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STRUCTURED ABSTRACTBackgroundPatients with localized prostate cancer have historically been assigned to clinical risk groups based on local disease extent, serum prostate specific antigen (PSA), and tumor grade. Clinical risk grouping is used to determine the intensity of treatment with external beam radiotherapy (EBRT) and androgen deprivation therapy (ADT), yet a substantial proportion of patients with intermediate and high risk localized prostate cancer will develop biochemical recurrence (BCR) and require salvage therapy. Prospective identification of patients destined to experience BCR would allow treatment intensification or selection of alternative therapeutic strategies.MethodsTwenty-nine individuals with intermediate or high risk prostate cancer were prospectively recruited to a clinical trial designed to profile the molecular and imaging features of prostate cancer in patients undergoing EBRT and ADT. Whole transcriptome cDNA microarray and whole exome sequencing were performed on pretreatment targeted biopsy of prostate tumors (n=60). All patients underwent pretreatment and 6-month post EBRT multiparametric MRI (mpMRI), and were followed with serial PSA to assess presence or absence of BCR. Genes differentially expressed in the tumor of patients with and without BCR were investigated using pathways analysis tools and were similarly explored in alternative datasets. Differential gene expression and predicted pathway activation were evaluated in relation to tumor response on mpMRI and tumor genomic profile. A novel TGF-β gene signature was developed in the discovery dataset and applied to a validation dataset.FindingsBaseline MRI lesion volume andPTEN/TP53status in prostate tumor biopsies correlated with the activation state of TGF-β signaling measured using pathway analysis. All three measures correlated with the risk of BCR after definitive RT. A prostate cancer-specific TGF-β signature discriminated between patients that experienced BCR vs. those that did not. The signature retained prognostic utility in an independent cohort.InterpretationTGF-β activity is a dominant feature of intermediate-to-unfavorable risk prostate tumors prone to biochemical failure after EBRT with ADT. TGF-β activity may serve as a prognostic biomarker independent of existing risk factors and clinical decision-making criteria.FundingThis research was supported by the Prostate Cancer Foundation, the Department of Defense Congressionally Directed Medical Research Program, National Cancer Institute, and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

High-confidence fusion transcripts detected in CRPC.

PCR scheme employed for comparing relative levels of spliced and unspliced species.

Intron retention is not a sequencing or amplification artifact.

Supplementary Data from Modeling Androgen Deprivation Therapy–Induced Prostate Cancer Dormancy and Its Clinical Implications

Supplementary Methods and Materials

Supplementary Figure Legends

Supplementary Table 1. Normalized RPKM expression values and normalized Affymetrix microarray intensity values for each CRPC sample used for correlation analysis. Supplementary Table 2. Complete listing of high probability, protein-coding somatic mutations detected by RNA-seq in CRPC samples. Mutations are classified as either a nonsense or missense single nucleotide variant (SNV) or as a >1 base insertion (INS) or deletion (DEL). The gene symbol and Genbank ID is provided for each predicted amino acid change given. Supplementary Table 3. High confidence putative fusions detected by ChimeraScan (at least one breakpoint-spanning read) and deFuse (greater than 50% probability score) for each CRPC sample. Supplementary Table 4. RPKM values for approximately 45,500 transcripts for all 8 CRPC samples. Supplementary Table 5. Top 100 expressed genes as measured by the mean RPKM across all 8 CRPC samples. Supplementary Table 6. Top 100 expressed genes as measured by the median RPKM across all 8 CRPC samples. Supplementary Table 7. Novel candidate lncRNAs observed CRPC. List is sorted by average FPKM for expression of the lncRNA. Supplementary Table 8. Transcripts, gene names, and gene description for the 1,465 transcripts with the widest range of expression used for unsupervised clustering of TCGA and CRPC samples. The average RPKM difference between CRPC and TCGA primary prostate cancers is shown. Supplementary Table 9. RNA-SeQC mapping statistics for 8 CRPC RNA-seq samples. For each sample, the Illumina instrument model, RIN score, type of run, and the number of total and uniquely-mapping reads is shown. In addition, it is indicated which percentage of quality-filtered reads mapped to exon, intron or intergenic coordinates, the number of Ensembl genes determined as expressed, and the percentage of reads mapping to ribosomal RNA. Supplementary Tables 10-11. Intronic RPB values for genes in CRPC 49 and 66. Supplementary Table 12. Global analysis of splicing in CRPC. For each CRPC sample, the number of sequence reads uniquely mapping to an exon-exon splice junction or overlapping an exon-intron splice boundary is shown. The percentage of unspliced out of the total is indicated. Supplementary Table 13. Primer sequences for the detection of correctly or incorrectly spliced portions of AR, KLK2, KLK3, STEAP2, CPSF6, and CDK19. Supplementary Table 14. Complete results for ChimeraScan on CRPC samples. Supplementary Table 15. Complete results for deFuse on CRPC samples.

Clustering and pathway analysis of CRPC samples.

PHF20L1:LRRC6 fusion transcript detected in CRPC 66.

Men with metastatic prostate cancer who are treated with androgen deprivation therapies (ADT) usually relapse within 2 to 3 years with disease that is termed castration-resistant prostate cancer (CRPC). To identify the mechanism that drives these advanced tumors, paired-end RNA-sequencing (RNA-seq) was performed on a panel of CRPC bone marrow biopsy specimens. From this genome-wide approach, mutations were found in a series of genes with prostate cancer relevance, including AR, NCOR1, KDM3A, KDM4A, CHD1, SETD5, SETD7, INPP4B, RASGRP3, RASA1, TP53BP1, and CDH1, and a novel SND1:BRAF gene fusion. Among the most highly expressed transcripts were 10 noncoding RNAs (ncRNAs), including MALAT1 and PABPC1, which are involved in RNA processing. Notably, a high percentage of sequence reads mapped to introns, which were determined to be the result of incomplete splicing at canonical splice junctions. Using quantitative PCR (qPCR), a series of genes (AR, KLK2, KLK3, STEAP2, CPSF6, and CDK19) were confirmed to have a greater proportion of unspliced RNA in CRPC specimens than in normal prostate epithelium, untreated primary prostate cancer, and cultured prostate cancer cells. This inefficient coupling of transcription and mRNA splicing suggests an overall increase in transcription or defect in splicing.Implications: Inefficient splicing in advanced prostate cancer provides a selective advantage through effects on microRNA networks but may render tumors vulnerable to agents that suppress rate-limiting steps in splicing. Mol Cancer Res; 13(1); 98–106. ©2014 AACR.

Extensive intronic coverage in a subset of genes.

Heatmap representation ERG expression in CRPC samples.

Supplementary Table Legends

Positive correlation between intron and exon expression in CRPCsamples.

Prostate cancer (PCa) frequently metastasizes to bone, and analyses of DNA from bone biopsies are exceptionally challenging. A recent report in Nature demonstrates that whole-genome sequencing (WGS) of circulating tumor DNA (ctDNA) from patients with metastatic PCa can inform on the subclonal composition of metastatic disease and infer patterns of gene expression.

Purpose: Therapies targeting the androgen receptor (AR) have improved the outcome for patients with castration-sensitive prostate cancer (CSPC). Expression of the constitutively active AR splice variant-7 (AR-V7) has shown clinical utility as a predictive biomarker of AR-targeted therapy resistance in castration-resistant prostate cancer (CRPC), but its importance in CSPC remains understudied. Experimental Design: We assessed different approaches to quantify AR-V7 mRNA and protein in prostate cancer cell lines, patient-derived xenograft (PDX) models, publicly available cohorts, and independent institutional clinical cohorts, to identify reliable approaches for detecting AR-V7 mRNA and protein and its association with clinical outcome. Results: In CSPC and CRPC cohorts, AR-V7 mRNA was much less abundant when detected using reads across splice boundaries than when considering isoform-specific exonic reads. The RM7 AR-V7 antibody had increased sensitivity and specificity for AR-V7 protein detection by immunohistochemistry (IHC) in CRPC cohorts but rarely identified AR-V7 protein reactivity in CSPC cohorts, when compared with the EPR15656 AR-V7 antibody. Using multiple CRPC PDX models, we demonstrated that AR-V7 expression was exquisitely sensitive to hormonal manipulation. In CSPC institutional cohorts, AR-V7 protein quantification by either assay was associated neither with time to development of castration resistance nor with overall survival, and intense neoadjuvant androgen-deprivation therapy did not lead to significant AR-V7 mRNA or staining following treatment. Neither pre- nor posttreatment AR-V7 levels were associated with volumes of residual disease after therapy. Conclusions: This study demonstrates that further analytical validation and clinical qualification are required before AR-V7 can be considered for clinical use in CSPC as a predictive biomarker.

Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors. Implications: We have established highly clinically relevant PDXs of ADT-induced dormant prostate cancer and identified two dormancy subtypes, leading to the development of a novel predicative gene signature that allows robust risk stratification of patients with prostate cancer to ADT or androgen-receptor pathway inhibitors.

Purpose: Mechanisms mediating androgen receptor (AR) reactivation in prostate cancer that progresses after castration (castration-resistant prostate cancer; CRPC) and subsequent treatment with abiraterone (CYP17A1 inhibitor that further suppresses androgen synthesis) remain unclear.Experimental Design: Prostate cancer xenografts were examined to identify mechanism of progression after castration and abiraterone.Results: AR reactivation in abiraterone-resistant VCaP xenografts was not associated with restoration of intratumoral androgens or alterations in AR coregulators. In contrast, mRNA encoding full-length AR (AR-FL) and a constitutively active splice variant (AR-V7) were increased compared with xenografts before castration, with an increase in AR-V7 relative to AR-FL. This shift toward AR-V7 was due to a feedback mechanism whereby the androgen-liganded AR stimulates expression of proteins that suppress generation of AR-V7 relative to AR-FL transcripts. However, despite the increases in AR-V7 mRNA, it remained a minor transcript (Conclusions: AR-V7 at these low levels is not adequate to restore AR activity, but its rapid induction after androgen deprivation allows tumors to retain basal AR activity that may be needed for survival until more potent mechanisms emerge to activate AR. Agents targeting AR splice variants may be most effective when used very early in conjunction with therapies targeting the AR ligand-binding domain. Clin Cancer Res; 20(6); 1590–600. ©2014 AACR.

Supplemental Methods, Figure Legends, Table Legends, References

Supplementary Figure S3. Dutasteride directly represses DHT and progesterone stimulated AR activity.

Somatic mutations in organoids and PDXs determined by WES

Supplementary Data from A Subset of Localized Prostate Cancer Displays an Immunogenic Phenotype Associated with Losses of Key Tumor Suppressor Genes

Wnt signaling driven by genomic alterations in genes including APC and CTNNB, which encodes β-catenin, have been implicated in prostate cancer development and progression to metastatic castration-resistant prostate cancer (mCRPC). However, nongenomic drivers and downstream effectors of Wnt signaling in prostate cancer and the therapeutic potential of targeting this pathway in prostate cancer have not been fully established. Here we analyzed Wnt/β-catenin signaling in prostate cancer and identified effectors distinct from those found in other tissues, including aryl hydrocarbon receptor and RUNX1, which are linked to stem cell maintenance, and ROR1, a noncanonical Wnt5a coreceptor. Wnt/β-catenin signaling–mediated increases in ROR1 enhanced noncanonical responses to Wnt5a. Regarding upstream drivers, APC genomic loss, but not its epigenetic downregulation commonly observed in prostate cancer, was strongly associated with Wnt/β-catenin pathway activation in clinical samples. Tumor cell upregulation of the Wnt transporter Wntless (WLS) was strongly associated with Wnt/β-catenin pathway activity in primary prostate cancer but also associated with both canonical and noncanonical Wnt signaling in mCRPC. IHC confirmed tumor cell WLS expression in primary prostate cancer and mCRPC, and patient-derived prostate cancer xenografts expressing WLS were responsive to treatment with Wnt synthesis inhibitor ETC-1922159. These findings reveal that Wnt/β-catenin signaling in prostate cancer drives stem cell maintenance and invasion and primes for noncanonical Wnt signaling through ROR1. They further show that autocrine Wnt production is a nongenomic driver of canonical and noncanonical Wnt signaling in prostate cancer, which can be targeted with Wnt synthesis inhibitors to suppress tumor growth.Significance:This work provides fundamental insights into Wnt signaling and prostate cancer cell biology and indicates that a subset of prostate cancer driven by autocrine Wnt signaling is sensitive to Wnt synthesis inhibitors.

Supplementary Table S4. Mutations found only in block S tumor focus.

Figure S1: Assessment for neuroendocrine features by histology and IHC; Figure S2: Scatter plots depicting relationships between the 267-gene AR activity ssGSEA score and the 131-gene proliferation ssGSEA score for multiple genesets; Figure S3: Plots depicting correlations with AR activity ssGSEA score, and assessing GR expression; Figure S4: Segmented read depth array CGH-style plots for chromosomes showing alterations to cancer-related genes; Figure S5: Euler diagrams depicting shared and distinct somatic genomic events for cases 003, 007, 501, 503, 506, 507, 509, 512, and 520.

Mutations in 3 cases with only 1 focus dissected

Supplementary Figure from Autocrine Canonical Wnt Signaling Primes Noncanonical Signaling through ROR1 in Metastatic Castration-Resistant Prostate Cancer

Purpose:Therapies targeting the androgen receptor (AR) have improved the outcome for patients with castration-sensitive prostate cancer (CSPC). Expression of the constitutively active AR splice variant-7 (AR-V7) has shown clinical utility as a predictive biomarker of AR-targeted therapy resistance in castration-resistant prostate cancer (CRPC), but its importance in CSPC remains understudied.Experimental Design:We assessed different approaches to quantify AR-V7 mRNA and protein in prostate cancer cell lines, patient-derived xenograft (PDX) models, publicly available cohorts, and independent institutional clinical cohorts, to identify reliable approaches for detecting AR-V7 mRNA and protein and its association with clinical outcome.Results:In CSPC and CRPC cohorts, AR-V7 mRNA was much less abundant when detected using reads across splice boundaries than when considering isoform-specific exonic reads. The RM7 AR-V7 antibody had increased sensitivity and specificity for AR-V7 protein detection by immunohistochemistry (IHC) in CRPC cohorts but rarely identified AR-V7 protein reactivity in CSPC cohorts, when compared with the EPR15656 AR-V7 antibody. Using multiple CRPC PDX models, we demonstrated that AR-V7 expression was exquisitely sensitive to hormonal manipulation. In CSPC institutional cohorts, AR-V7 protein quantification by either assay was associated neither with time to development of castration resistance nor with overall survival, and intense neoadjuvant androgen-deprivation therapy did not lead to significant AR-V7 mRNA or staining following treatment. Neither pre- nor posttreatment AR-V7 levels were associated with volumes of residual disease after therapy.Conclusions:This study demonstrates that further analytical validation and clinical qualification are required before AR-V7 can be considered for clinical use in CSPC as a predictive biomarker.

Supplementary Data from Autocrine Canonical Wnt Signaling Primes Noncanonical Signaling through ROR1 in Metastatic Castration-Resistant Prostate Cancer

Figure S1. The expressions of ERBB2 and ERBB3 are not increased by androgen deprivation; Figure S2. AR mRNA expression was not significantly decreased by lapatinib in LAPC4-CR cells; Figure S3. Lapatinib can block heregulin stimulated ErbB2 activation in C4-2 cells; Figure S4. Lapatinib decreased AR protein expression at 8h treatement; Figure S5. AR mRNA expression was not significantly decreased by lapatinib or AKT-x in C4-2 cells; Figure S6. AR protein nuclear expression was decreased by siRNA against ERBB2; Figure S7. AKT inhibitor treatment decreased AR protein expression and activity; Figure S8. Silencing PIK3CB expression decreased AR protein expression.

PDF file - 97KB, Supplementary Table 1. List of genes up-regulated in abiraterone-resistant VCaP xenograft samples. Supplementary Table 2. List of genes down-regulated in abiraterone-resistant VCaP xenograft samples. Supplementary Table 3. AR mutation detected in RNA-Seq analysis.

Supplementary Figure from Assessment of Androgen Receptor Splice Variant-7 as a Biomarker of Clinical Response in Castration-Sensitive Prostate Cancer

Prostate cancer responds to therapies that suppress androgen receptor (AR) activity (androgen deprivation therapy, ADT) but invariably progresses to castration-resistant prostate cancer (CRPC). The Tec family nonreceptor tyrosine kinase BMX is activated downstream of PI3K and has been implicated in regulation of multiple pathways and in the development of cancers including prostate cancer. However, its precise mechanisms of action, and particularly its endogenous substrates, remain to be established. Here, we demonstrate that BMX expression in prostate cancer is suppressed directly by AR via binding to the BMX gene and that BMX expression is subsequently rapidly increased in response to ADT. BMX contributed to CRPC development in cell line and xenograft models by positively regulating the activities of multiple receptor tyrosine kinases through phosphorylation of a phosphotyrosine-tyrosine (pYY) motif in their activation loop, generating pYpY that is required for full kinase activity. To assess BMX activity in vivo, we generated a BMX substrate–specific antibody (anti-pYpY) and found that its reactivity correlated with BMX expression in clinical samples, supporting pYY as an in vivo substrate. Inhibition of BMX with ibrutinib (developed as an inhibitor of the related Tec kinase BTK) or another BMX inhibitor BMX-IN-1 markedly enhanced the response to castration in a prostate cancer xenograft model. These data indicate that increased BMX in response to ADT contributes to enhanced tyrosine kinase signaling and the subsequent emergence of CRPC, and that combination therapies targeting AR and BMX may be effective in a subset of patients.Significance: The tyrosine kinase BMX is negatively regulated by androgen and contributes to castration-resistant prostate cancer by enhancing the phosphorylation and activation of multiple receptor tyrosine kinases following ADT. Cancer Res; 78(18); 5203–15. ©2018 AACR.

Figure S1. Inducible BMX knockdown suppresses CWR22RV1 tumor growth in vitro and in vivo; Figure S2. Schematic picture of BMX signaling pathway; Figure S3. The effect of BMX SiRNA smart pool on CWR22RV1 cell response to serum stimulation; Figure S4. The specificity of BMX antibody; Figure S5. Affymetrix microarray expression of BMX in PCa patients; Figure S6. BMX mRNA expression is repressed by DHT; Figure S7. BMX knockdown in CWR22RV1 cells; Figure S8. AR binding site sequence in BMX loci; Figure S9. DHT effect on expression of AR and associated proteins.

Purpose: The CYP17A1 inhibitor abiraterone markedly reduces androgen precursors and is thereby effective in castration-resistant prostate cancer (CRPC). However, abiraterone increases progesterone, which can activate certain mutant androgen receptors (AR) identified previously in flutamide-resistant tumors. Therefore, we sought to determine if CYP17A1 inhibitor treatment selects for progesterone-activated mutant ARs.Experimental Design: AR was examined by targeted sequencing in metastatic tumor biopsies from 18 patients with CRPC who were progressing on a CYP17A1 inhibitor (17 on abiraterone, 1 on ketoconazole), alone or in combination with dutasteride, and by whole-exome sequencing in residual tumor in one patient treated with neoadjuvant leuprolide plus abiraterone.Results: The progesterone-activated T878A-mutant AR was present at high allele frequency in 3 of the 18 CRPC cases. It was also present in one focus of resistant tumor in the neoadjuvant-treated patient, but not in a second clonally related resistant focus that instead had lost one copy of PTEN and both copies of CHD1. The T878A mutation appeared to be less common in the subset of patients with CRPC treated with abiraterone plus dutasteride, and transfection studies showed that dutasteride was a more potent direct antagonist of the T878A versus the wild-type AR.Conclusions: These findings indicate that selection for tumor cells expressing progesterone-activated mutant ARs is a mechanism of resistance to CYP17A1 inhibition. Clin Cancer Res; 21(6); 1273–80. ©2014 AACR.See related commentary by Sharifi, p. 1240

Tables of compounds, recombinant proteins, antibodies, and primers used