Epigenetic control of telomeric RNA maintains heterochromatin in telomerase-driven cancers [ChIP-Seq]

Tumor heterogeneity presents a major clinical challenge. Reactivation of telomerase (hTERT) is a near-universal hallmark of cancer, yet direct inhibition of hTERT has shown limited therapeutic benefit. To uncover tractable telomerase-related vulnerabilities, we applied a synthetic dosage lethality (SDL) strategy to identify genes required only when hTERT is overexpressed. We performed genome-wide CRISPR/Cas9 and shRNA screens across multiple isogenic cell line pairs differing in hTERT expression. From these datasets, 100 high-confidence candidates were prioritized and validated using an arrayed in vitro CRISPR screen and a pooled in vivo CRISPR screen across diverse cancer models, non-malignant cells, and patient-derived organoids. Through this pipeline, we identified FTSJ3, an RNA 2′-O-methyltransferase, as a top SDL target of hTERT. Depletion of FTSJ3 selectively impaired the viability of hTERT-positive cancer cells while sparing normal cells. Mechanistically, FTSJ3 installs 2′-O-methylation on the telomeric RNA TERRA, a modification essential for TERRA stability and function. Loss of FTSJ3 destabilizes TERRA, disrupts recruitment of the histone methyltransferase SUV39H1, and diminishes H3K9 trimethylation and HP1 assembly at sub-telomeric regions. This breakdown of repressive telomeric chromatin leads to genome instability and apoptosis specifically in hTERT-positive cells. These findings highlight SDL as a powerful strategy for uncovering telomerase-dependent liabilities and establish FTSJ3 as a central regulator of telomere stability in telomerase-active cancers. Targeting FTSJ3 enzymatic activity offers a promising therapeutic entry point, acting upstream of TERRA to eliminate telomerase-driven malignancies selectively.