Loredana Puca

Emerging Variants of Castration-Resistant Prostate Cancer

Metastatic castration-resistant prostate cancer (CRPC) is associated with substantial clinical, pathologic, and molecular heterogeneity. Most tumors remain driven by androgen receptor (AR) signaling, which has clinical implications for patient selection for AR-directed approaches. However, histologic and clinical resistance phenotypes can emerge after AR inhibition, in which the tumors become less dependent on the AR. In this review, we discuss prostate cancer variants including neuroendocrine (NEPC) and aggressive variant (AVPC) prostate cancers and their clinical implications. Improvements in the understanding of the biologic mechanisms and molecular features underlying prostate cancer variants may help prognostication and facilitate the development of novel therapeutic approaches for subclasses of patient with CRPC.

An emerging role for cytopathology in precision oncology

Precision medicine is an emerging field in medicine for disease prevention and treatment that takes into account the individual variability in genes, environment, and lifestyle for each individual patient. The authors have developed a special program as part of the Englander Institute for Precision Medicine to grow patient‐derived, 3‐dimensional tumor organoids for tumor‐specific drug testing, tailoring treatment strategies, and as models for studying drug resistance. Routine cytology preparations represent a cost‐effective and powerful tool to aid in performing molecular testing in the age of personalized medicine. In this commentary, the platforms used for the characterization and validation of patient‐derived, 3‐dimensional tumor organoids are outlined and discussed, and the role of cytology as a cost‐effective and powerful quality‐control measure is illustrated. Cancer Cytopathol 2016;124:167–173.

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 3093: Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC)

Expression of DLL3 was examined in additional tumor types, as it was found to be highly expressed in tumor-initiating cells (TIC) in small cell lung cancer (SCLC), where a DLL3-targeted antibody drug conjugate (ADC), rovalpituzumab tesirine (Rova-T; SC16LD6.5; Saunders et al. 2015 Sci Transl Med 7:302ra136)) exerted clinically meaningful anti-tumor effects in a phase I trial (Spigel et al. 2016 Lancet Oncology; In Press). DLL3 expression was profiled by qRT-PCR, ELISA and immunohistochemistry in multiple tumor types. Patient-derived xenografts (PDX) from melanoma and ovarian small cell carcinoma were established and used for efficacy studies to determine the ability of Rova-T to impact tumor growth and TIC frequency. DLL3 expression was seen in metastatic melanoma (55%), low grade gliomas (90%), glioblastoma (70%), medullary thyroid cancer (65%), carcinoids (33%), dispersed neuroendocrine tumors in the pancreas (9%), bladder (57%) and prostate (24%), testicular cancer (90%), and lung adenocarcinomas with neuroendocrine features (80%). Unlike SCLC, where DLL3 does not predict clinical outcome on standard therapies, DLL3 expression negatively correlates with overall survival in melanoma and small cell bladder cancer. In mice bearing DLL3 positive melanoma PDX, treatment with a single dose of Rova-T resulted in effective and durable responses (>100 days), which correlated with a significant impact on TIC frequency. Similarly, in mice bearing DLL3-positive ovarian small cell PDX, a single dose of Rova-T resulted in effective and durable responses (>100 days). Our results show that DLL3 is expressed in many neuroendocrine tumors (lung, ovarian, prostate, bladder, etc), metastatic melanoma, medullary thyroid cancer, low-grade gliomas and glioblastoma. Given pre-clinical results showing efficacy of Rova-T in melanoma and ovarian small cell carcinoma, as well as encouraging clinical data with Rova-T in patients with recurrent/refractory SCLC, clinical evaluation of Rova-T in DLL3-positive melanoma, glioblastoma, medullary thyroid cancer and other high-grade neuroendocrine carcinomas is warranted. A “basket” trial enrolling patients with DLL3-positive solid tumors is now recruiting patients (NCT02709889). Citation Format: Laura R. Saunders, Samuel A. Williams, Sheila Bheddah, Kumiko Isse, Sarah Fong, Marybeth A. Pysz, Himisha Beltran, Loredana Puca, Verena Sailer, Juan M. Mosquera, Yu Yin, Jiaoti Huang, Andrew J. Armstrong, Jorge Garcia, Cristina Magi-Galluzzi, Vadim Koshkin, Petros Grivas, Farhad Kosari, John Cheville, Justin C. Moser, Thomas J. Flotte, Thorvardur Halfdanarson, Aaron Mansfield, Konstantinos N. Leventakos, Julian R. Molina, Douglas W. Ball, Barry D. Nelkin, Jill E. Shea, Courtney L. Scaife, Scott J. Dylla. Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2017-3093

Neuroendocrine Differentiation in Prostate Cancer: Emerging Biology, Models, and Therapies

Although a de novo clinical presentation of small cell neuroendocrine carcinoma of the prostate is rare, a subset of patients previously diagnosed with prostate adenocarcinoma may develop neuroendocrine features in later stages of castration-resistant prostate cancer (CRPC) progression as a result of treatment resistance. Despite sharing clinical, histologic, and some molecular features with other neuroendocrine carcinomas, including small cell lung cancer, castration-resistant neuroendocrine prostate cancer (CRPC-NE) is clonally derived from prostate adenocarcinoma. CRPC-NE therefore retains early prostate cancer genomic alterations and acquires new molecular changes making them resistant to traditional CRPC therapies. This review focuses on recent advances in our understanding of CRPC-NE biology, the transdifferentiation/plasticity process, and development and characterization of relevant CRPC-NE preclinical models.

Abstract B037: N-Myc driven cell plasticity in castrate-resistant prostate cancer

An emerging concept of anti-AR therapy resistance is the induction of epithelial plasticity in castration-resistant prostate adenocarcinoma (CRPC) cells that eventually evolve to a neuroendocrine phenotype (e.g., low to no AR signaling and expression of neuroendocrine markers). Neuroendocrine prostate cancer (NEPC) is clinically aggressive and carries a poor prognosis with an average survival of less than one year. We and others have identified and validated new therapeutic targets and drivers of cell transformation from CRPC to NEPC. These include induction of MYCN (encodes N-Myc) or RB1/TP53 loss of function. While N-Myc is a bona fide driver oncogene in several rare tumor types, the N-Myc-driven molecular reprogramming and acquisition of cell plasticity remains poorly understood. We have previously shown that intact N-Myc+/Pten-null genetically engineered mice (GEM) develop poorly differentiated, highly proliferative, invasive prostate cancer that is molecularly similar to human NEPC tumor (1). Similar morphologic features were observed in organoids derived from our GEM model. Flow analysis of these organoids revealed that N-Myc+/Pten-null GEM-derived organoids are comprised of a heterogeneous population of cells with luminal, basal, or features of both lineages compared to more homogeneous population of cells from Pten-null control organoids. Recently, we also found that N-Myc overexpression induced a faster recurrence in the context of castration in vivo, based on the GEM model and 22Rv1 isogenic xenografts. This is consistent with the significant N-Myc induced increased castrate-resistant tumor growth based on data from GEM organoids grown in vivo as allografts. In the GEM, we found that castration led to invasive prostate tumors that metastasize to multiple locations including the liver and that express the EMT marker vimentin and the NEPC marker neural cell adhesion molecule (NCAM1). Based on RNAseq data from 22Rv1 xenografts in castrated mice, we identified stem cell signatures including neural crest stem genes (GSEA q value = 7.0E-19) that were significantly enriched in the N-Myc upregulated genes including SOX2, SOX10, and SOX11, genes implicated in neural development and NEPC associated genes (e.g., NCAM1 and chromogranin A [CHGA]). N-Myc ChIP-Seq data confirmed N-Myc occupancy enriched at promoters of genes implicated in stem cell-associated pathways. RNAseq data from human NEPC organoid following MYCN depletion revealed similar N-Myc-associated gene expression changes. Altogether, these data led to a model in which N-Myc overexpression induces a castrate-resistant, lineage-plastic phenotype that would give rise to NEPC, consistent with the phenotype induced by RB1/TP53/PTEN loss [2-4]. References: 1. Dardenne E., et al. N-Myc induces an EZH2-mediated transcriptional program driving neuroendocrine prostate cancer. Cancer Cell 2016:30(4):563-77. 2. Ku SY, et al. Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance. Science 2017:355(6320):78-83. 3. Mu P, et al., SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer. Science 2017;355(6320):84-8. 4. Zou M, et al. Transdifferentiation as a mechanism of treatment resistance in a mouse model of castration-resistant prostate cancer. Cancer Discov 2017. Citation Format: Etienne Dardenne, Adeline Berger, Katie Gayvert, Loredana Puca, Jamal Elkader, Brian Robinson, Olivier Elemento, Beltran Himisha, David Rickman. N-Myc driven cell plasticity in castrate-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B037.

Abstract 992: Patient-derived tumor organoids of neuroendocrine prostate cancer

Background: The development of neuroendocrine prostate cancer (NEPC) is one mechanism of treatment resistance to androgen receptor (AR)-targeted therapies for a subset of patients with advanced prostate cancer. This is associated with transition from a prostate adenocarcinoma to small cell/NEPC histology, low AR signaling signaling, and expression of neuroendocrine markers as Chromogranin A (CGHA), Synaphophysin (SYP) and CD56). Patient derived preclinical models recapitulating the NEPC phenotype may be used to address NEPC pathogenesis and test emerging therapeutic targets. Methods: Tumor organoids were developed according to protocols previously described (Gao et al, Cell 2015). Briefly the tissue biopsies (liver and bone biopsy) were washed, enzymatically digested and then seeded in Matrigel (BD) droplets. Organoids were characterized at genomic (WES), RNA and protein level (IHC) to confirm the expression of specific markers. Lentiviral infections were performed using shRNAs against EZH2 to knock down EZH2 in organoids. Organoids were also subcutaneously injected in NSG mice to generate patient derived xenografts (PDXs) for drug treatment in vivo. Results: We developed and characterized two NEPC tumor organoids from tumor biopsies (liver and bone) of two patients both in vitro and in vivo (as PDXs). NEPC tumor organoid models retained the molecular and histological characteristic of their matched patient samples. We successfully manipulated the activity of the histone methyltransferase EZH2 by using a catalytic inhibitor and its expression by infecting organoids with shEZH2. We showed that the absence of EZH2 affects the expression of neuroendocrine-associated programs as stem cell and neuronal pathway. Moreover treatment with EZH2 inhibitor decreased tumor organoids viability and PDXs tumor volume. Drug screening approaches on NEPC organoids were used to discovery novel drug targets and combinations that could potentially benefit NEPC patients. Top single agent hits included previously identified targets such as EZH2, AURKA, as well as novel synergies. Conclusions NEPC patient tumor organoids are clinically relevant tumor models to study the NEPC phenotype in advanced prostate cancer and may be used to elucidate novel drug targets. Citation Format: Loredana Puca, Rohan Bareja, Reid Shaw, Wouter Karthaus, Dong Gao, Chantal Pauli, Juan Miguel Mosquera, Joanna Cyrta, Rachele Rosati, Rema Rao, Andrea Sboner, Carla Grandori, Giorgio Inghirami, Yu Chen, Mark A. Rubin, Himisha Beltran. Patient-derived tumor organoids of neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 992. doi:10.1158/1538-7445.AM2017-992

Abstract A20: Personalized models to guide precision medicine

Background: Precision oncology is a clinical approach aimed towards tailoring treatment strategies for patients based on the genetic profile of each patient9s cancer. Available cell line models alone often do not accurately recapitulate the genetic profile of individual patient tumors and therefore limit preclinical evaluation of newly targeted agents. Furthermore, a high failure rate of drug candidates can be attributed in part to the use of monolayer cultures as the initial screening method that is associated with highly variable responses and does not predict clinically observed chemoresistance. In our Englander Institute for Precision Medicine we developed a program utilizing patient derived tumor organoids, in combination with individualized genomic sequencing, targeted and/or high throughput drug screenings to nominate drug candidates in a precision patient care setting. Drug candidates are further validated with personalized in vivo models. Utilizing these various genomic and biological platforms for pharmacological screenings, we can more closely recapitulate the in vivo tumor of individual patients and can more accurately model personalized therapeutic response and resistance in vitro and in vivo. Design: Fresh tissue samples were collected, washed and mechanically or enzymatically dissociated and then plated in a Matrigel (BD) scaffold with primary culture media. Primary spheres were characterized according to our cytology, histology and genomic platforms. Established and characterized tumor organoids were expanded, cryopreserved for banking, used for in vitro studies and implanted in nude mice for patient derived xenografts (PDXs) to further validate potential drug candidates. Results: Our success rate in generating patient derived pan-cancer tumor organoids is 30%, depending on specimen quality and tumor type (e.g. endometrial cancer 70%, metastatic prostate cancer 15%). Morphology and molecular profiles show good concordance among tumor organoids and native tumor tissues. The success rate in establishing PDXs from organoid cultures is currently at 70-80%. In vitro and in vivo drug screenings show tumor specific drug sensitivity. Conclusion: We have developed protocols for the generation and characterization of individual patient-derived tumor organoids. Cytopathology, histopathology and molecularpathology represent important platforms in our Precisicon Medicine Program. Tumor organoid characterization, pharmacological screenings and drug validation in PDX models are effective models which can be used to tailor standard of care treatment, study drug resistance, and nominate novel therapeutic targets unique to the individual genomic landscape and biology of each tumor. Citation Format: Chantal Pauli, Loredana Puca, Benjamin Hopkins, Brooke M. Emerling, Andrea Sboner, Olivier Elemento, Terra J. McNary, Yelena Churakova, Himisha Beltran, Mark A. Rubin. Personalized models to guide precision medicine. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A20.

Abstract 619: Personalized models to guide precision medicine

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA BACKGROUND Precision oncology is a clinical approach aimed towards tailoring treatment strategies for patients based on the genetic profile of each patients cancer. Available cell line models alone often do not accurately recapitulate the genetic profile of individual patient tumors and therefore limit preclinical evaluation of newly targeted agents. Furthermore, a high failure rate of drug candidates can be attributed in part to the use of monolayer cultures as the initial screening method that is associated with highly variable responses and does not predict clinically observed chemoresistance. In our Englander Institute for Precision Medicine we developed a program utilizing patient derived tumor organoids, in combination with individualized genomic sequencing, targeted and/or high throughput drug screenings to nominate drug candidates in a precision patient care setting. Drug candidates are further validated with personalized in vivo models. Utilizing these various genomic and biological platforms for pharmacological screenings, we can more closely recapitulate the in vivo tumor of individual patients and can more accurately model personalized therapeutic response and resistance in vitro and in vivo. DESIGN Fresh tissue samples were collected, washed and mechanically or enzymatically dissociated and then plated in a Matrigel (BD) scaffold with primary culture media. Primary spheres were characterized according to our cytology, histology and genomic platforms. Established and characterized tumor organoids were expanded, cryopreserved for banking, used for in vitro studies and implanted in nude mice for patient derived xenografts (PDXs) to further validate potential drug candidates. RESULTS Our success rate in generating patient derived pan-cancer tumor organoids is 30%, depending on specimen quality and tumor type (e.g. endometrial cancer 70%, metastatic prostate cancer 15%). Morphology and molecular profiles show good concordance among tumor organoids and native tumor tissues. The success rate in establishing PDXs from organoid cultures is currently at 70-80%. In vitro and in vivo drug screenings show tumor specific drug sensitivity. CONCLUSION We have developed protocols for the generation and characterization of individual patient-derived tumor organoids. Cytopathology, histopathology and molecular pathology represent important platforms in our Precisicon Medicine Program. Tumor organoid characterization, pharmacological screenings and drug validation in PDX models are effective models which can be used to tailor standard of care treatment, study drug resistance, and nominate novel therapeutic targets unique to the individual genomic landscape and biology of each tumor. Citation Format: Chantal Pauli, Loredana Puca, Brooke M. Emerling, Benjamin Hopkins, Andrea Sboner, Olivier Elemento, Juan Miguel Mosquera, Himisha Beltran, Mark A. Rubin. Personalized models to guide precision medicine. [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 619.

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.