Abstract 1286: Comprehensive phenotypic mapping of alternative lengthening of telomeres (ALT) regulation expands the landscape of targetable suppressors

Abstract Background: Alternative lengthening of telomeres (ALT) is used by 10-15% of all cancers as a telomerase-independent mechanism of telomere maintenance. Despite the identification of several ALT regulators, targeting has been challenging due to incomplete penetrance across cell lines and a limited understanding of the broader regulatory landscape. A major barrier to systematic discovery has been the lack of scalable assays compatible with high-throughput screening approaches. Methods: Native fluorescence in situ hybridization (FISH) detecting ALT-specific single-stranded telomeric DNA was adapted and optimized for hybridization in-suspension with a fluorescent G-rich telomere PNA probe (TelG-647) and fluorescence-activated cell sorting (ALT-FlowFISH). Genome-wide CRISPR knockout screens using the TKOv3 library were performed, followed by selection and ALT-FlowFISH at one week following transduction and selection. Cells with the highest and lowest Native FISH signals were sorted (candidate ALT suppressors and promoters, respectively) and integrated sgRNA sequences were amplified and sequenced. Significantly enriched guides were identified as candidate ALT suppressors and promoters and were validated using targeted depletion (sgRNA/Cas9 RNP) and pharmacological inhibition followed by ALT assay validation (Native FISH and C-circle assay). Results: ALT-FlowFISH was validated using depletion of established regulators FANCM (suppressor) and BLM (promoter), which resulted in expected increase and decrease of native FISH signal, respectively. Genome-wide phenotypic screens were performed in ALT-positive cell lines U2OS and RPE-ALT, followed by ALT-FlowFISH. These identified established ALT promoters, including components of the BTR complex, and suppressors, including FANCM, validating the screening approach. Overall, ALT suppressors were significantly enriched for known essential genes compared to ALT promoters. Importantly, we discovered and validated multiple novel ALT suppressor pathways, including components of the proteasome, the polyadenylation complex, RNA polymerase II machinery, and previously unrecognized members of the Fanconi anemia pathway. To confirm the functional relevance of these findings, we treated multiple ALT-positive cell lines with the proteasome inhibitor bortezomib, which recapitulated the suppressor phenotype observed in the genetic screens, demonstrating therapeutic potential. Conclusions: This study establishes the first scalable ALT activity assay and provides the most comprehensive functional map of ALT regulation to date. Our identification of the proteasome and transcriptional machinery as novel ALT suppressors reveals potential precision medicine opportunities for targeting ALT-positive malignancies. Citation Format: Bill Diplas, Oluchi Ezekwenna, Madison Rex, Mingxuan Wei, Agnel Sfeir. Comprehensive phenotypic mapping of alternative lengthening of telomeres (ALT) regulation expands the landscape of targetable suppressors [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 1286.