Search

Current treatments for retinoblastoma, the most common intraocular malignancy, rely on a limited number of drugs repurposed from other pediatric cancer therapies. Drug toxicity and relapse of the disease necessitate new therapeutic strategies. The implementation of drug candidates in the clinic is usually followed by in vitro validation, which requires accurate and clinically relevant in vitro models. We report here a robust 3D tumoroid-based platform, for testing various clinical protocols, e.g. therapeutic agents following a single or repeated exposure, alone or in combination with focal therapy (thermotherapy). The platform, generated from the Y79 retinoblastoma cell line, consists of matrix-embedded tumoroids, which retain the key features of the disease and allow prolonged culture time. Following the optimization of growth conditions, the effect of single and repeated exposure to chemotherapeutic agents has been measured by high-throughput automated fluorescence imaging followed by quantification. The clinical conditions of chemothermotherapy have been recapitulated with a setup consisting of: an infrared diode laser (810 nm, 0.3 W) that heats the tumoroids in the presence of indocyanine green (50 µg/mL); a thermal camera to enable real-time temperature measurement; and an environment-controlled inverted microscope to ensure physiological conditions and precise focusing of the laser. Melphalan and carboplatin, two drugs currently used to treat retinoblastoma in clinical protocols of chemotherapy and chemothermotherapy respectively, have been applied to validate the system.Tumoroid viability and size progressively decreased over 4 weeks of repeated drug exposure with melphalan, reflecting the response of retinoblastoma in advanced clinical cases. Carboplatin treatment showed an activity enhancement in the tumoroid model similar to that observed in the clinic, when combined with focal therapy, demonstrating that the platform system closely recapitulates the clinical conditions. Inversely, the thermotherapy experimental protocol designed for 2D in vitro cell culture and commonly used to date, i.e. an incubation temperature increased to 42 °C for 1 h, did not show an improvement of the carboplatin cytotoxicity, confirming that our laser-based thermotherapy protocol provides a more accurate and clinically relevant drug response. Such an approach, combining physiologically relevant in vitro cancer models and investigation of drugs in well-established treatment modalities, can significantly reduce the number of new drug candidates that give false-positives or overestimated efficacies when evaluated in relevant in vivo models. It is expected that the platform will contribute to the implementation of new therapeutic strategies to treat retinoblastoma, and could also be adapted to other diseases for which repeated drug exposure and/or chemothermotherapy is relevant. Citation Format: Irina Sinenko, Fabien Kuttler, Valentin Simeonov, Alexandre Moulin, Christina Stathopoulos, Gerardo Turcatti, Adeline Berger, Francis Munier, Paul Dyson. Tumoroid-based screening platform to test focal, chemo- and combination therapy for retinoblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3541.

Background: Despite recent advances in the development of highly effective androgen receptor (AR)-directed therapies for the treatment of prostate cancer, nearly 37% of patients develop resistance. A further third of these patients progress to develop aggressive neuroendocrine prostate cancer (NEPC) for which no effective therapies exist. Lineage plasticity, a process by which differentiated cells lose their identity and acquire alternative lineage programs, has been proposed as a mechanism of resistance to targeted therapies in epithelial tumors such as prostate cancer. Although epigenetic deregulation has been implicated as a driver of the epithelial to neuronal switch observed in NEPC, the molecular programs are poorly understood. Previously, we observed that the majority of NEPC and 20% of castration-resistant prostate cancer (CRPC) aberrantly overexpress the transcription factor MYCN (N-Myc). Despite this frequent occurrence, the role of N-Myc in driving lineage plasticity and the epigenetic mechanisms which regulate disease progression remain to be elucidated. Methods: We analyzed overall survival and whole transcriptome data from a cohort of over 200 prostate cancer patients, including the largest-to-date population of NEPC patients. We also analyzed epigenetic modifications along with the N-Myc transcriptome, cistrome and chromatin-bound interactome by performing ChIP-seq, RNA-seq and RIME in a combination of mouse models, human prostate cancer cell lines, and NEPC patient-derived organoids. Results: Expression of MYCN in CRPC and NEPC patients correlates with reduced overall survival. NEPC tumors are significantly enriched for stem cell genes associated with normal neuroendocrine cell precursors, embryonic stem cells and neural lineage-defining genes from activated neural stem cells. The integration of next-generation sequencing data revealed that the N-Myc cistrome is androgen-dependent and drives a transcriptional program leading to epithelial plasticity and the acquisition of clinically relevant neuronal lineage markers. Our data also reveal that N-Myc interacting proteins as well as AR co-factors and pioneering factors (e.g. FOXA1 and HOXB13) explain the observed dynamic binding of N-Myc. Interestingly, histone marks and EZH2 activity specifically associated with lineage-defining genes were reprogrammed by N-Myc. Finally, we demonstrated that N-Myc-induced gene expression and epigenetic changes accurately classify our patient cohort. Conclusions: We describe a novel role for N-Myc in prostate cancer, characterized by changes in the N-Myc cistrome and cooperation with AR co-factors, as well as reprogramming of the epigenome in an androgen context-dependent manner. This reprogramming is associated with induction of a lineage plastic state and a switch towards a neural identity that favors the development of AR independence and NEPC. Citation Format: Adeline Berger, Nicholas Brady, Rohan Bareja, Brian Robinson, Vincenza Conteduca, Michael Augello, Loredana Puca, Adnan Ahmed, Xiaodong Lu, Inah Hwang, Alyssa Bagadion, Andrea Sboner, Olivier Elemento, Jihye Paik, Jindan Yu, Christopher Barbieri, Noah Dephoure, Himisha Beltran, David S. Rickman. N-Myc-mediated epigenetic reprogramming drives lineage plasticity in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2099.

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.

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