Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Multiplexed live-cell imaging for drug responses in patient-derived organoid models of cancer.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to simultaneously quantitate cell health and apoptosis. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Critical role of antioxidant programs in enzalutamide-resistant prostate cancer.

Therapy resistance to second-generation androgen receptor (AR) antagonists, such as enzalutamide, is common in patients with advanced prostate cancer (PCa). To understand the metabolic alterations involved in enzalutamide resistance, we performed metabolomic, transcriptomic, and cistromic analyses of enzalutamide-sensitive and -resistant PCa cells, xenografts, patient-derived organoids, patient-derived explants, and tumors. We noted dramatically higher basal and inducible levels of reactive oxygen species (ROS) in enzalutamide-resistant PCa and castration-resistant PCa (CRPC), in comparison to enzalutamide-sensitive PCa cells or primary therapy-naive tumors respectively. Unbiased metabolomic evaluation identified that glutamine metabolism was consistently upregulated in enzalutamide-resistant PCa cells and CRPC tumors. Stable isotope tracing studies suggest that this enhanced glutamine metabolism drives an antioxidant program that allows these cells to tolerate higher basal levels of ROS. Inhibition of glutamine metabolism with either a small-molecule glutaminase inhibitor or genetic knockout of glutaminase enhanced ROS levels, and blocked the growth of enzalutamide-resistant PCa. The critical role of compensatory antioxidant pathways in maintaining enzalutamide-resistant PCa cells was validated by targeting another antioxidant program driver, ferredoxin 1. Taken together, our data identify a metabolic need to maintain antioxidant programs and a potentially targetable metabolic vulnerability in enzalutamide-resistant PCa.

B7-H3 as a Therapeutic Target in Advanced Prostate Cancer.

BACKGROUND: B7-H3 is a cell surface immunomodulatory glycoprotein overexpressed in prostate cancers (PCs). Understanding its longitudinal expression at emergence of castration resistance and association with tumour genomics are critical to the development of and patient selection for B7-H3 targeted therapies. OBJECTIVE: To characterise B7-H3 expression in same-patient hormone-sensitive (HSPC) and castration-resistant (CRPC) PC biopsies, associating this with PC genomics, and to evaluate the antitumour activity of an anti-B7-H3 antibody-drug conjugate (ADC) in human CRPC in vitro and in vivo. DESIGN, SETTING, AND PARTICIPANTS: We performed immunohistochemistry and next-generation sequencing on a cohort of 98 clinically annotated CRPC biopsies, including 72 patients who also had HSPC biopsies for analyses. We analysed two CRPC transcriptome and exome datasets, and PC scRNASeq datasets. PC organoids (patient-derived xenograft [PDX]-derived organoids [PDX-Os]) were derived from PDXs generated from human CRPC biopsies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We evaluated B7-H3 mRNA expression in relation to a panel of 770 immune-related genes, compared B7-H3 protein expression between same-patient HSPC and CRPC biopsies, determined associations with PC genomic alterations, and evaluated the antitumour activity of DS-7300a, a topoisomerase-1 inhibitor payload anti-B7-H3 ADC, in human PC cell lines, organoids (PDX-Os), and xenografts (PDXs) of different histologies, B7-H3 expressions, and genomics. RESULTS AND LIMITATIONS: B7-H3 was among the most highly expressed immunomodulatory genes in CRPCs. Most CRPCs (93%) expressed B7-H3, and in patients who developed CRPC, B7-H3 expression was frequently expressed at the time of HSPC diagnosis (97%). Conversion from B7-H3 positive to negative, or vice versa, during progression from HSPC to CRPC was uncommon. CRPC with neuroendocrine features were more likely to be B7-H3 negative (28%) than adenocarcinomas. B7-H3 is overexpressed in tumours with defective DNA repair gene (ATM and BRCA2) alterations and is associated with ERG expression, androgen receptor (AR) expression, and AR activity signature. DS7300a had antitumour activity against B7-H3 expressing human PC models including cell lines, PDX-Os, and PDXs of adenocarcinoma and neuroendocrine histology. CONCLUSIONS: The frequent overexpression of B7-H3 in CRPC compared with normal tissue and other B7 family members implicates it as a highly relevant therapeutic target in these diseases. Mechanisms driving differences in B7-H3 expression across genomic subsets warrant investigation for understanding the role of B7-H3 in cancer growth and for the clinical development of B7-H3 targeted therapies. PATIENT SUMMARY: B7-H3, a protein expressed on the surface of the most lethal prostate cancers, in particular those with specific mutations, can be targeted using drugs that bind B7-H3. These findings are relevant for the development of such drugs and for deciding which patients to treat with these new drugs.

Prostate-Specific Membrane Antigen Expression and Response to DNA Damaging Agents in Prostate Cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA) targeting therapies such as Lutetium-177 (177Lu)-PSMA-617 are affecting outcomes from metastatic castration-resistant prostate cancer (mCRPC). However, a significant subset of patients have prostate cancer cells lacking PSMA expression, raising concerns about treatment resistance attributable at least in part to heterogeneous PSMA expression. We have previously demonstrated an association between high PSMA expression and DNA damage repair defects in mCRPC biopsies and therefore hypothesized that DNA damage upregulates PSMA expression. EXPERIMENTAL DESIGN: To test this relationship between PSMA and DNA damage we conducted a screen of 147 anticancer agents (NCI/NIH FDA-approved anticancer "Oncology Set") and treated tumor cells with repeated ionizing irradiation. RESULTS: The topoisomerase-2 inhibitors, daunorubicin and mitoxantrone, were identified from the screen to upregulate PSMA protein expression in castration-resistant LNCaP95 cells; this result was validated in vitro in LNCaP, LNCaP95, and 22Rv1 cell lines and in vivo using an mCRPC patient-derived xenograft model CP286 identified to have heterogeneous PSMA expression. As double-strand DNA break induction by topoisomerase-2 inhibitors upregulated PSMA, we next studied the impact of ionizing radiation on PSMA expression; this also upregulated PSMA protein expression in a dose-dependent fashion. CONCLUSIONS: The results presented herein are the first, to our knowledge, to demonstrate that PSMA is upregulated in response to double-strand DNA damage by anticancer treatment. These data support the study of rational combinations that maximize the antitumor activity of PSMA-targeted therapeutic strategies by upregulating PSMA.

HER3 Is an Actionable Target in Advanced Prostate Cancer.

It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow-derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody-drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. SIGNIFICANCE: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials.

Commensal bacteria promote endocrine resistance in prostate cancer through androgen biosynthesis.

The microbiota comprises the microorganisms that live in close contact with the host, with mutual benefit for both counterparts. The contribution of the gut microbiota to the emergence of castration-resistant prostate cancer (CRPC) has not yet been addressed. We found that androgen deprivation in mice and humans promotes the expansion of defined commensal microbiota that contributes to the onset of castration resistance in mice. Specifically, the intestinal microbial community in mice and patients with CRPC was enriched for species capable of converting androgen precursors into active androgens. Ablation of the gut microbiota by antibiotic therapy delayed the emergence of castration resistance even in immunodeficient mice. Fecal microbiota transplantation (FMT) from CRPC mice and patients rendered mice harboring prostate cancer resistant to castration. In contrast, tumor growth was controlled by FMT from hormone-sensitive prostate cancer patients and Prevotella stercorea administration. These results reveal that the commensal gut microbiota contributes to endocrine resistance in CRPC by providing an alternative source of androgens.

JMJD6 Is a Druggable Oxygenase That Regulates AR-V7 Expression in Prostate Cancer.

Endocrine resistance (EnR) in advanced prostate cancer is fatal. EnR can be mediated by androgen receptor (AR) splice variants, with AR splice variant 7 (AR-V7) arguably the most clinically important variant. In this study, we determined proteins key to generating AR-V7, validated our findings using clinical samples, and studied splicing regulatory mechanisms in prostate cancer models. Triangulation studies identified JMJD6 as a key regulator of AR-V7, as evidenced by its upregulation with in vitro EnR, its downregulation alongside AR-V7 by bromodomain inhibition, and its identification as a top hit of a targeted siRNA screen of spliceosome-related genes. JMJD6 protein levels increased (P < 0.001) with castration resistance and were associated with higher AR-V7 levels and shorter survival (P = 0.048). JMJD6 knockdown reduced prostate cancer cell growth, AR-V7 levels, and recruitment of U2AF65 to AR pre-mRNA. Mutagenesis studies suggested that JMJD6 activity is key to the generation of AR-V7, with the catalytic machinery residing within a druggable pocket. Taken together, these data highlight the relationship between JMJD6 and AR-V7 in advanced prostate cancer and support further evaluation of JMJD6 as a therapeutic target in this disease. SIGNIFICANCE: This study identifies JMJD6 as being critical for the generation of AR-V7 in prostate cancer, where it may serve as a tractable target for therapeutic intervention.

Advanced Prostate Cancer with ATM Loss: PARP and ATR Inhibitors.

BACKGROUND: Deleterious ATM alterations are found in metastatic prostate cancer (PC); PARP inhibition has antitumour activity against this subset, but only some ATM loss PCs respond. OBJECTIVE: To characterise ATM-deficient lethal PC and to study synthetic lethal therapeutic strategies for this subset. DESIGN, SETTING, AND PARTICIPANTS: We studied advanced PC biopsies using validated immunohistochemical (IHC) and next-generation sequencing (NGS) assays. In vitro cell line models modified using CRISPR-Cas9 to impair ATM function were generated and used in drug-sensitivity and functional assays, with validation in a patient-derived model. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: ATM expression by IHC was correlated with clinical outcome using Kaplan-Meier curves and log-rank test; sensitivity to different drug combinations was assessed in the preclinical models. RESULTS AND LIMITATIONS: Overall, we detected ATM IHC loss in 68/631 (11%) PC patients in at least one biopsy, with synchronous and metachronous intrapatient heterogeneity; 46/71 (65%) biopsies with ATM loss had ATM mutations or deletions by NGS. ATM IHC loss was not associated with worse outcome from advanced disease, but ATM loss was associated with increased genomic instability (NtAI:number of subchromosomal regions with allelic imbalance extending to the telomere, p = 0.005; large-scale transitions, p = 0.05). In vitro, ATM loss PC models were sensitive to ATR inhibition, but had variable sensitivity to PARP inhibition; superior antitumour activity was seen with combined PARP and ATR inhibition in these models. CONCLUSIONS: ATM loss in PC is not always detected by targeted NGS, associates with genomic instability, and is most sensitive to combined ATR and PARP inhibition. PATIENT SUMMARY: Of aggressive prostate cancers, 10% lose the DNA repair gene ATM; this loss may identify a distinct prostate cancer subtype that is most sensitive to the combination of oral drugs targeting PARP and ATR.