Abstract A major obstacle in the efficacy of targeted therapies of oncogene-driven tumors are drug-tolerant persister cells (DTPs) that build the basis for the outgrowth of drug-resistant clones and ultimately limit patient survival. During treatment, DTPs enter a reversible senescent state to survive therapy while cell death is induced in non-DTPs. Here, using RNA-Seq and proteomic analyses, we identified drug-induced TGFβ2 secretion in DTPs derived from different oncogene-driven lung cancer cell lines. As expected, TGFβ2 induces epithelial-to-mesenchymal transition (EMT) that over time promotes the ability of cells to survive targeted therapy. Using CRISPR/Cas9-mediated loss-of-function and reconstitution experiments we show that downstream of TGFβ2, expression of the transcription factor SNAI2 (SLUG) is essential for the outgrowth of DTPs during targeted treatment. Unsupervised RNA-Seq data analyses in combination with Cut&Tag profiling revealed that EMT signaling is in part a SLUG regulated process but that is also independent of direct SLUG binding at the transcription start sites (TSS) of EMT signature genes. In contrast, we observed high occupancy of SLUG at TSS of genes involved in the regulation of cell cycle, being repressed in DTPs and at TSS of genes that regulate sphingolipid metabolism and MAPK signaling, being induced in DTPs. In line with our previous findings, a motif-based analysis of the Cut&Tag data (TOBIAS) furthermore uncovered a tight connection between DTP outgrowth and inflammatory signaling induced through IRF7, IRF4 or STAT1 activation. In vivo, EGFR-mutant SLUG deficient cells showed a significantly prolonged tumor onset and a higher response rate to osimertinib treatment. Overall, we uncover a major role of TGFβ2/SLUG-mediated EMT signaling that may be induced indirectly through SLUG-dependent reprogramming of cell cycle directed and metabolic processes in drug tolerant cells. These insights may offer unique therapeutic opportunities to limit the outgrowth of drug resistant tumors in cancer patients. Citation Format: Jenny Ostendorp, Hannah Lea Tumbrink, Pascal Hunold, Michaela Hoehne, Philipp Jurmeister, Anastasia Dekker, Felix Heisel, Johannes Brägelmann, Frederick Klauschen, Robert Haensel-Hertsch, Martin L. Sos. Essential role of Slug during the evolution of drug-tolerance in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5626.

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit enhancer of zeste homolog 2 (EZH2) stimulates its activity by an unknown mechanism. Here, we show that human PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosteric trans-autoactivation via EED, suggesting a previously unknown mechanism mediating context-dependent activation of PRC2. Our work showcases the molecular mechanism of auto-modification-coupled dimerization in the regulation of chromatin-modifying complexes.

Breast cancer is characterised by genetic and epigenetic alterations, such as G-quadruplex (G4) DNA secondary structures. Here, we uncover differentially enriched G4 structure-forming regions (∆G4Rs) and interlinked transcriptomes in the tumour microenvironment (TME) of breast cancer PDX models in vivo . We show that well-defined breast cancer cell models non-autonomously instruct ∆G4Rs and transcriptomes in the epigenomes of primary macrophages in vitro . Mechanistically, we uncover that TNBC secretes, amongst other factors, glucocorticoids to promote G4-linked activation of octamer-binding transcription factor 1 (OCT-1) and thereby reprogramme macrophages into an immunosuppressed and immunosuppressive state. This epigenetic mechanism is of clinical importance since instructed macrophages selectively associate with the triple-negative breast cancer (TNBC) basal-like 2 (BL2) subtype and with the distinct TNBC molecular signature derived from 2,000 primary breast cancer samples. Altogether, our data suggest that G4 formation is not only prevalent in breast cancer genomes but relevant in their TMEs as well, which is of clinical importance for cancer stratification and the discovery of novel actionable drivers.