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Supplementary Figure S1. Generation of ENZR Xenografts and Cell Lines. Supplementary Figure S2. AR non-driven ENZR cells display a NE differentiation signature. Supplementary Figure S3. BRN2 expression is associated with PCa progression and a NE phenotype in human tumors. Supplementary Figure S4. BRN2 expression is inversely related to AR activity and is required for ENZ-induced NE marker expression in CRPC. Supplementary Figure S5. BRN2 overexpression induces NE marker expression and reciprocally regulates AR. Supplementary Figure S6. BRN2 activity and markers of NE differentiation are suppressed by androgens in CRPC and ENZR cells. Supplementary Figure S7. SOX2 expression is induced by ENZ and suppressed by androgens. Supplementary Figure S8. BRN2 is required for neuroendocrine marker expression and aggressive growth of CRPC cells in vitro.

Castrate-resistant prostate cancer (CRPC) and neuroendocrine carcinoma of the prostate are invariably fatal diseases for which only palliative therapies exist. As part of a prostate tumor sequencing program, a patient tumor was analyzed using Illumina genome sequencing and a matched renal capsule tumor xenograft was generated. Both tumor and xenograft had a homozygous 9p21 deletion spanning the MTAP, CDKN2, and ARF genes. It is rare for this deletion to occur in primary prostate tumors, yet approximately 10% express decreased levels of methylthioadenosine phosphorylase (MTAP) mRNA. Decreased MTAP expression is a prognosticator for poor outcome. Moreover, it seems that this deletion is more common in CRPC than in primary prostate cancer. We show for the first time that treatment with methylthioadenosine and high dose 6-thioguanine causes marked inhibition of a patient-derived neuroendocrine xenograft growth while protecting the host from 6-thioguanine toxicity. This therapeutic approach can be applied to other MTAP-deficient human cancers as deletion or hypermethylation of the MTAP gene occurs in a broad spectrum of tumors at high frequency. The combination of genome sequencing and patient-derived xenografts can identify candidate therapeutic agents and evaluate them for personalized oncology. Mol Cancer Ther; 11(3); 775–83. ©2012 AACR.

PDF file - 29K, Overlapping adult stem cell signature genes. List of overlapping genes between our dataset (this study) and our adult stem cell signature, which is downregulated in many epithelial cancers relative to normal tissue. Primary MECs overexpressing Znf217 significantly repressed genes of the adult stem cell signature (P=1.89x10-10)

PDF file - 12MB, Movie of SCp2 cells infected with vector (left movie) or Znf217 (right movie) following a scratch with a pipette tip. This movie ran for 20.25 hours

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Supplementary methods, figure legends, and primer lists.

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PEG10 levels are elevated in chemotherapy-resistant cells.

Primary antibodies used in this study

PDF file - 233K, This file contains supplemental materials and methods, including cell lines, antibodies and staining procedures used in this study

PDF file - 50K, Human microarray summary. Summary of correlations between ZNF217 and selected genes from ChIP-chip and gene expression microarray datasets. ZNF217 expression levels correlated with expression of K19 and K8/18, genes that have ZNF217 enriched at their promoters, in human breast cancer cell lines, tumors, and after knockdown in MCF7 cells

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PDF file - 576K, Immunohistochemical staining of patient #946's urethra metastasis (A) and LTL352 xenograft (B) for AR protein. Panels C and D show staining of the patient's urethra metastasis and xenograft respectively for TP63 protein.

RNAseq of ENZ-resistant cells.

Supplementary Methods

Probes used in the PCR assays

PDF file - 37K, Drug list identified in silico. List of drugs identified in silico to inhibit breast cancer cell lines that overexpress ZNF217. Correlation of ZNF217 expression in the cell line panel with the drug panel of ~50,000 drugs generated by the NCI Developmental Therapeutics Program (dtp.nci.nih.gov) identified several drugs that selectively inhibited growth of cells, assessed by GI50, expressing high levels of ZNF217 with a low drug concentration

PDF file - 46K, Gene ontology for microarray gene sets. List of gene ontology (GO) terms from the microarray gene expression datasets of primary MECs � Znf217 using DAVID software

PDF file - 117K, Microarray target genes from MCF7 siRNA. Target genes identified by microarray expression analysis from MCF7 cells � Znf217 siRNA

Supplementary Figure 1. Comparison of AR, MYC, CCND1 and CCNE1 copy number status in 5 patients with matched circulating cell-free DNA and tumor biopsies subjected to array comparative genomic hybridization

Figure S4

Supplementary Table 1. Genome copy number calls for all patients.

Supplemental Figure 3. AR full length was highly expressed in nucleus of ENZ-R compared to CR LNCaP xenograft tumors.

Supplemental Figure 5. AR-V7 enhances AR transactivation in ENZ-R cells.

Serum PSA (ng/ml) per unit tumour volume (mm3) in individual mice and in Scrb and ASO-6w2 groups

Gene expression changes in matrisome genesets associated with ASO-6w2 treatment of LTL-313BR xenografts

List of Antisense Oligonucleotides (ASOs) and qPCR Primer Sequences used.

Supplemental Figure 4. Effects of AR-ASO on AR full length and AR-V7 levels in parental LNCaP and ENZ-R MR1F cells.

Table S5

Excel file with all five supplemental tables, each contained on a separate tab. Table S1 describes the patient cohort and clinical characteristics. Table S2 describes the targets of our custom sequencing approach. Table S3 relates cfDNA yields and subsequent ctDNA fractions from all samples. Table S4 describes the somatic mutations identified. Table S5 describes the chromosomal rearrangements identified.

List of patient fusions

Purpose: Enzalutamide resistance has emerged as a major problem in the management of castration-resistant prostate cancer (CRPC). Research on therapy resistance of CRPCs has primarily focused on the androgen receptor pathway. In contrast, there is limited information on antiapoptotic mechanisms that may facilitate the treatment resistance. The inhibitor of apoptosis proteins (IAP) family is well recognized for its role in promoting treatment resistance of cancers by inhibiting drug-induced apoptosis. Here, we examined whether BIRC6, an IAP family member, has a role in enzalutamide resistance of CRPCs and could provide a therapeutic target for enzalutamide-resistant CRPC.Experimental Design: Use of enzalutamide-resistant CRPC models: (i) the transplantable, first high-fidelity LTL-313BR patient-derived enzalutamide-resistant CRPC tissue xenograft line showing primary enzalutamide resistance, (ii) MR42D and MR49F CRPC cells/xenografts showing acquired enzalutamide resistance. Specific BIRC6 downregulation in these models was produced using a BIRC6-targeting antisense oligonucleotide (ASO-6w2). Gene expression was determined by qRT-PCR and gene expression profiling. Molecular pathways associated with growth inhibition were assessed via gene enrichment analysis.Results: Of eight IAPs examined, BIRC6 was the only one showing elevated expression in both enzalutamide-resistant CRPC models. Treatment with ASO-6w2 markedly suppressed growth of LTL-313BR xenografts and increased tumor apoptosis without inducing major host toxicity. Pathway enrichment analysis indicated that GPCR and matrisome signaling were the most significantly altered pathways. Furthermore, ASO-6w2 inhibited expression of prosurvival genes that were upregulated in the LTL-313BR line.Conclusions: BIRC6 targeting inhibited the growth of enzalutamide-resistant CRPC models and may represent a new option for clinical treatment of advanced, enzalutamide-resistant prostate cancer. Clin Cancer Res; 23(6); 1542–51. ©2016 AACR.

Supplementary Methods, Figures, Table Legends Supplementary Figure 1: Representative Hematoxylin and Eosin stained photomicrographs of prostatectomy specimens demonstrating the effects of neoadjuvant chemohormonal therapy. Supplementary Figure 2: Quality metrics for targeted DNA sequencing of FFPE biopsy and RP specimens. Supplementary Figure 3: Somatic mutations detected pre- and post-treatment through targeted DNA sequencing. Supplementary Figure 4: Differences in the number and allelic frequency of somatic mutations between treated and untreated specimens. Supplementary Figure 5: Persistence of TP53 mutations in post-treated radical prostatectomy (RP) specimens. Supplementary Figure 6: Representative copy number plots from targeted sequencing of diagnostic biopsies and RP specimens from the untreated arm. Supplementary Figure 7: Pearson's correlation comparing the Nanostring platform (x axis) and RNA-seq (y axis) performed in 13 matched cases. Supplementary Figure 8: Scatterplots showing Pearson correlation between Nanostring data obtained from matched FFPE and fresh/frozen tissue in 7 patients. Supplementary Figure 9: Gene expression of PTEN (y axis) relative to genomic status (PTEN deletion or wild type (WT). Supplementary Figure 10: Left: Correlation of TMPRSS2-ERG fusion transcript expression (x axis) and ERG gene expression (y axis). Right: ERG expression vs. TMPRSS2-ERG fusion status with positive defined as normalized count> 50. Supplementary Figure 11: Principal component analysis plotted using the Biological Coefficient of Variation (BCV) distance as part of the edgeR package. Supplementary Figure 12: Unsupervised analysis of expression of all genes within the panel in radical prostatectomies from treated (green) and untreated (yellow) arms. Red= high expression; blue= low expression.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. HP1α knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management.Significance: Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(10); 2691–704. ©2018 AACR.

Purpose:An increasing number of castration-resistant prostate cancer (CRPC) tumors exhibit neuroendocrine (NE) features. NE prostate cancer (NEPC) has poor prognosis, and its development is poorly understood.Experimental Design: We applied mass spectrometry–based proteomics to a unique set of 17 prostate cancer patient–derived xenografts (PDX) to characterize the effects of castration in vivo, and the proteome differences between NEPC and prostate adenocarcinomas. Genome-wide profiling of REST-occupied regions in prostate cancer cells was correlated to the expression changes in vivo to investigate the role of the transcriptional repressor REST in castration-induced NEPC differentiation.Results:An average of 4,881 proteins were identified and quantified from each PDX. Proteins related to neurogenesis, cell-cycle regulation, and DNA repair were found upregulated and elevated in NEPC, while the reduced levels of proteins involved in mitochondrial functions suggested a prevalent glycolytic metabolism of NEPC tumors. Integration of the REST chromatin bound regions with expression changes indicated a direct role of REST in regulating neuronal gene expression in prostate cancer cells. Mechanistically, depletion of REST led to cell-cycle arrest in G1, which could be rescued by p53 knockdown. Finally, the expression of the REST-regulated gene secretagogin (SCGN) correlated with an increased risk of suffering disease relapse after radical prostatectomy.Conclusions:This study presents the first deep characterization of the proteome of NEPC and suggests that concomitant inhibition of REST and the p53 pathway would promote NEPC. We also identify SCGN as a novel prognostic marker in prostate cancer.

Purpose: Targeted agents and immunotherapies promise to transform the treatment of metastatic bladder cancer, but therapy selection will depend on practical tumor molecular stratification. Circulating tumor DNA (ctDNA) is established in several solid malignancies as a minimally invasive tool to profile the tumor genome in real-time, but is critically underexplored in bladder cancer.Experimental Design: We applied a combination of whole-exome sequencing and targeted sequencing across 50 bladder cancer driver genes to plasma cell-free DNA (cfDNA) from 51 patients with aggressive bladder cancer, including 37 with metastatic disease.Results: The majority of patients with metastasis, but only 14% of patients with localized disease, had ctDNA proportions above 2% of total cfDNA (median 16.5%, range 3.9%–72.6%). Twelve percent of estimable samples had evidence of genome hypermutation. We reveal an aggressive mutational landscape in metastatic bladder cancer with 95% of patients harboring deleterious alterations to TP53, RB1, or MDM2, and 70% harboring a mutation or disrupting rearrangement affecting chromatin modifiers such as ARID1A. Targetable alterations in MAPK/ERK or PI3K/AKT/mTOR pathways were robustly detected, including amplification of ERBB2 (20% of patients) and activating hotspot mutations in PIK3CA (20%), with the latter mutually exclusive to truncating mutations in TSC1. A novel FGFR3 gene fusion was identified in consecutive samples from one patient.Conclusions: Our study demonstrates that ctDNA provides a practical and cost-effective snapshot of driver gene status in metastatic bladder cancer. The identification of a wide spectrum of clinically informative somatic alterations nominates ctDNA as a tool to dissect disease pathogenesis and guide therapy selection in patients with metastatic bladder cancer. Clin Cancer Res; 23(21); 6487–97. ©2017 AACR.

Purpose: Gene fusions are frequently found in prostate cancer and may result in the formation of unique chimeric amino acid sequences (CASQ) that span the breakpoint of two fused gene products. This study evaluated the potential for fusion-derived CASQs to be a source of tumor neoepitopes, and determined their relationship to patterns of immune signatures in prostate cancer patients.Experimental Design: A computational strategy was used to identify CASQs and their corresponding predicted MHC class I epitopes using RNA-Seq data from The Cancer Genome Atlas of prostate tumors. In vitro peptide-specific T-cell expansion was performed to identify CASQ-reactive T cells. A multivariate analysis was used to relate patterns of in silico–predicted tumor-infiltrating immune cells with prostate tumors harboring these mutational events.Results: Eighty-seven percent of tumors contained gene fusions with a mean of 12 per tumor. In total, 41% of fusion-positive tumors were found to encode CASQs. Within these tumors, 87% gave rise to predicted MHC class I–binding epitopes. This observation was more prominent when patients were stratified into low- and intermediate/high-risk categories. One of the identified CASQ from the recurrent TMPRSS2:ERG type VI fusion contained several high-affinity HLA-restricted epitopes. These peptides bound HLA-A*02:01 in vitro and were recognized by CD8+ T cells. Finally, the presence of fusions and CASQs were associated with expression of immune cell infiltration.Conclusions: Mutanome analysis of gene fusion-derived CASQs can give rise to patient-specific predicted neoepitopes. Moreover, these fusions predicted patterns of immune cell infiltration within a subgroup of prostate cancer patients. Clin Cancer Res; 23(24); 7596–607. ©2017 AACR.

Methods for blood processing and DNA isolation.

Supplementary Table 5. Univariate and multivariate analysis examining association between pre-treatment clinico-pathological factors and progression-free survival (radiographic and/or clinical) in patients switched onto enzalutamide after collection of circulating cell-free DNA (n = 39)

Supplemental Figure Legends and Mathods. Description of supplemental figure contents and methods used in the related studies.