Abstract PR010: In-“speck”-ting the nucleus: Nuclear speckles are a critical layer of gene regulation that predict outcomes in clear cell renal cell carcinoma

Abstract Nuclear speckles are dynamic substructures within the nucleus that contain a myriad of factors involved in gene activation. While nuclear speckles were discovered over 100 years ago, how they vary in human health and disease and the consequences of such variation remain enigmatic. Using RNA-seq data to estimate nuclear speckle content, we found that nuclear speckle phenotypes vary reproducibly in >20 cancer types. Of these cancers, nuclear speckle variation predicted patient outcomes specifically in clear cell renal cell carcinoma (ccRCC). This ccRCC speckle sensitivity was confirmed in a separate cohort using immunofluorescence to independently assess speckle phenotypes, and was specific to ccRCC with VHL mutation or copy number loss, suggesting involvement of the VHL-degraded HIF stress-responsive transcription factors. Given our previous findings linking nuclear speckles to another stress-responsive transcription factor, p53, we hypothesized that the speckle sensitivities we observed in ccRCC may be a consequence of as-of-yet uncharacterized speckle functions of HIF1A or HIF2A. Previously, we found that p53 drives DNA-speckle contacts of a subset of target genes to boost expression, revealing a novel method of transcription-factor-based gene regulation. However, the extent that DNA-speckle positioning is regulated by transcription factors other than p53, including the HIF transcription factors, is currently unclear. Critically, p53 and HIF2A, but not HIF1A, share a homologous protein motif, now coined the speckle targeting motif, that precisely overlaps with the region where we had mapped p53 DNA-speckle targeting abilities. Using genomic and imaging-based methods, we identified HIF2A as a second transcription factor that drives association between a subset of its target genes and nuclear speckles. Deletion of the HIF2A speckle targeting motifs ablated its DNA-speckle targeting functions, led to lower induction of speckle-associating genes, and resulted in a HIF2A-mediated gene expression program that more closely resembled the better-outcome ccRCC speckle patient group. Beyond p53 and HIF2A, we found that the speckle targeting motif occurs within hundreds of proteins and is specifically enriched among transcriptional regulators. These findings suggest that DNA-speckle targeting by transcription factors is potentially a wide-spread mechanism of gene regulation that may be manipulated by changing speckle-targeting abilities of transcription factors or by altering speckles themselves. For ccRCC, our findings reveal nuclear speckle phenotypes as novel prognostic indicators and illuminate nuclear speckles and HIF2A speckle-targeting abilities as new avenues for therapeutic development. Citation Format: Katherine A. Alexander, Ruofan Yu, Christine Faunce, Nicolas Skuli, Nathan Coffey, Son Nguyen, Nicholas Biddle, Catherine Li, Eric Joyce, Arjun Raj, M. Celeste Simon, Shelley Berger. In-“speck”-ting the nucleus: Nuclear speckles are a critical layer of gene regulation that predict outcomes in clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR010.