Localized prostate cancer (PC) is curable, but options are limited for recurrent or metastatic tumors developing resistance to androgen-deprivation therapy (ADT) or AR targeted therapy (ART), known as metastatic castration-resistant prostate cancer (mCRPC). Overexpressed tumor antigens, such as PSMA, are targets of novel PET imaging approaches1 as well as precision therapeutics (177Lu-PSMA-617) in mCRPC (https://www.accessdata.fda.gov/drugsatfda_docs/label/2022/215833s000lbl.pdf). Identifying additional mCRPC tumor antigens contributes to new strategies to develop precision antibody-drug conjugates that permit immuno- or cellular therapies2,3.
B7-H3 is a transmembrane glycoprotein in the B7 immune checkpoint superfamily4. Other well-known members, such as PD-L1 and CTLA-4, are targets in various hematologic and solid tumors5,6,7. B7-H3 is overexpressed in several cancers including prostate cancer, with minimal expression in normal prostatic tissue8,9,10,11,12. Higher expression of B7-H3 correlates with poor cancer prognosis8. B7-H3 has implications for cancer cell transformation and metastasis, and is thought to have a significant effect on the tumor microenvironment and immune suppression11,13. New strategies have been developed to target B7-H3 through antibody-dependent cell-mediated cytotoxicity14, antibody–drug conjugates15 and linking with immunotherapy such as chimeric antigen receptor-T cell16 or NK cell therapies17. However, little is known about the molecular features and regulatory mechanisms of B7-H3 in mCRPC, which prevents the optimal design of such targeted interventions and precludes rational patient selection. To address these barriers, we characterized the genomic, transcriptomic and epigenomic features of B7-H3 expression in mCRPC.
We first evaluated transcript expression profiles of B7-H3 (CD276) and other immune-regulatory genes in mCRPC. We conducted bioinformatic interrogations on whole-exome (WES) and whole-transcriptome sequencing (WTS) data from the datasets including MSKCC 2010 (n = 131, primary PC; n = 19, CRPC)18, SU2C/PCF (n = 208, mCRPC)19, SUWC (n = 101, mCRPC)20, and GTEx (n = 245, benign prostate tissue) datasets21. The MSKCC samples included both primary and metastatic tumors processed through the same platform. mRNA expression of B7-H3 was significantly increased in metastatic PC compared to primary PC (p = 0.004) (Fig. 1a). In other mCRPC datasets, expression of B7-H3 was significantly elevated in both mCRPC datasets compared to benign prostate tissues (median TPM 115, 87 vs. 60, p < 0.0001) (Fig. 1b). We also evaluated the association of the expression of B7-H3 mRNA with protein levels and found significant association in 369 cancer cell lines (p = 1.03E-72) (Supplementary Fig. 1a). In 10 patient-derived xenograft (PDX) models of castration-resistant prostate cancer (LuCaP PDX series)22, we detected B7-H3 expression in each PDX tumor pair and found a positive trend between mRNA and protein expression (n = 10, r = 0.52, p = 0.06) (Supplementary Figs. 1b–c and 4). Based on TPM, other B7 family members including PD-L1 (CD274), PD-L2 (PDCD1LG2), and CTLA-4, exhibited reduced expression in both mCRPC datasets and had limited overall transcript abundance compared to B7-H3 (Fig. 1c–e). Other immunological markers exhibited low abundance or were not overexpressed in mCRPC (Supplementary Fig. 2). The expression of B7-H3 in mCRPC was independent of that of PSMA, and exhibited robust expression even in PSMA-low tumors (Fig. 1f–g). This suggests that targeting B7-H3 could be an attractive alternative for PSMA-negative/low mCRPC patients.
To examine how mCRPC tumor cells regulate B7-H3 expression in response to the ART, enzalutamide, we analyzed single-cell mRNA sequencing (scRNA-seq) data of paired biopsy samples from one patient (pre- and post-enzalutamide). We found an increased proportion of B7-H3-expressing tumor cells post-enzalutamide (38.5%) relative to pre-enzalutamide (17.9%) (Fig. 1h). Based on the genomic alterations in mCRPC with high B7-H3 expression, B7-H3 was associated with several known resistance markers including PTEN inactivation and AR-V7 detection19,20,23 (Supplementary Fig. 3). We conducted Gene Set Enrichment Analysis (GSEA)24 on mCRPC datasets (SU2C/PCF, n = 208 and SUWC, n = 101) and identified B7-H3 was enriched of TGF-beta, WNT, and Epithelial-to-Mesenchymal Transition (EMT) signaling pathways (Fig. 1i); each has been associated with resistance to enzalutamide25,26,27. Altogether, we found robust B7-H3 expression in mCRPC patients with existing molecular or signaling features that promote resistance to ADT and/or ART.
To enhance our mechanistic understanding of B7-H3 expression in mCRPC, we developed a machine-learning algorithm that quantitatively measures the degree of all gene-to-gene interactions to construct gene networks for all detectable genes. We used this algorithm to compare the degree of gene-network interactions between B7-H3 and all other detectable gene networks in the 208 mCRPC patients from the SU2C/PCF study. The overarching degree of gene-network association was visualized on UMAP, depicted through distances on an x-y plane. Remarkably, B7-H3 networks were closely clustered with those of AR, as well as with FOXA1, HOXB13, SPOP, MYC, and ERG (Fig. 2a). CTLA-4, PD-L1, PD-L2, and other immune markers were in distinct clusters (Fig. 2a), which indicated a lack of association with AR-signaling genes. We further examined the similarities of gene networks of B7-H3 and key regulators of AR signaling on a violin plot, in which the degree of overlap represents similarity. We observed that the B7-H3 gene network overlapped with those of AR, HOXB13, and FOXA1, and to a lesser degree with SPOP, but exhibited no intersection with PD-L1 (Fig. 2b). Altogether, these analyses suggest a robust convergence between B7-H3 and multiple genes with known functions in AR signaling.
B7-H3 is regulated epigenetically in nasopharyngeal carcinoma28 and glioblastoma29, via histone acetylation and DNA methylation at the promotor, respectively. The convergence between B7-H3 and AR signaling that we identified agreed with prior studies30. We thus sought to interrogate this mechanism of regulation through CHIP-seq data from both primary prostate cancer and mCRPC xenograft samples31,32. Remarkably, we observed enhanced histone-3-lysine-27 acetylation (H3K27ac) marks at the B7-H3 promoter and at putative B7-H3 distal enhancers in mCRPC as compared to primary prostate cancer (Fig. 2c, d), which reflected molecular mechanisms that increased transcription of B7-H3 in mCRPC. Further, we found that AR (and its co-regulators HOXB13 and FOXA1) were directly bound to B7-H3 enhancers (Fig. 2e). Notably, we found that AR exhibited selective binding to one of the B7-H3 putative enhancers in mCRPC as opposed to primary tumors (Fig. 2f). Although AR signaling is active at all stages of prostate cancer, our ChIP-seq analysis illustrated differential epigenetic regulation of B7-H3 transcripts in mCRPC compared to primary prostate cancer.
Our findings provide support that B7-H3-targeting therapies can fulfill an unmet medical need for ADT/ART-resistant mCRPC patients. Strategies to target B7-H3 with checkpoint inhibitors (NCT03729596), monoclonal antibodies (NCT02923180), antibody-drug conjugates (NCT03729596, NCT04145622), or tri-specific killer engager (TriKE) agents, are currently under investigation33,34. These therapeutics could be rationally designed for mCRPC patients that harbor ADT/ART-resistant biomarkers (e.g., PTEN loss, AR-V7 or ERG fusion) or other oncogenic signaling pathways (WNT, EMT, TGF-Beta). Further, targeting B7-H3 may be relevant in mCRPC patients with limited expression of PSMA, although our analysis did not address the status of B7-H3 in neuroendocrine/ small-cell prostate cancers. Our findings established a mechanistic connection between B7-H3 expression and AR-related signaling in mCRPC. This may also hold true in high-risk localized prostate tumors, since B7-H3 immunostaining is reduced after intense neoadjuvant ADT given before radical prostatectomy30. Finally, the epigenetic modifications we found may act as surrogates to measure B7-H3 levels from noninvasive liquid biopsies that include circulating-tumor DNA from mCRPC patients27.