The N6-methyladenosine (m 6 A) is an abundant internal RNA modification 1,2 catalysed predominantly by the METTL3-METTL14 methyltransferase complex 3,4 . The m 6 A writer METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but its true therapeutic importance is still unknown [5][6][7] . Here we present the identification and characterisation of a highly potent and selective first-in-class catalytic inhibitor of METTL3 (STM2457) and its co-crystal structure bound to METTL3/METTL14. Treatment with (Extended Data Fig. 2f). These data demonstrate that STM2457 is a highly potent, specific and bioavailable inhibitor of METTL3, suitable for in vivo investigations. Cellular and molecular effects of STM2457To study the anti-leukaemic potential of STM2457 we examined the proliferation of a panel of human AML cell lines post-treatment and detected significant growth reduction in a concentration-dependent manner (Fig. 2a) while we found that STM2457 did not affect the colony-forming ability of normal human cord blood CD34 + cells (Extended Data Fig. 3a). We also observed no impact on the proliferation of MOLM-13 cells treated with the control small molecule STM2120, unlike our observations with STM2457 (Extended Data Fig. 3b). Additionally, treatment with STM2457 significantly reduced the clonogenic potential of primary murine AML cells (Fig. 2b and Extended Data Fig. 3c), while having no effect on normal haematopoietic stem and progenitor cells (HSPCs) (Fig. 2c). Pharmacological inhibition of METTL3 also caused significant myeloid differentiation 6,11 and cell cycle arrest in MOLM-13 and primary murine AML cells (Fig. 2d, e). In contrast, the same effects were not identified using the non-leukaemic haemopoietic cell line HPC7 (Fig. 3e and Extended Data Fig. 3d). Moreover, treatment with STM2457 induced apoptosis in human and mouse AML models but not in normal non-leukaemic haemopoietic cells (Fig. 2f and Extended Data Fig. 3e). To assess the impact of pharmacological inhibition of METTL3 on two known METTL3 biomarkers associated with AML, SP1 6,12 and BRD4 13,14 , we treated MOLM-13 cells with STM2457 and observed a dose-dependent reduction of SP1 and BRD4 protein levels (Fig. 2g). Notably, ectopic expression of SP1 significantly reduced the sensitivity of MOLM-13 cells to STM2457 (Extended Data Fig. 3f, g). These data establish that the catalytic function of METTL3 is important for leukaemia growth, in line with previous findings 6,7,15 . We next sought to investigate the molecular mechanism by which STM2457 affects AML. RNAseq analysis of MOLM-13 cells treated with STM2457 revealed 1,338 up-regulated and 489 down-regulated genes (Extended Data Fig. 4a and Supplementary Table 1). Gene ontology (GO) analysis of the differentially expressed genes showed enrichment in pathways related to myeloid differentiation, cell cycle and leukaemia progression (Extended Data Fig. 4b, c) in close agreement with our phenotypic observations (Supplementary Table 2). To examine the impact of the pharmac...
Mutation of the POLH gene encoding DNA polymerase H (pol H) causes the UV-sensitivity syndrome xeroderma pigmentosumvariant (XP-V) which is linked to the ability of pol H to accurately bypass UV-induced cyclobutane pyrimidine dimers during a process termed translesion synthesis. Pol H can also bypass other DNA damage adducts in vitro, including cisplatininduced intrastrand adducts, although the physiological relevance of this is unknown. Here, we show that independent XP-V cell lines are dramatically more sensitive to cisplatin than the same cells complemented with functional pol H. Similar results were obtained with the chemotherapeutic agents, carboplatin and oxaliplatin, thus revealing a general requirement for pol H expression in providing tolerance to these platinum-based drugs. The level of sensitization observed was comparable to that of XP-A cells deficient in nucleotide excision repair, a recognized and important mechanism for repair of cisplatin adducts. However, unlike in XP-A cells, the absence of pol H expression resulted in a reduced ability to overcome cisplatin-induced S phase arrest, suggesting that pol H is involved in translesion synthesis past these replicationblocking adducts. Subcellular localization studies also highlighted an accumulation of nuclei with pol H foci that correlated with the formation of monoubiquitinated proliferating cell nuclear antigen following treatment with cisplatin, reminiscent of the response to UV irradiation and further indicating a role for pol H in dealing with cisplatin-induced damage. Together, these data show that pol H represents an important determinant of cellular responses to cisplatin, which could have implications for acquired or intrinsic resistance to this key chemotherapeutic agent. (Cancer Res 2005; 65(21): 9799-806)
Colon cancer cells frequently display minisatellite instability (MIN) or chromosome instability (CIN). While MIN is caused
N 6 -methyladenosine (m 6 A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m 6 A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m 6 A-modified. How the cellular m 6 A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human β-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m 6 A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m 6 A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m 6 A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m 6 A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m 6 A-modified and host m 6 A pathway components control β-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m 6 A pathway to restrict coronavirus reproduction.
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