Feng, Wenjia

Abstract Purpose: Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor signaling inhibitors (ARSI) is often lethal. Liquid biopsy biomarkers for this deadly form of disease remain under investigation, and underpinning mechanisms remain ill-understood. Experimental Design: We applied targeted cell-free DNA (cfDNA) sequencing to 126 patients with mCRPC from three academic cancer centers and separately performed genome-wide cfDNA methylation sequencing on 43 plasma samples collected prior to the initiation of first-line ARSI treatment. To analyze the genome-wide sequencing data, we performed nucleosome positioning and differential methylated region analysis. We additionally analyzed single-cell and bulk RNA sequencing data from 14 and 80 patients with mCRPC, respectively, to develop and validate a stem-like signature, which we inferred from cfDNA. Results: Targeted cfDNA sequencing detected AR/enhancer alterations prior to first-line ARSIs that correlated with significantly worse progression-free survival (P = 0.01; HR = 2.12) and overall survival (P = 0.02; HR = 2.48). Plasma methylome analysis revealed that AR/enhancer lethal mCRPC patients have significantly higher promoter-level hypomethylation than AR/enhancer wild-type mCRPC patients (P < 0.0001). Moreover, gene ontology and CytoTRACE analysis of nucleosomally more accessible transcription factors in cfDNA revealed enrichment for stemness-associated transcription factors in patients with lethal mCRPC. The resulting stemness signature was then validated in a completely held-out cohort of 80 patients with mCRPC profiled by tumor RNA sequencing. Conclusions: We analyzed a total of 220 patients with mCRPC, validated the importance of cell-free AR/enhancer alterations as a prognostic biomarker in lethal mCRPC, and showed that the underlying mechanism for lethality involves reprogramming developmental states toward increased stemness. See related commentary by Nawfal et al., p. 7

Abstract Background: Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor (AR)-targeted agents is often lethal. Unfortunately, biomarkers for this deadly disease remain under investigation, and underpinning mechanisms are ill-understood. Here, we applied epigenomic approaches to identify cell-free (cf) DNA features associated with these lethal AR-resistant mCRPC patients. Methods: Plasma from 99 mCRPC patients was collected from two independent institutions, either prior to first-line AR-targeted therapy (n = 63) or during treatment (n = 36). We applied EnhanceAR-Seq to detect alterations in the AR locus including the upstream enhancer. We then applied genome-wide cfDNA epigenomics to 43 pre-treatment plasma samples and performed both fragmentomics and methylation sequencing to delineate the biology of AR/enhancer-altered lethal mCRPC. Finally, we identified stemness genes by applying CytoTRACE to external single-cell RNA-seq data from mCRPC, and applied these to cfDNA via promoter-level methylation analysis. We then assessed these stem-associated genes in tumor bulk RNA-seq data from an independent mCRPC cohort. Results: AR/enhancer alterations were detected in 44% of pre-treatment cfDNA samples and correlated with significantly worse progression-free survival (PFS) (HR = 2.12, P = 0.01) and overall survival (OS) (HR = 2.48; P = 0.02). AR/enhancer alterations were detected in 19% of on-treatment cfDNA samples and were also associated with profoundly worse PFS (HR = 15.38, P = 0.0003) and OS (HR = 15.53, P = 0.002). Further, genome-wide cfDNA fragmentomic analysis revealed that binding sites for developmentally relevant transcription factors such as FOXA1, NKX-3 and HOXB13 were significantly more accessible in AR/enhancer-altered lethal mCRPC patients. Gene set enrichment analysis of the 20 most accessible transcription factors revealed several pathways associated with stem cell development. Interestingly, the 10 most stem-associated genes obtained using CytoTRACE applied to an external cohort of single-cell RNA-seq data from mCRPC (He et al. 2021) were promoter-hypomethylated in the plasma cfDNA of AR/enhancer-altered lethal mCRPC patients (P = 0.005). Further, metagene analysis of these stem-associated genes revealed worse PFS by Kaplan-Meier analysis (P = 0.04), which was confirmed by multivariate cox proportional hazards modeling. Our stemness metagene further stratified overall survival (P = 0.03) when applied to an external cohort of 80 mCRPC patients from Abida et al. 2019. Conclusions: Alterations in AR including the upstream enhancer are remarkably prognostic in mCRPC with pre-treatment predictive potential. The mechanism can be inferred via cfDNA epigenomic analysis and appears to involve increased tumor cell stemness in AR-altered lethal mCRPC. Citation Format: Pradeep Singh Chauhan, Irfan Alahi, Savar Sinha, Ryan Mueller, Alexander L. Shiang, Jace Webster, Ha X. Dang, Debanjan Saha, Lilli Greiner, Breanna Yang, Elisa M. Ledet, Ramandeep K. Babbra, Wenjia Feng, Peter K. Harris, Faridi Qaium, Ellen B. Jaeger, Patrick J. Miller, Sydney A. Caputo, Oliver A. Sartor, Russell K. Pachynski, Christopher A. Maher, Aadel A. Chaudhuri. Epigenomic analysis of plasma cell-free DNA identifies stemness features associated with worse prognosis in AR-altered lethal metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 983.