Early chemotherapy-induced gonad toxicity threatens future fertility in boys, yet early in-vivo testicular responses are poorly defined. To characterize acute effects of cyclophosphamide (CPA) on the prepubertal testis and explore whether combined antioxidants (AO;L-carnitine [LC] and N-acetyl cysteine [NAC]) modulate these changes. CBA/B6 F1 male pups (postnatal day 7–9) were randomized to saline control, CPA (100 mg/kg i.p.), AO, or CPA+AO. Testes were collected every 8 h to 48 h for histology/immunostaining and were pooled (n = 3 per group/time point) for bulk RNA-seq per group/time point. Histology showed emerging degeneration from ∼32 h with prominent effects by 48 h after CPA, including reduced germ cell layers, increased γH2AX/CC3, and decreased Ki67. Transcriptionally, CPA perturbed apoptosis/developmental pathways as early as 16 h, preceding overt histological change. AO and CPA+AO groups displayed partial transcriptional shifts toward control profiles, consistent with mitigation of CPA-associated signatures, but not full normalization. In neonatal mouse testis, CPA elicits rapid transcriptomic reprogramming within 16 h, before morphological injury at ∼32–48 h. Concomitant AO shows preliminary, partial protective transcriptional effects. These proof-of-concept data support transcriptomics as an early, sensitive readout of testicular toxicity and motivate follow-up studies with independent validation and long-term outcomes prior to clinical translation.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal disease that resists therapy targeting androgen signaling, the primary driver of prostate cancer. mCRPC resists androgen receptor (AR) inhibitors by amplifying AR signaling or by evolving into therapy-resistant subtypes that do not depend on AR. Elucidation of the epigenetic underpinnings of these subtypes could provide important insights into the drivers of therapy resistance. In this study, we produced chromatin accessibility maps linked to the binding of lineage-specific transcription factors (TF) by performing assay for transposase-accessible chromatin sequencing on 70 mCRPC tissue biopsies integrated with transcriptome and whole-genome sequencing. mCRPC had a distinct global chromatin accessibility profile linked to AR function. Analysis of TF occupancy across accessible chromatin revealed 203 TFs associated with mCRPC subtypes. Notably, ZNF263 was identified as a putative prostate cancer TF with a significant impact on gene activity in the double-negative subtype (AR− neuroendocrine−), potentially activating MYC targets. Overall, this analysis of chromatin accessibility in mCRPC provides valuable insights into epigenetic changes that occur during progression to mCRPC.

Tumors escape immune detection and elimination through a variety of mechanisms. Here, we used prostate cancer as a model to examine how androgen-dependent tumors undergo immune evasion through downregulation of the major histocompatibility complex class I (MHCI). We report that response to immunotherapy in late-stage prostate cancer is associated with elevated MHC expression. To uncover the mechanism, we performed a whole genome CRISPRi screen and identified AR as a repressor of the MHCI pathway. Syngeneic mouse models of aggressive prostate cancer deficient in AR also demonstrated increased tumor immunogenicity and promoted T cell mediated tumor-control. Notably, the increase in MHCI expression upon androgen receptor blockade is transient and correlates with resistance to AR inhibition. Mechanistic studies identified androgen response elements upstream of MHCI transcription start sites which increased MHCI expression when deleted. Together, this body of work highlights another mechanism by which hormones can promote immune escape.

The widespread use of potent androgen receptor signaling inhibitors (ARSIs) has led to an increasing emergence of AR-independent castration-resistant prostate cancer (CRPC), typically driven by loss of AR expression, lineage plasticity, and transformation to prostate cancers (PCs) that exhibit phenotypes of neuroendocrine or basal-like cells. The anti-apoptotic protein BCL2 is upregulated in neuroendocrine cancers and may be a therapeutic target for this aggressive PC disease subset. There is an unmet clinical need, therefore, to clinically characterize BCL2 expression in metastatic CRPC (mCRPC), determine its association with AR expression, uncover its mechanisms of regulation, and evaluate BCL2 as a therapeutic target and/or biomarker with clinical utility. Here, using multiple PC biopsy cohorts and models, we demonstrate that BCL2 expression is enriched in AR-negative mCRPC, associating with shorter overall survival and resistance to ARSIs. Moreover, high BCL2 expression associates with lineage plasticity features and neuroendocrine marker positivity. We provide evidence that BCL2 expression is regulated by DNA methylation, associated with epithelial-mesenchymal transition, and increased by the neuronal transcription factor ASCL1. Finally, BCL2 inhibition had antitumor activity in some, but not all, BCL2-positive PC models, highlighting the need for combination strategies to enhance tumor cell apoptosis and enrich response.

Transcription factor (TF) proteins regulate gene activity by binding to regulatory regions, most importantly at gene promoters. Many genes have alternative promoters (APs) bound by distinct TFs. The role of differential TF activity at APs during tumour development is poorly understood. Here we show, using deep RNA sequencing in 274 biopsies of benign prostate tissue, localized prostate tumours and metastatic castration-resistant prostate cancer, that AP usage increases as tumours progress and APs are responsible for a disproportionate amount of tumour transcriptional activity. Expression of the androgen receptor (AR), the key driver of prostate tumour activity, is correlated with elevated AP usage. We identified AR, FOXA1 and MYC as potential drivers of AP activation. DNA methylation is a likely mechanism for AP activation during tumour progression and lineage plasticity. Our data suggest that prostate tumours activate APs to magnify the transcriptional impact of tumour drivers, including AR and MYC.

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.

Genomic alterations resulting in loss of control over the cell cycle is a fundamental hallmark of human malignancies. Whilst pan-cancer studies have broadly assessed tumour genomics and their impact on oncogenic pathways, analyses taking the baseline signalling levels in normal tissue into account are lacking. To this end, we aimed to reclassify the cell cycle activity of tumours in terms of their tissue of origin and determine if any common DNA mutations, chromosome arm-level changes or signalling pathways contribute to an increase in baseline corrected cell cycle activity. Combining normal tissue and pan-cancer data from over 13,000 samples we demonstrate that tumours of gynaecological origin show the highest levels of corrected cell cycle activity, partially owing to hormonal signalling and gene expression changes. We also show that normal and tumour tissues can be separated into groups (quadrants) of low/high cell cycle activity and propose the hypothesis of an upper limit on these activity levels in tumours.

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease.