The Genomic and Epigenomic Landscape of Double-Negative Metastatic Prostate Cancer

Abstract

Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen signaling. Androgen deprivation therapy and second-generation androgen receptor (AR)-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by AR and neuroendocrine (NE) markers. Molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterized treatment-emergent mCRPC by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing from 210 tumors. AR-/NE- tumors were clinically and molecularly distinct from other mCRPC subtypes, with the shortest survival, amplification of the chromatin remodeler CHD7, and PTEN loss. Methylation changes in CHD7 candidate enhancers were linked to elevated CHD7 expression in AR-/NE+ tumors. Genome-wide methylation analysis nominated Krüppel-like factor 5 (KLF5) as a driver of the AR-/NE- phenotype, and KLF5 activity was linked to RB1 loss. These observations reveal the aggressiveness of AR-/NE- mCRPC and could facilitate the identification of therapeutic targets in this highly aggressive disease.

Significance: Comprehensive characterization of the five subtypes of metastatic castration-resistant prostate cancer identified transcription factors that drive each subtype and showed that the double-negative subtype has the worst prognosis.

Figure 1.

mCRPC tumors cluster into five groups using the expression of androgen, neuroendocrine, and squamous gene panels. A and B, Heat map representing RNA-seq gene expression level of AR, NE, and squamous (SQUAM) gene panels of mCRPC tumors from the WCDT cohort (A; refs. 5, 11) and the Beltran and colleagues cohort (B; ref. 7). Results are expressed as log₂ TPM (z-score) and colored from low (blue) to high (yellow) expression level. AR gene panel includes AR and AR-regulated genes, NE gene panels (NE1 and NE2) include NE related genes, and SQUAM panel includes genes associated with squamous cell differentiation. The expression levels of genes included in neuroendocrine prostate cancer (NEPC) panel from Beltran and colleagues cohort (7) were used to assign a binary classification (Binary class) of the samples based on their gene expression. White, adenocarcinoma tumors; black, small cell NEPC. AR and NEPC signature scores were calculated based on the AR and NEPC-related gene expression values as reported previously (7). The tumor subtypes can be read as follows: AR+/NE−, dark turquoise; ARL/NE−, dark orange; AR−/NE−, light purple; AR−/NE+, pink; AR+/NE+, light green.

Figure 2.

Distinct clinical outcomes associated with the five subtypes of mCRPC. A, Heat map representing results of ssGSEAs and colored according to the legends. B, Kaplan–Meier curves representing clinical outcome of patients in the WCDT cohort, using survival from date of biopsy acquisition as the clinical outcome. Pairwise test conducted between AR−/NE− and other subtypes. The tumor subtypes can be read as follows: AR+/NE−, dark turquoise; ARL/NE−, dark orange; AR−/NE−, light purple; AR−/NE+, pink; AR+/NE+, light green.

Figure 3.

Somatic and structural alterations associated with subtypes of mCRPC. A, Top rows show mCRPC subtypes, ETS family fusions, TMPRSS2-ERG fusions, tumor purity, and tumor ploidy in the WCDT cohort. Bottom rows show occurrence of AR, AR enhancer, PTEN, RB1, TP53, MYC, BRCA2, and CHD7 alterations in each sample. Tumors are sorted by their subtypes. Alteration frequency is shown to the right. B, Bar plots representing alteration frequency (%) of AR, PTEN, RB1, TP53, MYC, BRCA2, and CHD7 genes within each subtype. In both panels, types of alterations are colored (and/or marked with symbols) according to the legends.

Figure 4.

Hypomethylation in the putative enhancer regions of CHD7 is correlated with elevated gene expression in AR−/NE+. Integration of gene expression and DNA-methylation data for the CHD7 gene. A, Box plots representing CHD7 gene expression in the five mCRPC subtypes, colored according to the key below the plot. B, Top, chromosomal location of the CHD7 gene along with H3K27ac ChIP-seq marker, DHS, and DMRs in AR−/NE+ tumors compared with AR+/NE−. Bottom, ChIP-seq data for ASCL1 in different cell lines as indicated in the panels. The vertical dashed green and red lines show the transcription start site and transcription end site of the CHD7 gene, respectively. The yellow bar indicates the canonical promoter region of CHD7. C–F, Box plots showing mean methylation level per sample in DMR1 (C), DMR2 (D), DMR3 (E), and DMR4 (F) for AR−/NE−, AR−/NE+, and AR+/NE− subtypes. Pearson correlations were calculated between CHD7 gene expression and mean methylation of each sample at DMRs1–4. Box plots should be interpreted as follows: horizontal lines, median values; boxes extend from the 25th to the 75th percentile of each group's distribution of values; vertical extending lines, adjacent values (the most extreme values within 1.5 interquartile range of the 25th and 75th percentile of each group). Differences between groups were assessed by the Kruskal–Wallis test. Significance is indicated as follows: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. G, Venn diagram representing the overlap between the top 10 TF motifs enriched at each DMR location. Neuroendocrine-lineage motifs found in DMRs are labeled in the panel.

Figure 5.

Gene expression and DNA methylation analysis converges on KLF5 TF in AR−/NE− tumors. A, Heat map representing differentially expressed TFs in five subtypes of mCRPC. B, Heat map representing top 20 enriched TFs in hypomethylated regions of the five mCRPC subtypes. TFs are ranked by log (P value). The color intensity indicates the rank of the TFs from most enriched (dark red) to least enriched (white).

Figure 6.

Association between KLF5 TF enrichment and RB1 gene loss in AR−/NE− tumors. A, Rank order plots show the enrichment rank of KLF5 in AR−/NE− and AR−/NE+ subtypes on the left to right. Dashed red color indicates rank 20. B, Bar plots showing the gene set enrichment analyses for genes mapped to the KLF5 motif. Dashed line, FDR = 0.05. C, Scatter plots representing Spearman correlation between KLF5 gene expression and KRT5, KRT8, RB1, and CCNB2 genes. D, Scatter plots showing the relation between RB1 gene expression and RB1 copy numbers (top row), KLF5 gene expression and KLF5 copy numbers (middle row), and KLF5 gene expression and RB1 copy number (bottom row).