Human DEAH-box RNA helicase 8 regulates HSF1-mediated stress response and cancer-associated pre-mRNA splicing

Background: Heat Shock Factor 1 (HSF1) drives stress tolerance, oncogenic transformation, and survival in diverse human cancers. Although pharmacologic inhibition of HSF1 remains challenging, identifying druggable upstream regulators of HSF1 offers a promising alternative strategy to disrupt stress adaptation in tumours. Methods: A focused siRNA screen targeting 7,598 druggable genes was performed in human osteosarcoma cells to identify modulators of HSF1-activation. Transcriptomic, RNA immunoprecipitation (RIP), and enhanced CLIP (eCLIP) analyses were used to define the function and RNA-binding sites of the DEAH-box RNA helicase 8 (DHX8). Genetic rescue with wild-type versus ATPase- and RNA-binding-defective DHX8 mutants validated the mechanistic requirements for HSF1 mRNA processing. Functional assays (viability, cell cycle, apoptosis) assessed the effects of DHX8 depletion across tumorigenic and non-tumorigenic cell lines. Results: DHX8 was identified and validated as a potent regulator of the HSF1 stress response. DHX8 silencing led to the accumulation of intron-retained HSF1 transcripts, decreased HSF1 protein levels, and suppression of HSF1 target genes. Genome-wide RNA-seq revealed broad DHX8-dependent splicing changes, dominated by intron retention, affecting>1,300 mRNAs. eCLIP profiling identified DHX8 binding between the lariat branch site and 3′ splice junction, consistent with its role in late-stage mRNA splicing. Rescue experiments demonstrated that both ATPase and RNA-binding activities are essential for HSF1 mRNA maturation. Acute degradation of DHX8 using a dTAG system phenocopied siRNA knockdown, confirming on-target effects. Functionally, DHX8 depletion in tissue culture experiments was generally tolerated by non-tumorigenic lines, but induced G₂/M arrest, apoptosis, and loss of viability in cancer cells. Conclusions: DHX8 is a key regulator of HSF1 mRNA processing and cancer-associated splicing programs. Loss of DHX8 disrupts the oncogenic stress response, suppresses heat shock gene induction, and preferentially kills tumour cells. These findings establish DHX8 as a potentially druggable node linking RNA splicing to HSF1-driven cancer-survival pathways, offering a new therapeutic entry point for targeting stress resilience in malignancy.