Abstract 678: Identification of an E3 ligase regulating the catalytic subunit of RNA polymerase I

Abstract RNA polymerase I (Pol I) is responsible for the transcription of ribosomal DNA (rDNA), the first and rate-limiting step of ribosome biogenesis. Pol I is the most active RNA polymerase, and it comprises over 60% of transcriptional activity in cancer cells. The robust transcriptional activity, coupled with the high density of elongating Pol I transcription complexes on the rDNA, renders Pol I vulnerable to transcriptional blocks. In healthy cells, this type of transcriptional stress activates inhibitory downstream cascades such as the p53 pathway. However, cancer cells elude this regulatory mechanism to sustain high levels of growth. Further, oncogenic drivers upregulate Pol I activity and achieve abnormally high rates of ribosome biogenesis. This makes Pol I a highly relevant target for cancer therapeutics. Yet little is known about the stability and regulation of the enzyme complex and whether this knowledge can be used for therapeutic purposes. Our lab recently discovered a first-in-class small molecule, BMH-21, that inhibits Pol I transcription and induces the degradation of the catalytic subunit of Pol I, RPA194. We showed that the inducible degradation of RPA194 is cancer cell-specific, correlated with cell death, and is mediated by the proteasome system. A detailed understanding of the degradation process will provide essential knowledge about the stability of the Pol I complex and benefit therapeutic implementation of Pol I inhibitory strategies. To identify the E3 ligases involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes associated with RPA194 degradation. This screen identified a Skp-Cullin-F-box (SCF) complex as a promising candidate E3 ligase. We focused on validating the role of the identified F-box protein, FBXL14, in RPA194 degradation. To do this, we conducted knockout, knockdown, and overexpression analyses of FBXL14 in melanoma, osteosarcoma, breast cancer and chronic myelogenous leukemia cancer cell lines. We show here that both the basal expression of RPA194 and the BMH-21-induced degradation depend on FBXL14. The expression of FBXL14 significantly affected the half-life of RPA194. Knockdown of FBXL14 increases RPA194 turnover upon BMH-21 treatment, while its overexpression reduces RPA194 half-life. Using co-immunoprecipitation analyses, we demonstrate that FBXL14 interacts with RPA194 both in cell lines and in vitro. To investigate the correlation between RPA194 degradation and cell death, we analyzed how the expression of FBXL14 affects sensitivity to BMH-21 using cell growth, viability, and colony formation assays. We found that FBXL14 overexpression increased the sensitivity to the drug. Our results have revealed novel regulatory aspects of Pol I, showing that the stability of the catalytic subunit is controlled by the F-box protein FBXL14. We discuss the implications of these findings on Pol I transcription activity and its regulation. Citation Format: Stephanie Pitts, Hester Liu, Wenjun Fan, Brittany Ford, Ronald Hay, Marikki Laiho. Identification of an E3 ligase regulating the catalytic subunit of RNA polymerase I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 678.