Matteo Benelli

SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer

Evading cancer drugs by identity fraud Prostate cancer growth is fueled by male hormones called androgens. Drugs targeting the androgen receptor (AR) are initially efficacious, but most tumors eventually become resistant (see the Perspective by Kelly and Balk). Mu et al. found that prostate cancer cells escaped the effects of androgen deprivation therapy through a change in lineage identity. Functional loss of the tumor suppressors TP53 and RB1 promoted a shift from AR-dependent luminal epithelial cells to AR-independent basal-like cells. In related work, Ku et al. found that prostate cancer metastasis, lineage switching, and drug resistance were driven by the combined loss of the same tumor suppressors and were accompanied by increased expression of the epigenetic regulator Ezh2. Ezh2 inhibitors reversed the lineage switch and restored sensitivity to androgen deprivation therapy in experimental models. Science, this issue p. 84, p. 78; see also p. 29 Prostate cancer cells escape androgen deprivation therapy by morphing into a cell type that does not require androgens. Some cancers evade targeted therapies through a mechanism known as lineage plasticity, whereby tumor cells acquire phenotypic characteristics of a cell lineage whose survival no longer depends on the drug target. We use in vitro and in vivo human prostate cancer models to show that these tumors can develop resistance to the antiandrogen drug enzalutamide by a phenotypic shift from androgen receptor (AR)–dependent luminal epithelial cells to AR-independent basal-like cells. This lineage plasticity is enabled by the loss of TP53 and RB1 function, is mediated by increased expression of the reprogramming transcription factor SOX2, and can be reversed by restoring TP53 and RB1 function or by inhibiting SOX2 expression. Thus, mutations in tumor suppressor genes can create a state of increased cellular plasticity that, when challenged with antiandrogen therapy, promotes resistance through lineage switching.

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.

Differential impact of RB status on E2F1 reprogramming in human cancer

The tumor suppressor protein retinoblastoma (RB) is mechanistically linked to suppression of transcription factor E2F1-mediated cell cycle regulation. For multiple tumor types, loss of RB function is associated with poor clinical outcome. RB action is abrogated either by direct depletion or through inactivation of RB function; however, the basis for this selectivity is unknown. Here, analysis of tumor samples and cell-free DNA from patients with advanced prostate cancer showed that direct RB loss was the preferred pathway of disruption in human disease. While RB loss was associated with lethal disease, RB-deficient tumors had no proliferative advantage and exhibited downstream effects distinct from cell cycle control. Mechanistically, RB loss led to E2F1 cistrome expansion and different binding specificity, alterations distinct from those observed after functional RB inactivation. Additionally, identification of protumorigenic transcriptional networks specific to RB loss that were validated in clinical samples demonstrated the ability of RB loss to differentially reprogram E2F1 in human cancers. Together, these findings not only identify tumor-suppressive functions of RB that are distinct from cell cycle control, but also demonstrate that the molecular consequence of RB loss is distinct from RB inactivation. Thus, these studies provide insight into how RB loss promotes disease progression, and identify new nodes for therapeutic intervention.

Tumor purity quantification by clonal DNA methylation signatures

Motivation: Controlling for tumor purity in molecular analyses is essential to allow for reliable genomic aberration calls, for inter‐sample comparison and to monitor heterogeneity of cancer cell populations. In genome wide screening studies, the assessment of tumor purity is typically performed by means of computational methods that exploit somatic copy number aberrations. Results: We present a strategy, called Purity Assessment from clonal MEthylation Sites (PAMES), which uses the methylation level of a few dozen, highly clonal, tumor type specific CpG sites to estimate the purity of tumor samples, without the need of a matched benign control. We trained and validated our method in more than 6000 samples from different datasets. Purity estimates by PAMES were highly concordant with other state‐of‐the‐art tools and its evaluation in a cancer cell line dataset highlights its reliability to accurately estimate tumor admixtures. We extended the capability of PAMES to the analysis of CpG islands instead of the more platform‐specific CpG sites and demonstrated its accuracy in a set of advanced tumors profiled by high throughput DNA methylation sequencing. These analyses show that PAMES is a valuable tool to assess the purity of tumor samples in the settings of clinical research and diagnostics. Availability and implementation: https://github.com/cgplab/PAMES Contact: matteo.benelli@uslcentro.toscana.it or f.demichelis@unitn.it Supplementary information: Supplementary data are available at Bioinformatics online.

Abstract IA03: Differential impact of RB pathway status on E2F1 reprogramming and disease progression in human prostate cancer

The retinoblastoma tumor suppressor (RB) is mechanistically linked to suppression of E2F1-mediated cell cycle regulation. Abrogation of RB function is associated with poor clinical outcome across various tumor types, which frequently elicit a preference for either RB depletion or functional inactivation, yet the basis for selectivity is unknown. Here, examination of RB pathway alterations in advanced prostate cancer revealed that cyclin dependent kinase (CDK)/cyclin/CDKi alterations are infrequent, and identify RB loss as the major mechanism of pathway disruption in human disease. Furthermore, RB status was readily traced through cell-free DNA analyses in human specimens, thus identifying new ways to assign RB status in the clinical setting. Strikingly, RB depletion in human disease was not associated with a higher Ki67 index, indicating a role for the RB/E2F1 pathway in regulating processes distinct from cell cycle control and associated with lethal-stage disease. Subsequent mechanistic investigation utilized isogenic prostate cancer models, wherein RB could be differentially inactivated through depletion or through hormone-induced, CDK-mediated phosphorylation. Unbiased molecular interrogation uncovered a novel E2F1 cistrome and downstream engagement of transcriptional networks exclusively observed after RB loss, with binding specificity divergent from canonically described E2F1 binding patterns. Additionally, E2F1 cistrome alterations elicited by RB depletion were seen to be distinct from those after phosphorylation-induced RB functional inactivation, providing needed insight into the basis of selectivity for RB loss versus CDK-mediated inactivation observed in human disease. Analyses of human CRPC tumor samples further underscored the clinical relevance of RB loss-induced gene expression programs, which were significantly correlated with reprogrammed E2F1 binding identified herein. Taken together, the studies presented are the first to identify the consequences of RB loss, demonstrating molecular distinction from RB inactivation and illustrating the clinical relevance of RB loss-induced E2F rewiring. Citation Format: Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A Rubin, Myles Brown, Karen E. Knudsen. Differential impact of RB pathway status on E2F1 reprogramming and disease progression in human 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 IA03.

Abstract B040: Differential impact of RB status on E2F1 reprogramming in human cancer

Recent examination of advanced prostate cancer (PCa) has suggested a major mechanism of progression to castration-resistant disease (CRPC) to be loss of the retinoblastoma (RB) protein. Along with its critical role in controlling cell cycle progression, RB is known to have important tumor-suppressor functions, and has been shown in PCa to be lost exclusively in late-stage disease. Additionally, loss of RB has been shown to correlate with increased E2F1 transcript and protein expression, via E2F-dependent mechanisms. Despite the vital role RB loss has been shown to play in this fatal stage of disease, the molecular underpinnings remain undefined. Thus, in order to elucidate these CRPC specific alterations, the current study utilizes isogenic models of RB loss in combination with genome-wide binding and transcriptional studies. Data presented herein demonstrate that loss of RB is frequent in CRPC, and represents the main mechanism of RB pathways disruption in PCa as detected through analyses of tumor samples and cell-free DNA. However, this phenomenon is not correlated with changes in proliferative indices, suggesting a role for RB loss outside of canonical cell cycle control. Further, RB loss induces significant genome-wide transcriptional alterations, including upregulation in Myc, E2F, and DNA-repair related pathways. Additionally, loss of RB significantly expands E2F1 binding capacity in castrate conditions, while largely maintaining the RB-intact E2F1 cistrome. Strikingly, while the current RB/E2F1 paradigm suggests that E2F1 exclusively occupies promoter regions of DNA in order to regulate transcriptional changes, RB loss induces marked reprogramming of E2F1 occupied regions, with a distinct increase in enhancer-bound E2F1. Further, motif analyses suggest divergence away from canonical E2F1 binding motifs after RB loss, specifically in regions of expanded E2F1 binding, and additionally suggest likely interaction of novel E2F1 cofactors under RB loss conditions. Interestingly, changes in E2F1 binding capacity after RB loss were seen to be distinct from those detected after androgen-induced RB inactivation, suggesting that the molecular alterations underlying RB loss are discrete from those resulting from functional inactivation. With respect to putative mechanism, it is of note that chromatin accessibility was not significantly altered to sufficiently explain the widespread changes in E2F1 cistrome, regardless of RB status, suggesting a mechanism outside simple opportunistic E2F1 binding after RB loss. Finally, interrogation of a CRPC patient tumor cohort showed predictive capacity for an “Expanded E2F1 Signature,” resulting from genes exhibiting gained E2F1 binding and differential expression after RB loss, in predicting loss of RB in patient samples, and indicating a novel E2F1-driven set of targets vital for CRPC transition in human disease. Together, these data present the first insight into E2F1 activity resulting from RB loss, and the role these changes play in progression to CRPC. Citation Format: Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Mateus Crespo, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A. Rubin, Myles Brown, Karen E. Knudsen. Differential impact of RB status on E2F1 reprogramming in human 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 B040.

Abstract IA19: Phenotype plasticity—a novel mechanism of targeted therapy resistance

Prostate cancer (PCa) is the most commonly diagnosed cancer and the third highest cause of cancer-related death in men in Europe, where it is responsible for over 90,000 deaths a year. The mainstay of treatment for metastatic PCa is androgen-deprivation therapy (ADT). Although initially effective, the treatment ultimately fails and progression to castration-resistant prostate cancer (CRPC) occurs. Given that CRPC is still driven by hormonal signaling through aberrant activation of the androgen receptor (AR), improved, more potent AR-targeting therapies have been developed for CRPC patients. However, resistance to these therapies ultimately occurs as well. One form of resistance identified by my group results in a phenotypic switch leading to androgen receptor indifference and progression to neuroendocrine prostate cancer (NEPC), which shows a distinct histomorphology and expresses neural-like markers. Unlike more commonly recognized mechanisms of ADT resistance due to AR mutations or amplification, NEPC no longer responds to AR-targeting therapy and has a mean survival of 7 months. There is mounting evidence supporting the role of epigenetic events as a mechanism for transdifferentiation of PCa to an androgen signaling-indifferent state under specific genomic conditions, involving but not limited to TP53, RB1, and PTEN loss. However, the epigenetic regulators at work and the specific changes in the epigenetic landscape are unknown. The SWI/SNF complex is a major epigenetic player, both in regulating normal cell differentiation and in cancer biology. This presentation will focus on novel data supporting the role of alterations in this complex that could contribute to PCa phenotype plasticity. Citation Format: Joanna Cyrta, David Wilkes, Sung Suk Chae, Matteo Benelli, Rohan Bareja, Davide Prandi, Paola Maria Giovanna Cavaliere, Himisha Beltran, Andrea Sboner, Francesca Demichelis, Mark A. Rubin. Phenotype plasticity—a novel mechanism of targeted therapy resistance [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 IA19.

Abstract A078: Towards understanding noncanonical phosphatidylinositol kinases in the maintenance of prostate metabolism

An estimated 1 in 7 men will develop prostate cancer (PCa) with many progressing to advanced castrate-resistant disease. Unlike other tissue types, normal prostate cell growth and development is heavily dependent on the androgen receptor (AR) signaling pathway. While the introduction of novel AR antagonists for clinical treatment has improved outcomes, most castration-resistant prostate cancer (CRPC) patients ultimately develop resistance to these therapies. A need exists to better understand the mechanisms that control the transition of prostate cells from a hormone-dependent to castrate-resistant state. Androgens strongly influence the metabolic state of PCa cells to favor sustained cellular growth. We hypothesize there are effectors working in conjunction with AR to coordinate alterations to androgen-dependent metabolism that are linchpins in the orchestration of the transition to CRPC. Leading candidates are members of phosphoinositol (PI) pathways, which have a high frequency of alteration in PCa (i.e phosphoinositide 3-kinase (PI3K)). Herein we explore a family of poorly understood lipid kinases called the type II phosphatidylinositol-5-phosphate 4-kinases (PI5P4Ks) and predict them to be critical regulators of cancer cell survival. PI5P4Ks are druggable targets that act by phosphorylating the lipid phosphatidylinositol-5-phosphate (PI 5-P) at the 4 position of the inositol ring to generate phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2; PIP2). We implicate the three PI5P4K isoforms (PI5P4Kα, PI5P4Kβ, and PI5P4Kγ) encoded by the genes PIP4K2A, B, and C, to be important regulators of cancer metabolism that play a role in the maintenance of prostate biology and oncogenesis. Analysis of transcript data revealed expression of PIP4K2A, B, and C in primary PCa patient samples, which was correlated with an AR activation gene signature and hotspot tumor suppressor deletion. As well, isoform expression was assessed for differential expression in relation to an integrated neuroendocrine prostate cancer mRNA score (TCGA; n=333). PI5P4Kα and PI5P4Kβ protein was detected in primary and advanced prostate cancer using optimized antibodies of patient tissue TMAs (n= 72). Using in vitro LNCaP cell models, siRNA knockdown systems were tested to evaluate the molecular consequence of targeting PIP4K2A and PIP4K2B in androgen-dependent systems. Stable knockdown using fluorescently labeled lentiviral shRNA constructs significantly reduced proliferation of shPIP4K2 treated cells. As well, we have produced a prostate-specific PI5P4K knockout mouse model by expressing probasin-driven Cre in a homozygous 129/SvEv Pip4k2aflx/flx murine strain. Finally, implementation of a discovery-based metabolomic platform (Metabolon HD4) was used to profile the overall shift in metabolite species that results from downregulating the expression of PIP4K2A in androgen-dependent cell models. In summary, we have developed novel insights into the role of a family of noncanonical PI kinases in prostate biology. There are a growing number of PI3K/AKT inhibitors being tested in combination with androgen deprivation therapy in clinical trials, but there is still almost nothing known about the potential crosstalk of the greater PI kinase network. These data convincingly implicate a fundamental role for PI5P4Ks in PCa androgen signaling and metabolism, as well as lay the foundation of phenotypic understanding of what PI5P4K is responsible for in the prostate. Citation Format: Joanna Triscott, Matteo Benelli, Verena Sailer, Davide Prandi, Brooke Emerling, Francesca Demichelis, Lewis Cantley, Mark A. Rubin. Towards understanding noncanonical phosphatidylinositol kinases in the maintenance of prostate metabolism [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 A078.

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 B39: Exome sequencing in primary melanoma identifies novel drivers of melanoma progression

Melanoma is the most malignant and lethal among skin cancers, due to its high infiltration and invasion ability and resistance to therapy. Whereas early stages melanoma can be cured in the majority of cases by surgical excision, metastatic melanoma is a highly lethal condition. The most frequent known oncogenic mutation in melanoma is BRAF-V600E and several full exome sequencing studies have revealed numerous other alterations (Wei et al, 2011; Nikolaev et al, 2011; Stark et al, 2011; Krauthammer et al, 2012). A crucial issue in understanding melanoma progression is to identify which mutations are specifically involved in making an individual melanoma competent for metastatic spread. It is well established that this behavior is highly correlated with histological features, such as the thickness of the primary tumor and the mitotic index. Here we performed full exome sequencing of 5 thin ( 4mm in thickness) primary melanomas compared to matched-normal DNA. We confirmed recurrent somatic mutations in known melanoma-related genes, including BRAF, c-KIT, EGFR, PPP6C, MLL3 and several components of the glutamate signaling. In addition, we discovered mutations in genes not previously linked to this tumor, such as CSMD1, FGFR4 and components of the Hedgehog (HH) signaling pathway. In particular, in a thick melanoma we found an novel activating mutation in the transcription factor GLI1, one of the final effectors of the HH signaling. Notably, in the only 3 thick melanomas that produced metastasis, we identified candidate metastasis-driving mutations in six genes (ADAMTS6, ADAMTS7, CHD9, MLL3, NALCN and TSC2). Interestingly, we identified several regions of focal somatic copy-number alterations (SCNAs) that were altered at significantly higher frequency in thick compared to thin melanomas. Several gene families are comprised among these regions of focal SCNAs, including components of Notch, HH and Wnt/s-catenin signaling pathways, BRAF, c-MYC and its cofactor PIM1, several ADAMs, EGFR and the HOX genes. Our preliminary results identify potential drivers of melanoma progression, enhancing our understanding of the genomic complexity underlying melanoma. Citation Format: Valentina Montagnani, Matteo Benelli, Alessandro Apollo, Gianni Gerlini, Lorenzo Borgognoni, Barbara Stecca. Exome sequencing in primary melanoma identifies novel drivers of melanoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B39.