Abstract 2603: Targeting METTL3 as a novel therapeutic strategy for H3 G34-mutant gliomas

Abstract Background: Diffuse hemispheric glioma, H3 G34-mutant, is a pediatric-type high-grade glioma with limited treatment options and a poor prognosis. RNA-modifying enzymes, including METTL3, are critical for the initiation and maintenance of various cancers, including brain tumors. We previously presented STM2457, a first-in-class RNA-modifying enzyme inhibitor targeting the catalytic activity of METTL3, preventing m6A deposition on mRNA. Here, we show small-molecule inhibition of METTL3 as a novel therapeutic strategy for H3 G34-mutant gliomas. Aims: To study the therapeutic potential of pharmacological inhibition of METTL3 in preclinical models of H3 G34-mutant diffuse hemispheric gliomas (DHG). Results: Using genome-wide CRISPR/Cas9 screening, we identified METTL3 as a genetic vulnerability in H3.3-G34-mutant primary murine glioma models. We confirmed that METTL3 is essential for the maintenance of glioma through genetic and pharmacological approaches in primary mouse and human glioma models, harboring H3.3-G34 mutations. Notably, METTL3 inhibition did not affect the proliferation of normal murine neuronal stem cells or isogenic primary murine glioma models with H3.3-K27M mutations. m6A-immunoprecipitation-sequencing revealed downregulation of several stemness-related m6A substrates, including Nestin and Sox2, upon pharmacological inhibition of METTL3. In H3.3-G34-mutant human and mouse glioma models, METTL3 inhibition disrupted key glioma pathways, suppressing translation of stem cell-related markers, like Nestin and Sox2, while upregulating glioma differentiation markers, such as Tubb3. In parallel, the transcriptional levels of these markers remained unaltered, highlighting that the effects are primarily on the mRNA translation of glioma-associated m6A substrates. Notably, pharmacological inhibition in patient-derived samples with H3.3-G34 mutations impaired growth and suppressed the same molecular pathways consistent with our epitranscriptomic and proteomic analyses. Orthotopic transplantation of primary murine H3.3-G34R glioma cells with CRISPR/Cas9-mediated METTL3 knockout into NSG mice significantly extended survival compared to controls. Importantly, pharmacological inhibition of METTL3 in an H3.3-G34V patient-derived xenograft model recapitulated this effect, significantly prolonging mouse lifespan and validating METTL3 as a therapeutic target in these gliomas. Conclusions: In summary, here we demonstrate that pharmacological inhibition of METTL3 is a potent therapeutic strategy for H3 G34-mutant gliomas. To our knowledge, this is the first study that reveals that the catalytic function of METTL3 is a critical epitranscriptomic factor in H3 G34-mutant gliomagenesis. Our findings provide strong proof-of-concept that targeting RNA-modifying enzymes offers a promising therapeutic approach for this aggressive glioma subtype. Citation Format: Maria Eleftheriou, Eliza Yankova, Rebecca D. Haase, Antonella De Cola, Michael McNicholas, Siân Evans, João M. Dias, Natalie Webster, Georgia Tsagkogeorga, James Russell, Manav Pathania, Oliver Rausch, Mariella G. Filbin, Konstantinos Tzelepis. Targeting METTL3 as a novel therapeutic strategy for H3 G34-mutant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2603.