Abstract The RNA methyltransferase METTL3, catalyzing N6-methyladenosine (m6A) modification, is implicated in oncogenesis. METTL3 inhibitors have shown potent anti-tumor efficacy across diverse preclinical models and are being tested in early-phase clinical trials. However, how cells fundamentally and dynamically respond to disruption of the m6A RNA methylation machinery and what drives resistance to the catalytic inhibition of METTL3 remains unknown. Using genome-wide CRISPR sensitization and resistance screens in sensitive and resistant cancer models, we identify the nuclear m6A-reader YTHDC1 as a critical determinant of METTL3 inhibition response. Genetic depletion of YTHDC1 markedly sensitizes cells to METTL3 inhibitor (METTL3i) treatment in vitro and in vivo across solid and hematologic cancers, and its overexpression drives primary resistance. A YTHDC1-condensate imaging screen identified YTHDC1 interactor PABPN1, a PAXT complex component and a resistance hit from our CRISPR screen, as a negative regulator of YTHDC1 condensate dynamics. PABPN1 overexpression restores METTL3i sensitivity in resistant models, whereas its loss induces resistance, highlighting that the state of YTHDC1 nuclear condensates functionally mediate METTL3i response. Mechanistically, catalytic blockade of METTL3 remodels YTHDC1 condensates, increasing their intensity, size, and number while reducing their biophysical dynamics in sensitive cancer models and patient samples. In contrast, healthy human blood cells retain dynamic condensates, and resistant cancers display static condensates. Integrating direct RNA nanopore m6A mapping, bulk-RNAseq and quantitative proteomics in YTHDC1-overexpression model, we find that residual m6A modification persist on transcripts, and YTHDC1 drives restoration of MYC/BCL-2 programs upon METTL3 inhibition. Furthermore, YTHDC1 enhances binding to MYC and BCL-2 mRNAs under METTL3i treatment, preserving their translation despite global m6A loss. Accordingly, MYC overexpression phenocopies YTHDC1-mediated resistance to METTL3i, confirming its functional relevance. Therapeutically, co-targeting with a newly developed YTHDC1 inhibitor or the clinically approved BCL-2 inhibitor venetoclax synergistically enhances METTL3i efficacy in vitro and in vivo in clinically relevant models including AML patient-derived xenografts. Together, these findings uncover adaptive reorganization of the m6A machinery across hematologic and solid cancers. The YTHDC1 pathway and its condensate state act as predictive biomarkers and enhancers of therapeutic efficacy in METTL3-targeted cancers. Combinatorial targeting of METTL3 and YTHDC1/BCL-2 as a strategy to overcome therapeutic resistance. Citation Format: Xuejing Yang, Maria Eleftheriou, Eliza Yankova, Siân Evans, Theodore M. Nelson, Isaac Wakiro, Emily Batchelor, Kathryn Chang, Joao M. Dias, Aspen Pierson, Genevieve Girard, Rhoshini Raghuraman, Demetrios Aspris, Vidur Tandon, Grace Han, Lydia P. Tsamouri, Hanzhi Luo, James Russell, Maria T. Bejar, Maria P. Alcolea, Ajay Edakkara, Muxin Gu, Rupert Öllinger, Malgorzata Gozdecka, Brian J. Huntly, Roland Rad, Lina Vasiliauskaitė, Yaara Ofir-Rosenfeld, George S. Vassiliou, Yuanming Cheng, Mansi Arora, Richard C. Centore, Christopher E. Mason, Oliver Rausch, Shilpi Arora, Konstantinos Tzelepis, Michael G. Kharas. Condensate dynamics drive adaptive METTL3 inhibitor resistance abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3983.
Yang et al. (Fri,) studied this question.