Reproducible preclinical models of androgen receptor driven human prostate cancer bone metastasis.

BACKGROUND: Preclinical models recapitulating the metastatic phenotypes are essential for developing the next-generation therapies for metastatic prostate cancer (mPC). We aimed to establish a cohort of clinically relevant mPC models, particularly androgen receptor positive (AR+) bone metastasis models, from LuCaP patient-derived xenografts (PDX) that reflect the heterogeneity and complexity of mPC. METHODS: PDX tumors were dissociated into single cells, modified to express luciferase, and were inoculated into NSG mice via intracardiac injection. The progression of metastases was monitored by bioluminescent imaging. Histological phenotypes of metastases were characterized by immunohistochemistry and immunofluorescence staining. Castration responses were further investigated in two AR-positive models. RESULTS: Our PDX-derived metastasis (PDM) model collection comprises three AR+ adenocarcinomas (ARPC) and one AR- neuroendocrine carcinoma (NEPC). All ARPC models developed bone metastases with either an osteoblastic, osteolytic, or mixed phenotype, while the NEPC model mainly developed brain metastasis. Different mechanisms of castration resistance were observed in two AR+ PDM models with distinct genotypes, such as combined loss of TP53 and RB1 in one model and expression of AR splice variant 7 (AR-V7) expression in another model. Intriguingly, the castration-resistant tumors displayed inter- and intra-tumor as well as organ-specific heterogeneity in lineage specification. CONCLUSION: Genetically diverse PDM models provide a clinically relevant system for biomarker identification and personalized medicine in metastatic castration-resistant prostate cancer.

Stem cell dynamics and cellular heterogeneity across lineage subtypes of castrate-resistant prostate cancer.

To resist lineage-dependent therapies such as androgen receptor inhibition, prostate luminal epithelial adenocarcinoma cells often adopt a stem-like state resulting in lineage plasticity and phenotypic heterogeneity. Castrate-resistant prostate adenocarcinoma can transition to neuroendocrine (NE) and occasionally to amphicrine, co-expressed luminal and NE, phenotypes. We developed castrate-resistant prostate cancer (CRPC) patient-derived organoid models that preserve heterogeneity of the originating tumor, including an amphicrine model displaying a range of luminal and NE phenotypes. To gain biological insight and to identify potential treatment targets within heterogeneous tumor cell populations, we assessed the lineage hierarchy and molecular characteristics of various CRPC tumor subpopulations. Transcriptionally similar stem/progenitor (St/Pr) cells were identified for all lineage populations. Lineage tracing in amphicrine CRPC showed that heterogeneity originated from distinct subclones of infrequent St/Pr cells that produced mainly quiescent differentiated amphicrine progeny. By contrast, adenocarcinoma CRPC progeny originated from St/Pr cells and self-renewing differentiated luminal cells. Neuroendocrine prostate cancer (NEPC) was composed almost exclusively of self-renewing St/Pr cells. Amphicrine subpopulations were enriched for secretory luminal, mesenchymal, and enzalutamide treatment persistent signatures that characterize clinical progression. Finally, the amphicrine St/Pr subpopulation was specifically depleted with an AURKA inhibitor, which blocked tumor growth. These data illuminate distinct stem cell (SC) characteristics for subtype-specific CRPC in addition to demonstrating a context for targeting differentiation-competent prostate SCs.

Localized high-risk prostate cancer harbors an androgen receptor low subpopulation susceptible to HER2 inhibition.

Patients diagnosed with localized high-risk prostate cancer have higher rates of recurrence, and the introduction of neoadjuvant intensive hormonal therapies seeks to treat occult micrometastatic disease by their addition to definitive treatment. Sufficient profiling of baseline disease has remained a challenge in enabling the in-depth assessment of phenotypes associated with exceptional vs. poor pathologic responses after treatment. In this study, we report comprehensive and integrative gene expression profiling of 37 locally advanced prostate tumors prior to six months of androgen deprivation therapy (ADT) plus the androgen receptor (AR) inhibitor enzalutamide prior to radical prostatectomy. A robust transcriptional program associated with HER2 activity was positively associated with poor outcome and opposed AR activity, even after adjusting for common genomic alterations in prostate cancer including PTEN loss and expression of the TMPRSS2:ERG fusion. Patients experiencing exceptional pathologic responses demonstrated lower levels of HER2 and phospho-HER2 by immunohistochemistry of biopsy tissues. The inverse correlation of AR and HER2 activity was found to be a universal feature of all aggressive prostate tumors, validated by transcriptional profiling an external cohort of 121 patients and immunostaining of tumors from 84 additional patients. Importantly, the AR activity-low, HER2 activity-high cells that resist ADT are a pre-existing subset of cells that can be targeted by HER2 inhibition alone or in combination with enzalutamide. In summary, we show that prostate tumors adopt an AR activity-low prior to antiandrogen exposure that can be exploited by treatment with HER2 inhibitors.

Tumor-derived biomarkers predict efficacy of B7H3 antibody-drug conjugate treatment in metastatic prostate cancer models.

Antibody-drug conjugates (ADCs) are a promising targeted cancer therapy; however, patient selection based solely on target antigen expression without consideration for cytotoxic payload vulnerabilities has plateaued clinical benefits. Biomarkers to capture patients who might benefit from specific ADCs have not been systematically determined for any cancer. We present a comprehensive therapeutic and biomarker analysis of a B7H3-ADC with pyrrolobenzodiazepine(PBD) payload in 26 treatment-resistant, metastatic prostate cancer (mPC) models. B7H3 is a tumor-specific surface protein widely expressed in mPC, and PBD is a DNA cross-linking agent. B7H3 expression was necessary but not sufficient for B7H3-PBD-ADC responsiveness. RB1 deficiency and/or replication stress, characteristics of poor prognosis, and conferred sensitivity were associated with complete tumor regression in both neuroendocrine (NEPC) and androgen receptor positive (ARPC) prostate cancer models, even with low B7H3 levels. Non-ARPC models, which are currently lacking efficacious treatment, demonstrated the highest replication stress and were most sensitive to treatment. In RB1 WT ARPC tumors, SLFN11 expression or select DNA repair mutations in SLFN11 nonexpressors governed response. Importantly, WT TP53 predicted nonresponsiveness (7 of 8 models). Overall, biomarker-focused selection of models led to high efficacy of in vivo treatment. These data enable a paradigm shift to biomarker-driven trial designs for maximizing clinical benefit of ADC therapies.

Radiogenomic profiling of prostate tumors prior to external beam radiotherapy converges on a transcriptomic signature of TGF-ß activity driving tumor recurrence.

Background: Patients with localized prostate cancer have historically been assigned to clinical risk groups based on local disease extent, serum prostate specific antigen (PSA), and tumor grade. Clinical risk grouping is used to determine the intensity of treatment with external beam radiotherapy (EBRT) and androgen deprivation therapy (ADT), yet a substantial proportion of patients with intermediate and high risk localized prostate cancer will develop biochemical recurrence (BCR) and require salvage therapy. Prospective identification of patients destined to experience BCR would allow treatment intensification or selection of alternative therapeutic strategies. Methods: Twenty-nine individuals with intermediate or high risk prostate cancer were prospectively recruited to a clinical trial designed to profile the molecular and imaging features of prostate cancer in patients undergoing EBRT and ADT. Whole transcriptome cDNA microarray and whole exome sequencing were performed on pretreatment targeted biopsy of prostate tumors (n=60). All patients underwent pretreatment and 6-month post EBRT multiparametric MRI (mpMRI), and were followed with serial PSA to assess presence or absence of BCR. Genes differentially expressed in the tumor of patients with and without BCR were investigated using pathways analysis tools and were similarly explored in alternative datasets. Differential gene expression and predicted pathway activation were evaluated in relation to tumor response on mpMRI and tumor genomic profile. A novel TGF-ß gene signature was developed in the discovery dataset and applied to a validation dataset. Findings: Baseline MRI lesion volume and PTEN/TP53 status in prostate tumor biopsies correlated with the activation state of TGF-ß signaling measured using pathway analysis. All three measures correlated with the risk of BCR after definitive RT. A prostate cancer-specific TGF-ß signature discriminated between patients that experienced BCR vs. those that did not. The signature retained prognostic utility in an independent cohort. Interpretation: TGF-ß activity is a dominant feature of intermediate-to-unfavorable risk prostate tumors prone to biochemical failure after EBRT with ADT. TGF-ß activity may serve as a prognostic biomarker independent of existing risk factors and clinical decision-making criteria. Funding: This research was supported by the Prostate Cancer Foundation, the Department of Defense Congressionally Directed Medical Research Program, National Cancer Institute, and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Defining cellular population dynamics at single-cell resolution during prostate cancer progression.

Advanced prostate malignancies are a leading cause of cancer-related deaths in men, in large part due to our incomplete understanding of cellular drivers of disease progression. We investigate prostate cancer cell dynamics at single-cell resolution from disease onset to the development of androgen independence in an in vivo murine model. We observe an expansion of a castration-resistant intermediate luminal cell type that correlates with treatment resistance and poor prognosis in human patients. Moreover, transformed epithelial cells and associated fibroblasts create a microenvironment conducive to pro-tumorigenic immune infiltration, which is partially androgen responsive. Androgen-independent prostate cancer leads to significant diversification of intermediate luminal cell populations characterized by a range of androgen signaling activity, which is inversely correlated with proliferation and mRNA translation. Accordingly, distinct epithelial populations are exquisitely sensitive to translation inhibition, which leads to epithelial cell death, loss of pro-tumorigenic signaling, and decreased tumor heterogeneity. Our findings reveal a complex tumor environment largely dominated by castration-resistant luminal cells and immunosuppressive infiltrates.

Progression of prostate cancer reprograms MYC-mediated lipid metabolism via lysine methyltransferase 2A.

BACKGROUND: The activities of MYC, the androgen receptor, and its associated pioneer factors demonstrate substantial reprogramming between early and advanced prostate cancer. Although previous studies have shown a shift in cellular metabolic requirements associated with prostate cancer progression, the epigenetic regulation of these processes is incompletely described. Here, we have integrated chromatin immunoprecipitation sequencing (ChIP-seq) and whole-transcriptome sequencing to identify novel regulators of metabolism in advanced prostate tumors characterized by elevated MYC activity. RESULTS: Using ChIP-seq against MYC, HOXB13, and AR in LNCaP cells, we observed redistribution of co-bound sites suggestive of differential KMT2A activity as a function of MYC expression. In a cohort of 177 laser-capture microdissected foci of prostate tumors, KMT2A expression was positively correlated with MYC activity, AR activity, and HOXB13 expression, but decreased with tumor grade severity. However, KMT2A expression was negatively correlated with these factors in 25 LuCaP patient-derived xenograft models of advanced prostate cancer and 99 laser-capture microdissected foci of metastatic castration-resistant prostate cancer. Stratified by KMT2A expression, ChIP-seq against AR and HOXB13 in 15 LuCaP patient-derived xenografts showed an inverse association with sites involving genes implicated in lipid metabolism, including the arachidonic acid metabolic enzyme PLA2G4F. LuCaP patient-derived xenograft models grown as organoids recapitulated the inverse association between KMT2A expression and fluorine-18 labeled arachidonic acid uptake in vitro. CONCLUSIONS: Our study demonstrates that the epigenetic activity of transcription factor oncogenes exhibits a shift during prostate cancer progression with distinctive phenotypic effects on metabolism. These epigenetically driven changes in lipid metabolism may serve as novel targets for the development of novel imaging agents and therapeutics.

Assessment of Androgen Receptor Splice Variant-7 as a Biomarker of Clinical Response in Castration-Sensitive Prostate Cancer.

PURPOSE: Therapies targeting the androgen receptor (AR) have improved the outcome for patients with castration-sensitive prostate cancer (CSPC). Expression of the constitutively active AR splice variant-7 (AR-V7) has shown clinical utility as a predictive biomarker of AR-targeted therapy resistance in castration-resistant prostate cancer (CRPC), but its importance in CSPC remains understudied. EXPERIMENTAL DESIGN: We assessed different approaches to quantify AR-V7 mRNA and protein in prostate cancer cell lines, patient-derived xenograft (PDX) models, publicly available cohorts, and independent institutional clinical cohorts, to identify reliable approaches for detecting AR-V7 mRNA and protein and its association with clinical outcome. RESULTS: In CSPC and CRPC cohorts, AR-V7 mRNA was much less abundant when detected using reads across splice boundaries than when considering isoform-specific exonic reads. The RM7 AR-V7 antibody had increased sensitivity and specificity for AR-V7 protein detection by immunohistochemistry (IHC) in CRPC cohorts but rarely identified AR-V7 protein reactivity in CSPC cohorts, when compared with the EPR15656 AR-V7 antibody. Using multiple CRPC PDX models, we demonstrated that AR-V7 expression was exquisitely sensitive to hormonal manipulation. In CSPC institutional cohorts, AR-V7 protein quantification by either assay was associated neither with time to development of castration resistance nor with overall survival, and intense neoadjuvant androgen-deprivation therapy did not lead to significant AR-V7 mRNA or staining following treatment. Neither pre- nor posttreatment AR-V7 levels were associated with volumes of residual disease after therapy. CONCLUSIONS: This study demonstrates that further analytical validation and clinical qualification are required before AR-V7 can be considered for clinical use in CSPC as a predictive biomarker.

Prostate-Specific Membrane Antigen Is a Biomarker for Residual Disease following Neoadjuvant Intense Androgen Deprivation Therapy in Prostate Cancer.

PURPOSE: Neoadjuvant intense androgen deprivation therapy (iADT) can exert a wide range of histological responses, which in turn are reflected in the final prostatectomy specimen. Accurate identification and measurement of residual tumor volumes are critical for tracking and stratifying patient outcomes. MATERIALS AND METHODS: The goal of this current study was to evaluate the ability of antibodies against prostate-specific membrane antigen (PSMA) to specifically detect residual tumor in a cohort of 35 patients treated with iADT plus enzalutamide for 6 months prior to radical prostatectomy. RESULTS: Residual carcinoma was detected in 31 patients, and PSMA reacted positively with tumor in all cases. PSMA staining was 96% sensitive for tumor, with approximately 82% of benign regions showing no reactivity. By contrast, PSMA positively reacted with 72% of benign regions in a control cohort of 37 untreated cases, resulting in 28% specificity for tumor. PSMA further identified highly dedifferentiated prostate carcinomas including tumors with evidence of neuroendocrine differentiation. CONCLUSIONS: We propose that anti-PSMA immunostaining be a standardized marker for identifying residual cancer in the setting of iADT.

Modeling Androgen Deprivation Therapy-Induced Prostate Cancer Dormancy and Its Clinical Implications.

Treatment-induced tumor dormancy is a state in cancer progression where residual disease is present but remains asymptomatic. Dormant cancer cells are treatment-resistant and responsible for cancer recurrence and metastasis. Prostate cancer treated with androgen-deprivation therapy (ADT) often enters a dormant state. ADT-induced prostate cancer dormancy remains poorly understood due to the challenge in acquiring clinical dormant prostate cancer cells and the lack of representative models. In this study, we aimed to develop clinically relevant models for studying ADT-induced prostate cancer dormancy. Dormant prostate cancer models were established by castrating mice bearing patient-derived xenografts (PDX) of hormonal naïve or sensitive prostate cancer. Dormancy status and tumor relapse were monitored and evaluated. Paired pre- and postcastration (dormant) PDX tissues were subjected to morphologic and transcriptome profiling analyses. As a result, we established eleven ADT-induced dormant prostate cancer models that closely mimicked the clinical courses of ADT-treated prostate cancer. We identified two ADT-induced dormancy subtypes that differed in morphology, gene expression, and relapse rates. We discovered transcriptomic differences in precastration PDXs that predisposed the dormancy response to ADT. We further developed a dormancy subtype-based, predisposed gene signature that was significantly associated with ADT response in hormonal naïve prostate cancer and clinical outcome in castration-resistant prostate cancer treated with ADT or androgen-receptor pathway inhibitors. IMPLICATIONS: We have established highly clinically relevant PDXs of ADT-induced dormant prostate cancer and identified two dormancy subtypes, leading to the development of a novel predicative gene signature that allows robust risk stratification of patients with prostate cancer to ADT or androgen-receptor pathway inhibitors.

Comparison of approaches to transcriptomic analysis in multi-sampled tumors.

Intratumoral heterogeneity is a well-documented feature of human cancers and is associated with outcome and treatment resistance. However, a heterogeneous tumor transcriptome contributes an unknown level of variability to analyses of differentially expressed genes (DEGs) that may contribute to phenotypes of interest, including treatment response. Although current clinical practice and the vast majority of research studies use a single sample from each patient, decreasing costs of sequencing technologies and computing power have made repeated-measures analyses increasingly economical. Repeatedly sampling the same tumor increases the statistical power of DEG analysis, which is indispensable toward downstream analysis and also increases one's understanding of within-tumor variance, which may affect conclusions. Here, we compared five different methods for analyzing gene expression profiles derived from repeated sampling of human prostate tumors in two separate cohorts of patients. We also benchmarked the sensitivity of generalized linear models to linear mixed models for identifying DEGs contributing to relevant prostate cancer pathways based on a ground-truth model.

A Subset of Localized Prostate Cancer Displays an Immunogenic Phenotype Associated with Losses of Key Tumor Suppressor Genes.

PURPOSE: A subset of primary prostate cancer expresses programmed death-ligand 1 (PD-L1), but whether they have a unique tumor immune microenvironment or genomic features is unclear. EXPERIMENTAL DESIGN: We selected PD-L1-positive high-grade and/or high-risk primary prostate cancer, characterized tumor-infiltrating lymphocytes with multiplex immunofluorescence, and identified genomic alterations in immunogenic and nonimmunogenic tumor foci. RESULTS: One quarter of aggressive localized prostate cancer cases (29/115) had tumor PD-L1 expression more than 5%. This correlated with increased density of CD8+ T cells, a large fraction coexpressing PD-1, versus absent PD-1 expression on sparse CD8 T cells in unselected cases. Most CD8+PD-1+ cells did not express terminal exhaustion markers (TIM3 or LAG3), while a subset expressed TCF1. Consistent with these CD8+PD-1+TCF1+ cells being progenitors, they were found in antigen-presenting cell niches in close proximity to MHC-II+ cells. CD8 T-cell density in immunogenic prostate cancer and renal cell carcinoma (RCC) was nearly identical. Shallow RB1 and BRCA2 losses, and deep deletions of CHD1, were prevalent, the latter being strongly associated with a dendritic cell gene set in The Cancer Genome Atlas. Tumor mutation burden was variable; neither high microsatellite instability nor CDK12 alterations were present. CONCLUSIONS: A subset of localized prostate cancer is immunogenic, manifested by PD-L1 expression and CD8+ T-cell content comparable with RCC. The CD8+ T cells include effector cells and exhausted progenitor cells, which may be expanded by immune checkpoint inhibitors (ICI). Genomic losses of RB1, BRCA2, and CHD1 may be drivers of this phenotype. These findings indicate that immunotherapies may be effective in biomarker-selected subpopulations of patients with localized prostate cancer.

Harnessing Androgen Receptor Pathway Activation for Targeted Alpha Particle Radioimmunotherapy of Breast Cancer.

PURPOSE: The impact of androgen receptor (AR) activity in breast cancer biology is unclear. We characterized and tested a novel therapy to an AR-governed target in breast cancer.Experimental Design: We evaluated the expression of prototypical AR gene products human kallikrein 2 (hK2) and PSA in breast cancer models. We screened 13 well-characterized breast cancer cell lines for hK2 and PSA production upon in vitro hormone stimulation by testosterone [dihydrotestosterone (DHT)]. AR-positive lines were further evaluated by exposure to estrogen (17ß-Estradiol) and the synthetic progestin D-Norgestrel. We then evaluated an anti-hK2-targeted radiotherapy platform (hu11B6), labeled with alpha (α)-particle emitting Actinium-225, to specifically treat AR-expressing breast cancer xenografts under hormone stimulation. RESULTS: D-Norgestrel and DHT activated the AR pathway, while 17ß-Estradiol did not. Competitive binding for AR protein showed similar affinity between DHT and D-Norgestrel, indicating direct AR-ligand interaction. In vivo production of hK2 was sufficient to achieve site-specific delivery of therapeutic radionuclide to tumor tissue at >20-fold over background muscle uptake; effecting long-term local tumor control. CONCLUSIONS: [225Ac]hu11B6 targeted radiotherapy was potentiated by DHT and by D-Norgestrel in murine xenograft models of breast cancer. AR activity in breast cancer correlates with kallikrein-related peptidase-2 and can be activated by D-Norgestrel, a common contraceptive, and AR induction can be harnessed for hK2-targeted breast cancer α-emitter radiotherapy.

Internalization of secreted antigen-targeted antibodies by the neonatal Fc receptor for precision imaging of the androgen receptor axis.

Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen-targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue-specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers.

Increased KIT inhibition enhances therapeutic efficacy in gastrointestinal stromal tumor.

PURPOSE: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and a model of targeted molecular therapy. GIST depends on oncogenic KIT signaling and responds to the tyrosine kinase inhibitor imatinib. However, imatinib is rarely curative. We hypothesized that PLX3397, which inhibits KIT and colony-stimulating-factor-1 receptor (CSF1R), would be more efficacious than imatinib in GIST by also depleting tumor-associated macrophages, which are generally thought to support tumor growth. EXPERIMENTAL DESIGN: We treated Kit(V558del/+) mice that develop GIST or mice with subcutaneous human GIST xenografts with imatinib or PLX3397 and analyzed tumor weight, cellular composition, histology, molecular signaling, and fibrosis. In vitro assays on human GIST cell lines were also performed. RESULTS: PLX3397 was more effective than imatinib in reducing tumor weight and cellularity in both Kit(V558del)(/+) murine GIST and human GIST xenografts. The superiority of PLX3397 did not depend on depletion of tumor-associated macrophages, because adding CSF1R inhibition did not improve the effects of imatinib. Instead, PLX3397 was a more potent KIT inhibitor than imatinib in vitro. PLX3397 therapy also induced substantial intratumoral fibrosis, which impaired the subsequent delivery of small molecules. CONCLUSIONS: PLX3397 therapy has greater efficacy than imatinib in preclinical GIST models and warrants study in patients with GIST. The resultant intratumoral fibrosis may represent one of the barriers to achieving complete tumor eradication.