonia (R.C.H. and K.M.L.). 517

518




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Table 1. Phenotypic distinctions amongst p4, UV, MMC, and CDK4/6i cells. 651

Culture condition

Edu Labelinga

% SA-β-galb

p16INK4a mRNAc

% 53BP1d

% γH2AXd

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 a Average and standard deviation from 3 independent assays are shown. 652 b Average and standard deviation from at least three views in two biological replicates. 653 c p16INK4a mRNA levels in UV, MMC, CDK4/6i cells were normalized to p4. Data indicate the average and standard 654 deviation of 3 biological replicates. 655 d Cells with >3 nuclear foci were scored as positive. Average and standard deviation from 3 independent assays are 656 shown. Bolded numbers indicate CDK4/6i values that are statistically different from both UV and MMC MEFs (p<0.001). 657 658 659 660

FIGURE LEGENDS 661

Figure 1. Prolonged PD-0332991 treatment induces senescence in primary fibroblasts. A, Representative 662

histograms of MEFs labeled with EdU for 24 hours in normal growth medium. The percentage of EdU 663

positive cells is indicated in each histogram, with quantification from 3 biological replicates shown in Table 1. 664

B, Representative images of pre-senescent (p4), UV, MMC and CDK4/6i-treated cells stained for SA-β-gal. 665

Scale bar = 100 µm. Percent positivity from 3 biological replicates appears in Table 1. C, 18s normalized 666

p16INK4a mRNA levels measured using a Taqman-based strategy. Fold differences relative to p4 were 667

calculated using the ∆∆Ct method. Data bars represent the mean of three biological replicates performed in 668

triplicate. Error bars show the standard deviation. D-E, Representative immunofluorescent staining for 669

53BP1 (C) or γH2AX (D) foci. Scale bar =100 µm. Quantification of 3 biological replicates appears in Table 1. 670

671

Figure 2. Loss of Mdm2 induces senescence in CDK4/6i fibroblasts and is accompanied by a robust SASP. A, 672

Heatmap depicting pro-inflammatory gene expression levels relative to actively proliferating fibroblasts (p4). 673




of 31

Transcripts were measured by real-time PCR in 3 biological replicates and fold change values 674

log2-transformed. Blue hues indicate decreased gene expression relative to p4. Red hues represent genes 675

with elevated expression compared to p4. B, Top, Representative immunoblots of Mdm2, p53, p21Cip1/Waf1 676

and β-Actin expression in fibroblasts. Bottom, Quantification of Mdm2, p53, and p21Cip1/Waf1 signal intensity 677

relative to β-Actin. Bars represent the average fold change over p4 from three biological replicates. Error 678

bars depict the standard deviation. * p< 0.05, ** p< 0.01 compared to p4 679

680

Figure 3. NF-κB activity triggers the SASP in CDK4/6i fibroblasts. A, Top, Representative immunoblots 681

showing p-p65 and p65 expression in fibroblast lysates. Bottom, Quantification of p-p65 a signal intensity 682

relative to p65. Bars represent the average fold change over p4 from three biological replicates. Error bars 683

depict the standard deviation. * p< 0.05, ** p< 0.01, *** p< 0.001 compared to p4. B, Representative images 684

of vehicle or PD-03329991 treated pBabe-NFκB-SR or pBabe-EV transduced fibroblasts stained for SA-β-gal. 685

Scale bar = 100 µm. C-D, Gusb normalized gene expression levels measured by real-time PCR. Fold 686

differences relative to vehicle treated pBabe-EV cells were calculated using the ∆∆Ct method. Shown is one 687

representative of two independent biological replicates. Data bars represent the mean of one biological 688

experiment performed in triplicate. Error bars show the standard deviation. * p< 0.05, ** p< 0.01, *** p< 689

0.001. 690

691

Figure 4. CDK4/6i fibroblasts exert genotype-dependent effects on melanoma growth in vitro. A, 692

Procedure for direct 2D co-culture experiments. B-H, GFP labeled Ras mutant (TRIA, NL212, NL216), Braf 693

mutant (4434 and 21015), and wild type (B16-F1 and B16-F10) melanoma cells were cultured for 4 days on 694

pre-senescent (p4) or senescent (UV, MMC, CDK4/6i) MEF monolayers. Relative cell numbers were 695




of 31

determined by measuring GFP intensity. Individual dots represent the mean of a single biological replicate 696

performed in triplicate. Error bars represent the standard deviation from ≥ 4 independent experiments 697

performed in triplicate. *Statistical comparison with no MEFs, p < 0.001; ┼ Statistical comparison with p4 cells, 698

p < 0.001; ∆ Statistical comparison with CDK4/6i cells, p < 0.001. 699

700

Figure 5. The response of melanoma cells to fibroblast conditioned media is genotype dependent. A, 701

Procedure for indirect co-culture experiments. B-H, Melanoma cells were cultured for 4 days in fresh (FR) or 702

conditioned media from pre-senescent (p4) or senescent (UV, MMC, CDK4/6i) fibroblasts, after which 703

relative cell numbers were determined by measuring GFP intensity. Individual dots represent the mean of a 704

single biological replicate performed in triplicate. Error bars represent the standard deviation from ≥ 4 705

experiments performed in triplicate. *Statistical comparison with no MEFs, p < 0.001; ┼ Statistical 706

comparison with p4 cells, p < 0.001; ∆ Statistical comparison with CDK4/6i cells, p < 0.001. 707

708

Figure 6. CDK4/6i senescent fibroblasts recruit Gr-1 positive immune cells to promote syngeneic tumor 709

growth. A-C, 5 × 105 NL212(A), 4434 (B) or B16-F10 (C) melanoma cells were injected alone into C57BL/6 710

mice, or co-injected with 5 × 105 pre-senescent (p4) or senescent (UV, MMC, CDK4/6i) fibroblasts. Tumor 711

growth was measured until exclusion criteria were met by any study group. Average tumor sizes, relative to 712

the nadir, are plotted with error bars representing the standard error of the mean. For NL212 tumors, n=8 in 713

the no MEFs and p4 groups and n=10 for UV, MMC and CDK4/6i groups. For 4434 and B16-F10 tumors, n=12 714

except for the 4434 no MEFs group where n=4 due to decreased tumor take. D-F, The percentage of 715

tumor-associated cells that stain Gr-1 positive under each experimental condition is indicated. Five views per 716

tumor were quantified, with each dot representing an individual tumor. Error bars represent the SEM. * p< 717




of 31

0.05, ** p< 0.01. G-I, The percentage of tumor-associated cells that stain Gr-1 positive under each 718

experimental condition is indicated. Five views per tumor were quantified, with each dot representing an 719

individual tumor. Error bars represent the SEM. * p< 0.05, ** p< 0.01. 720

721




Published OnlineFirst December 30, 2016.Mol Cancer Res Xiangnan Guan, Kyle M LaPak, Rebecca C Hennessey, et al. tumor growthStromal senescence by prolonged CDK4/6 inhibition potentiates

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