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Differentially Expressed Proteins Deleted versus non-deleted clones

Supp Fig. S1-6, Supp Tables S1-S3, Supp Methods and References

Deletion of chromosome 6q is a well-recognized abnormality found in poor-prognosis T-cell acute lymphoblastic leukemia (T-ALL). Using integrated genomic approaches, we identified two candidate haploinsufficient genes contiguous at 6q14, SYNCRIP (encoding hnRNP-Q) and SNHG5 (that hosts snoRNAs), both involved in regulating RNA maturation and translation. Combined silencing of both genes, but not of either gene alone, accelerated leukemogeneis in a Tal1/Lmo1/Notch1-driven mouse model, demonstrating the tumor-suppressive nature of the two-gene region. Proteomic and translational profiling of cells in which we engineered a short 6q deletion by CRISPR/Cas9 genome editing indicated decreased ribosome and mitochondrial activities, suggesting that the resulting metabolic changes may regulate tumor progression. Indeed, xenograft experiments showed an increased leukemia-initiating cell activity of primary human leukemic cells upon coextinction of SYNCRIP and SNHG5. Our findings not only elucidate the nature of 6q deletion but also highlight the role of ribosomes and mitochondria in T-ALL tumor progression.Significance:The oncogenic role of 6q deletion in T-ALL has remained elusive since this chromosomal abnormality was first identified more than 40 years ago. We combined genomic analysis and functional models to show that the codeletion of two contiguous genes at 6q14 enhances malignancy through deregulation of a ribosome–mitochondria axis, suggesting the potential for therapeutic intervention.This article is highlighted in the In This Issue feature, p. 1494

Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor GCN2 represses the expression of the precursor of ribosomal RNA, 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, GCN2 activity supports cell proliferation through the transcription stimulation of 47S rRNA, independently of the canonical ISR axis. However, impairment of GCN2 activity prevents nuclear translocation of the methionyl tRNA synthetase (MetRS) underlying the generation of a nucleolar stress, mTORC1 inhibition and autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA pol I inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls the ribosome biogenesis according the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and the RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically-stressed COAD cell.

Resistance of advanced hormone-dependent endometrial carcinoma to endocrine therapy remains a worldwide clinical issue. We recently reported that the combination of Vistusertib (V, mTOR inhibitor) and Anastrozole (A, aromatase inhibitor) improves the progression-free rate compared to Anastrozole alone. However, a better patient selection based on biomarkers would improve patient outcome. We evaluate for the first time the usage of ribosome biogenesis (RiBi) factors as a source of innovative markers. Using 47 FFPE tumours (A n = 18; V + A n = 29), 32 blood samples (A n = 13; V + A n = 19) and 30 samples of total RNAs (A n = 12; V + A n = 18) from the VICTORIA clinical trial, we observed an association between RiBi-associated markers and drug activity or prediction of treatment response. NOP10 and NHP2 mRNA levels were significantly higher in non-responders compared to responders in the Vistusertib + Anastrozole arm (P = 0.0194 and P = 0.0002 respectively; i.e. 8 weeks progression-free survival as endpoint). This study provides RiBi-based markers relevant for a better selection of patients with advanced endometrial carcinoma by predicting the response of endocrine therapy combined with mTOR inhibitor.

5-Fluorouracil (5-FU) is an anticancer drug extensively used for different cancers. Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes, DNA and RNA. In this paper, we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites (nucleoside, nucleotide and sugar nucleotide) of 5-FU. The chromatographic separation was optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides. The detection was performed on an Orbitrap® tandem mass spectrometer. Linearity of the method was verified in intracellular content and in RNA extracts. The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono- and tri-phosphate nucleotide metabolites. Matrix effect was evaluated in cellular contents, DNA and RNA extracts for nucleoside and nucleotides metabolites. The method was successfully applied to i) measure the proportion of each anabolic metabolite of 5-FU in cellular contents, ii) follow the consequence of inhibition of enzymes on the endogenous nucleotide pools, iii) study the incorporation of metabolites of 5-FU into RNA and DNA, and iv) to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA., Graphical abstract Image 1, Highlights • The LC-MS-HRMS method allows the analysis of the ten anabolic metabolites of 5-FU. • The present method is useful to study the incorporation of 5-FU into RNA and DNA. • Method to determine the incorporation rate of 5-FU into subunit of rRNA is innovative.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q− syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle–arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Embryonic stem cell (ESC) fate decisions are regulated by a complex molecular circuitry that requires tight and coordinated gene expression regulations at multiple levels from chromatin organization to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key pathways that efficiently control stem cell homeostasis. However, the molecular mechanisms underlying the regulation of these pathways remain largely unknown to date. Here, we analyzed the expression, in mouse ESCs, of over 300 genes involved in ribosome biogenesis and we identified RSL24D1 as the most differentially expressed between self-renewing and differentiated ESCs. RSL24D1 is highly expressed in multiple mouse pluripotent stem cell models and its expression profile is conserved in human ESCs. RSL24D1 is associated with nuclear pre-ribosomes and is required for the maturation and the synthesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors, including POU5F1 and NANOG, as well as components of the polycomb repressive complex 2 (PRC2). Consistently, RSL24D1 is required for mouse ESC self-renewal and proliferation. Taken together, we show that RSL24D1-dependant ribosome biogenesis is required to both sustain the expression of pluripotent transcriptional programs and silence developmental programs, which concertedly dictate ESC homeostasis.

Bone is a frequent site of metastasis. Bone metastasis is associated with a short-term prognosis in cancer patients, and current treatments aim to slow its growth, but are rarely curative. Thus, revealing molecular mechanisms that explain why metastatic cells are attracted to the bone micro-environment, and how they successfully settle in the bone marrow—taking advantage over bone resident cells—and grow into macro-metastasis, is essential to propose new therapeutic approaches. MicroRNAs and snoRNAs are two classes of small non-coding RNAs that post-transcriptionally regulate gene expression. Recently, microRNAs and snoRNAs have been pointed out as important players in bone metastasis by (i) preparing the pre-metastatic niche, directly and indirectly affecting the activities of osteoclasts and osteoblasts, (ii) promoting metastatic properties within cancer cells, and (iii) acting as mediators within cells to support cancer cell growth in bone. This review aims to highlight the importance of microRNAs and snoRNAs in metastasis, specifically in bone, and how their roles can be linked together. We then discuss how microRNAs and snoRNAs are secreted by cancer cells and be found as extracellular vesicle cargo. Finally, we provide evidence of how microRNAs and snoRNAs can be potential therapeutic targets, at least in pre-clinical settings, and how their detection in liquid biopsies can be a useful diagnostic and/or prognostic biomarker to predict the risk of relapse in cancer patients.

Partial response to chemotherapy leads to disease resurgence. Upon treatment, a subpopulation of cancer cells, called drug-tolerant persistent cells, display a transitory drug tolerance that lead to treatment resistance 1,2. Though drug-tolerance mechanisms remain poorly known, they have been linked to non-genomic processes, including epigenetics, stemness and dormancy 2–4. 5-fluorouracil (5-FU), the most widely used chemotherapy in cancer treatment, is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways 5–9. Here, we show that 5-FU treatment leads to the unexpected production of fluorinated ribosomes, exhibiting altered mRNA translation. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts and human tumours. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs according to the nature of their 5’-untranslated region. As a result, we found that sustained translation of IGF-1R mRNA, which codes for one of the most potent cell survival effectors, promoted the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that “man-made” fluorinated ribosomes favour the drug-tolerant cellular phenotype by promoting translation of survival genes. This could be exploited for developing novel combined therapies. By unraveling translation regulation as a novel gene expression mechanism helping cells to survive a drug-challenge, our study extends the spectrum of molecular mechanisms driving drug-tolerance.

5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat patients with solid tumours, such as colorectal and pancreatic cancers. Colorectal cancer (CRC) is the second leading cause of cancer-related death and half of patients experience tumour recurrence. Used for over 60 years, 5-FU was long thought to exert its cytotoxic effects by altering DNA metabolism. However, 5-FU mode of action is more complex than previously anticipated since 5-FU is an extrinsic source of RNA modifications through its ability to be incorporated into most classes of RNA. In particular, a recent report highlighted that, by its integration into the most abundant RNA, namely ribosomal RNA (rRNA), 5-FU creates fluorinated active ribosomes and induces translational reprogramming. Here, we review the historical knowledge of 5-FU mode of action and discuss progress in the field of 5-FU-induced RNA modifications. The case of rRNA, the essential component of ribosome and translational activity, and the plasticity of which was recently associated with cancer, is highlighted. We propose that translational reprogramming, induced by 5-FU integration in ribosomes, contributes to 5-FU-driven cell plasticity and ultimately to relapse., Graphical Abstract Graphical Abstract5-FU integration in ribosomes contributes to cell plasticity through translational reprogramming.

Simple Summary Translation occurs in the cell both through cytoplasmic and mitochondrial ribosomes, respectively translating mRNAs encoded by the nuclear and the mitochondrial genome. Here we found that the silencing of DHX30, an RNA-binding protein that we previously studied for its role in p53-dependent apoptosis, enhances the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing that of the mRNAs encoding for mitoribosomal proteins. This coordination of the cytoplasmic and mitochondrial translation machineries affected both cell proliferation and energy metabolism, suggesting an important role for this mechanism in determining the fitness of cancer cells. By integrating multiple datasets, we identified a gene signature that will represent a starting point to assess the prognostic value of this mechanism in cancer. We thus propose DHX30 as a potential vulnerability in cancer cells that could be exploited to develop novel therapeutic strategies. Abstract DHX30 was recently implicated in the translation control of mRNAs involved in p53-dependent apoptosis. Here, we show that DHX30 exhibits a more general function by integrating the activities of its cytoplasmic isoform and of the more abundant mitochondrial one. The depletion of both DHX30 isoforms in HCT116 cells leads to constitutive changes in polysome-associated mRNAs, enhancing the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing the translational efficiency of the nuclear-encoded mitoribosome mRNAs. Furthermore, the depletion of both DHX30 isoforms leads to higher global translation but slower proliferation and lower mitochondrial energy metabolism. Isoform-specific silencing supports a role for cytoplasmic DHX30 in modulating global translation. The impact on translation and proliferation was confirmed in U2OS and MCF7 cells. Exploiting RIP, eCLIP, and gene expression data, we identified fourteen mitoribosome transcripts we propose as direct DHX30 targets that can be used to explore the prognostic value of this mechanism in cancer. We propose that DHX30 contributes to cell homeostasis by coordinating ribosome biogenesis, global translation, and mitochondrial metabolism. Targeting DHX30 could, thus, expose a vulnerability in cancer cells.

Simple Summary Pancreatic cancer is an aggressive disease characterized by its invasiveness, rapid progression, and resistance to conventional therapy. There is a need to identify new molecules to improve current therapies. The aim of this study was to analyze how the pancreatic cancer cells react to the treatment with an inhibitor of nucleolin, N6L. To this end, we analyzed how the translation was affected in the cells during the treatment. We discovered that in response to N6L, a signaling pathway called the mTOR pathway was activated and was involved in the activation of translation of a subset of mRNA that could be involved in the resistance of the cells to the treatment. Indeed, we showed that the combined action of inhibitors of the mTOR pathway with N6L synergistically inhibited the cancer cells’ proliferation. We propose that this new combination of molecules could be a novel therapeutic option for pancreatic cancer. Abstract We previously showed that N6L, a pseudopeptide that targets nucleolin, impairs pancreatic ductal adenocarcinoma (PDAC) growth and normalizes tumor vessels in animal models. In this study, we analyzed the translatome of PDAC cells treated with N6L to identify the pathways that were either repressed or activated. We observed a strong decrease in global protein synthesis. However, about 6% of the mRNAs were enriched in the polysomes. We identified a 5′TOP motif in many of these mRNAs and demonstrated that a chimeric RNA bearing a 5‘TOP motif was up-regulated by N6L. We demonstrated that N6L activates the mTOR pathway, which is required for the translation of these mRNAs. An inhibitory synergistic effect in PDAC cell lines, including patient-derived xenografts and tumor-derived organoids, was observed when N6L was combined with mTOR inhibitors. In conclusion, N6L reduces pancreatic cells proliferation, which then undergoes translational reprogramming through activation of the mTOR pathway. N6L and mTOR inhibitors act synergistically to inhibit the proliferation of PDAC and human PDX cell lines. This combotherapy of N6L and mTOR inhibitors could constitute a promising alternative to treat pancreatic cancer.

Background A current critical need remains in the identification of prognostic and predictive markers in early breast cancer. It appears that a distinctive trait of cancer cells is their addiction to hyperactivation of ribosome biogenesis. Thus, ribosome biogenesis might be an innovative source of biomarkers that remains to be evaluated. Methods Here, fibrillarin (FBL) was used as a surrogate marker of ribosome biogenesis due to its essential role in the early steps of ribosome biogenesis and its association with poor prognosis in breast cancer when overexpressed. Using 3,275 non-metastatic primary breast tumors, we analysed FBL mRNA expression levels and protein nucleolar organisation. Usage of TCGA dataset allowed transcriptomic comparison between the different FBL expression levels-related breast tumours. Results We unexpectedly discovered that in addition to breast tumours expressing high level of FBL, about 10% of the breast tumors express low level of FBL. A correlation between low FBL mRNA level and lack of FBL detection at protein level using immunohistochemistry was observed. Interestingly, multivariate analyses revealed that these low FBL tumors displayed poor outcome compared to current clinical gold standards. Transcriptomic data revealed that FBL expression is proportionally associated with distinct amount of ribosomes, low FBL level being associated with low amount of ribosomes. Moreover, the molecular programs supported by low and high FBL expressing tumors were distinct. Conclusion Altogether, we identified FBL as a powerful ribosome biogenesis-related independent marker of breast cancer outcome. Surprisingly we unveil a dual association of the ribosome biogenesis FBL factor with prognosis. These data suggest that hyper- but also hypo-activation of ribosome biogenesis are molecular traits of distinct tumors.

IMPORTANCE: Endometrial cancer is often hormone-dependent and treated with aromatase inhibitors. The PI3K-AKT-mTOR pathway deregulation observed in endometrial cancer drives hormonal resistance, thus supporting the rationale of combining mTOR inhibitor with endocrine therapy. OBJECTIVE: To evaluate the safety and efficacy of vistusertib in combination with anastrozole in the treatment of women with hormone receptor−positive recurrent or metastatic endometrial cancer. DESIGN, SETTINGS, AND PARTICIPANTS: The VICTORIA study was a multicenter, open-label, randomized clinical trial that accrued 75 patients with hormone receptor−positive recurrent or metastatic endometrial cancer from 12 cancer centers in France in April 2016 to October 2019. After a safety run-in period, a Simon 2-stage design was used. Data analyses were performed from December 11, 2020, to March 11, 2021. INTERVENTIONS: Patients were randomized in a 2:1 ratio to oral vistusertib (125 mg twice daily 2 days per week) and oral anastrozole (1 mg daily) in the combination vistusertib with anastrozole arm (V+A arm) or oral anastrozole alone (A arm). MAIN OUTCOMES AND MEASURES: The primary end point was serious adverse events for the safety run-in period and progression-free rate at 8 weeks (8wk-PFR)—assessed with a blinded independent central review in phase 2. The secondary end points were objective response rate, duration of response, progression-free survival (PFS), overall survival, and incidence of adverse events. RESULTS: Of the 75 patients who were randomized, 73 (median [range] age, 69.5 [37-88] y; all female) were treated: V+A arm, 49 patients; A arm, 24 patients. In the V+A arm, the 8wk-PFR was 67.3% (unilateral 95% CI, 54.7%) and in the A arm, 39.1% (unilateral 95% CI, 22.2%). No significant serious adverse events were reported during the safety run-in period (n = 6 in V+A arm). The overall response rate was 24.5% (95% CI, 13.3%-38.9%) in the V+A arm vs 17.4% (95% CI, 5.0%-38.8%) in the A arm. With a median follow-up of 27.7 months, median PFS was 5.2 (95% CI, 3.4-8.9) in the V+A arm and 1.9 (95% CI, 1.6-8.9) months in the A arm. Fatigue, lymphopenia, hyperglycemia, and diarrhea were the most common (grade ≥2) adverse events associated with vistusertib. CONCLUSIONS AND RELEVANCE: This multicenter, open-label, phase 1/2 randomized clinical trial demonstrated that adding vistusertib to anastrozole improved 8wk-PFR, overall response rate, and PFS for patients with endometrial cancer and had manageable adverse events. Identification of molecular subgroups would allow for more precise selection of patients who may be most likely to experience favorable outcomes. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02730923

FANCA is a multitasking protein with essential functions beyond DNA repair in nucleolar homeostasis and ribosome biogenesis., Fanconi anemia (FA), the most common inherited bone marrow failure and leukemia predisposition syndrome, is generally attributed to alterations in DNA damage responses due to the loss of function of the DNA repair and replication rescue activities of the FANC pathway. Here, we report that FANCA deficiency, whose inactivation has been identified in two-thirds of FA patients, is associated with nucleolar homeostasis loss, mislocalization of key nucleolar proteins, including nucleolin (NCL) and nucleophosmin 1 (NPM1), as well as alterations in ribosome biogenesis and protein synthesis. FANCA coimmunoprecipitates with NCL and NPM1 in a FANCcore complex–independent manner and, unique among the FANCcore complex proteins, associates with ribosomal subunits, influencing the stoichiometry of the translational machineries. In conclusion, we have identified unexpected nucleolar and translational consequences specifically associated with FANCA deficiency that appears to be involved in both DNA damage and nucleolar stress responses, challenging current hypothesis on FA physiopathology.

Growing evidence exposes translation and its translational machinery as key players in establishing and maintaining physiological and pathological biological processes. Examining translation may not only provide new biological insight but also identify novel innovative therapeutic targets in several fields of biology, including that of epithelial-to-mesenchymal transition (EMT). EMT is currently considered as a dynamic and reversible transdifferentiation process sustaining the transition from an epithelial to mesenchymal phenotype, known to be mainly driven by transcriptional reprogramming. However, it seems that the characterization of EMT plasticity is challenging, relying exclusively on transcriptomic and epigenetic approaches. Indeed, heterogeneity in EMT programs was reported to depend on the biological context. Here, by reviewing the involvement of translational control, translational machinery and ribosome biogenesis characterizing the different types of EMT, from embryonic and adult physiological to pathological contexts, we discuss the added value of integrating translational control and its machinery to depict the heterogeneity and dynamics of EMT programs.

International audience; Many studies have focused on understanding the regulation and functions of aberrant protein synthesis in colorectal cancer (CRC), leaving the ribosome, its main effector, relatively underappreciated in CRC. The production of functional ribosomes is initiated in the nucleolus, requires coordinated ribosomal RNA (rRNA) processing and ribosomal protein (RP) assembly, and is frequently hyperactivated to support the needs in protein synthesis essential to withstand unremitting cancer cell growth. This elevated ribosome production in cancer cells includes a strong alteration of ribosome biogenesis homeostasis that represents one of the hallmarks of cancer cells. None of the ribosome production steps escape this cancer-specific dysregulation. This review summarizes the early and late steps of ribosome biogenesis dysregulations described in CRC cell lines, intestinal organoids, CRC stem cells and mouse models, and their possible clinical implications. We highlight how this cancer-related ribosome biogenesis, both at quantitative and qualitative levels, can lead to the synthesis of ribosomes favoring the translation of mRNAs encoding hyperproliferative and survival factors. We also discuss whether cancer-related ribosome biogenesis is a mere consequence of cancer progression or is a causal factor in CRC, and how altered ribosome biogenesis pathways can represent effective targets to kill CRC cells. The association between exacerbated CRC cell growth and alteration of specific steps of ribosome biogenesis is highlighted as a key driver of tumorigenesis, providing promising perspectives for the implementation of predictive biomarkers and the development of new therapeutic drugs.

DHX30 was recently implicated in the translation control of mRNAs involved in p53-dependent apoptosis. Here we show that DHX30 exhibits a more general function by integrating the activities of its cytoplasmic isoform and of the more abundant mitochondrial one. The depletion of both DHX30 isoforms in HCT116 cells leads to constitutive changes in polysome-associated mRNAs, enhancing the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing the translational efficiency of the nuclear-encoded mitoribosome mRNAs. Furthermore, depletion of both DHX30 isoforms exhibits higher global translation but slower proliferation, and reduced mitochondrial energy metabolism. Isoform-specific silencing established a role for cytoplasmic DHX30 in modulating global translation. The impact on global translation and proliferation were confirmed in U2OS and MCF7 cells, although the effect of DHX30 depletion on mitochondrial gene expression was observed only in MCF7 cells. Exploiting RIP, eCLIP, and gene expression data, we identified a gene signature comprising DHX30 and fourteen mitoribosome transcripts that we candidate as direct targets: this signature shows prognostic value in several TCGA cancer types, with higher expression associated with reduced overall survival. We propose that DHX30 contributes to cell homeostasis by coordinating ribosome biogenesis, global translation, and mitochondrial metabolism. Targeting DHX30 could, thus, expose a vulnerability in cancer cells.Author summaryTranslation occurs in the cell both through cytoplasmic and mitochondrial ribosomes, respectively translating mRNAs encoded by the nuclear and the mitochondrial genome. Here we found that DHX30, an RNA-binding protein implicated in p53-dependent apoptosis, enhances the translation of mRNAs coding for cytoplasmic ribosomal proteins while reducing that of the mitoribosome mRNAs when silenced. This coordination of the cytoplasmic and mitochondrial translation machineries affected both cell proliferation and energy metabolism, suggesting an important role for this mechanism in determining the fitness of cancer cells. Indeed, the analysis of publicly available cancer datasets led us to define a 15-genes signature that is able to affect the prognosis of a subset of cancer types. In this subset, we found that higher expression of the genes composing the signature is associated with a worse prognosis. We thus propose DHX30 as a potential vulnerability in cancer cells, that could be targeted to develop novel therapeutic strategies.

International audience; BRCA1 inactivation is a hallmark of familial breast cancer, often associated with aggressive triple negative breast cancers. BRCA1 is a tumor suppressor with known functions in DNA repair, transcription regulation, cell cycle control, and apoptosis. In the present study, we demonstrate that BRCA1 is also a translational regulator. We previously showed that BRCA1 was implicated in translation regulation. Here, we asked whether translational control could be a novel function of BRCA1 that contributes to its tumor suppressive activity. A combination of RNA-binding protein immunoprecipitation, microarray analysis, and polysome profiling, was used to identify the mRNAs that were specifically deregulated under BRCA1 deficiency. Western blot analysis allowed us to confirm at the protein level the deregulated translation of a subset of mRNAs. A unique and dedicated cohort of patients with documented germ-line BRCA1 pathogenic variant statues was set up, and tissue microarrays with the biopsies of these patients were constructed and analyzed by immunohistochemistry for their content in each candidate protein. Here, we show that BRCA1 translationally regulates a subset of mRNAs with which it associates. These mRNAs code for proteins involved in major programs in cancer. Accordingly, the level of these key proteins is correlated with BRCA1 status in breast cancer cell lines and in patient breast tumors. ADAT2, one of these key proteins, is proposed as a predictive biomarker of efficacy of treatments recently recommended to patients with BRCA1 deficiency. This study proposes that translational control may represent a novel molecular mechanism with potential clinical impact through which BRCA1 is a tumor suppressor.

Background Dysregulation of ribosome biogenesis and alteration of ribosome composition, including alteration in ribosomal RNA (rRNA) 2'-O-ribose methylation, can play a role in malignant transformation and cancer progression. Several studies recently reported that components of the rRNA methylation complex are associated with leukemogenesis. However, no study ever investigated the alteration of ribosome biogenesis factors in the most common pediatric malignancy - B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Objectives The objective of this study was to examine the expression of factors building the rRNA methylation complex, either the protein components (1 methyl-transferase (FBL), NOP56, NOP58, NHP2L1) or some RNA components (box C/D snoRNAs: SNORD35B, SNORD65, SNORD46, SNORD50A, SNORD38B), as well as CMYC, and nucleolin (NCL) - a protein involved in rRNA synthesis. Clinical effects in children with BCP-ALL were also investigated. Material and methods The factors involved in ribosome biogenesis were studied in 28 children with BCP-ALL with the use of real-time polymerase chain reaction (RT-PCR) using the BioMark HD System (Fluidigm, San Francisco, USA) in cDNA prepared from the bone marrow samples collected at diagnosis. Results Strong correlations were observed between NOP56, NOP58 and NCL, and multiple weaker correlations were observed in the box C/D snoRNA category, and between box C/D snoRNA and transcripts coding proteins of the rRNA methylation complex. The expression of analyzed transcripts did not correlate with the initial white blood cells count (WBC) or with bone marrow blast percentage. Ribosome biogenesis upregulation with overexpression of FBL and NOP56, and CMYC was found in patients who subsequently relapsed and the upregulation signature was not associated with known risk predictors. Conclusions This is the first report on the clinical aspect of ribosome biogenesis in pediatric BCP-ALL, and it shows that overexpression of CMYC and C/D box nucleoproteins FBL and NOP56 is an antecedent event in patients who subsequently relapse. The dysregulation pattern is different from the previous reports in acute myeloid leukemia (AML), suggesting that dysregulation of ribosome biogenesis is specific for BCP-ALL.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs localized in the nucleolus, where they participate in the cleavage and chemical modification of ribosomal RNAs. Their biogenesis and molecular functions have been extensively studied since their identification in the 1960s. However, their role in cancer has only recently started to emerge. In lung cancer, efforts to profile snoRNA expression have enabled the definition of snoRNA-related signatures, not only in tissues but also in biological fluids, exposing these small RNAs as potential non-invasive biomarkers. Moreover, snoRNAs appear to be essential actors of lung cancer onset and dissemination. They affect diverse cellular functions, from regulation of the cell proliferation/death balance to promotion of cancer cell plasticity. snoRNAs display both oncogenic and tumor suppressive activities that are pivotal in lung cancer tumorigenesis and progression. Altogether, we review how further insight into snoRNAs may improve our understanding of basic lung cancer biology and the development of innovative diagnostic tools and therapies.

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International audience; Direct stochastic optical reconstruction microscopy (dSTORM), developed in the last decade, has revolutionised optical microscopy by enabling scientists to visualise objects beyond the resolution provided by conventional microscopy (200 nm). We developed an innovative method based on blinking particle standards and conditions for long-lived imaging over several weeks. Stable localisation precisions within the 10 nm-range were achieved for single virions and in cellulo 2D imaging of centrosomes, as well as their reliable reconstruction in 3D dSTORM.