Concurrent Targeting of HDAC and PI3K to Overcome Phenotypic Heterogeneity of Castration-resistant and Neuroendocrine Prostate Cancers.

Castration-resistant prostate cancer (CRPC) consists of multiple phenotypic subtypes including androgen receptor (AR)-active prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). Tumor cells with these phenotypes can coexist between metastases within a patient and within an individual tumor. Treatments that are effective across CRPC subtypes are currently lacking. Histone deacetylation is crucial for the regulation of chromatin structure and maintenance of cancer cell state and activation of the PI3K/AKT/mTOR signaling cascade is a tumor growth-promoting pathway. We therefore investigated combined targeting of histone deacetylase (HDAC) and PI3K using a rationally designed dual inhibitor, fimepinostat, in CRPC subtypes in vitro and in vivo. Dual HDAC1/2 and PI3K/AKT pathway inhibition by fimepinostat led to robust tumor growth inhibition in both ARPC and NEPC models including cell line- and patient-derived xenografts. HDAC1/2 inhibition combined with PI3K/AKT inhibition was more effective than targeting each pathway alone, producing growth inhibitory effects through cell-cycle inhibition and apoptosis. Molecular profiling revealed on-target effects of combined HDAC1/2 and PI3K/AKT inhibition independent of tumor phenotype. Fimepinostat therapy was also associated with the suppression of lineage transcription factors including AR in ARPC and Achaete-scute homolog 1 (ASCL1) in NEPC. Together, these results indicate that fimepinostat represents a novel therapeutic that may be effective against both ARPC and NEPC through CRPC subtype-dependent and -independent mechanisms. SIGNIFICANCE: CRPC is a heterogeneous disease constituting multiple phenotypic subtypes that often co-occur within tumors or across metastases in patients. Existing targeted therapies for CRPC do not take this into account. Here we show that fimepinostat, a dual HDAC1/2 and PI3K/AKT inhibitor investigated clinically in other cancer types but not prostate cancer, may overcome this heterogeneity by effectively inhibiting both ARPC and NEPC subtypes of CRPC.

Nucleosome Patterns in Circulating Tumor DNA Reveal Transcriptional Regulation of Advanced Prostate Cancer Phenotypes.

Advanced prostate cancers comprise distinct phenotypes, but tumor classification remains clinically challenging. Here, we harnessed circulating tumor DNA (ctDNA) to study tumor phenotypes by ascertaining nucleosome positioning patterns associated with transcription regulation. We sequenced plasma ctDNA whole genomes from patient-derived xenografts representing a spectrum of androgen receptor active (ARPC) and neuroendocrine (NEPC) prostate cancers. Nucleosome patterns associated with transcriptional activity were reflected in ctDNA at regions of genes, promoters, histone modifications, transcription factor binding, and accessible chromatin. We identified the activity of key phenotype-defining transcriptional regulators from ctDNA, including AR, ASCL1, HOXB13, HNF4G, and GATA2. To distinguish NEPC and ARPC in patient plasma samples, we developed prediction models that achieved accuracies of 97% for dominant phenotypes and 87% for mixed clinical phenotypes. Although phenotype classification is typically assessed by IHC or transcriptome profiling from tumor biopsies, we demonstrate that ctDNA provides comparable results with diagnostic advantages for precision oncology. SIGNIFICANCE: This study provides insights into the dynamics of nucleosome positioning and gene regulation associated with cancer phenotypes that can be ascertained from ctDNA. New methods for classification in phenotype mixtures extend the utility of ctDNA beyond assessments of somatic DNA alterations with important implications for molecular classification and precision oncology. This article is highlighted in the In This Issue feature, p. 517.

Unchecked oxidative stress in skeletal muscle prevents outgrowth of disseminated tumour cells.

Skeletal muscle has long been recognized as an inhospitable site for disseminated tumour cells (DTCs). Yet its antimetastatic nature has eluded a thorough mechanistic examination. Here, we show that DTCs traffic to and persist within skeletal muscle in mice and in humans, which raises the question of how this tissue suppresses colonization. Results from mouse and organotypic culture models along with metabolomic profiling suggested that skeletal muscle imposes a sustained oxidative stress on DTCs that impairs their proliferation. Functional studies demonstrated that disrupting reduction-oxidation homeostasis via chemogenetic induction of reactive oxygen species slowed proliferation in a more fertile organ: the lung. Conversely, enhancement of the antioxidant potential of tumour cells through ectopic expression of catalase in the tumour or host mitochondria allowed robust colonization of skeletal muscle. These findings reveal a profound metabolic bottleneck imposed on DTCs and sustained by skeletal muscle. A thorough understanding of this biology could reveal previously undocumented DTC vulnerabilities that can be exploited to prevent metastasis in other more susceptible tissues.

Genomic attributes of homology-directed DNA repair deficiency in metastatic prostate cancer.

Cancers with homology-directed DNA repair (HRR) deficiency exhibit high response rates to poly(ADP-ribose) polymerase inhibitors (PARPi) and platinum chemotherapy. Though mutations disrupting BRCA1 and BRCA2 associate with HRR deficiency (HRRd), patterns of genomic aberrations and mutation signatures may be more sensitive and specific indicators of compromised repair. Here, we evaluated whole-exome sequences from 418 metastatic prostate cancers (mPCs) and determined that one-fifth exhibited genomic characteristics of HRRd that included Catalogue Of Somatic Mutations In Cancer mutation signature 3. Notably, a substantial fraction of tumors with genomic features of HRRd lacked biallelic loss of a core HRR-associated gene, such as BRCA2. In this subset, HRRd associated with loss of chromodomain helicase DNA binding protein 1 but not with mutations in serine-protein kinase ATM, cyclin dependent kinase 12, or checkpoint kinase 2. HRRd genomic status was strongly correlated with responses to PARPi and platinum chemotherapy, a finding that supports evaluating biomarkers reflecting functional HRRd for treatment allocation.

Selective androgen receptor modulators activate the canonical prostate cancer androgen receptor program and repress cancer growth.

Prostate cancer (PC) is driven by androgen receptor (AR) activity, a master regulator of prostate development and homeostasis. Frontline therapies for metastatic PC deprive the AR of the activating ligands testosterone (T) and dihydrotestosterone (DHT) by limiting their biosynthesis or blocking AR binding. Notably, AR signaling is dichotomous, inducing growth at lower activity levels, while suppressing growth at higher levels. Recent clinical studies have exploited this effect by administration of supraphysiological concentrations of T, resulting in clinical responses and improvements in quality of life. However, the use of T as a therapeutic agent in oncology is limited by poor drug-like properties as well as rapid and variable metabolism. Here, we investigated the antitumor effects of selective AR modulators (SARMs), which are small-molecule nonsteroidal AR agonists developed to treat muscle wasting and cachexia. Several orally administered SARMs activated the AR program in PC models. AR cistromes regulated by steroidal androgens and SARMs were superimposable. Coregulatory proteins including HOXB13 and GRHL2 comprised AR complexes assembled by both androgens and SARMs. At bioavailable concentrations, SARMs repressed MYC oncoprotein expression and inhibited the growth of castration-sensitive and castration-resistant PC in vitro and in vivo. These results support further clinical investigation of SARMs for treating advanced PC.

Correction: A phase I study of niclosamide in combination with enzalutamide in men with castration-resistant prostate cancer.

[This corrects the article DOI: 10.1371/journal.pone.0198389.].

A phase I study of niclosamide in combination with enzalutamide in men with castration-resistant prostate cancer.

BACKGROUND: Niclosamide, an FDA-approved anti-helminthic drug, has activity in preclinical models of castration-resistant prostate cancer (CRPC). Potential mechanisms of action include degrading constitutively active androgen receptor splice variants (AR-Vs) or inhibiting other drug-resistance pathways (e.g., Wnt-signaling). Published pharmacokinetics data suggests that niclosamide has poor oral bioavailability, potentially limiting its use as a cancer drug. Therefore, we launched a Phase I study testing oral niclosamide in combination with enzalutamide, for longer and at higher doses than those used to treat helminthic infections. METHODS: We conducted a Phase I dose-escalation study testing oral niclosamide plus standard-dose enzalutamide in men with metastatic CRPC previously treated with abiraterone. Niclosamide was given three-times-daily (TID) at the following dose-levels: 500, 1000 or 1500mg. The primary objective was to assess safety. Secondary objectives, included measuring AR-V expression from circulating tumor cells (CTCs) using the AdnaTest assay, evaluating PSA changes and determining niclosamide's pharmacokinetic profile. RESULTS: 20 patients screened and 5 enrolled after passing all screening procedures. 13(65%) patients had detectable CTCs, but only one was AR-V+. There were no dose-limiting toxicities (DLTs) in 3 patients on the 500mg TID cohort; however, both (N = 2) subjects on the 1000mg TID cohort experienced DLTs (prolonged grade 3 nausea, vomiting, diarrhea; and colitis). The maximum plasma concentration ranged from 35.7 to 182 ng/mL and was not consistently above the minimum effective concentration in preclinical studies. There were no PSA declines in any enrolled subject. Because plasma concentrations at the maximum tolerated dose (500mg TID) were not consistently above the expected therapeutic threshold, the Data Safety Monitoring Board closed the study for futility. CONCLUSIONS: Oral niclosamide could not be escalated above 500mg TID, and plasma concentrations were not consistently above the threshold shown to inhibit growth in CRPC models. Oral niclosamide is not a viable compound for repurposing as a CRPC treatment. CLINICAL TRIAL REGISTRY: Clinicaltrials.gov: NCT02532114.

Androgen Receptor Pathway-Independent Prostate Cancer Is Sustained through FGF Signaling.

Androgen receptor (AR) signaling is a distinctive feature of prostate carcinoma (PC) and represents the major therapeutic target for treating metastatic prostate cancer (mPC). Though highly effective, AR antagonism can produce tumors that bypass a functional requirement for AR, often through neuroendocrine (NE) transdifferentiation. Through the molecular assessment of mPCs over two decades, we find a phenotypic shift has occurred in mPC with the emergence of an AR-null NE-null phenotype. These "double-negative" PCs are notable for elevated FGF and MAPK pathway activity, which can bypass AR dependence. Pharmacological inhibitors of MAPK or FGFR repressed the growth of double-negative PCs in vitro and in vivo. Our results indicate that FGF/MAPK blockade may be particularly efficacious against mPCs with an AR-null phenotype.

Substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer.

Tumor heterogeneity may reduce the efficacy of molecularly guided systemic therapy for cancers that have metastasized. To determine whether the genomic alterations in a single metastasis provide a reasonable assessment of the major oncogenic drivers of other dispersed metastases in an individual, we analyzed multiple tumors from men with disseminated prostate cancer through whole-exome sequencing, array comparative genomic hybridization (CGH) and RNA transcript profiling, and we compared the genomic diversity within and between individuals. In contrast to the substantial heterogeneity between men, there was limited diversity among metastases within an individual. The number of somatic mutations, the burden of genomic copy number alterations and aberrations in known oncogenic drivers were all highly concordant, as were metrics of androgen receptor (AR) activity and cell cycle activity. AR activity was inversely associated with cell proliferation, whereas the expression of Fanconi anemia (FA)-complex genes was correlated with elevated cell cycle progression, expression of the E2F transcription factor 1 (E2F1) and loss of retinoblastoma 1 (RB1). Men with somatic aberrations in FA-complex genes or in ATM serine/threonine kinase (ATM) exhibited significantly longer treatment-response durations to carboplatin than did men without defects in genes encoding DNA-repair proteins. Collectively, these data indicate that although exceptions exist, evaluating a single metastasis provides a reasonable assessment of the major oncogenic driver alterations that are present in disseminated tumors within an individual, and thus may be useful for selecting treatments on the basis of predicted molecular vulnerabilities.

Exome sequencing identifies a spectrum of mutation frequencies in advanced and lethal prostate cancers.

To catalog protein-altering mutations that may drive the development of prostate cancers and their progression to metastatic disease systematically, we performed whole-exome sequencing of 23 prostate cancers derived from 16 different lethal metastatic tumors and three high-grade primary carcinomas. All tumors were propagated in mice as xenografts, designated the LuCaP series, to model phenotypic variation, such as responses to cancer-directed therapeutics. Although corresponding normal tissue was not available for most tumors, we were able to take advantage of increasingly deep catalogs of human genetic variation to remove most germline variants. On average, each tumor genome contained ~200 novel nonsynonymous variants, of which the vast majority was specific to individual carcinomas. A subset of genes was recurrently altered across tumors derived from different individuals, including TP53, DLK2, GPC6, and SDF4. Unexpectedly, three prostate cancer genomes exhibited substantially higher mutation frequencies, with 2,000-4,000 novel coding variants per exome. A comparison of castration-resistant and castration-sensitive pairs of tumor lines derived from the same prostate cancer highlights mutations in the Wnt pathway as potentially contributing to the development of castration resistance. Collectively, our results indicate that point mutations arising in coding regions of advanced prostate cancers are common but, with notable exceptions, very few genes are mutated in a substantial fraction of tumors. We also report a previously undescribed subtype of prostate cancers exhibiting "hypermutated" genomes, with potential implications for resistance to cancer therapeutics. Our results also suggest that increasingly deep catalogs of human germline variation may challenge the necessity of sequencing matched tumor-normal pairs.

Differential gene expression in benign prostate epithelium of men with and without prostate cancer: evidence for a prostate cancer field effect.

BACKGROUND: Several malignancies are known to exhibit a "field effect," whereby regions beyond tumor boundaries harbor histologic or molecular changes that are associated with cancer. We sought to determine if histologically benign prostate epithelium collected from men with prostate cancer exhibits features indicative of premalignancy or field effect. EXPERIMENTAL DESIGN: Prostate needle biopsies from 15 men with high-grade (Gleason 8-10) prostate cancer and 15 age- and body mass index-matched controls were identified from a biospecimen repository. Benign epithelia from each patient were isolated by laser capture microdissection. RNA was isolated, amplified, and used for microarray hybridization. Quantitative PCR was used to determine the expression of specific genes of interest. Alterations in protein expression were analyzed through immunohistochemistry. RESULTS: Overall patterns of gene expression in microdissected benign prostate-associated benign epithelium (BABE) and cancer-associated benign epithelium (CABE) were similar. Two genes previously associated with prostate cancer, PSMA and SSTR1, were significantly upregulated in the CABE group (false discovery rate <1%). Expression of other prostate cancer-associated genes, including ERG, HOXC4, HOXC5, and MME, were also increased in CABE by quantitative reverse transcription-PCR, although other genes commonly altered in prostate cancer were not different between the BABE and CABE samples. The expression of MME and PSMA proteins on immunohistochemistry coincided with their mRNA alterations. CONCLUSION: Gene expression profiles between benign epithelia of patients with and without prostate cancer are very similar. However, these tissues exhibit differences in the expression levels of several genes previously associated with prostate cancer development or progression. These differences may comprise a field effect and represent early events in carcinogenesis.