Cynthia Cheung

ERG induces taxane resistance in castration-resistant prostate cancer

Taxanes are the only chemotherapies used to treat patients with metastatic castration-resistant prostate cancer (CRPC). Despite the initial efficacy of taxanes in treating CRPC, all patients ultimately fail due to the development of drug resistance. In this study, we show that ERG overexpression in in vitro and in vivo models of CRPC is associated with decreased sensitivity to taxanes. ERG affects several parameters of microtubule dynamics and inhibits effective drug-target engagement of docetaxel or cabazitaxel with tubulin. Finally, analysis of a cohort of 34 men with metastatic CRPC treated with docetaxel chemotherapy reveals that ERG-overexpressing prostate cancers have twice the chance of docetaxel resistance than ERG-negative cancers. Our data suggest that ERG plays a role beyond regulating gene expression and functions outside the nucleus to cooperate with tubulin towards taxane insensitivity. Determining ERG rearrangement status may aid in patient selection for docetaxel or cabazitaxel therapy and/or influence co-targeting approaches.

Characterization of a cellular denitrase activity that reverses nitration of cyclooxygenase

Protein 3-nitrotyrosine (3-NT) formation is frequently regarded as a simple biomarker of disease, an irreversible posttranslational modification that can disrupt protein structure and function. Nevertheless, evidence that protein 3-NT modifications may be site selective and reversible, thus allowing for physiological regulation of protein activity, has begun to emerge. We have previously reported that cyclooxygenase (COX)-1 undergoes heme-dependent nitration of Tyr(385), an internal and catalytically essential residue. In the present study, we demonstrate that nitrated COX-1 undergoes a rapid reversal of nitration by substrate-selective and biologically regulated denitrase activity. Using nitrated COX-1 as a substrate, denitrase activity was validated and quantified by analytic HPLC with electrochemical detection and determined to be constitutively active in murine and human endothelial cells, macrophages, and a variety of tissue samples. Smooth muscle cells, however, contained little denitrase activity. Further characterizing this denitrase activity, we found that it was inhibited by free 3-NT and may be enhanced by endogenous nitric oxide and exogenously administered carbon monoxide. Finally, we describe a purification protocol that results in significant enrichment of a discrete denitrase-containing fraction, which maintains activity throughout the purification process. These findings reveal that nitrated COX-1 is a substrate for a denitrase in cells and tissues, implying that the reciprocal processes of nitration and denitration may modulate bioactive lipid synthesis in the setting of inflammation. In addition, our data reveal that denitration is a controlled process that may have broad importance for regulating cell signaling events in nitric oxide-generating systems during oxidative/nitrosative stress.

A Computational Drug Repositioning Approach for Targeting Oncogenic Transcription Factors

Mutations in transcription factor (TF) genes are frequently observed in tumors, often leading to aberrant transcriptional activity. Unfortunately, TFs are often considered undruggable due to the absence of targetable enzymatic activity. To address this problem, we developed CRAFTT, a computational drug-repositioning approach for targeting TF activity. CRAFTT combines ChIP-seq with drug-induced expression profiling to identify small molecules that can specifically perturb TF activity. Application to ENCODE ChIP-seq datasets revealed known drug-TF interactions, and a global drug-protein network analysis supported these predictions. Application of CRAFTT to ERG, a pro-invasive, frequently overexpressed oncogenic TF, predicted that dexamethasone would inhibit ERG activity. Dexamethasone significantly decreased cell invasion and migration in an ERG-dependent manner. Furthermore, analysis of electronic medical record data indicates a protective role for dexamethasone against prostate cancer. Altogether, our method provides a broadly applicable strategy for identifying drugs that specifically modulate TF activity.

Physical evidence for substrate binding in preventing cyclooxygenase inactivation under nitrative stress

Prostaglandin biosynthesis is catalyzed by two spatially and functionally distinct active sites in cyclooxygenase (COX) enzymes. Despite the crucial role of COXs in biology, molecular details regarding the function and regulation of these enzymes are incompletely defined. Reactive nitrogen species, formed during oxidative stress, produce modifications that alter COX functionalities and prostaglandin biosynthesis. We previously established that COX-1 undergoes selective nitration on Tyr385 via a mechanism that requires the presence of bound heme cofactor. As this is a critical residue for COX-1 catalysis, nitration at this site results in enzyme inactivation. We now show that occupancy of the COX-1 active site with substrate protects against Tyr385 nitration and redirects nitration to alternative Tyr residues on COX-1, preserving catalytic activity. This study reveals a novel role for the substrate in protecting COX-1 from inactivation by nitration in pathophysiological settings.

Patient derived organoids to model rare prostate cancer phenotypes

A major hurdle in the study of rare tumors is a lack of existing preclinical models. Neuroendocrine prostate cancer is an uncommon and aggressive histologic variant of prostate cancer that may arise de novo or as a mechanism of treatment resistance in patients with pre-existing castration-resistant prostate cancer. There are few available models to study neuroendocrine prostate cancer. Here, we report the generation and characterization of tumor organoids derived from needle biopsies of metastatic lesions from four patients. We demonstrate genomic, transcriptomic, and epigenomic concordance between organoids and their corresponding patient tumors. We utilize these organoids to understand the biologic role of the epigenetic modifier EZH2 in driving molecular programs associated with neuroendocrine prostate cancer progression. High-throughput organoid drug screening nominated single agents and drug combinations suggesting repurposing opportunities. This proof of principle study represents a strategy for the study of rare cancer phenotypes.There are few available models to study neuroendocrine prostate cancer. Here they develop and characterize patient derived organoids from metastatic lesions, use these models to show the role of EZH2 in driving neuroendocrine phenotype, and perform high throughput organoid screening to identify therapeutic drug combinations.

Abstract 362: Computational drug repositioning identifies dexamethasone as potential ERG inhibitor

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Mutations in genes coding for transcription factors (TFs) are frequently observed in tumors, many of which lead to aberrant transcriptional activity. Unfortunately, transcription factors are often considered “undruggable” due to the absence of targetable enzymatic activity and the large surface contacting DNA. To address the transcription factor “druggability” problem, we developed a computational repositioning approach to identify small molecules that can perturb the activity of transcription factors. Our approach involves identifying drugs that mobilize many of the target genes of a transcription factor. This approach uses Gene Set Enrichment Analysis to integrate genomewide binding data (ChIP-seq) with drug perturbation differential gene expression profiles. When applied to ENCODE ChIP-seq and the Connectivity Map expression profiles, our approach predicted 38,000 disruptive drug-TF interactions (FWER<0.1). These predictions included the known inhibitory effect of a BRD4 inhibitor (JQ1, FWER=0.000) on MYC and of HSP90 inhibitors (e.g.17-AAG, FWER=0.031) on HSF1. We used an integrated biological network with 22k genes and 7k drugs to identify predicted disruptive drug-TF interactions. Based on this approach network path length of predicted drug-TF was significantly shorter than non-predictions (p=2.2e-16). Many predicted drug-TF interactions involved only one protein intermediary between the drug and the TF, indicating that our predictions are not random and suggesting that many drugs might disrupt TFs by targeting their regulatory or interacting co-factors. We then decided to apply our approach to identifying candidate molecules that can inhibit ERG, a pro-invasive, oncogenic TF over-expressed in as many as 50% of prostate cancer patients. Using ERG ChIP-seq peaks in prostate cells, dexamethasone (FWER=0.086) was predicted to inhibit ERG transcriptional activity. Using cell invasion and migration assays, we found that dexamethasone significantly decreased cell invasion and migration in DU145 prostate cancer cells over-expressing ERG, but not in isogenic control cells. Dexamethasone also abrogated expression of PLAU, a known ERG target, and substantially decreased binding of ERG at the PLAU promoter. Analysis of ERG ChIP-seq peaks revealed a highly enriched AP-1 DNA motif and preferential mobilization by dexamethasone of genes near peaks containing the AP-1 motif (p=0.06). ChIP-seq experiments showed that dexamethasone reduced AP-1 binding at ERG-JUN promoter sites. These results suggest that dexamethasone inhibits ERG by disruption of AP1, a key co-factor. Altogether, this method provides a novel, broadly applicable strategy to computationally identify drugs that indirectly target transcription factors. This may be of further interest for other factors with oncogenic activity, such as FOXA1 or for reactivating deactivated tumor suppressive transcription factors such as p53. Citation Format: Kaitlyn Gayvert, Cynthia Cheung, David Rickman, Olivier Elemento. Computational drug repositioning identifies dexamethasone as potential ERG inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 362. doi:10.1158/1538-7445.AM2014-362

Abstract 887: N-Myc drives neuroendocrine prostate cancer

Emerging observations from clinical trials suggest that a subset of castration resistant prostate adenocarcinomas (CRPC) eventually evolve or progress to a predominantly neuroendocrine phenotype (NEPC). This transition is emerging as an important mechanism of treatment resistance. This cell plasticity is characterized by loss of androgen receptor (AR) and prostate specific antigen (PSA), and significant over-expression and gene amplification of MYCN (encoding N-Myc). While N-Myc is a bona fide driver oncogene in several rare tumor types, the molecular mechanisms that underlie N-Myc driven NEPC have yet to be characterized. Integrating a novel genetically engineered mouse (GEM) model of prostate specific N-Myc overexpression, human prostate cancer cell line modeling, and human prostate cancer transcriptome data, we found that N-Myc over-expression leads to the development of poorly differentiated, invasive prostate cancer that is molecularly similar to human NEPC tumors. To determine if N-Myc plays a causal role in driving the NEPC phenotype, we generated GEM lines that carry a CAG-driven lox-stop-lox human MYCN gene integrated into the ROSA26 (LSL-MYCN) locus and either a Tmprss2 driven tamoxifen-activated Cre recombinase (T2-Cre) or probasin (Pb)-Cre. Since PTEN deletion is a frequent alteration in CRPC and PI3K/AKT signaling can enhance N-Myc protein stability we also engineered the mice with a floxed Pten locus. N-Myc over-expression in the context of Ptenf/+ at 3 months post-induction leads to focal mouse high-grade prostatic intraepithelial neoplasia (mHGPIN). T2-Cre; Ptenf/f; LSL-MYCN+/+ mice develop highly proliferative, diffuse mHGPIN which consists of proliferations of cells with nuclear atypia that expand the glands, imparting irregular borders and inducing a mild stromal response, mitotic figures, and incipient necrosis. RNAseq data from N-Myc these mHGPIN lesions show they are molecularly similar to NEPC based on RNAseq data from 203 human CRPC and NEPC samples. At 6 months, Pb-Cre; Ptenf/f; LSL-MYCN+/+ mice develop poorly differentiated, highly proliferative, invasive prostate cancer. Based on the RNAseq data from the N-Myc GEM line, GEM-derived mouse prostate cancer organoid cultures and isogenic cell lines, we found that N-Myc regulates a specific NEPC-associated molecular program that includes a repression of AR signaling, enhanced AKT signaling and repression of Polycomb Repressive Complex 2 target genes. We further showed that N-Myc interacts with AR and this interaction depends on Enhancer of Zeste Homolog 2 (EZH2). Finally, N-Myc expressing cell lines and organoids displayed an enhanced sensitivity to inhibitors targeting the AKT pathway, EZH2 and Aurora-A. Altogether, our data shows that N-Myc drives the neuroendocrine phenotype in prostate cancer and provides rationale for the development of new therapeutic strategies for treating this aggressive subtype of prostate cancer. Citation Format: Etienne Dardenne, Himisha Beltran, Kaitlyn Gayvert, Matteo Benelli, Adeline Berger, Loredana Puca, Joanna Cyrta, Andrea Sboner, Zohal Noorzad, Theresa MacDonald, Cynthia Cheung, Dong Gao, Yu Chen, Martin Eilers, Juan Miguel Mosquera, Brian D. Robinson, Mark A. Rubin, Olivier Elemento, Francesca Demichelis, David S. Rickman. N-Myc drives neuroendocrine prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 887.

Abstract PR08: The N-Myc transcriptional program driving the neuroendocrine prostate cancer phenotype

Emerging observations from clinical trials suggest that a subset of castration resistant prostate adenocarcinomas (CRPC) eventually evolve or progress to a predominantly small cell carcinoma or neuroendocrine phenotype [1]. Neuroendocrine prostate cancer (NEPC) does not typically express the androgen receptor (AR) or secrete prostate specific antigen (PSA), and often expresses markers of neuroendocrine lineage [2]. NEPC is clinically aggressive and carries a poor prognosis with an average survival of less than one year [3, 4]. Identification of effective treatment strategies for this lethal subtype of prostate cancer represents a significant unmet need in the clinic. We have previously discovered significant over-expression and gene amplification of AURKA (encoding Aurora-A) and MYCN (encoding N-Myc) in NEPC as compared to prostate adenocarcinoma [5, 6]. As in neuroblastoma [7], N-Myc interacts with Aurora-A in NEPC which leads to a co-stabilization of both proteins and that ectopic expression of N-Myc or Aurora-A induces neuroendocrine transformation of prostate adenocarcinoma cells [5]. However, the molecular mechanisms that underlie N-Myc driven NEPC have yet to be characterized. We performed RNA-sequencing (RNAseq) and ChIP-sequencing from multiple stable prostate adenocarcinoma cells with and without N-Myc over-expression. RNAseq reads were aligned to the hg19 reference genome using TopHat. Based on GSEA , pathway analysis and further validation of our N-Myc signature, we found that N-Myc is recruited to AR-bound enhancers and AR target genes, dramatically reduces AR signaling and induces a profile enriched in pro-metastatic, dedifferentiation and Polycomb Repressive Complex deregulated genes as well as other genes encoding targetable proteins. RNAseq data from 128 clinical samples (17 NEPC, 10 castrate resistant prostate cancer, 68 prostate adenocarcinoma patient tumors and 33 matched benign prostate samples) were used to assess clinical relevance and a novel Nanostring assay, targeted RT-PCR and ChIP-PCR to validate our findings. We used DESeq and MISO to identify differential expressed genes and differential exon expression. We have identified a signature of deregulated genes including specific alternatively spliced mRNA variants and splicing factors associated with N-Myc over-expression that are both biologically and clinically relevant. This included several variant transcripts from genes that are implicated in cancer-related signaling (e.g.PIK3C2A,ATM,HUWE1) and an up-regulation of the splicing factor NOVA1 (neuro-oncological ventral antigen 1). In conclusion, our findings have the potential to ultimately lead to the identification of a new class of disease specific biomarkers and therapeutic alternatives for this aggressive subgroup of prostate cancer. References: 1. Beltran, H., S. Tomlins, et al., Clin Cancer Res, 2014. 20(11): p. 2846-50. 2. Wang, W. and J.I. Epstein, Am J Surg Pathol, 2008. 32(1): p. 65-71. 3. Palmgren, J.S., S.S. Karavadia, et al., Semin Oncol, 2007. 34(1): p. 22-9. 4. Wang, H.T., Y.H. Yao, et al., J Clin Oncol, 2014. 5. Beltran, H., D.S. Rickman, et al., Cancer Discovery, 2011. 1(6): p. 487-495. 6. Mosquera, J.M., H. Beltran, et al., Neoplasia, 2013. 15(1): p. 1-10. 7. Otto, T., S. Horn, et al., Cancer Cell, 2009. 15(1): p. 67-78. Citation Format: Etienne Dardenne, Kaitlyn Gayert, Andrea Sboner, Cynthia Cheung, Martin Eilers, Mark Rubin, Himisha Beltran, Olivier Elemento, David Rickman. The N-Myc transcriptional program driving the neuroendocrine prostate cancer phenotype. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR08.

Abstract LB-072: The N-Myc transcriptional program driving the neuroendocrine prostate cancer phenotype

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Although neuroendocrine prostate cancer (NEPC) rarely arises de novo, up to 30% of patients with prostate adenocarcinoma (PCa) develop NEPC features in later stages of their disease as a mechanism of treatment resistance to hormonal therapies including abiraterone and enzalutamide. NEPC is clinically more aggressive than PCa, commonly metastases to visceral organs such as liver and brain, and can be suspected in patients with progressive disease and a disproportionately low serum PSA. There is currently no effective therapy for NEPC, and most patients with NEPC survive less than one year. The development of novel treatment strategies for patients with NEPC represents a significant clinical unmet need. We have previously discovered significant over-expression and gene amplification of AURKA (encoding Aurora-A) and MYCN (encoding N-Myc) in NEPC as compared to prostate adenocarcinoma. As in neuroblastoma, N-Myc interacts with Aurora-A in NEPC which leads to a co-stabilization of both proteins that is targetable with allosteric inhibitors of Aurora A. We have also shown that ectopic expression of N-Myc induces neuroendocrine transformation of prostate adenocarcinoma cells. However, the molecular mechanisms that underlie N-Myc driven NEPC phenotype have yet to be characterized. To address this, we performed RNA-sequencing (RNAseq) and ChIP-sequencing from multiple stable prostate adenocarcinoma cells with and without N-Myc over-expression. We have identified a signature of N-Myc deregulated genes that are both biologically and clinically relevant. Based on RNAseq data from 128 clinical samples (17 NEPC, 10 castrate resistant prostate cancer 68 prostate adenocarcinoma patient tumors and 33 matched benign prostate samples) we found that majority (70%) of the N-Myc deregulated genes identified in our in vitro models distinguish the NEPC from PCa tumor samples. Based on GSEA and pathway analysis we found that N-Myc induces a profile enriched in pro-metastatic, dedifferentiation and Polycomb Repressive Complex deregulated genes and dramatically reduces AR signaling. Furthermore we show that N-Myc is recruited to AR-bound enhancers of AR target genes. To further determine the role of N-Myc in driving the NEPC phenotype we have generated transgenic mice that carry an integrated MYCN gene behind a lox-stop-lox (LSL) cassette at the ROSA26 locus, a floxed Pten locus and a tamoxifen-activated Cre recombinase driven by Tmprss2. N-Myc over-expression in the context of Ptenfl/+ leads to focal high levels of activated AKT pathway that accompany mouse high grade prostatic intraepithelial neoplasia (mHGPIN) at 3 months post-induction and other clinically relevant molecular changes. Littermates that harbor Ptenfl/+ alone do not display mHGPIN or AKT activation. In the context of Pten homozygous loss (Ptenfl/fl), N-Myc is associated with diffuse mHGPIN in the ventral and dorsolateral prostate lobes and irregular gland borders, nuclear atypia and high levels of AKT activity at 3 months (MYCN homozygous) or 6 months (MYCN heterozygous) post-induction. In conclusion, our findings support the role of N-Myc as one of the drivers of the NEPC phenotype and have the potential to ultimately lead to the identification of a new class of disease specific biomarkers and therapeutic alternatives for this aggressive subgroup of prostate cancer. Citation Format: Etienne Dardenne, Kaitlyn Gayvert, Adeline Berger, Andrea Sboner, Brian Robinson, Kenneth Hennrick, Juan Miguel Mosquera, Cynthia Cheung, Martin Eilers, Himisha Beltran, Mark A. Rubin, Olivier Elemento, David S. Rickman. The N-Myc transcriptional program driving the neuroendocrine prostate cancer phenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-072. doi:10.1158/1538-7445.AM2015-LB-072