Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma.

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.

RNA in cardiovascular disease: A new frontier of personalized medicine.

Personalized medicine has witnessed remarkable progress with the emergence of RNA therapy, offering new possibilities for the treatment of various diseases, and in particular in the context of cardiovascular disease (CVD). The ability to target the human genome through RNA manipulation offers great potential not only in the treatment of cardiac pathologies but also in their diagnosis and prevention, notably in cases of hyperlipidemia and myocardial infarctions. While only a few RNA-based treatments have entered clinical trials or obtained approval from the US Food and Drug Administration, the growing body of research on this subject is promising. However, the development of RNA therapies faces several challenges that must be overcome. These include the efficient delivery of drugs into cells, the potential for immunogenic responses, and safety. Resolving these obstacles is crucial to advance the development of RNA therapies. This review explores the newest developments in medical studies, treatment plans, and results related to RNA therapies for heart disease. Furthermore, it discusses the exciting possibilities and difficulties in this innovative area of research.

Assessing disparities in medical students' knowledge and attitude about monkeypox: a cross-sectional study of 27 countries across three continents.

Background and aims: The recent monkeypox (Mpox) outbreak confirmed by the World Health Organization (WHO) underscores the importance of evaluating the knowledge and attitude of medical students toward emerging diseases, given their potential roles as healthcare professionals and sources of public information during outbreaks. This study aimed to assess medical students' knowledge and attitude about Mpox and to identify factors affecting their level of knowledge and attitude in low-income and high-income countries. Methods: A cross-sectional study was conducted on 11,919 medical students from 27 countries. A newly-developed validated questionnaire was used to collect data on knowledge (14 items), attitude (12 items), and baseline criteria. The relationship between a range of factors with knowledge and attitude was studied using univariate and multivariate analyses. Results: 46% of the study participants were males; 10.7% were in their sixth year; 54.6% knew about smallpox; 84% received the coronavirus disease 2019 (COVID-19) vaccine; and 12.5% had training on Mpox. 55.3% had good knowledge of Mpox and 51.7% had a positive attitude towards it. Medical students in their third, fifth, or sixth year high- income countries who obtained information on Mpox from friends, research articles, social media and scientific websites were positive predictors for good knowledge. Conversely, being male or coming from high-income countries showed a negative relation with good knowledge about Mpox. Additionally, a positive attitude was directly influenced by residing in urban areas, being in the fifth year of medical education, having knowledge about smallpox and a history of receiving the coronavirus disease 2019 (COVID-19) vaccine. Receiving information about Mpox from social media or scientific websites and possessing good knowledge about Mpox were also predictors of a positive attitude. On the other hand, being male, employed, or receiving a training program about Mpox were inversely predicting positive attitude about Mpox. Conclusion: There were differences in knowledge and attitude towards Mpox between medical students in low and high-income countries, emphasizing the need for incorporating epidemiology of re-emerging diseases like Mpox into the medical curriculum to improve disease prevention and control.

Genome-wide pooled CRISPR screening in neurospheres.

Spheroid culture systems have allowed in vitro propagation of cells unable to grow in canonical cell culturing conditions, and may capture cellular contexts that model tumor growth better than current model systems. The insights gleaned from genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening of thousands of cancer cell lines grown in conventional culture conditions illustrate the value of such CRISPR pooled screens. It is clear that similar genome-wide CRISPR screens of three-dimensional spheroid cultures will be important for future biological discovery. Here, we present a protocol for genome-wide CRISPR screening of three-dimensional neurospheres. While many in-depth protocols and discussions have been published for more typical cell lines, few detailed protocols are currently available in the literature for genome-wide screening in spheroidal cell lines. For those who want to screen such cell lines, and particularly neurospheres, we provide a step-by-step description of assay development tests to be performed before screening, as well as for the screen itself. We highlight considerations of variables that make these screens distinct from, or similar to, typical nonspheroid cell lines throughout. Finally, we illustrate typical outcomes of neurosphere genome-wide screens, and how neurosphere screens typically produce slightly more heterogeneous signal distributions than more canonical cancer cell lines. Completion of this entire protocol will take 8-12 weeks from the initial assay development tests to deconvolution of the sequencing data.

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma.

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets.

Verteporfin-induced proteotoxicity impairs cell homeostasis and survival in neuroblastoma subtypes independent of YAP/TAZ expression.

Neuroblastoma (NB) is a highly aggressive extracranial solid tumor in children. Due to its heterogeneity, NB remains a therapeutic challenge. Several oncogenic factors, including the Hippo effectors YAP/TAZ, are associated with NB tumorigenesis. Verteporfin (VPF) is an FDA-approved drug shown to directly inhibit YAP/TAZ activity. Our study aimed to investigate VPF's potential as a therapeutic agent in NB. We show that VPF selectively and efficiently impairs the viability of YAP/TAZ-expressing NB GI-ME-N and SK-N-AS cells, but not of non-malignant fibroblasts. To investigate whether VPF-mediated NB cell killing is YAP-dependent, we tested VPF potency in CRISPR-mediated YAP/TAZ knock-out GI-ME-N cells, and BE(2)-M17 NB cells (a MYCN-amplified, predominantly YAP-negative NB subtype). Our data shows that VPF-mediated NB cell killing is not dependent on YAP expression. Moreover, we determined that the formation of higher molecular weight (HMW) complexes is an early and shared VPF-induced cytotoxic mechanism in both YAP-positive and YAP-negative NB models. The accumulation of HMW complexes, involving STAT3, GM130 and COX IV proteins, impaired cell homeostasis and triggered cell stress and cell death mechanisms. Altogether, our study shows significant in vitro and in vivo VPF-induced suppression of NB growth, making VPF a potential therapeutic candidate against NB.

FOXR2 Is an Epigenetically Regulated Pan-Cancer Oncogene That Activates ETS Transcriptional Circuits.

Forkhead box R2 (FOXR2) is a forkhead transcription factor located on the X chromosome whose expression is normally restricted to the testis. In this study, we performed a pan-cancer analysis of FOXR2 activation across more than 10,000 adult and pediatric cancer samples and found FOXR2 to be aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors. The majority of tumors (78%) aberrantly expressed FOXR2 through a previously undescribed epigenetic mechanism that involves hypomethylation of a novel promoter, which was functionally validated as necessary for FOXR2 expression and proliferation in FOXR2-expressing cancer cells. FOXR2 promoted tumor growth across multiple cancer lineages and co-opted ETS family transcription circuits across cancers. Taken together, this study identifies FOXR2 as a potent and ubiquitous oncogene that is epigenetically activated across the majority of human cancers. The identification of hijacking of ETS transcription circuits by FOXR2 extends the mechanisms known to active ETS transcription factors and highlights how transcription factor families cooperate to enhance tumorigenesis. SIGNIFICANCE: This work identifies a novel promoter that drives aberrant FOXR2 expression and delineates FOXR2 as a pan-cancer oncogene that specifically activates ETS transcriptional circuits across human cancers. See related commentary by Liu and Northcott, p. 2977.

PPM1D mutations are oncogenic drivers of de novo diffuse midline glioma formation.

The role of PPM1D mutations in de novo gliomagenesis has not been systematically explored. Here we analyze whole genome sequences of 170 pediatric high-grade gliomas and find that truncating mutations in PPM1D that increase the stability of its phosphatase are clonal driver events in 11% of Diffuse Midline Gliomas (DMGs) and are enriched in primary pontine tumors. Through the development of DMG mouse models, we show that PPM1D mutations potentiate gliomagenesis and that PPM1D phosphatase activity is required for in vivo oncogenesis. Finally, we apply integrative phosphoproteomic and functional genomics assays and find that oncogenic effects of PPM1D truncation converge on regulators of cell cycle, DNA damage response, and p53 pathways, revealing therapeutic vulnerabilities including MDM2 inhibition.

Mitogenic and progenitor gene programmes in single pilocytic astrocytoma cells.

Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) programme that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway.

Mutations in the Non-Structural Protein-Coding Sequence of Protoparvovirus H-1PV Enhance the Fitness of the Virus and Show Key Benefits Regarding the Transduction Efficiency of Derived Vectors.

Single nucleotide changes were introduced into the non-structural (NS) coding sequence of the H-1 parvovirus (PV) infectious molecular clone and the corresponding virus stocks produced, thereby generating H1-PM-I, H1-PM-II, H1-PM-III, and H1-DM. The effects of the mutations on viral fitness were analyzed. Because of the overlapping sequences of NS1 and NS2, the mutations affected either NS2 (H1-PM-II, -III) or both NS1 and NS2 proteins (H1-PM-I, H1-DM). Our results show key benefits of PM-I, PM-II, and DM mutations with regard to the fitness of the virus stocks produced. Indeed, these mutants displayed a higher production of infectious virus in different cell cultures and better spreading capacity than the wild-type virus. This correlated with a decreased particle-to-infectivity (P/I) ratio and stimulation of an early step(s) of the viral cycle prior to viral DNA replication, namely, cell binding and internalization. These mutations also enhance the transduction efficiency of H-1PV-based vectors. In contrast, the PM-III mutation, which affects NS2 at a position downstream of the sequence deleted in Del H-1PV, impaired virus replication and spreading. We hypothesize that the NS2 protein-modified in H1-PM-I, H1-PM-II, and H1-DM-may result in the stimulation of some maturation step(s) of the capsid and facilitate virus entry into subsequently infected cells.

Surface marker profiling of SH-SY5Y cells enables small molecule screens identifying BMP4 as a modulator of neuroblastoma differentiation.

Neuroblastoma is the most common extra-cranial solid tumor in children. Its broad spectrum of clinical outcomes reflects the underlying inherent cellular heterogeneity. As current treatments often do not lead to tumor eradication, there is a need to better define therapy-resistant neuroblastoma and to identify new modulatory molecules. To this end, we performed the first comprehensive flow cytometric characterization of surface molecule expression in neuroblastoma cell lines. Exploiting an established clustering algorithm (SPADE) for unbiased visualization of cellular subsets, we conducted a multiwell screen for small molecule modulators of neuroblastoma phenotype. In addition to SH-SY5Y cells, the SH-EP, BE(2)-M17 and Kelly lines were included in follow-up analysis as in vitro models of neuroblastoma. A combinatorial detection of glycoprotein epitopes (CD15, CD24, CD44, CD57, TrkA) and the chemokine receptor CXCR4 (CD184) enabled the quantitative identification of SPADE-defined clusters differentially responding to small molecules. Exposure to bone morphogenetic protein (BMP)-4 was found to enhance a TrkAhigh/CD15-/CD184- neuroblastoma cellular subset, accompanied by a reduction in doublecortin-positive neuroblasts and of NMYC protein expression in SH-SY5Y cells. Beyond yielding novel marker candidates for studying neuroblastoma pathology, our approach may provide tools for improved pharmacological screens towards developing novel avenues of neuroblastoma diagnosis and treatment.

The Hippo pathway member YAP enhances human neural crest cell fate and migration.

The Hippo/YAP pathway serves as a major integrator of cell surface-mediated signals and regulates key processes during development and tumorigenesis. The neural crest is an embryonic tissue known to respond to multiple environmental cues in order to acquire appropriate cell fate and migration properties. Using multiple in vitro models of human neural development (pluripotent stem cell-derived neural stem cells; LUHMES, NTERA2 and SH-SY5Y cell lines), we investigated the role of Hippo/YAP signaling in neural differentiation and neural crest development. We report that the activity of YAP promotes an early neural crest phenotype and migration, and provide the first evidence for an interaction between Hippo/YAP and retinoic acid signaling in this system.