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Zusammenfassung. Die 78-jährige Patientin wurde nach dreimonatiger Verschlechterung ihres Allgemeinzustands bei B-Symptomatik, anhaltend erhöhten Entzündungswerten, progredienter Anämie und Niereninsuffizienz von ihrem Hausarzt zur Tumorsuche überwiesen. Angesichts der zuvor entdeckten pulmonalen Noduli und der neu diagnostizierten Mikrohämaturie vermuteten wir eine Vaskulitis, die wir serologisch (erhöhte MPO-ANCA) und bioptisch bestätigen konnten. Unter Steroidtherapie und Cyclophosphamid konnte eine Remission erzielt werden.

Supplementary Figures

Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy resulting from improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles have been shown to be associated with the mutually exclusive SPOP-mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of prostate cancer brain metastases (PCBM) from 42 patients, with matched primary tumors for 17 patients, we carried out a DNA methylation analysis to examine the epigenetic distinction between primary prostate cancer and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM. Multiregion sampling of PCBM revealed epigenetic stability within metastases. Aberrant methylation in PCBM was associated with mutational background and PRC2 complex activity, an effect that is particularly pronounced in SPOP-mutant PCBM. While PCBM displayed a CpG island hypermethylator phenotype, hypomethylation at the promoters of genes involved in neuroactive ligand–receptor interaction and cell adhesion molecules such as GABRB3, CLDN8, and CLDN4 was also observed, suggesting that cells from primary tumors may require specific reprogramming to form brain metastasis. This study revealed the DNA methylation landscapes of PCBM and the potential mechanisms and effects of PCBM-associated aberrant DNA methylation.Significance:DNA methylation analysis reveals the molecular characteristics of PCBM and may serve as a starting point for efforts to identify and target susceptibilities of these rare metastases.

Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy resulting from improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles have been shown to be associated with the mutually exclusive SPOP-mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of prostate cancer brain metastases (PCBM) from 42 patients, with matched primary tumors for 17 patients, we carried out a DNA methylation analysis to examine the epigenetic distinction between primary prostate cancer and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM. Multiregion sampling of PCBM revealed epigenetic stability within metastases. Aberrant methylation in PCBM was associated with mutational background and PRC2 complex activity, an effect that is particularly pronounced in SPOP-mutant PCBM. While PCBM displayed a CpG island hypermethylator phenotype, hypomethylation at the promoters of genes involved in neuroactive ligand–receptor interaction and cell adhesion molecules such as GABRB3, CLDN8, and CLDN4 was also observed, suggesting that cells from primary tumors may require specific reprogramming to form brain metastasis. This study revealed the DNA methylation landscapes of PCBM and the potential mechanisms and effects of PCBM-associated aberrant DNA methylation. Significance: DNA methylation analysis reveals the molecular characteristics of PCBM and may serve as a starting point for efforts to identify and target susceptibilities of these rare metastases.

Supplementary Table 2: Transcriptomic analyses of the GEMMs A. GEMMs Interactome B. Protein activity C. Cluster analyses D. Pathway analyses

Index of Supplementary Data

Prioritizing treatments for individual patients with cancer remains challenging, and performing coclinical studies using patient-derived models in real time is often unfeasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework that predicts drug sensitivity in human tumors and their preexisting high-fidelity (cognate) model(s) by leveraging drug perturbation profiles. As a proof of concept, we applied OncoLoop to prostate cancer using genetically engineered mouse models (GEMM) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of human prostate cancer cohorts by Master Regulator (MR) conservation analysis revealed that most patients with advanced prostate cancer were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated in allograft, syngeneic, and patient-derived xenograft (PDX) models of tumors and metastasis. Furthermore, OncoLoop-predicted drugs enhanced the efficacy of clinically relevant drugs, namely, the PD-1 inhibitor nivolumab and the AR inhibitor enzalutamide.Significance:OncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround coclinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available.This article is highlighted in the In This Issue feature, p. 247

Detailed Materials and Methods

Figure S1: Genomic alterations in prostate cancer represented in the GEMMs (related to Fig. 2). Figure S2: Additional phenotypic analyses of the GEMMs (related to Fig. 2). Figure S3: Phenotypic analysis of allograft and organoid models (related to Fig. 2). Figure S4: Additional transcriptomic analyses of the GEMMs (related to Fig. 3). Figure S5: Analyses of AR activity in GEMMS (related to Fig. 3). Figure S6: Regulatory sub-networks of the GEMM clusters (related to Fig. 3). Figure S7. MR-match of prostate cancer cells lines to human PCa (related to Figs. 5). Figure S8. Drug perturbation protein activity profiles from DU145 cells (related to Figs. 5, 6, 7). Figure S9. LNCaP Pharmacotyping to patients and GEMMs (related to Figs. 5). Figure S10: Additional validation of drug candidates (related to Figs 6, 7). Figure S11: Additional validation of drug candidates (related to Figs 6).

List of all supplementary items included with the manuscript.

Detailed materials and methods including references.

Supplementary Figures and Figure legends

Datasets reported in the manuscript including gene expression, pathway analyses, and mass spectrometry data.

This files contains all of the supplementary tables included in the manuscript.

Mitochondria provide the first line of defense against the tumor-promoting effects of oxidative stress. Here we show that the prostate-specific homeoprotein NKX3.1 suppresses prostate cancer initiation by protecting mitochondria from oxidative stress. Integrating analyses of genetically engineered mouse models, human prostate cancer cells, and human prostate cancer organotypic cultures, we find that, in response to oxidative stress, NKX3.1 is imported to mitochondria via the chaperone protein HSPA9, where it regulates transcription of mitochondrial-encoded electron transport chain (ETC) genes, thereby restoring oxidative phosphorylation and preventing cancer initiation. Germline polymorphisms of NKX3.1 associated with increased cancer risk fail to protect from oxidative stress or suppress tumorigenicity. Low expression levels of NKX3.1 combined with low expression of mitochondrial ETC genes are associated with adverse clinical outcome, whereas high levels of mitochondrial NKX3.1 protein are associated with favorable outcome. This work reveals an extranuclear role for NKX3.1 in suppression of prostate cancer by protecting mitochondrial function.Significance:Our findings uncover a nonnuclear function for NKX3.1 that is a key mechanism for suppression of prostate cancer. Analyses of the expression levels and subcellular localization of NKX3.1 in patients at risk of cancer progression may improve risk assessment in a precision prevention paradigm, particularly for men undergoing active surveillance.See related commentary by Finch and Baena, p. 2132.This article is highlighted in the In This Issue feature, p. 2113

Many human cancers manifest the capability to circumvent attack by the adaptive immune system. In this work, we identified a component of immune evasion that involves frequent up-regulation of fragile X mental retardation protein (FMRP) in solid tumors. FMRP represses immune attack, as revealed by cancer cells engineered to lack its expression. FMRP-deficient tumors were infiltrated by activated T cells that impaired tumor growth and enhanced survival in mice. Mechanistically, FMRP’s immunosuppression was multifactorial, involving repression of the chemoattractant C-C motif chemokine ligand 7 (CCL7) concomitant with up-regulation of three immunomodulators—interleukin-33 (IL-33), tumor-secreted protein S (PROS1), and extracellular vesicles. Gene signatures associate FMRP’s cancer network with poor prognosis and response to therapy in cancer patients. Collectively, FMRP is implicated as a regulator that orchestrates a multifaceted barrier to antitumor immune responses.

Prioritizing treatments for individual patients with cancer remains challenging, and performing coclinical studies using patient-derived models in real time is often unfeasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework that predicts drug sensitivity in human tumors and their preexisting high-fidelity (cognate) model(s) by leveraging drug perturbation profiles. As a proof of concept, we applied OncoLoop to prostate cancer using genetically engineered mouse models (GEMM) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of human prostate cancer cohorts by Master Regulator (MR) conservation analysis revealed that most patients with advanced prostate cancer were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated in allograft, syngeneic, and patient-derived xenograft (PDX) models of tumors and metastasis. Furthermore, OncoLoop-predicted drugs enhanced the efficacy of clinically relevant drugs, namely, the PD-1 inhibitor nivolumab and the AR inhibitor enzalutamide. Significance: OncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround coclinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available. This article is highlighted in the In This Issue feature, p. 247

Introduction: Interpatient heterogeneity is one of the causes of treatment failure in bladder cancer (BLCa) patients, who would therefore profit from tailor-made therapies. Toward the direction of precision medicine, patient-derived organoids (PDOs), which retain parental tumor (PT) features, have been successfully tested in different cancer types and shown to predict patients’ responses. Therefore, our study aimed to explore the ability of BLCa PDOs to retain PT features and to determine patient drug sensitivity. We correlated PDO drug response profiles to their genomic profiles and the patient's clinical history. Methods: PDOs were derived from the suspension culture of single cells. Whole exome sequencing was performed on genomic DNA and scRNA sequencing on 3 PT/PDO pairs. Histological evaluation of PDO and PT marker expression and morphology was performed. PDOs were treated for 48h and drug response was evaluated by viability assay. Results: We derived PDOs from non-muscle-invasive and muscle-invasive BLCa. Genomic analysis of 15 representative PT/PDOs pairs showed high similarity in terms of copy number variations and single nucleotide variants (SNVs) with preservation of peculiar BLCa alterations. PT genomic heterogeneity was preserved in PDOs as demonstrated by the correlation between SNVs clonality profiles of PT/PDOs pairs. Moreover, marker analyses at the scRNA and protein levels supported that PDOs retained the main tumor phenotype. Interestingly, PDO grew accordingly to three morphologies linked to marker expression and PT stage.PDOs were used in drug screen assays for testing standard-of-care (SOC) and FDA-approved drugs. PDO responses were highly heterogeneous highlighting interpatient heterogeneity. In 2 case, PDO sensitivity mimicked the patient’s response to SOC and in a few cases correlated with the sample-specific genomic background. Due to the high relapse rate of BLCa, we performed 2 longitudinal studies. In the first one, PDOs from a relapse collected after epirubicin treatment showed less sensitivity to the drug compared to the baseline sample. Moreover, the SNVs' mean clonality of relapse PDOs was significantly higher than the baseline PDOs which were characterized by mutations in DNA damage repair genes (e.g., ATM, FANC1, ERCC2, and BRCA2). We hypothesize a drug-induced selection of a pre-existing epirubicin-resistant population. In the second study, PDOs from the cystectomy and the following relapse showed similar genomic and drug sensitivity profiles suggesting that the first sample could be informative for adjuvant therapy. Conclusion: We established PDOs from different BLCa stages and grades which preserve PT features and can be implemented in drug screening assays for personalized approaches. The power of PDOs in precision medicine was highlighted by the longitudinal studies that showed that PDOs mirrored patient response and tumor evolution in vitro. Citation Format: Martina Minoli, Thomas Cantore, Daniel Hanhart, Mirjam Kiener, Tarcisio Fedrizzi, Federico La Manna, Sofia Karkampouna, Panagiotis Chouvardas, Vera Genitsch, José Antonio Rodriguez-Calero, Eva Compérat, Irena Klima, Paola Gasperini, Bernhard Kiss, Roland Seiler-Blarer, Francesca Demichelis, George N. Thalmann, Marianna Kruithof-de Julio. Bladder cancer organoids as a functional system to model different disease stages and therapy response [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 182.

Purpose of Review Classification and nomenclature of neuroendocrine neoplasms (NEN) have frequently changed over the last years. These changes reflect both increasing knowledge and international standardisation. Recent Findings The most recent changes in the Gastro-Entero-Pancreatic system induced the concept of well-differentiated NET with high proliferation rate (NET G3), explaining partially the heterogeneity of G3 NEN. Even if the nomenclature in pulmonary NEN is still different, the terms ‘carcinoid’ and ‘atypical carcinoid’ are widely overlapping with NET G1 and NET G2. Molecular data shows an additional heterogeneity both in well-differentiated NET and poorly differentiated NEC. However, no studies are available demonstrating clinical usefulness yet. Summary The heterogeneity of NEN regarding the organ of origin, differentiation and molecular subtypes make development of personalised therapy a challenge needing more international and interdisciplinary collaborations and clinical trials allowing stratification according to biological subgroups.

Introduction: Improved survival in prostate cancer through more effective therapies has also led to an increase in the diagnosis of metastases to infrequent locations such as the brain. In the largest cohort of prostate cancer brain metastases (PCBM) yet studied, we investigated the DNA methylation landscape of PCBM. Methods: Using Illumina EPIC arrays, we profiled the DNA methylation landscapes of 95 metastatic samples from 42 patients, with multi-regional analysis of the metastases from 31 patients. We compared DNA methylation profiles from metastases to the primary prostate cancer (PCa) samples of 18 of these patients. Multi-regional data was obtained for 15 of these. We performed unsupervised analyses of DNA methylation profiles across sample types, and identified differentially methylated CpG sites and regions (DMRs) between PCBM and PCa samples, and PCBM/PCa samples and normal prostate tissue. We integrated our data with targeted-RNA sequencing and whole exome sequencing (WES). Results: We highlight the epigenetic heterogeneity present in different regions of the PCBM from patients for which multi-regional data were available. While substantial DNA methylation changes were detected in PCa compared to normal prostate tissue, epigenetic dysregulation was strikingly more pronounced in PCBM samples. DNA methylation changes in PCBM involved striking hypermethylation particularly enriched at CpG islands, and associated with activity of the PRC2 complex. We observed a marked distinction of the methylation profiles of SPOP mutant and TMPRSS2-ERG samples. SPOP mutant samples acquired substantially more epigenetic aberrations, particularly enriched at CpG islands which were hypermethylated genome-wide compared to TMPRSS2-ERG PCBM samples, as well as SPOP mutant PCa samples. These changes were strongly associated with the activity of EZH2 and MYC, both of which were overexpressed specifically in SPOP mutant PCBM. TMPRSS2-ERG samples showed fewer DNA methylation changes, but those detected were associated with targets of the SP1 transcription factor. Conclusion: We show that epigenetic heterogeneity is present in different regions of PCBM. While the distinct methylation landscapes of SPOP mutant and TMPRSS2-ERG PCa has been previously reported, we show that metastasis-specific DNA methylation changes emerge in each of these settings. These observations suggest that the acquisition of DNA methylation changes may be an important feature of PCBM, and may help to improve our understanding of the processes driving the occurrence of these rare metastases. Citation Format: John Gallon, Antonio Rodriguez-Calero, Sina Maletti, Charlotte Ng, Mark Rubin, Salvatore Piscuoglio. The DNA methylation landscape of prostate cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5708.

Mitochondria provide the first line of defense against the tumor-promoting effects of oxidative stress. Here we show that the prostate-specific homeoprotein NKX3.1 suppresses prostate cancer initiation by protecting mitochondria from oxidative stress. Integrating analyses of genetically engineered mouse models, human prostate cancer cells, and human prostate cancer organotypic cultures, we find that, in response to oxidative stress, NKX3.1 is imported to mitochondria via the chaperone protein HSPA9, where it regulates transcription of mitochondrial-encoded electron transport chain (ETC) genes, thereby restoring oxidative phosphorylation and preventing cancer initiation. Germline polymorphisms of NKX3.1 associated with increased cancer risk fail to protect from oxidative stress or suppress tumorigenicity. Low expression levels of NKX3.1 combined with low expression of mitochondrial ETC genes are associated with adverse clinical outcome, whereas high levels of mitochondrial NKX3.1 protein are associated with favorable outcome. This work reveals an extranuclear role for NKX3.1 in suppression of prostate cancer by protecting mitochondrial function. Significance: Our findings uncover a nonnuclear function for NKX3.1 that is a key mechanism for suppression of prostate cancer. Analyses of the expression levels and subcellular localization of NKX3.1 in patients at risk of cancer progression may improve risk assessment in a precision prevention paradigm, particularly for men undergoing active surveillance. See related commentary by Finch and Baena, p. 2132. This article is highlighted in the In This Issue feature, p. 2113