BET inhibitors as a therapeutic intervention in gastrointestinal gene signature-positive castration-resistant prostate cancer.

A subgroup of castration-resistant prostate cancer (CRPC) aberrantly expresses a gastrointestinal (GI) transcriptome governed by two GI-lineage-restricted transcription factors, HNF1A and HNF4G. In this study, we found that expression of GI transcriptome in CRPC correlates with adverse clinical outcomes to androgen receptor signaling inhibitor treatment and shorter overall survival. Bromo- and extra-terminal domain inhibitors (BETi) downregulated HNF1A, HNF4G, and the GI transcriptome in multiple CRPC models, including cell lines, patient-derived organoids, and patient-derived xenografts, while AR and the androgen-dependent transcriptome were largely spared. Accordingly, BETi selectively inhibited growth of GI transcriptome-positive preclinical models of prostate cancer. Mechanistically, BETi inhibited BRD4 binding at enhancers globally, including both AR and HNF4G bound enhancers while gene expression was selectively perturbed. Restoration of HNF4G expression in the presence of BETi rescued target gene expression without rescuing BRD4 binding. This suggests that inhibition of master transcription factors expression underlies the selective transcriptional effects of BETi. SIGNIFICANCE: GI transcriptome expression in CRPC is regulated by the HNF1A-HNF4G-BRD4 axis and correlates with worse clinical outcomes. Accordingly, BET inhibitors significantly reduce tumor cell growth in multiple GI-transcriptome-positive preclinical models of CRPC. Our studies point that expression of GI transcriptome could serve as a predictive biomarker to BETi therapy response.

Dual targeting of the androgen receptor and PI3K/AKT/mTOR pathways in prostate cancer models improves antitumor efficacy and promotes cell apoptosis.

Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.

UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas.

Neuroendocrine carcinomas, such as neuroendocrine prostate cancer and small-cell lung cancer, commonly have a poor prognosis and limited therapeutic options. We report that ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is elevated in tissues and plasma from patients with neuroendocrine carcinomas. Loss of UCHL1 decreases tumor growth and inhibits metastasis of these malignancies. UCHL1 maintains neuroendocrine differentiation and promotes cancer progression by regulating nucleoporin, POM121, and p53. UCHL1 binds, deubiquitinates, and stabilizes POM121 to regulate POM121-associated nuclear transport of E2F1 and c-MYC. Treatment with the UCHL1 inhibitor LDN-57444 slows tumor growth and metastasis across neuroendocrine carcinomas. The combination of UCHL1 inhibitors with cisplatin, the standard of care used for neuroendocrine carcinomas, significantly delays tumor growth in pre-clinical settings. Our study reveals mechanisms of UCHL1 function in regulating the progression of neuroendocrine carcinomas and identifies UCHL1 as a therapeutic target and potential molecular indicator for diagnosing and monitoring treatment responses in these malignancies.

Targeting the fibroblast growth factor pathway in molecular subtypes of castration-resistant prostate cancer.

BACKGROUND: Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. However, castration-resistant prostate cancer (CRPC) tumors can resist AR signaling inhibitors through AR amplification and AR splice variants in AR-positive CRPC (ARPC), and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We have shown previously that DNPC can bypass AR-dependence through fibroblast growth factor receptor (FGFR) signaling. However, the role of the FGFR pathway in other CRPC phenotypes has not been elucidated. METHODS: RNA-Seq analysis was conducted on patient metastases, LuCaP patient-derived xenograft (PDX) models, and CRPC cell lines. Cell lines (C4-2B, VCaP, and 22Rv1) and ex vivo LuCaP PDX tumor cells were treated with enzalutamide (ENZA) and FGFR inhibitors (FGFRi) alone or in combination and sensitivity was determined using cell viability assays. In vivo efficacy of FGFRi in ARPC, DNPC, and SCNPC were evaluated using PDX models. RESULTS: RNA-Seq analysis of FGFR signaling in metastatic specimens, LuCaP PDX models, and CRPC cell lines revealed significant FGF pathway activation in AR-low PC (ARLPC), DNPC, and SCNPC tumors. In vitro/ex vivo analysis of erdafitinib and CH5183284 demonstrated robust and moderate growth suppression of ARPC, respectively. In vivo studies using four ARPC PDX models showed that combination ENZA and CH5183284 significantly suppressed tumor growth. Additional in vivo studies using four ARPC PDX models revealed that erdafitinib monotherapy was as effective as ENZA in suppressing tumor growth, and there was limited combination benefit. Furthermore, two of three DNPC models and two of four SCNPC models responded to CH5183284 monotherapy, suggesting FGFRi responses were model dependent. RNA-Seq and gene set enrichment analysis of end-of-study ARPC tumors treated with FGFRi displayed decreased expression of E2F and MYC target genes and suppressed G2M checkpoint genes, whereas end-of-study SCNPC tumors had heterogeneous transcriptional responses. CONCLUSIONS: Although FGFRi treatments suppressed tumor growth across CRPC phenotypes, our analyses did not identify a single pathway or biomarker that would identify tumor response to FGFRi. This is very likely due to the array of FGFR1-4 expression and tumor phenotypes present in CRPC. Nevertheless, our data nominate the FGFR pathway as a clinically actionable target that promotes tumor growth in diverse phenotypes of treatment-refractory metastatic CRPC.

ACAA2 is a novel molecular indicator for cancers with neuroendocrine phenotype.

BACKGROUND: Neuroendocrine phenotype is commonly associated with therapy resistance and poor prognoses in small-cell neuroendocrine cancers (SCNCs), such as neuroendocrine prostate cancer (NEPC) and small-cell lung cancer (SCLC). Expression levels of current neuroendocrine markers exhibit high case-by-case variability, so multiple markers are used in combination to identify SCNCs. Here, we report that ACAA2 is elevated in SCNCs and is a potential molecular indicator for SCNCs. METHODS: ACAA2 expressions in tumour xenografts, tissue microarrays (TMAs), and patient tissues from prostate and lung cancers were analysed via immunohistochemistry. ACAA2 mRNA levels in lung and prostate cancer (PC) patients were assessed in published datasets. RESULTS: ACAA2 protein and mRNA levels were elevated in SCNCs relative to non-SCNCs. Medium/high ACAA2 intensity was observed in 78% of NEPC PDXs samples (N = 27) relative to 33% of adeno-CRPC (N = 86), 2% of localised PC (N = 50), and 0% of benign prostate specimens (N = 101). ACAA2 was also elevated in lung cancer patient tissues with neuroendocrine phenotype. 83% of lung carcinoid tissues (N = 12) and 90% of SCLC tissues (N = 10) exhibited medium/high intensity relative to 40% of lung adenocarcinoma (N = 15). CONCLUSION: ACAA2 expression is elevated in aggressive SCNCs such as NEPC and SCLC, suggesting it is a potential molecular indicator for SCNCs.

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.

Genetic dissection of crossover mutants defines discrete intermediates in mouse meiosis.

Crossovers (COs), the exchange of homolog arms, are required for accurate chromosome segregation during meiosis. Studies in yeast have described the single-end invasion (SEI) intermediate: a stabilized 3' end annealed with the homolog as the first detectible CO precursor. SEIs are thought to differentiate into double Holliday junctions (dHJs) that are resolved by MutLgamma (MLH1/MLH3) into COs. Currently, we lack knowledge of early steps of mammalian CO recombination or how intermediates are differentiated in any organism. Using comprehensive analysis of recombination in thirteen different genetic conditions with varying levels of compromised CO resolution, we infer CO precursors include asymmetric SEI-like intermediates and dHJs in mouse. In contrast to yeast, MLH3 is structurally required to differentiate CO precursors into dHJs. We verify conservation of aspects of meiotic recombination and show unique features in mouse, providing mechanistic insight into CO formation.

Enhancer profiling identifies epigenetic markers of endocrine resistance and reveals therapeutic options for metastatic castration-resistant prostate cancer patients.

Androgen Receptor (AR) signaling inhibitors, including enzalutamide, are treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC), but resistance inevitably develops. Using metastatic samples from a prospective phase II clinical trial, we epigenetically profiled enhancer/promoter activities with H3K27ac chromatin immunoprecipitation followed by sequencing, before and after AR-targeted therapy. We identified a distinct subset of H3K27ac-differentially marked regions that associated with treatment responsiveness. These data were successfully validated in mCRPC patient-derived xenograft models (PDX). In silico analyses revealed HDAC3 as a critical factor that can drive resistance to hormonal interventions, which we validated in vitro . Using cell lines and mCRPC PDX tumors in vitro , we identified drug-drug synergy between enzalutamide and the pan-HDAC inhibitor vorinostat, providing therapeutic proof-of-concept. These findings demonstrate rationale for new therapeutic strategies using a combination of AR and HDAC inhibitors to improve patient outcome in advanced stages of mCRPC.

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.

Oxidation of p-[125I]Iodobenzoic Acid and p-[211At]Astatobenzoic Acid Derivatives and Evaluation In Vivo.

The alpha particle-emitting radionuclide astatine-211 (211At) is of interest for targeted radiotherapy; however, low in vivo stability of many 211At-labeled cancer-targeting molecules has limited its potential. As an alternative labeling method, we evaluated whether a specific type of astatinated aryl compound that has the At atom in a higher oxidation state might be stable to in vivo deastatination. In the research effort, para-iodobenzoic acid methyl ester and dPEG4-amino acid methyl ester derivatives were prepared as HPLC standards. The corresponding para-stannylbenzoic acid derivatives were also prepared and labeled with 125I and 211At. Oxidization of the [125I]iodo- and [211At]astato-benzamidyl-dPEG4-acid methyl ester derivatives provided materials for in vivo evaluation. A biodistribution was conducted in mice with coinjected oxidized 125I- and 211At-labeled compounds. The oxidized radioiodinated derivative was stable to in vivo deiodination, but unfortunately the oxidized [211At]astatinated benzamide derivative was found to be unstable under the conditions of isolation by radio-HPLC (post animal injection). Another biodistribution study in mice evaluated the tissue concentrations of coinjected [211At]NaAtO3 and [125I]NaIO3. Comparison of the tissue concentrations of the isolated material from the oxidized [211At]benzamide derivative with those of [211At]astatate indicated the species obtained after isolation was likely [211At]astatate.

Response to supraphysiological testosterone is predicted by a distinct androgen receptor cistrome.

The androgen receptor (AR) is a master transcription factor that regulates prostate cancer (PC) development and progression. Inhibition of AR signaling by androgen deprivation is the first-line therapy with initial efficacy for advanced and recurrent PC. Paradoxically, supraphysiological levels of testosterone (SPT) also inhibit PC progression. However, as with any therapy, not all patients show a therapeutic benefit, and responses differ widely in magnitude and duration. In this study, we evaluated whether differences in the AR cistrome before treatment can distinguish between SPT-responding (R) and -nonresponding (NR) tumors. We provide the first preclinical evidence to our knowledge that SPT-R tumors exhibit a distinct AR cistrome when compared with SPT-NR tumors, indicating a differential biological role of the AR. We applied an integrated analysis of ChIP-Seq and RNA-Seq to the pretreatment tumors and identified an SPT-R signature that distinguishes R and NR tumors. Because transcriptomes of SPT-treated clinical specimens are not available, we interrogated available castration-resistant PC (CRPC) transcriptomes and showed that the SPT-R signature is associated with improved survival and has the potential to identify patients who would respond to SPT. These findings provide an opportunity to identify the subset of patients with CRPC who would benefit from SPT therapy.

Sequencing SARS-CoV-2 from antigen tests.

Genomic surveillance empowers agile responses to SARS-CoV-2 by enabling scientists and public health analysts to issue recommendations aimed at slowing transmission, prioritizing contact tracing, and building a robust genomic sequencing surveillance strategy. Since the start of the pandemic, real time RT-PCR diagnostic testing from upper respiratory specimens, such as nasopharyngeal (NP) swabs, has been the standard. Moreover, respiratory samples in viral transport media are the ideal specimen for SARS-CoV-2 whole-genome sequencing (WGS). In early 2021, many clinicians transitioned to antigen-based SARS-CoV-2 detection tests, which use anterior nasal swabs for SARS-CoV-2 antigen detection. Despite this shift in testing methods, the need for whole-genome sequence surveillance remains. Thus, we developed a workflow for whole-genome sequencing with antigen test-derived swabs as an input rather than nasopharyngeal swabs. In this study, we use excess clinical specimens processed using the BinaxNOW™ COVID-19 Ag Card. We demonstrate that whole-genome sequencing from antigen tests is feasible and yields similar results from RT-PCR-based assays utilizing a swab in viral transport media.

Magneto-Endosomalytic Therapy for Cancer.

A remarkably simple yet effective mode of cancer treatment is reported by repurposing clinically approved magnetic nanoparticles (MNPs). Intracellular nanoparticle self-assembly directed by static parallel magnetic fields leads to cell death in targeted tissues while leaving other cells and organs intact. This simple concept opens a new avenue to treat cancer, capitalizing on nanosciences and the nanoparticle (NP) design principles accumulated in the past decades.

Caring for the Animal Caregiver-Occupational Health, Human-Animal Bond and Compassion Fatigue.

Laboratory Animal Professionals experience many positive and rewarding interactions when caring for and working with research animals. However, these professionals also may experience conflicting feelings and exhaustion when the work is stressful due to factors such as limited resources, making end of life decisions, dealing with conflicting priorities, and negotiating animal care priorities with colleagues. These stresses may be further complicated by each individual's self-understanding and emotional investment in the human-animal bond. The term used for this type of complex emotional conflict and exhaustion is Compassion Fatigue. Compassion Fatigue in the Laboratory Animal Science setting is a combination of physical, emotional and psychological depletion associated with working with and caring for animals and their well-being in a research environment. The University of Washington has developed a Compassion in Science Program called Dare2Care which emphasizes self-care and helps Laboratory Animal Professionals identify stress factors and work toward a personal solution to relieve stress. The first step in developing a resiliency program is to assess the current culture and needs of the organization. At an institutional level we identified that we needed increased communication concerning study endpoints, as well as identified individuals with whom affected personnel can talk about personal concerns. We also implemented community events to reflect on the positive aspects of this field of work. We improved the physical work environment, and provided outlets established for personnel to express feelings via written word or artistically. Lastly, we started working with our Center for One Health to encompass a holisitic approach to the occupational health of our animal caregivers. One health is the relationship and interplay between people, animals and the environment and we needed to include emotional well-being in our assessment of the health of our personnel. A question was added to our occupational health screening form to include additional health or workplace concerns (e.g., Compassion Fatigue) not covered by the questionnaire, and we added a component of Compassion Fatigue awareness in our training program. Here we review the importance of identifying Compassion Fatigue in the animal research setting, focus on developing a compassion resiliency culture and provide tools and coping strategies to validate and strengthen the human-animal bond with research animals and to sustain the care that is necessary for both people and research animals.

Subtype heterogeneity and epigenetic convergence in neuroendocrine prostate cancer.

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyze cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single-cell analysis of human clinical samples exhibits a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall, our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy.

RNA Splicing Factors SRRM3 and SRRM4 Distinguish Molecular Phenotypes of Castration-Resistant Neuroendocrine Prostate Cancer.

Neuroendocrine (NE) differentiation in metastatic castration-resistant prostate cancer (mCRPC) is an increasingly common clinical feature arising from cellular plasticity. We recently characterized two mCRPC phenotypes with NE features: androgen receptor (AR)-positive NE-positive amphicrine prostate cancer (AMPC) and AR-negative small cell or neuroendocrine prostate cancer (SCNPC). Here, we interrogated the regulation of RE1-silencing transcription factor (REST), a transcriptional repressor of neuronal genes, and elucidated molecular programs driving AMPC and SCNPC biology. Analysis of prostate cancer cell lines, mCRPC specimens, and LuCaP patient-derived xenograft models detected alternative splicing of REST to REST4 and attenuated REST repressor activity in AMPC and SCNPC. The REST locus was also hypermethylated and REST expression was reduced in SCNPC. While serine/arginine repetitive matrix protein 4 (SRRM4) was previously implicated in alternative splicing of REST in mCRPC, we detected SRRM3 expression in REST4-positive, SRRM4-negative AMPC, and SCNPC. In CRPC cell lines, SRRM3 induced alternative splicing of REST to REST4 and exacerbated the expression of REST-repressed genes. Furthermore, SRRM3 and SRRM4 expression defined molecular subsets of AMPC and SCNPC across species and tumor types. Two AMPC phenotypes and three SCNPC phenotypes were characterized, denoted either by REST attenuation and ASCL1 activity or by progressive activation of neuronal transcription factor programs, respectively. These results nominate SRRM3 as the principal REST splicing factor expressed in early NE differentiation and provide a framework to molecularly classify diverse NE phenotypes in mCRPC. SIGNIFICANCE: This study identifies SRRM3 as a key inducer of cellular plasticity in prostate cancer with neuroendocrine features and delineates distinct neuroendocrine phenotypes to inform therapeutic development and precision medicine applications.

MCM2-7 complex is a novel druggable target for neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer that rarely develops de novo in primary tumors and is commonly acquired during the development of treatment resistance. NEPC is characterized by gain of neuroendocrine markers and loss of androgen receptor (AR), making it resistant to current therapeutic strategies targeting the AR signaling axis. Here, we report that MCM2, MCM3, MCM4, and MCM6 (MCM2/3/4/6) are elevated in human NEPC and high levels of MCM2/3/4/6 are associated with liver metastasis and poor survival in prostate cancer patients. MCM2/3/4/6 are four out of six proteins that form a core DNA helicase (MCM2-7) responsible for unwinding DNA forks during DNA replication. Inhibition of MCM2-7 by treatment with ciprofloxacin inhibits NEPC cell proliferation and migration in vitro, significantly delays NEPC tumor xenograft growth, and partially reverses the neuroendocrine phenotype in vivo. Our study reveals the clinical relevance of MCM2/3/4/6 proteins in NEPC and suggests that inhibition of MCM2-7 may represent a new therapeutic strategy for NEPC.

Reprogramming of the FOXA1 cistrome in treatment-emergent neuroendocrine prostate cancer.

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. Here, we investigate the epigenomic basis of this resistance mechanism by profiling histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identify a vast network of cis-regulatory elements (N~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying cancer dependencies through epigenomic profiling.

A bladder cancer patient-derived xenograft displays aggressive growth dynamics in vivo and in organoid culture.

Bladder cancer is among the most prevalent cancers worldwide. Currently, few bladder cancer models have undergone thorough characterization to assess their fidelity to patient tumors, especially upon propagation in the laboratory. Here, we establish and molecularly characterize CoCaB 1, an aggressive cisplatin-resistant muscle-invasive bladder cancer patient-derived xenograft (PDX) and companion organoid system. CoCaB 1 was a subcutaneous PDX model reliably transplanted in vivo and demonstrated an acceleration in growth upon serial transplantation, which was reflected in organoid and 2D cell culture systems. Transcriptome analysis revealed progression towards an increasingly proliferative and stem-like expression profile. Gene expression differences between organoid and PDX models reflected expected differences in cellular composition, with organoids enriched in lipid biosynthesis and metabolism genes and deprived of extracellular components observed in PDXs. Both PDX and organoid models maintained the histological fidelity and mutational heterogeneity of their parental tumor. This study establishes the CoCaB 1 PDX and organoid system as companion representative tumor models for the development of novel bladder cancer therapies.

Cabozantinib can block growth of neuroendocrine prostate cancer patient-derived xenografts by disrupting tumor vasculature.

With the advent of potent second-line anti-androgen therapy, we and others have observed an increased incidence of androgen receptor (AR)-null small cell or neuroendocrine prostate cancer (SCNPC) in metastatic castration-resistant prostate cancer (mCRPC). Our study was designed to determine the effect of cabozantinib, a multi-targeted tyrosine kinase inhibitor that inhibits VEGFR2, MET and RET on SCNPC. Transcriptome analysis of the University of Washington rapid autopsy and SU2C mCRPC datasets revealed upregulated MET and RET expression in SCNPCs relative to adenocarcinomas. Additionally, increased MET expression correlated with attenuated AR expression and activity. In vitro treatment of SCNPC patient-derived xenograft (PDX) cells with the MET inhibitor AMG-337 had no impact on cell viability in LuCaP 93 (MET+/RET+) and LuCaP 173.1 (MET-/RET-), whereas cabozantinib decreased cell viability of LuCaP 93, but not LuCaP 173.1. Notably, MET+/RET+ LuCaP 93 and MET-/RET- LuCaP 173.1 tumor volumes were significantly decreased with cabozantinib treatment in vivo, and this activity was independent of MET or RET expression in LuCaP 173.1. Tissue analysis indicated that cabozantinib did not inhibit tumor cell proliferation (Ki67), but significantly decreased microvessel density (CD31) and increased hypoxic stress and glycolysis (HK2) in LuCaP 93 and LuCaP 173.1 tumors. RNA-Seq and gene set enrichment analysis revealed that hypoxia and glycolysis pathways were increased in cabozantinib-treated tumors relative to control tumors. Our data suggest that the most likely mechanism of cabozantinib-mediated tumor growth suppression in SCNPC PDX models is through disruption of the tumor vasculature. Thus, cabozantinib may represent a potential therapy for patients with metastatic disease in tumor phenotypes that have a significant dependence on the tumor vasculature for survival and proliferation.