Rarγ -Foxa1 signaling promotes luminal identity in prostate progenitors and is disrupted in prostate cancer.

Retinoic acid (RA) signaling is a master regulator of vertebrate development with crucial roles in directing body axis orientation and tissue differentiation, including in the reproductive system. However, a mechanistic understanding of how RA signaling promotes cell lineage identity in different tissues is often missing. Here, leveraging prostate organoid technology, we demonstrated that RA signaling orchestrates the commitment of adult mouse prostate progenitors to glandular identity, epithelial barrier integrity, and ultimately, proper specification of the prostatic lumen. Mechanistically, RA-dependent RARγ activation promotes the expression of the pioneer factor Foxa1, which synergizes with the androgen pathway for proper luminal expansion, cytoarchitecture and function. FOXA1 nucleotide variants are common in human prostate and breast cancers and considered driver mutations, though their pathogenic mechanism is incompletely understood. Combining functional genetics experiments with structural modeling of FOXA1 folding and chromatin binding analyses, we discovered that FOXA1 F254E255 is a loss-of-function mutation leading to compromised transcriptional function and lack of luminal fate commitment of prostate progenitors. Overall, we define RA as a crucial instructive signal for glandular identity in adult prostate progenitors. We propose deregulation of vitamin A metabolism as a risk factor for benign and malignant prostate disease, and identified cancer associated FOXA1 indels affecting residue F254 as loss-of-function mutations promoting dedifferentiation of adult prostate progenitors. Summary: Retinoic acid signaling orchestrates luminal differentiation of adult prostate progenitors.

Increased genomic instability and reshaping of tissue microenvironment underlie oncogenic properties of Arid1a mutations.

Oncogenic mutations accumulating in many chromatin-associated proteins have been identified in different tumor types. With a mutation rate from 10 to 57%, ARID1A has been widely considered a tumor suppressor gene. However, whether this role is mainly due to its transcriptional-related activities or its ability to preserve genome integrity is still a matter of intense debate. Here, we show that ARID1A is largely dispensable for preserving enhancer-dependent transcriptional regulation, being ARID1B sufficient and required to compensate for ARID1A loss. We provide in vivo evidence that ARID1A is mainly required to preserve genomic integrity in adult tissues. ARID1A loss primarily results in DNA damage accumulation, interferon type I response activation, and chronic inflammation leading to tumor formation. Our data suggest that in healthy tissues, the increased genomic instability that follows ARID1A mutations and the selective pressure imposed by the microenvironment might result in the emergence of aggressive, possibly immune-resistant, tumors.

Germline determinants of aberrant signaling pathways in cancer.

Cancer is a complex disease influenced by a heterogeneous landscape of both germline genetic variants and somatic aberrations. While there is growing evidence suggesting an interplay between germline and somatic variants, and a substantial number of somatic aberrations in specific pathways are now recognized as hallmarks in many well-known forms of cancer, the interaction landscape between germline variants and the aberration of those pathways in cancer remains largely unexplored. Utilizing over 8500 human samples across 33 cancer types characterized by TCGA and considering binary traits defined using a large collection of somatic aberration profiles across ten well-known oncogenic signaling pathways, we conducted a series of GWAS and identified genome-wide and suggestive associations involving 276 SNPs. Among these, 94 SNPs revealed cis-eQTL links with cancer-related genes or with genes functionally correlated with the corresponding traits' oncogenic pathways. GWAS summary statistics for all tested traits were then used to construct a set of polygenic scores employing a customized computational strategy. Polygenic scores for 24 traits demonstrated significant performance and were validated using data from PCAWG and CCLE datasets. These scores showed prognostic value for clinical variables and exhibited significant effectiveness in classifying patients into specific cancer subtypes or stratifying patients with cancer-specific aggressive phenotypes. Overall, we demonstrate that germline genetics can describe patients' genetic liability to develop specific cancer molecular and clinical profiles.

Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

Analysis of Genetic Ancestry from NGS Data Using EthSEQ.

Next-generation sequencing (NGS) is widely utilized both in translational cancer genomics studies and in the setting of precision medicine. Identification and stratification of an individual's ancestry is fundamental for the correct interpretation of genetic and genomic profiling. EthSEQ provides an easy and effective computational workflow to determine the ancestry of individuals, exploiting single nucleotide polymorphism genotypes computed from NGS data of whole-exome and targeted sequencing experiments. Genotypes are determined by EthSEQ from sequencing alignment files (BAM files) or can be provided as input in Variant Call Format (VCF) or CoreArray Genomic Data Structure (GDS) format. Ancestry is determined and assigned to individuals by EthSEQ exploiting a reference model and a standard or multi-step refinement approach based on Principal Component Analysis (PCA). A complete and detailed set of textual and graphical output files are generated by EthSEQ as result. EthSEQ is easy to use and can be integrated into any NGS-based processing pipeline also exploiting multi-core capabilities. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Perform ancestry analysis using a pre-computed reference model Alternate Protocol: Perform ancestry analysis using a user-specified GDS file as reference model Basic Protocol 2: Perform ancestry analysis using multi-step refinement Support Protocol 1: Create a reference model from multiple VCF genotype data files Support Protocol 2: Create VCF genotype data files from a BAM file.

LSD1/PRMT6-targeting gene therapy to attenuate androgen receptor toxic gain-of-function ameliorates spinobulbar muscular atrophy phenotypes in flies and mice.

Spinobulbar muscular atrophy (SBMA) is caused by CAG expansions in the androgen receptor gene. Androgen binding to polyQ-expanded androgen receptor triggers SBMA through a combination of toxic gain-of-function and loss-of-function mechanisms. Leveraging cell lines, mice, and patient-derived specimens, we show that androgen receptor co-regulators lysine-specific demethylase 1 (LSD1) and protein arginine methyltransferase 6 (PRMT6) are overexpressed in an androgen-dependent manner specifically in the skeletal muscle of SBMA patients and mice. LSD1 and PRMT6 cooperatively and synergistically transactivate androgen receptor, and their effect is enhanced by expanded polyQ. Pharmacological and genetic silencing of LSD1 and PRMT6 attenuates polyQ-expanded androgen receptor transactivation in SBMA cells and suppresses toxicity in SBMA flies, and a preclinical approach based on miRNA-mediated silencing of LSD1 and PRMT6 attenuates disease manifestations in SBMA mice. These observations suggest that targeting overexpressed co-regulators can attenuate androgen receptor toxic gain-of-function without exacerbating loss-of-function, highlighting a potential therapeutic strategy for patients with SBMA.

Synggen: fast and data-driven generation of synthetic heterogeneous NGS cancer data.

SUMMARY: Whole-exome and targeted sequencing are widely utilized both in translational cancer genomics and in the setting of precision medicine. The benchmarking of computational methods and tools that are in continuous development is fundamental for the correct interpretation of somatic genomic profiling results. To this aim we developed synggen, a tool for the fast generation of large-scale realistic and heterogeneous cancer whole-exome and targeted sequencing synthetic datasets, which enables the incorporation of phased germline single nucleotide polymorphisms and complex allele-specific somatic genomic events. Synggen performances and effectiveness in generating synthetic cancer data are shown across different scenarios and considering different platforms with distinct characteristics. AVAILABILITY AND IMPLEMENTATION: synggen is freely available at https://bitbucket.org/CibioBCG/synggen/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Divergent HLA variations and heterogeneous expression but recurrent HLA loss-of- heterozygosity and common HLA-B and TAP transcriptional silencing across advanced pediatric solid cancers.

The human leukocyte antigen (HLA) system is a major factor controlling cancer immunosurveillance and response to immunotherapy, yet its status in pediatric cancers remains fragmentary. We determined high-confidence HLA genotypes in 576 children, adolescents and young adults with recurrent/refractory solid tumors from the MOSCATO-01 and MAPPYACTS trials, using normal and tumor whole exome and RNA sequencing data and benchmarked algorithms. There was no evidence for narrowed HLA allelic diversity but discordant homozygosity and allele frequencies across tumor types and subtypes, such as in embryonal and alveolar rhabdomyosarcoma, neuroblastoma MYCN and 11q subtypes, and high-grade glioma, and several alleles may represent protective or susceptibility factors to specific pediatric solid cancers. There was a paucity of somatic mutations in HLA and antigen processing and presentation (APP) genes in most tumors, except in cases with mismatch repair deficiency or genetic instability. The prevalence of loss-of-heterozygosity (LOH) ranged from 5.9 to 7.7% in HLA class I and 8.0 to 16.7% in HLA class II genes, but was widely increased in osteosarcoma and glioblastoma (~15-25%), and for DRB1-DQA1-DQB1 in Ewing sarcoma (~23-28%) and low-grade glioma (~33-50%). HLA class I and HLA-DR antigen expression was assessed in 194 tumors and 44 patient-derived xenografts (PDXs) by immunochemistry, and class I and APP transcript levels quantified in PDXs by RT-qPCR. We confirmed that HLA class I antigen expression is heterogeneous in advanced pediatric solid tumors, with class I loss commonly associated with the transcriptional downregulation of HLA-B and transporter associated with antigen processing (TAP) genes, whereas class II antigen expression is scarce on tumor cells and occurs on immune infiltrating cells. Patients with tumors expressing sufficient HLA class I and TAP levels such as some glioma, osteosarcoma, Ewing sarcoma and non-rhabdomyosarcoma soft-tissue sarcoma cases may more likely benefit from T cell-based approaches, whereas strategies to upregulate HLA expression, to expand the immunopeptidome, and to target TAP-independent epitopes or possibly LOH might provide novel therapeutic opportunities in others. The consequences of HLA class II expression by immune cells remain to be established. Immunogenetic profiling should be implemented in routine to inform immunotherapy trials for precision medicine of pediatric cancers.

A slow-cycling/quiescent cells subpopulation is involved in glioma invasiveness.

Pediatric and adult high-grade gliomas are the most common primary malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after therapy. Quiescent cells have been implicated in tumor recurrence and treatment resistance, but their direct visualization and targeting remain challenging, precluding their mechanistic study. Here, we identify a population of malignant cells expressing Prominin-1 in a non-proliferating state in pediatric high-grade glioma patients. Using a genetic tool to visualize and ablate quiescent cells in mouse brain cancer and human cancer organoids, we reveal their localization at both the core and the edge of the tumors, and we demonstrate that quiescent cells are involved in infiltration of brain cancer cells. Finally, we find that Harmine, a DYRK1A/B inhibitor, partially decreases the number of quiescent and infiltrating cancer cells. Our data point to a subpopulation of quiescent cells as partially responsible of tumor invasiveness, one of the major causes of brain cancer morbidity.

Transient rapamycin treatment during developmental stage extends lifespan in Mus musculus and Drosophila melanogaster.

Lifespan is determined by complex and tangled mechanisms that are largely unknown. The early postnatal stage has been proposed to play a role in lifespan, but its contribution is still controversial. Here, we show that a short rapamycin treatment during early life can prolong lifespan in Mus musculus and Drosophila melanogaster. Notably, the same treatment at later time points has no effect on lifespan, suggesting that a specific time window is involved in lifespan regulation. We also find that sulfotransferases are upregulated during early rapamycin treatment both in newborn mice and in Drosophila larvae, and transient dST1 overexpression in Drosophila larvae extends lifespan. Our findings unveil a novel link between early-life treatments and long-term effects on lifespan.

Allele-informed copy number evaluation of plasma DNA samples from metastatic prostate cancer patients: the PCF_SELECT consortium assay.

Sequencing of cell-free DNA (cfDNA) in cancer patients' plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA.

Intra-epithelial non-canonical Activin A signaling safeguards prostate progenitor quiescence.

The healthy prostate is a relatively quiescent tissue. Yet, prostate epithelium overgrowth is a common condition during aging, associated with urinary dysfunction and tumorigenesis. For over thirty years, TGF-ß ligands have been known to induce cytostasis in a variety of epithelia, but the intracellular pathway mediating this signal in the prostate, and its relevance for quiescence, have remained elusive. Here, using mouse prostate organoids to model epithelial progenitors, we find that intra-epithelial non-canonical Activin A signaling inhibits cell proliferation in a Smad-independent manner. Mechanistically, Activin A triggers Tak1 and p38 ΜAPK activity, leading to p16 and p21 nuclear import. Spontaneous evasion from this quiescent state occurs upon prolonged culture, due to reduced Activin A secretion, a condition associated with DNA replication stress and aneuploidy. Organoids capable to escape quiescence in vitro are also able to implant with increased frequency into immunocompetent mice. This study demonstrates that non-canonical Activin A signaling safeguards epithelial quiescence in the healthy prostate, with potential implications for the understanding of cancer initiation, and the development of therapies targeting quiescent tumor progenitors.

ETS-related gene (ERG) undermines genome stability in mouse prostate progenitors via Gsk3ß dependent Nkx3.1 degradation.

21q22.2-3 deletion is the most common copy number alteration in prostate cancer (PCa). The genomic rearrangement results in the androgen-dependent de novo expression of ETS-related gene (ERG) in prostate cancer cells, a condition promoting tumor progression to advanced stages of the disease. Interestingly, ERG expression characterizes 5-30% of tumor precursor lesions - High Grade Prostatic Intraepithelial Neoplasia (HGPIN) - where its role remains unclear. Here, by combining organoids technology with Click-chemistry coupled Mass Spectrometry, we demonstrate a prominent role of ERG in remodeling the protein secretome of prostate progenitors. Functionally, by lowering autocrine Wnt-4 signaling, ERG represses canonical Wnt pathway in prostate progenitors, and, in turn, promotes the accumulation of DNA double strand breaks via Gsk3ß-dependent degradation of the tumor suppressor Nkx3.1. On the other hand, by shaping extracellular paracrine signals, ERG strengthens the pro-oxidative transcriptional signature of inflammatory macrophages, which we demonstrate to infiltrate pre-malignant ERG positive prostate lesions. These findings highlight previously unrecognized functions of ERG in undermining adult prostate progenitor niche through cell autonomous and non-autonomous mechanisms. Overall, by supporting the survival and proliferation of prostate progenitors in the absence of growth stimuli and promoting the accumulation of DNA damage through destabilization of Nkx3.1, ERG could orchestrate the prelude to neoplastic transformation.

YES1 and MYC Amplifications as Synergistic Resistance Mechanisms to Different Generation ALK Tyrosine Kinase Inhibitors in Advanced NSCLC: Brief Report of Clinical and Preclinical Proofs.

INTRODUCTION: ALK tyrosine kinase inhibitors (TKIs) are the standard treatment for advanced ALK-positive NSCLC. Nevertheless, drug resistance inevitably occurs. Here, we report a case of a patient with metastatic ALK-positive lung adenocarcinoma with an impressive resistance to sequential treatment with ALK TKIs mediated by YES1 and MYC amplification in a contest of epithelial-to-mesenchymal transition and high progressive chromosomal instability. METHODS: The patient received, after chemotherapy and 7 months of crizotinib, brigatinib and lorlatinib with no clinical benefit to both treatments. A study of resistance mechanisms was performed with whole exome sequencing on different biological samples; primary cell lines were established from pleural effusion after lorlatinib progression. RESULTS: At whole exome sequencing analysis, YES1 and MYC amplifications were observed both in the pericardial biopsy and the pleural effusion samples collected at brigatinib and lorlatinib progression, respectively. Increasing chromosomal instability from diagnostic biopsy to pleural effusion was also observed. The addition of dasatinib to brigatinib or lorlatinib restored the sensitivity in primary cell lines; data were confirmed also in H3122_ALK-positive model overexpressing both YES1 and MYC. CONCLUSIONS: In conclusion, YES1 and MYC amplifications are candidates to justify a rapid acquired resistance to crizotinib entailing primary brigatinib and lorlatinib resistance. In this context, a combination strategy of ALK TKI with dasatinib could be effective to overcome a rapid resistance.

Polympact: exploring functional relations among common human genetic variants.

In the last years, many studies were able to identify associations between common genetic variants and complex diseases. However, the mechanistic biological links explaining these associations are still mostly unknown. Common variants are usually associated with a relatively small effect size, suggesting that interactions among multiple variants might be a major genetic component of complex diseases. Hence, elucidating the presence of functional relations among variants may be fundamental to identify putative variants' interactions. To this aim, we developed Polympact, a web-based resource that allows to explore functional relations among human common variants by exploiting variants' functional element landscape, their impact on transcription factor binding motifs, and their effect on transcript levels of protein-coding genes. Polympact characterizes over 18 million common variants and allows to explore putative relations by combining clustering analysis and innovative similarity and interaction network models. The properties of the network models were studied and the utility of Polympact was demonstrated by analysing the rich sets of Breast Cancer and Alzheimer's GWAS variants. We identified relations among multiple variants, suggesting putative interactions. Polympact is freely available at bcglab.cibio.unitn.it/polympact.

Plasma tumor DNA is associated with increased risk of venous thromboembolism in metastatic castration-resistant cancer patients.

Cancer is a risk factor for venous thromboembolism (VTE). Plasma tumor DNA (ptDNA) is an independent predictor of outcome in metastatic castration-resistant prostate cancer (mCRPC). We aimed to investigate the association between ptDNA and VTE in mCRPC. This prospective biomarker study included 180 mCRPC patients treated with abiraterone and enzalutamide from April 2013 to December 2018. We excluded patients with a previous VTE history and/or ongoing anticoagulation therapy. Targeted next-generation sequencing was performed to determine ptDNA fraction from pretreatment plasma samples. VTE risk based on survival analysis was performed using cumulative incidence function and estimating sub-distributional hazard ratio (SHR). At a median follow-up of 58 months (range 0.5-111.0), we observed 21 patients who experienced VTE with a cumulative incidence at 12 months of 17.1% (95% confidence interval [CI] 10.3-23.9). Elevated ptDNA, visceral metastasis, prior chemotherapy and lactate dehydrogenase (LDH) were significantly associated with higher VTE incidence compared to patients with no thrombosis (12-month estimate, 18.6% vs 3.5%, P = .0003; 44.4% vs 14.8%, P = .015; 24.7% vs 4.5%, P = .006; and 30.0% vs 13.5%, P = .05, respectively). In the multivariate analysis including ptDNA level, visceral metastases, number of lesions and serum LDH, high ptDNA fraction was the only independent factor associated with the risk of thrombosis (HR 5.78, 95% CI 1.63-20.44, P = .006). These results first suggest that baseline ptDNA fraction in mCRPC patients treated with abiraterone or enzalutamide may be associated with increased VTE risk. These patients may be followed-up more closely for the VTE risk, and the need for a primary thromboprophylaxis should be taken into account in mCRPC with elevated ptDNA.

TranSNPs: A class of functional SNPs affecting mRNA translation potential revealed by fraction-based allelic imbalance.

Few studies have explored the association between SNPs and alterations in mRNA translation potential. We developed an approach to identify SNPs that can mark allele-specific protein expression levels and could represent sources of inter-individual variation in disease risk. Using MCF7 cells under different treatments, we performed polysomal profiling followed by RNA sequencing of total or polysome-associated mRNA fractions and designed a computational approach to identify SNPs showing a significant change in the allelic balance between total and polysomal mRNA fractions. We identified 147 SNPs, 39 of which located in UTRs. Allele-specific differences at the translation level were confirmed in transfected MCF7 cells by reporter assays. Exploiting breast cancer data from TCGA we identified UTR SNPs demonstrating distinct prognosis features and altering binding sites of RNA-binding proteins. Our approach produced a catalog of tranSNPs, a class of functional SNPs associated with allele-specific translation and potentially endowed with prognostic value for disease risk.

One-shot analysis of translated mammalian lncRNAs with AHARIBO.

A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (AHARIBO), a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs, and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds of lncRNAs.