TMPRSS2-ERG Controls Luminal Epithelial Lineage and Antiandrogen Sensitivity in PTEN

Author Manuscript Published OnlineFirst on May 29, 2018; DOI: 10.1158/1078-0432.CCR-18-0653 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 TMPRSS2-ERG controls luminal epithelial lineage and antiandrogen sensitivity in PTEN

2 and TP53-mutated prostate cancer

4 Alexandra M. Blee1,2,10, Yundong He1,10, Yinhui Yang1,3,10, Zhenqing Ye4, Yuqian Yan1,

5 Yunqian Pan1, Tao Ma4, Joseph Dugdale1, Emily Kuehn1, Manish Kohli5, Rafael Jimenez6, Yu

6 Chen9, Wanhai Xu3, Liguo Wang4, and Haojie Huang1,7,8

8 1Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine,

9 Rochester, MN 55905, USA;

10 2Biochemistry and Molecular Biology Graduate Program, Mayo Clinic Graduate School of

11 Biomedical Sciences, Rochester, MN 55905, USA;

12 3Department of Urology, the Fourth Hospital of Harbin Medical University, Harbin,

13 Heilongjiang 150001, China;

14 4Division of Biomedical Statistics and Informatics, Department of Health Sciences Research,

15 Mayo Clinic College of Medicine, Rochester, MN 55905, USA;

16 5Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA;

17 6Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine,

18 Rochester, MN 55905, USA;

19 7Department of Urology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA;

20 8Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

21 9Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New

22 York, New York, 10065, USA.

23 10These authors contributed equally to this work.

Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 29, 2018; DOI: 10.1158/1078-0432.CCR-18-0653 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

25 Running title: ERG controls luminal lineage and antiandrogen sensitivity

26 Keywords: lineage plasticity, prostate cancer, TMPRSS2-ERG, PTEN, TP53

28 Corresponding authors: Haojie Huang, PhD, Department of Biochemistry and Molecular

29 Biology, Mayo Clinic College of Medicine, 200 First St SW, Rochester, MN, 55905, USA.

30 Email: [email protected]; LiguoWang, PhD, Division of Biomedical Statistics and

31 Informatics, Department of Health Sciences Research, Mayo Clinic College of Medicine, 200

32 First St SW, Rochester, MN 55905, USA. Email: [email protected]; or Wanhai Xu,

33 Department of Urology, the Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang

34 150001, China. Email: [email protected].

36 Conflict of Interest: The authors declare no conflicts of interest.

38 Word count: 4,539 without Materials and Methods; 6,676 with Materials and Methods

39 Total number of figures and tables: 6 Main Figures, 8 Supplementary Figures, 5

40 Supplementary Tables

42 Author contributions

43 H.H. conceived the study. A.M.B., Y.H., Y.Yang, Y.Yan, Y.P., J.D., E.K., W.X., and Y.C.

44 performed the experiments and data analyses. Z.Y., T.M., and L.W. performed bioinformatics

45 analyses. M.K. and R.J. supervised histological and IHC data analyses. A.M.B., L.W., and H.H.

46 wrote the paper.

47 Abstract

48 Purpose: Deletions or mutations in PTEN and TP53 tumor suppressor genes have been linked to

49 lineage plasticity in therapy-resistant prostate cancer. Fusion-driven overexpression of the

50 oncogenic transcription factor ERG is observed in approximately 50% of all prostate cancers,

51 many of which also harbor PTEN and TP53 alterations. However, the role of ERG in lineage

52 plasticity of PTEN/TP53-altered tumors is unclear. Understanding the collective effect of

53 multiple mutations within one tumor is essential to combat plasticity-driven therapy resistance.

54 Experimental design: We generated a Pten-negative/Trp53-mutated/ERG-overexpressing

55 mouse model of prostate cancer and integrated RNA-sequencing with ERG chromatin-

56 immunoprecipitation sequencing (ChIP-seq) to identify pathways regulated by ERG in the

57 context of Pten/Trp53 alteration. We investigated ERG-dependent sensitivity to the antiandrogen

58 enzalutamide and cyclin dependent kinases 4 and 6 (CDK4/6) inhibitor palbociclib in human

59 prostate cancer cell lines, xenografts, and allografted mouse tumors. Trends were evaluated in

60 TCGA, SU2C, and Beltran 2016 published patient cohorts and a human tissue microarray.

61 Results: Transgenic ERG expression in mice blocked Pten/Trp53 alteration-induced decrease of

62 AR expression and downstream luminal epithelial genes. ERG directly suppressed expression of

63 cell cycle-related genes, which induced RB hypophosphorylation and repressed E2F1-mediated

64 expression of mesenchymal lineage regulators, thereby restricting adenocarcinoma plasticity and

65 maintaining antiandrogen sensitivity. In ERG-negative tumors, CDK4/6 inhibition delayed tumor

66 growth.

67 Conclusion: Our studies identify a previously undefined function of ERG to restrict lineage

68 plasticity and maintain antiandrogen sensitivity in PTEN/TP53-altered prostate cancer. Our

69 findings suggest ERG fusion as a biomarker to guide treatment of PTEN/TP53-altered, RB1-

70 intact prostate cancer.

72 Translational significance

73 Prostate cancer resistance to androgen deprivation and AR-targeted therapies remains a pressing

74 clinical obstacle, partly explained by lineage plasticity and transition to AR-independent tumor

75 types in response to these therapies. A comprehensive understanding of genetic prostate tumor

76 subtypes and the unique response of each mutational subtype to AR-targeted therapies is

77 necessary to develop new, subtype-specific therapeutic strategies that overcome therapy-induced

78 lineage plasticity. Our results demonstrate that E-twenty-six transformation specific (ETS)-

79 related gene (ERG) prevents phosphatase and tensin homolog (PTEN)- and tumor protein 53

80 (TP53)-negative tumor cell lineage plasticity and antiandrogen resistance by blocking E2F1-

81 mediated expression of lineage switch genes. These findings also reveal the efficacy of targeting

82 retinoblastoma (RB)/E2F1 activity with palbociclib in ERG-negative, PTEN/TP53-altered

83 tumors. This study redefines the role of ERG in a specific tumor subtype and may guide

84 evaluation of the status of concomitant ERG fusion, PTEN/TP53 alteration, and RB1 when

85 selecting therapeutic strategies.

87 Introduction

88 Castration resistant prostate cancers respond to current antiandrogen therapies with variable

89 levels of success (1), in part due to extensive genetic heterogeneity (2-4). While mechanisms of

90 androgen receptor (AR) pathway restoration and compensation are well-documented,

91 adenocarcinoma cell lineage plasticity and reprogramming to AR-independence represents an

92 additional resistance mechanism (5). Interestingly, the incidence of AR-independent tumor

93 progression after castration and antiandrogen treatment has increased since the advent of

94 enzalutamide and abiraterone use in the clinic, highlighting that prostate cancer lineage plasticity

95 is an increasingly important barrier to overcome (6). Recent studies have identified a few key

96 molecular events involved in AR-independent tumor progression, such as RB1/PTEN/TP53 loss,

97 MYCN/AURKA amplification, and altered epigenetic regulators including EZH2 (7). However,

98 the molecular basis underlying prostate cancer lineage plasticity and antiandrogen resistance

99 remains poorly understood due to extensive patient tumor heterogeneity and model limitations.

100

101 PTEN loss frequently overlaps with TP53 mutation or loss in drug-resistant, morphologically

102 distinct, reprogrammed tumors (8-11). A significant proportion of both primary and castration

103 resistant tumors with PTEN/TP53 alteration also have AR-dependent, TMPRSS2 fusion-driven

104 overexpression of the ETS family transcription factor ERG (2-4). ERG alone has been shown to

105 repress a neural gene expression signature (12) as well as partially rescue the AR pathway under

106 PTEN loss conditions (13), but the mechanistic role of ERG in the clinically-relevant context of

107 both PTEN/TP53 alteration remains uncharacterized.

108

109 To address these gaps in the field, we generated a mouse model of prostate cancer that

110 encompasses Pten deletion, Trp53 mutation, and ERG overexpression. Notably, we revealed a

111 novel function of ERG to repress expression of a subset of cell cycle-related genes and block RB

112 hyperphosphorylation in Pten/Trp53-altered, Rb1-intact tumors. As a result, ERG-positive,

113 Pten/Trp53-altered tumors had minimal expression of E2F1 downstream targets involved in a

114 mesenchymal cell lineage switch. We extended these findings to both preclinical xenograft and

115 allograft models of tumor progression and demonstrated that ERG overexpression maintained

116 AR-positivity and sensitivity to enzalutamide. In stark contrast, ERG-negative, Pten/Trp53-

117 altered tumors were resistant to enzalutamide treatment and instead developed a reliance on the

118 RB/E2F1 pathway, which was effectively targeted with a CDK4/6 inhibitor, palbociclib. This

119 study emphasizes the importance of evaluating the individual genetic profile of tumors when

120 designing therapeutic strategies, with particular emphasis on ERG fusion, RB1, and PTEN/TP53

121 status.

122

123 Materials and Methods

124 Cell lines, cell culture and drug treatment

125 LNCaP, HEK293T, VCaP, and PC-3 cells were obtained from the American Type Culture

126 Collection (ATCC). C4-2 cells were purchased from Uro Corporation. LNCaP-RF cells were

127 described previously (14). HEK293T cells were maintained in Dulbecco’s modified eagle’s

128 medium (DMEM) supplemented with 10% fetal bovine serum (FBS). VCaP cells were

129 maintained in DMEM supplemented with 13% FBS. C4-2, LNCaP, LNCaP-RF, and PC-3 cells

130 were maintained in RPMI 1640 medium supplemented with 10% FBS. All cell lines were

131 authenticated (karyotyping, mutations in p53 and ERG fusions, and AR, PTEN, p53, and ERG

132 protein expression) and used within 6 months of thawing. No mycoplasma contamination was

133 detected in these cell lines by testing with the Lookout Mycoplasma PCR Detection Kit (Sigma-

134 Aldrich). Charcoal-stripped serum (CSS) was purchased from Thermo Fisher Scientific-Gibco

135 (#12676029). Enzalutamide was kindly provided by Medivation. LNCaP-RF cells were treated

136 with 10 µM of enzalutamide for 72 hours unless otherwise noted. Palbociclib (PD-0330991) was

137 obtained from ApexBio. LNCaP-RF cells were treated with 1 µM of palbociclib for 72 hours

138 unless otherwise noted. For combination treatment, LNCaP-RF cells were treated with 10 µM

139 enzalutamide and 1 µM palbociclib for 72 hours.

140

141 Cell transfection and lentivirus transduction

142 For lentiviral shRNA or stable plasmid expression, HEK293T cells were transiently transfected

143 with pTsin-HA-ERG FL, pTsin-HA-ERG-T1-E4, pTsin-EV, pLKO-shNT, pLKO-shRB, pLKO-

144 shERG, pLKO-shPTEN, or pLKO-shE2F1 as indicated using Lipofectamine 2000 (Thermo

145 Fisher Scientific) following manufacturer’s instructions. Virus-containing supernatant was

146 collected 48 hours post-transfection and indicated cells were infected with virus-containing

147 supernatant and 8 g/ml polybrene. Selection was performed with 1.5 g/ml puromycin.

148 Sequences of gene-specific shRNAs are listed in Supplementary Table 1. Two shRNAs per

149 gene were tested.

150

151 Co-immunoprecipitation and Western blotting

152 Co-immunoprecipitation and subsequent Western blotting was performed as described

153 previously (15). Antibodies include: anti-ERG (ab92513, Abcam; CM421C, Biocare Medical),

154 anti-PTEN (CST9559L, Cell Signaling Technology), anti-p53 (sc126, Santa Cruz

155 Biotechnology), anti-AR (sc816, Santa Cruz Biotechnology), anti-NKX3.1 (NB100-1828, Novus

156 Biologicals), anti-RB (554136, BD Biosciences), anti-pRB S795 (CST9301S, Cell Signaling

157 Technology), anti-SKP2 (32-3300, Life Technologies), anti-CCND1 (sc718, Santa Cruz

158 Biotechnology), anti-CDK1 (sc54, Santa Cruz Biotechnology), anti-TWIST (sc6269, Santa Cruz

159 Biotechnology), anti-CDH1 (610181, BD Biosciences), anti-VIM (sc73258, Santa Cruz

160 Biotechnology), anti-ERK2 (sc1647, Santa Cruz Biotechnology), anti-CDK2 (sc6248, Santa

161 Cruz Biotechnology), anti-E2F1 (sc193, Santa Cruz Biotechnology), anti-pAKT S473

162 (CST4060L, Cell Signaling Technology), anti-AKT (CST9272, Cell Signaling Technology).

163

164 qRT-PCR

165 qRT-PCR was performed as described previously (15). All quantifications were normalized to

166 the level of endogenous GAPDH gene. Primers used are listed in Supplementary Table 2.

167

168 Chromatin immunoprecipitation and qPCR

169 ChIP was performed as described previously (16). DNA was pulled down with indicated primary

170 antibodies (anti-ERG, ab92513; anti-E2F1, sc193) or nonspecific IgG. Primers to amplify DNA

171 by real-time qPCR are listed in Supplementary Table 3.

172

173 Cell proliferation assays

174 LNCaP-RF, VCaP, or PC-3 cells were seeded in 96-well plates (~3,000 cells/wells) and treated

175 as indicated. Cells were fixed at indicated timepoints (day 0 – 5) and cell growth was measured

176 using sulfohodamine B (SRB) assay (n = 5) as described previously (17).

177

178 Hematoxylin and eosin (H&E) staining

179 Four micron-thick sections were cut from formalin-fixed paraffin-embedded (FFPE) tumor

180 samples from indicated samples. Xylene washes were used to deparaffinize the tissue, followed

181 by graded ethanol washes to rehydrate tissue. Tissue was stained with hematoxylin, washed, and

182 counterstained with 1% eosin. Stained tissue was dehydrated with graded ethanol washes and a

183 final xylene wash before mounting and sealing with coverslips.

184

185 Immunohistochemistry and immunofluorescent cytochemistry

186 Four micron-thick sections were cut from FFPE tumor samples from indicated mice, xenografts,

187 or human tissue microarrays. Tissue was deparaffinized with xylene and rehydrated through

188 graded ethanol washes. Antigen retrieval and immunostaining was performed as described

189 previously (18,19). Antibodies for IHC and IFC include: anti-AR (ab108341), anti-ERG

190 (ab92513), anti-CD31 (ab28364), anti-CKAE1/3 (ab27988), anti-RB pS795 (ab85607), anti-

191 Ki67 (ab15580), anti-pAKT S473 (CST4060L), anti-CK8/18 (ab531826), anti-CK5 (ab52635),

192 anti-Vimentin (CST5741S). Ki67 and pRB S795 staining of mouse and xenograft tissues was

193 quantified by counting the number of positive cells out of 100 cells in five random fields of view

194 at 400X per mouse. Staining intensity and percentage for ERG and AR staining of human tissue

195 microarrays were graded using a set of criteria. Intensity was graded 0 to 3: 0 no staining, 1 low

196 staining, 2 medium staining, 3 strong staining. A staining index score for each tissue biopsy was

197 obtained by multiplying the staining intensity and percentage values, and used for Pearson’s

198 product-moment correlation analysis.

199

200 Gene Set Enrichment Analysis

201 Gene Set Enrichment Analysis (GSEA) was performed with a pre-ranked list of the target genes

202 identified by integrated analysis of RNA-seq and ChIP-seq data against curated datasets

203 including HALLMARK_E2F_TARGETS,

204 HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION,

205 CHARAFE_BREAST_CANCER_LUMINAL_VS_MESENCHYMAL_DOWN,

206 CHARAFE_BREAST_CANCER_LUMINAL_VS_MESENCHYMAL_UP from the Broad

207 Institute (20).

208

209 Samples from patients with prostate cancer

210 The advanced prostate cancer dataset was generated from patients undergoing standard of care

211 clinical biopsies at Mayo Clinic. A tissue microarray was constructed from the formalin-fixed,

212 paraffin-embedded (FFPE) samples of metastatic prostate cancer, identified after a search of

213 pathologic and clinical databases of archival tissues. The Mayo Clinic institutional review board

214 approved the experimental protocols for retrieving pathology blocks/slides and for accessing

215 electronic medical records. The human tissue microarray contained 157 cores (16 0.6 mm and

216 141 1.0 mm cores) resulting from 53 samples (20 bone metastases and 33 non-bone metastases)

217 from 51 patients. Cores in which greater than 50% of the tissue was lost during IHC were

218 excluded from analysis.

219

220 Meta-analysis of publicly available datasets

221 ERG fusion and genetic alterations of PTEN and TP53 for TCGA (n = 333) and SU2C (n = 150)

222 cohorts were downloaded from CBioPortal (http://www.cbioportal.org/) (21,22). ERG fusion

223 status for Beltran’s cohort (metastatic tumor specimens = 114) was downloaded from Table S5

224 of the original study (23). Only ERG fusions with RNA-seq or Nanostring evidence were

225 included into the analysis. Odds ratios were calculated in CBioPortal where odds ratio > 1

226 indicates co-occurrence and odds ratio < 1 indicates mutual exclusivity, followed by two-tailed

227 Fisher’s exact tests to determine significance of the co-occurrence or mutual exclusivity, as

228 described previously (24).

229

230 RNA-seq and data analysis

231 Total RNA was isolated from mouse prostates by homogenization of frozen tissue and purified

232 using the RNeasy Plus Mini Kit (Qiagen). 200 ng high quality total RNA was used to generate

233 the RNA sequencing library. cDNA synthesis, end-repair, A-base addition, and ligation of the

234 Illumina indexed adapters were performed according to the TruSeq RNA Sample Prep Kit v2

235 (Illumina, San Diego, CA). The concentration and size distribution of the completed libraries

236 was determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit

237 fluorometry (Invitrogen, Carlsbad, CA). Paired-end libraries were sequenced on an Illumina

238 HiSeq 4000 following Illumina’s standard protocol using the Illumina cBot and HiSeq

239 3000/4000 PE Cluster Kit. Samples were sequenced in biological triplicates and each sample

240 yielded 60-90 million paired-end reads (2×50 nucleotide read length). Base-calling was

241 performed using Illumina’s RTA software (version 2.5.2). Paired-end RNA-seq reads were

242 aligned to the mouse reference genome (GRCm38/mm10) using RNA-seq spliced read mapper

243 Tophat2 (v2.0.6) (25). Pre- and post-alignment quality controls, gene level raw read count and

244 normalized read count (i.e. FPKM) were performed using RSeQC package (v2.3.6) with NCBI

245 mouse RefSeq gene model (26). Differential gene expression analyses were conducted using

246 edgeR (version 3.6.8) and the built-in “TMM” (trimmed mean of M-values) normalization

247 method were used (27). Differentially expressed genes were determined based on the false

248 discovery rate (FDR) threshold 0.01.

249

250 ChIP-seq data analysis

251 ERG, H3K4me1, and H3K4me3 ChIP-seq data in mouse prostate tissue was downloaded from

252 NCBI Gene Expression Omnibus (GEO) with accession number GSE47119 (13). To be

253 compatible with our RNA-seq analysis results, raw reads were realigned to the mouse reference

254 genome (GRCm38/mm10) using bowtie2 (version 2.2.9) (28). MACS2 (version 2.0.10) was used

255 to identify peaks with input samples used as background and a P-value cutoff 1E-5 (macs2

256 callpeak --bdg --SPMR -f BAM) (29). ChIP-seq tag intensity tracks (bedGraph files) were

257 generated by MACS2, and then were converted into bigWig files using UCSC “wigToBigWig”

258 tool. The association of peaks to target genes was performed by Genomic Regions Enrichment of

259 Annotations Tool (GREAT) (30). ERG ChIP-seq data in VCaP cells (GSE14092) (31), E2F1

260 ChIP-seq data in PC-3 cells (GSE77448) (32), and H3K4me3 ChIP-seq data in LNCaP cells

261 (GSE43791) (33) were downloaded from GEO. ChIP-seq analysis procedure was the same as

262 described above after mapping reads to the human reference genome (GRCh37/hg19).

263

264 Generation of Pten/Trp53/ERG-altered mouse model and genotyping

265 All animal study was approved by the Mayo Clinic Institutional Animal Care and Use

266 Committee (IACUC). All mice were housed in standard conditions with a 12 hour light/12 hour

267 dark cycle and access to food and water ad libitum. The indicated groups of mice were generated

268 by crossing the following mice: Probasin (Pb)-driven Cre4 recombinase transgenic mice,

269 acquired from the National Cancer Institute (NCI) Mouse Repository and originally generated in

270 the laboratory of Dr. Pradip Roy-Burnam at University of Southern California, Los Angeles, CA

271 (34); transgenic ERG mice purchased from the Jackson Laboratory (010929), originally

272 generated in the laboratory of Dr. Valeri Vasioukhin at Fred Hutchinson Cancer Research

273 Center, Seattle, WA (35); Pten loxp/loxp conditional mice, acquired from Jackson Laboratory

274 (004597) and originally generated in the laboratory of Dr. Hong Wu at University of California,

275 Los Angeles, CA (36); Trp53 loxp/loxp conditional mice, acquired from the NCI Mouse

276 Repository and originally generated in the laboratory of Dr. Tyler Jacks at Massachusetts

277 Institute of Technology at Cambridge, MA (37); and Trp53 loxp-STOP-loxp-R172H conditional

278 mice, acquired from the NCI Mouse Repository and originally generated in the laboratory of Dr.

279 Tyler Jacks at Massachusetts Institute of Technology at Cambridge, MA (37). PCR genotyping

280 primers are listed in Supplementary Table 4.

281

282 Generation and treatment of prostate cancer cell line xenografts and mouse-derived

283 allografts

284 All animal study was approved by the Mayo Clinic IACUC. All mice were housed in standard

285 conditions with a 12 hour light/12 hour dark cycle and access to food and water ad libitum.

286 NOD-SCID IL-2 receptor -null (NSG) mice were generated in house and at six weeks of age,

287 were randomly divided into different experimental treatment groups as indicated (six mice per

288 group). For prostate cancer cell line xenografts, 5x10^6 LNCaP-RF cells per injection were

289 suspended in 0.1 ml of 50% PBS and 50% Corning Matrigel Matrix and implanted by

290 subcutaneous injection into the left flank of each NSG mouse (one implantation per mouse)

291 using a 16 gauge needle. LNCaP-RF cells were tested and ensured to be mycoplasma-free prior

292 to injection using the Lookout Mycoplasma PCR Detection Kit purchased from Sigma-Aldrich,

293 and were stably expressing either pTsin-EV or pTsin-HA-ERG-T1-E4. For mouse-derived

294 allografts, three ARlow/KRTlow DMT prostate tumors and three ARhigh/KRThigh TMT prostate

295 tumors were homogenized and 200 l of tissue per NSG mouse was implanted by subcutaneous

296 injection into the left flank of each NSG mouse (one implantation per mouse) using a 16 gauge

297 needle. Once the implanted cells grew to reach a size of 100mm3 measured externally with

298 calipers (approximately four to five weeks post-transplantation), drug treatment began. Mice

299 were treated with vehicle (100 l sodium lactate), enzalutamide (30 mg/kg/day), palbociclib (100

300 mg/kg/day), or combination by oral gavage, once daily five days per week for three weeks.

301 Mouse weight and tumor size was measured every three days by measuring tumor length (L) and

302 width (W) using a caliper, and tumor volume (TV) was calculated with the following formula:

303 TV = (L x W2)/2. Post-treatment, xenografted tissue was harvested and collected for subsequent

304 study.

305

306 Data Availability

307 The datasets generated and/or analyzed during the current study are available in the following

308 repositories. The Cancer Genome Atlas (TCGA) and Stand Up To Cancer (SU2C) datasets

309 analyzed in Fig. 1 and Supplementary Fig. 1 were accessed from CBioPortal

310 http://www.cbioportal.org/ (21,22). The Beltran cohort analyzed in Fig. 5 was downloaded from

311 Table S5 of the original study (23). The ERG, H3K4me1, and H3K4me3 ChIP-seq datasets

312 analyzed in Fig. 3 were accessed from the NCBI Gene Expression Omnibus (GEO) with

313 accession number GSE47119 (13), the ERG and H3K4me3 ChIP-seq datasets analyzed in Fig. 4

314 were accessed from the NCBI GEO with the accession numbers GSE14092 (31) and GSE43791

315 (33), and the E2F1 and H3K4me3 ChIP-seq datasets analyzed in Supplementary Fig. 5 were

316 accessed from the NCBI GEO with the accession number GSE77448 (32) and GSE43791 (33)

317 https://www.ncbi.nlm.nih.gov/geo/. The HALLMARK_E2F,

318 HALLMARK_EPITHELIAL_TO_MESENCHYMAL, and CHARAFE_BREAST_CANCER

319 datasets for Gene Set Enrichment Analysis in Fig. 3 were accessed from the Broad Institute (20)

320 http://software.broadinstitute.org/gsea/index.jsp. The RNA-seq data generated from mouse

321 prostate tissues in Fig. 3 is accessible from the NCBI GEO with the accession number

322 GSE103871.

323

324 Statistical Analysis

325 All data are shown as mean values  s.e. for experiments performed with at least three replicates.

326 Differences between two groups were analyzed using paired Student’s t-tests unless otherwise

327 noted. P values < 0.05 were considered significant.

328

329 Acknowledgements

330 This work was supported in part by grants from NIH (CA134514, CA130908, CA203849 and

331 CA193239 to H. Huang) and DOD (W81XWH-14-1-0486 to H. Huang).

332

333 Results

334 Generation and characterization of a clinically relevant Pten/Trp53/ERG triple mutant

335 mouse model

336 By mining the whole exome sequencing data from TCGA patients with primary prostate cancer

337 (PRPC) (N = 333) (3), we revealed significant co-occurrence (P = 1.11×10-6, odds ratio = 3.01,

338 95% confidence interval = 1.89 – 4.84) of PTEN/TP53 deletions or mutations with ERG gene

339 fusions, one of the most frequent genetic alterations in prostate cancer (38) (Fig. 1A, B). In

340 contrast, while a similar trend was observed in the SU2C metastatic castration-resistant prostate

341 cancer (mCRPC) patients (N = 150) (4), the correlation (P = 0.043, odds ratio = 2.04, 95%

342 confidence interval = 0.98 – 4.33) was much weaker than that in TCGA PRPC patients (Fig. 1A,

343 B). Given that AR is more commonly expressed in PRPC compared to mCRPC, especially

344 neuroendocrine CRPC (NEPC) (23,39), these data suggest that ERG fusions are prone to

345 cooperate with PTEN and TP53 gene alterations in the pathogenesis of AR-positive prostate

346 cancer. It is important to note that in the mCRPC SU2C cohort, only 3.6% of samples displayed

347 neuroendocrine (ARlow/KRTlow) features (4), which may partly explain the apparent under-

348 representation of AR loss samples in the SU2C dataset (Fig. 1A). To genetically test this

349 hypothesis in vivo we generated four cohorts of mice recapitulating the genetic alterations most

350 frequently occurring in human prostate cancers (such as R175H in TP53) (3,4,40)

351 (Supplementary Fig. 1): 1) “wild-type” (Cre-negative littermates); 2) ERG transgenic alone,

352 with Met33 N-terminally truncated ERG driven by the AR-dependent probasin (Pb) promoter

353 (hereafter termed Pb-ERG); 3) prostate-specific Pten deletion and Trp53 deletion and mutation

354 (Ptenpc-/-;Trp53pcR172H/-, hereafter termed double mutant or DMT) where Trp53 R172H is the

355 mouse equivalent to human TP53 R175H; and 4) Ptenpc-/-;Trp53pcR172H/-;Pb-ERG (hereafter

356 termed triple mutant or TMT) (Supplementary Fig. 2A). We generated these four groups of

357 mice by using Pb-driven Cre recombinase (Pb-Cre4) (34), Pb-ERG (35), Ptenloxp/loxp (36), and

358 Trp53loxp-stop-loxp-R172H/loxp (37) as breeders. For comparison, we also generated prostate-specific

359 Pten deletion (Ptenpc-/-), Pten and Trp53 double deletion (Ptenpc-/-;Trp53pc-/-) (hereafter termed

360 double knockout or DKO) mice, as well as prostate-specific Pten and Trp53 double deletion plus

361 ERG transgenic (Ptenpc-/-;Trp53pc-/-;Pb-ERG) mice (Supplementary Fig. 2A-C). Pten/Trp53

362 DKO mice have been shown to develop plastic, de-differentiated tumors (8-10,41) and served as

363 controls for comparison purposes with the Trp53 mutant lines, which represent an unstudied

364 portion of patients with PTEN deletion/TP53 mutation.

365

366 At the age of 8-10 weeks, 100% of both Ptenpc-/-;Trp53pc-/- (DKO) and Ptenpc-/-;Trp53pcR172H/-

367 (DMT) mice developed well-differentiated adenocarcinomas with high expression of AR

368 proteins (ARhigh) (Supplementary Fig. 2B). In contrast, AR expression was dramatically

369 reduced (ARlow) in prostate tumors in approximately 90% of DKO and DMT mice at the age of

370 16-20 weeks (Fig. 1C and Supplementary Fig. 2B). Consistent with the reduced AR expression,

371 the level of pan-keratin (pan-KRT) in tumors, used as an indicator of epithelial cells as opposed

372 to mesenchymal cells, was also markedly reduced (KRTlow) in both DKO and DMT mice at the

373 age of 16-20 weeks compared to mice 8-10 weeks younger (Supplementary Fig. 2B). In

374 addition, DMT tumor cells from mice at the age of 16-20 weeks were also negative for both

375 luminal epithelial cell marker KRT8/18 and basal epithelial cell marker KRT5, but positive for

376 vimentin (VIM), a mesenchymal cell marker (Fig. 1C). These results suggest that tumors in

377 DKO and DMT mice at the age of 16-20 weeks transitioned to minimal luminal epithelial

378 phenotypes and were less differentiated compared to tumors in mice at younger ages, as

379 indicated by the comparatively weak but detectable pan-keratin and AR levels. These data

380 provide support to the previous observation that loss of Pten and Trp53 induces lineage plasticity

381 in mouse prostate cancer (8-10,41).

382

383 In striking contrast, at the same age (16-20 weeks), approximately 50% of Ptenpc-/-;Trp53pcR172H/-

384 ;Pb-ERG (TMT) mice developed well-differentiated ARhigh/KRThigh adenocarcinomas while the

385 other 50% developed ARlow/KRTlow tumors reminiscent of those in DMT mice (Fig. 1C).

386 Notably, ARlow/KRTlow tumors in TMT mice lacked transgenic ERG expression in the majority

387 of tumor cells, but as expected, endogenous ERG was highly expressed in CD31-positive

388 endothelial cells of blood vessels (Fig. 1C). Importantly, lack of epithelial expression of

389 transgenic ERG correlated with decreased expression of AR proteins in these Pten/Trp53-altered

390 tumors (Fig. 1C and Supplementary Fig. 2C). This observation is supported by the previous

391 report that ERG knockdown decreases the AR-positive luminal cell population in TMPRSS2-

392 ERG-expressing VCaP prostate cancer cells (12). Together, these findings reveal that

393 PTEN/TP53 alteration induces loss of the ARhigh luminal epithelial cell lineage in prostate cancer

394 and this phenomenon is disrupted in the presence of ERG expression.

395

396 Compared to Pten wild-type prostate tissues (“wild-type” and Pb-ERG genotypes), Pten-null

397 PIN lesions in Ptenpc-/- mice or tumors in DMT and TMT mice had increased, but comparable

398 levels of phosphorylated AKT (pAKT S473) (Fig. 2A, B and Supplementary Fig. 3A),

399 reinforcing the concept that PTEN loss is a key driver of initial tumorigenesis in these models

400 (42-44). Intriguingly, plasma membrane expression of pAKT S473 was detected in the luminal

401 epithelial cells of ARhigh/KRThigh tumors in TMT mice whereas no typical plasma membrane

402 staining of pAKT S473 was detected in ARlow/KRTlow tumor cells in DMT and TMT mice (Fig.

403 2A and Supplementary Fig. 3A, B), a phenomenon reminiscent of prostate-specific Pten/Rb1

404 double KO tumors (8). At present, the exact cause-and-effect of altered cellular localization of

405 phosphorylated AKT remains to be elucidated. Previous study has shown that in the presence of

406 PTEN loss, ERG partially rescues AR function (13). However, further analyses showed that

407 ARhigh/KRThigh tumors in TMT mice had lower levels of phosphorylated RB (pRB S795) and

408 lower expression of a subset of cell cycle-promoting genes compared to ARlow/KRTlow tumors in

409 DMT and TMT mice (Fig. 2C, D, see Fig. 3 below). Furthermore, expression of cell lineage

410 regulators commonly associated with epithelial-to-mesenchymal transition (EMT) and

411 neuroendocrine cell lineage was also much lower in ARhigh/KRThigh TMT tumors than that in

412 ARlow/KRTlow tumors (Fig. 1C, see Fig. 3 below). These data argue that ERG-induced

413 preservation of the late-stage ARhigh/KRThigh phenotype was not solely mediated by restored AR

414 activity in the PTEN loss context, but may require additional drivers.

415

416 Loss of RB function has been implicated in development of plastic, antiandrogen resistant

417 prostate tumors in Pten and Trp53-deficient mice (8,10). In agreement with functional loss of RB

418 as reflected by increased RB phosphorylation (Fig. 2C, D), cell proliferation as indicated by

419 Ki67 staining was much higher in ARlow/KRTlow de-differentiated tumors in both DMT and TMT

420 mice compared to that in ARhigh/KRThigh tumors in TMT mice (Fig. 2E, F). It is worth noting

421 that proliferation in ARhigh/KRThigh tumors in TMT mice was still much higher than that in

422 malignant and nonmalignant prostate tissues in Pten KO alone and ERG transgenic alone mice,

423 respectively (Fig. 2E, F), reinforcing the concept that ERG is an oncogenic protein that promotes

424 prostate tumorigenesis by cooperating with other lesions. Nevertheless, these data suggest that

425 ERG may regulate the cell cycle and subsequently, RB activity.

426

427 ERG downregulates a subset of cell cycle-promoting genes in Pten/Trp53-altered mouse

428 prostate tumors

429 To define the molecular mechanisms by which ERG modulates prostate cancer cell lineage

430 plasticity, we performed RNA sequencing (RNA-seq) analysis in three ARlow/KRTlow tumors

431 from DMT mice and three ARhigh/KRThigh tumors in TMT mice. We selected DMT

432 ARlow/KRTlow tissues rather than TMT ARlow/KRTlow tissues for this analysis to ensure no

433 possible contamination from any tumor cells that may have low levels of ERG expression.

434 Although we did not observe strongly positive ERG-expressing tumor cells by IHC in the TMT

435 ARlow/KRTlow tissues (Fig. 1C), the presence of the Pb-ERG transgene in these mice would not

436 allow us to eliminate that possibility. RNA-seq data for one DMT tumor was excluded from

437 further analysis due to its poor correlation with the other two biological replicates

438 (Supplementary Fig. 4A). Differential gene expression analyses revealed 1,281 and 1,598 genes

439 that were significantly down- and up-regulated by ERG, respectively, in ARhigh/KRThigh TMT

440 prostate tumors in comparison to ARlow/KRTlow DMT tumors (Fig. 3A and Supplementary Fig.

441 4B). After integrating RNA-seq data with ERG chromatin immunoprecipitation-coupled

442 sequencing (ChIP-seq) data obtained from prostate tumors in Rosa26 TMPRSS2-ERG mice (13),

443 we found 76% (972 out of 1,281) of ERG-downregulated genes and 82% (1,314 out of 1,598) of

444 ERG-upregulated genes contained ERG ChIP-seq peaks in their promoter and/or enhancer

445 regions (Fig. 3A), suggesting that they are putative ERG target genes.

446

447 Gene ontology (GO) analysis of the 972 ERG-downregulated genes demonstrated a significant

448 enrichment of genes that regulate the cell cycle (Fig. 3B), in agreement with our finding that

449 ARhigh/KRThigh tumors in TMT mice display decreased RB phosphorylation and cell proliferation

450 in comparison to ARlow/KRTlow tumors in DMT and TMT mice (Fig. 2C-F). Gene set

451 enrichment analysis (GSEA) revealed that ERG-downregulated genes significantly overlap with

452 hallmark E2F targets and EMT genes (Fig. 3C). Additional comparison demonstrated that ERG-

453 downregulated genes also correlated with luminal epithelial-to-mesenchymal changes in other

454 cancer types. For examples, these genes were also significantly overlapped with genes

455 downregulated in the luminal breast cancer cell type as compared to the mesenchymal-like breast

456 cancer cell type, while ERG-upregulated genes were significantly overlapped with genes

457 upregulated in the luminal breast cancer cell type (45) (Fig. 3C). Further analysis of RNA-seq

458 profiles between ARlow/KRTlow tumors in DMT mice and ARhigh/KRThigh tumors in TMT mice

459 revealed that ERG expression resulted in drastic upregulation of AR pathway genes (e.g. Ar and

460 Nkx3.1) and luminal epithelial lineage genes (e.g. Cdh1 and Krt8), and robust downregulation of

461 cell cycle genes (e.g. Ccnd1 and Cdk1) and non-luminal epithelial (mesenchymal and

462 neuroendocrine) lineage regulatory genes (11) (e.g. Twist1 and Sox11) (Fig. 3D).

463

464 ERG-downregulated genes are exemplified in Fig. 3E and were further confirmed by reverse

465 transcription-coupled quantitative polymerase chain reaction (RT-qPCR) (Fig. 3F). RT-qPCR

466 analysis of key cell cycle and EMT genes and Western blot analysis of AR proteins further

467 confirmed that ARlow/KRTlow tumors in DMT and TMT mice shared similar molecular traits

468 (Fig. 2B and Supplementary Fig. 4C). It is important to note that although TMT ARlow/KRTlow

469 tissues were not analyzed by RNA-seq, the trends in gene expression observed in DMT

470 ARlow/KRTlow tissues were seemingly conserved in the TMT ARlow/KRTlow tissues (Fig. 2B and

471 Supplementary Fig. 4C). ERG ChIP-seq data clearly showed ERG binding peaks in the

472 promoter region of cell cycle genes such as Ccnd1, and Cdk1, but not cell lineage regulatory

473 genes such as Twist1 and Sox11 (Fig. 3E), suggesting that cell cycle-related genes are likely

474 direct targets of ERG while Twist1 and Sox11 are not. These data suggest that in the context of

475 Pten/Trp53 alteration, ERG transcriptionally downregulates a subset of key cell cycle-promoting

476 genes and maintains AR signaling.

477

478 VCaP cells harbor intact PTEN, one allele loss of TP53 and a gain-of-function mutation R248W,

479 which is a hotspot mutation in CRPC (40) (Supplementary Fig. 1). ERG is also overexpressed

480 in this cell line due to TMPRSS2-ERG fusion (TMPRSS2 exon 1 fused with ERG exon 4 or

481 termed T1-E4 ERG). Importantly, knockdown of ERG in the presence of PTEN depletion

482 increased CCND1, CDK1, and SKP2 protein levels in VCaP cells (Supplementary Fig. 5A).

483 Thus, these data, provide futher support to the hypothesis and the validation of ERG regulation

484 of a few representative gene targets defined by the integrated RNA-seq and ChIP-seq analyses.

485 Our data that ERG bound to the promoter of Ccnd1 and Cdk1 genes and repressed their

486 expression suggests ERG is a potent upstream regulator of RB hypophosphorylation and

487 activation. This notion is further supported by a recent report that in spite of the androgen-

488 stimulating effect of RB hyperphosphorylation in TMPRSS2-ERG-negative LNCaP cells, RB

489 remains hypophosphorylated in TMPRSS2-ERG-positive VCaP cells even after androgen

490 stimulation (46).

491

492 Human prostate cancer cell lines recapitulate ERG-mediated repression of the cell cycle

493 through the RB pathway

494 To delineate the relationship between ERG expression and PTEN/TP53 alteration in tumor cell

495 proliferation, cellular identity, and antiandrogen resistance, we surveyed human prostate cancer

496 cell lines including VCaP, C4-2, LNCaP, LNCaP-RF, PC-3 (Fig. 4A). Among the cell lines

497 surveyed, VCaP cells had the highest level of AR protein, hypophosphorylated RB, minimal

498 expression of cell cycle-promoting proteins CCND1, CDK1, and SKP2, and low levels of

499 mesenchymal-related proteins TWIST1 and VIM (Fig. 4A), similar to ARhigh/KRThigh tumors in

500 TMT mice (Fig. 1C). Consistent with ARlow/KRTlow DMT or DKO tumors, PTEN- and ERG-

501 negative CRPC cell lines PC-3 and LNCaP-RF, which lack or express very low levels of

502 functional p53 respectively, displayed little to no expression of AR, but increased

503 hyperphosphorylated RB and augmented expression of cell cycle-driven proteins and

504 mesenchymal-specific proteins (Fig. 4A). Overexpression of full-length or fusion (T1-E4) ERG

505 in LNCaP-RF and PC-3 cells partially reversed these trends in a dose-dependent manner (Fig.

506 4B and Supplementary Fig. 5B) and decreased cell proliferation (Fig. 4C), as detected in ERG-

507 positive, Pten/Trp53-mutated mouse prostate tumors (Fig. 2E, F). Conversely, concomitant

508 knockdown of endogenous TMPRSS2-ERG and PTEN in TP53-mutated VCaP cells, mimicking

509 the situation in ARlow/KRTlow tumors in DMT or TMT mice, resulted in increased expression of

510 cell cycle-related proteins, hyperphosphorylation of RB, upregulation of non-epithelial cell

511 markers TWIST1 and VIM, and decreased expression of AR and the epithelial cell markers

512 CDH1 and NKX3.1 (Supplementary Fig. 5A, C).

513

514 Previous study has indicated that hypophosphorylated RB can be recruited by AR to repress cell

515 cycle genes (46). Co-immunoprecipitation assay in VCaP cells demonstrated that similar to

516 previous study (31), ERG interacted with AR (Fig. 4D). However, no interaction was detected

517 between ERG and RB, and similar results were obtained in PC-3 cells stably expressing T1-E4

518 ERG (Fig. 4D), excluding the possibility that ERG may recruit RB to repress cell cycle genes in

519 a manner similar to AR (46). However, because key cell cycle regulators such as CCND1 and

520 CDK1 were identified as transcriptionally-repressed target genes of ERG, it is possible that ERG

521 causes a reduction in RB phosphorylation and cell cycle progression by directly downregulating

522 cell cycle genes. This hypothesis is consistent with decreased expression of a subset of cell cycle

523 genes in ARhigh/KRThigh adenocarcinomas in TMT mice (Fig. 3) and in human LNCaP-RF and

524 PC-3 cells stably expressing ERG (Fig. 4B). Moreover, ERG-mediated upregulation of AR and

525 downregulation of EMT genes were reversed by depletion of RB in ERG-expressing LNCaP-RF

526 cells (Fig. 4E). Similar effects were observed in PC-3 cells (Fig. 4E). These results along with

527 the ERG ChIP-seq data (Fig. 3) suggest that ERG functions as an upstream activator of RB by

528 specifically binding to the promoter and repressing expression of a subset of cell cycle-driving

529 genes.

530

531 E2F1 activates expression of EMT-promoting factors in ERG-negative, PTEN/TP53-altered

532 tumor cells

533 It has been reported recently that E2F1 promotes prostate cancer cell metastasis and enhanced

534 mesenchymal-like phenotypes (increased migration and invasion) by binding to the promoter and

535 upregulating expression of the RHAMM gene (HMMR) (47). By analyzing E2F1 ChIP-seq data

536 obtained in PC-3 cells (32), we found robust binding of E2F1 proteins in the loci of ERG-

537 suppressed mesenchymal lineage-driving genes including SNAI1, TGFB2, TWIST1, TWIST2, and

538 HMMR (Supplementary Fig. 5D). This observation was further confirmed by ChIP-qPCR

539 (Supplementary Fig. 5E). Most importantly, the effect of ERG and PTEN double knockdown

540 on expression of EMT-promoting genes and epithelial and mesenchymal cell markers in VCaP

541 cells was abrogated by concomitant knockdown of E2F1 by two independent shRNAs

542 (Supplementary Fig. 5C). This data suggests E2F1 mediates repression of downstream

543 mesenchymal lineage genes in the context of ERG+/PTEN-/p53null/mutant cells. In further support

544 of the transcriptome results in DMT and TMT mouse tumors, ectopic expression of T1-E4 ERG

545 in PC-3 cells reduced expression of CCND1, CDK1, TWIST1, and SOX11, as well as other key

546 cell cycle, EMT, and neuroendocrine-related genes (Fig. 4F and Supplementary Fig. 6A-C).

547 ERG ChIP-seq in VCaP cells and ChIP-qPCR in PC-3 cells stably expressing T1-E4 ERG

548 confirmed cell cycle genes such as CCND1 and CDK1 as direct ERG targets in human prostate

549 cancer cells, but E2F1 ChIP-seq and ChIP-qPCR in PC-3 cells demonstrated TWIST1 and other

550 cell lineage-regulatory factors as downstream gene targets of E2F1 (Fig. 4G, H, Supplementary

551 Fig. 5D, E, and Supplementary Fig. 6D, E). It should be noted that there was an observed ERG

552 ChIP-seq peak at the TGFB2 locus, although this binding could not be validated by ChIP-qPCR

553 (Fig. 4G, H). Thus, our data cannot completely rule out the possibility that ERG may also

554 potentially regulate expression of this locus. Together, these data suggest that ERG directly binds

555 to and regulates expression of a subset of cell cycle genes in human PTEN/TP53-mutated

556 prostate cancer cells, which in turn leads to RB hypophosphorylation and inhibition of E2F1-

557 mediated transcription of mesenchymal-promoting genes.

558

559 ERG and AR expression are positively associated in human prostate tumors

560 In agreement with our observations in mouse models and human prostate cancer cell lines,

561 genome analysis of CRPC adenocarcinomas (CRPC-Ad) and neuroendocrine tumors (CRPC-

562 NE) (23) revealed a significant association of ERG fusion gene expression with CRPC-Ad

563 (ARhigh), but not CRPC-NE tumors (ARlow) (P value = 0.0485, odds ratio = 0.14, 95%

564 confidence interval = 0.003 – 1.06) (Fig. 5A). Because CRPC-NE tumors have low or absent AR

565 expression and TMPRSS2-ERG gene fusion expression is driven by AR, our analyses were only

566 focused on those samples with expression of ERG gene fusion as demonstrated by RNA-seq or

567 Nanostring data (23,39). Additionally, we performed IHC analysis on a human tissue microarray

568 with 157 cores constructed from 51 metastatic CRPC patients undergoing standard of care

569 clinical biopsies at Mayo Clinic. This analysis confirmed a strong association between AR and

570 ERG expression (P value = 2.98e-7, correlation = 0.41, 95% confidence interval = 0.263 –

571 0.536) (Fig. 5B, C and Supplementary Table 5).

572

573 ERG expression in PTEN/TP53 tumors regulates prostate tumor response to antiandrogen

574 and anti-RB/E2F1 pathway drugs

575 The above findings prompted us to hypothesize that ERG+/ARhigh/KRThigh (TMT)

576 adenocarcinoma cells would respond to enzalutamide treatment, but ERG-/ARlow/KRTlow (DMT)

577 tumor cells would not. Instead, ERG-/ARlow/KRTlow (DMT) tumor cells may rely heavily on RB

578 hyperphosphorylation to maintain cell proliferation, de-differentiation and antiandrogen resistant

579 phenotypes, and therefore this type of tumor may be highly responsive to RB-targeted therapy

580 such as CDK4/6 inhibitors. Palbociclib (PD-0332991) is a CDK4/6 inhibitor that has been shown

581 to be effective in preclinical models of prostate and other cancer types, and was recently

582 approved by the United States Food and Drug Administration (FDA) for treatment of breast

583 cancer (46-50). Treatment of control LNCaP-RF cells (ERG-/ARlow) with enzalutamide alone

584 had no overt effect on expression of cell cycle genes, RB phosphorylation, and cell proliferation

585 (Fig. 6A, B and Supplementary Fig. 7A, B), confirming the antiandrogen resistant nature of

586 ERG-/ARlow cells. However, palbociclib treatment, either alone or in combination with

587 enzalutamide, significantly decreased expression of cell cycle genes and inhibited proliferation in

588 ERG-/ARlow cells (Fig. 6A, B and Supplementary Fig. 7A, B). It is interesting to note that

589 combination of enzalutamide and palbociclib significantly inhibited proliferation of the ERG-

590 /ARlow LNCaP-RF cells compared to palbociclib treatment alone (Fig. 6B), suggesting that

591 palbociclib treatment may resensitize these cells to enzalutamide treatment. As expected,

592 LNCaP-RF cells stably expressing ERG (ERG+/ARhigh) responded favorably to enzalutamide

593 alone, and such effect was not enhanced in combination with palbociclib (Fig. 6A, B). In further

594 support of the finding that RB knockdown in ERG-positive cells abrogates the subsequent down-

595 regulation of cell lineage genes (Fig. 4E), RB knockdown in LNCaP-RF-ERG T1-E4 cells also

596 abolished enzalutamide sensitivity (Supplementary Fig. 7C, D). Sensitivity to enzalutamide in

597 ERG+/ARhigh cells (LNCaP-RF-ERG T1-E4 and VCaP), but not ERG-/ARlow (LNCaP-RF-EV)

598 cells, was abrogated by androgen deprivation of culture media (Supplementary Fig. 7E, F),

599 confirming AR pathway dependence in ERG-positive cells.

600

601 We further examined the responsiveness of ERG-positive prostate cancer to antiandrogen

602 therapy using in vivo models. Similar to the findings in vitro, ERG-/ARlow LNCaP-RF xenograft

603 tumors were resistant to enzalutamide treatment (Fig. 6C and Supplementary Fig. 8A-C). In

604 contrast, treatment of these tumors with palbociclib significantly decreased tumor volume, Ki67

605 staining, and RB phosphorylation (Fig. 6C and Supplementary Fig. 8A-C). Similar to the

606 LNCaP-RF cell line study, combination of enzalutamide and palbociclib significantly decreased

607 ERG-/ARlow LNCaP-RF xenograft tumor volume compared to palbociclib treatment alone (Fig.

608 6C and Supplementary Fig. 8A), which further highlights the potential efficacy of combination

609 treatment in these tumors. In ERG+/ARhigh LNCaP-RF xenograft tumors, palbociclib treatment

610 alone exerted little to no effect but both enzalutamide treatment alone and in combination with

611 palbociclib significantly reduced the tumor volume, Ki67 staining, and expression of pRB S795

612 (Fig. 6C and Supplementary Fig. 8A-C).

613

614 We attempted to perform similar studies using DMT and TMT spontaneous tumor models.

615 However, we found it was quite challeging to crossbreed five different alleles together to

616 simultaneously generate large cohorts of DMT and TMT mice at the same ages. Due to this

617 technical difficulty, we performed similar drug treatment studies using allografts derived from

618 ERG-/ARlow/KRTlow DMT and ERG+/ARhigh/KRThigh TMT tumors and confirmed the findings

619 from LNCaP-RF xenografts (Fig. 6D, E and Supplementary Fig. 8D, E). Collectively, these

620 data highlight that PTEN/TP53-altered tumors with hyperphosphorylated RB are resistant to

621 enzalutamide, but are sensitive to CDK4/6 inhibition alone or in combination with enzalutamide.

622 In contrast, ERG expression maintains antiandrogen sensitivity in tumors even with PTEN/TP53

623 alteration and this effect is related to ERG-induced inhibition of cell cycle gene expression and

624 restored AR signaling.

625

626 Discussion

627 The findings in the current study emphasize that the unique combination of genetic mutations

628 present within a single prostate tumor can greatly affect response to androgen- and AR-targeted

629 therapies. In particular, our study of the novel Pten/Trp53/ERG triple mutant mouse model of

630 prostate cancer recapitulates a trio of genetic events that co-occur in a significant subtype of

631 prostate tumors. Previous studies demonstrated that loss of Pten and Trp53 induces lineage

632 plasticity in mouse prostate cancer, where prostate-specific Pten and Trp53 double KO mice

633 develop prostate adenocarcinoma at a young age and further evolve into ARlow/KRTlow tumors

634 (8-10,41). These data and ours support the hypothesis that Pten/Trp53-altered tumors may

635 transition from an ARhigh/KRThigh adenocarcinoma to an altered ARlow/KRTlow state (Fig. 6F,

636 left).

637

638 Further analysis of the novel Pten/Trp53/ERG model revealed that ERG binds to chromatin loci

639 of a subset of cell cycle-driving genes and decreases their expression in Pten/Trp53-altered

640 mouse prostate tumors, thereby preventing loss of RB activity and E2F1-mediated cellular

641 reprogramming (Fig. 6F, right). Studies in human prostate cancer cell lines also supported these

642 findings. Most importantly, similar results were obtained through analysis of patient datasets and

643 clinical samples. Although previous studies have suggested a potential role for ERG in

644 repressing neuroendocrine differentiation and partially rescuing AR function (12,13), this study

645 represents the first to demonstrate ERG-mediated protection of the epithelial adenocarcinoma

646 cell lineage in a clinically-relevant mouse model with Pten/Trp53 mutations (Fig. 6F).

647

648 We further demonstrated in Pten/Trp53-mutated mouse prostate cancer and xenograft models

649 that while ERG-positive tumors are sensitive to antiandrogen treatment, ERG-negative tumors

650 have no overt response to antiandrogens and instead respond well to the CDK4/6 inhibitor

651 palbociclib. These findings were recapitulated in human cell lines. Together, these data reveal a

652 previously undefined role of ERG in maintaining neoplastic epithelial cell identity and

653 antiandrogen sensitivy in PTEN/TP53-mutated prostate cancer and highlight that different

654 therapeutic strategies are needed for PTEN/TP53-altered tumors with or without ERG (Fig. 6F).

655

656 Despite a previous finding that ERG overexpression alone is sufficient for focal prostatic

657 intraepithelial neoplasia (PIN) formation in mice with 129/Sv background (35), we did not

658 observe any PIN lesions in Pb-ERG mice during the course of our previous study (51) and this

659 report (Fig. 1C) perhaps due to the different genetic (C57BL6/129) background of mice we used.

660 ERG expression levels in Pb-ERG mouse prostate tissue were comparable to that in the

661 TMPRSS2-ERG fusion-positive human VCaP cell line (see Supplementary Fig. 2D).

662 Nevertheless, our findings are consistent with other reports that ERG alone is not sufficient to

663 promote prostate tumorigenesis in mice within the studied time frame (51-53). The Pb-ERG

664 transgenic mouse model also provides the unique ability to study AR-dependent transgenic

665 expression of ERG, which mimics AR-driven TMPRSS2-ERG expression in human prostate

666 tumors (35). For these reasons, this model system is particularly relevant and analogous to

667 human prostate cancer. However, it is important to note that the reduced AR expression observed

668 in approximately 50% of the tumors in TMT mice could contribute to absence of AR-dependent,

669 Pb-promoter-driven expression of ERG. The exact underlying molecular mechanism warrants

670 further investigation, and future studies will explore the exact cause-and-effect of reduced AR

671 expression and absence of ERG expression in the ARlow/KRTlow subset of TMT mice. Additional

672 studies with a more robust knock-in model of ERG (13) would be particularly useful to better

673 characterize this mechanism, although slightly less physiologically relevant.

674

675 These findings in prostate cancer also raise the larger question of whether the mechanism defined

676 in the current study might be applicable to other RB alteration-related cancer types such as lung

677 cancer (genomic loss of RB promotes the transition from adenocarcinoma to small cell lung

678 cancer) (54) and breast cancer (functional loss of RB due to HER2 amplification leads to

679 formation of non-luminal breast cancer). Nevertheless, our findings support the evaluation of

680 ERG fusion as a viable biomarker to guide antiandrogen and RB pathway-targeted therapies for

681 PTEN/TP53-mutated, RB1-intact prostate cancer. Studies such as these will be essential to

682 combat lineage plasticity-mediated therapy resistance in prostate cancer as well as other cancers.

683

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849

850

851 Figure Legends

852

853 Figure 1. ERG tempers PTEN/TP53 alteration-induced loss of ARhigh luminal epithelial

854 cells.

855 A, Oncoprint image with percentage of ERG, PTEN, TP53, and AR genetic alterations in 333

856 primary prostate cancer patient samples (top, TCGA cohort (3)) and 150 advanced mCRPC

857 patient samples (bottom, SU2C cohort (4)). B, Contingency tables used by Fisher’s exact test

858 (two-tailed) to examine association between ERG fusion and PTEN/TP53 alterations in primary

859 TCGA (left) and mCRPC SU2C (right) cohorts. C, Histological characterization of mouse

860 prostate tissue from 16-20 weeks of age. Wild-type n=8, Pb-ERG n=9, DMT n=10, TMT n=12.

861 Top, hematoxylin and eosin (H&E) staining. Subsequent rows, immunohistochemistry (IHC) for

862 AR, ERG, CD31, Pan-KRT, KRT8/18, KRT5, Vimentin. CD31 as an endothelial cell marker to

863 distinguish between endogenous endothelial versus transgenic ERG. Asterisk in Vimentin IHC

864 tissue indicates a stromal compartment that is distinct from the Vimentinlow adenocarcinoma.

865

866

867 Figure 2. ERG prevents Pten/Trp53 alteration-induced proliferation and loss of membrane-

868 localized phosphorylated AKT in mouse prostate tumors.

869 A, IHC for pAKT S473 in mouse prostate tissues from 16-20 weeks of age. Wild-type n=8, Pb-

870 ERG n=9, DMT n=10, TMT n=12, Ptenpc-/- n= 8. B, Protein levels of pAKT S473, total AKT,

871 and AR in mouse prostate tissues at 16-20 weeks of age. Both blots for each protein of interest

872 were exposed and developed on the same piece of film. ERK2 as a loading control. Band

873 intensity was quantified and normalized to ERK2 for each lane. Asterisk = outlier samples with

874 significantly low levels of total protein. C, IHC for pRB S795 in mouse prostate tissues from 16-

875 20 weeks of age as described in (A). D, Quantification of pRB S795 staining as shown in (C). E,

876 IHC for Ki67 in mouse prostate tissues from 16-20 weeks of age as described in (A). F,

877 Quantification of Ki67 IHC as shown in (E).

878

879 Figure 3. ERG expression down-regulates key cell cycle-driving genes and maintains both

880 AR pathway and epithelial gene expression in mouse prostate tumors.

881 A, Venn diagram indicating overlap between up- or down-regulated genes in DMT

882 ARlow/KRTlow (n=2) versus TMT ARhigh/KRThigh (n=3) tumors and ERG target genes identified

883 by ChIP-sequencing (13). Fisher’s exact test (assuming human genome code for 27,000 genes,

884 estimated from RefSeq) for ERG ChIP-seq vs down-regulated genes: P < 0.001; for ERG ChIP-

885 seq vs up-regulated genes: P < 9.088e-23. B, Gene Ontology analysis of 972 ERG target genes

886 down-regulated in TMT ARhigh/KRThigh tumors, ranked by P-value. C, Gene Set Enrichment

887 Analysis (GSEA) for all 2,595 up- or down-regulated ERG target genes (mouse gene names

888 converted to human homologues using NCBI Homology Map). D, Heatmap showing

889 differentially expressed genes between two DMT ARlow/KRTlow and three TMT ARhigh/KRThigh

890 tumors, highlighting a subset of genes involved in cell cycle, AR pathway, and epithelial-to-

891 mesenchymal transition (EMT). E, RNA-seq and ChIP-seq track views from UCSC Genome

892 Browser for two ERG predicted target genes (Ccnd1, Cdk1) with ERG binding peaks and two

893 predicted passenger genes (Sox11, Twist1) without ERG binding peaks. Peaks underlined with

894 black bars and boxed with a dashed red line indicate significant ERG ChIP-seq peaks with P

895 value = 1e-5 or lower as determined by MACS. ERG ChIP-seq input tracks shown as a control

896 for true ERG peaks. H3K4me3 peaks shown to indicate gene promoter regions. H3K4me1 peaks

897 shown to indicate gene enhancer regions. F, RT-qPCR of n=2 DMT ARlow/KRTlow and n=3

898 TMT ARhigh/KRThigh tumors for Ccnd1, Cdk1, Twist1, and Sox11. Relative to Gapdh.

899

900

901 Figure 4. ERG binds to the promoter and regulates expression of cell cycle genes in human

902 PTEN/TP53-altered prostate cancer cells.

903 A, Western blot analysis of expression of key AR pathway, cell cycle, and EMT-related proteins

904 in five prostate cancer cell lines. B, Western blot analysis of expression of key AR pathway, cell

905 cycle, and EMT-related proteins in LNCaP-RF and PC-3 cell lines after lentiviral-mediated

906 expression of full-length (ERG-FL) or ERG (T1-E4). C, Cell proliferation as measured by SRB

907 assay for LNCaP-RF and PC-3 cell lines with lentiviral-mediated expression of ERG-FL or ERG

908 (T1-E4). D, Top, co-immunoprecipitation of endogenous ERG and RB in VCaP cells. ERG and

909 AR co-immunoprecipitation shown as positive control. Bottom, co-immunoprecipitation of ERG

910 and RB in PC-3 cells stably expressing ERG (T1-E4). E, Western blot analysis of expression of

911 key AR pathway and EMT-related genes in LNCaP-RF and PC-3 cell lines after lentiviral-

912 mediated expression of ERG (T1-E4) with or without RB knockdown. F, RT-qPCR of CCND1,

913 CDK1, SKP2, FOXM1, TWIST1, TWIST2, SOX11, and TGFB2 in PC-3 cells with or without

914 lentiviral-mediated expression of ERG (T1-E4). Relative to GAPDH. G, ERG ChIP-seq tracks in

915 VCaP cells (GSE14092) (31) and H3K4me3 (histone mark of promoters) ChIP-seq tracks in

916 LNCaP cells (GSE43791) (33) from UCSC genome browser for CCND1, CDK1, SKP2, FOXM1,

917 TWIST1, TWIST2, SOX11, and TGFB2. ERG ChIP-seq input tracks shown as a control for true

918 ERG peaks. Peaks underlined with black bars and boxed with a dashed red line indicate

919 significant ERG ChIP-seq peaks with P value = 1e-5 or lower as determined by MACS. Asterisk

920 indicates ERG peak that could not be validated by ChIP-qPCR. H, ERG ChIP-qPCR of CCND1,

921 CDK1, SKP2, FOXM1, TWIST1, TWIST2, SOX11, and TGFB2 in PC-3 cells with lentiviral-

922 mediated expression of ERG (T1-E4).

923

924 Figure 5. ERG expression correlates with AR expression in human patient datasets.

925 A, Fisher’s exact test to determine association between ERG fusion and CRPC-adenocarcinoma

926 (CRPC-Ad) or CRPC-neuroendocrine (CRPC-NE) tumor subtypes from the Beltran cohort (23).

927 B, Representative IHC images for AR and ERG from human tissue microarray of metastic CRPC

928 obtained from Mayo Clinic. C, Pearson’s product-moment correlation between AR and ERG

929 IHC staining in clinical biopsies of metastatic CRPC in (B).

930

931

932 Figure 6. Differential responses of ERG-positive and ERG-negative human xenograft and

933 mouse allograft tumors with PTEN/TP53 alterations to enzalutamide and palbociclib.

934 A, Western blot analysis of expression of key AR pathway, cell cycle, and EMT-related proteins

935 in LNCaP-RF cells with or without lentiviral-mediated ERG (T1-E4) expression after treatment

936 with vehicle, enzalutamide (ENZ, 10 µM), palbociclib (PD, 1 µM), or combination (ENZ + PD).

937 B, Cell proliferation as measured by SRB assay for LNCaP-RF cells with or without lentiviral-

938 mediated ERG (T1-E4) expression after treatment with vehicle, ENZ (10 µM), PD (1 µM), or

939 combination. C, LNCaP-RF xenograft tumor volume with or without lentiviral-mediated ERG

940 (T1-E4) expression during three weeks treatment with vehicle, ENZ (30 mg/kg/day), PD (100

941 mg/kg/day), or combination. Six xenografts (n = 6) per cell line, per drug treatment. D, ERG-

942 /ARlow/KRTlow DMT and ERG+/ARhigh/KRThigh TMT allograft tumor volume during three weeks

943 of treatment with vehicle, ENZ (30 mg/kg/day), PD (100 mg/kg/day), or combination. Five

944 allografts (n = 5) per genotype, per drug treatment. E, Characterization of allograft tumors from

945 (D) after three weeks of treatment. Top, H&E. Subsequent rows, IHC for ERG, AR, pRB S795,

946 and Ki67. F, A hypothetical model. In prostate cancer cells without the TMPRSS2-ERG fusion,

947 PTEN deletion/mutation and TP53 deletion/mutation favor cell cycle gene expression, CDK

948 activation, and RB inhibition (hyperphosphorylation), which in turn leads to E2F1 activation and

949 luminal-epithelial-to-mesenchymal cell identity transition, antiandrogen resistance and increased

950 CDK4/6 inhibitor sensitivity. In contrast, in prostate cancer cells harboring the TMPRSS2-ERG

951 fusion, overexpression of ERG results in decreased expression of a subset of cell cycle-

952 promoting genes and RB activation (hypophosphorylation), thereby leading to E2F1 inhibition

953 and maintenance of luminal epithelial cell identity, increased antiandrogen sensitivity, but

954 CDK4/6 inhibitor resistance.

TMPRSS2-ERG controls luminal epithelial lineage and antiandrogen sensitivity in PTEN and TP53-mutated prostate cancer

Alexandra M Blee, Yundong He, Yinhui Yang, et al.

Clin Cancer Res Published OnlineFirst May 29, 2018.

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