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Supplementary Figure S4. Event-free survival (EFS) probability curve for STAT1. The curve depicts the predicted EFS probability together with the observed pCR responses in the bottom part. Each segment on the abscissa indicates single patientachieving (in green) or not achieving (in red) pCR.

Table S3. Multivariate logistic model in the study cohort (n=226; 80 pCR and 64 events).

Supplementary Figure S5. Patient-level heatmap of the 27 putative biomarkers assessed in the paper with annotation showing pCR (No; Yes), Event (No; Yes), TRT, Treatment (Combination; Single agent ), ER status (Negative; Positive), TRAR (Low; High), PAM50 subtype (HER2; Other)

Supplementary Figure S1. Correlation of immune-related metagenes, tumor-infiltrating immune cells, and expression of ERBB2, ESR1 and TRAR. The color indicates the direction of the correlation (yellow: negative-correlation; light blue: positive correlation) according to Spearman's correlation coefficient values.

Table S1. Association of clinico-pathological variables and the molecular classifiers TRAR and PAM50 with pathological complete response (pCR) and Event Free survival (EFS) in the study cohort (n=226; 80 pCR and 64 events).

Purpose:Little is known about the efficacy of HER2-targeted therapy in patients with breast cancer showing different HER2-pathway dependence and immune phenotypes. Herein, we report a NeoALTTO exploratory analysis evaluating the clinical value of 22 types of tumor-infiltrating immune cells by CIBERSORT and 5 immune-related metagenes in the overall patient population, and in subgroups defined by the TRAR classifier as HER2-addicted (TRAR-low) or not (TRAR-high).Patients and Methods:Association of baseline TRAR, immune-related metagenes, and CIBERSORT data with pathologic complete response (pCR) and event-free survival (EFS) were assessed using logistic and Cox regression models. Corrections for multiple testing were performed by the Bonferroni method.Results:A total of 226 patients were analyzed: 80 (35%) achieved a pCR, and 64 (28%) experienced a relapse with a median follow-up of 6.7 (interquartile range 6.1–6.8) years; 108 cases were classified as TRAR-low, and 118 TRAR-high. Overall, γδ T-cell fraction [OR = 2.69; 95% confidence interval (CI), 1.40–5.18], and no immune-related metagenes were predictive of pCR. Notably, lymphocyte-specific kinase (LCK) predicted pCR to combination (OR = 2.53; 95% CI, 1.12–5.69), but not to single-agent trastuzumab or lapatinib [OR = 0.74; 95% CI, 0.45–1.22 (Pinteraction = 0.01)]. Integrating LCK with γδ T cells in a multivariate model added to the discriminatory capability of clinical and molecular variables with a shift in AUC from 0.80 (95% CI, 0.74–0.86) to 0.83 (95% CI, 0.78–0.89). In TRAR-low cases, activated mast cells, IFN and MHCII were reduced, and STAT1, HCK1, and γδ T cells were associated with pCR. STAT1 was broadly associated with improved EFS regardless of pCR, and nodal status in overall (HR = 0.68; 95% CI, 0.49–0.94) and in TRAR-low cases (HR = 0.50; 95% CI, 0.30–0.86).Conclusions:Immuno-phenotyping holds the promise to complement current predictive models in HER2-positive breast cancer and to assist in new therapeutic development.

Table S2. Association of tumor infiltrating immune cells with pathological complete response (pCR) and event free survival (EFS) in TRAR groups.

Supplementary Figure S3. Interaction between tumor infiltrating immune cells and treatment arm in the study cohort (n=226; 80 pCR). Red diamond and line indicate Odds Ratio (OR) and its 95% confidence interval (CI) in the combination arm; blue diamond and line indicate OR and its 95% CI in single agent arm (trastuzumab or lapatinib).

Supplementary Figure S2. Interaction between immune-related metagenes and treatment arm in the study cohort (n=226; 80 pCR). Red diamond and line indicate Odds Ratio (OR) and its 95% confidence interval (CI) in the combination arm; blue diamond and line indicate OR and its 95% CI in single agent arm (trastuzumab or lapatinib).

Background Papillary thyroid cancer (PTC) is the most frequent endocrine tumor. Radioiodine (RAI) treatment is highly effective in these tumors, but up to 60% of metastatic cases become RAI-refractory. Scanty data are available on either the molecular pattern of radioiodine refractory papillary thyroid cancers (PTC) or the mechanisms responsible for RAI resistance. Methods We analyzed the molecular profile and gene/miRNA expression in primary PTCs, synchronous and RAI-refractory lymph node metastases (LNMs) in correlation to RAI avidity or refractoriness. We classified patients as RAI+/D+ (RAI uptake/disease persistence), RAI−/D+ (absent RAI uptake/disease persistence), and RAI+/D- (RAI uptake/disease remission), and analyzed the molecular and gene/miRNA profiles, and the expression of thyroid differentiation (TD) related genes. Results A different molecular profile according to the RAI class was observed: BRAFV600E cases were more frequent in RAI−/D+ (P = 0.032), and fusion genes in RAI+/D+ cases. RAI+/D- patients were less frequently pTERT mutations positive, and more frequently wild type for the tested mutations/fusions. Expression profiles clearly distinguished PTC from normal thyroid. On the other hand, in refractory cases (RAI+/D+ and RAI−/D+) no distinctive PTC expression patterns were associated with either tissue type, or RAI uptake, but with the driving lesion and BRAF−/RAS-like subtype. Primary tumors and RAI-refractory LNMs with BRAFV600E mutation display transcriptome similarity suggesting that RAI minimally affects the expression profiles of RAI-refractory metastases. Molecular profiles associated with the expression of TPO, SLC26A4 and TD genes, that were found more downregulated in BRAFV600E than in gene fusions tumors. Conclusions The present data indicate a different molecular profile in RAI-avid and RAI-refractory metastatic PTCs. Moreover, BRAFV600E tumors displayed reduced differentiation and intrinsic RAI refractoriness, while PTCs with fusion oncogenes are RAI-avid but persistent, suggesting different oncogene-driven mechanisms leading to RAI refractoriness.

Simple Summary In population-based screens, tissue biopsy remains the standard practice for women with imaging that suggests breast cancer. We examined circulating microRNAs as minimally invasive diagnostic biomarkers to discriminate malignant from benign breast lesions. A retrospective cohort of plasma samples divided into training and testing sets and a prospective cohort of women with suspicious imaging findings who underwent tissue biopsy were investigated through a global microRNA profile by OpenArray. Seven signatures, involving 5 specific miRNAs (miR-625, miR-423-5p, miR-370-3p, miR-181c, and miR-301b), were identified and validated in the testing set. Among the 7 signatures, the discriminatory performances of 5 of them were confirmed in the prospective cohort. Abstract In population-based screens, tissue biopsy remains the standard practice for women with imaging that suggests breast cancer. We examined circulating microRNAs as minimally invasive diagnostic biomarkers to discriminate malignant from benign breast lesions. miRNAs were analyzed by OpenArray in a retrospective cohort of plasma samples including 100 patients with malignant (T), 89 benign disease (B), and 99 healthy donors (HD) divided into training and testing sets and a prospective cohort (BABE) of 289 women with suspicious imaging findings who underwent tissue biopsy. miRNAs associated with disease status were identified by univariate analysis and then combined into signatures by multivariate logistic regression models. By combining 16 miRNAs differentially expressed in the T vs. HD comparison, 26 signatures were also able to significantly discriminate T from B disease. Seven of them, involving 5 specific miRNAs (miR-625, miR-423-5p, miR-370-3p, miR-181c, and miR-301b), were statistically validated in the testing set. Among the 7 signatures, the discriminatory performances of 5 were confirmed in the prospective BABE Cohort. This study identified 5 circulating miRNAs that, properly combined, distinguish malignant from benign breast disease in women with a high likelihood of malignancy.

BRAFV600E is the most frequent oncogenic mutation identified in papillary thyroid cancer (PTC). In PTC patients who do not respond to standard treatment, BRAF inhibitors are currently tested as alternative strategies. However, as observed for other targeted therapies, patients eventually develop drug resistance. The mechanisms of BRAF inhibitors response are still poorly understood in a thyroid cancer (TC) context. In this study, we investigated in BRAFV600E mutated TC cell lines the effects of Vemurafenib and Dabrafenib, two BRAF inhibitors currently used in a clinical setting. We assessed cell proliferation, and the expression and activity of the thyroid function related transporter NIS following the treatment with BRAF inhibitors. In addition, we investigated the global gene expression by microarray, the relevant modulated biological processes by gene set enrichment analysis (GSEA), and TC specific gene signatures related to MAPK pathway activation, thyroid differentiation, and transcriptional profile associated with BRAFV600E or RAS mutation. We found that both inhibitors induce antiproliferative and redifferentiative effects on TC cells, as well as a rewiring of the MAPK pathway related to RAS signaling. Our results suggest a possible mechanism of drug response to the BRAF inhibitors Vemurafenib or Dabrafenib, supporting very recent findings in TC patients treated with targeted therapies.

e16507 Background: The phase II BONSAI trial ( n = 25 ; NCT 03354884) met the primary endpoint demonstrating activity of cabozantinib in untreated metastatic collecting ducts carcinoma (mCDC), a rare and biologically poorly characterized disease. Here we report on molecular analyses of baseline tissue samples. Methods: Formalin-fixed paraffin-embedded (FFPE) samples from 18 mCDC patients enrolled in BONSAI were sequenced by TruSeq RNA Exome kit (Illumina). The data were mapped and quantified using STAR and htseq, respectively. Globaltest and edgeR packages in R were used to assess the correlation between transcriptional profiles and survival data. Nineteen samples underwent DNA Sequencing with the Oncomine Comprehensive Assay Plus (ThermoFisher Scientific). The reads were aligned to the human genome reference (hg19) and analyzed with Opencravat and IonReporter software. Germline variants were excluded based on 1000 Genomes, GnomAd and Exac databases, and Clinical annotation of somatic variants was performed using ClinVar. Results: The global expression levels of thirty-one genes have been found significantly associated with overall survival (OS). The natural grouping of the 18 tumor samples based on the 31-gene signature identified a main group of 11 cases, showing global higher expression levels in 22 out of the 31 genes. This group displayed overall a significant higher OS rate in comparison to the remaining 7 patients, carrying opposite expression trend and mostly undergoing to disease progression. The identified signature was enriched in biological processes like cell junction, cytoskeleton organization and methylation. FOS oncogene was among the 9 genes negatively associated to OS, showing common higher expression in the poorer survival rate group. Furthermore, a 22-gene signature was found significantly associated with progression-free survival (PFS), involving mainly genes associated to cell cycle regulation or recognized as components of Golgi apparatus. Only three genes were globally up-regulated in the group of 9 patients characterized by shorter PFS. Finally, heterogeneous pattern of somatic mutations was identified in 19 tumor samples, with at least 8 genes recurrently affected in more than three patients. Notably, mutated genes were mostly involved in DNA repair and chromatin modification processes. Conclusions: Our findings for the first time define specific molecular signatures that differentiate therapy-specific outcomes in first-line mCDC, warranting further investigation of their involvement in the tumor biology. Clinical trial information: NCT 03354884.

Purpose: Little is known about the efficacy of HER2-targeted therapy in patients with breast cancer showing different HER2-pathway dependence and immune phenotypes. Herein, we report a NeoALTTO exploratory analysis evaluating the clinical value of 22 types of tumor-infiltrating immune cells by CIBERSORT and 5 immune-related metagenes in the overall patient population, and in subgroups defined by the TRAR classifier as HER2-addicted (TRAR-low) or not (TRAR-high). Patients and Methods: Association of baseline TRAR, immune-related metagenes, and CIBERSORT data with pathologic complete response (pCR) and event-free survival (EFS) were assessed using logistic and Cox regression models. Corrections for multiple testing were performed by the Bonferroni method. Results: A total of 226 patients were analyzed: 80 (35%) achieved a pCR, and 64 (28%) experienced a relapse with a median follow-up of 6.7 (interquartile range 6.1–6.8) years; 108 cases were classified as TRAR-low, and 118 TRAR-high. Overall, γδ T-cell fraction [OR = 2.69; 95% confidence interval (CI), 1.40–5.18], and no immune-related metagenes were predictive of pCR. Notably, lymphocyte-specific kinase (LCK) predicted pCR to combination (OR = 2.53; 95% CI, 1.12–5.69), but not to single-agent trastuzumab or lapatinib [OR = 0.74; 95% CI, 0.45–1.22 (Pinteraction = 0.01)]. Integrating LCK with γδ T cells in a multivariate model added to the discriminatory capability of clinical and molecular variables with a shift in AUC from 0.80 (95% CI, 0.74–0.86) to 0.83 (95% CI, 0.78–0.89). In TRAR-low cases, activated mast cells, IFN and MHCII were reduced, and STAT1, HCK1, and γδ T cells were associated with pCR. STAT1 was broadly associated with improved EFS regardless of pCR, and nodal status in overall (HR = 0.68; 95% CI, 0.49–0.94) and in TRAR-low cases (HR = 0.50; 95% CI, 0.30–0.86). Conclusions: Immuno-phenotyping holds the promise to complement current predictive models in HER2-positive breast cancer and to assist in new therapeutic development.

Lamin A/C is a major constituent of the nuclear lamina implicated in a number of genetic diseases, collectively known as laminopathies. The most severe forms of laminopathies feature, among other symptoms, congenital scoliosis, osteoporosis, osteolysis or delayed cranial ossification. Importantly, specific bone districts are typically affected in laminopathies. Spine is severely affected in LMNA-linked congenital muscular dystrophy. Mandible, terminal phalanges and clavicles undergo osteolytic processes in progeroid laminopathies and Restrictive Dermopathy, a lethal developmental laminopathy. This specificity suggests that lamin A/C regulates fine mechanisms of bone turnover, as supported by data showing that lamin A/C mutations activate non-canonical pathways of osteoclastogenesis, as the one dependent on TGF beta 2. Here, we review current knowledge on laminopathies affecting bone and LMNA involvement in bone turnover and highlight lamin-dependent mechanisms causing bone disorders. This knowledge can be exploited to identify new therapeutic approaches not only for laminopathies, but also for other rare diseases featuring bone abnormalities.

Simple Summary The treatment of collecting duct carcinoma (CDC) remains unsatisfactory since this highly aggressive kidney cancer has an unfavorable clinical behavior. Due to the rarity of CDC a complete biological characterization of this disease is still lacking. The aim of our study is to provide new insights into the molecular biology of CDC leveraging newly generated and publicly available gene expression profiles of CDC tumors. We identified unique gene expression programs and pathways that distinguished CDC from other renal malignancies. The CDC-specific expression signature predicted in vitro sensitivity to several small molecule inhibitors screened in cancer cell lines from multiple cancer types. Finally, we proved that CDC is a molecularly heterogeneous disease made up of at least two subtypes distinguished by cell signaling, metabolic and immune-related alterations. Altogether, these findings pave the way for future investigations with meaningful clinical implications aimed at improving the management of CDC patients. Abstract Collecting duct carcinoma (CDC) is a rare and highly aggressive kidney cancer subtype with poor prognosis and no standard treatments. To date, only a few studies have examined the transcriptomic portrait of CDC. Through integration of multiple datasets, we compared CDC to normal tissue, upper-tract urothelial carcinomas, and other renal cancers, including clear cell, papillary, and chromophobe histologies. Association between CDC gene expression signatures and in vitro drug sensitivity data was evaluated using the Cancer Therapeutic Response Portal, Genomics of Drug Sensitivity in Cancer datasets, and connectivity map. We identified a CDC-specific gene signature that predicted in vitro sensitivity to different targeted agents and was associated to worse outcome in clear cell renal cell carcinoma. We showed that CDC are transcriptionally related to the principal cells of the collecting ducts providing evidence that this tumor originates from this normal kidney cell type. Finally, we proved that CDC is a molecularly heterogeneous disease composed of at least two subtypes distinguished by cell signaling, metabolic and immune-related alterations. Our findings elucidate the molecular features of CDC providing novel biological and clinical insights. The identification of distinct CDC subtypes and their transcriptomic traits provides the rationale for patient stratification and alternative therapeutic approaches.

4571 Background: Metastatic collecting ducts carcinoma (mCDC) is a rare disease with bad prognosis and no standard treatments. Due to its rarity, mCDC is biologically poorly characterized and under-represented in prospective randomized trials. We recently identified two different molecular subtypes of mCDC based on relative expression levels of angiogenesis, metabolic and immune-related genes. Methods: : This prospective, monocentric, phase II trial evaluated cabozantinib (cabo) 60 mg orally once daily until progression or unacceptable toxicity in untreated mCDC patients (pts). Primary endpoint was objective response rate (ORR) as the proportion of pts with best overall response of confirmed complete (CR) or partial responses (PR) per RECIST 1.1. Secondary endpoints were progression-free survival (PFS), overall survival and safety profile. Exploratory objectives were: to identify somatic mutations by targeted NGS-based sequencing; to define molecular subtypes, signatures and transcript fusions genes by RNA sequencing; to monitor circulating immune cells and study the immunological context of tumor cells. A central pathological review was mandatory. The study was based on a Simon’s two stage optimal design: at least 2 responses in 9 pts in the first stage were needed to proceed to the second stage where at least 6 responses in 14 additional pts were needed to prove activity of cabo. Results: From January 2018 to November 2020, 25 pts were enrolled, of whom 23 started treatment. Median age was 66 years, 19 pts were male. 19 (83%) pts received a previous nephrectomy. 9 pts presented with only one metastatic site, 8 pts with two, while the remaining part with multiple sites. The most common metastatic sites were lymphnodes and bone (15 and 13 pts respectively), followed by lung and liver (10 and 4 pts respectively). Median follow up was 8 months. As best overall response, 6 pts presented a stable disease (26%),1 pt achieved a confimed CR and 7 a PR for an ORR of 35%. Median PFS was 6 months. Treatment was feasible and well tolerated. All pts reported at least one grade (G) 1-2 adverse event (AE): the most common were fatigue (43%), hypotiroidism (28%), stomatitis (28%), anorexia (26%), Hand-Foot Syndrome (13%), hypertension (17%), and diarrhea (13%). 5 pts reported G3 AEs (2 thromboembolic events, 2 arterial hypertension, 1 fatigue), while no G4-5 AEs were reported. 17% of pts required dose reduction. DNA sequencing on CDC showed to be feasible, finding 256 mutations in 119 genes (missens mutations the majority). Altered genes, molecular subtypes and signatures will be associated to different outcomes and responses to cabo. Conclusions: The study met its primary endpoint showing promising efficacy and acceptable tolerability of cabo in mCDC pts. Mature results according to mutational profiles and gene signatures will be presented. Clinical trial information: NCT03354884.

Adolescents and young adults (AYA) with rhabdomyosarcoma (RMS) form a subgroup of patients whose optimal clinical management and access to care remain a challenge and whose survival lacks behind that of children diagnosed with histologically similar tumors. Understanding the tumor biology that differentiates children from AYA-RMS could provide critical information and drive new initiatives to improve the final outcome. MicroRNA (miRNA) and gene expression profiling (GEP) was evaluated in a RMS cohort of 49 tumor and 15 non-neoplastic tissues. miRNAs analysis identified miR-223 over-expression and miR-431 down-regulation in AYA, validated by Real-Time PCR and miRNA in situ hybridization (ISH). GEP analysis detected 793 age-correlated genes in tumors, of which 194 were anti-correlated. NOTCH2, FGFR1/2 were significantly down-modulated in AYA-RMS. miR-223 was associated with up-regulation of epithelial mesenchymal translation (EMT) and inflammatory pathways, whereas miR-431 was correlated to myogenic differentiation and muscle metabolism. GEP showed an increase in genes associated with CD4 memory resting cells and a decrease in genes associated with &gamma, &delta, T-cells in AYA-RMS. Immunohistochemistry (IHC) analysis demonstrated an increase of infiltrated CD4, CD8, and neutrophils in AYA-RMS tumors. Our results show that aggressiveness of AYA-RMS could be explained by differences in microenvironmental signal modulation mediated by tumor cells, suggesting a fundamental role of immune contexture in AYA-RMS development.

714 Background: Collecting duct carcinoma is a rare, highly aggressive and molecularly uncharacterized kidney disease with poor outcome. To date, no standard therapies are available and treatment choice is based on retrospective data. Identifying molecular features of this tumor could provide a biologic rationale for novel targeted systemic therapies. Methods: Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissues of 6 CDCs, 5 clear cell renal carcinomas (ccRCCs) and 4 adjacent normal tissues. Samples were profiled by GeneChip Human Transcriptome Array 2.0 (Affymetrix) and constituted the Istituto Nazionale dei Tumori (INT) dataset. Data were pre-processed using the robust multi-array average method and differentially expressed genes were identified according to the limma pipeline. Genes with a false discovery rate < 0.25 were selected. Pathway analysis was performed by gene set enrichment analysis (GSEA), single sample (ss) GSEA, DAVID Bioinformatics Resources 6.8 and Pathfinder Bioconductor package. Moreover, a list of genes representing angiogenesis and immune biology, previously used in the IMmotion150 study, was chosen to explore further functional subcategories of CDC. Available RNA-Seq data of 7 CDCs and 5 matched normal tissue from GSE89122 dataset were used for validation. Results: Comparison of transcriptome profiles of CDCs and ccRCCs versus normal tissue led to the identification of a core of 14 genes specifically up-regulated in CDC, 5 of which (SFN, S100A11, CKAP5, CDH1 and CD44) were significantly involved in extracellular matrix organization. Other deregulated genes were related to immune system, cell cycle progression, glucose and pentose phosphate metabolism and EGFR-signaling. In addition, ssGSEA permitted to identify two major distinct biological subgroups in CDC of both INT and GSE89122 datasets, based on relative expression levels of angiogenesis and immune genes. Conclusions: Our study reveals novel insights into the biology and heterogeneity of CDCs. Although further validation using larger samples is needed, the current findings suggest that this heterogeneity could have clinical implications for the treatment of patients with this rare kidney cancer.

Background Circulating microRNAs (cmiRNAs) are emerging as promising non-invasive biomarkers in cancer for their diagnostic, prognostic and predictive potential. Several platforms have been developed to determine the presence of miRNAs in biological fluids, including specific focus panels. In this pilot study, we aimed at comparing different platforms for detection of cmiRNAs against the same cohort of plasma samples alongside RT-qPCR and NGS technologies. Methods RNA was isolated from 24 plasma samples obtained from lung cancer patients (n = 12, including one replicate) and healthy heavy smokers (n = 12, including two replicates). Six different platforms were used to detect human cmiRNAs. Five of them were high-throughput technologies, three RT-qPCR-based (TaqMan OpenArray and TaqMan OpenArray Advanced MicroRNA Panels by ThermoFisher; miRCURY LNA miRNA miRNome PCR panel by Qiagen) and two NGS-based (EdgeSeq miRNA byHTG and QiaSeq miRNA by Qiagen). The sixth platform, RT-qPCR-based and serum/plasma focused, was TaqMan Advanced miRNA Human Card by ThermoFisher. Samples were handled according to manufacturer’s instructions and data from each platform were analyzed using specific recommended thresholds. Results Selecting cmiRNAs yielding signals above background, we highlighted a subset commonly detected by all platforms. Strong concordance and correlation coefficients (>0.8) between the three replicate samples were obtained in most platforms, from independent RNA extractions. For each sample, the number of cmiRNAs detected ranged from 90 to 140. We found that inter-platform correlations were highest for platforms based on similar chemistry and assay design. Between lung cancer patients and healthy donors, few deregulated miRNAs could be however commonly detected by all platforms and studies are ongoing to elucidate their role. Conclusions This study is one of the first comparing RT-qPCR, NGS-based high-throughput and serum/plasma focused platforms for human cmiRNA biomarker detection. Advantages and limits of all platforms and data on intra- and inter- reproducibility will be presented to help investigators in identifying technologies better suited for cmiRNAs screening or validation in clinical cohorts. Legal entity responsible for the study The authors. Funding Italian Association for Cancer Research (AIRC, Special Programme "5 X 1000", ED No12162) to GS and MS and by Italian Ministry of Health (funds obtained through an Italian law that allows tax-payers to allocate the “5 × 1000” share of their payments to research) to LDC. Disclosure All authors have declared no conflicts of interest.

Long-term memory is accompanied by changes in neuronal morphology and connectivity. These alterations are thought to depend upon new gene expression and protein synthesis over a distributed network of brain structures. Although much evidence supports the idea that the creation of stable, persistent memory traces requires synthesis of new proteins, the role of rRNA transcription and nucleolar activity in learning and memory has hardly been explored. rRNAs needed for protein synthesis result from the activity of two different RNA polymerases, RNA polymerase I and RNA polymerase III, transcribing for 47S RNA and 5S RNA, respectively. In this study, we first investigated the effects of spatial training in the Morris water maze on 47S RNA transcription in the central nervous system, demonstrating bidirectional modulation of its expression over a distributed neural network. We found learning-induced increases in the nucleolar organizer regions in the hippocampus. Finally, we demonstrated that intrahippocampal administrations of CX-5461 (0.6 μg/side), the specific RNA Polymerase I inhibitor, impair the ability of mice to locate the platform in the same task. These results suggest that de novo rRNA transcription is a necessary step for spatial memory consolidation, and that after learning, it occurs in several brain regions with a complex spatiotemporal dynamic. In this study, we demonstrate for the very first time that spatial learning modulates ribosomal RNA transcription in a wide brain circuit, with anatomical specificities in the dynamic of modulation. Together with pharmacological evidences, data presented here support the hypothesis of a necessary role of RNA Pol-I transcription during spatial memory formation. Read the Editorial Highlight for this article on page 673.