Abstract 4413: Combined transcriptomic and epitranscriptomic profiling identifies THBS1 as a regulator of enzalutamide resistance in prostate cancer

Background: Cancer drug resistance arises not only from selection of resistant clones, but also through rapid activation of adaptive transcriptional programs. One mechanism of transcriptional regulation involves N6-methyladenosine (m6A) RNA modification, which dynamically regulates mRNA processing and alternative splicing, ultimately impacting cell fate and differentiation. In metastatic prostate cancer (PC), alternative splicing of the androgen receptor (AR) generates a truncated variant (ARV7) that is resistant to inhibition with AR antagonists like enzalutamide. Therefore, we hypothesized that m6A modifications are associated with gene expression changes that promote enzalutamide resistance in PC. Methods: We used methyl-RNA-immunoprecipitation followed by sequencing (MeRIP-seq) in parallel with RNA-seq to identify gene transcripts that were both differentially methylated and differentially expressed in enzalutamide-sensitive (C4-2B) vs. enzalutamide-resistant (MDVR) PC cell lines. Differentially methylated and expressed transcripts were further filtered and prioritized using clinical and functional database tools, including Gene Ontology (GO) enrichment analysis, Gene Set Enrichment Analysis (GSEA), The Cancer Genome Atlas (TCGA) and the Oncology Research Information Network (ORIEN) avatar. Top ranked candidates were validated by targeted MeRIP-PCR and qPCR, then tested functionally using cell viability assays combined with siRNA-depletion and enzalutamide treatment. Results: We identified 487 transcripts that were both differentially methylated and differentially expressed, then we functionally prioritized 46 genes based on PC relevance and validated 8 of the top 12 via targeted qPCR and MeRIP-PCR. In a stable enzalutamide-resistant cell line (MDVR), one such gene, THBS1, had significantly increased mRNA m6A level associated with a 41-fold decrease in transcript level. Consistent with this, short term enzalutamide treatment of C4-2B and LNCaP PC cell lines induced a 2-fold reduction in THBS1 transcript levels, accompanied by significant increases in AR and MALAT1 mRNA levels. Similarly, siRNA-mediated THBS1 depletion potentiated enzalutamide resistance in these cell lines, but it did not increase AR, ARV7, or MALAT1 levels, suggesting that alternate mechanisms mediate its effects. Conclusions: Unbiased transcriptome-wide profiling revealed a large set of differentially methylated and expressed gene transcripts associated with enzalutamide resistance in PC. One such gene, THBS1, has previously been implicated in aggressive PC phenotypes like neuroendocrine transdifferentiation. We now show that THBS1 downregulation directly contributes to a rapid transition to enzalutamide resistance, suggesting a novel role for this gene in PC hormonal therapy resistance. Citation Format: Emmanuelle Hodara, Lisa M. Swartz, Aubree Mades, Tong Xu, Suhn Rhie, Amir Goldkorn. Combined transcriptomic and epitranscriptomic profiling identifies THBS1 as a regulator of enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4413.