Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N⁶ position of internal adenosine (m⁶A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells, and the loss of TERRA m⁶A/METTL3 results in telomere damage. We observed that m⁶A modification is abundant in R-loop enriched TERRA, and the m⁶A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m⁶A drives telomere targeting of TERRA via R-loops, and this m⁶A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+ NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+ NB.

Insights into host–virus interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics.N⁶-Methyladenosine modification (m⁶A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during stress response. Gene expression profiles observed postinfection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m⁶A readers and erasers. We found that infection with SARS-CoV-2 variants causes a loss of m⁶A in cellular RNAs, whereas m⁶A is detected abundantly in viral RNA. METTL3, the m⁶A methyltransferase, shows an unusual cytoplasmic localization postinfection. The B.1.351 variant has a less-pronounced effect on METTL3 localization and loss of m⁶A than did the B.1 and B.1.1.7 variants. We also observed a loss of m⁶A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m⁶A loss is characteristic of SARS-CoV-2-infected cells. Further, transcripts with m⁶A modification are preferentially down-regulated postinfection. Inhibition of the export protein XPO1 results in the restoration of METTL3 localization, recovery of m⁶A on cellular RNA, and increased mRNA expression. Stress granule formation, which is compromised by SARS-CoV-2 infection, is restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m⁶A-dependent manner.

Tumor cells are hallmarked by their capacity to undergo unlimited cell divisions, commonly accomplished either by mechanisms that activate TERT or through the alternative lengthening of telomeres pathway. Neuroblastoma is a heterogeneous pediatric cancer, and the aim of this study was to characterize telomere maintenance mechanisms in a high-risk neuroblastoma cohort. All tumor samples were profiled with SNP microarrays and, when material was available, subjected to whole genome sequencing (WGS). Telomere length was estimated from WGS data, samples were assayed for the ALT biomarker c-circles, and selected samples were subjected to methylation array analysis. Samples with ATRX aberration in this study were positive for c-circles, whereas samples with either MYCN amplification or TERT re-arrangement were negative for c-circles. Both ATRX aberrations and TERT re-arrangement were enriched in 11q-deleted samples. An association between older age at diagnosis and 1q-deletion was found in the ALT-positive group. TERT was frequently placed in juxtaposition to a previously established gene in neuroblastoma tumorigenesis or cancer in general. Given the importance of high-risk neuroblastoma, means for mitigating active telomere maintenance must be therapeutically explored.

Neuroblastoma (NB) is the most common extracranial childhood cancer, caused by the improper differentiation of developing trunk neural crest cells (tNCC) in the sympathetic nervous system. The N ⁶ -methyladenosine (m ⁶ A) epitranscriptomic modification controls post-transcriptional gene expression but the mechanism by which the m ⁶ A methyltransferase complex METTL3/METTL14/WTAP is recruited to specific loci remains to be fully characterized. We explored whether the m ⁶ A epitranscriptome could fine-tune gene regulation in migrating/differentiating tNCC. We demonstrate that the m ⁶ A modification regulates the expression of HOX genes in tNCC, thereby contributing to their timely differentiation into sympathetic neurons. Furthermore, we show that posterior HOX genes are m ⁶ A modified in MYCN -amplified NB with reduced expression. In addition, we provide evidence that sustained overexpression of the MYCN oncogene in tNCC drives METTL3 recruitment to a specific subset of genes including posterior HOX genes creating an undifferentiated state. Moreover, METTL3 depletion/inhibition induces DNA damage and differentiation of MYCN overexpressing cells and increases vulnerability to chemotherapeutic drugs in MYCN -amplified patient-derived xenografts (PDX) in vivo, suggesting METTL3 inhibition could be a potential therapeutic approach for NB.

Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N ⁶ position of internal adenosine (m ⁶ A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells, and the loss of TERRA m ⁶ A/METTL3 results in telomere damage. We observed that m ⁶ A modification is abundant in R-loop enriched TERRA, and the m ⁶ A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m ⁶ A drives telomere targeting of TERRA via R-loops, and this m ⁶ A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+ NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+ NB.

Neuroblastoma (NB) is the most common extracranial childhood cancer, caused by the improper differentiation of developing trunk neural crest cells (tNCC) in the sympathetic nervous system. The N ⁶ -methyladenosine (m ⁶ A) epitranscriptomic modification controls post-transcriptional gene expression but the mechanism by which the m ⁶ A methyltransferase complex METTL3/METTL14/WTAP is recruited to specific loci remains to be fully characterized. We explored whether the m ⁶ A epitranscriptome could fine-tune gene regulation in migrating/differentiating tNCC. We demonstrate that the m ⁶ A modification regulates the expression of HOX genes in tNCC, thereby contributing to their timely differentiation into sympathetic neurons. Furthermore, we show that posterior HOX genes are m ⁶ A modified in MYCN-amplified NB with reduced expression. In addition, we provide evidence that sustained overexpression of the MYCN oncogene in tNCC drives METTL3 recruitment to a specific subset of genes including posterior HOX genes creating an undifferentiated state. Moreover, METTL3 depletion/inhibition induces DNA damage and differentiation of MYCN overexpressing cells and increases vulnerability to chemotherapeutic drugs in MYCN-amplified patient-derived xenografts (PDX) cells, suggesting METTL3 inhibition could be a potential therapeutic approach for NB.